Method of performing pcr with a mult-ilane cartridge

ES 2 587 007 T3

DESCRIPTION

Integrated system for processing microfluidic samples, and methods of using it

Technical field

The technology described herein refers to an integrated apparatus for processing samples containing polynucleotides and performing diagnostic analysis on them. More specifically, the technology relates to an apparatus for obtaining a diagnostic result on a biological sample using a microfluidic cartridge that receives the sample, in conjunction with a benchtop system. Methods for using the technology are also described in this document.

Background

The medical diagnostics industry is a critical element of today's healthcare infrastructure. Today, however, diagnostic tests, no matter how routine, have become a bottleneck in patient care. There are several reasons for this. First, there are usually several stages in a diagnostic analysis between collecting the sample and obtaining a diagnostic result, requiring different levels of skill on the part of the operators, and different levels of complexity of the equipment. For example, a biological sample, once extracted from a patient, must be put into a form suitable for a processing regimen that typically involves using polymerase chain reaction (PCR) to amplify a nucleotide of interest. Once amplified, the presence of a nucleotide of interest in the sample needs to be unequivocally determined. Sample preparation is a process that is capable of automation but is also carried out relatively routinely in almost any location. In contrast, steps such as PCR and nucleotide detection have generally been only available to specially trained individuals who have access to specialized equipment. Second, many diagnostic tests can only be performed with highly specialized equipment that is both expensive and manageable only by trained practitioners. Such equipment is found in only a few locations - often only one in any given urban area. This means that most hospitals have to outsource samples to those locations for analysis, thus incurring shipping costs and transportation delays, and possibly even loss or mixing of samples. Third, some specialized equipment is not normally available “on demand” but instead works in batches, thus delaying the processing time for many samples, as they must wait for a machine to fill beforehand. that they can be analyzed.

Analysis of a biological sample to achieve a particular diagnosis usually includes detecting one or more polynucleotides present in the sample. An example of detection is qualitative detection, which refers, for example, to the determination of the presence of the polynucleotide and / or the determination of information related to, for example, the type, size, presence or absence of mutations, and / or the sequence of the polynucleotide. Another example of detection is quantitative detection, which refers, for example, to determining the amount of polynucleotide present. Detection can therefore generally include both qualitative and quantitative aspects. Qualitatively detecting polynucleotides often involves establishing the presence of extremely small amounts in a sample. To improve sensitivity, therefore, the amount of polynucleotide in question is often amplified. For example, some detection methods include amplification of polynucleotides by polymerase chain reaction (PCR) or a related amplification technique. Such techniques use a cocktail of ingredients, which include one or more of an enzyme, a probe, and a labeling agent. Therefore, the detection of polynucleotides may require the use of a number of different reagents, many of which require sensitive handling to maintain their integrity, both during use and over time.

Understanding that the sample flow is broken down into several key stages, it would be desirable to consider ways to automate as many of these as possible and, desirably, facilitate the achievement of as many as possible with a single machine that can be made available, upon request. , from many users. There is, therefore, a need for a method and apparatus for carrying out sample preparation, PCR, and detection steps on biological samples in such a manner that as few independent steps as possible are carried out.

European Patent Application Publication No. 15745862 describes an apparatus and method for amplifying nucleic acids. The apparatus includes a substrate, a reaction vessel formed within the substrate, and a heater unit installed on the substrate. The heater unit includes a heater for heating the substrate to a predetermined temperature and for periodically heating and cooling the substrate. Figures 9A and 9B show a substrate with two reaction vessels.

The description of the technology background in this document is included to explain the technology context. This should not be taken as an admission that any of the aforementioned material was published, was known, or was part of common general knowledge as at the priority date of any of the claims.

ES 2 587 007 T3

Throughout the description and claims of the specification, the word "comprise" and variations thereof, such as "comprising / n" and "comprises", are not intended to exclude other additives, components, integers or steps.

Summary

The invention is defined in the independent claims, referred to below. Preferred features are set out in the dependent claims.

In accordance with one aspect of the present invention, an apparatus comprises a receiving compartment configured to receive an insertable multi-lane microfluidic cartridge, each lane comprising a PCR reaction zone configured to accept a sample containing polynucleotides. The apparatus further comprises a plurality of heater assemblies, each heater assembly thermally coupled to a PCR reaction zone on the multi-lane cartridge. Each set of heaters comprises a plurality of heaters together configured to cyclically heat the PCR reaction zone under thermal cycling conditions. The plurality of heaters are further configured to maintain a substantially uniform temperature throughout the PCR reaction zone during each phase of thermal cycling. The apparatus further comprises a detector configured to detect the presence of one or more amplified polynucleotides in the multilane cartridge. The apparatus further comprises a processor configured to independently control each heater to cyclically heat each pCr reaction zone of the multi-rail cartridge. The processor is further configured to perform independent PCR reactions on samples containing polynucleotides in the PCR reaction zones of the multi-lane cartridge.

The application also reveals:

An apparatus, comprising: a receiving compartment configured to receive an insertable microfluidic cartridge; at least one heat source thermally coupled to the cartridge and configured to apply heat to one or more selected regions of the cartridge at one or more selected times, in order to: create a microdroplet of a biological sample containing polynucleotides contained in the cartridge; causing the microdroplet to move between one or more positions on the microfluidic cartridge; lysing cells, where present in the biological sample, thereby releasing polynucleotides from the cells; preparing one or more of the polynucleotides for amplification; and amplifying one or more of the polynucleotides; a detector configured to detect the presence of the one or more amplified polynucleotides; and a processor coupled to the detector and the at least one heat source, wherein the processor is configured to control the application of heat to the one or more selected regions of the microfluidic cartridge at one or more selected times.

The system further comprises an integrated system, comprising an apparatus and a complementary cartridge, where together, the apparatus and the cartridge process a sample that has been injected into the cartridge, and provide a diagnostic result on the sample.

The receiving compartment of the apparatus may be configured to selectively receive the microfluidic cartridge, as further described herein and exemplified by the accompanying drawings. For example, the receiving compartment and the microfluidic cartridge can be complementary in shape, so that the microfluidic cartridge can be selectively received in, for example, a single orientation. The microfluidic cartridge may have a correct positioning member that fits a complementary feature of the receiving compartment. By selectively receiving the cartridge, the receiving compartment can assist a user in positioning the cartridge, so that the apparatus can properly operate on the cartridge. The receiving compartment can also be configured so that various components of the apparatus that can operate on the microfluidic cartridge (heat pumps, Peltier coolers, electronic elements that remove heat, detectors, force members, and the like) can be positioned to operate. appropriately on the microfluidic cartridge. For example, a contact heat source may be located in the receiving compartment so that it can be thermally coupled to one or more other locations on a microfluidic cartridge that may be selectively received in the receiving compartment.

The heat pump can be, for example, a heat source such as a resistor, a reversible heat pump such as a liquid-filled heat transfer circuit or a thermoelectric element, a radiation heat source such as a lamp xenon, and the like. The heat pump can be used not only to provide heat to microfluidic elements but also to remove heat from microfluidic elements such as to reduce the activity of certain reagents, freeze liquid in a microchannel to change its phase from liquid to solid, reduce pressure of an air chamber to create a partial vacuum, etc.)

In various embodiments of the apparatus: the apparatus may further include a correct positioning member that is complementary to the microfluidic cartridge, whereby the receiving compartment receives the microfluidic cartridge in a single orientation; The apparatus may further include a sensor coupled to a processor, the sensor configured to detect whether the microfluidic cartridge can be selectively received.

ES 2 587 007 T3

The processor may be programmable to operate the detector to detect a polynucleotide or a probe thereof in a microfluidic cartridge located in the receiving compartment.

The detector can be, for example, an optical detector. For example, the detector may include a light source that emits light in an absorption band of a fluorescent dye and a light detector that detects light in an emission band of the fluorescent dye, where the fluorescent dye corresponds to a fluorescent polynucleotide probe. or a fragment of it. For example, the optical detector may include a band-pass filter diode that selectively emits light in the fluorescent dye absorption band and a band-pass filter photodiode that selectively detects light in the fluorescent dye emission band; or for example, the optical detector may be configured to independently detect a plurality of fluorescent dyes having different fluorescent emission spectra, where each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof; or for example, the optical detector may be configured to independently detect a plurality of fluorescent dyes at a plurality of different locations on the cartridge, where each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof.

The processor can be, for example, programmable to operate the at least one heat pump.

In various embodiments, the at least one heat pump can be a contact heat source selected from a resistive heater, a radiator, a fluidic heat exchanger, and a Peltier device. The contact heat source may be configured in the receiving compartment to be thermally coupled to a different location on a microfluidic cartridge received in the receiving compartment, whereby the different location can be selectively heated. At least one additional contact heat source may be included, where the contact heat sources may each be configured in the receiving compartment to be independently thermally coupled to a different distinct location on a microfluidic cartridge received at the receiving compartment, so that different locations can be heated independently. The contact heat source may be configured to be in direct physical contact with a location other than a received microfluidic cartridge in the receiving compartment. In various embodiments, each contact heat source can be configured to heat a different location that has a 2-dimensional average diameter of from about 1 millimeter (mm) to about 15mm (typically from about 1mm to about 10mm), or a distinct location that has a surface area of ​​between approximately 1mm² and about 225 mm² (normally between about 1 mm² and about 100 mm², or in some embodiments between about 5mm² and about 50 mm²).

In various embodiments, the apparatus may include a flexible layer on the contact heat source, configured to thermally couple the contact heat source with at least a portion of a microfluidic cartridge received in the receiving compartment. The flexible layer can have a thickness of between about 0.05 and about 2 millimeters, and a Shore hardness of between about 25 and about 100.

In various embodiments, the at least one heat pump can be a radiation heat source configured to direct heat to a location other than a received microfluidic cartridge in the receiving compartment.

In various embodiments, the one or more force members are configured to apply force to at least a portion of a microfluidic cartridge received in the receiving compartment.

In various embodiments, the one or more force members may be configured to apply force to thermally couple the at least one heat pump to at least a portion of the microfluidic cartridge. The one or more force members may be configured to operate a mechanical member in the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir, valve, or pump.

In various embodiments, the one or more force members can be configured to apply force to a plurality of locations on the microfluidic cartridge. The force applied by the one or more force members can result in an average pressure at an interface between a part of the receiving compartment and a part of the microfluidic cartridge of between about 5 kilopascals and about 50 kilopascals, for example, the average pressure. it can be at least about 14 kilopascals. At least one force member can be operated manually. At least one force member can be mechanically coupled to a lid in the receiving compartment, whereby operation of the lid operates the force member.

In various embodiments, the apparatus may further include a lid in the receiving compartment, the lid being operable to at least partially exclude ambient light from the receiving compartment. The lid can be, for example, a sliding lid. The lid may include the optical detector. A fundamental face of the cap in the optical detector or in the receiving compartment can vary from flatness by less than about 100 microns, eg, less than about 25 microns. The lid can be configured to

ES 2 587 007 T3 be removable from the device. The cap can include a holding member.

In various embodiments, the apparatus may further include at least one input device coupled to the processor.

In various embodiments, the apparatus may further include a heating phase configured to be removable from the apparatus, where at least one heat pump may be located in the heating phase.

In various embodiments, the cartridge can further include a test port. The analysis port can be configured to allow an external sample system to analyze a sample in the microfluidic cartridge; for example, the analysis port can be a hole or window in the apparatus that can accept an optical detection probe that can analyze a sample in situ in the microfluidic cartridge.

In some embodiments, the test port may be configured to direct a sample from the microfluidic cartridge to an external sample system; for example, the analysis port may include a conduit in fluid communication with the microfluidic cartridge that directs a liquid sample to a chromatography apparatus, an optical spectrometer, a mass spectrometer, or the like.

In some embodiments, the apparatus may include a receiving compartment configured to receive a microfluidic cartridge in a single orientation; at least one radiation heat source thermally coupled to the receiving compartment; at least two contact heat sources configured in the receiving compartment to be thermally coupled to different locations, whereby the different locations can be selectively heated; one or more force members configured to apply force to at least a portion of the microfluidic cartridge received in the receiving compartment, where at least one of the one or more force members may be configured to apply force to thermally couple the heat sources by contact to the different locations, and at least one of the one or more force members may be configured to operate a mechanical member in the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir; a cover in the reception compartment, the cover being maneuverable to at least partially exclude ambient light from the reception compartment, the cover comprising an optical detector configured to independently detect one or more fluorescent dyes, optionally having different fluorescent emission spectra, where each fluorescent dye corresponds to a fluorescent polynucleotide probe or a fragment thereof; at least one input device selected from the group consisting of a keyboard, a touch-sensitive surface, a microphone and a mouse, at least one data storage medium selected from the group consisting of a hard disk drive, a optical disc drive, a communication interface selected from the group consisting of: a serial connection, a parallel connection, a wireless network connection, and a wired network connection, a sample identifier selected from an optical character reader, a barcode reader, and a radio frequency mark reader ; at least one output selected from a screen, a printer, a speaker; and a processor coupled to the detector, sensor, heat sources, input, and output.

A microfluidic cartridge can include a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor. In some embodiments, the microfluidic cartridge also includes a correct positioning member.

In various embodiments of the microfluidic cartridge, the microfluidic cartridge may further include a sample inlet valve in fluid communication with the microfluidic network. The sample inlet valve may be configured to accept a sample at a pressure differential compared to ambient pressure of between about 20 kilopascals and 200 kilopascals, for example between about 70 kilopascals and 110 kilopascals.

In various embodiments, the microfluidic network may include a filter in fluid communication with the sample inlet valve, the filter configured to separate at least one component of a sample mixture introduced into the sample inlet.

In various embodiments, the microfluidic network can include at least one thermally driven pump in fluid communication with the microfluidic network. The thermally driven pump can include a thermo-expansive material selected from a gas, a vaporizable liquid at a temperature between 25 ° C and 100 ° C at 1 atmosphere, and an expancel polymer.

In various embodiments, the microfluidic network can include at least one thermally actuated valve in fluid communication with the microfluidic network. The thermally actuated valve may include a material having a solid to liquid phase transition at a temperature between 25 ° C and 100 ° C at 1 atmosphere.

In various embodiments, the microfluidic network can include at least one sealed reservoir that contains a reagent, a buffer, or a solvent. The sealed reservoir can be, for example, a self-piercing blister package configured

ES 2 587 007 T3 to bring the reagent, buffer or solvent into fluid communication with the microfluidic network.

In various embodiments, the microfluidic network can include at least at least one hydrophobic vent.

In various embodiments, the microfluidic network can include at least one reservoir configured to receive and contain debris such as fluids and / or particulate material such as cellular debris.

In various embodiments, the retention member can include a polyalkyleneimine or a polycationic polyamide, for example, polyethyleneimine, poly-L-lysine, or poly-D-lysine. The retention member can be in the form of one or more particles. The retention member may be removable from the microfluidic cartridge.

In various embodiments, the microfluidic network can include a lysis reagent. The lysis reagent may include one or more lyophilized surfactant pellets, where the microfluidic network may be configured to contact the lyophilized surfactant pellet with a liquid to create a lysis reagent solution. The microfluidic network can be configured to contact a sample with the lysis reagent to produce a lysed sample.

In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to the sample to produce the lysed sample. For example, the microfluidic network can be configured to contact the retention member and the lysed sample to create a polynucleotide loaded retention member.

In various embodiments, the microfluidic cartridge may further include a filter configured to separate the polynucleotide laden retention member from the liquid.

In various embodiments, the microfluidic cartridge may further include a reservoir containing a wash buffer, where the microfluidic network may be configured to contact the polynucleotide loaded retention member with the wash buffer, eg, the buffer. Wash can have a pH of at least about 10.

In various embodiments, the microfluidic cartridge may include a reservoir containing a release buffer, where the microfluidic cartridge may be configured to contact the polynucleotide loaded retention member with the release buffer to create a sample of released polynucleotides.

In various embodiments, the microfluidic network can be configured to couple heat from an external heat source to the polynucleotide loaded retaining member to create the released polynucleotide sample.

In various embodiments, the microfluidic cartridge may include a reservoir containing a neutralization buffer, where the microfluidic network may be configured to contact the released polynucleotide sample with the neutralization buffer to create a neutralized polynucleotide sample.

In various embodiments, the microfluidic cartridge can include a PCR reagent mix comprising a polymerase enzyme and a plurality of nucleotides. The PCR reagent mix can be in the form of one or more lyophilized microbeads, and the microfluidic network can be configured to contact the PCR microbead with liquid to create a PCR reagent mix solution.

In various embodiments, the microfluidic network may be configured to couple heat from an external heat source with the mixture of PCR reagents and the sample with neutralized polynucleotides under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides.

In various embodiments, the PCR reagent mix may further include a positive control plasmid and a selective fluorogenic hybridization probe for at least a portion of the plasmid.

In various embodiments, the microfluidic cartridge can include a negative control polynucleotide, wherein the microfluidic network may be configured to independently contact each of the sample with neutralized polynucleotides and the negative control polynucleotide with the PCR reagent mix under suitable thermal cycling conditions to independently create PCR amplicons of the sample with polynucleotides neutralized and negative control polynucleotide PCR amplicons.

In various embodiments, the microfluidic cartridge can include at least one probe that can be selective for a polynucleotide sequence, where the microfluidic cartridge can be configured to contact the sample with neutralized polynucleotides or a PCR amplicon thereof with the probe. . The probe can be a fluorogenic hybridization probe. The fluorogenic hybridization probe can include a polynucleotide sequence coupled to a fluorescent reporter dye and a fluorescence quenching dye. The reagent mix of

ES 2 587 007 T3

PCR may further include a positive control plasmid and a plasmid fluorogenic hybridization probe selective for at least a portion of the plasmid and the microfluidic cartridge may be configured to allow independent optical detection of the fluorogenic hybridization probe and the hybridization probe. plasmid fluorogenic.

In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism, for example any organism that employs deoxyribonucleic acid or ribonucleic acid polynucleotides. Thus, the probe can be selective for any organism. Suitable organisms include mammals (including humans), birds, reptiles, amphibians, fish, domestic animals, farm animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for components of organisms that employ their own polynucleotides, for example mitochondria. In some embodiments, the probe may be selective for microorganisms, for example, organisms used in food production (for example, yeasts used in fermented products, molds or bacteria used in cheeses, and the like) or pathogens (for example, of human beings). humans, domestic or wild mammals, domestic or wild birds, and the like). In some embodiments, the probe can be selective for organisms selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of group B Streptococcus.

In various embodiments, the microfluidic cartridge can be configured to allow optical detection of the fluorogenic hybridization probe.

In various embodiments, the microfluidic cartridge may further include a computer-readable label. For example, the label may include a barcode, a radio frequency marking, or one or more computer-readable characters. The label can be formed of a mechanically flexible material. For example, the mechanically flexible label material can have a thickness of between about 0.05 and about 2 millimeters and a Shore hardness of between about 25 and about 100.

In various embodiments, the microfluidic cartridge may be further surrounded by a sealed pouch, during handling and storage, and prior to being inserted into the chamber. The microfluidic cartridge can be sealed in the sachet with an inert gas. The sealed sachet can also contain a desiccant packet. The microfluidic cartridge can be disposable.

In various embodiments, the microfluidic cartridge can contain one or more sample lanes. For example, a sample rail can include a thermally actuated pump, a thermally actuated valve, a sample inlet valve, a filter, and at least one reservoir. The lanes can be independent of each other, or they can be partially dependent, for example, the lanes can share one or more reagents such as the lysis reagent.

In some embodiments, the microfluidic cartridge may include a correct positioning member; and a microfluidic network. The microfluidic network includes, in fluidic communication: at least one thermally driven pump; at least one thermally actuated valve; a sample inlet valve configured to accept a sample at a pressure differential compared to an ambient pressure of between about 70 kilopascals and 110 kilopascals; a selective retention member for at least one polynucleotide over at least one polymerase chain reaction inhibitor, the retention member being in the form of a plurality of particles formed of a polyalkyleneimine or a polycationic polyamide; a filter configured to separate the polynucleotide laden retention member from the liquid; a plurality of reservoirs, at least one reservoir being a sealed, self-piercing blister pack reservoir. The plurality of reservoirs may contain among them: a lysis reagent, the microfluidic network being configured to contact a sample introduced at the sample inlet with the lysis reagent and the retention member to create a polynucleotide loaded retention member. ; a reservoir containing a wash buffer, the microfluidic network configured to contact the polynucleotide laden retention member with the wash buffer; a reservoir containing a release buffer, the microfluidic network configured to contact the polynucleotide loaded retention member with the release buffer to create a sample with released polynucleotides; a neutralization buffer, the microfluidic network being configured to contact the sample with released polynucleotides with the neutralization buffer to create a sample with neutralized polynucleotides; a PCR reagent mix comprising a polymerase enzyme, a positive control plasmid, a selective fluorogenic hybridization probe for at least a part of the plasmid and a plurality of nucleotides; and at least one probe that can be selective for a polynucleotide sequence, where the microfluidic network can be configured to contact the sample with neutralized polynucleotides or a PCR amplicon thereof with the probe. In addition, the microfluidic network can be configured to couple heat from an external heat source to the mixture of PCR reagents and the sample with neutralized polynucleotides under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides. .

ES 2 587 007 T3

In various embodiments, a polynucleotide analysis system can include both the microfluidic cartridge and the apparatus, as further described herein.

In various embodiments, a polynucleotide sample kit may include a microfluidic cartridge comprising a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one inhibitor. from the polymerase chain reaction, a sample container; and a liquid transfer member such as a syringe.

In various embodiments, the polynucleotide sample kit may further include instructions for using the liquid transfer member to transfer a sample from the sample container to the microfluidic network.

In various embodiments, the polynucleotide sample kit may further include instructions for using the liquid transfer member to direct a sample from the sample container, and a volume of air into the microfluidic network, the volume being air between about 0.5 ml and about 5 ml.

In various embodiments, the polynucleotide sample kit may further include a filter, and, for example, instructions for using the liquid transfer member to direct a sample from the sample container through the filter into the network. microfluidics.

In various embodiments, the polynucleotide sample kit may further include at least one computer-readable label on the sample container. The label may include, for example, a barcode, a radio frequency mark, or one or more computer-readable characters. The microfluidic cartridge can be sealed in a sachet with an inert gas.

In various embodiments, the polynucleotide sample kit may further include a sampling member; a transfer container; and instructions for contacting the sampling member with a biological sample and for placing the sampling member in the transfer container.

In various embodiments, the polynucleotide sample kit may further include a sample buffer, and, for example, instructions for contacting the sampling member and the sample buffer.

In various embodiments, the polynucleotide sample kit may further include at least one probe that may be selective for a polynucleotide sequence, for example, the polynucleotide sequence that is characteristic of a pathogen selected from the group consisting of bacteria. gram positive, gram negative bacteria, yeast, fungi, protozoa, and viruses.

In some embodiments, the polynucleotide sample kit may include a microfluidic cartridge comprising a microfluidic network, a retention member in fluid communication with the microfluidic network, and a fluorogenic probe, the retention member being selective for at least one polynucleotide with respect to to at least one polymerase chain reaction inhibitor, the fluorogenic probe being selective for a polynucleotide sequence that may be characteristic of a pathogen selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses; a sample container; a liquid transfer member; a sampling member; a transfer container; a sample buffer; and instructions. The instructions may include instructions to: use the liquid transfer member to transfer a sample from the sample container to the microfluidic network; employing the liquid transfer member to direct a sample from the sample container and a volume of air into the microfluidic network, the volume of air being between about 0.5 ml and about 5 ml; employing the liquid transfer member to direct a sample from the sample container through a filter into the microfluidic network; and contacting the sampling member with a biological sample and placing the sampling member in the transfer container.

A method of sampling a polynucleotide may include the steps of contacting the retention member on the microfluidic cartridge with a biological sample, the biological sample comprising at least one polynucleotide, thereby producing a polynucleotide-loaded retention member on the cartridge. microfluidic; separating at least a portion of the biological sample from the polynucleotide loaded retention member; and releasing at least a portion of a polynucleotide from the polynucleotide loaded retention member, thereby creating a sample with released polynucleotides.

In various embodiments, the method may further include one or more of the following steps: placing the microfluidic cartridge in the receiving compartment of the apparatus; operating the force member in the apparatus to apply pressure at an interface between a portion of the receiving compartment and a portion of the microfluidic cartridge (for example, creating a pressure between about 5 kilopascals and about 50 kilopascals, or in some embodiments, by minus about 14 kilopascals); use the force member to apply

ES 2 587 007 T3 forces a mechanical member in the microfluidic cartridge, the mechanical member selected from the group consisting of a pierceable reservoir, valve, or pump, to release at least one reagent, buffer, or solvent from a deposit on the microfluidic chip; and / or closing the lid to operate the force member, where the force member may be mechanically coupled to a lid in the receiving compartment.

In some embodiments, the method may further include employing a sample identifier to read a label on the microfluidic cartridge or a label on the biological sample.

In some embodiments, the method may further include introducing an impure biological sample into the microfluidic cartridge and separating the biological sample from the impure biological sample in the microfluidic cartridge, for example, using a filter on the cartridge, or the biological sample may Separate from an impure biological sample before introducing the biological sample into the microfluidic cartridge.

In some embodiments, the method may further include lysing the biological sample, for example, using heat, a lysis reagent, and the like. In some embodiments, where the microfluidic cartridge comprises one or more lyophilized pellets of lysis reagent, the method may further include reconstituting the lyophilized pellet of surfactant with liquid to create a lysis reagent solution.

In various embodiments, the method may further include one or more of the following: heating the biological sample in the microfluidic cartridge; pressurizing the biological sample in the microfluidic cartridge at a pressure differential compared to ambient pressure of between about 20 kilopascals and 200 kilopascals, or in some embodiments between about 70 kilopascals and 110 kilopascals.

In some embodiments, the part of the biological sample separated from the polynucleotide loaded retention member may include at least one polymerase chain reaction inhibitor selected from the group consisting of hemoglobin, peptide, fecal compounds, humic acid, compounds mucosal, DNA-binding proteins, or a saccharide. In some embodiments, the method may further include separating the polynucleotide loaded retention member from substantially all of the polymerase chain reaction inhibitor in the biological sample.

In various embodiments, the method may further include one or more of the following: directing a fluid in the microfluidic cartridge by operating a thermally actuated pump or thermally actuated valve; contacting the polynucleotide loaded retention member with a wash buffer; heating the polynucleotide loaded retention member to a temperature of at least about 50 ° C (in some embodiments, the temperature may be 100 ° C or less); heating the polynucleotide loaded retention member for less than about 10 minutes; contacting the polynucleotide loaded retention member with a release buffer to create a sample with released polynucleotides (e.g., in some embodiments, the release buffer may have a volume of less than about 50 microliters, the release buffer may include a detergent, and / or the release buffer can have a pH of at least about 10); and / or contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample.

In various embodiments, the method may further include one or more of the following: contacting the sample with neutralized polynucleotides with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides (in some embodiments, the mixture PCR reagents may further include a positive control plasmid and a selective fluorogenic hybridization probe for at least a part of the plasmid); In some embodiments, the PCR reagent mix may be in the form of one or more lyophilized microbeads, and the method may further include reconstituting the PCR microbead with liquid to create a PCR reagent mix solution; heating the mixture of PCR reagents and the sample with neutralized polynucleotides under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides; contacting the sample with neutralized polynucleotides or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; independently contacting each of the sample with neutralized polynucleotides and a negative control polynucleotide with the PCR reagent mixture under suitable thermal cycling conditions to independently create PCR amplicons of the sample with neutralized polynucleotides and PCR amplicons of the polynucleotide of negative control; and / or contacting the sample with neutralized polynucleotides or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence.

In various embodiments, the method may further include one or more of the following:

determining the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the sample with neutralized polynucleotides or a PCR amplicon thereof; determining a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof; and / or in some embodiments, where the PCR reagent mix further comprises a positive control plasmid and a

ES 2 587 007 T3 selective plasmid probe for at least a part of the plasmid, the method further including determining that a PCR reaction has occurred if the plasmid probe is detected.

In various embodiments, the method does not comprise spinning the polynucleotide loaded retention member.

In some embodiments, the method for sampling a polynucleotide can include:

placing a microfluidic cartridge in the receiving compartment of an apparatus; operating a force member in the apparatus to apply pressure at an interface between a part of the receiving compartment and a part of the microfluidic cartridge, the force operating to release at least one reagent, buffer, or solvent from a reservoir in the microfluidic cartridge; lysing a biological sample on the microfluidic cartridge to create a lysed biological sample; contacting a retention member in a microfluidic cartridge with the lysed biological sample, the biological sample comprising at least one polynucleotide, thereby producing a polynucleotide loaded retention member in the microfluidic cartridge, where the retention member is in the form of a plurality of particles of a polyalkyleneimine or a polycationic polyamide; contacting the polynucleotide loaded retention member with a wash buffer; heating the polynucleotide loaded retention member at a temperature of at least about 50 ° C for less than about 10 minutes; contacting the polynucleotide loaded retention member with a release buffer to create a sample with released polynucleotides. contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample; contacting the sample with neutralized polynucleotides with a PCR reagent mixture under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides, the PCR reagent mixture comprising a polymerase enzyme, a control plasmid positive, a selective fluorogenic hybridization probe for at least a part of the plasmid, and a plurality of nucleotides, contacting the sample with neutralized polynucleotides or a PCR amplicon thereof with at least one fluorogenic probe that can be selective for a polynucleotide sequence, where the probe can be selective for a polynucleotide sequence that can be characteristic of an organism selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses; and detecting the fluorogenic probe and determining the presence of the organism for which the fluorogenic probe can be selective.

In various embodiments, a computer program product includes computer-readable instructions therein for operating the apparatus.

In some embodiments, a computer program product includes computer-readable instructions therein to cause the system to create a sample with released polynucleotides from a biological sample. Computer-readable instructions may include instructions for contacting the retention member with the biological sample under conditions suitable to produce a polynucleotide loaded retention member; separating at least a portion of the biological sample from the polynucleotide loaded retention member; and releasing at least a portion of a polynucleotide from the polynucleotide loaded retention member, thereby creating a sample with released polynucleotides.

In various embodiments, the computer program product may include one or more instructions for causing the system to: output an indicator of the placement of the microfluidic cartridge in the receiving compartment; read a sample label or microfluidic cartridge label; issue prompts for a user to enter a sample identifier; issue prompts for a user to load a sample transfer member with the biological sample; issue prompts for a user to apply a filter to the sample transfer member; issue prompts for a user to insert the biological sample into the microfluidic cartridge; issue prompts for a user to bring the biological sample into contact with a lysis reagent on the microfluidic cartridge; issue prompts for a user to place the microfluidic cartridge in the receiving compartment; issue prompts for a user to operate a force member on the apparatus to apply pressure at an interface between a portion of the receiving compartment and a portion of the microfluidic cartridge; issue prompts for a user to close the lid to operate the force member; and / or issue prompts for a user to pressurize the biological sample in the microfluidic cartridge by injecting a volume of air between about 0.5 ml and about 5 ml into the biological sample.

In various embodiments, the computer program product can include one or more instructions for causing the system to: lize the biological sample; lise the biological sample with a lysis reagent; Reconstitute a lyophilized surfactant microgranule with liquid to create a lysis reagent solution; heat the biological sample; separate the polynucleotide loaded retention member from at least a portion of the biological sample; separate the polynucleotide laden retention member from substantially all polymerase chain reaction inhibitors in the biological sample; direct a fluid in the microfluidic cartridge by operating a thermally actuated pump or thermally actuated valve; contact the polynucleotide loaded retention member with a wash buffer; heat the retaining member loaded with

ES 2 587 007 T3 polynucleotides at a temperature of at least about 50 ° C (in some embodiments, the temperature may be about 100 ° C or lower); heat the polynucleotide loaded retention member for less than about 10 minutes; contacting the polynucleotide loaded retention member with a release buffer to create a sample with released polynucleotides; and / or contact the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample.

In various embodiments, the computer program product may include one or more instructions for causing the system to: contact the sample with neutralized polynucleotides with a PCR reagent mixture comprising a polymerase enzyme and a plurality of nucleotides; heat the mixture of PCR reagents and the sample with neutralized polynucleotides under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides; contact the sample with neutralized polynucleotides or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; independently contact each sample with neutralized polynucleotides and a negative control polynucleotide with the PCR reagent mixture under suitable thermal cycling conditions to independently create sample PCR amplicons with neutralized polynucleotides and PCR amplicons of the polynucleotide negative control; contact the sample with neutralized polynucleotides or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that can be selective for a polynucleotide sequence; issue a determination of the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the sample with neutralized polynucleotides or a PCR amplicon thereof; and / or issue a determination of a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof.

In various embodiments, the computer program product may include one or more instructions for causing the system to automatically carry out one or more of the steps of the method.

In various embodiments, where the microfluidic network comprises two or more sample lanes each including a thermally actuated pump, a thermally actuated valve, a sample inlet valve, a filter, and at least one reservoir, where the instructions Computer-readable may be configured to independently operate each said lane in the system.

In some embodiments, the computer program product includes computer-readable instructions therein for causing a system to create a sample with released polynucleotides from a biological sample. The system may include a microfluidic cartridge comprising a microfluidic network and a retention member in fluid communication with the microfluidic network, the retention member being selective for at least one polynucleotide over at least one polymerase chain reaction inhibitor. ; and an apparatus comprising a receiving compartment configured to selectively receive the microfluidic cartridge; at least one heat pump configured to be thermally coupled to the microfluidic cartridge in the receiving compartment; a detector; and a programmable processor coupled to the detector and heat pump. Computer-readable instructions can include instructions to: lyse a biological sample by contacting the biological sample with a lysis reagent and heating to produce a lysed sample; contacting the retention member with the biological sample and / or the lysed sample to produce a polynucleotide loaded retention member; separating at least a portion of the biological sample from the polynucleotide loaded retention member; contacting the polynucleotide loaded retention member with a wash buffer; contacting the polynucleotide loaded retention member with a release buffer and / or heating to release at least a portion of a polynucleotide from the polynucleotide loaded retention member, thereby creating a sample with released polynucleotides; contacting the released polynucleotide sample with a neutralization buffer to create a neutralized polynucleotide sample; independently contacting each of the sample with neutralized polynucleotides and a negative control polynucleotide with a PCR reagent mixture under suitable thermal cycling conditions to independently create PCR amplicons, the PCR reagent mixture comprising a polymerase enzyme, a plurality of nucleotides, a positive control plasmid and a selective plasmid probe for at least a part of the plasmid; determining that a PCR reaction has occurred if the plasmid probe is detected; contacting the sample with neutralized polynucleotides or a PCR amplicon thereof and the negative control polynucleotide or a PCR amplicon thereof with at least one probe that is selective for a polynucleotide sequence; determining the presence of a polynucleotide sequence in the biological sample, the polynucleotide sequence corresponding to the probe, if the probe is detected in the sample with neutralized polynucleotides or a PCR amplicon thereof; and determining a contaminated result if the probe is detected in the negative control polynucleotide or a PCR amplicon thereof.

Details of one or more embodiments of the technology are set forth in the accompanying drawings and description below. Other features, objectives, and advantages of the technology will be apparent from the description and drawings, and from the claims. Similar reference symbols throughout the various drawings indicate similar elements.

ES 2 587 007 T3

Description of the drawings

Figure 1 shows a schematic overview of an apparatus described herein.

Figures 2A-2E show perspective views of an exemplary apparatus, in various configurations, as further described herein.

Figure 3 is an exploded view of typical components of an apparatus.

Figure 4 is a block diagram of an apparatus.

Figure 5 is a diagram of a fluorescent detection module.

Figures 6A and 6B depict the location, in various embodiments, of a microfluidic cartridge after installation in an apparatus.

Figure 7 shows a perspective view of a removable heater module, as further described herein.

Figures 8A-8C show a plan view of heater circuits adjacent to a PCR reaction zone; and thermal images of heater circuits in operation.

Figure 9 shows a perspective view of a microfluidic cartridge, as further described herein.

Figure 10 is a perspective view of a microfluidic device.

Figure 11 is a cross-sectional view of a processing region for retaining polynucleotides and / or separating polynucleotides from inhibitors.

Figure 12A is a perspective view of a gate.

Figure 12B is a perspective view of a flexed gate.

Figure 13 is a cross-sectional view of an actuator.

Figure 14A is a perspective view of a microfluidic cartridge.

Figure 14B is a side cross-sectional view of the microfluidic cartridge of Figures 14A and 14B.

Figures 15A and 15B, taken together, illustrate a perspective view of a microfluidic network of the microfluidic cartridge of Figures 14A and 14B.

Figure 16 illustrates a series of heat sources for operating components of the microfluidic cartridge of Figures 14A and 14B.

Figures 17 and 18 illustrate a valve in the open and closed states respectively.

Figures 19A-19D illustrate a mixing gate of the microfluidic network of Figures 6A and 6B and adjacent regions of the network.

Figures 20A-20C illustrate a tank with a drive mechanism.

Figures 21A-21C illustrate a tank with a drive mechanism.

Figure 22 illustrates a tank with a drive mechanism.

Figures 23A-23B illustrate a tank with a drive mechanism.

Figures 24A-24B illustrate a tank with a drive mechanism.

Figure 25 illustrates a tank with a drive mechanism.

Figure 26 illustrates a tank with a drive mechanism.

ES 2 587 007 T3

Figures 27A and 27B illustrate embodiments of a reagent container with a piercing member.

Figure 28 depicts an apparatus that can function as a small, tabletop, real-time polynucleotide analysis system.

Figure 29 depicts a sample kit that can be used with an apparatus.

Figure 30 represents a sachet for components of a sample kit.

Figure 31 depicts an exemplary microfluidic cartridge.

Figure 32 depicts the use of a barcode reader to read a barcode on a microfluidic cartridge.

Figure 33 depicts the use of a barcode reader to read a barcode on a sample container.

Figure 34 represents the attachment of a filter to a lysis reservoir of a microfluidic cartridge by means of a sample inlet, whereby the contents of the syringe can be injected into the microfluidic cartridge through a filter.

Figure 35 depicts the addition of air to the syringe in order to pressurize the microfluidic cartridge.

Figure 36 depicts the placement of a microfluidic cartridge in a receiving compartment of an apparatus.

Figures 37 and 38 represent the closure of a lid of an apparatus.

Figures 39 and 40 depict the removal of a heater / sensor module from an appliance.

Fig. 41A is a graph of real-time heat sensor data from an apparatus.

Fig. 41B is an apparatus real-time optical detector data graph.

Figure 42 is a schematic representation of various exemplary chambers and / or subunits in an exemplary microfluidic cartridge.

Figure 43 is a schematic representation of the steps relating to PCR and detection that can be performed on an exemplary microfluidic cartridge.

Figure 44 is a schematic of an exemplary real-time PCR assay based on the TaqMan® assay.

Figure 45 is a schematic of positive internal control plasmids that can be employed.

Figure 46 represents the mixture of two fluids ("A" - blue and "B" - orange).

Figures 47A and 47B depict a thermally driven pump 500 based on a phase transition material (PTM) 510, in closed (Figure 47A) and open (Figure 47B) configurations.

Figures 47C and 47D show another example of a pump 501 with expandable polymer 511 in chamber 513 that can be actuated to operate gate 515.

Figure 48 represents components of a single, integrated, disposable cartridge based on microfluidic technology.

Figure 49 depicts DNA capture beads that can be used.

Figures 50A-50J highlight various elements of the microfluidic cartridge shown in Figures 15A and 15B.

Figure 51 is a side view of a lever assembly 1200, with lever 1210, gear unit 1212, and force member 1214.

Figure 52 shows a side view of lever assembly 1200, with a microfluidic cartridge in a receiving compartment.

Figure 53 shows a close-up of a receiving compartment.

ES 2 587 007 T3

Figure 54 shows a close-up of the interface between a microfluidic cartridge, thermally conductive, mechanically flexible layer, and a thermal phase.

Figure 55 shows a top view of assembly 1200.

Figure 56 is a close-up of Figure 55.

Figures 57-59 are a series of photos of the 1210 lever in action. Also shown on gear assembly 1212 is cam 1228, which allows lever 1210 to apply force to plate 1230 coupled to force members 1214.

Figures 60 and 61 show views of a microfluidic cartridge with self-piercing reservoirs 1228 and mechanical members 1230 to drive the self-piercing reservoirs.

Figures 62 and 63 show elements of optical detector elements 1220 including light sources 1232 (eg, light emitting diodes), lenses 1234, light detectors 1236 (eg, photodiodes), and filters 1238.

Figure 64 illustrates a microfluidic device.

Figure 65 is a cross section of the microfluidic device of Figure 64 taken along 5.

FIG. 66 illustrates the retention of herring sperm DNA.

Figure 67 illustrates the retention and release of DNA from group B streptococci;

Figure 68 illustrates the PCR response of a sample from which inhibitors had been removed and a sample from which inhibitors had not been removed.

Figure 69 illustrates the PCR response of a sample prepared according to the technology described herein and a sample prepared using a commercial DNA extraction method.

Figure 70A illustrates a flow chart showing steps performed during a method to separate polynucleotides and inhibitors.

Figure 70B illustrates DNA from samples subjected to the method of Figure 26A.

Figure 71 is a flow chart outlining a set of exemplary criteria that can be used by the decision algorithm in apparatus 800 to interpret results, as described in Example 13.

Detailed description

A system, microfluidic cartridge, kit, methods, and software product are further described below.

Analysis of biological samples often includes determining whether one or more polynucleotides (eg, a DNA, RNA, mRNA, or rRNA) may be present in the sample. For example, a sample can be analyzed to determine whether a polynucleotide indicative of the presence of a particular pathogen (such as a bacterium or virus) may be present. The polynucleotide can be a genomic DNA sample, or it can be a mitochondrial DNA sample. Biological samples can normally be complex mixtures. For example, a sample may be provided as a blood sample, a tissue sample (eg, a smear from, for example, nasal, buccal, anal, or vaginal tissue), a biopsy aspirate, a lysate, such as fungi, or like bacteria. The polynucleotides to be determined can be contained within particles (for example, cells (for example, white blood cells and / or red blood cells), tissue fragments, bacteria (for example, gram positive bacteria and / or gram negative bacteria), fungi, spores). One or more liquids (eg, water, a buffer, blood, blood plasma, saliva, urine, cerebrospinal fluid, or organic solvent) can typically be part of the sample and / or can be added to the sample during a processing step.

Methods for analyzing biological samples include providing a biological sample (eg, a smear), releasing polynucleotides from particles (eg, cells such as bacteria) from the sample, amplifying one or more of the released polynucleotides (eg, by polymerase chain reaction (PCR)), and determining the presence (or absence) of the one or more amplified polynucleotides (eg, by fluorescence detection). Biological samples, however, typically include inhibitors (eg, mucosal compounds, hemoglobin, fecal compounds, and DNA-binding proteins) that can inhibit the determination of the presence of polynucleotides in the sample. For example, such inhibitors can reduce the efficiency of polynucleotide amplification by PCR and other enzymatic techniques to determine the

ES 2 587 007 T3 presence of polynucleotides. If the concentration of inhibitors is not reduced relative to the polynucleotides to be determined, the test may produce false negative results. The methods and systems listed herein for processing biological samples (eg, samples having one or more polynucleotides to be determined) are typically capable of reducing the concentration of inhibitors relative to the concentration of polynucleotides to be determined by methods further described in This document.

Various aspects of a microfluidic cartridge and system are described herein. Additional disclosures of various components thereof can be found in US Application Serial No. 11 / 580,267 (published as US 2007/0184547 A1), and Provisional Application Serial No. 60 / 859,284.

System overview

A schematic overview of a system 981 for performing analysis described herein is shown in Figure 1. The geometric arrangement of the components of system 981 shown in Figure 1 is exemplary and is not intended to be limiting. A processor 980, such as a microprocessor, is configured to control functions of various components of the system as shown, and is thus in communication with each such component. In particular, the processor 980 is configured to receive data about a sample to be analyzed, for example, from a sample reader 990, which can be a barcode reader, an optical character reader, or an RFID scanner. (RF mark reader). For example, the sample identifier can be a handheld barcode reader. Processor 980 is configured to accept user instructions from input 984, where those instructions may include instructions to begin analyzing the sample, dry selections of operating conditions. Processor 980 is also configured to communicate with a display 982, so that, for example, test results are transmitted to the display. Additionally, processor 980 may transmit one or more questions that will be displayed on screen 982 prompting the user to provide input in response to them. Therefore, in certain embodiments, input 984 and display 982 are integrated with each other. Processor 986 is optionally further configured to transmit test results to an output device such as a printer, visual display, or speaker, or a combination thereof. The processor 980 is furthermore optionally still connected via a communication interface such as a network interface to a computer network 988. The communication interface may be one or more interfaces selected from the group consisting of: a serial connection, a parallel connection, a wireless networking, and a wired networking. Thus, when the system is properly addressed on the network, a remote user can access the processor and transmit instructions, enter data, or retrieve data, such as may be stored in a memory (not shown) associated with the processor, or in some other computer-readable medium that is in communication with the processor.

Although not shown in Figure 1, in various embodiments, input 984 may include one or more input devices selected from the group consisting of: a keyboard, a touch-sensitive surface, a microphone, a touch panel, a scanner. of retina and a mouse.

Additionally, in various embodiments, the apparatus may further comprise a data storage medium configured to receive data from one or more of the processor, an input device, and a communication interface, the data storage medium being one or more media selected from the group consisting of: a hard disk drive, an optical disk drive, a flash card, and a CD-Rom.

Processor 980 is further configured to control various aspects of sample diagnostics, as follows in the overview, and as further described herein. The system is configured to work in conjunction with a companion cartridge 994, such as a microfluidic cartridge. The cartridge is, in turn, configured, as further described herein, to receive a biological sample 996 in a form suitable for diagnostic testing and analysis. The cartridge is received by a receiving bay 992 in the system. The receiving compartment is in communication with a heater 998 which is, in turn, controlled by processor 980 such that specific regions of the cartridge are heated at specific times during diagnostic testing and analysis of a sample. The processor is further configured to control a detector 999 that receives an indication of a diagnostic from cartridge 994. The diagnosis can be transmitted to the output device 986 and / or the display 982, as previously described herein.

A suitable processor 980 can be designed and manufactured in accordance with, respectively, design principles and semiconductor processing methods known in the art.

The system shown in schematic in Figure 1, as with other exemplary embodiments described herein, is advantageous in that it does not require locations within the system, properly configured for reagent storage. Nor does the system, or other exemplary embodiments herein, require inlet or outlet ports that are configured to receive reagents from, for example, externally stored containers such as bottles, cans, or reservoirs. Therefore, the system in the

ES 2 587 007 T3 figure 1 is self-contained and works in conjunction with a microfluidic cartridge, where the cartridge has locations within it dedicated to reagent storage.

The system of Figure 1 can be configured to carry out operation in a single location, such as a laboratory environment, or it can be portable so that it can accompany, for example, a physician, or other healthcare professional, who can visit patients in different locations. The system is typically fitted with a power cord so that it can accept AC power from a utility or generator network. An optional transformer (not shown) built into the system, or located externally between a power outlet and the system, transforms the AC input power into a DC output for use by the system. The system can also be configured to operate using one or more batteries and is therefore typically equipped with a battery recharging system, and various warning devices that alert a user if battery power is getting too high. low to reliably start or complete a diagnostic scan.

The system of Figure 1 may further be configured, in other embodiments, for multiplexed sample analysis. In such a configuration, multiple examples of a system, as outlined in Figure 1, are operated together to accept and process multiple cartridges, where each cartridge has been loaded with a different sample. Each component shown in Figure 1 may therefore be present as many times as there are samples, although the various components may be configured in a common housing.

In yet another configuration, a system is configured to accept and process multiple cartridges, but one or more components in Figure 1 are common to multiple cartridges. For example, a single device may be configured with multiple cartridge receiving bays, but a common processor and user interface appropriately configured to allow concurrent, consecutive, or simultaneous control of the various cartridges. It is also possible that said embodiment also uses a single sample reader, and a single output device.

In yet another configuration, a system as shown in Figure 1 is configured to accept a single cartridge, but where the single cartridge is configured to process more than 1, for example 2, 3, 4, 5 or 6, samples. in parallel, and independently of each other. Exemplary technology for creating cartridges that can handle multiple samples is described elsewhere, for example; in U.S. Application Serial No. 60 / 859,284.

It is further consistent with the present technology that a cartridge may be marked, for example, with a molecular barcode indicative of the sample, to facilitate sample tracking, and to minimize the risk of sample mixing. Methods for such marking are described elsewhere, for example, in United States Patent Application Publication No. 10 / 360,854 (published as US 2004/0157220 A1).

Exemplary systems

Figures 2A-2E show exterior perspective views of various configurations of an exemplary system, as further described herein. Figure 2A shows a perspective view of a system 2000 for receiving a microfluidic cartridge (not shown), and for causing and controlling various processing operations to be performed on a sample inserted into the cartridge. System 2000 items are not limited to those explicitly shown. For example, although not shown, the system 2000 may be connected to a handheld barcode reader, as further described herein.

The system 2000 comprises a housing 2002, which can be made of metal, or a hardened plastic. The shape of the housing shown in Figure 2A embodies both style and functional characteristics. Other embodiments of the invention may appear somewhat different, in their arrangement of components, as well as their overall appearance, in terms of smooth lines, and exterior finish, and texture. System 2000 further comprises one or more stabilization members 2004. Shown in Figure 2A is a stabilizing leg, several of which are normally present, located in various regions on the underside of the system 2000 to provide balance and support. For example, there may be three, four, five, six, or eight such stabilization legs. The legs can be molded from and made of the same material as the shell 2002, or they can be made of one or more different materials and attached to the underside of the system 2000. For example, the legs may comprise a rubber that makes it difficult for the System 2000 to slide on a surface on which it is located, and also protects the surface from scratches. The stabilizing member or members may assume different leg shapes, for example, rails, sliders, or one or more pads.

System 2000 further comprises a display 2006, which may be a liquid crystal display, such as an active matrix, an OLED, or some other suitable form. You can present images and other information in color or black and white. Screen 2006 can also be a touch-sensitive screen and therefore can be configured to accept input from a user in response to various displayed prompts. The 2006 display may have an anti-reflective coating over it to reduce glare and reflections from lights.

ES 2 587 007 T3 elevated in a laboratory environment. The 2006 screen can also be illuminated from, for example, a backlight, to facilitate easier viewing in a dark laboratory.

The system 2000, as shown in Figure 2A, also comprises a movable lid 202, which has a handle 2008. The lid 2010 can slide back and forth. In Figure 2A, the lid is in a forward position, thus it is "closed". In Figure 2B, the cap is shown in a rearward position, where the cap is "open" and reveals a receiving compartment 2014 that is configured to receive a microfluidic cartridge. Of course, as one skilled in the art would appreciate, the technology described herein is not limited to a lid that slides, or one that slides back and forth. Side-to-side movement is also possible, as is a configuration where the lid is "open" when positioned forward on the device. It is also possible that the lid is a hinged lid, or one that is fully removable.

The handle 2008 performs a role of allowing a user to move the lid 2010 from one position to another, and also performs a role of causing pressure to be pushed down on the lid, when it is in a closed position, so that it can apply pressure to a cartridge in the receiving compartment 2014. In Figure 2C, the handle 2008 is shown in a downwardly depressed position, where thus force is applied to the cap 2014, and thus pressure is applied to a cartridge received in the receiving compartment below the cap.

In one embodiment, the handle and cap assembly is also equipped with a mechanical sensor that does not allow the handle to be pressed down when there is no cartridge in the receiving compartment. In another embodiment, the handle and lid assembly is equipped with a mechanical latch that does not allow the handle to lift up when a test is in progress.

An additional configuration of system 2000 is shown in Figure 2D, where a door 2012 is in an open position. Door 2012 is shown in a closed position in the figures. 2A-C. The door is an optional component that allows a user to access a heater module 2020, and also a computer-readable media inbox 2022. System 2000 may operate without a door covering heater module 2020 and media inlet 2022, but such a door has associated convenience. Although the 2012 door is shown hinged at the bottom, it can also be hinged on one of its sides, or on its upper edge. The 2012 door can alternatively be a removable cover, rather than hinged. Door 2012 may also be located at the rear, or side of the system 2000 for example, if access to the heater module and / or the computer-readable media input is desired on a different face of the system. It is also consistent with the system herein that the heating module, and the entry of the computer-readable medium, is accessed through different doors on the same or on different sides of the device, and where said different doors can be independently hinged or removable. .

The heater module 2020 is preferably removable, and is further described hereinafter.

Computer-readable media input 2022 can accept one or more of several media. An exemplary form of input 2022 is shown in Figure 2D, a CD-Rom tray for accepting a CD, DVD, or mini-CD, or miniDVD, in any of the commonly used readable, readable-writable and writable formats. Also consistent with the description herein is an entry that can accept another form of medium, such as a floppy disk, flash memory such as memory stick, compact flash, smart data card, or secure data card, a memory removable laptop, portable USB drive, zip disk, and others. This input can also be configured to accept several different forms of media. Said input 2022 is in communication with a processor (as described in relation to Figure 1, but not shown in Figures 2A-E), which can read data from a computer-readable medium when properly inserted into the entry.

Figure 2E shows a plan view of a rear portion of the system 2000. An air vent 2024 is shown to allow excess heat to escape during an analysis. Normally, inside the system 2000, and through the air vent 2024 and not shown in FIG. 2E, there is a fan. Other ports shown in Figure 2E are as follows: a power outlet 2026 to accept a power cord that will connect the system 2000 to an electricity supply; an Ethernet connection 2028 to link the system 2000 to a computer network such as a local area network; a telephone jack connection 2032 to link the system 2000 to a communication network such as a telephone network; one or more USB ports 2030, to connect the system 2000 to one or more peripheral devices such as a printer, or a hard disk drive of a computer; an infrared port to communicate with, for example, a remote controller (not shown), to allow a user to control the system without using a touch screen interface. For example, a user could remotely send scheduling orders to system 2000 to have it start a scan at a specific time in the future.

Features shown on the back of the system 2000 may be arranged in any different way, depending on an internal configuration of various components. Additionally, features shown being on the back of the system 2000 may optionally be displayed on another face of the system 2000, depending on design preference. Exemplary connections are shown in Figure 2E. I know

ES 2 587 007 T3 would understand that various other features, including inputs, outputs, sockets and connections, may be present on the back of the System 2000, although not shown, or on other faces of the System 2000.

An exploded view of an exemplary embodiment of the apparatus is shown in Figure 3, particularly showing internal features of the apparatus 2000. Apparatus 2000 may comprise computer-readable media configured with hardware / firmware that can be used to drive and monitor operations on a cartridge used with it, as well as software to interpret, communicate, and store the results of a diagnostic analysis performed on a sample processed in the cartridge. Referring to Figure 3, typical components of apparatus 2000 are shown and include, for example, electronic control components 2005, removable sensor / heater module 2020, detector 2009 such as a fluorescent detection module, display screen, or optionally display screen. 2006 Combined User Interface (for example, a medical-grade touch-sensitive liquid crystal display (LCD)). In some embodiments, the lid 2010, the detector 2009, and the handle 2008 may be collectively referred to as the slide module 2007. Additional components of the apparatus 2000 may include one or more mechanical fasteners such as the frame 2019 to contain the various modules (e.g. , the heater / sensor module 2020, and / or the sliding module 2007) in alignment, and to provide structural rigidity. Detector module 2009 may be positioned on rails to facilitate opening and positioning of cartridge 2060 in apparatus 2000, and to facilitate alignment of the optical elements at closure. The 2020 Sensor / Heater Module can also be mounted on rails for easy assembly removal and insertion.

Embodiments of apparatus 2000 also include software (eg, to interface with users, perform analysis, and / or analyze analysis results), firmware (eg, to control hardware during analysis on cartridge 812), and one or more peripheral communication interfaces collectively shown as 2031 for peripherals (for example , communication ports such as USB / Serial / Ethernet for connecting to storage such as compact disc or hard drive, for connecting input devices such as a barcode reader and / or keyboard, for connecting to other computers or storage via a network, and the like).

The 840 electronic control components, shown schematically on the block diagram in figure

4, may include one or more functions in various embodiments, for example for main control 900, multiplexing 902, display control 904, detector control 906, and the like. The main control function can serve as the core of the electronic control components 840 in the apparatus 2000 and can manage the communication and control of the various electronic functions. The main control function can also support the electrical and communication interface 908 with a user or an output device such as a printer 920, as well as security and optical diagnostic functions. Together with the main control function 900, the multiplexer function 902 can control sensor data 914 and output current 916 to help control the heater / sensor module 2020. The screen control function 904 can control the output to and, if applicable, interpret the input from the LCD touch screen 846, which can thus provide a graphical user interface in certain embodiments. The function of detector 906 can be implemented in control electronics 840 using typical processing and control circuitry to collect, digitize, filter and / or transmit data from a detector 2009 such as one or more fluorescence detection modules.

In various embodiments, Fluorescent Detection Module 2009 may be a miniaturized highly sensitive fluorescence detection system that may, for example, be capable of real-time analysis of a fluorescent signal emanating from a suitably positioned microfluidic cartridge, as shown shown in the figure

5. Detection module 2009 may employ one or more 2850 light sources (eg, light-emitting diodes (LEDs)), one or more 2852 detectors (eg, photodiodes), and one or more 2851 filters and / or 2853 lenses. In some embodiments, detection module 2009 may contain multiple (eg, six) detection elements, where each element can detect one or more fluorescent probes.

In various embodiments, the sliding module 2007 of the apparatus 2000 can house the detection module 2009 (eg, optical detection system) as well as mechanical assembly / optical component holder 856 to press down on the microfluidic cartridge 2020 when the handle 2008 of the slide module 2007 is pressed down. Figures 6A and 6B depict the location, in various embodiments, of the 2060 microfluidic cartridge after insertion into apparatus 2000. Optical component carrier 856 may be suspended from the cover of slide module 2007 at one or more (eg, 4 ) points. At the moment of closing the sliding module 2007 and the turning of the handle 2008 of the apparatus 2000 downwards, one or more mechanical actuators 858 (for example, four cams) can push down the plate 860 against one or more (for example, 4 ) springs 862. At the moment of compression, springs 862 can supply force on detector module 2009. A bottom surface 864 of the detector module 2009 can be flattened (eg, within 250 microns, typically within 100 microns, more typically within 25 microns), and the surface 864 can press on the cartridge 2060, which may have a flexible layer 868 (eg Shore hardness about 50-70) with a thickness of 0.1-2.5mm in the absence of compression, typically about 1.5mm thick in the absence of compression. Consequently, the compression of the cartridge 2060, in combination with the flat surface 864, can make the pressure, and therefore the thermal contact, more or

ES 2 587 007 T3 less uniform over the 2060 microfluidic cartridge. One or more springs 862 in the 2007 slide module can supply a force (eg, 5 - 500 N, typically about 200 - 250 N) to generate a pressure ( for example, 2 psi) on the bottom of the 2060 microfluidic cartridge. Figure 6B also shows that, when the slide module 2007 can be closed, mechanical characteristics 863 of the slide module 2007 can press down on self-piercing reservoirs 866 of the microfluidic cartridge 2060, causing the contents of the reservoir (e.g., DI water, PCR) are released.

Removable heating module

An exemplary removable heater module 2020 is shown in Figure 7. The module is configured to deliver localized heat to various selected regions of a cartridge received in receiving compartment 2014. A heater module having a recessed surface is shown in Figure 7. 2044 which provides a platform to support a cartridge when it is in the receiving bay 2014. In one embodiment, the cartridge rests directly on surface 2044. Surface 2044 is shown recessed, in Figure 7, but need not be.

Area 2044 is configured to accept a microfluidic cartridge in a single orientation. Therefore, the area 2044 can be equipped with a correct positioning member such as a mechanical key that prevents a user from placing a cartridge in the receiving compartment 2014 in the wrong configuration. In Figure 7, an exemplary mechanical key 2045 is shown a diagonally cut corner of area 2044 into which a complementary cut corner of a microfluidic cartridge fits (see, for example, Figure 9). Other correct positioning members are consistent with the apparatus described herein: for example, a feature designed on one or more edges of a cartridge including but not limited to: several, such as two or more, cut corners, one or more notches cut into one or more edges of the cartridge of Figure 9; or one or more protrusions made on one or more edges of the cartridge of Figure 9. Alternate correct positioning members include one or more studs or protrusions designed on a bottom side of a cartridge, complementary to one or more recessed taps or holes in the 2044 surface. a lower side of a cartridge, complementary to one or more lugs or protrusions on surface 2044. In general, the pattern of features is such that the cartridge has at least one asymmetry element, so that it can only be inserted in a single orientation in the receiving compartment.

Also shown in Figure 7 is a hand grip 2042 that facilitates removal and insertion of the heater module by a user. The 2048 cutout allows a user to easily remove a cartridge from the 2014 receiving bay after a series of processing where, for example, a user's thumb or finger when the top of the cartridge is grasped, is provided comfortable space by clipping 2048. Both cutouts 2042 and 2048 are shown as semi-circular recesses in the embodiment of Figure 7, but it would be understood that they are not limited in shape in this way. Therefore, rectangular, square, triangular, oval, and otherwise shaped recesses are also consistent with a heater module as described herein.

In the embodiment of Figure 7, which is designed to be compatible with the system of Figures 2A-E, the front of the heater module is to the left of the figure. On the back of the 2020 heater module there is a 2050 electrical connection, such as an RS-232 connection, which allows electrical signals to be routed to heaters located in specific regions of the 2044 area during sample processing and analysis, as described additionally in this document. Therefore, below the area 2044 and not shown in Figure 7 may be a series of heat sources, such as resistive heaters, that are configured to align with specified locations of a microfluidic cartridge properly inserted into the receiving compartment. The 2044 surface is capable of being periodically cleaned to ensure that any liquid spills that may occur during sample handling do not cause a short circuit.

Other non-essential features of the 2020 heater module are as follows. One or more air vents 2052 may be located on one or more sides (such as front, back, or flanks) or faces (such as top or bottom) of the heater module 2020, to allow excess heat Heat escape, when the heaters under the receiving compartment 2014, are in operation. The configuration of the air vents in Figure 7 is exemplary and other numbers and shapes thereof would be understood to be consistent with routine manufacture and use of a heater module. For example, although 5 square air vents are shown, other numbers such as 1, 2, 3, 4, 6, 8 or 10 air vents are possible, arranged on one side, or distributed in two or more sides and / or faces of the heating module. In further embodiments, the air vents can be circular, rectangular, oval, triangular, polygonal, and have curved or square corners, or still other shapes, including irregular shapes.

The heater module 2020 may further comprise one or more guide members 2047 that facilitate insertion of the heater module into an appliance as further described herein for an embodiment in which the heater module 2020 is removable by a user. . The heating module is advantageously removable, since it allows the 2000 system to be easily reconfigured for one type of analysis.

ES 2 587 007 T3 different, such as employing a different cartridge with a different correct positioning member and / or microfluidic network, along with the same or a different sequence of processing operations. In other embodiments, the heater module 2020 is designed to be attached and removable only, eg, for cleaning, replacement, or maintenance, by the manufacturer or an authorized maintenance agent, and not routinely by the user. Guiding members can play one or more roles of ensuring that the heater module is properly aligned in the apparatus, and ensuring that the heater module establishes a tight fit and does not move significantly during sample processing and analysis, or during transportation of the appliance. The guide members shown in the embodiment of Figure 7 are on either side of the receiving compartment 2044 and stretch along a substantial fraction of the length of the module 2020. Other guide members are consistent with current use. document, and include, but are not limited to, other guide member numbers such as 1, 3, 4, 5, 6, or 8, and other positions thereof, including located in area 2051 of module 2020. Guide members 2047 are shown as having non-constant thickness throughout their lengths. It is consistent herein that other guide members may have essentially constant thicknesses throughout their lengths.

Adjacent to the receiving compartment 2014 is a non-contact heating element 2046, such as a lamp, installed in a recessed area 2053. The recessed area 2053 can also be configured with a reflector, or a reflective coating, so that as much thermal energy and optics from non-contact heating element 2046 as possible be directed outwardly toward receiving compartment 2014. Element 2046 is a thermal lamp in certain embodiments. Element 2046 is configured to receive electrical energy and thus heat up from the effects of electrical resistance. Element 2046 provides a way to heat an elevated region of a cartridge received in receiving compartment 2014. The raised region of the cartridge (see, for example, FIG. 9) may contain a lysis chamber, and the application of heat from non-contact heating element 2046 may have the effect of lysing cells within the lysis chamber.

Also shown in Figure 7 is an optical region of fluorescent material, such as optically fluorescent material, 2049 in area 2051 of heater module 2020. The region of fluorescent material is configured to be detected by a detection system further described herein. document. Region 2049 is used to verify the status of the optics in the detection system prior to sample processing and analysis and therefore acts as a control, or a standard. For example, in one embodiment a lid of the apparatus (see, for example, Figure 2A) when in an open position allows ambient light to reach the 2049 region and thus causes the fluorescent material to emit a frequency or characteristic light spectrum that can be measured by the detector for, for example, standardization or calibration purposes. In another embodiment, instead of relying on ambient light to cause the fluorescent material to fluoresce, the light source from the detection system itself is used, such as one or more LEDs. Region 2049 is therefore positioned to align with a position of a detector. Region 2049 is shown as rectangular, but can be configured in other shapes such as square, circular, elliptical, triangular, polygonal, and having curved or square corners. It will also be understood that region 2049 may be located elsewhere in heater module 2020, according to convenience and in order to be complementary to the deployed detection system.

The heater module 2020 also comprises a series of heaters, located below area 2044 and not shown in Figure 7. As further described herein, such heaters may be resistive heaters, configured to heat specifically and at specific times, according to received electrical signals.

In particular and not shown in Figure 7, the heater / sensor module 2020 may include, for example, a multiplexing function 902 on a discrete multiplexing circuit board (MUX board), one or more heaters (for example, a micro-heater ), one or more temperature sensors (optionally combined with each other as a single heater / sensor unit with one or more respective micro-heaters, for example, as fabricated photolithographically on fused silica substrates), and a non-contact heating element 2046. The micro-heaters and the non-contact heating element can provide thermal energy that can drive various microfluidic components in a suitably located microfluidic cartridge. A sensor (for example, such as a resistive temperature sensor (RTD)) can allow real-time monitoring of the micro-heaters and the one or more 2046 non-contact heaters, for example through a feedback-based mechanism to enable control temperature. One or more micro-heaters can be aligned with corresponding microfluidic components (eg, valves, pumps, gates, reaction chambers) to be heated in a suitably located microfluidic cartridge. A microheater can be designed to be slightly larger than the one or more corresponding microfluidic components in the microfluidic cartridge so that, even though the cartridge may be slightly misaligned, such as off-center, of the heater, the individual components can be effectively heated.

The non-contact heater 2046 can also serve as a radiation heat source to heat a section of a suitably positioned microfluidic cartridge. For example, a 20 W xenon lamp can be used as a non-contact heating element 2046. In various embodiments, the heater / sensor module 2020 can

ES 2 587 007 T3 be specific to particular cartridge designs and can be easily replaceable through the front panel of the apparatus 800. The heater / sensor module 2020 can be configured to allow cleaning of the heating surface 2044 with common cleaning agents. (for example, a 10% bleach solution).

Referring to Figures 8A and 8B, an exemplary heater assembly configured to cyclically heat the PCR reaction zone 1001 is shown. It should be understood that heater configurations to drive other regions of a microfluidic cartridge such as other gates, valves and actuators, may be designed and deployed in accordance with principles similar to those that govern the heaters shown in Figures 8A and 8B. An exemplary PCR reaction zone 1001, typically a chamber or channel having a volume ~ 1.6 µ !, is configured with a long side and a short side, each with an associated heating element. A PCR reaction zone may also be referred to as a PCR reactor, herein. The apparatus, therefore, preferably includes four heaters arranged along the sides of, and configured to heat, a given PCR reaction zone, as shown in the exemplary embodiment of Figure 8A: long top heater 1005 , bottom heater long 1003, left heater short 1007, and right heater short 1009. The small space between the long upper heater 1005 and the long lower heater 1003 results in a negligible temperature gradient (less than 1 ° C across the width of the PCR channel at any point along the length of the zone reaction zone) and therefore an effectively uniform temperature throughout the PCR reaction zone. The heaters on the short edges of the PCR reactor provide heat to counteract the gradient created by the two long heaters from the center of the reactor to the edge of the reactor.

It would be understood by one skilled in the art that still other configurations of one or more heaters located around a PCR reaction zone are consistent with the methods and apparatus described herein. For example, a "long" side of the reaction zone can be configured to be heated by two or more heaters. Specific heater orientations and settings are used to create uniform heating zones even on substrates that have poor thermal conductivity due to poor thermal conductivity of glass, or quartz, or fused silica substrates are used to aid independent operation of various components microfluidics such as valves and the independent operation of the various PCR lanes. It would be further understood by one of ordinary skill in the art that the principles underlying the configuration of heaters around a PCR reaction zone are similarly applicable to the arrangement of heaters adjacent to other components of the microfluidic cartridge, such as actuators, valves and gates.

In certain embodiments, each heater has an associated temperature sensor. In the embodiment of Figure 8A, a single temperature sensor 1011 is used for both long heaters. A 1013 temperature sensor for the short left heater, and a 1015 temperature sensor for the short right heater are also shown. The temperature sensor in the middle of the reactor is used to provide feedback and control the amount of power supplied to the two long heaters, while each of the short heaters has a dedicated temperature sensor placed adjacent to it in order to control it. . The temperature sensors are preferably configured to transmit information about the temperature in their vicinity to the processor at times such that the heaters are not receiving current causing them to heat up. This can be achieved with proper control of current cycling.

In order to reduce the number of heater or sensor elements required to control a PCR heater, the heaters can be used for sensing as well as for heating, thus obviating the need to have a different dedicated sensor for each heater. In another embodiment, each of the four heaters can be designed to have an appropriate wattage, and connect the four heaters in series or parallel to reduce the number of electronically controllable elements from 4 to only 1, thereby reducing the load on associated electronic circuits.

Figure 8B shows exploded views of heaters and temperature sensors used in conjunction with a PCR reaction zone of Figure 8A. The 1001 and 1013 temperature sensors are designed to have a resistance at room temperature of approximately 200-300 ohms. This resistance value is determined by controlling the thickness of the deposited metal layer (for example, a 400 A TiW / 3,000A Au / 400 A TiW sandwich), and chemically etching the winding metal line to have a width of approximately 10-25 μιτι and 20-40 mm in length. The use of metal in this layer gives it a temperature resistivity coefficient in the order of 0.5 - 20 ° C / ohms, preferably in the range of 1.5 - 3 ° C / ohms. Measuring resistance at higher temperatures allows the exact temperature determination of the location of these sensors.

The configuration for uniform heating, shown in Figure 8A for a single PCR reaction zone, can also be applied to a multi-lane PCR cartridge in which multiple independent PCR reactions occur.

Each heater can be independently controlled by a processor and / or control circuits used in conjunction with the apparatus described herein. Figure 8C shows thermal images, from the top surface of a microfluidic cartridge when heated by heaters configured as in Figures 8A and

ES 2 587 007 T3

8B, when each heater is activated, in turn, as follows: (A): upper long only; (B) long bottom only; (C) short left only; (D) short right only; and (E) all four heaters on. Panel (F) shows a view of the reaction zone and heaters on the same scale as the other image panels in Figure 8C. A temperature bar is also shown in the figure.

Microfluidic cartridge

Figure 9 shows a perspective view of an exterior of an exemplary microfluidic cartridge 2060 for use in conjunction with the system described herein. Present in cartridge 2060 is at least one reagent container 2062. Four such reagent containers are shown, although other numbers of such containers, such as but not limited to one, two, three, five, six, eight, ten and twelve are possible, depending on the application. Cartridge 2060 further comprises a tower 2064 containing reagents, and is equipped with an inlet 2066, such as a Luer, through which a portion of a biological sample can be introduced. The tower 2064 may also comprise one or more chambers such as a bulk lysis chamber 2065 and a waste chamber 2067. The waste chamber 2067 may have a vent 2069 to release gases such as air.

Aside from tower 2064, cartridge 2060 is substantially flat so that it can be easily manipulated by an operator and can be easily paired with a complementary receiving compartment of an apparatus, as shown in Figure 1.

The cartridge 2060 further comprises a port 2068 through which a detector can receive a signal directly or indirectly from one or more polynucleotides in the sample, during processing or amplification, in order to provide a user with a diagnostic result on the sample.

Cartridge 2060 may further comprise a correct positioning member such as a mechanical key, complementary to a corresponding correct positioning member in the receiving compartment. An exemplary correct positioning member 2071 is shown in FIG. 9, a corner cut away from the cartridge.

The integrated system, as described herein, comprises an apparatus configured to receive a microfluidic cartridge, and a microfluidic cartridge. This is consistent with the system described herein as a number of different microfluidic cartridge configurations, and purposes thereof, are compatible with suitably configured apparatus. Thus, for example, although benefits are described where a single cartridge is capable of accepting a collected biological sample, preparing the sample, including lysing cells to release and collecting polynucleotides contained within, applying preparative pre-amplification steps to the polynucleotides, amplifying polynucleotides, and cause the amplified polynucleotides to be detected, it is also consistent with the descriptions herein that other microfluidic cartridges may be used. Said other cartridges may be configured to carry out fewer, such as one or more, of the steps mentioned above and, correspondingly, the apparatus for use with them is configured to cause fewer of said steps to be carried out. It should therefore be understood that when various exemplary microfluidic cartridge configurations are presented herein, the various components thereof may be used interchangeably (for example, an exemplary valve described in connection with a cartridge may also be used in a network described in relation to another cartridge) both without modification, and with appropriate adjustments or modifications of geometry or size, as appropriate.

Microfluidic Cartridge Aspects

Accordingly, the technology herein also comprises a microfluidic cartridge having the following attributes. Thus, the technology includes a microfluidic cartridge that is configured to process one or more polynucleotides, for example, to concentrate the one or more polynucleotides and / or to separate the one or more polynucleotides from inhibitory compounds, (for example, hemoglobin, peptides, fecal compounds, humic acids, mucosal compounds, DNA-binding proteins, or a saccharide) that could inhibit the detection and / or amplification of the polynucleotides.

The microfluidic cartridge can be configured to contact the polynucleotides and a relatively immobilized compound that preferentially associates with (eg, retains) the polynucleotides as opposed to inhibitors. An exemplary compound is a poly-cationic polyamide (eg, poly-L-lysine and / or poly-D-lysine), or polyethyleneimine (PEI), which can be attached to a surface (eg, surfaces of one or more particles ). The compound retains the polynucleotides, so that the polynucleotides and inhibitors can be separated, such as by washing the surface to which the compound and associated polynucleotides are attached. At the time of separation, the association between the polynucleotide and the compound can be altered to release (eg, separate) the polynucleotides from the compound and the surface.

In some embodiments, the surface (eg, surfaces of one or more particles) can be modified with a polycationic substance such as a polyamide or PEI, which can be covalently attached to the surface. The polycationic polyamide can include at least one of poly-L-lysine and poly-D-lysine. In some embodiments, the polyamide

ES 2 587 007 T3 polycationic (eg, the at least one of poly-L-lysine and poly-D-lysine) has an average molecular weight of at least about 7500 Da. Polycationic polyamide (for example, the at least one of poly-L-lysine and poly-D-lysine) can have an average molecular weight of less than about 35,000 Da (for example, an average molecular weight of less than about 30,000 Da (eg an average molecular weight of about 25,000 Da)). Polycationic polyamide (eg, the at least one of poly-L-lysine and poly-Dlysine) can have a median molecular weight of at least about 15,000 Da. Polycationic polyamide (for example, the at least one of poly-L-lysine and poly-D-lysine) can have a median molecular weight of less than about 25,000 Da (for example, a median molecular weight of less of about 20,000 Da (eg median molecular weight of about 20,000 Da) If the polycationic material is PEI, its molecular weight is preferably in the range of 600-800 Daltons.

In other embodiments, the microfluidic cartridge includes a surface having a polycationic polyamide or PEI attached thereto and a sample introduction passage in communication with the surface for contacting the surface with a fluidic sample.

In some embodiments, the apparatus includes a heat source configured to heat an aqueous liquid in contact with the surface to at least about 65 ° C.

In some embodiments, the cartridge includes a reservoir of liquid that has a pH of at least about 10 (eg, about 10.5 or more). The cartridge may be configured to contact the surface with the liquid (for example, by actuating a source of pressure to move the liquid).

Another aspect of the microfluidic cartridge relates to a retention member, eg, a plurality of particles such as beads, comprising PEI, or bound poly-lysine, eg, poly-L-lysine, and related methods and systems. An exemplary method of processing a sample includes contacting a retention member with a mixture which includes providing a mixture that includes a liquid and an amount of polynucleotide. The retention member may be configured to preferably retain polynucleotides as compared to the polymerase chain reaction inhibitor. Substantially all of the liquid in the mixture can be removed from the retention member. The polynucleotides can be released from the retention member. The polynucleotide can be less than about 7.5 Mb in size.

The liquid may be a first liquid, and removing substantially all of the liquid from the retention member may include contacting the retention member with a second liquid.

Contacting the retention member with a second liquid may include operating a thermally actuated pressure source to apply a pressure to the second liquid. Contacting the retention member with a second liquid may include opening a thermally actuated valve to place the second liquid in fluid communication with the retention member.

The second liquid can have a volume of less than about 50 microliters, and can include a detergent (eg, SDS).

The retention member may include a surface having a compound configured to bind polynucleotides, preferably, to polymerase chain reaction inhibitors (such inhibitors including, for example, hemoglobin, peptides, fecal compounds, humic acids, mucosal compounds, DNA-binding proteins or a saccharide).

The surface can include a poly-lysine (eg, poly-L-lysine and / or poly-D-lysine) or PEI.

Releasing polynucleotides from the retention member may include heating the retention member to a temperature of at least about 50 ° C (eg, to about 65 ° C). The temperature may be insufficient to boil the liquid in the presence of the retaining member during heating. The temperature can be 100 ° C or less (eg, less than 100 ° C, about 97 ° C or less). The temperature can be held for less than about 10 minutes (eg, less than about 5 minutes, less than about 3 minutes). Release can be done without spinning the retention member.

In certain embodiments, the PCR inhibitors can be rapidly removed from clinical samples to create a PCR-ready sample. The methods herein may therefore comprise preparing a sample containing polynucleotides that may be substantially free of inhibitors. Such samples can be prepared from, for example, impure lysates resulting from thermal, chemical, ultrasonic, mechanical, electrostatic, and other lysing techniques. Samples can be prepared without centrifugation. Samples can be prepared using other microfluidic devices or on a larger scale.

The retention member can be used to prepare polynucleotide samples for further processing, such as amplification by polymerase chain reaction. In certain embodiments, more than 90% of a

ES 2 587 007 T3 polynucleotide present in a sample can be attached to the retention member, released and recovered.

In certain embodiments, a polynucleotide can be attached to the retention member, released, and recovered, in less than about 10 minutes (eg, less than about 7½ minutes, less than about 5 minutes, or less than about 3 minutes).

A polynucleotide can be attached to a retention member, released, and recovered without subjecting the polynucleotide, retention member, and / or inhibitors to centrifugation.

Separating the polynucleotides and inhibitors generally excludes subjecting the polynucleotides, inhibitors, processing region, and / or retention member to sedimentation (eg, centrifugation).

In various embodiments, the microfluidic cartridge can include a PCR reagent mix comprising a polymerase enzyme and a plurality of nucleotides. The PCR reagent mix can be in the form of one or more lyophilized microbeads, and the microfluidic network can be configured to contact the PCR microbead with liquid to create a PCR reagent mix solution.

In various embodiments, the microfluidic cartridge may be configured to couple heat from an external heat source with the mixture of PCR reagents and the sample with neutralized polynucleotides under suitable thermal cycling conditions to create PCR amplicons from the sample with neutralized polynucleotides.

In various embodiments, the PCR reagent mix may further include a positive control plasmid and a selective fluorogenic hybridization probe for at least a portion of the plasmid.

In various embodiments, the microfluidic cartridge can include a negative control polynucleotide, wherein the microfluidic network may be configured to independently contact each of the sample with neutralized polynucleotides and the negative control polynucleotide with the PCR reagent mix under suitable thermal cycling conditions to independently create PCR amplicons of the sample with polynucleotides neutralized and negative control polynucleotide PCR amplicons.

In various embodiments, the microfluidic cartridge can include at least one probe that can be selective for a polynucleotide sequence, where the microfluidic cartridge can be configured to contact the sample with neutralized polynucleotides or a PCR amplicon thereof with the probe. . The probe can be a fluorogenic hybridization probe. The fluorogenic hybridization probe can include a polynucleotide sequence coupled to a fluorescent reporter dye and a fluorescence quenching dye. The PCR reagent mix may further include a positive control plasmid and a plasmid fluorogenic hybridization probe selective for at least a portion of the plasmid and the microfluidic cartridge may be configured to allow independent optical detection of the hybridization probe. fluorogenic and the fluorogenic plasmid hybridization probe.

In various embodiments, the probe can be selective for a polynucleotide sequence that can be characteristic of an organism, for example any organism that employs deoxyribonucleic acid or ribonucleic acid polynucleotides. Therefore, the probe can be selective for any organism. Suitable organisms include mammals (including humans), birds, reptiles, amphibians, fish, domestic animals, farm animals, wild animals, extinct organisms, bacteria, fungi, viruses, plants, and the like. The probe can also be selective for components of organisms that employ their own polynucleotides, for example mitochondria. In some embodiments, the probe may be selective for microorganisms, for example, organisms used in food production (for example, yeasts used in fermented products, molds or bacteria used in cheeses, and the like) or pathogens (for example, of human beings). humans, domestic or wild mammals, domestic or wild birds, and the like). In some embodiments, the probe can be selective for organisms selected from the group consisting of gram positive bacteria, gram negative bacteria, yeast, fungi, protozoa, and viruses.

In various embodiments, the probe may be selective for a polynucleotide sequence that may be characteristic of an organism selected from the group consisting of Staphylococcus spp., Eg, S. epidermidis, S. aureus, methicillin-resistant Staphylococcus aureus (MRSA ), Vancomycin-resistant Staphylococcus; Streptococcus (eg, α, β, or γ-hemolytics, Group A, B, C, D, or G) such as S. pyogenes, S. agalactiae; E. faecalis, E. durans, and E. faecium (formerly S. faecalis, S. durans, S. faecium); non-enterococcal group D streptococci, eg, S. bovis and S. equines; Streptococci viridans, eg, S. mutans, S. sanguis, S. salivarius, S. mitior, A. milleri, S. constellatus, S. intermedius, and S. anginosus; S. iniae; S. pneumoniae; Neisseria, eg, N. meningitides, N. gonorrhoeae, Neisseria sp saprolytic; Erysipelothrix, for example, E. rhusiopathiae; Listeria spp., For example L. monocytogenes, rarely L. ivanovii and L. seeligeri; Bacillus, for example, B. anthracis, B. cereus, B. subtilis, B. subtilus niger, B. thuringiensis; Nocardia asteroids; Legionella, eg, L. pneumonophilia, Pneumocystis, eg, P. carinii; Enterobacteriaceae such as Salmonella, Shigella, Escherichia (eg, E. coli, E. coliO157: H7); Klebsiella, Enterobacter, Serratia, Proteus, Morganella,

ES 2 587 007 T3

Providencia, Yersinia, and the like, eg, Salmonella, eg, S. typhi S. paratyphi A, B (S. schottmuelleri), and C (S. hirschfeldii), S. dublin S. choleraesuis, S. enteritidis, S typhimurium, S. heidelberg, S. newport, S. infantis, S. agona, S. montevideo, and S. saint-paul; Shigella, for example, subgroups: A, B, C and D, such as S. flexneri, S. sonnei, S. boydii, S. dysenteriae; Proteus (P. mirabilis, P. vulgaris and P. myxofaciens), Morganella (M. morganii); Providencia (P. rettgeri, P. alcalifaciens and P. stuartii); Yersinia, eg, Y. pestis, Y. enterocolitica, Haemophilus, eg, H. influenzae, H. parainfluenzae H. aphrophilus, H. ducreyi; Brucella, for example, B. abortus, B. melitensis, B. suis, B. canis; Francisella, for example, F. tularensis; Pseudomonas, for example, P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, P. acidovorans, Burkholderia (Pseudomonas) pseudomallei, Burkholderia mallei, Burkholderia cepacia, and Stenotrophomonas maltophilia; Campylobacter, for example, C. fetus fetus, C. jejuni, C. pylori (Helicobacter pylori); Vibrio, eg, V. cholerae, V. parahaemolyticus, V. mimicus, V. alginolyticus, V. hollisae, V. vulnificus, and the non-agglutinable vibrios; Clostridia, eg, C. penfringens, C. tetani, C. difficile, C. botulinum; Actinomyces, for example, A. israelii; Bacteroides, eg, B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. caccae, and B. merdae; Prevotella, for example, P. melaninogenica; genus Fusobacterium; Treponema, for example T. pallidum subspecies endemicum, T. pallidum subspecies pertenue, T. carateum and T. pallidum subspecies pallidum; genus Borrelia, for example, B burgdorferi; genus Leptospira; Streptobacillus, for example S. moniliformis; Spirillum, for example, S. minus; Mycobacterium, for example, M. tuberculosis, M. bovis, M. africanum, M. avium, M. intracellulare, M. kansasii, M. xenopi, M. marinum, M. ulcerans, the M. fortuitum complex (M. fortuitum and M. chelonei), M. leprae, M. asiaticum, M. chelonei subsp. abscessus, M. fallax, M. fortuitum, M. malmoense, M. shimoidei, M. simiae, M. szulgai, M. xenopi; Mycoplasma, for example, M. hominis, M. orale, M. salivarium, M. fermentans, M. pneumoniae, M. bovis, M. tuberculosis, M. avium, M. leprae; Mycoplasma, for example, M. genitalium; Ureaplasma, for example, U. urealyticum; Trichomonas, for example, T. vaginalis; Cryptococcus, for example, C. neoformans; Histoplasma, eg, H. capsulatum; Candida, for example, C. albicans; Aspergillus sp; Coccidioides, for example, C. immitis; Blastomyces, for example B. dermatitidis; Paracoccidioides, for example, P. brasiliensis; Penicillium, for example, P. marneffei; Sporothrix, for example, S. schenckii; Rhizopus, Rhizomucor, Absidia and Basidiobolus; diseases caused by Bipolaris, Cladophialophora, Cladosporium, Drechslera, Exophiala, Fonsecaea, Phialophora, Xylohypha, Ochroconis, Rhinocladiella, Scolecobasidium and Wangiella; Trichosporon, for example, T. beigelii; Blastoschizomyces, for example, B. capitatus; Plasmodium, for example, P. falciparum, P. vivax, P. ovale, and P. malariae; Babesia sp; protozoa of the genus Trypanosoma, for example, T. cruzi; Leishmania, for example, L. donovani, L. major L. tropica, L. mexicana, L. braziliensis, L. viannia braziliensis; Toxoplasma, for example, T. gondii; Amoebas of the genera Naegleria or Acanthamoeba; Entamoeba histolytica; Giardia lamblia; genus Cryptosporidium, for example, C. parvum; Isospora belli; Cyclospora cayetanensis; Ascaris lumbricoides; Trichuris trichiura; Ancylostoma duodenale or Necator americanus; Strongyloides stercoralis Toxocara, eg, T. canis, T. cati; Baylisascaris, for example, B. procyonis; Trichinella, for example, T. spiralis; Dracunculus, for example, D. medinensis; genus Filarioidea; Wuchereria bancrofti; Brugia, for example, B. malayi, or B. timori; Onchocerca volvulus; Loa loa; Dirofilaria immitis; genus Schistosoma, for example S. japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium; Paragonimus, for example, P. Westermani, P. Skriabini; Clonorchis sinensis; Fasciola hepatica; Opisthorchis sp; Fasciolopsis buski; Diphyllobothrium latum; Taenia, for example, T. saginata, T. solium; Echinococcus, for example, E. granulosus, E. multilocularis; Picornavirus, rhinovirus ecovirus, coxsackievirus, influenza virus; paramyxoviruses, eg, types 1, 2, 3, and 4; adnovirus; Herpesvirus, eg, HSV-1 and HSV-2; varicella-zoster virus; human T lymphotrophic virus (type I and type II); Arbovirus and Arenavirus; Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae; Flavivirus; Hantavirus; viral encephalitis (alphavirus eg, Venezuelan equine encephalitis, eastern equine encephalitis, western equine encephalitis); viral hemorrhagic fevers (filovirus, eg, Ebola, Marburg, and arenavirus, eg, Lassa, Machupo); Smallpox (variola); retroviruses eg human immunodeficiency virus 1 and 2; human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33 and 35.

In various embodiments, the probe may be selective for a polynucleotide sequence that may be characteristic of an organism selected from the group consisting of Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumoniae, Escherichia coli, Acinetobacter Baumannii, Serratia marcescens, Enterobacteria. aerogenes, Enterococcus faecium, Vancomycin resistant enterococci (VRE), Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Streptococcus viridans, Listeria monocytogenes, Enterococcus spp., Group B Streptococcus, Group C Streptococcus, Group G Streptococcus, Group F Streptococcus, Enterococum faecalismidis, Streptococcus vaginalis, Streptococcus vaginalis, Staiderisptocmidis, Streptococcus vaginalis, Staiderisptoecalismidis , Micrococcus sps., Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrahlis, Salmonella sps., Chlamydia trachomatis, Peptostreptococcus productus, Peptostreptococcus anaerobius, Lactobacillus fermentum, Eubacterium lentum, Candida glabrata, Candida albicans, Chlamydia spp., Camplobacter spp., Salmonella spp., Smallpox (variola major), Yersinia Pestis, Herpes simplex virus I (HSV I), and Herpes simplex virus I (HSV I), and Herpes simplex virus I (HSV I), and (VHS II).

In various embodiments, the probe can be selective for a polynucleotide sequence that is characteristic of group B Streptococcus.

The technology herein also comprises a microfluidic cartridge that has a component to inhibit fluid movement. The component comprises a channel, a first mass of a thermally sensitive substance (TRS) arranged on a first side of the channel, a second mass of a TRS arranged on a second side of the channel opposite the first side of the channel, a source of gas pressure

ES 2 587 007 T3 associated with the first mass of the TRS. Actuation of the gas pressure source drives the first TRS mass into the second TRS mass and clogs the channel.

The microfluidic cartridge may include a second source of gas pressure associated with the second mass of the TRS. Actuation of the second gas pressure source drives the second TRS mass into the first TRS mass. At least one (eg, both) of the first and second TRS masses can be a wax.

Another aspect of the microfluidic cartridge includes a component for plugging a channel of a microfluidic cartridge. A mass of one TRS can be heated and driven through the channel (eg by gas pressure) into a second mass of TRS. The second mass of TRS can also be propelled (eg by gas pressure) towards the first mass of TRS.

Another aspect of the microfluidic cartridge is an actuator. The actuator includes a channel, a chamber connected to the channel, at least one encapsulated liquid reservoir disposed in the chamber, and a gas surrounding the reservoir within the chamber. Heating the chamber expands the encapsulated liquid reservoir and pressurizes the gas. Normally the liquid has a boiling point of about 90 ° C or less. The liquid can be a hydrocarbon having about 10 carbon atoms or less. The liquid can be encapsulated by a polymer.

An actuator can include multiple reservoirs of encapsulated liquid disposed in the chamber. The multiple deposits can be dispersed within a solid (eg, a wax). The multiple reservoirs may be arranged within a flexible enclosure (eg, a flexible bag).

Another aspect of the microfluidic cartridge includes pressurizing a gas within a chamber of the device to create a gas pressure sufficient to move a liquid within a channel of the microfluidic device. Pressurizing the gas typically expands at least one encapsulated liquid reservoir disposed within the chamber. Expanding the at least one reservoir may include heating the chamber. Pressurizing the gas can include expanding multiple reservoirs of encapsulated liquid.

Another aspect of a microfluidic cartridge for use herein includes combining (eg, mixing) first and second volumes of liquid. The device includes a mass of a temperature sensitive substance (TRS) that separates the first and second channels of the device. The device can be configured to move a first liquid along the first channel so that a part (for example, a central part) of the first liquid can be adjacent to the TRS, and to move a second liquid along the second channel so that a part (eg, a central part) of the second liquid can be adjacent to the TRS. A heat source can be actuated to move the TRS (eg, melting, scattering, fragmenting). The central parts of the first and second liquids normally combine without being separated by a gaseous interface. Normally, only a subset of the first liquid and a subset of the second liquid can be combined. Liquids are mixed after having moved through a mixing channel. Liquids, when combined, must move at least two-droplet lengths to achieve good mixing by interstratification and transverse diffusion (perpendicular to the length of the microchannel) without having to rely on longitudinal diffusion alone for mixing (see, also, for example, "Mathematical modeling of drop mixing in 'a slit-type microchannel", K Handique, et al., J. Micromech. Microeng., 11 548-554, (2001)). By moving the combined drop the length of one drop, the liquid in the middle of the receding drop is caused to move to the front of the leading drop and then toward the channel wall. At the back end of the drop, the fluid moves from the wall toward the center of the drop. This movement of the drop results in interstratification between the two liquids. Additional interstratification can be achieved by repeating the method additional times, such as over additional drop lengths.

The microfluidic cartridge further includes a lyophilized reagent particle. In some embodiments, the lyophilized particles include multiple smaller particles each having a plurality of ligands that preferentially associate with polynucleotides as compared to PCR inhibitors. The lyophilized particles can also (or alternatively) include lysis reagents (eg, enzymes) configured to lyse cells to release polynucleotides. The lyophilized particles can also (or alternatively) include enzymes (eg, proteases) that degrade proteins.

Cells can be lysed by combining a solution of the cells, eg, in a microfluidic droplet, with the lyophilized particles thereby reconstituting the particles. Reconstituted lysis reagents lyse cells. Polynucleotides associate with ligands on the smallest particles. During lysis, the solution can be heated (eg, by radiation using a lamp such as a heat lamp, or by a contact heat source.

In some embodiments, the lyophilized particles include reagents (eg, primers, control plasmids, polymerase enzymes) to perform PCR.

ES 2 587 007 T3

Another aspect of the microfluidic cartridge includes a liquid reservoir capable of containing a liquid (eg, a solvent, a buffer, a reagent, or combinations thereof). In general, the reservoir may have one or more of the following characteristics, as further described in International Application Publication No. WO2006 / 079082.

The reservoir may include a wall that can be manipulated (eg, pressed or depressed) to reduce a volume within the reservoir. For example, the reservoir may include a piercing member (eg, a needle-like or pointed or otherwise sharp member) that breaks another part of the reservoir (eg, a wall portion) to release liquid. The piercing member may be internal to the reservoir, such that the piercing member breaks the wall from an internal surface of the reservoir (eg, the wall) outward.

In general, the wall resists the passage of liquid or vapor through it. In some embodiments, the wall lacks the ability to stretch. The wall can be flexible. The wall can be, for example, a metallic layer, for example a foil layer, a polymer, or a laminate that includes a combination thereof. The wall can be formed by vacuum forming (eg, applying a vacuum and heat to a layer of material to stretch the layer against a molding surface). The molding surface can be concave so that the wall can be provided with a generally convex surface.

Exemplary liquids contained by the reservoir include water and aqueous solutions that include one or more salts (eg, magnesium chloride, sodium chloride, Tris buffer, or a combination thereof). The reservoir can retain the liquid (eg, without substantial evaporation thereof) for a period of time (eg, at least 6 months or at least one year). In some embodiments, less than 10% (eg, less than about 5%) by weight of the liquid evaporates over the course of a year.

The piercing member can be an integral part of a wall of the reservoir. For example, the reservoir may include a wall having an internal projection, which may be in contact with liquid in the reservoir. The reservoir also includes a second wall opposite the piercing member. During actuation, the piercing member can be pushed through the second wall (eg, from the inside out) to release the liquid.

In some embodiments, a maximum amount of liquid held by a reservoir can be less than about 1 ml. For example, a reservoir may contain approximately 500 microliters or less (eg, 300 microliters or less). In general, a reservoir contains at least about 25 microliters (eg, at least about 50 microliters). The reservoir can introduce approximately 10% of the desired amount of liquid (for example, 50 ± 5 μ!).

The reservoir can supply a predetermined quantity of liquid that can be substantially free of air (eg, substantially free of gas). At the time of introduction of the liquid, the substantially air and / or gas-free liquid produces few or no bubbles large enough to obstruct the movement of the liquid within the microfluidic device. The use of a piercing member internal to the reservoir can improve an ability of the reservoir to deliver liquids substantially free of air and / or gas.

In some embodiments, the reservoir can be actuated to release liquid by pressing (eg, with one's finger or thumb or by actuation by mechanical pressure). The pressure can be applied directly to a wall of the reservoir or to a plunger having a piercing member. In various embodiments, minimal pressure may be required to actuate the reservoir. An automated system can be used to actuate (eg, press on) a plurality of reservoirs simultaneously or in sequence.

Actuation of the reservoir may include urging a piercing member through a wall of the reservoir. In some embodiments, the reservoir does not include a piercing member. Instead, the internal pressure generated within the reservoir breaks a wall of the reservoir allowing liquid to enter the microfluidic device.

After actuating a reservoir to introduce liquid into the microfluidic device, the liquid is generally not drawn back into the reservoir. For example, at the time of actuation, the volume of the reservoir may decrease to a certain minimum but generally does not increase to draw liquid back into the reservoir. For example, the tank can remain folded down after actuation. In such embodiments, the flexible wall can be flexible but lacks hysteresis, elasticity, or stretchability. Alternatively or in combination, the reservoir can draw in air from a vent without removing any of the liquid.

The reservoir preserves the reactivity and composition of the reagents within it (for example, the chemicals within the reservoir may show little or no change in reactivity for 6 months or a year).

The flexible wall of the tank can limit or prevent the leakage of chemicals through it. The reservoir can be assembled independently of a microfluidic cartridge and then attached to the microfluidic cartridge.

ES 2 587 007 T3

Exemplary microfluidic cartridge for processing polynucleotides

Referring to Figure 10, a portion of an exemplary microfluidic cartridge 200 suitable for use with the system described herein includes first, second, and third layers 205, 207, and 209 that define a microfluidic network 201 having various components configured to processing a sample that includes one or more polynucleotides to be detected. Cartridge 200 typically processes the sample, among other things, increasing the concentration of a polynucleotide to be determined and / or reducing the concentration of inhibitors relative to the concentration of polynucleotide to be determined. The various features of cartridge 200 can be incorporated together, or with suitable modification, into alternative cartridge configurations that perform polynucleotide processing in conjunction with various other operations.

Cartridge manufacturing

The microfluidic cartridge 200 can be manufactured as desired. Typically, layers 205, 207, and 209 can be formed of a polymeric material. The components of the network 201 can typically be formed by molding (eg, by injection molding) the layers 207, 209. Layer 205 can typically be a flexible polymeric material (eg, a laminate) that can be attached (eg, adhesively and / or thermally) to layer 207 to seal the components of network 201. Layers 207 and 209 can fix to each other using adhesive. Other methods of making cartridges suitable for application herein can be found in US Provisional Patent Application Serial No. 60 / 859,284, filed November 14, 2006.

Microfluidic network

An exemplary arrangement of components of network 201 is as follows, as further described in United States Patent Application Publication No. 2006/0166233.

The net 201 includes an inlet 202 through which sample material can be introduced into the net and an outlet 236 by which a processed sample can be withdrawn (eg, ejected by or drawn from) the net 201. A channel 204 extends between inlet 202 and a junction 255. A valve 206 may be located along channel 204. A reservoir channel 240 extends between junction 255 and an actuator 244. Gates 242 and 246 may be located along channel 240. A channel 257 extends between junction 255 and a junction 259. A valve 208 may be located along channel 257. A reservoir channel 246 extends between junction 259 and an actuator 248. Gates 250 and 252 may be located along channel 246. A channel 261 extends between junction 259 and junction 263. A valve 210 and a hydrophobic vent hole 212 may be located along channel 261: A channel 256 extends between junction 263 and an actuator 254. A gate 258 may be located along channel 256.

A channel 214 extends between the junction 263 and a processing chamber 220, having an inlet 265 and an outlet 267. A channel 228 extends between the outlet of the processing chamber 267 and a waste reservoir 232. A valve 234 it may be located along channel 228. A channel 230 extends between the outlet of the processing chamber 267 and the outlet 236.

Particular components of the microfluidic network 201 are further described as follows.

Processing chamber

Referring also to Figure 11, processing chamber 220 includes a plurality of particles (eg, beads, microspheres) 218 ​​configured to retain sample polynucleotides under a first set of conditions (eg, a first temperature and / or a first pH) and to release the polynucleotides under a second set of conditions (eg, a second higher temperature and / or a second more basic pH). Typically, the polynucleotides may be retained, preferably in comparison to inhibitors that may be present in the sample. The particles 218 may be configured as a retention member 216 (eg, a column) through which sample material (eg, polynucleotides) must pass as it moves between the inlet 265 and the outlet 267 of the region of processing 220.

A filter 219 prevents particles 218 from passing downstream of the processing region 220. A channel 287 connects the filter 219 with the outlet 267. The filter 219 has a surface area within the processing region 220 that may be greater than the cross-sectional area of ​​inlet 265. For example, in some embodiments, the ratio of the surface area of ​​the filter 219 within the processing chamber 220 to the cross-sectional area of ​​the inlet 265 (a cross-sectional area that is typically approximately the same as the cross-sectional area of ​​the channel 214) can be at least about 5 (eg, at least about 10, at least about 20, at least about 30). In some embodiments, the surface area of ​​filter 219 within processing region 220 can be at least about 1mm.²(for example, at least about 2 mm², at least about 3 mm²). In some embodiments, the cross-sectional area of ​​inlet 265 and / or channel 214 can be approximately 0.25mm.² or less (for

ES 2 587 007 T3 example, approximately 0.2 mm² or less, about 0.15mm² or less, about 0.1mm² or less). The larger surface area presented by filter 219 to material flowing through processing chamber 220 helps prevent coagulation of the processing region while avoiding significant increases in void volume (described hereinafter). from the processing region.

Particles 218 can be modified with at least one polynucleotide-retaining ligand (eg, preferably compared to inhibitors). Typically, ligands retain polynucleotides from liquids that have a pH of about 9.5 or less (eg, about 9.0 or less, about 8.75 or less, about 8.5 or less). As a sample solution moves through the processing chamber 220, the polynucleotides may be retained while the liquid and other components of the solution (eg, inhibitors) may be less retained (eg, not retained) and exit the processing region. In general, ligands release polynucleotides when the pH can be about 10 or higher (eg, about 10.5 or higher, about 11.0 or higher). Consequently, the polynucleotides can be released from the ligand-modified particles into the surrounding liquid.

Exemplary particulate ligands 218 include, for example, polyamides (eg, polycationic polyamides such as poly-L-lysine, poly-D-lysine, poly-DL-ornithine) and PEl. Other ligands include, for example, intercalators, polyintercalators, minor groove-binding polyamines (eg, spermidine), homopolymers and copolymers comprising a plurality of amino acids, and combinations thereof. In some embodiments, the ligands have an average molecular weight of at least about 5,000 Da (eg, at least about 7,500 Da, of at least about 15,000 Da). In some embodiments, the ligands have an average molecular weight of about 50,000 Da or less (eg, about 35,000, or less, about 27,500 Da or less). In some embodiments, the ligand can be a polylysine ligand attached to the surface of the particle via an amide bond.

In certain embodiments, the ligands in particles 218 may be resistant to enzymatic degradation, such as degradation by protease enzymes (eg, mixtures of endo- and exo-proteases such as pronase) that cleave peptide bonds. Exemplary protease resistant ligands include, for example, poly-D-lysine and other ligands that may be enantiomers of ligands susceptible to enzymatic attack.

The particles 218 can normally be formed of a material to which the ligands can associate. Exemplary materials from which particles 218 can be formed include polymeric materials that can be modified to bind a ligand. Typical polymeric materials provide or can be modified to provide carboxylic groups and / or amino groups available to bind ligands. Exemplary polymeric materials include, for example, polystyrene, latex polymers (eg, polycarboxylate coated latex), polyacrylamide, polyethylene oxide, and derivatives thereof. Polymeric materials that can be used to form particles 218 are described in US Patent No. 6,235,313 to Mathiowitz et al. Other materials include glass, silica, agarose, and amino-propyl-tri-ethoxy-silane (APES) modified materials.

Exemplary particles that can be modified with suitable ligands include carboxylate particles (e.g., carboxylate modified magnetic beads (Sera-Mag carboxylate modified magnetic beads, Part No. 3008050250, Seradyn) and Polybead carboxylate modified microspheres available from Polyseience, Catalog # 09850). In some embodiments, the ligands include poly-D-lysine and the beads comprise a polymer (eg, polycarboxylate coated latex). In other embodiments, the ligands include PEl.

In general, the ratio of the mass of particles to the mass of polynucleotides retained by the particles may be no more than about 25 or more (eg, no more than about 20, no more than about 10). For example, in some embodiments, about 1 gram of particles retain about 100 milligrams of polynucleotides.

Typically, the total volume of processing chamber 220 (including particles 218) between inlet 265 and filter 219 can be about 15 microliters or less (e.g., about 10 microliters or less, about 5 microliters or less, about 2 , 5 microliters or less, about 2 microliters or less). In an exemplary embodiment, the total volume of processing region 220 can be about 2.3 microliters. In some embodiments, the particles 218 occupy at least about 10 percent (eg, at least about 15 percent) of the total volume of the processing region 220. In some embodiments, the particles 218 occupy about 75 percent. or less (eg, about 50 percent or less, about 35 percent or less) of the total volume of the processing chamber 220.

In some embodiments, the volume of the processing chamber 220 that may be free to be occupied by liquid (for example, the void volume of the processing chamber 220 that includes interstices between the particles 218) may be approximately equal to the total volume minus the volume occupied by the particles. Typically, the void volume of the processing region 220 can be about 10

ES 2 587 007 T3 microliters or less (for example, about 7.5 microliters or less, about 5 microliters or less, about 2.5 microliters or less, about 2 microliters or less). In some embodiments, the void volume can be about 50 nanoliters or more (eg, about 100 nanoliters or more, about 250 nanoliters or more). For example, in an exemplary embodiment, the total volume of the processing chamber 220 may be about 2.3 microliters, the volume occupied by the particles may be approximately 0.3 microliters, and the free volume to be occupied by liquid ( void volume) can be about 2 microliters.

Particles 218 typically have an average diameter of about 20 microns or less (eg, about 15 microns or less, about 10 microns or less). In some embodiments, the particles 218 have an average diameter of at least about 4 microns (eg, at least about 6 microns, at least about 8 microns).

In some embodiments, a volume of channel 287 between filter 219 and outlet 267 may be substantially smaller than the void volume of processing chamber 220. For example, in some embodiments, the volume of channel 287 between filter 219 and the outlet 267 may be about 35% or less (eg, about 25% or less, about 20% or less) of the void volume. In an exemplary embodiment, the volume of channel 287 between filter 219 and outlet 267 can be approximately 500 nanoliters.

The particle density can normally be at least about 10⁸ particles per milliliter (for example, about 10⁹ particles per milliliter). For example, a processing region with a total volume of about 1 microliter may include about 10³ pearls.

Filter 219 typically has pores with a diameter smaller than the diameter of particles 218. In an exemplary embodiment, filter 219 has pores that have an average width of about 8 microns, where particles 218 have an average diameter of about 10 microns.

In some embodiments, at least some (eg, all) of the particles may be magnetic. In alternative embodiments, few (eg, none) of the particles are magnetic.

In some embodiments, at least some (eg, all) of the particles may be solid. In some embodiments, at least some (eg, all) of the particles may be porous (eg, the particles may have channels that extend at least partially into them).

Additional components that can be found in the microfluidic network 201 are as follows.

Channels

The channels of the microfluidic network 201 typically have at least one sub-millimeter cross-sectional dimension. For example, the channels of the lattice 201 may have a width and / or a depth of about 1mm or less (eg, about 750 microns or less, about 500 microns, or less, about 250 microns or less).

Valves

A valve can be a component that has a normally open state that allows material to pass along a channel from a position on one side of the valve (for example, upstream of the valve) to a position on the other side of the valve. the valve (for example, downstream of the valve). Upon actuation, the valve changes to a closed state that prevents material from passing along the channel from one side of the valve to the other. For example, in Figure 10, valve 206 includes a mass 251 of a thermally sensitive substance (TRS) that may be relatively immobile at a first temperature and more mobile at a second temperature (e.g., a phase transition material ( PTM) of a known melting point, typically about 60 ° C, or about 75 ° C, or about 90 ° C, such as a paraffin wax, solder, etc.). A chamber 253 may be in gaseous communication with the mass 251. After heating the gas (eg, air) in chamber 253 and heating TRS mass 251 to the second temperature, the gas pressure within chamber 253 moves mass 251 into channel 204 obstructing the passage of material to throughout it. Other valves in network 201 have a similar structure and function in a similar way to valve 206.

A TRS mass can be an essentially solid mass or an agglomeration of smaller particles that cooperate to obstruct the passage. Examples of TRS include a eutectic alloy (eg, a solder), wax (eg, an olefin), polymers, plastics, and combinations thereof. The first and second temperatures may be insufficiently high to damage materials, such as polymer layers of cartridge 200. Generally, the second temperature can be less than about 90 ° C and the first temperature can be less than the second temperature (eg, about 70 ° C or less).

ES 2 587 007 T3

A gate can be a component that can have a closed state that does not allow material to pass along a channel from a position on one side of the gate to another side of the gate, and an open state that allows material pass along a channel from a position on one side of the gate to the other side of the gate. Actuation of an open gate can cause the gate to change to a closed state in which material is not allowed to pass from one side of the gate (e.g. upstream of the gate) to the other side of the gate (e.g. downstream of the gate). Upon actuation, a closed gate can change to an open state in which material is allowed to pass from one side of the gate (for example, upstream of the gate) to the other side of the gate (for example, downstream of the gate). For example, gate 242 in FIG. 10 includes a TRS mass 271 positioned to obstruct the passage of material between junction 255 and channel 240. Upon heating mass 271 to a second temperature, the mass changes state (eg, by melting, by dispersion, by fragmentation, and / or dissolution) to allow material to pass between junction 255 and channel 240.

In various embodiments, a microfluidic network 201 can include a narrow gate 380 as shown in Figure 12A where a gate loading channel 382 used to load wax from a wax loading hole 384 to a gate junction 386 can be narrower (for example, approximately 150 μιτι wide and 100 microns deep). An upstream channel 388 as well as a downstream channel 390 from gate junction 386 can be made wide (eg, ~ 500 pm) and deep (eg, ~ 500 pm) to help ensure that the wax stops at the gate joint 386. The amount of gate material melted and moved out of gate joint 386 can be minimized for optimal opening of gate 380. Since a heater external to the cartridge can be used to melt the thermally sensitive substance in gate 380, misalignment of the heater could cause the wax in gate charging channel 382 to melt as well. Therefore, narrowing the dimension of the loading channel can increase the reliability of the gate opening. In the case of excessive amounts of molten wax at gate junction 386 and gate loading channel 382, ​​the increased cross-sectional area of ​​downstream channel 390 adjacent to gate junction 386 can prevent wax from clogging the channel. downstream 390 during the opening of gate 380. The dimensions of upstream channel 388 at gate joint 386 can be made similar to downstream channel 390 to ensure proper wax loading during gate fabrication.

In various embodiments, the gate can be configured to minimize the effective area or space occupied by the gate within the network, such as the folded gate 392 as shown in FIG. 12B. Minimizing the effective area or space occupied by the gate within the network can increase the density of a given microfluidic network and can thereby reduce the cost per part, provide a more compact network, minimize the length or volume of channels from the network, or the like. Still other configurations are possible, although not explicitly shown in the drawings, in accordance with specific arrangements of the microfluidic network.

In the microfluidic cartridge of Figure 10, the channel portion 240 between gates 242 and 246 forms a fluid reservoir 279 configured to contain a liquid (eg, water, an organic liquid, or a combination thereof). During storage, gates 242 and 246 limit (eg, prevent) evaporation of liquid within the fluid reservoir. During operation of cartridge 200, the liquid in reservoir 279 can typically be used as a wash liquid to remove inhibitors from processing region 220 while leaving polynucleotides associated with particles 218 (FIG. 11). Typically, the wash liquid can be a solution that has one or more additional components (eg, a buffer, chelator, surfactant, detergent, base, acid, or a combination thereof). Exemplary solutions include, for example, a 10-50 mM Tris solution at pH 8.0, 0.5-2 mM EDTA, and 0.5% - 2% SDS, a 10-50 mM Tris solution at pH 8.0, 0.5 to 2 mM EDTA, and 0.5% - 2% Triton X-100.

Channel portion 247 between gates 250 and 252 forms a fluid reservoir 281 configured as reservoir 279 to contain a liquid (eg, a solution) with limited or no evaporation. During the operation of the cartridge 200, the liquid in the reservoir 281 can normally be used as a release liquid into which polynucleotides that had been retained by the particles 218 can be released. An exemplary release liquid may be a hydroxide solution (e.g., a NaOH solution) having a concentration of, for example, between about 2 mM hydroxide (e.g., about 2 mM NaOH) and about 500 mM hydroxide (e.g. example, NaOH about 500 mM). In some embodiments, the liquid in reservoir 281 may be a hydroxide solution having a concentration of about 25 mM or less (eg, a hydroxide concentration of about 15 mM).

The reservoirs 279, 281 typically each independently contain at least about 0.375 microliters of liquid (eg, at least about 0.750 microliters, at least about 1.25 microliters, at least about 2.5 microliters). In some embodiments, reservoirs 279, 281 each independently contain about 7.5 microliters or less of liquid (eg, about 5 microliters or less, about 4 microliters or less, about 3 microliters or less).

ES 2 587 007 T3

Actuators

An actuator may be a component that provides a gas pressure that can move material (eg, sample material and / or reagent material) between one location in a network, eg, network 201, and another location. For example, referring to FIG. 13, actuator 244 includes a chamber 272 having a mass 273 of thermally expansive material (TEM) therein. When heated, TEM expands decreasing the free volume within chamber 272 and pressurizing gas (eg, air) surrounding mass 273 within chamber 272. Typically, gates such as gates 246 and 242 in network 201 can be actuated with actuator 244. Consequently, pressurized gas drives liquid in fluid reservoir 279 toward junction 255. In some embodiments, actuator 244 can generate a pressure differential of greater than about 3 psi (eg, at least about 4 psi, at least about 5 psi) between actuator and junction 255.

In one embodiment, shown in Figure 13, the TEM includes a plurality of sealed liquid reservoirs (eg, spheres) 275 dispersed within a carrier 277. Typically, the liquid may be a liquid with high vapor pressure (eg , isobutane and / or isopentane) sealing within a shell (eg, a polymeric shell made up of monomers such as vinylidene chloride, acrylonitrile, and methyl methacrylate). Carrier 277 has properties (eg, flexibility and / or an ability to soften (eg, melt) at higher temperatures) that allow expansion of reservoirs 275 without allowing reservoirs to pass along channel 240. In In some embodiments, carrier 277 can be a wax (eg, an olefin) or a polymer with a suitable glass transition temperature. Typically, the deposits make up at least about 25 weight percent (eg, at least about 35 weight percent, at least about 50 weight percent) of the TEM. In some embodiments, the deposits make up about 75 percent by weight or less (eg, about 65 percent by weight or less, about 50 percent by weight or less) of the TEM. Suitable sealed liquid reservoirs can be obtained from Expancel (available from Akzo Nobel).

When TEM can be heated (for example, to a temperature of at least about 50 ° C (for example, to at least about 75 ° C, or at least about 90 ° C)), the liquid vaporizes and increases the volume of each sealed reservoir and dough 273. Carrier 277 softens allowing dough 273 to expand. Typically, the TEM can heat up to a temperature of less than about 150 ° C (eg, about 125 ° C or less, about 110 ° C or less, about 100 ° C or less) during actuation. In some embodiments, the volume of the TEM is expanded at least about 5 times (eg, at least about 10 times, at least about 20 times, at least about 30 times).

Ventilation holes

A hydrophobic vent (eg, vent 212) can be a structure that allows gas to exit a channel while limiting (eg, preventing) liquid from exiting the channel. Hydrophobic vents typically include a layer of porous hydrophobic material (eg, a porous filter such as an Osmonics porous hydrophobic membrane) that defines a wall of the channel. As described hereinafter, hydrophobic vents can be used to position a sample microdroplet at a desired location within network 201.

The hydrophobic vents of the present technology are preferably constructed so that the amount of air that escapes through them can be maximized while minimizing the volume of the channel below the surface of the vent. Accordingly, it is preferable that the vent can be constructed to have a hydrophobic membrane of large surface area and a shallow cross-section of the microchannel below the surface of the vent.

Hydrophobic vents typically have a length of at least about 2.5mm (eg, at least about 5mm, at least about 7.5mm) along a channel. The length of the hydrophobic vent can typically be at least about 5 times (eg, at least about 10 times, at least about 20 times) greater than a channel depth within the hydrophobic vent. For example, in some embodiments, the depth of the channel within the hydrophobic vent can be about 300 microns or less (eg, about 250 microns or less, about 200 microns or less, about 150 microns or less).

The depth of the channel within the hydrophobic vent can typically be about 75% or less (eg, about 65% or less, about 60% or less) of the depth of the channel upstream and downstream of the hole. hydrophobic vent. For example, in some embodiments, the depth of the channel within the hydrophobic vent can be about 150 microns and the depth of the channel upstream and downstream of the hydrophobic vent can be about 250 microns.

ES 2 587 007 T3

A channel width within the hydrophobic vent can typically be at least about 25% wider (eg, at least about 50% wider) than a channel width upstream of the vent and downstream of the hole. ventilation. For example, in an exemplary embodiment, the width of the channel within the hydrophobic vent can be about 400 microns and the width of the channel upstream and downstream of the vent can be about 250 microns.

In use, cartridge 200 can typically be thermally associated with a series of heat sources configured to operate various components (eg, valves, gates, actuators, and processing region 220) of the cartridge. In some embodiments, the heat sources may be controlled by a processor in a system such as that further described herein, which functions to receive and monitor the cartridge during use. The processor (eg, a microprocessor) is configured to operate the heat sources individually and at different times, according to a desired protocol. Processors configured to operate microfluidic cartridges, suitable for use or modification for use herein, are described in U.S. Application No. 09 / 819,105, filed March 28, 2001 (now U.S. Patent No. .7,010,391). In other embodiments, the heat sources can be integral with the cartridge itself.

Cartridge 200 can be operated in the following manner. The valves of the network 201 can be manufactured in an open state. The gates of the network 201 can be manufactured in a closed state. A fluid sample, such as a biological sample as further described herein, comprising polynucleotides can be introduced into lattice 201 via inlet 202. For example, the sample can be introduced with a syringe having a Luer fitting. The syringe provides pressure to initially move the sample within network 201. The sample passes along channels 204, 257, 261, and 214 to the inlet 265 of processing region 220. The sample passes through the region of processing 220, exits via outlet 267, and passes along channel 228 to waste chamber 232. When the trailing edge (for example, the upstream liquid-gas interface) of the sample reaches the hydrophobic vent hole 212, the pressure provided by the introducer (for example, the syringe) can be released from the network 201 by stopping the additional movement of the sample.

Typically, the amount of sample introduced can be about 500 microliters or less (eg, about 250 microliters or less, about 100 microliters or less, about 50 microliters or less, about 25 microliters or less, about 10 microliters or less). In some embodiments, the amount of sample can be about 2 microliters or less (eg, about 0.5 microliters or less).

Polynucleotides entering processing region 220 pass through interstices between particles 218. Polynucleotides in the sample contact retention member 216 and can be retained preferentially compared to sample liquid, and certain other components of the sample (eg inhibitors). Typically, the retention member 220 retains at least about 50% of the polynucleotides (eg, at least about 75%, at least about 85%, at least about 90%) of the polynucleotides present in the sample that entered. in processing region 220. Sample liquid and inhibitors present in the sample exit processing region 220 via outlet 267 and enter waste chamber 232. Processing region 220 can typically be at a temperature of about 50 ° C or less (eg, 30 ° C or less) during sample introduction.

Processing continues by flushing retention member 216 with liquid from reservoir 279 to remove remaining inhibitors from polynucleotides retained by retention member 216. To flush retention member 216, valve 206 can be closed and gates 242, 246 of the first reservoir 240 can be opened. Actuator 244 may be actuated to move wash liquid within reservoir 279 along channels 257, 261, and 214, through processing region 220, and into waste reservoir 232. Wash liquid moves sample that may have remained within channels 204, 257, 261, and 214 through the processing region and into waste chamber 232. Once the trailing edge of the wash liquid reaches the vent hole 212, the gas pressure generated by the actuator 244 can be vented and the further movement of the liquid can be stopped.

The volume of wash liquid moved by actuator 244 through processing region 220 can typically be at least about 2 times the void volume of processing region 220 (e.g., at least about 3 times the void volume ) and can be about 10 times the void volume or less (for example, about 5 times the void volume or less). The processing region can typically be at a temperature of about 50 ° C or less (eg, 30 ° C or less) during washing. Exemplary wash fluids include fluids described with respect to reservoirs 279 and 281, herein.

ES 2 587 007 T3

Processing continues to release polynucleotides from retention member 216. Typically, the wash fluid from reservoir 279 can be replaced with release fluid (eg, a hydroxide solution) from reservoir 281 prior to releasing the polynucleotides. Valve 208 can be closed and gates 250, 252 can be opened. Actuator 248 can be actuated, thereby moving the release liquid within reservoir 281 along channels 261, 214 and into processing region 220 and in contact with retention member 216. When the trailing edge of the release liquid from reservoir 281 reaches hydrophobic vent hole 212, the pressure generated by actuator 248 can be vented stopping further movement of the liquid. The volume of liquid moved by actuator 248 through processing region 220 can typically be at least approximately equal to the volume of voids in processing region 220 (eg, at least about 2 times the volume of voids) and can be about 10 times the void volume or less (for example, about 5 times the void volume or less).

Once the retaining member 216 with retained polynucleotides has been contacted with liquid from reservoir 281, a release step can normally be performed. Usually; release includes heating release liquid present within processing region 216. Generally, the liquid can be heated to a temperature insufficient to bring the liquid to a boil in the presence of the retention member. In some embodiments, the temperature can be 100 ° C or less (eg, less than 100 ° C, about 97 ° C or less). In some embodiments, the temperature can be about 65 ° C or more (eg, about 75 ° C or more, about 80 ° C or more, about 90 ° C or more). In some embodiments, the temperature is held for about 1 minute or more (eg, about 2 minutes or more, about 5 minutes or more, about 10 minutes or more). In some embodiments, the temperature can be held for about 30 minutes (eg, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less). In an exemplary embodiment, the processing region 220 may be heated to between about 65 and 90 ° C (eg, about 70 ° C) for between about 1 and 7 minutes (eg, about 2 minutes). Said temperatures and times vary according to the sample and can be selected accordingly by one skilled in the art.

The polynucleotides can be released into the liquid present in the processing region 220 (eg, the polynucleotides can normally be released in an amount of delivery liquid that has a volume approximately equal to the void volume of the processing region 220). Typically, polynucleotides can be released in about 10 microliters or less (eg, about 5 microliters or less, about 2.5 microliters or less) of liquid.

In certain embodiments, the ratio of the original sample volume moved through processing region 220 relative to the volume of liquid into which the polynucleotides can be delivered may be at least about 10 (e.g., at least about 50, at least about 10). minus about 100, at least about 250, at least about 500, at least about 1000). In some embodiments, polynucleotides from a sample having a volume of about 2 ml can be retained within the processing region, and released in about 4 microliters or less (eg, about 3 microliters or less, about 2 microliters or less, about 1 microliter or less) of liquid.

The liquid into which the polynucleotides can be released typically includes at least about 50% (eg, at least about 75%, at least about 85%, at least about 90%) of the polynucleotides present in the sample. which entered processing region 220. The concentration of polynucleotides present in the delivery liquid may be higher than in the original sample since the volume of the delivery liquid can normally be less than the volume of the original liquid sample moved through the processing region. For example, the concentration of polynucleotides in the delivery liquid can be at least about 10 times higher (eg, at least about 25 times higher, at least about 100 times higher) than the concentration of polynucleotides in the sample introduced into the cartridge 200. The concentration of inhibitors present in the liquid into which the polynucleotides can be released may, in general, be less than the concentration of inhibitors in the original fluid sample in an amount sufficient to increase the efficiency of amplification for the polynucleotides.

The time interval between introduction of the sample containing polynucleotides into processing region 220 and the release of the polynucleotides into the delivery liquid can typically be about 15 minutes or less (eg, about 10 minutes or less, about 5 minutes or less).

Liquid including released polynucleotides can be removed from processing region 220 in the following manner. Valves 210 and 234 may be closed. Gates 238 and 258 may be open. Actuator 254 can be actuated to generate pressure that moves liquid and polynucleotides from processing region 220, into channel 230, and toward outlet 236. The polynucleotide fluid can be removed using, for example, a syringe or automated sampling device. Depending on the liquid in contact with the limb

ES 2 587 007 T3 retention 216 during polynucleotide release, the released polynucleotide solution can be neutralized with an amount of buffer (eg, an equal volume of 25-50 mM Tris-HCl buffer pH 8.0).

Although the release of the polynucleotides has been described to include a heating step, the polynucleotides can be released without heating. For example, in some embodiments, the liquid in reservoir 281 has an ionic strength, pH, surfactant concentration, composition, or combination thereof that releases polynucleotides from the retention member at room temperature, without the need for additional heating.

Although polynucleotides have been described being released into a single volume of liquid present within processing region 220, other configurations can be used. For example, polynucleotides can be released with the concomitant (staged or continuous) introduction of fluid into and / or through processing region 220. In such embodiments, the polynucleotides can be released into liquid having a volume of about 10 times or less (eg, about 7.5 times or less, about 5 times or less, about 2.5 times or less, about 2 times or less) than the void volume of the processing region 220.

Although reservoirs 279, 281 have been described containing liquids between first and second gates, other configurations can be used. For example, the liquid for each reservoir may be contained within a sachet (eg, a blister pack, as further described herein) isolated from the net 201 by a generally impermeable membrane. The sachet may be configured so that a user can rupture the membrane by pushing liquid into reservoirs 279, 281 where actuators 244, 248 can move the liquid during use.

Although processing regions have been described as having microliter scale dimensions, other dimensions can be used. For example, processing regions with surfaces (eg, particles) configured to preferentially retain polynucleotides as opposed to inhibitors can have large volumes (eg, many tens of microliters or more, at least about 1 milliliter or more). In some embodiments, the processing region is table-scale.

Although the processing region 220 has been described as having a retention member comprised of multiple surface modified particles, other configurations can be used. For example, in some embodiments, the processing region 220 includes a retention member configured as a porous member (eg, a filter, a porous membrane, or a gel matrix) that has multiple openings (eg, pores and / or or channels) through which polynucleotides pass. The surfaces of the porous member can be modified to preferentially retain polynucleotides. Filter membranes available from, for example, Osmonics, can be made up of polymers that can be surface modified and used to retain polynucleotides within the 220 processing region. In some embodiments, the processing region 220 includes a retention member configured as a plurality of surfaces (eg, walls or baffles) through which a sample passes. The walls or baffles can be modified to preferentially retain polynucleotides.

Although processing region 220 has been described as a component of a microfluidic network, other configurations can be used. For example, in some embodiments, the retention member can be removed from a processing region for processing elsewhere. For example, the retention member can be contacted with a mixture comprising polynucleotides and inhibitors at one location and then moved to another location where the polynucleotides can be removed from the retention member.

Although the reservoirs 275 have been shown to be dispersed within a carrier, other configurations can be used. For example, the reservoirs 275 may be enclosed within a flexible enclosure (eg, a membrane, eg, an enclosure such as a sack). In some embodiments, the reservoirs may be loose within chamber 272. In such embodiments, actuator 244 may include a porous member that has pores that are too small to allow passage of reservoirs 275 but large enough to allow gas to exit chamber 272.

Exemplary microfluidic cartridge having a lysis chamber

An additional microfluidic cartridge with various components is described in US Provisional Application No. 60 / 553,553 filed March 17, 2004 to Parunak, et al, and Patent Application Publication No. 2005-0084424.

Although microfluidic cartridges have been described that are configured to receive polynucleotides already released from cells, microfluidic cartridges for use herein may also be configured to release polynucleotides from cells (eg, lysing cells). For example, referring to Figures 14A, 14B, 15A, and 15B, a microfluidic cartridge 300 includes a sample lysis chamber 302 in which cells can be lysed to release polynucleotides therein. The microfluidic cartridge 300 further includes substrate layers L1-L3, a microfluidic network 304 (of which only parts are seen in Figure 14A), and R1-R4 liquid reagent reservoirs. The R1-R4 liquid reagent reservoirs contain liquid reagents (for example, for processing

ES 2 587 007 T3 sample material) and can be connected to network 304 via ports for reagent RP1-RP4. The microfluidic cartridge 300 can therefore be a self-contained environment comprising all the reagents and materials necessary to perform preparation steps, cell lysis, polynucleotide isolation, pre-amplification processing, amplification, and detection of a sample. In some embodiments, a sample is introduced that has one or more of the required reagents mixed with it; in which case the remaining reagents are stored in the cartridge. Reagents and other materials can be stored in cartridge 300 in R1-R4 liquid reagent reservoirs, channels or microfluidic chambers in a microfluidic network, and / or in a lysis chamber 302.

The lattice 304 may be substantially defined between layers L2 and L3 but extends in part between the three layers L1-L3. The microfluidic network 304 includes various microfluidic components as further described herein, including Ci channels, Vi valves, V'i double valves, Gi gates, MGi mixing gates, Hi vents, gas actuators (e.g. , pumps) Pi, a first processing region B1, a second processing region B2, detection zones Di, air vents AVi, and debris zones Wi.

Figures 15A, 15B show two complementary halves of an exemplary microfluidic network 304. It would be understood by one skilled in the art that the division of the network into two separate halves is arbitrary, and purely for ease of illustration. The arrangement of components shown in Figures 15A, 15B is exemplary; It would be understood by a person skilled in the art that other arrangements of this type, such as different geometric arrangements of the same components, or different arrangements of different components can be constructed by a person skilled in the art, to achieve the steps described in this document. .

Components of network 304 can typically be thermally actuated. As seen in FIG. 16, an exemplary heat source network 312 includes heat sources (eg, resistive heat sources) that have locations corresponding to various thermally actuated components of the microfluidic network 304. For example, HPi heat source locations correspond to Pi actuator locations, HGi heat source locations correspond to Gi gate locations and MGi mix gates, HVi heat source locations correspond to the locations of the Vi valves and double valves V'i, and the heat source locations HDi correspond to the locations of the Di processing chambers, all of the 304 network. In use, the components of the cartridge 300 may be arranged in thermal contact with corresponding heat sources of the network 312, which can be operated normally using a processor as described above for the cartridge 200. The network of heat sources 312 it may be integral with or independent of cartridge 300 as described for cartridge 200. For example, the heat source network 312 may be integrated into a heater module 2020, such as below the receiving compartment 2014 in a manner that aligns with a cartridge network disposed therein.

Additional components of the exemplary microfluidic cartridge 300 are as follows.

Air vents

The air vents AVi can allow gas (eg, air) displaced by the movement of liquids within the network 304 to be vented so that pressure build-up does not inhibit the desired movements of the liquids. For example, the air vent hole AV2 allows liquid to move along the channel C14 and into the channel C16 by venting gas downstream of the liquid through the vent hole AV2.

Valves

Vi valves can have a normally open state that allows material to pass along a channel from a position on one side of the valve (for example, upstream of the valve) to a position on the other side of the valve. (eg downstream of the valve). The Vi valves may have a similar structure to the valves in the microfluidic cartridge 200, as further described herein.

As seen in Figures 17 and 18, double valves V'i can also have a normally open state that allows material to pass along a channel from a position on one side of the valve (e.g., water above the valve) to a position on the other side of the valve (for example, downstream of the valve). Taking the double valve V11 'of figures 17 and 18 as an example, the double valves Vi' include first and second masses 314, 316 of a TRS (for example, a eutectic alloy or wax) separated from each other on each side of a channel (for example, channel C14). Typically, the TRS masses 314, 316 may be offset from one another (eg, a distance of about 50% of a width of the TRS masses or less). Material moving through the open valve passes between the first and second TRS masses 314, 316. Each TRS mass 314, 316 may be associated with a respective chamber 318, 320, which typically includes a gas (e.g. , air).

The masses of TRS 314, 316 and the chambers 318, 320 of the double valve Vi 'can be in thermal contact with a corresponding heat source HV11' of the network of heat sources 312. Activating the heat source HV11 'makes

ES 2 587 007 T3 that the TRS masses 314, 316 move to a second, more mobile state (eg, a partially molten state) and increases the gas pressure within chambers 318, 320. The pressure of the gas drives the masses of TRS 314, 316 through channel C11 and closes valve HV11 '(figure 18). Typically, the masses 314, 316 combine at least partially to form a mass 322 that obstructs the C11 channel.

Returning to Figures 15A, 15B, the gates Gi may have a normally closed state that does not allow material to pass along a channel from a position on one side of the gate to another side of the gate. The Gi gates may have a structure similar to that described for the gates of the cartridge 200.

As seen in the figures. 19A-19D For an exemplary part of a microfluidic network, MGi mixing gates can allow two volumes of liquid to combine (eg, mix) within network 304. MGi mixing gates are further described below.

Actuators

The actuators Pi can provide a gas pressure to move material (eg, sample material and / or reagent material) between one location on the network 304 and another location. The Pi actuators can be similar in shape to the cartridge actuators 200. For example, each Pi actuator includes a chamber with a mass 273 of TEM that can be heated to pressurize gas within the chamber. Each actuator Pi includes a corresponding gate Gi (eg gate G2 of actuator P1) that prevents liquid from entering the chamber of the actuator. The gate can be actuated normally (eg open) to allow the pressure created in the actuator chamber to enter the microfluidic network.

Waste Chambers

Waste chambers Wi can receive liquid waste (eg, from overflow) resulting from handling (eg, movement and / or mixing) of liquids within the network 304. Typically, each waste chamber Wi has an orifice of Associated air vent that allows gas displaced by the liquid entering the chamber to vent.

Processing regions

The first B1 processing region of lattice 304 may be a component that allows polynucleotides to be concentrated and / or separated from inhibitors in a sample. Processing region B1 may be configured and operated as processing region 220 of cartridge 200. In some embodiments, the first B1 processing region includes a retention member (eg, multiple particles (eg, microspheres or beads), a porous member, multiple walls) that have at least one surface modified with one or more ligands such as described for processing region 220. For example, the ligand can include one or more polyamides (eg, polycationic polyamides such as poly-L-lysine, poly-D-lysine, poly-DLornithine), or polyethyleneimine. In some embodiments, the retention member particles may be disposed in lysis chamber 302 and may move to processing region B1 along with sample material.

The second processing region B2 can be a component that allows material (eg, sample material) to combine with compounds (eg, reagents) to determine the presence of one or more polynucleotides. In some embodiments, the compounds include one or more PCR reagents (eg, primers, control plasmids, and polymerase enzymes).

Lyophilized particles

In some embodiments, compounds for determining the presence of one or more polynucleotides can be stored within a processing region such as B2 as one or more lyophilized particles (eg, microgranules). The particles generally have a shelf life at room temperature (eg, about 20 ° C) of at least about 6 months (eg, at least about 12 months). The liquid entering the second processing region B2 dissolves (eg reconstitutes) the lyophilized compounds.

Typically, the one or more lyophilized particles in the B2 processing region have an average volume of about 5 microliters or less (eg, about 4 microliters or less, about 3 microliters or less, about 2 microliters or less). In some embodiments, the one or more lyophilized particles from the B2 processing region have an average diameter of about 4mm or less (eg, about 3mm or less, about 2mm or less) In an exemplary embodiment, the one or more Various lyophilized particles have an average volume of about 2 microliters and an average diameter of about 1.35 mm. In other embodiments, the lyophilized particles may have a diameter of about 5mm or less (eg, about 2.5mm or less, about

ES 2 587 007 T3

1.75mm or less).

Lyophilized particles to determine the presence of one or more polynucleotides typically include multiple compounds. In some embodiments, the lyophilized particles include one or more compounds used in a reaction to determine the presence of a polynucleotide and / or to increase the concentration of the polynucleotide. For example, lyophilized particles can include one or more enzymes to amplify a polynucleotide, such as by PCR.

Exemplary lyophilized particles include exemplary reagents for the amplification of polynucleotides associated with group B Streptococcus (GBS) bacteria. In some embodiments, the lyophilized particles include one or more of a cryoprotectant, one or more salts, one or more primers (eg, GBS F Primer and / or GBS R Primer), one or more probes (eg, probe of GBS-FAM), one or more internal control plasmids, one or more specificity controls (e.g., Streptococcus pneumoniae DNA as a control for GBS PCR), one or more PCR reagents (e.g., dNTP and / or dUTP), one or more blocking or bulking agents (eg, nonspecific proteins (eg, bovine serum albumin (BSA), RNaseA, or gelatin), and a polymerase (eg, glycerol-free Taq Polymerase). Of course, others Components (eg, other primers and / or specificity controls) can be used for amplification of other polynucleotides.

Cryoprotectants generally help increase the stability of the lyophilized particles and help prevent damage to other compounds in the particles (eg, by preventing denaturation of enzymes during preparation and / or storage of the particles). In some embodiments, the cryoprotectant includes one or more sugars (eg, one or more disaccharides (eg, trehalose, melizito, raffinose)) and / or one or more polyalcohols (eg, mannitol, sorbitol).

The lyophilized particles can be prepared as desired. One method of making lyophilized particles includes forming a reagent solution of the particle and a cryoprotectant (eg, a sugar or poly-alcohol). Typically, the compounds in the lyophilized particles can be combined with a solvent (eg, water) to prepare a solution, which can then be placed (eg, dropwise, in discrete aliquots (eg, drops) such as by a pipette) onto a cooled hydrophobic surface (eg, a diamond film or polytetrafluoroethylene surface). In general, the surface temperature can be lowered to close to the temperature of liquid nitrogen (for example, about -150 ° F or less, about -200 ° F or less, about 275 ° F or less), such as by means of the use of a cooling bath of a cryogenic reagent directly below. The solution can be dispensed without contacting the cryogenic agent. The solution freezes as discrete particles. The frozen particles can be subjected to a vacuum, usually while still frozen, for sufficient pressure and time to remove the solvent (eg, by sublimation) from the microgranules. Such methods are further described in International Patent Publication No. WO 2006/119280.

In general, the concentrations of the compounds in the solution from which the particles are prepared can be higher than when they are reconstituted in the microfluidic cartridge. Typically, the ratio of solution concentration to reconstituted concentration can be at least about 3 (eg, at least about 4.5). In some embodiments, the ratio can be about 6.

An exemplary solution for preparing lyophilized microgranules for use in amplification of polynucleotides indicative of the presence of GBS can be prepared by combining a cryoprotectant (eg, 120 mg of trehalose as a dry powder), a buffer solution (eg, 48 microliters of a solution of 1 M Tris pH 8.4, 2.5 M KCl, and 200 mM MgCl2), a first primer (for example, 1.92 microliters of 500 micromolar GBS F Primer (Invitrogen)), a second primer (for example, 1.92 microliters of 500 micromolar GBS R Primer (Invitrogen)), a probe (for example, 1.92 microliters of 250 micromolar GBS-FAM Probe (IDT / Biosearch Technologies)), a control probe (for example, 1.92 microliters of Cal Orange 560 250 micromolar (Biosearch Technologies)), a template plasmid (for example, 0.6 microliters of a solution of 105 plasmid copies per microliter), a specificity control (for example, 1.2 microliters of a 10 nanograms per microliter solution (for example, approximately 5,000,000 copies per microliter) of streptococcus pneumoniae (ATCC) DNA), PCR reagents (for example, 4.8 microliters of a 100Q millimolar solution of dNTP (Epicenter) and 4 microliters of a 20 millimolar solution of dUTP (Epicenter)), a thickening agent (for example, 24 microliters of a 50 milligram per milliliter solution of BSA (Invitrogen)), a polymerase (for example, 60 microliters of a 5 U per microliter solution of Glycerol-free Taq Polymerase (Invitrogen / Eppendorf) and a solvent (eg, water) to prepare approximately 400 microliters of solution. Approximately 200 aliquots of approximately 2 microliters each of this solution can be frozen and desolvated as described above to prepare 200 microgranules. When reconstituted, the 200 particles make up a PCR reaction solution that has a total volume of approximately 2.4 milliliters.

Reagent reservoirs

As seen in Figure 14, the reagent reservoirs Ri can be configured to hold liquid reagents (eg, water, buffer, hydroxide solution) separated from network 304 until ready.

ES 2 587 007 T3 to use them. The reservoirs R1 include an enclosure 329 that defines a sealed space 330 for containing liquids. Each space 330 may be separated from the RPi reagent port and network 304 by a lower wall 333 of the enclosure 329. A covering material 341 (eg, a laminate, adhesive, or polymeric layer) can cover an upper wall of the enclosure.

A portion of the enclosure 329 may be formed as a drive mechanism (eg, a piercing member 331) facing the bottom wall 333 of each enclosure. When cartridge 300 can be used, reagent reservoirs Ri can be actuated by pressing down on piercing member 331 to pierce wall 333. Piercing member 331 may be depressed down by a user (eg, with a thumb) or by the operating system used to operate cartridge 300.

Wall 333 can typically be formed of a material that has a low vapor transmission rate (eg, Aclar, a metallized laminate (eg, aluminum), a plastic, or a metallized foil laminate) that can break or puncture. Reservoir 330 contains an amount of liquid suitable for cartridge 300. For example, the reservoir can hold up to about 200 microliters. Piercing member 331 can make up a portion (eg, up to about 25%) of that volume. Laminate material within the blister that can touch corrosive reagent such as basic sodium hydroxide should not run even after six to twelve months of exposure.

In general, the reservoirs Ri can be formed and filled as desired. For example, the upper wall of the enclosure may be sealed to the lower wall 333 (eg, by adhesive and / or heat sealing). Liquid can be introduced into the reservoir through, for example, an opening in the lower end of the piercing member 331. After filling, the opening can be sealed (eg, by heat sealing through localized application of heat or by application of a sealing material (eg, cover material 341)).

When wall 333 can be perforated, fluid from reservoir enters network 333. For example, as seen in Figures 14 and 15, liquid from reservoir R2 enters network 304 through port RP2 and travels to along a C2 channel. Gate G3 prevents liquid from passing along channel C8. The excess liquid passes through the channel C7 and into the waste chamber W2. When the trailing edge of the liquid from reservoir R2 passes through the hydrophobic vent hole H2, the pressure created within the reservoir can be vented stopping further movement of the liquid. Consequently, network 304 receives an aliquot of liquid reagent having a volume defined by the volume of the C2 channel between a J1 junction and a J2 junction. When actuator P1 can be actuated, this reagent aliquot can move further within network 304. Reagent reservoirs R1, R3, and R4 can be associated with channels, hydrophobic vents, and corresponding actuators.

In the configuration shown, the reagent reservoir R1 typically contains a release liquid (eg, a hydroxide solution as described above for cartridge 200) to release retained polynucleotides within the B1 processing region. The reagent reservoir R2 typically contains a wash liquid (eg, a buffer solution as described above for cartridge 200) to remove untreated compounds (eg, inhibitors) from the B1 processing region before releasing the polynucleotides. Reagent reservoir R3 typically contains a neutralization buffer (eg 25-50mM Tris-HCl buffer at pH 8.0). Reagent reservoir R 4 normally contains deionized water.

Although the tanks have been shown to have a piercing member formed by a wall of the tank, other configurations are possible. For example, in some embodiments, the reservoir includes a needle-like piercing member that extends through an upper wall of the reservoir into the sealed space toward a lower wall of the reservoir. The upper wall of the reservoir may be sealed to the needle-like piercing member (eg, with an adhesive, an epoxy). In use, the top wall can be pressed downward by urging the piercing member through the bottom wall pushing liquid into the sealed space to enter a microfluidic network.

Although the reservoirs have been described including a drive mechanism (eg, a piercing member), other configurations are possible. For example, in some embodiments, a bottom wall of the reservoir sealed space includes a weakened portion that lines an opening to a microfluidic network. The bottom wall material (for example, laminating, polymeric film, or foil) covering the opening may be thick enough to prevent loss of liquid into the sealed space but thin enough to rupture upon application of pressure to the liquid inside. Typically, the material lining the opening can be thinner than the adjacent material. Alternatively or in addition, the weakened material can be formed leaving this material relatively unsupported compared to the surrounding material of the bottom wall.

Although reservoirs have been described as having a sealed space formed in part by a wall of the sealed space, other configurations are possible. For example, referring to Figure 20A, a reservoir includes a plunger-like drive mechanism (e.g., a piercing member 342) and a gasket-like sealed space 343 having top and bottom layers 344, 345 respectively ( for example, layers of

ES 2 587 007 T3 laminate top and bottom). The liquid can be sealed between the upper and lower layers. The sealed space may be surrounded by a support structure 346 (eg, an O-ring) that supports the sealed space at its upper and lower peripheral surfaces.

Referring to Figure 20B, the piercing member 342 is shown being pressed down until the piercing member 342 has pierced both the top and bottom layers, placing the liquid in communication with the microfluidic network. A vent 346 adjacent to the plunger allows gas trapped between the piercing member and the top layer of the sealed space to escape without being pushed into the microfluidic network.

Referring to FIG. 20C, piercing member 342 is shown being fully actuated. A part of the piercing member has displaced a corresponding volume of liquid from the sealed space and has introduced the predetermined volume of liquid into the microfluidic cartridge.

Although reservoirs have been described as having a sealed space that can be stationary with respect to a piercing member, other configurations are possible. For example, Figure 21A illustrates a reservoir having a sealed space 347 that can be fixed (eg, integral) with respect to a drive mechanism having a movable member 348 (eg, a plunger) and a piercing member 349. supported by a piercing member support 350 that may be stationary with respect to the sealed space. Typically, the sealed space can be defined by a cavity within the movable member and a bottom wall 351 that seals the liquid within the sealed space. The piercing member can be configured to break the bottom wall when the movable member can be pressed downward. The piercing member support has a shape generally complementary to the cavity of the movable member. The piercing member support includes a channel 352 connected to a microfluidic network to allow fluid released from the closed space to enter the microfluidic network.

Referring to Figure 21B, the movable member has been pressed down so that the piercing member has just broken through the bottom layer of the sealed space. Referring to Figure 21C, the reservoir has been fully pressed down onto the piercing member and the piercing member holder. The volume of fluid displaced from the reservoir generally corresponds to the volume of the piercing member holder entering the enclosed space. A channel 353 allows the air displaced by the movable member to escape.

Although reservoirs have been described as having a piercing member that can be attached relative to some part of the reservoir, other configurations are possible. For example, referring to FIG. 22, a reservoir includes a drive mechanism 354 (eg, a piercing member such as a needle-like piercing member) that may not be fixed relative to the reservoir. A sealed space 355 of the reservoir may be defined by a top wall 356 and includes a channel 357 that extends through a portion of a substrate 361 in which a microfluidic network may be defined. A bottom wall 358 of the sealed space separates the sealed space from a channel 359 of the microfluidic network. The piercing member occupies the channel 357 of the sealed space, so that the piercing tip 360 of the piercing member rests against the bottom wall 358. Plunging the upper wall 356 of the reservoir urges the piercing member 354 through the lower wall and pushes the liquid into the sealed space into the microfluidic network.

As another example, Figures 23A and 23B illustrate a reservoir that includes a drive mechanism (eg, a piercing member) that may initially be attached to an interior of a top wall of the reservoir but is at least partially separated from the top wall after the actuation of the tank.

As yet another example, Figures 24A and 24B illustrate a reservoir that includes a piercing member 364 that may be initially attached to an interior 365 of a top wall 366 of the reservoir but substantially detaches (eg, fully detaches) from the wall. after the tank is activated.

Although reservoirs have been described as having an enclosed space that may be fixed or otherwise integral with a portion of the reservoir, other configurations are possible. For example, referring to Figure 25, a reservoir includes a closed capsule-like space 367 defined by an outer wall 368. The outer wall may generally be formed of a material having a low vapor transmission rate. . The reservoir also includes a drive mechanism having a movable member 369 with a piercing member 370 that pierces the closed space even to release the liquid therein. The liquid passes through a channel 372 that leads to a microfluidic network. A channel 371 allows gas (eg, air) otherwise trapped by the movable member to escape.

Although reservoirs have been described generally lining an entrance to a microfluidic network, other configurations are possible. For example, referring to Figure 26, a reservoir includes an enclosed space 373 in which liquid can be stored and a connecting portion 374 connected to an inlet 376 of a microfluidic network. The enclosed space 373 and the connecting portion 374 may be separated by a frangible joint 375 (eg, a weak joint). In general, breakable seal 375 prevents liquid or vapor from escaping from the enclosed space. However, upon application of pressure to the liquid (for example, sinking a wall 377 of the enclosed space), the frangible joint 375 breaks allowing the liquid to pass through the weak joint to the connection portion and into the network. microfluidics 378.

ES 2 587 007 T3

A still further embodiment of a reservoir with a piercing member is shown in Figure 27A, which shows a reservoir 2701 having an outer shell 2703 and a piercing element 2704 that can both be made from the same piece of material. Such a combined shell and piercing element can be formed from many processes known to a person skilled in the art. Especially preferred processes can be vacuum thermoforming and injection molding. Piercing element 2704 may be generally conical in shape, with the apex adjacent to a membrane 2702; its apex preferably does not exceed 0.040. The piercing element will pierce membrane 2702 and release liquid from reservoir 2701 when the outer shell can be pressed down. Representative dimensions are shown in Figure 27A. The reservoir can be constructed so that the top surface can be level, with a flat shield 2705 covering the base of the taper of piercing element 2704.

Yet another embodiment of a reservoir with a piercing member is shown in Figure 27B, which shows a reservoir 2711 having a one-piece outer shell 2712 and piercing element 2714. Such combined shell and piercing element can be formed from many processes known to a person skilled in the art. Especially preferred processes can be vacuum thermoforming and injection molding. Piercing element 2714 may be frusto-conical in shape, with its narrower side adjacent to membrane 2713. Alternatively, piercing element 2714 may comprise a number of different piercing elements, arranged within a conical space. Preferably there can be four of said piercing elements where multiple elements can be present.

It should be understood that the dimensions of the reservoir, piercing element, shell and molding shown in Figures 27A and 27B as decimal amounts in inches are exemplary. In particular, the dimensions may be such that the casing does not retract under its own weight and is normally not strong enough to prohibit collapse of the piercing member when required during operation of the cartridge.

Furthermore, the materials of the various embodiments can also be selected so that the cartridge has a shelf life of about one year. By this, it is understood that the thickness of the various materials may be such that they resist the loss, through means such as diffusion, of 10% of the volume of liquid contained therein over a desired shelf life.

Preferably, the volume of the reservoir can be about 150 µl before a shell is pressed down. After collapse of a shell, the volume can preferably deform to about half its original volume.

It will be appreciated that completely filling the blister pack with a liquid reagent - without any remaining space for an air bubble, results in a blister that requires application of a significantly greater force than is preferable. Accordingly, the one or more blisters are normally filled to about 80-95% of their volume, thus reserving about 5-20%, usually 10-15% of the volume for air. Thus, in one embodiment, a blister having a total volume of 200 µl is filled with 170 µl of liquid.

Lysis chamber

An exemplary lysis chamber 302, as shown in Figures 14A and 14B, is shown in a tower configuration, protruding from a plane of the microfluidic cartridge 300. The lysis chamber 302 can be divided into a primary lysis chamber 306 and a debris chamber 308. In one embodiment, the primary and waste chambers 306, 308 are spaced from each other such that material cannot pass from one of the chambers into the other chamber without passing through at least a portion of the web 304. Primary lysis chamber 306 includes a sample inlet port SP1 for introducing a sample into chamber 306, a sample outlet port SP2 that connects chamber 306 to network 304, and lyophilized reagent LP that interacts with sample material. sample within chamber 306, as described herein. Port SP2 is shown in Figure 14A as being at the bottom of chamber 302. Figure 15B shows a position of SP2 relative to the rest of microfluidic network 304. Inlet port SP1 includes a one-way valve that allows material (eg, sample material and gas) to enter chamber 306 but limits (for example, prevents) material from exiting chamber 308 through port SP1. Typically, the sP1 port includes a fitting (eg, a Luer fitting) configured to mate with a sample inlet device (eg, a syringe) to form a gas tight seal. Primary chamber 306 typically has a volume of about 5 milliliters or less (eg, about 4 milliliters or less). Before use, the primary chamber 306 can normally be filled with a gas (eg, air).

The waste chamber 308 includes a waste part W6 through which liquid can enter the chamber 308 from the network 304 and a vent 310 through which the gas displaced by the liquid entering the chamber 308 can exit. .

ES 2 587 007 T3

Lysis Reagent Particles

The lyophilized LP reagent particles from lysis chamber 302 include one or more compounds (eg, reagents) configured to release polynucleotides from cells (eg, lysing cells). For example, LP particles can include one or more enzymes configured to reduce (eg, denature) proteins (eg, proteinases, proteases (eg, pronase), trypsin, proteinase K, phage lytic enzymes (eg, PlyGBS). )), lysozymes (eg, a modified lysozyme such as ReadyLyse), cell-specific enzymes (eg, mutanolysin for lysing group B streptococci)).

In some embodiments, the LP particles alternatively or additionally include components to retain polynucleotides as compared to inhibitors. For example, LP particles can include multiple ligand-surface-modified particles 218, as described above for the cartridge processing chamber 200. The LP particles can include polynucleotide reducing enzymes that could compete with a polynucleotide to be determined for binding to sites on the surface modified particles. For example, to reduce RNA that could compete with DNA to be determined, LP particles can include an enzyme such as an RNase (eg, RNaseA ISC BioExpress (Amresco)).

In an exemplary embodiment, the LP particles include a cryoprotectant, ligand-modified particles configured to retain polynucleotides as compared to inhibitors, and one or more enzymes.

Typically, LP particles have an average volume of about 35 microliters or less (eg, about 27.5 microliters or less, about 25 microliters or less, about 20 microliters or less). In some embodiments, the LP particles have an average diameter of about 8mm or less (eg, about 5mm or less, about 4mm or less) In an exemplary embodiment, the one or more lyophilized particles have an average volume of about 20 microliters and an average diameter of approximately 3.5 mm.

The LP particles can be prepared as desired. Typically, the particles can be prepared using a cooled hydrophobic surface and cryoprotectant as described hereinbefore for other reagent particles. For example, a solution for preparing LP particles can be prepared by combining a cryoprotectant (eg, 6 grams of trehalose), a plurality of ligand-modified particles (eg, approximately 2 milliliters of a suspension of carboxylate-modified particles with poly ligands). -D-lysine), a protease (for example, 400 milligrams of pronase), an RNase (for example, 30 milligrams of RNaseA (activity of 120 U per milligram), an enzyme that digests peptidoglycan (for example, ReadyLyse (for example, 160 microliters of a 30,000 U solution per microliter of ReadyLyse)), a cell-specific enzyme (for example, mutanolysin (for example, 200 microliters of a solution of 50 U per microliter of mutanolysin), and a solvent (eg, water) to make about 20 milliters. Approximately 1,000 aliquots of approximately 20 microliters each of this solution can be frozen and desolvated as described above to prepare 1,000 microgranules. When reconstituted, the microgranules can normally be used to make a total of about 200 milliliters of solution.

Exemplary performance of the microfluidic cartridge

In use, various components of the cartridge 300 can be operated in the following manner. Valves Vi and Vi 'of network 304 may be configured in the open state. The Gi gates and MGi mix gates of network 304 may be configured in the closed state. The reagent ports R1-R4 may be depressed down, for example, by application of mechanical force, to introduce liquid reagents into network 304, as previously described herein. A sample can be introduced into lysis chamber 302 via port SP1 and combined with lyophilized LP particles within primary lysis chamber 306. Typically, the sample includes a combination of particles (eg, cells) and a buffer solution. For example, an exemplary sample includes about 2 parts of whole blood to about 3 parts of buffer solution (eg, a 20 mM Tris solution at pH 8.0, 1 mM EDTA, and 1% SDS). Another exemplary sample includes group B streptococci and a buffer solution (eg, a solution of 20 mM Tris pH 8.0, 1 mM EDTA, and 1% Triton X-100).

In general, the volume of sample introduced may be smaller than the total volume of the primary lysis chamber 306. For example, the volume of sample may be approximately 50% or less (eg, approximately 35% or less, about 30% or less) of the total volume of chamber 306. A typical sample has a volume of about 3 milliliters or less (eg, about 1.5 milliliters or less). A volume of gas (eg, air) can generally be introduced into the primary chamber 306 along with the sample. Typically, the volume of gas introduced can be about 50% or less (eg, about 35% or less, about 30% or less) of the total volume of chamber 306. The volume of sample and gas are combined to pressurize gas already present within chamber 306. Port valve 307 SP1 prevents gas from exiting chamber 306. Since gates G3, G4, G8, and G10 may be in the closed state, the pressurized sample can be prevented from entering network 304 via port SP2.

ES 2 587 007 T3

The sample dissolves the LP particles in chamber 306. Reconstituted lysis reagents (eg, ReadyLyse, mutanolysin) begin to lyse cells in the sample that release polynucleotides. Other reagents (eg, protease enzymes such as pronase) begin to reduce or denature inhibitors (eg, proteins) within the sample. Polynucleotides from the sample begin to associate with (eg, bind to) 218 ​​particle ligands released from LP particles. Typically, the sample within chamber 306 can be heated (eg, to at least about 50 ° C, at least about 60 ° C) for a period of time (eg, for about 15 minutes or less, about 10 minutes or less, about 7 minutes or less) while lysis occurs. In some embodiments, the optical energy may be used at least in part to heat the contents of the lysis chamber 306. For example, the operating system used to operate the cartridge 300 may include a light source 399 (eg, a lamp. emitting mainly light in the infrared) arranged in thermal and / or optical contact with the camera 306. Said light source may be that shown in connection with the heating module 2020, figure 7, reference number 2046. Chamber 306 includes a TS temperature sensor used to monitor the temperature of the sample within chamber 306. The lamp output can be increased or decreased based on the temperature determined with the TS sensor.

With continued operation of cartridge 300, G2 can be actuated (eg, opened) providing a path between port SP2 of primary lysis chamber 306 and port W6 of residue lysis chamber 308. The path extends along along the C9 channel, the C8 channel, through the B1 processing region, and the C11 channel. Pressure within chamber 306 propels lysed sample material (containing lysate, polynucleotides attached to particles 218, and other sample components) along the path. Particles 218 (with polynucleotides) can be retained within processing region B1 (eg, by a filter) while liquid and other sample components flow into waste chamber 308. After a period of time (eg, between about 2 and about 5 minutes), the pressure in lysis chamber 306 can be vented by opening gate G1 to create a second path between ports SP2 and W6. Dual valves V1 'and V8' can be closed to isolate lysis chamber 302 from network 304.

Cartridge 300 operation continues to drive pump P1 and open gates G2, G3, and G9. The pump P1 drives the washing liquid in channel C2 downstream of junction J1 through processing region B1 and into waste chamber W5. The wash fluid removes inhibitors and other compounds not retained by particles 218 from the B1 processing region. When the trailing edge of the scrubbing liquid (eg, the upstream interface) passes the hydrophobic vent hole H14, the pressure from the actuator P1 is vented from the network 304, stopping further movement of the liquid. The double valves V2 'and V9' can be closed.

Operation continues by driving pump P2 and opening gates G6, G4, and G8 to move release liquid from reagent reservoir R1 into processing region B1 and into contact with particles 218. Air vent hole AV1 vents pressure ahead of moving release liquid. The hydrophobic vent hole H6 vents pressure behind the trailing edge of the release liquid, stopping further movement of the release liquid. The double valves V6 'and V10' can be closed.

Operation continues by heating processing region B1 (eg, heating particles 218) to release polynucleotides from particles 218. The particles can be heated as described above for cartridge 200. Typically, the release liquid includes about 15 mM hydroxide (eg, NaOH solution) and the particles can be heated to about 70 ° C for about 2 minutes to release the polynucleotides from the 218 particles.

Operation continues by driving pump P3 and opening gates G5 and G10 to move the release liquid from downstream process region B1. The air vent hole AV2 vents the gas pressure downstream of the release liquid allowing the liquid to move into the channel C16. Hydrophobic vent hole H8 vents pressure from upstream of the release liquid stopping further movement. Double valve V11 'and valve V14 can be closed.

Referring to Figures 19A-19D, the MG11 mixing gate can be used to mix a portion of the release liquid that includes polynucleotides released from 218 particles and neutralization buffer from reagent reservoir R3. Figure 19A shows the region of the MG11 mix gate prior to pressing down the reagent reservoir R3 to introduce the neutralization buffer into network 304. Figure 19B shows the region of the MG11 mix gate, after the neutralization buffer has been introduced into the C13 and C12 channels. The double valve V13 'can be closed to isolate the network 304 from the reagent reservoir R3. The double valve V12 'can be closed to isolate the network 304 from the waste chamber W3. The neutralization buffer contacts one side of a TRS mass 324 of the MG11 gate.

Figure 19C shows the region of the MG11 mix gate after the release liquid has moved into the C16 channel. The dimensions of the microfluidic network 304 (for example, the dimensions of the channel and the position of the hydrophobic vent hole H8) can be configured so that the part of release liquid located between the junctions J3 and J4 of the channels C16 and C14 corresponds approximately to the volume of

ES 2 587 007 T3 liquid in contact with the particles 218 during the release step. In some embodiments, the volume of liquid between the J3 and J4 junctions may be less than about 5 microliters (eg, about 4 microliters or less, about 2.5 microliters or less). In an exemplary embodiment, the volume of release liquid between junctions J3 and J4 can be about 1.75 microliters. Typically, the liquid between the J3 and J4 junctions includes at least about 50% polynucleotides (at least about 75%, at least about 85%, at least about 90%) of the polynucleotides present in the sample that entered. in processing region B1. Valve V14 can be closed to isolate network 304 from air vent AV2.

Before operating the MG11 mixing gate, the release liquid at junction J4 and neutralization buffer at junction J6 between channels C13 and C12 may be separated by mass 324 of tRs (for example, liquids are normally not separated by a volume of gas). To combine the release liquid and the neutralization buffer, the pump P4 and the gates G12, G13 and MG11 can be operated. Pump P4 drives the volume of neutralization liquid between junctions J5 and J6 and the volume of release liquid between junctions J4 and J3 into mixing channel C15 (Figure 19D). The TRS mass 324 normally disperses and / or melts allowing the two liquids to combine. The combined liquids include a downstream interface 335 (formed by junction J3) and an upstream interface (formed by junction J5). The presence of these interfaces allows for more efficient mixing (eg recirculation of the combined liquid) than if the interfaces were not present. As seen in Figure 19D, mixing typically begins near the interface between the two liquids. Mixing channel C15 can typically be at least about as long (eg, at least about twice as long) as a total length of the combined liquids within the channel.

The volume of neutralization buffer combined with the release liquid can be determined by the dimensions of the channel between junctions J5 and J6. Typically, the combined neutralization liquid volume can be approximately the same as the combined release liquid volume. In some embodiments, the volume of liquid positioned between junctions J5 and J6 may be less than about 5 microliters (eg, about 4 microliters or less, about 2.5 microliters or less). In an exemplary embodiment, the volume of release liquid between the J5 and J6 junctions can be about 2.25 microliters (eg, the total volume of the release liquid and neutralization buffer can be about 4 microliters).

Returning to Figures 15A, 15B, the combined release liquid and neutralization buffer move along mixing channel C15 and into channel C32 (vented downstream by air vent AV8). The movement continues until the upstream interface of the combined liquids passes the hydrophobic vent H11, which vents pressure from the actuator P4 stopping further movement of the combined liquids.

Continuing the operation of the cartridge 300, the actuator P5 and the gates G14, G15 and G17 can be operated to dissolve the lyophilized PCR particles present in the second processing region B2 in water from the reagent reservoir R4. Hydrophobic vent hole H10 vents pressure from actuator P5 upstream of the water stopping further movement. Dissolution of a PCR reagent pellet typically occurs in about 2 minutes or less (eg, about 1 minute or less). Valve V17 can be closed.

Continuing the operation of the cartridge 300, the actuator P6 and gate G16 can be actuated to drive the dissolved compounds of the lyophilized particle from the processing region B2 into the channel C31, where the dissolved reagents mix to form a solution of particles. homogeneous dissolved lyophilized. Actuator P6 moves the solution into channels C35 and C33 (vented downstream by air vent AV5). The hydrophobic vent hole H9 vents the pressure generated by the actuator P6 upstream of the solution stopping further movement. Valves V18, V19, V20 'and V22' can be closed.

Continuing the operation of cartridge 300, actuator P7 and gates G18, MG20, and G22 can be operated to combine (for example, mix) a portion of the neutralized release liquid in channel 32 between gate MG20 and gate G22 and a part of the solution of lyophilized particles dissolved in channel C35 between gate G18 and MG20. The combined liquids travel along a mixing channel C37 and into the detection region D2. An AV3 air vent port vents gas pressure downstream of the combined liquids. When the upstream interface of the combined liquids passes the hydrophobic vent H13, the pressure from the actuator P7 can be vented and the combined liquids can be located within the detection region D2.

The actuator P8 and the gates MG2, G23 and G19 can be operated to combine a part of the water coming from the reagent reservoir R4 between MG2 and the gate G23 with a second part of the solution of freeze-dried particles dissolved in the channel C33 between the gate G19 and MG2. The combined liquids travel along a C41 mixing channel and into the D1 detection region. A ventilation hole

ES 2 587 007 T3 Air AV4 vents gas pressure downstream of the combined liquids. When the upstream interface of the combined liquids passes the hydrophobic vent H12, the pressure from the actuator P8 can be vented and the combined liquids can be located within the detection region D1.

Continuing the operation of the cartridge 300, the double valves V26 'and V27' can be closed to isolate the detection region D1 from the network 304 and the double valves V24 'and V25' can be closed to isolate the detection region D2 from the network 304. The content of each detection region (release liquid neutralized with sample polynucleotides in detection region D2 with PCR reagents from the solution of dissolved lyophilized particles and deionized water with PCR reagents from the solution of freeze-dried particles dissolved in detection region D1) can undergo heating and cooling steps to amplify polynucleotides (if present in detection region D2). Dual valves in each detection region prevent evaporation of the contents of the detection region during heating. Amplified polynucleotides can normally be detected using fluorescence detection. Therefore, usually above one or both of the detection regions D1, D2, there is a window (as, for example, in Figure 9) that allows the detection of fluorescence from a fluorescent substance in the reaction mixture when a detector hovers above the window.

Although cartridges for carrying out various stages of sample processing have been shown and described herein as having a generally flat configuration, other configurations can be used and are consistent with an integrated system as described herein. For example, a cartridge having a generally tube-like or vial-like configuration is described in US Patent Application Publication No. 2006-0166233.

Examples

The following examples are illustrative and are not intended to be limiting.

Example 1: Apparatus for Polynucleotide Processing

This non-limiting example describes various exemplary embodiments of an apparatus, system, microfluidic cartridge, kit, methods, and computer program product, as shown in Figures 28-40.

For example, FIG. 28 is a diagram of an apparatus 800 that can function as a small, benchtop, real-time polynucleotide analysis system. Such a system can perform various analyzes on polynucleotides, for example, analyzing patient samples for hallmarks of one or more infectious diseases. The apparatus can function, for example, as a real-time polynucleotide analysis system for use in the clinical diagnostic market to assist clinical personnel in analyzing and treating patients before they leave the medical environment. Apparatus 800 may include, for example, a housing having a display outlet 802, a lid 804 having a handle 805, and a barcode reader 806. Referring to Figures 28 and 36, the apparatus 800 may also include a receiving compartment 807, which may be covered by the lid 804. In various embodiments, the apparatus 800 may be portable, for example, the apparatus 800 may weigh approximately 10 kg and can have dimensions of approximately 25 cm wide by 40 cm deep by 33 cm high.

The 800 apparatus will be used with an 810 sample kit, shown in Figure 29. The 810 sample kit may include, for example, an 812 microfluidic cartridge with an optional 813 label (for example, a barcode label), an 814 sample container with an optional 815 label (for example, a barcode label), an optional 818 filter, an optional 820 pipette tip, and an optional 822 syringe. Referring to Figure 30, one or more components of sample kit 810 (eg, microfluidic cartridge 812) may be packaged, for example, in a sealed pouch 824 which may optionally be hermetically sealed with an inert gas such as argon or nitrogen.

Microfluidic cartridge 812, as depicted in FIG. 31, may include a sample inlet 826, a plurality of self-piercing reservoirs 828, a lysis reservoir 830, a waste reservoir 832, an optional label 813 (e.g., a barcode), and a correct positioning member 836 (eg, a beveled corner). Referring to Figures 31 and 36, correct positioning member 836 may engage a complementary correct positioning member feature 809 of receiving compartment 907 in apparatus 800, which can be used to facilitate orientation of cartridge 812 when inserted. on the 800 device. In some examples, the 812 microfluidic cartridge may be designed to be slightly smaller (for example, 50-300 microns, typically 200-300 microns) than the 807 well in the 842 heater / sensor module to facilitate placement and removal of the microfluidic cartridge 812.

With reference to Figures 32 and 33, labels, eg, barcodes 813 and 815, on microfluidic cartridge 812 and / or sample container 814 may be registered by apparatus 800, typically prior to performing an analysis, for example, entering a label code manually using an 806 barcode reader.

ES 2 587 007 T3

In preparation for analyzing a sample, the pipette tip 820 from kit 810 can be attached to syringe 822, and a sample can be drawn from sample container 814 into syringe 822. Referring to Figure 34, filter 818 can then be attached to lysis reservoir 830 of microfluidic cartridge 812 via sample inlet 826 (for example, using a Luer lock with a duckbill valve at sample inlet 826 ) and the contents (eg, a sample / air mixture) from syringe 822 can be injected into microfluidic cartridge 812 through filter 818. Additional air (for example, 1-3 ml) can be drawn into the syringe 822 (as shown in figure 35) so that the microfluidic cartridge 812 can be pressurized (for example, 5-50 pounds per square inch (psi) relative to ambient pressure, typically 5-25 psi, more typically 10-15 psi, relative to ambient pressure). Microfluidic cartridge 812 may contain buffers, reagents, and the like, for example, in lysis reservoir 830 in the form of liquids, solids, lyophilized reagent pellets, and the like. The 812 microfluidic cartridge can be shaken to mix the injected sample with buffers, reagents, etc.

Referring to Figure 35, the microfluidic cartridge 812 can be pressurized using the syringe 822 and filter 818 with added air. The microfluidic cartridge 812 can be placed in the receiving compartment 907 of the apparatus 800, as shown in Figure 36, and can be seated in a single orientation in the receiving compartment 907 of the apparatus 800 due to the interaction between the positioning member. correct 836 in the microfluidic cartridge 812 and the complementary correct positioning member 809 in the receiving compartment 907.

As shown in the figures. 37 and 38, the closure of the lid 804 of apparatus 800 may serve to block ambient light from the sample compartment. Additionally, the lid closure 804 may place an optical detector contained in the lid 804 in position with respect to the microfluidic cartridge 812. In addition, the lid 804 of the apparatus can be closed to apply pressure to the microfluidic cartridge 812 to ensure thermal contact in the well 807 with the heater / sensor module 842. Referring to Figures 39 and 40, the heater / sensor module 842 of the apparatus 800 can be removable for cleaning, maintenance, or for replacement with a custom warm-up phase for a particular 812 microfluidic cartridge.

Example 2: Apparatus for Polynucleotide Processing

This non-limiting example describes various embodiments of an apparatus, system, microfluidic cartridge, kit, methods, and computer program product, in particular, various aspects of the use of apparatus 800 as described in Example 1 related to exemplary aspects of the method. and the computer program product.

With reference to Figures 28-40, apparatus 800 may include or be configured with a computer program product in the form of control software. The software can provide the apparatus with a "READY MODE" (eg, standby mode) when it is not being used for analysis where the apparatus 800 can be plugged in and can wait for user input. An operator can slide open lid 804 using handle 805 to its fully open position. Software and apparatus 800 may be configured to indicate, at display output 802, that lid 804 is open and apparatus 800 is ready. The software and apparatus 800 may be configured to perform a hardware and / or software self-analysis. The software and apparatus 800 may be configured to request entry of a user ID and password screen, for example, to allow a user to log in using a touch screen interface (for example, by touching keys on an emulated keyboard in display output 802), scanning a barcode representing the user, such as that found on an ID plate, with the barcode reader 806, and the like.

A user can then remove the microfluidic cartridge 812 and the sample container 814 from the sealed pouch 824. The software and apparatus 800 may be configured to request that the barcode 813 on the microfluidic cartridge 812 and / or the code of bars 815 in sample container 814 are scanned using barcode reader 806, as in Figures 32 and 33, prior to performing any analysis. The software and apparatus 800 may be configured to perform barcode-based qualification analyzes (for example, to determine whether the 812 microfluidic cartridge and 814 sample container came from the same 824 sealed pouch, if the 810 kit has exceeded an expiration date, whether kit 810 can be configured for use with a particular analysis sequence to be carried out by software and apparatus 800 and the like) prior to performing nucleic acid analysis. The display output 802 may then advance to a screen that requires input, such as a patient identifier. Apparatus 800 may also allow patient information to be entered by scanning a barcode (eg, on a medical ID bracelet assigned to the patient) using barcode reader 806. Display output 802 may provide information to the patient. user regarding, for example, results of a previously performed analysis, selection options for analysis to be performed, and the like. Examples of information that may be provided include, but are not limited to: a test determination (eg, positive / negative result), an internal control result (eg, positive and / or negative), and / or test results. patient. In this example, the user may also be prompted to allow apparatus 800 to perform additional tasks with the analysis data, such as recording or transmitting the data to a printer, storage device, or computer, and the like.

ES 2 587 007 T3

Various embodiments of the software and apparatus 800 may be configured to allow a user to perform one or more optional functions (eg, adjustments to apparatus 800 or software) including, but not limited to: modify user settings, modify logout settings, adjust system clock, modify display settings, modify QC requirements, adjust notification preferences, configure a connected printer, configure a network connection, send data over a network connection, select or adapt data analysis protocols, or the like.

In various embodiments, the software can include a user interface and device firmware. User interface software may allow aspects of user interaction including, but not limited to, entering patient / sample information, monitoring analysis progress, error warnings, printing analysis results, uploading results to databases, software updates, and the like. The firmware of the device can operate the apparatus 800 during analytical analyzes and can have a generic part that can be independent of the analysis and a specific part of the analysis that is being performed. The specific part of the analysis ("protocol") can specify the microfluidic operations and their order to achieve the analysis. Figure 41A shows a screen shot of an exemplary interface displaying heat sensor data in real time. Figure 41B shows a screen shot of an exemplary interface displaying data from the optical detector in real time.

Example 3: Apparatus for Polynucleotide Processing

This non-limiting example describes various embodiments of the claimed apparatus, system, microfluidic cartridge, kit, methods, and software product. In one embodiment, apparatus 800, shown in FIG. 28, may be a self-contained, real-time PCR device based on microfluidic technology for rapid and accurate diagnosis of pathogens (eg, group B Streptococcus (GBS) colonization in prenatal women). In an exemplary embodiment, when the microfluidic cartridge 812 (FIG. 31) can be installed, the apparatus 800 can drive cartridge operations, detect and analyze the products of a PCR amplification, and / or display the results on a graphical user interface. Microfluidic cartridge 812 may include a plurality of chambers and / or subunits, to perform various tasks, with limited or no user intervention. Figure 42 is a schematic representation of the various chambers and / or subunits in an exemplary microfluidic cartridge 812. The microfluidic cartridge 812 can accept raw clinical specimens (eg, vaginal / rectal swab immersed in transport buffer in the case of GBS ) via sample inlet 826, which may be a Luer-style injection port. Clinical smear samples containing human and bacterial cells and debris can be routinely collected in 2 ml of transport buffer. However, small volumes (eg, on the order of a few microliters) can be easily processed in microfluidic devices. The incorporation of an interface between macro- and micro-operations may allow the adaptation of microfluidic technology for clinical diagnosis. After injection of a sample, the cartridge can be placed in apparatus 800 and further operations; for example, sample preparation, reagent measurement / mixing, and PCR amplification / detection, can be performed in an automated and hands-free manner.

Figure 43 is a schematic representation of the PCR and detection related steps that can be performed on an exemplary 812 microfluidic cartridge. In various embodiments, steps for processing samples for PCR-based pathogen detection and diagnosis (eg, GBS from vaginal / rectal swabs) may include: lysis to release DNA (eg, lysis of GBS cells to release polynucleotides ), DNA capture and concentration, and minimization of inhibitors and competitors to clean DNA for PCR compatibility. Inhibitors present in clinical samples may increase the risk of a false negative result for PCR-based diagnostic tests unless their influence can be mitigated through sample cleaning.

Cell lysis can be accomplished by methods known in the art, eg, heat and / or chemical activation. In some embodiments, after a 1.0 +/- 0.2 mL sample can be injected into lysis reservoir 830 via sample inlet 826, apparatus 800 can cause the sample to be mixed with lysis reagents from of Wet Storage Reagent 838 and heated (for example, for 7 minutes) in Lysis Tank 830. Using this protocol, more than 90% lysis efficiency has been achieved for GBS and other bacterial cells. The lysis reagents may also incorporate a DNA affinity matrix based on cationic polyamide modified polycarbonate-polystyrene latex beads (e.g., retention member 821) to capture negatively charged DNA that can be released during the lytic process. . Affinity beads can bind negatively charged DNA with very high affinity while potential pCr inhibitors may fail to bind or can be removed during subsequent washing steps.

In an exemplary embodiment, apparatus 800 can also automate the capture and cleanup of DNA from impure sample lysate (eg, GBS sample lysate) to generate "PCR ready" DNA. The contents of lysis reservoir 830 (eg 1.0 +/- 0.2 ml sample and reagents) can be transferred to DNA processing chamber 840. Affinity beads with bound DNA from the introduced sample can be trapped using an in-line bead column (e.g., a specific pore size filter) and a cartridge pump can be used to wash the affinity beads to remove non-specifically bound fractions. ,

ES 2 587 007 T3 as well as soluble inhibitors by performing a buffer exchange. Bound DNA can be released by known methods, eg, by heating the affinity beads (eg, at 80 ° C) and / or using a release buffer. Intact DNA can be recovered with this single step release in very small volume (3-4 µl) thereby achieving a significant concentration of the original target DNA. Other methods known in the art can be employed by the system to achieve cell lysis and DNA capture, washing and release.

In the example described in this case, the basis for the real-time PCR assay used is the TaqMan® assay, the schematic operation of which is as depicted in Figure 44. However, other assay techniques known in the art may be used. technique (eg SYBR-Green I fluorescence). The TaqMan® PCR reaction takes advantage of the 5 'nuclease activity of certain DNA polymerases to cleave a TaqMan® probe during PCR. The TaqMan® probe contains a reporter dye at the 5 'end of the probe and a quenching dye at the 3' end of the probe. During the reaction, cleavage of the probe can separate the reporter dye and quench dye, which can result in increased reporter fluorescence. Accumulation of the PCR products can be detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quenching dye can result in suppression of the reporter (fluorescence emission can be by Forster-type energy transfer). During PCR, if the target of interest can be present, the probe can hybridize between the forward and reverse primer sites. DNA polymerase can normally cleave the probe between the reporter and quencher if the probe hybridizes to the target. Probe fragments can then be displaced from the target, and chain polymerization can continue. The 3 'end of the probe can be blocked to prevent probe extension during PCR.

This process can occur normally, for example, in each thermal cycle and must not interfere with the exponential accumulation of product. Increased fluorescence signal can normally detect whether the target sequence can be complementary to the probe and can be amplified during PCR. The TaqMan® assay can offer a two-fold stringency (primer normally binds and probe normally binds to the target sequence) and therefore detection of any nonspecific amplification can be reduced or eliminated.

Real-time PCR probe and primer sets for GBS (Streptococcus agalactiae) have been designed and tested using clinical specimens. The PCR reagents can include a pair of hybridization primers specific to the part of the cfb gene between positions 328 and 451 that encode the CAMP factor (Christie, Atkins and Munch-Petersen, see, for example, Boll Ist Sieroter Milan, ( 1955 Jul-Aug); 34 (7-8): 441-52). CAMP factor is a diffusible extracellular protein and is produced by most GBS. The gene encoding the CAMP factor, the cfb gene (GenBank entry number: X72754), may be present in GBS isolate and has been used for the development of a PCR-based identification of GBS (Danbing K., and col., (2000), Clinical Chemistry, 46, 324-331). In addition, a specific TaqMan®-style fluorogenic probe has also been designed and tested, in one example, to recognize amplified sequence between primers to allow real-time detection using fluorescence measurements.

In order to evaluate the DNA clean-up process and monitor the performance of the primers of the invention at run time, positive internal control plasmids (eg, as depicted in Figure 45) can be employed. In various embodiments, specific GBS primers have been used to construct internal control plasmids as follows. A piece of random DNA sequence flanked by the specific PCR primers was generated by oligonucleotide synthesis. Any possible homology between this sequence and other DNA sequences especially Strep DNA. agalactiae, available from the Genbank, was carefully checked. A fluorogenic TaqMan® style probe was designed to recognize amplicons generated from this sequence and a fluorophore (Cal Orange 560 or analog) other than that used for GBS target sequence DNA (FAM or analog) was used for fluorescent detection. simultaneous double color. In certain embodiments, the amount of the internal control plasmid to be included in the PCR reaction was optimized to allow amplification of the internal control product without significant detrimental effects on GBS-specific amplification. In some examples, the specificity of the probes for internal controls was also analyzed and optimized by PCR using DNA purified from pathogens included in the previously specified cross-reactivity test list and GBS DNA.

In an exemplary embodiment, a robust system for performing rapid thermocycling using a microfluidic volume was designed, developed and implemented in a microfluidic format. The microfluidic volumes that can be accommodated range from about 0.01 µl to about 10 µί, where the main limitation at the lower limit is detection sensitivity. Exemplary volumes are in the range of 0.5 4.5 μΙ Still other exemplary volumes are 2 μι Figures 41A and 41B show screen shots of the output of an exemplary apparatus 800 (for example, as viewed on the LCD touch sensitive 846). Figure 41A shows a microfluidic PCR module undergoing rapid thermocycling and Figure 41B shows a real-time PCR assay. This data may be available to the user of apparatus 800 or it may be hidden. In this case, the GBS cartridges were also designed to accommodate two PCR chambers to allow incorporation of a negative control incorporated with each sample tested to improve the fidelity of the result. In some examples, the chemistry has been optimized and a compatible detection system developed to allow for two-color multiplex PCR, thus facilitating the use of internal positive controls to check efficiency.

ES 2 587 007 T3 of sample preparation and the appropriate performance of associated instrumentation. Due to very small thermal masses and efficient feedback control algorithms, it may be possible to perform ultra-fast thermocycling, so that a typical 50-cycle PCR can be completed in approximately 20 minutes. The heat required for thermocycling can be provided by the heater / sensor module 842 and multiplexed detection in real time can be carried out by the optical module (eg, the fluorescent detection module 844).

In various embodiments, any number (eg, 0, 1, 2, or all) of the reagents for performing the PCR can be incorporated into the cartridge in a lyophilized format. At the time of use, lyophilized PCR reagents can be reconstituted using, for example, deionized water, which can be stored in microfluidic cartridge 812 in a blister format (eg, self-piercing reservoir 828). The reconstituted PCR reagents can be aliquoted into, for example, two parts. In various embodiments, PCR-ready DNA (emitted from the sample preparation module) can be mixed with an aliquot and sent to the first PCR channel for real-time PCR (sample PCR). DI water containing non-target DNA can be mixed with the second aliquot of the PCR reagents and can be sent to the other PCR chamber (negative PCR) to serve as a negative control.

Example 4: Apparatus for Polynucleotide Processing

In various embodiments, a microfluidic system (e.g., microfluidic cartridge 812) may include components such as micropumps to move / mix liquid droplets, microreactors to perform thermally initiated biochemical reactions, and microvalves or microgates to allow control of pump operations. pumping of liquid, as well as to isolate regions of the cartridge such as the PCR chambers during thermocycling.

In some embodiments, a liquid droplet handling system can be used to produce injection and movement of liquid samples based on thermally actuated pumps (eg, thermopneumatic pumping) that can be actuated electronically without the use of mechanical valves. For example, by heating trapped air within chambers that may be connected to the main channel, significant air pressure can be generated for thermo-pneumatic pumping. Increasing the air temperature can cause the pressure within the chamber to rise until the pressure can be high enough to separate a drop (measure an aliquot) and move it to the desired location. This technique can be implemented as a drive mechanism in the cartridge and can use, for example, molded chambers, channels and heaters. Typically this can avoid mechanical moving parts and can facilitate manufacturing. Figure 46 shows photos of a demonstration showing mixing of two fluids ("A" - blue and "B" - orange) using the drop handling system described above. The pressure pumps P1 and P2 can be activated in a precisely controlled manner, which can push the liquids to move as alternating rolling discrete droplets along the M channel, where they can mix, and finally move into chamber C, where they can take place the PCR.

In some embodiments, thermally expansive materials such as gas, readily vaporizable liquid (eg, vaporizable between 25 ° C and 100 ° C at 1 atmosphere), and / or a thermally expanding polymer (eg, Expancel) can be introduced into thermally driven pumps (eg, thermo-pneumatic air chambers), which can minimize the size of the pumps to generate differential pressures greater than 5 psi. These materials can expand, for example, by more than 100% when a threshold temperature can be reached, causing it to partially or completely fill the therm-pneumatic chamber causing additional compression of the air.

For example, Figures 47A and 47B depict a thermally driven pump 500 based on a phase transition material (PTM) 510 (a PTM of a known melting point (e.g., 60 ° C / 75 ° C / 90 ° C). ) such as a paraffin wax, solder, Expancel, or the like), in a closed (Figure 47A) and an open (Figure 47B) configuration. The PTM 510 can be constrained to a specific location. The specific location can be a sealed chamber 512 with the PTM 510 (typically about 50-100 gl) deposited on a laminate. After heating the pump to a temperature above 120 ° C, the PTM 510 expands irreversibly (up to 40 times its original size), compressing the air within chamber 512, displacing the air in the chamber, and causing the adjacent gate 514 opens. Figures 47C and 47D show another example of a pump 501 with expandable polymer 511 in chamber 513 that can be actuated to operate gate 515.

An exemplary clinical sample introduced into microfluidic cartridge 812 may have a volume of approximately 1 milliliter. After enzymatic / thermal lysis of cells, the released DNA can be bound to affinity microbeads. These microbeads can be, for example, on the order of 10 microns in size. In various embodiments, a total number of beads in the range of a few million can be used per 812 microfluidic cartridge for DNA concentration. In some cases, a minimum pressure of 10 psi (for example, 10 psi, 11 psi, or 15 psi) can be used to concentrate the beads against an in-line filter area of ​​a few millimeters (8 micron pore size) within a few few minutes (for example 3 minutes). This pressure can be generated, for example, by injecting extra air (eg, 1-3 ml) into the bulk lysis chamber of the 812 microfluidic cartridge. In some embodiments, a one-way duckbill valve at the Luer inlet can be used.

ES 2 587 007 T3 to minimize or prevent air pressure from escaping through the inlet.

Reagents that can be used for sample preparation and PCR reactions can be pumped into the microfluidic network by pressing down the domes of the reagent blister via the instrument slide during instrument use.

In exemplary embodiments, the enzymes typically used for cell lysis, DNA capsule, and to perform real-time PCR can be lyophilized into pellets and stored in different locations on the cartridge. Air contact can be minimized by storing the microgranules in the microfluidic cartridge 812, for example, in a nitrogen purged chamber or in a channel structure sealed at both ends of the microchannel by heat gates. Buffers commonly used for sample preparation, reagent hydration, and PCR can be stored in hermetically sealed reagent blisters (eg, 828 self-piercing reservoirs). The materials used to prepare the reagent blister can have a high moisture vapor barrier and can minimize liquid loss during one year storage of the cartridge. The waste generated from the clinical specimen as well as the various wash buffers can be stored embedded in the cartridge in chambers and microchannels (eg, waste reservoir 832, which can normally be leak resistant).

Example 5 Real-time PCR aspects

A plurality of steps that can be used for accurate, real-time PCR-based diagnosis of pathogens (e.g., group B Streptococcus (GBS) colonization in prenatal women) were integrated into a single disposable cartridge based on microfluidic technology, such as as shown in figure 48. Exemplary stages that can be performed in a style operation “enter a sample; a result comes out "on said cartridge include: bulk lysis; capture, wash and release of DNA; and preparation and execution of PCR. In various embodiments of this cartridge, the sample and reagents may be contained incorporated into the microfluidic cartridge 812 (as shown in Figure 31) and there is normally no need for manual interaction with the operation except, for example, during the act. injecting the sample into the device. Strategically placed hydrophobic vents can be included to remove trapped air formed during processing and reagents normally used for the assay can be packaged as lyophilized beads in the cartridge. Liquids required for reconstitution can be stored in blister sachets that release them at the time of use.

In various embodiments, samples (eg, GBS samples) can be introduced through sample inlet 826, which may have a Luer fitting to accommodate a syringe. A pre-filter (eg attached to the syringe) can be used to remove at least a portion of crude impurities from the sample and, in some embodiments, the sample (eg 1 ml) can be lysed in the lysis chamber using heat and / or lytic enzymes. Enzymes such as pronase, proteinase K, and RNaseA can be used (eg, during the lysis step) to remove inhibitory proteinaceous matter and competing RNA molecules. Referring to Figure 49, DNA capture beads can also be included in the master mix. The lysed liquid sample (eg, containing the GBS and / or and other DNA bound to affinity beads) can flow back into the microfluidic cartridge from the outlet of the lysis chamber. In some examples, the captured DNA affinity beads can be retained by an in-line filter, while unwanted debris and excess liquid can be sent to the debris chamber. In such embodiments, the beads used can be non-magnetic, or magnetic, and the filter is selected to discriminate particles by size. In other embodiments, the beads are magnetic, and are concentrated in a particular location on the microfluidic cartridge, such as a chamber or channel, applying a magnetic field configured to focus streamlines at the location in question. The magnet used may be an electromagnet, such as that controlled by the processor to turn it on and off at specified times during sample analysis. The magnet can alternatively be a permanent magnet, such as one that moves in and out of place when required.

In some embodiments, the waste chamber is equipped with an antifoam agent, such as simethicone. Vigorous bubble formation can occur in the waste chamber as the liquid enters it at high speed and, after mixing with air in the waste chamber, foams. It is undesirable for the foam to overflow from the waste chamber. The presence of a defoaming agent can mitigate this phenomenon. The defoaming agent can be present in powder, tablet, microgranule, or particle form.

In some embodiments, the beads can be washed to remove non-bound and nonspecifically bound matter, and the cleaned DNA can be released into a small volume compartment (~ 3 gl) and concentrated (eg, by a factor of about 300). In various embodiments, the concentrated DNA can then be mixed with the appropriate PCR reagents and / or sent to a PCR channel for real-time PCR. An internal control plasmid (along with its analog probe) can also be included in the first PCR channel, which can act as a positive control. In a second PCR channel (if present), DI water containing non-target DNA and the internal control can be mixed together with the PCR reagents, to act as a negative control.

ES 2 587 007 T3

A user can introduce a sample into the bulk lysis sample through a Luer duckbill valve (for example, sample inlet 826), shake gently to dissolve pellets of the lysis reagent, and introduce an excess amount of air (eg 0.25-0.75 ml) into the lysis chamber to overpressurize the lysis chamber. The absolute pressure (P) generated in the lysis chamber having a chamber volume, Vchamber, is related to the amount of liquid sample injected, Vsample, and the volume of extra air injected, Vaire extra, by the formula:

P = __________^{Patm (V} chamber - V sample) __________ (V chamber + V sample + V extra air)

The microfluidic cartridge 812 can then be placed in the apparatus 800 and the slide module 848 closed. When closed, the slide module 848 can press reagent blisters (eg, 828 self-piercing reservoirs), causing them to burst and release reagents (eg, wash buffer, release buffer, neutralization buffer, and water) into the channels with reagents.

In various embodiments, apparatus 800 may perform any or all of the following steps. Referring to Figures 50A-50J hereinafter, various elements may also be located with the same indicators in Figures 15A and 15B. Referring to Figure 50A, valves (V3, V4, V5, V7, V12, V13, V15, V16, V23) on both sides of the channel containing the 4 reagents can be closed and the bulk lysis lamp can be activated to heat the bulk lysis chamber, eg, at 60 ° C for 7 minutes (eg, using the temperature sensor L in Figure 50B for feedback). Referring to Figure 50b, the G1 gate can be opened to drain, for a predetermined amount of time (e.g., 2-5 minutes depending on the type of sample), the liquid (containing lysate, DNA bound to affinity beads by DNA, etc.) through the bead capture filter into the waste chamber. The DNA beads can get trapped against the in-line filter while the other liquid flows into the waste chamber. Gate G7 can be opened to vent excess liquid and / or pressure in the lysis chamber into the waste chamber. Valves V1 and V8 can be closed to block the lysis chamber and the waste chamber, respectively.

Referring to Figure 50C, in some examples, trapped beads can be washed by pumping wash buffer through the bead column using pump E1 and opening gates G2, G3, and G9 to place wash buffer downstream of the port of hydrophobic ventilation H2. The valves that isolate the wash buffer channel (eg V2) and wash buffer residues (eg V9) can be closed.

Referring to Figure 50D, release buffer can be pumped, using pump E2 and opening gates G6, G4, and G8 to fill a column of beads and position release buffer downstream of air vent H1. The valves that block the release buffer channel (eg V6) and the channel downstream of the bead column (eg V10) can be closed and the beads heated to, eg 70 ° C for 2 minutes as long as possible. which can release DNA from DNA affinity beads.

Referring to Figure 50E, the released DNA can be pumped, using pump E3 and opening gates G5 and G10 to a position downstream of the hydrophobic vent H4, at which point valves V11 and V14 can be closed. Referring to Figure 50F, a part of the released DNA (between junction G11 and a1) and a part of the neutralization buffer (between G11 and a2) can be mixed using pump E4 and opening gates G12, G11 and G13. Mixing of the compound liquid plug between a1 and a2 can occur after it can be pumped through the neutralization mixing channel and located downstream of the hydrophobic vent hole H4. G11 can be a zero dead volume gate that carries two liquid channels close to each other without trapping air bubbles during the combination of the two liquid plugs. The two valves (V21 and V22) at the ends of the neutralized sample can be closed and, now referring to figure 50G, DI water can be pumped using the E5 pump and opening the gates G14, G15 and G17 to dissolve the reagent pellet. of PCR. The liquid can be located using the hydrophobic vent hole H12. After a period of time long enough for complete dissolution of the lyophilized PCR reagent pellet (eg, approximately one minute) valve V17 can be closed.

Referring to Figure 50H, dissolved enzyme can be pumped through the enzyme mixing channel using pump E6, opening gate G16, and using hydrophobic vent H5 to aid fluid placement. In various embodiments, the mixed enzyme can be distributed (eg, in 2 equal parts) for multiple reactions to the sample PCR mix section as well as the negative PCR mix section, at which time, the V18 valves , V20 and V19 can be closed.

In various embodiments, referring to Figure 50I, a part of the neutralized DNA (between the G20 and b1 junction) and a part of the enzyme (between G20 and b2) can be mixed using the E7 pump and opening the gates G18, G20 and G22. Mixing of the compound liquid plug between a1 and a2 can occur after it can be pumped through the neutralization mixing channel and placed downstream of the hydrophobic vent hole H6. A part of the DI water (between junction G21 and c1) and a part of the enzyme (between G21 and c2) can be mixed using pump E8 and opening gates G19, G21 and G23. Mixing of the compound liquid plug can occur between c1 and c2, after it can be pumped through the neutralization mixing channel and

ES 2 587 007 T3 located downstream of the hydrophobic vent hole H7.

Referring to Figure 50J, the PCR valves V24, 25, 26, 27, in some examples, may be closed to perform sample PCR and negative control PCR. In various embodiments, light (eg, fluorescence) can be detected using the optical system on the slide. In some examples, the software can determine the presence of target (eg, GBS) in the sample based on fluorescence data and can report the results.

Example 6: Apparatus for polynucleotide processing

This non-limiting example shows CAD views of various exemplary embodiments of the apparatus, system, microfluidic cartridge, kit, methods, and computer program product, as further described herein.

Figure 51 shows a side view of a lever assembly 1200, with lever 1210, gear unit 1212, and force member 1214. The assembly 1200 can be used to close the lid of the apparatus and (through the force members 1214) applying force to a microfluidic cartridge 1216 in receiving compartment 1217. A force member is visible in this cutaway view, but any number can be used, for example four. The force members may be, for example, a manual spring-operated actuator as shown, an automatic mechanical actuator, a material with sufficient mechanical flexibility and rigidity (eg, a hard elastomeric plug), and the like. The force applied to the microfluidic cartridge 1216 can result in a pressure on the surface of the microfluidic cartridge 1216 of at least about 0.7 psi to about 7 psi (between about 5 and about 50 kilopascals), or in some embodiments about 2 psi ( approximately 14 kilopascals.

Figure 52 shows a side view of the lever assembly 1200, with the microfluidic cartridge 1216 in the receiving compartment 1217. A heat pump 1219 (eg, a xenon bulb as shown) can function as a heat source. Radiant directed into a sample inlet reservoir 1218, where heat can lyse the cells in reservoir 1218. A mechanically flexible, thermally conductive layer 1222 may be at an interface between the microfluidic cartridge 1216 and the thermal phase 1224. Typically, the microfluidic cartridge 1216 and the thermal phase 1224 may be flat at their respective interface surfaces, eg, flat within about 100 microns, or more typically within about 25 microns. Layer 1222 can improve thermal coupling between microfluidic cartridge 1216 and thermal phase 1224. Optical sensing elements 1220 can be directed to the top surface of microfluidic cartridge 1216.

Figure 53 shows a close-up of receiving compartment 1217.

Figure 54 shows a close-up of the interface between the microfluidic cartridge 1216, the mechanically flexible, thermally conductive layer 1222, and the thermal phase 1224.

Figure 55 shows a top view of assembly 1200. In addition to mechanical members 1214, guide members 1226 may be employed.

Figure 56 is a close-up of Figure 55.

Figures 57-59 are a series of photos of the 1210 lever in action. Also shown on gear assembly 1212 is cam 1228, which allows lever 1210 to apply force to plate 1230 coupled to force members 1214.

Figures 60 and 61 show views of the microfluidic cartridge 1216 with self-piercing reservoirs 1228 and mechanical members 1230 for driving the self-piercing reservoirs.

Figures 62 and 63 show elements of optical detector elements 1220 that include 1232 light sources (eg, light emitting diodes), 1234 lenses, 1236 light detectors (eg, photodiodes), and 1238 filters. The filters may be, for example, band pass filters, the filters in the light sources corresponding to the absorption band of one or more fluorogenic probes and the filters in the detectors corresponding to the emission band of the fluorogenic probes.

Example 7: Preparation of the retention member

Carboxylate Surface Magnetic Beads (Carboxylate Modified Magnetic Sera-Mag, Part No. 3008050250, Seradyn) at a concentration of approximately 10¹¹ ml^-1 activated for 30 minutes using N-hydroxylsuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide

ES 2 587 007 T3 (EDAC) in a 500 mM 2- (N-morpholinium) -ethanesulfonic acid (MES) buffer solution at pH 6.1. The activated beads were incubated with poly-L-lysine (PLL) of 3,000 Da or 300,000 Da average molecular weight. After 2 washes to remove unbound PLL, the beads were ready for use.

Example 8: Microfluidic cartridge

Referring to Figures 64 and 65, a microfluidic cartridge 300 was manufactured to demonstrate the separation of polynucleotides from inhibitors. Cartridge 300 comprises first and second substrate parts 302 ', 304', comprising, respectively, first and second layers 302a ', 302b' and 304a ', 304b'. The first and second layers 302a ', 302b' define a channel 306 'comprising an inlet 310' and an outlet 312 '. The first and second layers 304a ', 304b' define a channel 308 'comprising an inlet 314' and an outlet 316 '. The first and second substrate portions 302 ', 304' were matched using adhesive 324 'so that outlet 312' communicated with inlet 314 'with a filter 318' positioned between them. A portion of the outlet 312 'was filled with the activated beads prepared above to provide a processing region 320' comprising a retention member (the beads). A pipette 322 '(Figure 66) attached by adhesive 326' facilitated the introduction of the sample.

In use, the sample introduced via inlet 310 'passed along the channel and through processing region 320'. Excess sample material passed through channel 308 'and exited device 300' via outlet 316 '. The polynucleotides were preferentially retained by the beads compared to the inhibitors. Once the sample had been introduced, additional liquids, eg, a wash liquid and / or a liquid for use to release the retained polynucleotides, were introduced via the 326 'inlet.

Example 9: DNA retention

The retention of polynucleotides by the poly-L-lysine modified beads of device 300 'was demonstrated by preparing respective devices comprising processing regions having a volume of about 1 µl including about 1000 beads. The beads were modified with poly-L-lysine of between about 15,000 and 30,000 Da. Each processing region was filled with a liquid comprising herring sperm DNA (approximately 20 µl of sample with a concentration of approximately 20 mg / ml) thereby placing the beads and the liquid in contact. After the liquid and the beads had been in contact for 10 minutes, the liquid was removed from each processing region and subjected to quantitative real-time PCR to determine the amount of herring sperm DNA present in the liquid.

Two controls were carried out. First, an otherwise identical processing region was filled with unmodified beads, that is, beads that were identical to the poly-L-lysine beads except for the poly-L-lysine activation and incubation steps. . The liquid comprising herring sperm DNA was contacted with these beads, allowed to stand for 10 minutes, removed, and subjected to quantitative real-time PCR. Second, the liquid comprising the herring sperm DNA ("the raw liquid") was subjected to quantitative real-time PCR.

Referring to Figure 66, the first and second controls showed essentially identical responses indicating the presence of herring sperm DNA in the liquid contacted with the unmodified beads and in the unprocessed liquid. The liquid that had been contacted with the 3,000 poly-Llysine beads showed a lower response indicating that the modified beads had retained substantially all of the herring sperm DNA. The PCR response of the liquid that had been contacted with the 300,000 Da poly-L-lysine beads showed an amplification response that was at least about 50% greater than for the 3,000 Da beads, indicating that the modification surface area of ​​the lower molecular weight was more efficient in retaining herring sperm DNA.

Example 10: Release of DNA from Modified Poly-L-Lysine Beads

Devices having processing regions were filled with 3,000 Da poly-L-lysine modified beads. The liquid containing polynucleotides derived from group B streptococci (GBS) was contacted with the beads and incubated for 10 minutes as above for herring sperm DNA. This liquid had been obtained by subjecting approximately 10,000 GBS bacteria in 10 µl of 20 mM Tris buffer pH 8, 1 mM EDTA, 1% Triton X-100 to thermal lysis at 97 ° C for 3 min.

After 10 minutes, the liquid in contact with the beads was removed by flowing approximately 10 µl of wash solution (Tris-EDTA pH 8.0 with 1% Triton X 100) through the processing region. Subsequently, approximately 1 µl of 5 mM NaOH solution was added to the processing region. This process left the processing region filled in, filled with the NaOH solution in contact with the beads. The solution in contact with the beads was heated to 95 ° C. After 5 minutes of heating at 95 ° C, the solution in contact with the beads was removed by eluting the processing region with a volume of solution equal to three times the void volume of the processing region.

ES 2 587 007 T3

Referring to Figure 67, five aliquots of solution were subjected to quantitative real-time PCR amplification. Aliquots E1, E2, and E3 each contained approximately 1 µl of liquid. Aliquot L corresponds to liquid from the original sample that had passed through the processing region. Aliquot W was liquid obtained from unheated wash solution. The E1 aliquot corresponds to the dead volume of the device 300, approximately equal to the volume of the channel 308. Therefore, the liquid in the E1 aliquot was present in the channel 308 and not in contact with the beads during heating. This liquid had passed through the processing region before heating. Aliquot E2 comprises liquid that was present within the processing region and in contact with the beads during heating. The E3 aliquot comprises liquid used to remove the E2 aliquot from the processing region.

As seen in Figure 67, more than 65% of the GBS DNA present in the initial sample was retained by and released from the beads (aliquot E2). The E2 aliquot also demonstrates the release of more than 80% of the DNA that had been retained by the beads. Less than about 18% of the GBS DNA passed through the processing region without being captured. The no-heat wash solution comprised less than 5% of the GBS DNA (aliquot W).

Example 11: Separation of polynucleotides and inhibitors

The buccal cells of the lining of the cheeks provide a source of human genetic material (DNA) that can be used for the detection of single nucleotide polymorphism (SNP). A sample containing buccal cells was thermally lysed to release DNA from within the cells. Device 300 was used to separate DNA from concomitant inhibitors as described above. A cleaned sample corresponding to aliquot E2 of Figure 67 was subjected to polymerase chain reaction. A control or impure sample such as that obtained from thermal lysis was also amplified.

Referring to Figure 68, the cleaned sample showed a substantially higher PCR response in fewer cycles than the control sample. For example, the cleaning sample exceeded a response of 20 within 32 cycles, while the control sample required approximately 45 cycles to reach the sample response.

Blood acts as a sample matrix in various diagnostic tests including detection of infectious disease agents, cancer markers, and other genetic markers. Hemoglobin present in blood samples is a documented potent inhibitor of CRP. Two 5 ml blood samples were lysed in 20 mM Tris buffer pH 8, 1 mM EDTA, 1% SDS and loaded into respective 300 devices, which were operated as described above to prepare two cleanup samples. A third 5 ml blood sample was lysed and prepared using a commercial Puregene DNA extraction method, Gentra Systems, MN. The respective cleaned samples and the sample subjected to the commercial extraction method were used for allelic discrimination analysis (CYP2D6 * 4 reagents, Applied Biosystems, CA). Each sample contained an amount of DNA corresponding to approximately 1 ml of blood.

Referring to Figure 69, commercially cleaned and extracted samples showed a similar PCR response, demonstrating that the processing region of device 300 'efficiently removed inhibitors from blood samples.

Example 12: Protease Resistant Retaining Member

Preparing polynucleotide samples for further processing often includes subjecting the samples to protease treatment in which a protease cleaves peptide bonds of proteins in the sample. An exemplary protease is pronase, a mixture of endo- and exo-proteases. Pronase clears most peptide bonds. Certain ligands, such as poly-L-lysine can be susceptible to cleavage by pronase and other proteases. Therefore, samples are generally not subjected to protease treatment in the presence of the retention member if the ligands attached to it are susceptible to proteases.

Poly-D-lysine, the dextrorotatory enantiomer of poly-lysine, resists cleavage by pronase and other proteases. The ability of a retention member comprising bound poly-D-lysine to retain DNA even when subjected to protease treatment was studied.

Eight (8) samples were prepared. A first group of 4 samples contained 1000 GBS cells in 10 µl of buffer. A second group of 4 samples contained 100 GBS cells in 10 µl of buffer. Each of the 8 samples was heated at 97 ° C for 3 min to lyse the GBS cells. Four (4) sample sets were created from the heated samples. Each set of samples contained 1 sample from each of the first and second groups. The samples from each set of samples were treated as follows.

Referring to Figure 70A, samples from Sample Set 1 were incubated with pronase to prepare respective protein cleaved samples, which were then heated to inactivate proteases. Heated, protein-cleaved samples were contacted with retention members

ES 2 587 007 T3, each comprising a set of poly-L-lysine modified beads. After 5 minutes, the respective bead sets were washed with 5 microliters of a 5 mM NaOH solution to remove inhibitors and protein cleavage products from bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated at 80 ° C for 2 minutes to release the DNA. Released DNA solutions were neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the DNA recovery efficiency. The results were averaged and are shown in Figure 70B.

Samples from Sample Set 2 were incubated with pronase to prepare respective protein cleaved samples, which were then heated to inactivate proteases. The heated, protein-cleaved samples were contacted with respective retention members each comprising a set of poly-D-lysine modified beads. After 5 minutes, the respective bead sets were washed with 5 microliters of a 5 mM NaOH solution to remove inhibitors and protein cleavage products from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated at 80 (eighty) ° C for 2 minutes to release the DNA. Released DNA solutions were neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and are shown in Figure 70B.

Samples from Sample Set 3 were incubated with pronase to prepare respective protein cleaved samples. The proteases were not thermally or chemically inactivated. The protein cleaved samples were contacted with respective retention members each comprising a set of poly-L-lysine modified beads. After 5 minutes, the respective bead sets were washed with 5 microliters of a 5 mM NaOH solution to remove inhibitors and protein cleavage products from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated at 80 (eighty) ° C for 2 minutes to release the DNA. The solutions with released polynucleotides were each neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and are shown in Figure 70B.

Samples from sample set 4 were incubated with pronase to prepare respective protein cleaved samples. The proteases were not thermally or chemically inactivated. The protein cleaved samples were contacted with respective retention members each comprising a set of poly-D-lysine modified beads. After 5 minutes, the respective bead sets were washed with 5 microliters of a 5 mM NaOH solution to remove inhibitors and protein cleavage products from the bound DNA. The respective sets of beads were each contacted with a second aliquot of NaOH solution and heated at 80 (eighty) ° C for 2 minutes to release the DNA. The solutions with released polynucleotides were each neutralized with an equal volume of buffer. The neutralized solutions were analyzed to determine the efficiency of DNA recovery. The results were averaged and are shown in Figure 70B.

As seen in Figure 70B, an average of more than 80% DNA from GBS cells was recovered using sample set 4 in which the samples were contacted with poly-D-lysine modified beads and were incubated with pronase in the presence of the beads without protease inactivation. The recovery efficiency for sample set 4 can be more than twice as high as for any of the other samples. Specifically, the recovery efficiencies for sample sets 1,2, 3, and 4 were 29%, 32%, 14%, and 81.5%, respectively. The efficiencies demonstrate that high recovery efficiencies can be obtained for samples incubated with protease in the presence of a DNA-retaining retention member.

Example 13: Operator's manual for apparatus for processing polynucleotides

This non-limiting example describes, in the form of an operator's manual, various embodiments of the claimed apparatus, microfluidic cartridge, kit, methods, and software product, in particular directed to a single-cartridge system that includes a microfluidic PCR assay for the qualitative detection of microorganisms, such as group B Streptococcus (GBS). Additional descriptions pertinent to the GBS analysis are presented in Example 14.

Sample preparation kit, which may include

Sample vial containing buffer and preservative

Collection swab with instructions for self-collection by the patient.

A number (for example, 25) of filter syringes

Vials containing buffer.

Patient ID labels for collection vials

3 cc syringes.

ES 2 587 007 T3

Microfluidic cartridge, as further described herein.

External control sample kits, which may include

Positive control smear specimen containing sample at the limit of detection of, for example, GBS bacteria in dehydrated form;

Vials containing buffer with POSITIVE identification on the vial;

Vials containing buffer with NEGATIVE identification on the vial; Y

Syringes

Team:

Bar code reader system, both of which as further described herein, equipped with, for example, a 115V or 220V power cord.

Additional optional equipment:

Printer with USB connection

Connection to the hospital network

Exemplary use and indications for use

The description in this example is suitable for analyzing samples for the presence of GBS, details of which are provided in Example 14.

Analysis application explained

The apparatus and materials can be used to screen for various pathogens and microorganisms, as further described herein. An example is testing for GBS, as further described in Example 14. Testing can be performed in the near-patient environment by clinicians who are not extensively trained in laboratory procedures. A QC routine can be built into the system user interface to provide continuous Quality Assurance for GBS analysis. The analysis can also be carried out in a statistical laboratory of a central hospital, provided that the analysis of the sample occurs within the period of time required by the physician requesting the analysis.

Exemplary Warnings and Cautions

Other GBS-specific warnings and precautions are presented in Example 14.

• In some embodiments, if a patient is currently being treated with antibiotics, the analysis may be an unreliable indicator of a pathology.

• Normally avoid using the cartridge / sample kit beyond the expiration date.

• Normally, avoid using a cartridge that was previously opened. Exposure to air and moisture can degrade reagents. Avoid opening the cartridge until after the sample is ready for injection.

• Typically use new syringe materials for sample preparation.

• Normally use the swabs and buffer provided in the test kit. In some embodiments, other brands of collection swabs may interfere with assay performance. • When a specimen must be stored, refrigeration at 4 ° C for up to 24 hours is usually indicated.

• Normally wear protective clothing and disposable gloves while handling specimen and system components.

• Normally use aseptic technique. It may be especially important to wear new disposable gloves to avoid contamination of the test specimen or test materials.

• Normally, avoid injecting the sample with high pressure. Gentle injection is preferred.

• Normally, handle specimens using universal precautions in accordance with safe hospital procedures for potentially infectious specimens.

• Normally, use recommended cleaning agents when cleaning the system.

• Normally avoid cleaning the test cartridge with any cleaning chemicals if spills occur. If necessary, use a dry laboratory cloth to remove liquids.

ES 2 587 007 T3

Typical storage and stability conditions

<td>Product description</td><td>Use / storage condition</td><td>Stability</td><td>Transport condition</td>

<td>System</td><td>15-35 ° C, operating range</td><td>NA</td><td>-10 ° C to 65 ° C</td>

<td>GBS Sampling Kits</td><td>4-40 ° C. Do not freeze.</td><td>Date of Expiry</td><td>Temp&gt; 4 ° C</td>

<td>Patient specimen or QC in buffer (wet)</td><td>at 15-30 ° C</td><td>8 hours</td><td></td>

<td>Patient specimen or QC in buffer (wet)</td><td>at 4 ° C</td><td>24 hours</td><td></td>

<td>Patient specimen in container (dry)</td><td>at 15-30 ° C</td><td>24 hours</td><td></td>

<td>Patient specimen in container (dry)</td><td>at 4 ° C</td><td></td><td></td>

<td>Unopened GBS cartridges</td><td>4-30 ° C. Do not freeze,</td><td>Date of Expiry</td><td>Temp&gt; 4 ° C</td>

<td>Unopened GBS cartridges</td><td>Do not use.</td><td>60 minutes maximum</td><td></td>

<td>GBS Control Kits</td><td>15-30 ° C</td><td>Date of Expiry</td><td>TBD protection</td>

Exemplary specimen collection

Caution: Avoid touching the Dacron® end of the swab with your fingers.

Remove the swab from the package

Clean excess vaginal secretions.

Insert the swab 2 cm into the vagina (front pass).

Insert the same swab 1 cm into the anus (posterior pass)

Place the swab in the shipping container if stored dry.

Exemplary specimen preparation

Use aseptic technique when handling specimen and test materials.

Confirm that the sample buffer has not expired.

Immerse the swab vigorously 20 times in the vial containing 1 cc of buffer

Remove and discard the swab.

Label the vial with the patient ID and collection time, if required by standard clinical procedure.

Caution: Avoid opening the cartridge package until you are ready to use it. Included reagents can be sensitive to light and moisture.

Exemplary System Operating Instructions • System Ready screen can be displayed.

• Touch the screen to stop the screensaver.

• Lift the handle and open the system lid to get started.

• A self-test of the system can begin once the lid is fully open and an “open” indicator, if present, is on.

• Inspect the system for any remaining used cartridges from a previous analysis.

• Login with the user ID and password.

• Scan the barcode on the sample collection vial.

• Open the analysis cartridge. Scan the barcode on the cartridge.

• The system may display an error message if the materials have expired and / or if the specimen vial and test materials are not paired properly. • Confirm the sample ID with patient information.

• Enter the patient's ID and other hospital identifying information, if requested.

Exemplary instructions for transferring the sample to the cartridge:

• Put on a new pair of gloves.

• Place the sample vial in the counter.

• Attach the tip to the syringe. Extract all the sample into the syringe. Draw an additional 2 cc of air into the syringe.

• Remove the tip and attach the filter.

• Inject 1 cc of sample into the cartridge using the syringe and an additional 2 cc of air.

ES 2 587 007 T3

Caution: Use gentle pressure to inject the sample to avoid splashing from the sample.

Exemplary instructions for conducting the analysis:

Gently swirl the cartridge back and forth until the pellets dissolve.

Place the cartridge in the system

Close the system cover (Analysis can start immediately)

Results

When the analysis is complete, the results can be available for viewing or printing. Disposal of materials: Used cartridge and collection kit can be treated as biohazard

Preparation of materials for shipment

The cartridge should normally be packaged in biohazard protective materials as described by International Standards.

Exemplary cleaning procedure for the system

A 10% bleach solution (0.5% sodium hypochlorite) followed by a clean water rinse can be used to disinfect as well as to reduce the potency of DNA contamination.

DNA contamination can usually be achieved by cleaning with bleach or suitable materials to remove DNA contamination. Chemicon ™ Nucleic Acid Scavengers can also be used after cleaning with ordinary disinfectants.

Normally alcohol or ordinary sanitary napkins will not reduce the DNA contamination of the instrument.

Exemplary reagent lot verification

Purpose - evaluation of cartridge and sample kit batches and verification of total system performance.

Recommendation: Upon receipt of a new lot of sample cartridges or kits, a quality control set can be run to confirm, for example, if the reagent set includes (1) external control and (1) negative external control.

Exemplary quality control routine

The external positive control is used to monitor and calibrate the sensitivity of the specimen preparation steps and the assay, and can be used to minimize the risk of false negative results. If the analysis fails, it may invalidate the results for that lot of cartridges, and the manufacturer must be notified.

Purpose - verification of total system performance including evaluation of sample handling technique. Analysis can be performed at a preset interval selected by the user.

Recommendation: When performing the analysis, run a QC set that includes (1) Positive External Control and (1) Negative External Control, if the QC analysis fails to provide the expected results, the manufacturer should be contacted.

Exemplary Routine Quality Control Instructions

Go to the quality control screen

Enter user ID

Select QC Technique Verification, New Reagent Lot, run daily QC on samples as indicated on screens.

System self-analysis

The system can perform a startup scan of the system when power is supplied to it. As a start-up routine for each patient test sample or QC sample, the system can run a self-test to determine, for example, that electronics, optics, heaters, and temperature sensors are working as they should.

Exemplary internal controls (on cartridge)

Reagents that can be used in the assay can be included in the cartridge to reduce the potential for user handling errors and contamination. Two types of positive and negative control strategies in the

ES 2 587 007 T3 cartridge can be incorporated into each microfluidic cartridge to monitor the performance of the individual PCR assay. Two examples are:

1) Positive internal control plasmid (ICP): an internal control plasmid can provide a control of the integrity of the PCR assay reagents as well as be an indicator of the presence of PCR inhibitory substances in the specimen, i.e. it can be a control for false negative results. During thermocycling, amplification of this region can produce a different fluorogenic signal in both lanes of PCR. Failure to amplify the internal control sequence, in the absence of a positive sample, may be indicative of a failure of the reagent mix or the presence of PCR inhibitors in the specimen. This can invalidate the analysis results as indicated by an error code on the instrument. "IND" can be displayed to report an indeterminate result.

2) Negative internal control PCR lane: A parallel lane can be used to run a second PCR with reagents from the same mix without any sample. This can provide a control against false positive results due to reagent contamination. A failed result in the negative control lane may invalidate the result and may be indicated by an error code on the instrument. "IND" can be displayed to report an indeterminate result.

Exemplary real-time PCR for GBS DNA detection

In various embodiments, the assay can use real-time polymerase chain reaction (PCR) for amplification of GBS cfb gene sequences recovered from clinical samples and fluorogenic target-specific hybridization for detection of amplified DNA. The cfb gene encodes the CAMP factor, a diffusible extracellular protein that is normally present in GBS isolates. The Group B Streptococcus (GBS) screening test can also be an integrated, raw sample to result, type of nucleic acid amplification assay. A TaqMan fluorogenic probe can be used to detect PCR amplicons. The reagents used in the assay can be included in the cartridge to reduce the potential for user handling errors and contamination.

Exemplary potential precautions

In some embodiments, the assay system may not be qualified to identify targets, such as GBS DNA, in specimens other than vaginal and / or rectal specimens. Urine and blood specimens may not be qualified.

In some embodiments, a patient undergoing antibiotic treatment may not be able to obtain a correct diagnosis with this or other diagnostic tests.

In some embodiments, the analysis may not produce a GBS culture suitable for direct identification of the bacteria by a microbiologist. In some embodiments, the test may not provide susceptibility results that are necessary to recommend a treatment for persons allergic to penicillin.

Exemplary Potential Interfering Substances

In some embodiments, urine and vaginal secretions, if present in very large amounts, can interfere with the analysis.

Normally, sample contaminants such as contamination with blood, meconium, and amniotic fluid are not likely to interfere with the analysis.

Normally, at this time it is not known that interference by drugs (such as those present in vaginal and rectal secretions), other than antibiotics, interfere with CRP.

Exemplary performance characteristics and interpretation

The flow chart in Figure 71 outlines an exemplary set of criteria that can be used by the decision algorithm in the instrument to interpret the results. PCR reactions (Sample and Control) can be interpreted as positive or negative for the target in question. A logical algorithm can be used to determine if the sample is definitely Positive or Negative or Indeterminate.

Exemplary cross-reactive substances

The specificity of the primers and probes can be analyzed with real-time PCR (Taqman assay) using genomic DNA isolated from the following organisms: nine GBS serotypes (serotype la, 1b, lc, II, III, IV, V, VI and VII; American Type Culture Collection and National Center for Streptococcus, Canada); 10 isolates of clinical GBS; 60 clinical samples; a wide variety of gram-positive and gram-negative bacterial strains as well as two strains

ES 2 587 007 T3 yeast and RSV type 1 and 2.

Exemplary microorganisms

<td>PATHOGEN</td><td>Kind</td>

<td>Pseudomones aeruginosa</td><td>Gram bacteria -</td>

<td>Proteus mirabilis</td><td>Gram bacteria -</td>

<td>Kiebsiella oxytoca</td><td>Gram bacteria -</td>

<td>Kiebsiella pneurnoniae</td><td>Gram bacteria -</td>

<td>Escherichia coli (clinical isolate 1)</td><td>Gram bacteria -</td>

<td>Escherichia coli (clinical isolate 2)</td><td>Gram bacteria -</td>

<td>Acinetobacter baumannd</td><td>Gram bacteria -</td>

<td>Serra. marcescens</td><td>Gram bacteria -</td>

<td>Entembacter aerugenes</td><td>Gram + bacteria</td>

<td>Enterococcus Maclean</td><td>Gram + bacteria</td>

<td colspan="2">Staphylococcus aureus (clinical isolate 1) Gram + bacteria</td>

<td colspan="2">Staphylococcus aureus (clinical isolate 2) Gram + bacteria</td>

<td>Streptococcus pyogenes</td><td>Gram + bacteria</td>

<td>Streptococcus viridans</td><td>Gram + bacteria</td>

<td>Listena monocytogenes</td><td>Gram + bacteria</td>

<td>Enterococcus sps.</td><td>Gram + bacteria</td>

<td>Cendida glabrata</td><td>Yeast</td>

<td>Candida albicans</td><td>Yeast</td>

<td>Streptococcus Group C</td><td>Gram + bacteria</td>

<td>Streptococcus Group G</td><td>Gram + bacteria</td>

<td>Streptococcus Group F</td><td>Gram + bacteria</td>

<td>Enterococcus faecalis</td><td>Gram + bacteria</td>

<td>Streptococcus pneumoniae</td><td>Gram + bacteria</td>

<td>Staphylococcus epidermidis (C-)</td><td>Gram + bacteria</td>

<td>Gardenerella vaginalis</td><td>Gram + bacteria</td>

<td>Micrococcus spa</td><td>Gram + bacteria</td>

<td>Haemophilus influenza "</td><td>Gram bacteria -</td>

<td>Neisseria gonorrhoeae</td><td>Gram bacteria -</td>

<td>Moraxella catarrahlis</td><td>Gram bacteria -</td>

<td>Salmonella sps.</td><td>Gram bacteria -</td>

<td>Chlamytha trechomatis</td><td>Gram bacteria -</td>

<td>Peptostreptococcus product.</td><td>Gram + bacteria</td>

<td>Peptostreptococcus anaerobe.</td><td>Gram + bacteria</td>

<td>Lactobacillus lennentum</td><td>Gram + bacteria</td>

<td>Eubacterium lentum</td><td>Gram + bacteria</td>

<td>Herpes simplex virus I (HSV I)</td><td>Virus</td>

<td>Herpes simplex virus II (HSV II)</td><td>Virus</td>

___________________________ Sample Problem Solving Diagram

<td>Problem</td><td>Possible cause</td><td>Possible action</td>

<td>The positive control reads a negative result. No other indication.</td><td>Sample not processed properly.</td><td>Review the sampling method. Analyze again.</td>

<td></td><td>Bulk lysis not performed, degraded reagents.</td><td>Analyze a new lot of cartridge. Check cartridge storage location (&lt;30C). Contact HandyLab.</td>

<td>The negative control reads a positive. No other indication.</td><td>Contamination</td><td>Clean the Instrument. Review the sampling method. Analyze again.</td>

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<td>Problem</td><td>Possible cause</td><td>Possible action</td>

<td>Error: IND (indeterminate result-CI failure)</td><td>IC failure, PCR not performed.</td><td>Analyze a new batch of cartridges.</td>

<td>Error: IND (indeterminate result)</td><td>Borderline result - IQ exceeded.</td><td>Reanalyze with new patient sample.</td>

Example 14: Operator's Manual for Apparatus for Processing Polynucleotides

This non-limiting example describes, in the form of instructions to the user, various embodiments of the claimed apparatus, microfluidic cartridge, kit, methods, and software product, in particular directed to a microfluidic cartridge for use in a microfluidic PCR assay including qualitative detection of microorganisms such as group B Streptococcus.

The presence of group B streptococci (GBS) remains a leading cause of serious neonatal infection despite the great advance in prevention since the 1990s with sepsis, pneumonia, and meningitis affecting the baby after birth. The GBS Test System can be used for the rapid qualitative detection of Group B Streptococcus (GBS) DNA in vaginal / rectal specimens.

Exemplary use and indications for use

In various embodiments, the GBS test system can be used for the qualitative and rapid detection of microorganisms in clinical samples, such as Group B Streptococcus (GBS) DNA in vaginal / rectal samples.

Typical indications for use of the GBS test include, for example, a rapid screening test in the antenatal care regimen for the maternity patient to determine the need for antibiotic treatment during labor, as described by the guidelines of the CDC (Centers for Disease Control and Prevention. Prevention of Perinatal Group B Streptococcal Disease: Revised Guideline from CDC. Morbidity and Mortality Weekly Report, 2002 Aug 16; 51 (No. RR-11); 1-24). The test can provide rapid results at the point of care or in a central laboratory with rapid result delivery service during the intrapartum and antepartum phase of the maternity patient. The test can also be used to detect GBS DNA in vaginal or rectal samples from any subject suspected of having GBS infection. See also Mark A. Bums, Brian N. Johnson, Sundaresh N. Brahmasandra, Kalyan Handique, James R. Webster, Madhavi Krishnan, Timothy S. Sammarco, Piu M. Man, Darren Jones, Dylan Heldsinger, Carlos H. Mastrangelo, David T. Burke “An Integrated Nanoliter DNA Analysis Device ”Science, Vol. 282, October 16, 1998.

Explanation of the application of the analysis

The Centers for Disease Control and Prevention recommends universal prenatal screening for vaginal / rectal GBS colonization of all women at 35-37 weeks' gestation to determine the need for prophylactic antibiotics during labor and delivery. The current CDC recommendation is the culture-based test method (Standard Culture Method), the results of which are typically available in 48-72 hours, compared to approximately 30 minutes for the present test. In analysis you can use automated sample preparation and real-time PCR to identify the cfb gene in the GBS genome which is an established identification sequence that encodes the CAMP factor. CAMP factor is an extracellular protein normally present in GBS isolates. The CAMP factor can be used for the credible identification of GBS bacteria in clinical samples by the culture method. This analysis can be performed in the environment close to the patient by physicians who are not extensively trained in laboratory procedures. A QC routine can be incorporated into the user interface to provide continuous Quality Assurance for gBs analysis. The analysis can also be performed in a central hospital statistical laboratory as long as the analysis of the sample occurs within the time period required by the maternity department.

Possible contraindications

GBS testing may be contraindicated for individuals with an allergy to the polyester contained in the specimen collection swab.

Exemplary Warnings and Cautions

The warnings herein may be in addition to those of general application shown in Example 13, herein.

• The GBS test may not provide susceptibility results that are recommended for women allergic to penicillin.

• Since it is usually necessary to start IV antibiotics at least 4 hours before delivery, and at

ES 2 587 007 T3 absence of antepartum GBS data, it may be important to collect samples and begin GBS analysis as soon as possible after the patient enters the Labor and Delivery area of ​​the hospital.

• In some embodiments, if a patient is currently being treated with antibiotics, the analysis may be an unreliable indicator of pathology.

• The buffer may contain sodium azide as a preservative. It poses a health risk if ingested.

• Typically, I test the specimen within, for example, 8 hours of sampling and storage in the buffer. In the situation where a specimen must be stored, refrigeration at 4 ° C can be used for a period of up to 24 hours.

• Normally avoid using test materials beyond the expiration date.

• Normally, avoid opening the cartridge until after the sample is ready to be injected. Normally, avoid using a cartridge if the protective foil pouch is open. In some embodiments, exposure to light, air, and moisture can degrade the reagents.

• Normally use swabs, syringe and buffer provided in the test kit. In some embodiments, other GBS collection swab marks may interfere with assay performance.

• Materials are typically single-use only; reusing materials can give wrong results.

• Avoid opening the cartridge package until ready for testing. Included reagents can be sensitive to light and moisture. Avoid using if the seal on the container is broken and the foil bag is no longer expanded (swollen).

Exemplary sample collection kit

A. Hyssop

B. Tubes containing sample collection buffer.

C. Cannula tips

D. Syringes (for example, 3cc)

E. Syringe filters

F. GBS Microfluidic Cartridge

Specimen Collection Instructions and Sample Buffer Suspension • Caution: Normally, use only the collection kit; avoid touching the end of the swab with your fingers.

• Remove the swab from the package.

• Clean excess vaginal secretions.

• Insert the swab 2 cm into the vagina (front pass).

• Insert the same swab 1 cm into the anus (posterior pass).

• Confirm that the Sample Buffer vial (B) has not expired.

• Dip the swab (A) vigorously up and down 20 times in the vial containing buffer.

• Remove and discard the swab.

• Label the vial with patient ID information

Exemplary sample preparation for analysis • The tip of the cannula (C) can be attached to the syringe (D) • Extract part or all of the sample into the syringe.

• Additional air can be extracted (eg 2 ml).

• The tip of the cannula (C) can be replaced by the filter (E).

• The cartridge package can be opened at the tear points marked on the package.

• The barcodes on the sample vial (B) and cartridge (F) can be scanned with the system scanner. The system can notify you if the materials have expired.

• Patient identification information can be entered, if required.

• Cartridge can be laid out on flat surface or held flat with Luer in upright position. Normally, make sure the cartridge remains label side up during the procedure.

• The sample (including excess air) can be injected into the cartridge using the syringe / filter assembly. Gentle injection pressure can be used to avoid splashing from the sample.

• The syringe / filter assembly can be removed from the cartridge.

• The cartridge can be gently shaken back and forth approximately 10 times until the pellets within the sample chamber dissolve and mix.

• The cartridge can be placed in the system • The system cover can be closed and the handle can be locked in the low position. (Analysis can start automatically)

ES 2 587 007 T3

Results

When the test is complete, the results can be clearly displayed. Results can be printed or stored as determined by laboratory procedures.

Discarded specimen of materials

The cartridge and the collection kit must be treated as a biohazard.

Exemplary Recommended Lab QC Routine

Verification of exemplary analysis

At weekly intervals, (1) positive external control and (1) negative external control can be run. A set of QCs to confirm the full performance of the system. This procedure is also recommended when training new users.

Exemplary Routine Quality Control Indications:

Samples can be analyzed as indicated on the display screens. If a QC analysis fails to produce the expected results, the manufacturer can be contacted.

The external positive control can include a lyophilized aliquot of Streptococcus agalactiae (GBS) cells that are reconstituted at the time of analysis with sample collection buffer. The number of GBS cells in the external positive control may be approximately equal to the lower detectable limit (LMD) of the assay.

Exemplary QC tests also include a system self-test QC, as described in Example 14.

Exemplary internal controls (on cartridge)

Typical reagents for the assay may be included in the cartridge to reduce the potential for user handling errors and contamination. Two types of cartridge positive and negative control strategies can be incorporated into each microfluidic cartridge to monitor the performance of individual PCR assays.

An exemplary positive internal control plasmid for GBS is a double-stranded circular DNA molecule containing a 96 bp region composed of a single 39 bp DNA sequence flanked by the forward and reverse sequences of the cfb gene, it may be included in the mix lyophilized master along with a second distinct fluorogenic probe specific for this unique sequence. Failure to amplify the internal control sequence, in the absence of a positive GBS sample, may be indicative of a reagent mix failure or the presence of PCR inhibitors in the specimen.

Exemplary real-time PCR for GBS DNA detection

The analysis can use real-time polymerase chain reaction (PCR) for the amplification of a GBS cfb gene sequence recovered from clinical samples. A fluorogenic target specific Tacman® probe can be used for the detection of amplified DNA. The cfb gene encodes factor cAmP, a diffusible extracellular protein that is normally present in GBS isolates. The Group B Streptococcus (GBS) screening test can be an integrated type, from raw sample to nucleic acid amplification assay result. Typical reagents for the assay may be included in the cartridge to reduce the potential for user handling errors and cross contamination.

Possible limitations of exemplary analysis

In some embodiments, the assay system may not be qualified to identify GBS DNA in specimens other than vaginal / rectal specimens. For example, urine and blood specimens may not be qualified in some embodiments.

In some embodiments, a patient undergoing antibiotic treatment may not be able to obtain a correct diagnosis of GBS.

In some embodiments, the analysis may not produce a GBS culture suitable for direct identification of the bacteria by a microbiologist.

ES 2 587 007 T3

Exemplary performance and interpretation characteristics

10-30% of pregnant women are colonized with GBS. The GBS colonization limit is determined to be approximately 1000 DNA copies / sample determined by amplification of the cfb gene sequence. The user is referred to the flow chart of Figure 71 for application of exemplary criteria for interpretation of the result, where the target in question is GBS and IC plasmid.

Possible exemplary interfering substances

In some embodiments, urine or vaginal secretions, or mucus, if present in large amounts, can interfere with the analysis.

In some embodiments, contamination of the samples with blood, meconium, or amniotic fluid is unlikely to interfere with the assay.

In some embodiments, interference by drugs (such as those present in vaginal and rectal secretions), other than antibiotics, is not known at this time to interfere with PCR.

Interpretation of results and exemplary expected values

10-30% of pregnant women may be colonized with GBS. The GBS colonization limit can be determined to be approximately 1000 DNA copies / sample, determined by amplification of the cfb gene sequence.

PCR reactions can be interpreted as positive or negative for GBS and internal control. A logical algorithm can be used to determine if the sample is Positive or Negative or Indeterminate.

Storage and stability information

<td>Description</td><td>Condition of use / storage</td><td>Stability</td>

<td>Patient specimen in buffer (wet)</td><td>at 15-30 ° C</td><td>8 hours</td>

<td>Patient specimen in buffer (wet)</td><td>at 4 ° C</td><td>24 hours</td>

<td>Patient specimen (dry storage)</td><td>at 15-30 ° C</td><td>24 hours</td>

<td>GBS Cartridges - Unopened</td><td>4-30 ° C</td><td>Date of Expiry</td>

<td>GBS Cartridges -Open</td><td>Do not use.</td><td>60 minutes max.</td>

A number of embodiments of the technology have been described. However, it will be understood that various modifications can be made without departing from the scope of the claims. Accordingly, other embodiments are within the scope of the following claims.