Assay system

Description of the invention

The present invention relates to an analysis apparatus comprising an analysis cartridge, its use, an analysis cartridge, and an analysis device.

The present invention relates to analyzers, particularly diagnostic analytical systems, in particular systems used at the site of treatment, e.g. at a doctor's workplace or at the patient's bedside.

Many diagnostic tests are available today, e.g. pregnancy tests, blood sugar, homocysteine, low-carbohydrate transferrin, blood clotting, blood cholesterol, etc. Some of these tests are performed by the patient and some by the patient's physician, but many, especially those that provide quantitative results, must now be performed in a laboratory remote from both the patient and the clinician, resulting in significant delays between sampling and sample collection. analysis and generally requires the patient to see the doctor again in order to know the test results. This is not only an inconvenience for the patient as it also increases the cost of the patient or the paying organization for the patient's health.

There is therefore a need for analyzers, particularly those that provide quantitative results, operated by a physician or physician's assistants at the patient's treatment site.

Quantitative analyzers often require the use of highly accurate volume measuring devices, a few reagents, and test-specific detectors for reading the result, and it is impractical to make specialized test equipment for a wide range of different analyzers at the treatment site, both due to space and cost. .

The object of the invention is to provide an analysis apparatus containing an analysis cartridge and its use.

The object of the invention is an analysis cartridge.

The object of the invention is an analysis device.

An analysis apparatus comprising an analysis cartridge having at least two wells mounted in the holder, a drive means by which the pipette is inserted into selected recesses of the cartridge, a gas pressure generating apparatus and a radiation detector, according to the invention, characterized in that the analysis cartridge further comprises a pipette which is inserted into at least two of the wells of the wells, the pipette having a proximal end and a distal end, the distal end is closed by a fluid-permeable membrane, and a gas pressure generating apparatus is connected to the pipette for causing fluid to flow through said membrane, and radiation from the analysis cartridge cavity or pipette is detected by a radiation detector.

An analysis apparatus comprising an analysis cartridge having at least one cavity mounted in a holder, a drive means by which the pipette is inserted into selected cavities of the cartridge, a gas pressure generating apparatus and a radiation detector according to the invention, characterized in that the analysis cartridge comprises a pipette which is inserted into at least one cavity, the pipette having a capillary end, and the gas pressure generating apparatus is connected to the pipette to cause fluid to flow through the pipette, and the radiation from the analysis cartridge cavity or from the pipette is detected by a radiation detector.

An analysis apparatus comprising an analysis cartridge having at least one cavity mounted in a holder, a drive means by which the pipette is inserted into selected cavities of the cartridge, a gas pressure generating apparatus, and a radiation detector according to the invention, characterized in that at least one cavity of the cartridge is for analysis, it has two parallel, flat, side walls connected by a base wall containing at least one flat surface, wherein the line perpendicular to the base wall and the line perpendicular to the sidewall lie in the same plane and form with each other an angle different from the right angle, the analysis cartridge further comprises a pipette located in at least one cavity, and the gas pressure generating apparatus is connected with a pipette causing the fluid to flow through the pipette, while the radiation from the well of the analysis cartridge or from the pipette is detected by a radiation detector.

Preferably, the assay cartridge comprises a capillary-tipped pipette and a membrane-tipped pipette.

PL 207 204 B1

Preferably, the assay cartridge comprises a pipette, the distal end of which is closed by an inclined fluid-permeable membrane.

Preferably, the tilted membrane lies in a plane which is at an angle of between 20 ° and 40 ° to the axis of the pipette to which it is attached.

Preferably, the analysis cartridge comprises a membrane-tipped pipette, the pipette tip having a rectangular cross-section.

Preferably, the analysis cartridge includes detachable base and cap elements, the recesses being located in the base and the cap supporting the pipette.

Preferably, the cap comprises means for receiving a pipette tipped with a capillary.

Preferably, at least one of the recesses is sealed at its upper end by a brittle seal, and the cap is provided with a cutter for cutting said seal.

Preferably, the base comprises an absorbing wiper for wiping the exterior of the capillary-tipped pipette provided in the wiper.

Preferably, the assay cartridge comprises a membrane tipped pipette, the proximal end of which is closed by a pierceable, self-sealing membrane.

Preferably, the recesses in the analysis cartridge are arranged in a linear pattern.

Preferably, the radiation detector comprises a digital camera.

Preferably, the base of at least one of the recesses in the analysis cassette is flat and not perpendicular to the continuous side walls of the well.

Preferably, the apparatus also comprises a light source for illuminating the analysis cassette.

Preferably, the apparatus also includes a magnet.

Preferably, the apparatus comprises a heater for heating the analysis cartridge.

Preferably, the apparatus also comprises a controller for controlling the execution of an analysis by said apparatus.

Preferably, the gas pressure generating apparatus comprises a piston disposed within the cylindrical housing and a drive motor for driving said piston.

An assay cartridge having at least two wells according to the invention is characterized in that it further comprises a pipette which is inserted into at least two wells among the wells, the pipette having a proximal end and a distal end, the distal end closed by a fluid-permeable membrane.

An analysis cartridge with at least one well according to the invention is characterized in that it further comprises a pipette which is inserted into at least one well, the pipette having a capillary tip.

Preferably, the capillary tip is provided with a removable sleeve.

An analysis cassette comprising at least one well according to the invention is characterized in that it further comprises a pipette which is positioned in at least one well, the at least one well having two parallel, flat side walls connected by a base, a line perpendicular to the base wall and a line perpendicular to the sidewall lie in the same plane and form an angle with each other different from the right angle.

Preferably, the cartridge comprises a capillary-tipped pipette and a membrane-tipped pipette.

Preferably, the cartridge comprises a pipette, the closed end of which is closed by an inclined fluid-permeable membrane.

Preferably, the membrane lies in a plane at an angle of 20 ° to 40 ° to the axis of the pipette to which it is attached.

Preferably, the cartridge comprises a membrane-tipped pipette whose membrane-tipped end has a rectangular cross section.

Preferably, the cassette includes detachable base and cap members, the recesses being located in the base and the cap supporting the pipette.

Preferably, the cap comprises means for receiving a pipette tipped with a capillary.

Preferably, at least one of the recesses is sealed at the upper end by a brittle seal, and the cap is provided with a cutter for cutting this seal.

Preferably, the base comprises an absorbent wiper for wiping the exterior of the capillary-tipped pipette inserted into the wiper.

Preferably, the cartridge comprises a membrane tipped pipette, the proximal end of which is closed by a pierceable, self-sealing membrane.

PL 207 204 B1

Preferably the recesses are arranged in a linear pattern.

Preferably, at least one of the wells contains the reagent to perform the analysis.

An analysis device comprising a cartridge holder, drive means, gas pressure generating apparatus and a radiation detector according to the invention is characterized in that the analysis cartridge described above is placed in the holder and that the pipette of the analysis cartridge is placed in selected recesses by means of the driving means said cartridge, while the gas pressure generating apparatus is connected to the pipette of said analysis cartridge by causing fluid to flow therethrough, and the radiation from the cavity of the cartridge or its pipette is detected by a radiation detector.

Preferably, the radiation detector comprises a digital camera.

Preferably, the device also comprises a light source for illuminating the analysis cassette.

Preferably, the device also includes a magnet.

Preferably, the device also includes a heater for heating the analysis cartridge.

Preferably, the device also includes a controller for controlling the performance of an analysis by the device.

Preferably, the gas pressure generating apparatus comprises a piston disposed within the cylindrical housing and a drive motor for driving the piston.

The use of an apparatus for analyzing an analyte in a biological sample or testing the properties of a biological sample.

The use of an apparatus to analyze the clotting time in a blood sample or blood derivative.

Use of an apparatus to analyze a protein analyte in a body fluid sample or a body fluid derivative.

The Applicant has therefore developed a test device which, in its preferred embodiments, can be used at a treatment site, can perform a number of different tests, can provide quantitative test results and is relatively cheap.

According to one aspect, the invention provides a test device, preferably a diagnostic test device, comprising:

i) an analysis cartridge comprising at least two wells and a pipette that can be placed in at least two of said wells, said pipette having a proximal end and a distal end, said distal end being closed by a fluid-permeable membrane;

ii) a holder adapted to receive said cassette;

iii) drive means for inserting said pipette into a selected well of said cartridge;

iv) a pressure generating device coupled to said pipette to cause fluid to flow through said membrane;

v) a radiation detector measuring the radiation intensity from a cavity of said cartridge or from said pipette and, optionally, but preferably, vi) a source of electromagnetic radiation.

According to another aspect, the invention provides an assay cartridge comprising at least two wells and a pipette that can be inserted into at least two of said wells, said pipette having a proximal end and a distal end, said distal end being closed by a fluid-permeable membrane. .

A pipette is a tube with an opening at one end (distal end) into which fluid can flow under reduced pressure at the other end (proximal end). In the device described in the previous paragraphs, the distal end of the pipette is terminated (closed) by a fluid-permeable membrane. The proximal end of this pipette may be open or closed, but if it is closed then it must be closed by certain means which enable the pressure necessary for the pipette to function as a pipette. In one embodiment described below, the proximal end of the membrane-tipped pipette is connected to a pierceable, self-sealing membrane (e.g., a rubber seal) and pressure may be applied by a hollow needle through the membrane. Alternatively, the proximal end may be closed by a removable cap or plug that is removed to generate pressure or by a brittle seal that is broken to allow for pressure generation.

According to yet another aspect, the invention provides an analysis apparatus, comprising a) a cassette holder into which an analysis cassette of the invention can be received; b) drive means for inserting a pipette of said cassette into selected wells of said cassette; c) a gas pressure generating device connected to the pipette of said cartridge to cause fluid to flow through the pipette; d) a radiation detector for measuring radiation from a cavity of said cassette or from a cassette pipette and, optionally, but preferably, e) a source of electromagnetic radiation.

Thus, the combination of device and cassette according to the invention provides the analyzer according to the invention.

An analysis cartridge is preferably provided to the user filled with the reagents needed to perform the test or tests in question with the cartridge. In the event that two or more reagents are needed and should not be mixed prior to testing, the reagents may be pre-loaded into different wells in the cassette. Generally, such agents are pre-introduced into the recesses in measured amounts. Such reagents can be, for example, liquids, powders, beads, coatings on the walls of wells, or materials impregnated into or immobilized in a pipette membrane. Where the reagents are liquids or where they are prone to degradation by exposure to air or moisture, the cassette may be sealed to avoid loss of fluid or the ingress of air or moisture into the sensitive reagent. Such a seal is preferably achieved by forming a cassette with a base containing the recesses and a cap covering the recesses, and if desired, placing a fluid impermeable seal, e.g. an O-ring, between the recess openings in the base and the cap covering the recesses, if desired, placing a removable seal, e.g. sealing tape around the outer joint between the cap and the base. In another, more preferred embodiment, one or more of the wells may be sealed with foil before use: in this embodiment, the cap covering the wells is preferably provided with foil seal cutting means to cut the foil seals covering the well to allow the pipette to be inserted into the recesses. recesses. Alternatively, a cap of flexible material may be provided at the tops of the recesses (or only the fluid containing recesses) such that when the cap is connected to the base, a fluid tight seal is formed at the tops of the recesses. Such material can be, for example, a layer covering the cap or the discs or gaskets attached (e.g. welded or glued) to the cap. In one embodiment, the lower surface of the crate wheels is provided with flexible tongues that may serve as plugs in the recesses. In this way, the plugs serve to maintain the connection between the cap and the base prior to using the cassette in a test, and after the test, the base and the cap can be sealed prior to disposal by simply pressing the cap against the base and causing the plugs to reseal the recesses. This is particularly advantageous when the cavities, after analysis, contain toxic or potentially infectious materials. In this case, the hubcaps may, if necessary, be removed prior to the test; however, in a preferred embodiment, the cap serves to support the pipette and optionally also provides fastening means for the pressure generating device. In such an embodiment, the driving means may serve to move the base relative to the cap so as to locate the pipette in the desired recesses at different stages of the analysis.

Generally, and particularly in the case where the cap of the cassette is provided with flexible plugs for recesses in the base of the cassette, the device of the invention preferably comprises means for separating the cap from the base so that the cassette can be installed in the device in a sealed state. In one embodiment, such separating means include a wedge that slides over the loaded cassette and cooperates with protrusions, e.g., flanges, on the cap and base to spread them apart. Preferably, the separation means is actuated automatically when the cassette is installed, e.g. in response to closing the lid of the compartment containing the installed cassette, or moving the cassette into the compartment, for example, using a conveyor which may similarly remove the cassette from the compartment after the analysis is performed.

Different cassettes may be provided for different analyzes, e.g. for testing different analytes; however, the cassettes may also be designed to perform two or more different tests. In the latter case, it is often desirable that the cartridge contain two or more pipettes closed with membranes, i.e., that different pipettes can be used for each analysis.

The depressions in the cassette may be any suitable two-dimensional set arrangement (e.g., as in traditional multi-cavity plates): a linear set or wheel set arrangement. The use of circular and especially linear assemblies is particularly advantageous as

This simplifies the mechanism needed to move the cassette between predetermined positions, i.e. the drive means may move the cassette along a linear path or may rotate the cassette.

Particularly preferred is the use of a linear set of wells, particularly a set comprising sequentially: a material processing well (optionally prior to use holding a capillary-tipped pipette not permanently mounted on the cartridge cap or adapted to receive, in use, a capillary-tipped pipette mounted in the cartridge cap); a well that holds a membrane-sealed pipette before use, or a second capillary-tipped pipette mounted in the cassette cap and one or a series of two or more (e.g. up to six) analysis wells and reading the analysis result - these wells may contain reagents and prior to use the well the reagents containing the reagents may be closed with a foil and one of the wells may have an open end or an open side to facilitate reading of the result. In such an arrangement, the cap and base can advantageously be separated before starting the analysis and reattached only after the analysis is completed. Thus, the reading of the result in such an arrangement takes place when the cap and the base are separated. In this arrangement, the cap and base are preferably snapped together, e.g. by a snap lock. The material processing well may, for example, contain a dry reagent to be mixed during the analysis, a filter for separating the sample (e.g. to remove red blood cells from a blood sample) or an additional pipette which can work with a pipette mounted in the cap (e.g. pipette tipped with a capillary).

Although the cassette may include at least two wells, one or more of the positions in the well set of the multi-well cassette may have open ends or open sides such that detection of radiation from the pipette when placed in such positions is facilitated. If the radiation from a pipette in a well is to be measured, then at least part of the wall of the well must be transparent to the type of radiation measured.

Cavities in the cassette may be stationary during analysis; however, as it may be desirable to use a detector to monitor the progress of the analysis, it is generally preferred that the drive means be able to move the cassette between two or more positions such that the detector can measure radiation from different wells of the cassette. Alternatively, but less preferably, the detector itself may be moved between specific positions or movable mirrors may be installed to allow the path of light to be changed from the cassette to the detector to achieve the same effect.

Thus, in a preferred embodiment, the driving means causes the cassette cap and pipette to rise above the base containing the recesses during analysis (or, more preferably, to move the base downwards away from the cap) in order to move the base relative to the cap (preferably by moving the base e.g. linearly or by rotating) to position the pipette against the desired well and to move the cap and base simultaneously to place the pipette in the desired well, and so on until the analysis is complete.

In some analyzes it may be desirable to tilt the recesses during fluid transfer or to agitate the fluid in the recess and, accordingly, it is desirable that the driving means also tilt or rock (e.g., by rocking or shaking) at least a portion of the cassette containing the recesses.

The driving means may be manually actuated, e.g. a mechanical drive or a motor drive may be actuated at any stage by an operator; however, it is preferred that the motor-drive is actuated to perform the desired actions by an external, and more preferably internal, computer that controls the operation of the analysis device.

The recesses in the cassette can have any desired shapes or volumes; however, they are preferably cylindrical with straight sides or, less preferably, conical. The cross-section of such cylindrical recesses may have any desired shape, e.g. circular, oval, polygonal (e.g. rectangular), semicircular, etc. The bottoms of the depressions can be flat or curved; however, for wells intended to be monitored from below during or after analysis, the bottom of the well is preferably flat. In a particularly preferred embodiment, the bottom of the recess is flat and inclined, i.e. not horizontal. The recesses can be made in a solid base, or alternatively and less preferably, the recess containers can be joined in the form of a tape, plate, disk, spinning head, etc. The walls of the recesses, for example a fixed base containing recesses, are preferably made of plastic, especially light transmitting plastic, e.g. acrylic, vinyl, styrene or olefin. The choice of the appropriate plastic, however, depends, as usual, on the nature of the reagents used. It has been found to be

It is particularly advantageous to use plastics with good optical properties and poor gas and / or fluid permeability. For this purpose, alpha-olefin copolymers (e.g. ethylene and propylene, especially ethylene) and ring olefins (e.g. norbornene) are particularly preferred, e.g. the product sold under the trade name Topas® 8007 by the company Ticona GmbH of Frankfurt, Germany (Topas® 8007 is a copolymer of ethylene and norbornene). Preferably such copolymers have a light transmission (measured according to ASTM D1003 for a 2 mm wall thickness) of at least 80%, more preferably at least 90% and a water vapor transmission (at 23 ° C and 85% relative humidity measured according to DIN 53122 in sample 80 x 80 x 1 mm) less than 0.2 g.mm.m^-2d^-1, more preferably less than 0.05 g.mm.m^-2d^-1.

Typically the recesses have internal diameters of 2 to 20 mm, more preferably 5 to 15 mm, and a volume of 0.1 to 5 ml, more preferably 0.5 to 1.5 ml.

The membrane-sealed pipette of the cartridge according to the invention is preferably cylindrical, and the membrane preferably is in, and more preferably covers, one end. The second, open end is preferably shaped for a gas-tight connection to the pressure-generating device. The pipette may be of any suitable material, although clear plastic or glass is preferred. The membrane can be attached to the pipette in any suitable way, e.g. by welding (e.g. ultrasonic or thermally), gluing, fusing granular membrane precursor, etc.

The membrane itself can be made of any suitable material, e.g. plastic (e.g. nylon, polysulfones etc.), glass (e.g. glass fiber), metal etc. However, cellulose membranes (e.g. reinforced nitrocellulose) are particularly preferred as it is relatively easy to immobilize antibodies or other test reagents on such materials.

In various embodiments of the invention, the membrane is preferably flat and perpendicular to the pipette axis; such membranes are particularly effective in removing fluid from recesses with flat, horizontal or concave bottoms.

The diaphragm, however, may alternatively and more preferably be flat, but angled with respect to the pipette axis, e.g. up to 85 ° from perpendicular to the axis, preferably from 10 ° to 80 ° from perpendicular, more preferably from 50 ° to 70 ° from the perpendicular, in in particular about 60 ° to perpendicular. In case the pipette and one or more wells have a rectangular cross-section (e.g. square), the membrane is preferably inclined and the bottom of one or more of these wells is also inclined such that it is substantially parallel to the membrane when the pipette is in the well.

The use of a tilted membrane is particularly advantageous when, for a given cross-sectional area of ​​the pipette, the surface area of ​​the membrane is increased as it is deviated further from horizontal, providing a larger area for reading or monitoring during analysis. Most unexpectedly, it was found that the tilted membranes not only allowed substantially all of the contents of a suitably shaped cavity to pass through the membrane, but also allowed the contents to pass through uniformly (i.e. if colored analyte was trapped on the membrane, the membrane was uniformly colored). Another advantage is that the membrane can be viewed from the side, without the risk of droplets of sample, reagent, etc. falling onto the optics of the device. Yet another advantage is that the membrane can be easily illuminated without the risk that strong light from the illuminating device is reflected towards the light detector. Another advantage is that even with a colored sample (e.g. blood), it is possible to monitor the membrane surface through the side wall of the well and terminate any reaction step when a desired change occurs in the membrane surface as the distance between the membrane and the well wall may be smaller than that of a horizontal membrane in the fluid containing well. Yet another advantage is that there is less gas bubble formation between the membrane and the cavity wall than with horizontal membranes, so that less need to tilt or shake the base of the cassette.

The use of pipettes with an angled diaphragm is considered novel, and according to another aspect, the invention provides a pipette the distal end of which is cylindrical and terminated by a porous diaphragm whose outer surface is deviated from a plane perpendicular to the cylindrical axis of said distal. end, said pipette preferably forming part of a diagnostic test cassette.

The use of recesses with a rectangular cross-section is particularly advantageous as it reduces the occurrence of trapping liquid reagents at the top of the wells due to

Capillary effect when inverting test cassettes during shipping or storage. The corners where the side walls of the recess meet should therefore be as sharp as possible at the top of the recesses, e.g. they should have a radius of curvature of 0.5mm or less, e.g. 0.1mm or less. However, in order to prevent fluid from creeping in the recess along the corners of the recess, it is desirable for the lower part of the recess to be chamfered or more rounded, e.g. have a radius of curvature of at least 0.5mm, preferably at least 0.8mm.

In the case where the well is used for the readings of the analysis result, e.g. the absorption of the light passing through the fluid in the cavity is measured, it is also particularly advantageous to use a well with a rectangular cross section with an inclined bottom. In this way, by appropriately masking the portion of the cavity visible to the detector, light transmitted across the entire width of the cavity or through a narrower width at the bottom of the cavity (i.e. between the side wall and the sloped bottom). Thus, the length of the light path in the cavity may be increased or decreased by moving the visible portion up or down. In this way, for example, when the optical density of the contents of the well is high, a shorter path can be chosen.

Moreover, by measuring the intensity of the transmitted light for two or more optical path lengths (e.g., in and over the conical bottom of the cavity), the effect of the cavity walls on the measured signal can be determined and corrected.

If the scattered light is to be measured (e.g. when the test sample contains particles or agglomerates, or is fluorescent or phosphorescent), it is again desirable to use cavities with a rectangular cross-section, where the incident light is directed perpendicular to a pair of cavity walls and the scattered light is measured. by a detector (e.g. a digital camera) directed at one of the other walls. If the cassette contains a linear set of wells, the scattering light measurement well is preferably at one end of the assembly.

This use of sloped wall recesses is also novel and constitutes further aspects of the invention.

According to another aspect, the invention thus provides an analysis apparatus, comprising:

i) an analysis cassette containing at least one well and a pipette, which can be placed in at least one of said wells, at least one of said wells having two parallel, flat, side walls connected by a bottom wall comprising at least one planar surface, the normal thereto is coplanar with respect to and not perpendicular to the plane normal to parallel surfaces of said side walls;

ii) a holder adapted to receive said cassette;

iii) drive means for inserting said pipette into selected wells of said cartridge;

iv) a pressure generating device connected to said pipette thereby causing a fluid to flow through said membrane; and

v) a radiation detector for detecting radiation from a cavity of said cartridge or from said pipette.

In this aspect, the bottom is preferably flat, inclined with respect to the horizontal, as described above, and the recess preferably has a rectangular cross-section. The cartridge further preferably comprises at least one capillary-tipped pipette and / or a membrane-closed pipette as described above.

According to yet another aspect, the invention provides an analysis cartridge comprising at least one well and a pipette that can be inserted into at least one said well, at least one said well having two parallel, flat sidewalls connected by a bottom wall having at least one bottom wall. one flat surface to which the normal is coplanar with respect to and is not perpendicular to the normals to parallel, the flat surfaces of said side walls.

In addition to the membrane-sealed pipette, the cassettes of the invention may contain one or more other pipettes, again preferably supported by the cap of the cartridge, for example for measuring the exact volume of a reagent or sample or for mixing reagents and samples. In one preferred embodiment, the cartridge comprises a capillary-tipped pipette which draws a desired amount of fluid from the sample by the action of its capillary. Particularly preferably, this involves placing the capillary in a chamber with a wider internal diameter such that the capillary action causes only the tip of the capillary to fill. After the tip is withdrawn from the fluid, the contents of the tip can then be dropped under pressure into the cartridge well or sucked further into the pipette past the capillary tip and chamber.

In another aspect of the invention, the cartridge may include a capillary tipped pipette in place of a membrane tipped pipette. As will be discussed below, such a cassette may, for example, be used for clotting time measurement.

The outer diameter of the membrane-tipped pipette is preferably at least 0.8 mm, e.g. 1 to 5 mm, in particular 1.5 to 2.5 mm, less than the inner diameter of the wells, so as to facilitate gas flow between the wall of the well. and pipette as fluid flows through the pipette membrane and ensure substantially complete withdrawal of fluid from the well. The slit also allows a fluid (e.g., 200 µΐ and a membrane-tipped pipette to be held in the well) prior to withdrawing the fluid into the pipette.

Although the pipette and wells may have the same cross-sectional shape (i.e. circular, square, etc.), it can sometimes be advantageous for the shapes to be slightly different, e.g. one round and the other elliptical, as this reduces the risk that the pipette is tipped over. the membrane will be sucked to the bottom of the cavity. For this purpose, the pipette tip or the bottom of the well can also be made somewhat irregularly, e.g. with indentations or projections.

In a particularly preferred embodiment, the cassette comprises: a base containing a plurality of, e.g. 2 to 8 or 10, wells of which at least two, preferably at least three, are free of liquid reagent and at least one of which is liquid reagent; and a cap supporting the diaphragm-tipped pipette so that its diaphragm end is positioned in one of the hollow recesses and its open end is accessible from the outer surface of the shield and the cap has an opening in the shield for introducing the sample communicating with other cavities in which there is no fluid. Preferably, removable seals are provided to cover the open ends of the pipette and the sample introduction opening. If the cap does not support the recess sealing plugs, or if the recesses are not sealed as described above, an additional removable seal is preferably provided surrounding the outer connection between the cap and the base, and an O-ring or other seals are placed between the cap and the base around at least those recesses that contain fluid. In any event, the inside of the cassette is insulated from air and moisture before it is used. The base and cap preferably have indentations or projections to cooperate with the cassette holder and drive means to ensure proper alignment of the cap with the base during analysis, and the cap supports cavity sealing plugs for cooperation with a separator as described above which causes the cap to separate. and basics to enable the analysis to be performed.

The base and cap are preferably such that the diaphragm tipped pipette can be placed inside the reading well or in a position outside the wells so that radiation from the pipette is available to the detector. Such a reading well may have, for example, a translucent flat bottom or a flat piece of the side wall through which light can pass to the detector. When the reading is taken outside of the wells, this could be, for example, an open hole in the base or part of the base where the side wall has been removed or recessed so that light from the pipette can reach the detector without passing through the material, with of which the base is made.

The use of a reading well is preferred because it reduces the risk that reagents or a sample fall onto the analyzer. In the event that a tilted diaphragm is to be analyzed, a separate reading well may not be used, as simply lifting the diaphragm above the fluid in the well or sucking the fluid through the diaphragm into the pipette allows the surface of the diaphragm to be analyzed.

In one embodiment, the base may be formed to form a mirror surface (e.g., a plastic prism surface) under the bottom of the reading cavity that reflects light from the bottom of the reading cavity, e.g., from a vertical to a horizontal direction. In this way, the detector does not need to be placed under the cartridge and problems with dust or liquid falling onto the detector can be avoided. As with a Fresnel lens, a prism can similarly be manufactured as an integral combination of parallel individual prism elements. Such a prism structure is referred to herein as a Fresnel prism and such prisms and their use, e.g. for modifying the light path in an optical device, e.g.

Aspects of the present invention. Image distortion due to surface distortion, often observed in extruded plastic parts with a thickness greater than a few millimeters, is reduced or removed by using a plastic Fresnel prism rather than a traditional plastic prism having the same incident light surface area. Thus, the use of a Fresnel prism formed in the base of the cassette to obtain light reflection is particularly advantageous in the devices of the invention. A typical Fresnel prism is a structure made of transparent material having a step on one side and a flat part on the other - light normally incident on the stepped horizontal portion is internally reflected by the flat surface and leaves the prism perpendicular through the vertical portion of the step. As a result, the prism acts like a mirror. However, with an inclined diaphragm such a Fresnel prism is usually not needed.

In the cartridges of the invention, the proximal or open end of the at least one pipette is preferably closed by a flexible, self-sealing membrane, e.g. a rubber membrane, which can be pierced by a hollow needle to generate a suitable gas pressure. In this embodiment, a buffer reservoir is preferably provided in the pipette between the pipette tip and the flexible membrane. In this embodiment, fluid in the cartridge may be drawn into the buffer reservoir during or at the end of the test so that the used cartridge can be removed and discarded without the risk of leakage of residues.

The device for generating gas pressure in the device of the invention may, for example, include a pump and a conduit from the pump to the cartridge joint, and optionally at least one reservoir and a valve with two or more positions. Addition of a tank with a capacity of e.g. one or more liters, preferably at least two reservoirs, enables the pipette to be delivered with pressures greater and / or less than the surrounding pressure for a short period of time with negligible variations in pressure build-up time due to the ability to isolate the pipette from the pump and due to a relatively small pressure change inside the reservoir during pressure build-up (thanks to the relatively large volume of the reservoir). Between pressurization, the pump may be used to restore the pressure in the reservoir to the desired level. As it may be desirable to vent the pipette to the atmosphere and / or to provide the pipette with pressures greater than or less than the ambient pressure, it is desirable to locate the valve with multiple positions in the conduit upstream of the pipette to allow such various pressures to be generated. The valve, which should preferably also have a closed position to prevent gas flow into or out of the pipette, is preferably computer controlled. The use of pressure vessels as described above, however, causes the devices according to the invention to be relatively bulky. As the device should preferably be portable, it is preferable to use a piston pump (e.g. syringe) connected by a conduit (preferably of minimal volume) to the cartridge connector. In fact, it is particularly advantageous to use a set of coupled piston pumps, each connected to a separate cartridge connector such that when the cartridge is installed, operation of the pump motor causes all the pumps to operate. In this embodiment, the cassette is preferably provided with plugs or active means for cooperating with the connectors, the plugs allowing ventilation of the respective piston pump. In certain example embodiments, for example, when measuring the clotting time, or when it is required to bind the analyte to a ligand immobilized on a pipette membrane, it may be desirable to accelerate or slow the fluid flow under the influence of a pressure generating device; under these conditions this can be achieved, for example, by accelerating or slowing down the movement of the pistons in piston pumps.

The pressure generating device is preferably connected directly to the open end of the pipette; however, alternatively, and much less preferably, it may be directly connected to a well in the cartridge with the open end of the pipette at ambient pressure.

In one particular embodiment, a (preferably movable) connector of the pressure generating device is provided for each recess in the cassette or position outside the recesses, and the cassette is provided with plugs or active means for cooperating with each of these connectors. In this way, the need to accurately align the cassette when placed in the holder can be avoided - the cassette can be placed in any of the provided positions and the lid of the device is closed, automatically connecting the connectors to the plugs and the active means of the cassette. Identification of the cassette (as will be discussed below) by the device allows the cassette to be automatically moved to the correct position for analysis. However, this is only desirable if it is essential to reduce the time required for insertion of the cassette or if the cassette is intended for use in multiple analyzes (i.e. has multiple pipettes).

The detector in the device according to the invention may be any suitable radiation detector, e.g. a radioactive emission detector or an electromagnetic radiation detector. Alternatively, the device may include two or more detectors that can detect different types of radiation. However, when used at a treatment site, it is preferred that the detector is an electromagnetic radiation detector, more particularly a detector that can measure light intensity in at least part of the ultraviolet or infrared range, in particular in the near ultraviolet or near infrared range. in particular in the visible range (the term light here means electromagnetic radiation ranging from ultraviolet to infrared). For this purpose, it is particularly advantageous to use a digital camera as a detector.

The use of a digital camera as a detector is particularly advantageous since it can function not only as a light detector but as an image structure analyzer. Thus, for example, irregularities in the membrane image in the pipette can be detected and taken into account.

Between the detector and the cassette it may be desirable to place, permanently or temporarily, elements that either select the radiation energy that may reach the detector (e.g. filters, prisms, etc.) or to block diffuse radiation from reaching the detector (e.g. openings). and light traps).

Scattered radiation blocking elements are especially important when the measured radiation intensity is low (e.g. due to chemoluminescence or fluorescence) or the radiation is forced or results from the transmission or reflection of the radiation measured by the detector. Under these conditions, light barriers or collimators may also be placed elsewhere on the device or inside the cassette.

Generally, the device of the invention is equipped with electromagnetic radiation sources (e.g. visible light sources or near infrared or near ultraviolet radiation) arranged such that radiation emitted, reflected, or transmitted through the appropriate cartridge cavities or pipette is directed to the detector. Consequently, it is also preferred that the cassette, cassette holder and detector are placed in the device in a light-tight chamber and that the device is provided with a closable cassette insertion opening, e.g.

It is particularly advantageous if a light source is provided which, after installation of the cassette, has a recess between it and the detector, e.g. so that the light transmission through the recess can be determined. To this end, the cassette may be provided with an opening into which a light source may be inserted when installing the cassette, preferably an axially disposed opening, the recesses in the cassette being arranged around a central axis.

It should be noted that the detector may be positioned relative to the cavity and the light source to measure transmitted, reflected, scattered, or emitted light.

If the detector is a digital camera (or is a scanning laser), it can also be used to identify the test. Thus, a barcode or similar machine readable code may be placed on the analysis cartridge and, by reading it, the apparatus control computer may identify the type of test, and therefore also the test steps, that must be performed. The user may likewise apply a barcode or device readable code to an analysis cartridge to identify the patient, such that the device may generate a patient and test identification report or may generate entries in a computerized patient file. Arrangements for reading the code and reading the result of an analysis of this type are discussed, for example, in WO 98/32004.

As mentioned above, cassettes in which the pipette is capillary-tipped rather than membrane-tipped may advantageously be used for measuring coagulation time in blood or plasma (preferably blood). The pipette preferably comprises, in sequence, a capillary, a chamber and a second capillary, which may be non-linear, e.g. sinusoidal, if desired. Opening the cassette and submerging the capillary tip in the blood sample causes the capillary to fill to the junction with the chamber, i.e. collecting a sample of a certain volume. The cassette can then be closed and placed in the analyzer. The second capillary, or one of the cavities in the cartridge, is covered with a clotting accelerator (e.g., tissue factor) and the sample fluid may contact it by creating a pressure above or below the ambient pressure at the open end of the pipette, respectively. In the first case, the pressure causes the sample to be drawn through the chamber into the second capillary and thus into contact

With a clotting accelerator. In the latter case, the pressure generated ejects the sample into the coated cavity. If desired, in the latter case, the sample and the clotting agent may be mixed by pulling it back into the pipette and ejecting it one or more times. The sample is then pulled through the capillary and the chamber into the second capillary. In both cases, movement of the sample under pressure in the second capillary is monitored by the detector until clotting has reached such a state that movement is no longer detected. This may involve passing the sample back and forth in a second capillary by alternately creating a pressure below and above the surrounding pressure.

It should therefore be noted that the same capillary may be used to collect a sample (e.g., blood) and mix it with one or more reagents (e.g., by pumping it into and out of a cavity in a cartridge).

In any case, it is important to measure the clotting time that the temperature of the sample is controlled, and thus it is desirable that the device, e.g. in a cassette holder, be equipped with a temperature controller, e.g. air, etc.

In an alternative embodiment, the clotting time of the blood or plasma may be determined by placing the sample in a cavity containing an effervescent agent and monitoring the rate of rising of the gas bubbles with a digital camera.

In case a capillary-tipped pipette is used, it may be desirable to provide the pipette separate from the cartridge so that it can be placed in the well and connected to a pressure generating device.

Such capillary-tipped pipettes and their use in conjunction with assay cartridges constitute further aspects of the invention.

Thus, according to another aspect, the invention provides an analysis apparatus, comprising:

i) an analysis cartridge comprising at least one, preferably at least two wells and a pipette capable of being placed in at least one, and preferably at least two, said wells, said pipette having a capillary end;

ii) a holder for receiving said cassette;

iii) driving means for inserting said pipette into selected wells of said cartridge;

iv) a pressure generating device connected to said pipette to cause fluid to flow through said membrane and

v) a radiation detector for measuring the radiation intensity from a cavity of said cartridge or from said pipette. According to yet another aspect, the invention also provides an assay cartridge comprising at least one, and preferably at least two, wells and a pipette that can be inserted into at least one, and preferably at least two, said wells, said pipette having a capillary end.

By using pipettes in the analysis cassettes according to the invention, it is therefore possible to place test samples in the wells of the cartridge, mix the reagents or the reagents and the sample in the wells, transfer fluids from one well to another, etc. By pumping the fluids into and out of the pipette in one well, the homogeneity of the mixture can be improved. and by pumping the fluids back and forth through the membrane of the reagent pipette, the extent of reaction with the reagent can be increased. By varying the rate at which fluid is pumped through the membrane of the reagent-containing pipette, the degree to which the reagent participates in the reaction can be altered. Correspondingly, the shapes of the pipette and cassette provide great versatility for testing.

Where the assay cartridge includes a capillary-tipped pipette, e.g. for transferring blood samples, it is often desirable to remove excess fluid from the outer surface of the capillary. In such cases, it is preferable that one of the wells is provided with an absorbing pipette wiper over which the capillary tip can be moved so that the wiper absorbs any liquid from the outer surface of the capillary. The squeegee may, for example, take the form of an absorbent pad placed on or near the upper end of a cavity, e.g. a U-shaped insert, preferably a cut in the base U. In such an embodiment, when the capillary is withdrawn from the cavity, it may be moved sideways to rub the capillary tip against the notch. Since such a shift may occur before the membrane-tipped pipette is fully pulled out of the well into which it is inserted, it may be necessary to design the wells to prevent the membrane-tipped pipette from coming closer to the side wall of the well. Thus, the well for a membrane tipped pipette may be made wider, or alternatively its side wall may be partially removed at the top of the well.

Instead of wiping the tip of the capillary to remove excess sample from the outside of the tip, alternatively, the capillary tip may be placed in an absorption assembly that is parallel to the axis of the capillary tip, e.g. in the form of absorbent fibers lying parallel to the tip, or sheets of absorbent material (e.g. paper ), with surfaces parallel to the axis of the capillary. Since the open end of the capillary is not in contact with the absorbent material, the capillary contents are not removed as the exterior of the capillary is purged of excess fluid. This is especially important with blood samples. Thus, for example, a 1 µΐ capillary gives poor accuracy unless blood adhering to the outside of the capillary is removed. On average, a 1 μl capillary carries 0.25 μl on the outside. Without removing the blood adhering to the outer part, a CV (fluctuation coefficient) of 7% -8% (blood volume delivered) was found. With the efficient removal of blood carried on the exterior, the CV was reduced to 1.0% - 1.5%.

In the event that cleaning the capillary is part of the test, time to purge may cause the blood on the outside of the capillary to dry out. If this happens, not all blood will be absorbed and it may be dissolved during the next dilution step. If the user waits one minute from drawing blood into the capillary to starting the device, wiping is somewhat inefficient. After three minutes, no blood will be absorbed at all.

It is therefore highly advantageous if wiping of the capillary takes place immediately after the blood sample is taken into the capillary. This can be achieved by placing in a given cavity of the cassette where the capillary is placed an absorption assembly as described above, e.g. a strip of V-bent paper with an open V-end intended to contact the capillary tip. The paper may be positioned and held in the cavity either by applying paper forces to the outside of the cavity wall or, if desired, by mounting the paper in the support frame. As the user introduces the capillary holder into the cartridge, the capillary opens the two upper arms to the sides and the capillary moves downward in contact with the paper on both opposite sides. This design, with the paper parallel to the capillary, ensures that no blood is absorbed from the inside of the capillary, and furthermore, the capillary never touches the bottom of the bent paper. Using a 1 μl capillary and whole blood, a CV (blood volume) of 0.75% was obtained in this construction.

In another preferred embodiment, the assay cartridge is provided to the user with a capillary tipped pipette for use in sampling either loosely or unstuck to the cartridge, e.g. in an end cavity of a linear set of wells. In this embodiment, releasably attached to the capillary tip, i.e. the distal end of the pipette, is a sleeve that snugly embraces and preferably ends flush with the open end of the capillary. When a sample is taken through a capillary, excess external fluid suitably adheres to the exterior of the sleeve rather than to the exterior of the capillary. The sleeve is preferably provided, e.g. on the outer surface, with means for cooperating with the inner or top surface of a cavity in the cartridge (e.g. a deformable flange, etc.) so that when a filled capillary-tipped pipette is inserted into the well, the pipette can then be removed. from a recess (e.g. after starting the automatic analysis), leaving the cuff and excess fluid in the cavity. Experiments have shown that by transferring 1 µl of a blood sample using such a sleeve shrouded capillary, a CV (blood volume) as low as using the folded paper wiper described in the previous paragraph can be achieved.

With certain tests, it may be desirable to perform a sample separation, e.g., to generate a plasma sample from an original blood sample. In such cases, it may be desirable to place the filter in one of the recesses. It may be removable or alternatively may form part of an integral pipette extension positioned in the cavity. Such a pipette extension may, for example, comprise a cylinder which is open at the upper end, the shape of which is adapted to cooperate with a pipette mounted in the cap of the cartridge and filled at the lower end with glass fibers. In one embodiment, the sample may be taken into a capillary-tipped pipette mounted in the cartridge cap when the cap and base are separated, or into a capillary-tipped pipette that may be mounted in the cartridge cap. Then, when the cap and base are connected, a sample can be discharged under air pressure into the barrel of the pipette extension; after filtering, it flows to the bottom of the cavity. The second, capillary-tipped, pipette-tipped pipette can then be used to collect the filtered sample after the pipette has been removed and reinserted.

From the recess. In this way, starting with a blood sample, an undiluted plasma sample can be produced.

Like pipette extensions, capillary wipers, etc., other items can also be placed inside the cartridge wells. Thus, for example, the well for receiving the sample capillary therein may contain another fixed or removable well containing the dry reagent, so that the sample and the given reagent may be mixed at the start of the test.

The device, apparatus and cassettes according to the invention are intended to perform analyzes. Such analyzes, using the apparatus, device or cassettes of the invention, form further aspects of the invention. While the invention is particularly suited to medical diagnostic analyzes, it can also be applied to other analyzes, e.g. environmental pollution, food, etc., including analysis of samples from production processes. It is particularly advantageous for such applications that the cassettes and devices may be small enough to be fully portable, e.g. with a maximum device size (excluding any connection to external device or power sources) of no more than 30cm, more preferably not greater than 20 cm.

The use of membrane tipped pipettes in testing is also novel and forms a further aspect of the invention. According to this aspect, the invention provides an analysis method in which fluid is transferred from the container to the pipette, characterized in that the end of said pipette through which fluid is introduced is closed by a fluid-permeable membrane.

The invention also provides a method of using the device of the invention to analyze an analyte in a biological sample or a property of a biological sample, e.g. for measuring clotting time in a blood sample or blood derivative or for measuring a protein analyte in a body fluid sample or body fluid derivative.

The subject of the invention has been illustrated in an embodiment in the drawing, in which Fig. 1 schematically shows a cross-section through a cassette according to the invention; Fig. 2 is a schematic partial cross-sectional view through a cassette according to the invention; Figure 3 is a schematic partial cross-sectional view through a cassette according to the invention; Fig. 4 is a schematic drawing of an apparatus according to the invention; fig. 5 schematically shows a cross section through a cassette according to the invention; Figures 6 and 7 show the radiation intensity curves for the tests of Examples 1 and 2; Figure 8 shows the test results for Example 3; Figures 9 to 19 schematically show other embodiments of cassettes according to the invention in which the recesses are arranged in a linear pattern; fig. twenty shows schematically how a movable magnet can be used to separate magnetic polymer beads from a sample in a cavity of a cassette according to the invention; Figure 21 shows schematically how a strip of paper can be used to wipe excess fluid from the exterior of the capillary-tipped pipette in a cartridge according to the invention; fig. 22A and 22B, 22C, 22D show schematically how a membrane-closed buffer reservoir can form part of a pipette in a cartridge according to the invention, and Fig. 23 schematically shows a side view of a capillary tipped pipette for use in an analysis cartridge according to the invention.

1, the transparent cylindrical base 1 of the plastic cassette 23 is shown, including cylindrical recesses 2 (only two of them are shown) arranged in a circular pattern about the cassette axis 3. Above the base 1 of the cassette 23 is a cassette cover 5. The outlets of each cavity 2 are sealed by plugs 4, attached to the cover 5. The casing 5 also supports a pipette 6 having, on the outside of the casing 5, an extension 7 of the pressure generating device connector and a diaphragm 8 - closing the end of the pipette 6, located in the recess 2 of the base 1 of the cartridge. In the sheath 5 there is also a port 9 for introducing the sample. Port 9 and pipette 6 are held against wells 2 by mating tabs and cutouts 10, 11, 12, 13. Similar mating protrusions and / or cutouts 14 (shown here as cutout) are provided in the base 1 and in the housing 5 to allow cooperation between the base 1 and the housing 5 and the cassette holder and driving means (not shown) of the analysis apparatus. The base 1 and the cover 5 are provided with flanges 15 to cooperate with a separator (not shown) that separates the seals of the base 1 and covers 5 before starting the test. The essence of the test for which the cartridge is intended is identified by a barcode label 17 on the side of the base 1. The pipette 6 and the sample introduction port 9 are shown closed sealed by removable tape seals 16. They are removed before use cassettes.

PL 207 204 B1

In Fig. 2, the cassette of Fig. 1 is shown in a different orientation for reading the test result upon completion of the analysis. In this arrangement, depicted recesses 18 and 19 are different from recesses 2 in Fig. 1. Recess 18 is a reading cavity containing a plastic prism 20 located at its bottom, and part of the light path from the membrane 8 to the detector is shown in dashed line. 21. Pipette 6 is shown with Reagent 22. The light source 44 is shown within the axial channel 45 in the base of the cassette.

Fig. 3 shows another embodiment of the cassette of Fig. 2 in which the bottom of the reading well 18 is staggered and the base below the reading well 18 is inclined to form a Fresnel prism 29. The light source 46 is positioned to illuminate the diaphragm 8. W in this embodiment, the pipette 6 is also shown with a relatively large volume chamber 47. This makes it easier to keep the 6 test fluids in the pipette.

Figure 4 shows schematically the components of the apparatus according to the invention. Cartridge 23 (with base 1, skirt 5 and pipette 6) is supported by holder 24 and moved by driving means 25. Pipette 6 is connected by lines 26 to piston pumps 27 driven by motor 28. The detector, digital camera 32, is positioned to measure the light intensity from the reading cavity of the cartridge 23 after the test is completed, and the powered light sources 44 and 46 are arranged to illuminate the reading cavity.

The drive means 25, the motor 28, the camera 32 and the power 34 are controlled by a computer 35 which sends the information to the monitor and printer 36 or to a remote computer 37 (e.g. via a wireless infrared connection). The camera 32 of the light sources 44 and 46, the handle 24 and the cassette 23 are housed inside a light tight chamber 38 provided with a port 39 for installing and removing the cassette 23.

Fig. 5 shows a cross-section through an alternative capillary-tipped pipette 6 used in cartridges according to the invention.

The open end 39 of the pipette 6 is adapted to be connected to a pressure-generating device. The other end of the pipette 6 is provided with a capillary tip 40 which is connected to the chamber 41 and then, via a distal sinusoidal capillary 42, with an open end 39. Part 43 of the bottom of the well 2 is covered with a coagulation accelerating agent, e.g. a tissue factor. Immersion of the capillary tip 40 in blood or plasma causes a predetermined volume of sample to be drawn in due to the capillary effect. Retracting the pipette 6 from the sample and then either discharging the contents into a well coated with a clotting accelerator and then sucking the sample back into the capillary 42, or sucking the tissue factor sample into the capillary 42, accelerates the onset of clotting and a digital camera 32 can be used to determine the time it takes for sample flow along the capillary 42 to effectively cease, i. e. clotting time.

Figures 9 to 19 show alternative assay cartridge systems in which the wells are arranged in a linear fashion.

Figure 9 shows a detached capillary-tipped pipette 50 that can be immersed in a fluid to collect a sample. The filled pipette 50 may then be inserted into the opening 51 in the cap 52 of the cartridge whereby the tip of the capillary will be placed in an end cavity in the base 53 of the cartridge. The open top end of the pipette 50 is provided with cutouts 54 so that if an operator places the pipette 50 in the cap 52 and in the cartridge base 53 by pressing against the pipette tip, this will not increase the pressure of the pipette 50 and will not prematurely eject some or all of the sample contents.

Fig. 10 shows the cassette of Fig. 9 assembled after the sample pipette 50 has been inserted, i.e. the stage when the cassette is ready to be inserted into the device of the invention.

During test execution, cap 52 and cassette base 53 are separated by disengaging the latch mechanism 84. The disassembled cassette is shown in Figure 11. The cassette cap 52 is shown with a capillary-tipped pipette and a diaphragm-tipped pipette 55. The membrane-tipped pipette 55 has a rectangular cross-section and has a sloping end 56. For the sake of clarity of the drawing, the membrane 8 covering the open, lower end of the pipette 55 is not shown in Figure 11. This membrane 8 is shown in Fig. 1, which shows the connection of the membrane 8 to the tip of the pipette. The base 53 of the cassette is shown with six depressions 57, 58, 59, 60, 61, 62, all of which are rectangular in cross-section. In order to allow wiping of the capillary tip, there is no top wall between recesses 57 and 58.

As shown in Fig. 12, the bottoms 63 of the wells 59, 60, 61, 62 are inclined so as to be parallel to the inclined tip 56 of the membrane-tipped pipette 55. The depressions 59, 60, 61, 62 are 16

The top ends are covered with foil. The foil closures are pierced during the test by piercing members 64 mounted in the cassette cap 52 (see FIG. 13). The individual piercing elements 64 are connected to each other by the tape 65 shown in Fig. 14. Each piercer 64, which may be metal, but preferably plastic, is a hollow cylinder of rectangular cross-section with a blade edge 66 at the lower edge and flanges 67 at the upper edge that retain the piercer 64 by the base 53 of the cartridge. after it has been placed in the base 53 (as shown in Fig. 15). The internal cross-section of the piercing members 64 is shaped such that it serves as a guide for the pipettes.

Fig. 16 shows cartridge cap 52 and base 53 when separated by sliding sideways to bring the capillary pipette tip 50 into contact with an absorption wiper 68 positioned above cavity 57. As shown, the membrane tipped pipette 55 is partially transferred from cavity 58 to cavity. recesses 57.

Figures 17 and 18 show the exploded views of the cap 52 and cartridge base 53 with pipette extensions 69 and 70 which, in operation, are placed into a cavity 57 into which the sample is initially introduced by means of a pipette 50. In the case shown in Fig. 18, the pipette extension 70 serves to transfer the sample to a membrane-tipped pipette 55, e.g. for sample filtration.

Fig. 19 shows the lower ends of three wells adapted to a blood clotting test with a steel ball 72 as shown in Figs. 19a and 19b that can be moved along the bottom of the well, and Fig. 19c shows a polymer ball 73 that can float. on the surface of the sample while the sample is still liquid.

After testing with the cassette of Figs. 9 to 19, an absorbent strip is preferably inserted into the opening 51 in the cap 52 of the cassette to avoid leakage of fluid that remains in the recesses 58 to 62. Alternatively, the opening 51 may be closed using an oblong plunger. which is used to drive the piercing members 64 into the foil that closes the recesses 58, 59, 60, 61, 62.

Referring to Fig. 20, a recess 75 in a cassette according to the invention is shown. In the cavity 75 there is a fluid 76 with magnetic polymer beads. In order to separate the beads from the fluid during the test (e.g. as in Example 12 below), a magnet 77 is provided that is moved from a position (A) away from the well to a position (B) where it contacts the cavity. wall of cavity 75. The membrane tipped pipette 55 can then be placed in the cavity 75 to draw out any fluid that remains outside the magnetic beads.

Fig. 21 shows schematically a cartridge 78 according to the invention with a linear set of wells 79, 80, 81, 82, 83, 84, the end recess 79 of which is adapted to receive a capillary, the tip 85 of which is shown. A V-shaped strip of absorbent paper 86 is disposed within the cavity 79 such that insertion of the capillary tip 85 into the cavity 79 causes the sides of the capillary to be wiped.

Figure 22 shows partially and schematically a cartridge 87 of the invention having a capillary-tipped pipette 88 and a membrane-tipped pipette 89 in the cap 90 of the cartridge. The diaphragm-terminated pipette 89 has a fluid buffer reservoir 91 near its proximal end, and when placed in the cartridge cap 90, the reservoir 91 is closed by a self-sealing rubber gasket 92. If pressure is required at the proximal end of the diaphragm-tipped pipette 89, this is achieved by piercing a seal 92 with a recessed needle 93 (Fig. 22D) connected to a pressure generating device (not shown).

Fig. 23 shows a capillary tipped pipette 94 that is provided as part of an analysis cartridge according to the invention. The pipette 94 is loosely inserted into one of the wells, e.g., like the pipette 50 in the cavity 57 of the embodiment of Fig. 11. The distal end 95 of the pipette 94 is provided with a sleeve 96 which includes and fits snugly the end of the pipette 94 and ends at the end 95 of the pipette. flush with capillary tip. The upper edge of sleeve 96 is provided with a deformed collar 97 that can be pushed through a mating collar in the recess so as to lock the sleeve in the recess. In practice, the capillary-tipped pipette 94 is removed from the attached sleeve 96 cartridge, immersed in the sample fluid to draw fluid into the capillary tip, and reseated into the cavity and pushed to lock the sleeve 96 into the cavity. The cartridge 78 may then be installed in the analysis apparatus, and the operation of separating the cap and base of the cartridge during the test serves to detach the sleeve 96 from the capillary.

PL 207 204 B1

Example 1

Serum C-reactive protein content test

Human blood samples of 1 µl, admixed with purified C-reactive protein (CRP) to concentrations ranging from 0 to 160 mg / L, were placed in a round bottom cavity with an internal diameter of 9 mm (in a cassette-like assay cassette). of Figure 1) containing 200 g of an aqueous dilution fluid (30 mM borate buffer pH 8.0 containing 0.01 wt% sodium citrate, 0.02 wt% NaN3 and deoxycholate).

A membrane-tipped pipette, having an outer diameter of 7.2 mm, was placed into the well containing the sample, and a pressure less than surrounding pressure was applied to the open end of the pipette, causing the contents of the well to flow through the membrane into the pipette. In this example, the pipette membrane was a sheet of nitrocellulose on which an anti-CRP monoclonal antibody (prepared by conventional techniques) had been immobilized.

The pipette was then removed from the well and placed in a second well of the same configuration containing 200 g of an aqueous suspension of gold micro-beads [4.5 nm in diameter, with a concentration (optical density at 540 nm) of about 3, corresponding to an antibody concentration of about 50 gg / ml in 50 mM borate buffer, pH 8.05, containing 20 mM NaCl, 0.05 wt.% NaN3 and 0.1 wt.% BSA)], conjugated in the traditional way with an anti-CRP monoclonal antibody. A pressure less than surrounding pressure was created at the open end of the pipette, causing fluid to flow from the well into the pipette, thereby saturating the membrane with conjugated gold.

The pipette was then removed from the second well and placed in the third well, again of the same configuration, containing 200 g of aqueous diluent (supra). A pressure less than surrounding pressure was applied to the open end of the pipette in order to draw the washing reagent into the pipette; thus, unbound conjugated gold was removed from the membrane.

The pipette was then removed from the third well and placed in a fourth empty well having an internal diameter of 9 mm and a flat bottom. For the purposes of this test, the fourth well was the test result reading well. The diaphragm of the pipette was illuminated (e.g. green light from a light emitting diode (LED) through a transparent base containing an analysis cartridge cavity and 540 nm light reflected by the membrane was measured using a detector (e.g. digital camera or photodiode).

Fig. 6 in the accompanying drawings shows a linear plot of the light intensity in this test using a green LED.

The test procedure requires a waiting time of approximately 40 seconds after the addition of serum to measure the intensity of the reflected radiation.

Example 2

Analysis of human serum albumin in urine

Human urine was stripped of human serum albumin (HSA) by ultrafiltration and then doped with purified HSA to concentrations between 0 and 200 mg / L.

A 10 g urine sample was transferred in a capillary into a round bottom 9 mm internal diameter well (in an assay cartridge similar to that of Figure 1) containing 200 g of aqueous sodium phosphate buffer, pH 5.6, containing 4 .0wt% propanol1, 0.05wt% NaN3, 0.003wt% tropeolin-O and 0.5wt% BSA. Urine was mixed with the dilution buffer by pumping into and out of the capillary three times. The capillary was removed and a membrane-tipped pipette was placed in the well. In this test, the membrane was a sheet of nitrocellulose with an anti-HSA monoclonal antibody immobilized thereon. The diluted sample was drawn into the pipette as in Example 1.

The pipette was then removed from the well and placed in a second well having the same configuration but containing 200 µl of a suspension of antibody conjugated gold beads (as in Example 1, but anti-HSA antibody rather than anti-CRP, 50 mM borate buffer with pH 7.8, 0.05wt% NaN3 and 0.2wt% BSA). The contents of the well were drawn into the pipette as in Example 1, and as in Example 1, the pipette was then transferred to the third (washing) and fourth (reading) wells. In this test, the washing reagent was PBS, pH 7.4.

Fig. 7 of the accompanying drawings shows a plot of the radiation intensity for this assay.

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Example 3

Blood glycohemoglobin test

A 1 µΙ whole blood sample was collected from the blood sample using a capillary mounted on the tip of an inverted cone-shaped container with a volume of approximately 500 µl, ie a funnel-shaped device to the upper end of which a pressure device was attached.

The capillary was placed in a well with a rounded bottom and an internal diameter of 9 mm in an assay cartridge (as described in the previous examples) containing 200 µl of an aqueous solution of conjugated boric acid.

The conjugate solution contained 0.25 mM xylene cyanol conjugated with boric acid (example 18 in US-A-5631364), 0.07 wt% Triton X-100, 9 mM zinc chloride and 100 mM HEPES buffer at pH 8.15.

The blood sample was pumped into the well and mixed with the boric acid conjugate solution by pumping the solution into and out of the conical container three times. The capillary was removed and the contents of the well were left for two minutes for incubation. This allowed the detergent to break down the blood cells, precipitate the hemoglobin with zinc and bind the conjugated boric acid to glycohemoglobin.

The membrane-tipped pipette was then placed into the well and a pressure less than surrounding pressure was created, causing fluid to flow from the well into the pipette and depositing hemoglobin on the membrane. In this test, the membrane was a porous filter having a pore size of 1 µm.

The pipette was removed from the well and placed in a second well of the same configuration containing 200 µL of aqueous wash (50 mM morpholine buffer pH 9.5, containing 200 mM NaCl, 0.5 wt% Triton X-100, 0.1 % glycerin by weight and 0.05% by weight NaN3 The pipette was pressurized to below ambient pressure, causing the flushing reagent and unbound conjugated boric acid to be drawn into the pipette.

The pipette was then removed and inserted into an empty 9 mm internal diameter flat bottom reading cavity for reflectometric measurement of the hemoglobin retained by the pipette membrane. Total hemoglobin was measured using 460 nm blue light and glycohemoglobin was measured using 620 nm red light (e.g. using red and blue LEDs). The ratio of glycohemoglobin to total hemoglobin (sometimes called% Hb1Ac) was determined by calculating the ratio of measured reflected light intensities, calibrated against samples with known% Hb1Ac.

Figure 8 in the accompanying figures shows the test results for this example for 6 blood samples where the% Hb1Ac content was analyzed 24 hours previously by HPLC (Variant, by BioRad).

Example 4

Capacity of fluid collection by membrane-tipped pipettes

The collection efficiency of wells of various configurations was checked for a pipette tipped with a flat nitrocellulose membrane as described in Example 1 against a standard open-tip conical pipette.

In any case, 200 µl of fluid should be drawn from a recess with a flat or rounded bottom and an internal diameter of 9 mm in a soft or hard plastic base (LDPE and polystyrene, respectively). The results are shown in Table 1 below.

Table 1

<td rowspan="2">The recess</td><td colspan="2">Part of the fluid taken (%)</td>

<td>Pipette with an open tip</td><td>Pipette tipped with a membrane</td>

<td>Soft, round</td><td> 98,9</td><td> 99,8</td>

<td>Hard, round</td><td> 99,5</td><td> 99,7</td>

<td>Hard, flat</td><td> 84,0</td><td> 99,5</td>

Example 5

Blood coagulation time test

The pipette of Figure 5 was used to sample approximately 2 µl of blood. The cartridge was then reassembled and pressure was applied to the pipette to discharge the blood sample into the pipette

The wells of the cassette, the bottom of which has been coated with a coagulation-enhancing agent (e.g., tissue factor). A pressure below ambient was then applied to draw the sample back into the pipette, through the chamber, into the sinusoidal capillary. The sample was then passed back and forth in a sinusoidal capillary by creating a pressure higher and lower than the surrounding pressure and, using a digital camera, the time from the contact of the blood sample with the coagulation accelerator to the effective cessation of movement of the blood sample was measured. This usually takes around 40 seconds.

Example 6

Whole blood or plasma coagulation time test

An assay cartridge of the type shown in Fig. 11 was used. One of the wells 59 to 62 contained dry tissue factor and calcium chloride or calcium gluconate and a steel ball, e.g., 2 mm in diameter (see Fig. 19a).

The device into which the cassette was inserted was equipped with a heating element to maintain the temperature of the cassette contents at about 37 ° C and a magnet for moving the steel ball along the bottom of the cavity in which it was placed.

A capillary-tipped removable pipette is placed in the cavity 57 and can withdraw a predetermined volume of sample, e.g., 1 to 15 g, preferably 10 g, whole blood, venous blood with citric acid, plasma or plasma with citric acid.

The sample was taken by a capillary-tipped pipette and placed with it in a cartridge that was installed in the analyzer. The sample was then transferred to the well containing the steel ball and mixed.

The cassette was then moved against the magnet in a horizontal direction, parallel to the tip of the recess containing the ball (the cassette as a whole or the magnet may be moved, but preferably the cassette is moved and the magnet initially serves to hold the steel ball stationary).

A digital camera was used to monitor the position of a steel ball. When the mixture started to coagulate, the ball ceased to be stationary against the magnet, which was detected by the camera, making it possible to determine the clotting time (from the contact of the sample with the calcium salt solution).

In an alternative, less preferred embodiment, the magnet under the cassette has been removed and the ball is placed in a recess with a sloped bottom (e.g., as shown in Fig. 19b). Rapid movement of the cartridge towards the lower end of the bottom, e.g. by mechanical impact or by actuation of an electromagnet from the side of the cavity, caused the ball to move up the sloped bottom and, before solidification occurred, the ball returned to the lower end of the bottom by gravity.

Example 7

Coagulation time test for whole blood or plasma

The assay cartridge as in Example 6 was used with a low density polymer ball (e.g., a polystyrene ball 3-5 mm in diameter) instead of a steel ball. The ball is preferably placed in a recess with a flat or concave bottom and a circular cross section (see Fig. 19c).

The sample was taken and mixed as in Example 6, then placed in the well containing the ball, the ball began to float on the surface of the sample. The ball was then repeatedly pushed beneath the sample surface and released to allow it to return to the surface. When the sample coagulated, the ball returned to the surface much more slowly and then did not return at all.

The ball may be pushed beneath the surface by pressing with the tip of a pipette, or alternatively a magnetically movable ball may be used and the magnetic field may be turned on and off to pull the ball down and release it, respectively. Such magnetic field responsive spheres can be prepared, for example, by embedding superparamagnetic crystals in a polymeric ball (e.g. as in magnetic beads sold by Dynal Biotech of Oslo, Norway).

Example 8

Plasma clotting time test

An assay cartridge similar to that shown in Figure 11 was used. As in Example 6, one of the wells 59 to 62 contains citrate buffer, the other contains fibrinogen and coagulation factor V, and the third contains a calcium salt solution. Recess 57 includes a capillary-tipped pipette and recess 58 includes a filter extension as shown in Fig. 18.

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The sample was taken into a capillary-tipped pipette which was then placed in the well 57, and the cartridge was installed in the analyzer and then heated to 37 ° C. The sample was then transferred to a well containing buffer and mixed. All or a predetermined portion of the mixture was then transferred to a filter pipette extension and the cell free diluted plasma was pumped to the bottom of the well. A predetermined volume of cell free plasma was then transferred to the fibrinogen-containing well using another capillary-tipped pipette, the other pipette also being used to transfer a predetermined volume of calcium salt solution to the fibrinogen / plasma well to initiate a clotting reaction. The well was illuminated and a digital camera was used to record the turbidity of the mixture in the well. The time from adding calcium to increasing the turbidity to a predetermined level is taken as the clotting time.

Example 9

A whole blood or plasma coagulation test

An assay cartridge similar to that shown in Figure 11 and described in Example 8 was used. As in Example 8, one of the wells 59 to 62 contained citrate buffer and the other contained calcium salt solution, however the well containing the bead was omitted and instead of the reagent, V coagulation and fibrinogen, the reagent well contained a dry specific thrombin-based chromogenic substance (e.g. Nycotest Chrom (described in Janson et al. Thrombostasis and Haemostasis 62: 530 (poster 1677) (1989) and Jonker et al. Research in Clinic and Laboratory 20: 45-57 (1990)) or one of the chromogenic substances discussed in DE-A- 3113350, DE-A-3413311, DE-A-3311287, US-A-4458015 or US-A-4784944).

A sample was taken and mixed analogously to the procedure described in Example 7. The coagulation process induced thrombin formation and thus the release of the dye from the chromogenic substance (e.g. yellow para-nitroaniline from Nycotest Chrom).

The color change of the sample was observed using a digital camera and the clotting time was calculated as the time from calcium addition to the predetermined color change.

Example 10

C-reactive protein test (CRP in whole blood, conjugated enzyme test (ELISA)

Using a capillary-tipped pipette from the cartridge, 1 μL of whole blood was added to a well (e.g., well 59) of a cartridge, similar to that shown in Figure 11 and containing 200 μL of a dilution and decomposition fluid (30 mM borate buffer pH 8.0 containing 0.01 wt% sodium citrate, 0.02 wt% NaN3 and deoxycholate). The cassette recesses had a rectangular cross-section with internal dimensions of 6.0 by 6.5 mm. The flat bottom of the well was inclined at a 30 degree angle to the longitudinal axis of the well.

The diaphragm-tipped rectangular pipette (which had external dimensions of 3.7 by 4.2 mm and was equipped with a nitrocellulose membrane coated with an anti-CRP antibody mounted at an angle of 30 degrees to the longitudinal axis of the membrane tube) was lowered into the cavity and the solution with degraded blood cells was absorbed by the membrane by creating a pressure lower than that of the interior of the membrane-tipped pipette. When all the fluid was absorbed, a pressure higher than surrounding pressure was created to force the fluid back through the membrane and back into the cavity. Running the CRP solution twice through the membrane increases the CRP capture efficiency.

The membrane-tipped pipette was then transferred to a similar well (e.g., well 60) in a cartridge that contained an alkaline phosphatase (ALP) solution conjugated with an anti-CRP antibody (approximately 40 μg / ml ALP and 40 μg / ml antibody in 50 mM borate buffer) pH 8.0 containing 0.02 wt.% NaN3 and 0.5 wt.% BSA). Conjugated phosphatase solution was absorbed by the membrane and pumped back into the well by creating a downstream and upstream pressure inside the membrane-tipped pipette as described above for antigen capture.

In the next step, the membrane-tipped pipette was transferred to the next well (e.g. well 61) in the cassette which contained 200 μL of washing solution (50 mM borate buffer pH 8.0 containing 0.01 wt% NaN3, 0.5 wt%). BSA and deoxycholate) that was absorbed and then pumped back into the cavity. The washing step was repeated twice by transferring the membrane-tipped pipette to two additional wells (not shown in Fig. 11, but similar to well 61) which also contained the washing solution. Three washing cycles ensured efficient removal of unbound conjugated phosphatase.

Finally, the membrane-tipped pipette was transferred to yet another well (e.g., well 62) in the cassette that contained 300 μl of para-nitrophenyl phosphate alkaline phosphatase substrate (1.0 mg / ml pNPP in 1.0 M diethanolamine buffer). pH 9.6 containing 0.5 mM MgCl2 and 0.025 wt% NaN3). The yellow enzyme, the para-nitrophenol product, was prepared by pumping a substrate solution into and out of a membrane-tipped pipette over a two minute period. Incubation was terminated by pumping all fluid back into the well and removing the membrane-tipped pipette from the substrate solution. With 300 μl of substrate solution, the fill height was approximately 3 mm above the curved portion of the well, which allows color analysis through the parallel walls of the well.

With the membrane tipped pipette removed, the absorbance was measured using a blue LED as light source and a digital camera to measure the transmitted light.

Example 11

Whole blood C-reactive protein (CRP) assay using scattered light measurement on aggregated latex beads

Using a capillary-tipped pipette from a cartridge, 2 μl of whole blood was added to a well (e.g., well 62) of a cartridge similar to that shown in Figure 11 and containing latex beads 120 nm in diameter (0.2 wt%) suspended in 300 μl of 50 mM borate buffer, pH 8.0, containing 0.01% by weight of sodium citrate, 0.02% by weight of NaN3 and deoxycholate. The beads were coated by simple adsorption with anti-CRP antibodies. The recess had a rectangular cross-section and was at the end of the cassette to facilitate light scattering measurement. The light was directed to one side of the wall of the cavity. After the initial phase of cell decomposition, which lasted about 10 seconds, the increase in light scattering at an angle of 90 degrees to the incident light was measured. The increase in light scattering due to the aggregation of the latex beads under the influence of CRP was measured by a digital camera at a wavelength of 425 nm.

Example 12

Urine albumin test using magnetic beads, colored latex beads and reflectometry

Using a capillary-tipped pipette from the cartridge, 2 μL of urine was added to a well (e.g., well 62) of the cartridge, similar to that shown in Figure 11 and containing 1000 nm magnetic polymer beads (0.2 wt%) and 1000 nm blue latex beads. (0.2 wt%) in 200 μl of 30 mM sodium phosphate buffer, pH 5.7 containing 0.5 wt% BSA and 0.05 wt% NaN3. Magnetic beads (e.g. of the type available from Dynal Biotech of Oslo, Norway) were coated with an antibody that reacts with an antigenic determinant on the albumin molecule other than the antigenic determinant recognized by the antibody covering the latex beads.

After incubating for 60 seconds, the neodymium magnet (10 x 7 x 2 mm) was moved from its rest position (20 mm from the nearest wall of the well) towards the well, bringing the magnet into direct contact with the side of the well. The magnet was in contact with the wall opposite to the sloped side and encircled the fluid-filled portion of the cavity (200 µ. The cavity and the position of the magnet are shown schematically in Fig. 20. In the resting position, the magnetic field acting on the magnetic beads was too weak to move them. In contact with the cavity, the distance from the magnet to the nearest and farthest walls of the cavity was 0.8 mm and 6.3 mm, respectively. At this distance, the beads were collected quantitatively on the wall after 30 seconds. In the presence of the analyte, the blue latex was bonded to the magnetic particles and some of the reacted latex beads were collected on the wall, while the remaining unreacted latex particles remained in the suspension.

Leaving the magnet in contact with the wall of the well, a capillary-tipped pipette was used to withdraw the fluid containing unreacted latex particles. The magnet was then moved away from the recess to its rest position.

The capillary-tipped pipette was then transferred to an empty cavity (e.g., cavity 61) and fluid was introduced into the cavity by creating a pressure higher than surrounding pressure inside the pipette.

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The capillary-tipped pipette was then transferred to the next well (e.g., well 60) which contained 500 µL of washing solution (PBS, pH 7.4) and 200 µL of the solution was withdrawn. The capillary-tipped pipette was then returned to the well containing the magnetic beads and the beads were suspended by pumping the wash solution into and out of the well five times. The magnet was then moved to the position where it was in contact with the wall, and the magnetic beads were collected on the wall of the cavity. After 30 seconds, the washing solution was collected through a capillary-tipped pipette. The magnet was then moved back to its rest position.

The capillary-tipped pipette was in the next step transferred to the well containing the first supernatant (well 61) and its contents pumped out into this well.

The capillary-tipped pipette was then transferred to the well containing the washing solution (well 60) and 200 µl of the solution was withdrawn.

The capillary-tipped pipette was transferred to a cavity containing the magnetic beads (cavity 62) and the beads were resuspended by pumping the washing solution into and out of the cavity five times.

The membrane-tipped pipette, equipped with a 0.45 µm microporous membrane, was transferred to a well containing a magnetic bead slurry (well 62) and the beads were collected on the membrane by creating a suction.

The membrane-tipped pipette was removed from cavity 62 and the amount of blue latex particles and yellow-brown magnetic beads was estimated by reflectometry, using a red light emitting diode for the blue latex beads and a blue light emitting diode for the magnetic beads. The ratio of the amount of absorbed red light to the amount of absorbed blue light was a measure of the proportion of blue latex in the mixture and thus a measure of the albumin content of the sample.

The same cassette can also be used to determine the creatinine content of the urine and therefore also the ratio of albumin to creatinine in the urine sample. The albumin content of the urine indicates that the kidneys are working properly, and the ratio of albumin to creatinine can be used to correct the effect of diuresis. The measurement of the ratio of albumin to creatinine is described, for example, in US-A-5385847.

In this embodiment, a portion of the urine sample was mixed with a dilution reagent and an enzyme or enzyme mixture that reacts with creatinine to produce a colored analyte that was detected using a digital camera by measuring light transmission through a well containing urine, enzymes and the dilution reagent.