Apparatuses and methods for manipulating droplets on a printed circuit board

p00001Apparatus and methods for handling droplets on a printed circuit board.

Related Requests

p00002This application is related to U.S. Patent Application Serial No. 10 / 253,342, filed on September 24, 2002 and U.S. Provisional Patent Application Serial No. 60 / 648,051, filed on January 28, 2005.

p00003Technical field

p00004The material disclosed herein refers, in general, to apparatus and methods for performing micromanipulation of droplets on a printed circuit board (PCB) substrate. More particularly, the matter disclosed herein refers to apparatus and methods for manufacturing and operating microfluidic systems based on droplets based on conventional PCB technology in which the droplets move on the surface of the PCB by applying potentials. electrical to electrodes defined on the PCB. The material disclosed herein also describes the use of a welding mask as an electrode insulator for the handling of droplets, as well as techniques for adapting other layers and traditional PCB materials for microfluidic based droplets.

Background

p00005Microfluidics is a rapidly advancing field that deals with the study of fluids with a volume below the microliter. Microfluidic devices are increasingly applied and are more accepted in many fields of biology, chemistry, medicine, environmental monitoring, drug discovery and electronic components for the consumer. Miniaturization of traditional devices, particularly analytical devices, is expected to lead to many benefits including reduced consumption (and cost) of reagents and samples, higher performance and automation, faster analysis times and more reliable, economical and portable instrumentation . Since more functionality is included in these devices, the analysis microsystems Total (TAS) fully integrated or "laboratories on a chip" are becoming a reality and are increasingly important. The laboratory on a chip is an emerging paradigm that aims to miniaturize and integrate fluid management into a chip. A laboratory on a chip must allow dispensing, transport, mixing, incubation, detection / separation of fluid and waste disposal to be a true autonomous unit. Laboratory systems on a microfluidic chip can be broadly classified into continuous flow and discrete flow systems. A continuous flow system is self-descriptive and in discrete flow systems the fluid separates into droplets. A common limitation of continuous flow systems is that fluid transport is physically confined to fixed channels while droplet (or discrete flow) based systems can be confined to physical channels or operate in flat systems without channels. The transport mechanisms generally used in continuous flow systems are pressure driven by external pumps or electrokinetically driven by high voltages. Continuous flow systems can involve complex channeling and require large support instruments in the form of valves or external power supplies. In another approach to channel-based systems, centrifugal forces propel the fluids to flow unidirectionally in the channels. The microfluidic continuous flow paradigm has limitations of versatility, making it difficult to achieve high degrees of integration and functional control.

p00006Discrete or droplet-based microfluidic systems have been progressing steadily to fulfill the promise of the laboratory concept on a chip to handle all stages of analysis, including sample extraction, sample preparation, sample processing including transport, mixing and incubation, detection and waste management. These stages have been designed to be performed on a chip without significant support systems outside the chip. Several discrete flow approaches have recently been developed to manipulate droplets based on multilayer soft lithography, hydrodynamic multiphase flows, continuous electrowetting technique, dielectric electrowetting (EWOD), dielectrophoresis, electrostatics and acoustic surface waves. Some of the above techniques manipulate droplets or ingots in physically confined channels while other techniques allow the handling of droplets on flat surfaces without physically channels

p00007defined some. Droplet-based approaches without channels have been referred to as "digital microfluidics," given

p00008that the liquid is separated and manipulated programmatically.

p00009Droplet-based protocols are very similar to laboratory-scale biometric protocols that also generally run in discrete volumes of fluids. Therefore, established protocols can be easily adapted to the digital microfluidic format. Some of the distinctive features of digital microfluidic systems include: reconfigurability (the operations and trajectories of the droplets are selected through a software control panel to allow users to create any combination of microfluidic operations on the fly); Software programmability also results in design flexibility where a generic microfluidic processor chip can be designed and reprogrammed for different applications; conditional execution stages can be implemented, since each microfluidic operation can be performed under direct control of a computer to allow maximum operational flexibility; multidirectional droplet transport since channels only exist in virtual sense and can be reconfigured instantly through the software; small volumes of droplet (<1 l); fully electronic operation without using external pumps or valves; simultaneous and independent control of many droplets; and operation without channels (where priming is not required).

p00010Many current laboratory technologies on a chip (including both continuous flow and discrete flow devices) are relatively inflexible and are designed to perform only a single test or a small set of very similar tests. Due to the fixed provisions of current microfluidic chips, a new chip design is required for each application, which makes it expensive to develop new applications. In addition, many of these devices are manufactured using expensive microfabrication techniques derived from the manufacture of integrated semiconductor circuits. As a result, applications for microfluidic devices expand relatively slowly due to the cost and effort required to develop new devices for each specific application. Although batch manufacturing allows microfabricated devices to be economical when mass produced, the development of new devices can be prohibitively expensive and time-consuming due to high prototype development costs and the long response time associated with the techniques. of conventional semiconductor microfabrication. To expand the range of applications and the impact of microfluidics in medicine, drug discovery, environmental and food monitoring, and other areas including electronic components for the consumer, there has been a long-standing need for both microfluidic approaches that provide more reconfigurable and flexible integrated devices, as well as techniques to develop and manufacture these chips more economically and quickly.

p00011In the last several years there have been advances using different approaches to microfluidics based on the manipulation of individual nanoliter size droplets by direct electrical control. Examples of such systems can be found in U.S. Patent No. 6,911,132 and U.S. Patent Application Publication No. 2004/0058450, both of Pamula et al., (And usually assigned to the Assignee of the present subject). .

p00012Document US-A-2004/0055891 also discloses an apparatus for handling droplets comprising a single-sided electrode design in which all conductive elements are contained on a surface on which the droplets are handled, although a surface can be provided additional parallel to the first surface to contain the droplets to be handled. The droplets are manipulated by enhancing electrowetting-based techniques, in which electrodes contained in or embedded in the first surface are supplied and sequentially deprived of energy in a controlled manner.

p00013Paik et al., In an article entitled "Thermal Effects on droplet transport in digital microfluidics with applications to chip cooling". Ninth Intersociety Conference on Thermal and Thermochemical Phenomena in Electronic Systems, 2004 (ITHERM '04), Las Vegas, NV, United States, 1-4 June 2004, Piscataway, NJ, United States, IEEE, 2004, pages 649-654 , reveal a cooling technique in which discrete liquid droplets with a size of one nanoliter immersed in oil are handled on a series of electrodes by electrowetting, and describes the effect of temperature dependent system parameters on the transport of the droplets.

p00014These various techniques offer many advantages in the implementation of the digital microfluidic paradigm, as described above, but current manufacturing techniques to produce these microfluidic chips continue to depend on quite complex and expensive manufacturing techniques. These microfluidic chips are currently produced in microfabrication smelters using costly processing steps based on semiconductor processing techniques routinely used in the integrated circuit manufacturing (IC) industry. In addition to the higher cost of semiconductor manufacturing techniques, semiconductor smelters are not easily accessible and typically do not offer manufacturing response times or prototype development as fast as 24 hours.

p00015Microfluidic chips are generally manufactured using custom processes based on traditional semiconductor microfabrication procedures. The devices are manufactured on glass substrates by repeated stages of thin film deposition and modeling using conventional photolithographic techniques. Typically, at least two layers of metal (one for electrodes and one for wiring) are required in addition to two or three insulating layers, as well as layers to form the separator between the upper and lower plates. Due to the high cost of photo mask manufacturing and chip manufacturing, a single prototype development run that produces up to 100 devices can cost up to $ 10,000 and take three months to complete depending on the number of photolithographic levels. In addition, since the process flow is not standardized, the device yields tend to be very low during the first several attempts to manufacture any new design.

p00016The expenses and time required for the development of prototypes have been a serious impediment to the development and optimization of droplet-based microfluidics. In addition, high chip costs and the inability to quickly customize or improve device designs are expected to reduce the commercial prospects of this versatile technology. In the short term, faster, more reliable and low-cost manufacturing technology is required to accelerate the development and acceptance by the user of these devices. Since microfluidic structures tend to be relatively large and rarely test the limits of semiconductor manufacturing techniques, batch manufacturing methods of lower resolution and lower cost should be considered.

p00017In particular, printed circuit board (PCB) technology offers many capabilities and materials similar to traditional semiconductor manufacturing although at a much lower resolution. The layers of conductors and insulators are deposited and modeled photolithographically and stacked together to create intricate multilevel structures. For the manufacture of digital microfluidic systems, PCB technology is believed to offer an excellent compromise in terms of resolution, availability, cost and ease of manufacturing. It is also believed that an additional advantage of using a PCB as a substrate is that electronic devices for detecting, controlling or analyzing the device can be easily integrated at a very low cost.

p00018Typically, the width of the copper tube and the separation of the tube are measured in thousandths of an inch (25.4 m) in a PCB process, which is several orders of magnitude greater than the submicron characteristics generally achieved in a semiconductor manufacturing. Typically, PCB processing does not require an expensive ultra-clean environment such as that required for the manufacture of a semiconductor IC. The plates are generally made, in addition, of reinforced plastic, epoxy with fiberglass, TEFLON®, polyimide, etc., in comparison with silicon or glass that are used as substrates for microfluidic microfabricated devices in the manufacture of semiconductors. In addition, instead of a semiconductor mask aligner, alignment can usually be done manually for PCB processing. Economical masks made of transparencies or sheets of MYLAR are used instead of the expensive chromium on glass photomasks used in the manufacture of semiconductors. In PCB processing, transit gaps (via holes) are drilled mechanically or with a laser and then electroplated instead of etched and steam deposition used in semiconductor processing that needs vacuum processing. Generally multiple wiring layers are obtained are generally obtained by joining single plates individually modeled together in opposition to the use of a single substrate and the accumulation of multiple layers or joining of disks in the manufacture of a semiconductor. Broadly, these are the main differences between a PCB manufacturing process and a semiconductor manufacturing process, even though high quality PCB processes move towards the adoption of some of the semiconductor processes (such as physical deposition steam).

p00019In today's highly competitive business environment, it is imperative that products reach the market quickly and economically, particularly in consumer electronics and medical diagnostic companies. The present subject relates to the use of manufacturing techniques for printed circuit boards (PCBs) that are widely available, reliable, economical and well defined. By manufacturing reconfigurable microfluidic platforms with reliable, easily accessible and low-cost manufacturing technology, the development and acceptance of laboratory devices on a chip for many potential applications in biomedicine and in other areas will be more widespread and will be faster.

p00020The attractiveness of PCB technology as an economical, well established, flexible and easily accessible manufacturing process for the development of microfluidic systems has already been recognized by researchers working with more traditional continuous flow microfluidic systems. For example, researchers have previously demonstrated a series of microfluidic continuous flow devices based on PCB technology including a bubble detector, a pH regulation system, a micropump and a capacitive pressure sensor. More recently, PCB devices for manipulation and analysis of single cells by dielectrophoresis have also been described, since they have hybrid approaches in which a PCB is used to monolithically integrate silicon-based microfluidic devices. However, there remains a long-standing need for an economical, flexible and reconfigurable system for discrete flow droplet handling.

Summary

p00021The present invention is defined by independent claim 1.

p00022The apparatus and methods, as disclosed herein, comprise a new way of using a substrate manufactured in a conventional printed circuit board (PCB) process for handling digital microfluidic droplets. This unconventional use of well-established PCB processes incorporates several new aspects including: (1) the use of copper tracks and solder points (PCBs) as electrodes for the handling of droplets in which fluids in the form of droplets they are routed (ie transported) to the external surfaces of a PCB substrate by electrical means (as opposed to the placement of electronic components and the routing of electronic signals); (2) the use of welding mask material as a dielectric electrode to act as an insulator for droplet manipulation induced by electric field (as opposed to the use of welding mask, as the name implies, to protect copper wires of welding); (3) the use of photosensitive liquid or dry welding mask to manufacture physical structures to contain fluids; (4) the use of transit gaps in the PCB to electrically connect droplet control electrodes to each other or to contact welding points; (5) the use of transit gaps filled with inner welding mask with respect to the electrodes on the PCB to allow close filling of droplet control electrodes; (6) the filling of interior transit gaps with respect to electrodes with conductive epoxies to allow close filling of electrodes without loss of electrode conductive area; (7) filling of interior transit gaps with respect to electrodes with optically transparent epoxies for optical measurements through the droplet; (8) the use of copper tracks adjacent to the electrodes, on the same surface of the PCB to provide a common reference potential (coplanar arrangement); (9) the use of copper embedded in the control electrodes and at the top of the dielectric layer to serve as reference electrodes; (10) the use of perforations in the PCB to establish a fluidic interface to allow the entry and exit of fluids in / out of the PCB; and (11) the use of copper pipes as heating elements.

p00023Therefore, in accordance with the present invention, an apparatus for handling droplets is provided, the apparatus comprising:

<dl><dt>(to)</dt><dd> a printed circuit board substrate comprising a first lateral surface and a second lateral surface; </dd></dl>

<dl><dt>(b)</dt><dd> a series of electrodes arranged on the first lateral surface of the substrate; </dd></dl>

<dl><dt>(c)</dt><dd> a dielectric layer disposed on the first lateral surface of the substrate and modeled to cover the electrodes; and</dd></dl>

<dl><dt>(d)</dt><dd> an electrode selector to dynamically create an activation sequence of the series electrodes, whereby a droplet disposed on the first lateral surface of the substrate is electrically manipulated, </dd></dl>

p00024wherein the substrate of the printed circuit board comprises a plurality of substrate openings defined therein and extending from the first lateral surface of the substrate to the second lateral surface of the substrate and each electrode comprises an electrode opening, in which each electrode opening is aligned with one of the plurality of substrate openings to define a plurality of transit gaps through the apparatus.

p00025In another embodiment, an apparatus for handling droplets is provided that includes a printed circuit board substrate comprising a first lateral surface and a second lateral surface. A series of driving electrodes is disposed on the first lateral surface of the substrate and a series of one or more reference elements adjustable to a common reference potential is arranged in at least substantially co-planar with the series of driving electrodes. A layer of dielectric substance is disposed on the first lateral surface of the substrate and is modeled to cover the driving electrodes. An electrode selector is also included to dynamically create an activation sequence of the series electrodes, whereby a droplet disposed on the first lateral surface of the substrate is electrically manipulated.

p00026In yet another embodiment, an apparatus for handling droplets is provided that includes a printed circuit board substrate comprising a first lateral surface and a second lateral surface. A series of driving electrodes is disposed on the first lateral surface of the substrate and an elongated reference element is provided that is substantially parallel and is separated from the first lateral surface of the substrate a distance to define a space between the reference element and the first lateral surface of the substrate, in which the distance is sufficient to contain a droplet arranged in space. A layer of dielectric substance is disposed on the first lateral surface of the substrate and is modeled to cover the driving electrodes. An electrode selector is also included to dynamically create an activation sequence of the series electrodes, whereby a droplet disposed on the first lateral surface of the substrate is electrically manipulated.

p00027In a further embodiment, an apparatus for handling droplets is provided that includes a first printed circuit board substrate comprising a first side surface and a second side surface, a series of driving electrodes disposed on the first side surface of the first plate substrate printed circuit, and a layer of dielectric substance disposed on the first lateral surface of the first printed circuit board substrate and modeled to cover the driving electrodes. The apparatus also includes a second printed circuit board substrate comprising a first lateral surface and a second lateral surface, the second printed circuit board substrate being substantially parallel to and separated from the first printed circuit board substrate a distance to define a space between the second side surface of the second printed circuit board substrate and the first side surface of the first substrate of printed circuit board, in which the distance is sufficient to contain a droplet arranged in space. A series of driving electrodes and a series of one or more reference elements are arranged on the second side surface of the second printed circuit board substrate. The apparatus also includes an electrode selector to dynamically create an activation sequence of the series electrodes, whereby a droplet disposed between the first lateral surface of the first printed circuit board substrate and the second lateral surface of the second Printed circuit board substrate is electrically manipulated.

p00028Methods for moving a droplet in relation to the apparatus of the present invention are also provided.

p00029In one case, a method of moving a droplet includes the step of providing a droplet on a surface of a printed circuit board substrate. The surface comprises a series of electrodes and the droplet is initially disposed on a first of the electrodes and is adjacent to a second of the electrodes separated from the first electrode by a first space. The method also includes the step of polarizing the first electrode at a first voltage and the second electrode at a second voltage different from the first voltage, whereby the drop moves towards the second electrode.

p00030In another case, a method of moving a droplet includes the step of providing a droplet on a surface of a printed circuit board substrate. The surface comprises a series of driving electrodes and at least substantially coplanar series of one or more reference elements and the droplet is disposed on a first of the driving electrodes. The method further includes polarizing the first driving electrode to move the droplet from the first driving electrode to a second driving electrode.

p00031In a further case, a method of moving to a droplet includes the step of providing a droplet between a surface of a printed circuit board substrate and an elongated reference element substantially parallel to and separated from the surface of the printed circuit board. The surface of the printed circuit board comprises a series of driving electrodes and the droplet is disposed on a first of the driving electrodes. The method further includes polarizing the first driving electrode to move the droplet from the first driving electrode to a second driving electrode.

p00032In a further additional case, a method of moving to a droplet includes the step of providing a droplet between a surface of a first printed circuit board substrate and a surface of a second printed circuit board substrate substantially parallel to and separated from the Printed circuit board. The first surface of the printed circuit board comprises a series of driving electrodes and the droplet is disposed on a first of the driving electrodes, and the second surface of the printed circuit board comprises a series of driving electrodes and a series of one or more reference elements. The method further includes the step of polarizing the first driving electrode to move the droplet from the first driving electrode to a second driving electrode.

p00033In another case, a method for moving a droplet in a series of driving electrodes on the surface of a printed circuit board without some physically distinct reference elements, requires that the droplet be arranged on a first of the driving electrodes and move towards a second or third drive electrode. The method also includes polarizing the second and third driving electrodes, so that the droplet will be driven to or away from any of the second or third electrodes due to non-homogeneity in the electric field applied between them. In this case, the droplet cannot move gradually, that is, moving from an electrode to its adjacent electrode, but instead the droplet can move continuously in the non-homogeneous electric field gradient towards or away from the electrode of destination using a phenomenon known as dielectrophoresis.

p00034Methods are also provided to fuse two or more droplets into a droplet and divide a droplet into two or more droplets.

p00035In one case, a method of fusing two or more droplets into a droplet includes the step of providing first and second droplets on a surface of a printed circuit board substrate. The surface comprises a series of electrodes in which the electrode series comprises at least three electrodes comprising a first external electrode, a middle electrode adjacent to the first external electrode and a second external electrode adjacent to the middle electrode. The first droplet is disposed on the first external electrode and is adjacent to the middle electrode, and the second droplet is disposed on the second external electrode and is adjacent to the middle electrode. The method also includes selecting one of the three electrodes as the target electrode and selecting two or more of the three electrodes for activation and deactivation based on the selection of the destination electrode. The method also includes activating and deactivating the selected electrodes to move one of the first and second droplets towards the other droplet or both of the first and second droplets towards each other, whereby the first and second droplets are fused together to form a combined droplet on the target electrode.

p00036In another case, a method of dividing a droplet into two or more droplets includes the step of providing a starting droplet on a surface of a printed circuit board substrate. The surface comprises a series of electrodes, in which the electrode series comprises at least three electrodes comprising a first external electrode, a middle electrode adjacent to the first external electrode and a second external electrode adjacent to the middle electrode. The starting droplet is initially disposed on at least one of the three electrodes and is adjacent to at least one of the three electrodes. The method also includes polarizing each of the three electrodes at a first voltage to place the starting droplet across the three electrodes. The method further includes polarizing the middle electrode to a second voltage different from the first voltage to divide the starting droplet into first and second divided droplets, whereby the first divided droplet is formed on the first external electrode and the second divided droplet is form on the second external electrode.

p00037In another case, a method of dividing a droplet into two or more droplets includes the step of providing a starting droplet on a surface of a printed circuit board substrate. The surface comprises a series of electrodes, in which the series of electrodes comprises at least three electrodes comprising a first external electrode, a middle electrode adjacent to the first external electrode, and a second external electrode adjacent to the middle electrode. The starting droplet is initially arranged on at least one of the three electrodes and at least partially overlaps with at least one of the three electrodes. The method also includes polarizing the middle electrode at a first voltage to place the starting droplet, whereby the starting droplet overlaps at least partially with the three electrodes. The method also includes polarizing the middle electrode at a second voltage and at least one of the first and second external electrodes at a third voltage, the second and third voltages different from the first voltage, to divide the initial droplet into first and second droplets. divided, whereby the first divided droplet is formed on the first external electrode and the second divided droplet is formed on the second external electrode.

p00038It is, therefore, an object of the present invention to provide apparatus for manufacturing and operating microfluidic systems based on droplets based on conventional printed circuit board (PCB) technology in which the droplets move toward the surface of the PCB by application of electrical potentials to electrodes defined on the PCB.

p00039Having previously indicated in this document an object of the matter disclosed in this document, and which is fully or partially addressed by the matter disclosed in this document, other objects will become apparent as the description progresses when taken together with the drawings attachments, as described in the best way below in this document.

p00040Brief description of the drawings

p00041Figure 1A is a top plan view and Figures 1B-1D are profile views of an embodiment of the present matter representing a strictly co-planar arrangement on a PCB with filled or unfilled transit gaps in the electrodes;

p00042Figure 2A is a top plan view and Figure 2B is a profile view of an embodiment of the present matter representing a substantially co-planar arrangement on a PCB with filled or unfilled transit gaps in the electrodes;

p00043Figure 3A is a top plan view and Figure 3B is a profile view of an embodiment of the present matter representing a co-planar arrangement embedded on a PCB with filled or unfilled transit gaps in the electrodes;

p00044Figure 4A is a top plan view and Figure 4B is a profile view of an embodiment of the present matter depicting a parallel or bi-planar plate arrangement on a PCB with full or unfilled transit gaps in the electrodes. ;

p00045Figure 5 is a perspective view of a droplet located on a dense series of electrodes with transit gaps on a PCB for handling droplets in accordance with the present subject matter (liquid deposits are not shown);

p00046Figure 6 is a photograph depicting the front part of a PCB chip used to test the transport performance of droplets of different shapes and sizes of driving electrodes in accordance with the present subject matter;

p00047Figures 7A-7D are photographs depicting various forms of electrode in accordance with the present subject,

p00048Figures 8A-8B are photographs of an embodiment of the present material representing front and rear views, respectively, of a PCB chip design featuring a three-phase droplet conveyor and other structures for dispensing, storing and mixing droplets;

p00049Figures 9A-9B are photographs of another embodiment of the present material depicting front and rear views, respectively, of a PCB chip design that presents a three-phase droplet conveyor and other structures for dispensing, storing and mixing droplets;

p00050Figure 10 is a graph depicting droplet transport characteristics (frequency versus threshold voltage) of 1.5 mm electrodes in different ways in accordance with the present matter;

p00051Figure 11 is a graph depicting droplet transport characteristics (frequency versus threshold voltage) of 1.0 mm electrodes in different ways in accordance with the present matter;

p00052Figure 12 is a graph depicting the voltage stability of the droplet transport over time in accordance with the present matter;

p00053Figure 13 is a graph depicting minimum voltage requirements for the transport of droplets at a given switching frequency in accordance with the present matter; and

p00054Figures 14A-14D are photographs representing images at time intervals demonstrating the transport and mixing of droplets in accordance with the present subject.

p00055Detailed description

p00056A printed circuit board (PCB), also sometimes called a printed wiring board (PCB), is a substrate used to interconnect electronic components using solder points and conductive tracks modeled on the surface. Typically, PCBs are manufactured by adhering a layer of copper over the entire substrate, sometimes on both sides (known as creating a "blank PCB") then removing unwanted copper (for example, etching on an acid) after of applying a temporary mask, leaving

p00057only the desired copper tracks. Electrical connections ("tracks") between opposite sides of the substrate can

p00058be formed by drilling holes through the substrate mechanically or with a laser and metallizing the inside of the perforation to provide a continuous electrical connection between the two sides. Multi-layer plates can be created by joining together individually processed substrates. The electrode lines in the copper layer are usually defined by engraving copper from a blank PCB in a subtractive process while some smelters use semi-additive and completely additive processes where copper is accumulated on the substrate by electroplating or other techniques

p00059As described above, digital microfluidics is a microfluidic approach in which discrete droplets of fluid are electrically manipulated on a substrate containing a series of electrodes. In a commonly used configuration, the droplets are sandwiched between two parallel plates where the upper plate physically contains the droplets and the lower plate contains a series of individually manageable driving or control electrodes (or elements) that are electrically isolated. Typically, one or more reference electrodes (or elements) are also required to control the electrical potential of the droplet. The reference electrodes may be provided on the same substrate as the driving electrodes (co-planar) or on an opposite plate (bi-planar). The space between the two plates surrounding the droplet is generally open and can be filled with air or with an immiscible liquid to prevent evaporation. Examples of immiscible liquids that can be used with aqueous droplets include silicone oil, fluorosilicone oil or hydrocarbon oils. When the reference electrodes and the driving electrodes are provided on the same substrate, the opposite plate does not serve as part of the electrical circuit, but only serves as a cover plate to physically contain the liquids and may not be required for operation. Of the device.

p00060The movement of the droplet is achieved by applying a potential between the reference electrode and one or more of the driving electrodes. The applied potential may be DC or AC and it is not necessary that the reference electrodes be physically different from the driving electrodes. A droplet adjacent to an activated control electrode will be attracted to that control electrode and will move towards it. Control electrodes can be activated sequentially using user-defined patterns (possibly using an electrode selector) to transport droplets along arbitrary paths defined by contiguous control electrodes. In addition to transportation, other operations including melting, splitting, mixing, deformation and dispensing of droplets can be performed based on the design of the control electrodes and activation patterns.

p00061A digital microfluidic processor consists essentially of a series of control electrodes with one or more reference electrodes. A complete chip can include many other types of structures including channels, liquid reservoirs, top plates, sensors, inputs, outputs, etc. The electrode series requires interconnections to electrically connect some electrodes together and to connect electrodes to contact welding points for connection to external circuits. Previously, digital microfluidic chips were manufactured on glass or silicon substrates using thin film deposition and photolithography techniques taken from semiconductor fabrication. Multiple levels of electrical interconnection for wiring were accumulated by depositing and modeling successive layers of conductors and insulators on a single starting substrate. The present subject relates to apparatus and methods with which digital microfluidic processors can be advantageously constructed in a conventional PCB process as opposed to a custom glass or silicon based process.

p00062The material disclosed in this document takes advantage of the ease with which multiple layers of conductors can be generated in a PCB process versus a process based on glass or silicon. This is essentially the case, since in the PCB processing the metal layers are manufactured on different substrates that are laminated together at the end instead of accumulating sequentially on a single substrate.

p00063The conductor wiring patterns are transferred to the PCB substrate by subtractive plating, panel plating, pattern plating or additive plating. Generally, two or more layers of wiring will be required for complex droplet handling operations that require the use of multi-layer plates. Multi-layer plates are assembled by joining several double-sided plates or by means of accumulation / sequential plates that do not require mechanical hole drilling (for example, transit gaps are chemically etched or laser drilled and then plated without electricity). By definition, double-sided plates have wiring on both sides of the plates that can be further classified into plates with no through hole metallization and plates with through hole metallization. Plates with through hole metallization are further classified into plated through hole metallization and full through hole metallization. In the metallization of plated through holes, the holes are metallized by copper plating (for example, electroplating or plating without electricity or a combination thereof) and in metallization of full through holes, the holes can be filled with conductive pastes such as copper plast , silver paste, conductive epoxy, etc.

p00064In digital microfluidic chips, through holes (or transit gaps) are drilled through the center of the driving electrodes on one side of a multilayer plate to establish electrical connections on the opposite side of the plate. The footprint of a droplet is defined by the area of ​​a driving electrode. To obtain small droplet volumes, it is necessary to minimize the area of ​​the driving electrodes. Since the transit gaps are drilled through the driving electrodes, it is important to minimize the diameter of the transit gaps including the diameter of the weld / ground point. Therefore, transit gaps play an important role in defining the minimum volumes of droplets obtainable in a PCB process. The PCB industry is reducing the size of the traffic gaps for a different reason, which is to avoid blocking the conduction channels of the cables and maximizing the surface area of ​​the PCB available for the tracks. Many accumulation processes use small pathways that are formed by drilling through using an excimer laser. There are a number of variations of the accumulation processes used in the PCB industry including, but not limited to, Superficial Laminar Circuits (SLC) where the tracks are photoformed; DYCOstrate ™ where the tracks are etched with dry plasma in parallel; Film Redistribution (FRL) layer where the outermost dielectric is photosensitive while the inner layers constitute a regular multi-layer plate; Flexible Conductive Adhesive United (Z-Link); Structural Accumulation System (IBSS) where the dielectric is photosensitive; Sequential Junction Cores / Internal Transit Lagoon of Any Layer (ALIVH) where a CO2 laser is used to drill the tracks and the holes are then filled with silver paste; Carrier Formed Circuits where different circuits are prepared on stainless steel carriers and then laminated in a prepreg FR-4; Accumulation of sheets with rollers where single-sided epoxy coated sheets are laminated by heat and pressure applied with rollers; and Sheet Accumulation that is similar to the accumulation of sheets with rollers but double-sided or multi-layer circuits are laminated. In an embodiment of the use of an accumulated plate (Z-Link) for digital microfluidic chips, multiple flexible plates consisting of polyimide reinforced copper sheets can be laminated together and then on a rigid plate to form a multi-layer plate. In this case, the holes in each flexible layer can be perforated, laser drilled or plasma perforated. The holes that interconnect various layers can then be filled with conductive adhesive.

GENERAL REALIZATIONS

p00066Referring to Figures 1A-1D, 2A-2B, 3A-3B, 4A-4B and 5, the requirements for adapting processed PCB substrates for droplet handling will be described in more detail below. As will be described in more detail below, Figures 1A-1D refer to a digital microfluidic PCB chip 10 that includes a strictly co-planar arrangement on a PCB with filled or unfilled transit gaps in the electrodes; Figures 2A-2B refer to a digital microfluidic PCB chip 20 that includes a substantially coplanar arrangement on a PCB with filled or unfilled transit gaps in the electrodes; Figures 3A-3B refer to a digital microfluidic PCB chip 30 that includes a co-planar arrangement embedded on a PCB with filled or unfilled transit gaps in the electrodes; Figures 4A-4B refer to a digital microfluidic chip of PCB 40 that includes a parallel or bi-planar plate arrangement on a PCB with filled or unfilled transit gaps in the electrodes; and Figure 5 represents a droplet located on a dense series of electrodes with transit gaps on a PCB for droplet handling. Figure 5 shows in a general way the concept of the present matter in which liquid samples are digitized in droplets or discrete droplets D which can then be dispensed, transported, incubated, detected or reacted with other droplets

p00067independently (the “digital microfluidic” approach).

p00068In each of the embodiments shown in Figures 1A-1D, 2A-2B, 3A-3B and 4A-4B, and as will be described individually in more detail below, a PCB substrate plate 12 is provided, having the plate a first upper lateral surface 13 and a second lower lateral surface 14. Impulse control electrodes (or elements) 16, such as copper track impeller electrodes, may be provided on the upper surface 13 of the PCB 12, and reference electrodes (or elements) 18, such as copper track reference electrodes or a parallel plate reference electrode, can also be provided in various configurations for droplet handling. Welding masks, such as a photosensitive liquid solder mask (LPI), are typically used in traditional PCB processes as an outer layer to protect copper tubes from the action of engraving or plating or welding agents during the placement of electronic components . However, in its usefulness for driving droplets in accordance with the present matter, this outer layer is an insulator 22 which serves to isolate the droplets from the potentials applied on driving and reference electrodes 16, 18. The driving electrodes 16 are completely isolated. by insulator 22, preferably an LPI welding mask or some other dielectric including temporary welding masks. Complete isolation means that the impeller electrode 16 is covered on all sides including the edges. Insulator 22 (LPI welding mask) is applied using conventional processes that include, but are not limited to, curtain coating, centrifugal coating, spray coating or screen coating. In the case where a reference electrode 18 is needed, some of the copper elements may be left bare and not insulated to provide a direct reference potential to the droplets.

p00069This exposed part is the closest to the driving electrodes 16 that allows the PCB process that is defined by the resolution of the copper elements as well as the resolution of the welding mask and the registration of the welding mask layer to the copper layer The exposed portion of the reference electrode 18 may have an optional conductive surface finish that usually includes immersion silver, immersion gold and nickel without electricity / immersion gold (ENIG).

p00070Substrate material

p00071As described above, the electrostatic microfluidic devices of the present matter include a substrate plate 12 that can be manufactured in almost any plate material that is commonly used for PCB manufacturing. These materials include, but are not limited to, FR-2, FR-4, FR-5, polyimide, Kapton ™, Rogers ™, Duroid ™, BT, cyanate esters and Polytetrafluoroethylene (PTFE). Rigid, rigid flexible or flexible substrates can be used as base material 12 for the manufacture of these devices.

p00072Electrode formation

p00073The outermost conductive copper layer of the PCB is modeled to form the driving electrodes required to manipulate liquid droplets by electric fields. The driving electrodes 16 can assume a wide variety of contours and shapes depending on the particular application. For example, square-shaped electrode series, circular-shaped electrode series, hexagon-shaped electrode series, star-shaped electrode series and other interlocking or interdigitated electrode forms, as well as elongated electrode structures can be used. Reference electrodes 18 may also be modeled on the same conductive layer or on a different conductive layer on the same (co-planar) substrate or may be provided on a different (bi-planar) substrate.

p00074In one embodiment as shown in Figures 1A-1D, the reference electrodes 18 may be modeled on the same conductive copper layer as the driving control electrodes 16 in which the insulator 22 is removed on reference electrode parts 18 to expose the underlying conductive layer. This pattern allows simultaneous electrical contact between the reference electrodes 18 and the liquid droplet. In this embodiment, the reference electrodes 18 may be located adjacent to or between the drive control electrodes 16.

p00075In another embodiment, as shown in Figures 2A-2B, the reference elements 18 may be formed as a different conductive layer modeled directly on the insulator 22. The conductive layer may be a thin metal film deposited by vacuum processing, non-electrical plating, electroplating, lamination or other means and modeling to form the reference elements 18. The reference elements 18 can assume various contours and shapes and can be directly above and / or next to the driving elements 16 (i.e., it is not necessary that the reference elements 18 be precisely aligned with the driving control elements 16). In one arrangement, the reference elements 18 may form a grid or mesh of conductive tubes superimposed on driving elements 16. In this arrangement, the reference elements 18 could electrically protect the control electrodes 16 where they overlap, so that the overlap should ideally be minimized when the reference elements 18 are dimensioned and located with respect to the driving control elements 16 In another arrangement, the grid spacing is selected to be less than, but not an integer factor of, electrode spacing. This different conductive layer arrangement can be made using additive metal PCB processes where the metal is deposited on the insulator 22 or alternatively it could be done using subtractive processes where the reference elements 18 and the driving elements 16 are formed on opposite sides of a thin flexible circuit substrate. In the latter case, the flexible circuit substrate serves as an insulator for the drive control elements 16 and the flexible circuit can be laminated to a rigid substrate to provide mechanical rigidity and to provide electrical interconnections for the electrodes.

p00076In a further embodiment, as shown in Figures 3A-3B, the reference elements 18 may be provided in a co-planar arrangement embedded within the drive control elements 16. In said arrangement, the traffic gaps 25 with veneer 26 can function as reference elements 18 in areas not covered by insulator 22. Other transit gaps 24 with plating 26 covered by the insulator 22 may also be provided and operated as described hereinbelow.

p00077In another embodiment, as shown in Figures 4A-4B, the reference elements 18 may be provided on a different substrate such as a parallel plate. Typically, the substrate containing driving electrodes 16 and the substrate containing reference elements 18 are placed opposite each other with a space G between them

p00078to contain the liquids creating, in this way, a "sandwich" structure. A parallel plate arrangement

p00079Additional may include two opposing surfaces that are electrostatic PCB microfluidic devices themselves (The upper "plate" may be a PCB that has a first upper side surface 13 'and a second lower side surface 14') and have drive elements 16 in both surfaces and reference elements 18 on at least one surface.

p00080Since very little current is needed to charge the driving electrodes 16 for droplet manipulation based on an electric field, the conductive material that forms the electrodes may be substantially more resistive than is typically acceptable for PCB applications. Therefore, a wide range of different types of conductors can be used, in addition to copper. This includes conductors that are typically considered unsuitable for forming solder points and PCB tracks. Similarly, the conductive layer may be substantially thinner than is typically preferred for PCBs: Ideally, the conductive layer should be as thin as possible to minimize the topography of the conductive elements that should subsequently be covered with the insulating layer. Additionally, minimization of conductor thickness promotes the flatness of the PCB surface that is desirable for consistent and reliable handling of droplets on the surface. Conductor thickness can be minimized by using a starting substrate material with a minimum conductive thickness (for example, ¼ oz. Layer or 5 m copper cladding) or by adding a polishing or etching stage to reduce conductor thickness before of the deposition of the insulator.

p00081Interconnection and electrode paths

p00082The conductive tracks on the PCB substrate 12 are used to establish electrical connections with the driving electrodes 16 and the reference elements 18. Each driving electrode 16 or reference element 18 may be connected to one or more driving electrodes 16 or reference elements. 18 different, to other electronic components in the same PCB 12 substrate, or to solder points for external connection. In one arrangement, the solder points for the external connection are provided along an edge of the PCB and the PCB is adapted for use in a card edge connector receptacle 28 (see Figures 8A-8B). In another arrangement, a series of welding points are disposed on the surface of the PCB and contact is made with the welding points using spring-loaded pins, test clips or a strip of anisotropically shaped conductor material 29 (see figure 9A). In yet another arrangement, a plug connector, receptacle connector or other discrete electronic component is connected to the PCB to facilitate connection to an external circuit.

p00083As shown in Figures 1A-1D, 2A-2B, 3A-3B and 4A-4B, the electrical connections between different conductive substrate layers 12 can be established by PCB methods as are known in the art, whereby a hole or transit gap 24 is drilled through the substrate 12 from the two conductive regions (upper surface 13 and lower surface 14) on either side of the substrate 12 and which must be electrically connected. Although they are shown as circles in the drawings, it is understood that the transit gaps 24 can be of any shape such as squares, oval, etc., that could be formed in the substrate material 12. The interior of the hole 24 can also be metallized by non-electrical or electroplated plating or by using other methods to form a plating 26 (plating through hole metallization) so that an electrical continuity is established between the two opposite sides at the location of the lagoon of transit. As described above, conductive pastes (metallization of full through holes) could also be used in place of plating through holes to establish electrical continuity.

p00084Several approaches are available to establish electrical connections between electrodes and tracks. In one approach, a cable or track moves away from the electrode on the same side of the PCB, the cable can then be routed if necessary through the substrate at a track location away from the electrode. In another approach, paths are made in the electrodes. In this case it may be necessary to provide means to fill or cover the perforation to prevent liquid from entering or evaporating through the transit perforation. The transit gap 24 can be closed by plating using non-electric or electroplating plating or it can be filled or covered using various techniques and various materials (conductive epoxy, non-conductive epoxy, transparent epoxy or any other material). After filling the transit gaps with any of these filling materials, the surface of the PCB can then be covered with copper by non-electric plating or electroplating to completely hide the transit gap to the droplets moving on the surface.

p00085In one approach, the hole is made small enough so that an insulator deposited in liquid form, such as a traditional liquid solder mask material, is prevented from penetrating the hole through viscosity or surface tension effects, or could be made large enough so that the liquid welding mask can enter the transit lagoon, thereby forming a transit lagoon filled with welding mask 24 '(see Figure 1B). Alternatively, an extra process step can be added to fill the perforations with an epoxy or similar material before depositing the insulator, thereby forming a transit lagoon filled with epoxy 24 "(see Figure 1C), or a lagoon of transit filled with transparent epoxy 24 "'

p00086(see figure 1D). Another approach is to use a dry film insulating material that "covers" the perforation,

p00087effectively covering it and sealing the surface of the chip. A possible disadvantage of several of these approaches is that it results in the formation of a non-conductive region at the edge of the otherwise conductive electrode that reduces the area of ​​that electrode that can be used for the generation of the electric field. To address this problem, several techniques are available to produce a conductive filler, including the use of conductive epoxies to fill the hole and the use of non-electrical or electroplated plating to provide a conductive surface coating on a non-conductive filler method. Another alternative is to electroplate the perforation so that it becomes completely filled with metal. This approach may require a planarization step to remove excess metal deposited on the substrate surface by means of the greatest amount of electroplating. Planarization and control of the thickness of the conductor on the surface of the substrate can

p00088Simplified in this case by using a "button plating" process on the additional metal is added only in the region surrounding the track. The resulting “buttons” can then be removed by polishing the

p00089PCB surface. In this method, substantial amounts of metal can be deposited inside the perforations without increasing the final thickness of the metal on the surface of the PCB.

p00090Electrode insulation

p00091Referring further to Figures 1A-1D, 2A-2B, 3A-3B and 4A-4B, the driving electrodes 16 are typically electrically isolated by the insulator 22 to prevent the flow of direct electric current between the electrodes and the liquid conductor when a DC potential is applied to the drive electrodes. It should be noted that AC potentials could also be applied to the driving electrodes to allow droplet manipulation induced by an electric field. Although any dielectric can be used, the welding mask is typically used in traditional PCB processes to protect copper pipes over a PCB and to expose copper only where electronic components eventually weld. The most direct approach to isolating the driving electrodes 16 is to use welding mask material (or other dielectric) as electrical insulator 22. Both liquid and dry film welding masks are suitable for use as electrode insulators 22. Photosensitive welding masks are generally preferred, since they can be easily modeled to provide electrical access to the reference elements 18 or contact welding points under insulation 22.

p00092Welding masks are available in two varieties: photosensitive liquid welding mask (LPI) or dry film (DFSS). The LPI is not compliant. The DFSS offers almost vertical side walls and has been described to manufacture electroplating molds, seal fluid channels and as a mask for treatment: with powder jet “powderblasting” of microchannels. However, DFSS has not been used to form liquid deposits or as a gasket material to provide a separator or seal between two parallel plates as provided herein.

p00093In some applications, welding mask materials may not exist with the desired combination of thermal, mechanical, electrical or optical properties. In these cases, the materials of the welding mask can be replaced by or combined with other types of insulating materials. For example, disposable materials such as polyimide, materials that can be coated by immersion or by centrifugation or by spraying or by brushing such as TEFLON® AF and Cytop ™, vapor deposited or cathodic spray materials such as silicon dioxide, and polymers such as parylene they can be applied to the PCB substrate.

p00094As an alternative to welding masks for insulator 22, a thin layer of parylene could be deposited in a physical vapor deposition (PVD) process as a dielectric. Parylene is the generic name for a family of poly (para-xylylene) polymers that includes C, D, and N. parylene. As used in this description, parylene refers to any poly (para-xylylene) composition and mixtures from the same. A fundamental advantage with parylene is that it can be deposited as a compliant layer and a much smaller thickness than both LPI and DFSS. In PCB methods, the LPI can form a coating of a fineness of up to 0.5 mils (1 mil = 25.4 while the holeless parylene can form a coating of a fineness of up to 0.5 thin insulator reduces the potential required for droplet impulse. In some applications, the dielectric will have to be modeled to expose copper electrodes. Parylene can be modeled by reactive ionic etching, plasma calcination, chemical etching or by laser ablation. Alternatively, parylene can also be deposited selectively by masking the regions that need to be exposed by a tape (for example, 3M® Mask Plus II Water Soluble Wave Solder Tape No. 5414 which is used to mask golden connectors on PCB during wave solder). Other representative examples of materials that could be used as dielectrics include silicones, polyurethanes, acrylics and other dielectrics that can form a coating by centrifugation or depositing.

p00095Generally, it is desirable to minimize the thickness of the insulator 22 to reduce the voltage required for the movement of the liquid.

p00096Separator layers

p00097It is also provided that additional layers of welding mask material can be deposited and modeled to create physical structures on the surface of the PCB such as wells and channels (not shown) for use to gather or direct the flow of liquid.

ADDITIONAL PROCESSES

p00099Combination of subtractive and additive processing

p00100In a further embodiment, a combination of subtractive and additive processes can be used to manufacture PCB droplet handling plates of the present matter. Subtractive processes can be used to make a multilayer plate that defines all electrical routing and interconnections to the droplet control electrodes. A modelable dielectric layer can be applied next. Paths in this dielectric layer can be modeled by laser drilling or photo masking. In one embodiment, LPI can be used as a dielectric. The welding point of the electrode exposed in the hole may optionally be plated to make it planar with the dielectric surface. At this point, an additive process can be used to define all electrodes using non-electric copper, since a smaller tube separation could be obtained.

p00101Post processing

p00102A finished device may include a combination of conventional PCB processes and unconventional processes. For example, a single stage hydrophobic coating can be applied to a finished PCB to facilitate the transport of droplets. In addition, the use of a welding mask as a dielectric may not be desirable for some applications, in which PCBs not insulated with a housing could subsequently be coated with specialty materials not available in a conventional PCB process. However, in such cases, the use of a PCB as a starting substrate and PCB processes to form the conductive tracks continues to provide many, if not most, of the benefits of a completely PCB compatible process.

p00103In one embodiment, all conductive tubes required for electrical routing can be manufactured on a multilayer PCB. Part or all of the outer copper layer can then be removed by polishing or chemical etching. This PCB, which contains all the electrical wiring required for droplet handling, can then serve as a substrate for further processing to model impulse and reference electrodes with finer tube separation. To obtain finer tube separation, control electrodes can be modeled using semiconductor processing techniques including thin film deposition and photolithography.

p00104Plating of coplanar reference elements

p00105In an embodiment where the reference electrodes 18 are also modeled on the same layer as the driving electrodes 16 (see, for example, Figures 1A-1D), there may be a significant depression in the LPI welding mask since it only covers the drive electrodes and leave the reference electrodes open. This depression could affect the reliability of operation since the droplet may not be in contact with the reference element. In this case, the reference electrodes can be plated so that the surface of the reference element is planar with the LPI welding mask (not shown). This plating stage could be done before the surface is finished with copper or nickel.

p00106Reference electrodes on the external surface

p00107In one embodiment, after all copper electrodes have been formed as described hereinbefore, the LPI coating can then be used as an interlevel dielectric and another copper layer can be modeled on the LPI to serve as electrodes. reference. The dielectric can also be a thin (2 thousand or less) prepreg PCB board in a typical multilayer construction or it could be a flexible plate with copper elements to serve as reference electrodes on the outermost layer. The copper layer just below this outermost copper layer has copper elements that define the driving electrodes.

p00108Integration of electronic components and detection on the PCB

p00109In a further embodiment, it is envisioned that the PCB of the present matter may also be constituted by electronic components in the areas that are not used for liquid handling. Electronic components may include microcontrollers, relays, high voltage multiplexers, voltage converters (DC-DC to set the voltage, DC-AC, AC-DC, etc.), electro-optical elements such as LEDs, photodiodes, photomultiplier tubes (PMT), heating elements, thermistors, temperature resistance devices (RTD) and other electrodes for electrochemical measurements. Copper tracks can also be used for droplet impedance measurements. The resistive heating elements are made by winding copper tracks and the resistive heating characteristics will depend on the dimensions of the copper pipes. In one embodiment, a PCB containing an optical detector, such as a PMT or a photodiode, can be used as a parallel plate for

p00110form a "sandwich" with the droplet handling PCB board. In another embodiment, electrodes may be used.

p00111Gold-plated obtained in a conventional PCB process, for electrochemical measurements.

p00112Perforations for fluid inlet / outlet

p00113Mechanically drilled holes in a PCB are typically used to glue or fix the board to another surface. It is further provided in the microfluidic PCB chip of the present matter that these perforations can be used to serve as fluid inlet / outlet holes for the addition and removal of liquids to or from the surface of the PCB substrate. It is also provided that these perforations can be coupled with a liquid source including, but not limited to, flexible tubes, syringes, pipettes, glass capillary tubes, intravenous tube or microdialysis light. The liquid in these tubes can be driven by pressure or any other means. A continuous flow of liquid from the tubes can establish communication with the PCB through these perforations that can be separated into droplets directly from the flow or through an intermediate reservoir in the PCB.

p00114For example, in one embodiment, metallic perforations (see, for example, perforations 32 in Figures 9A-9B) may be located adjacent to control electrodes to serve as fluid inlet / outlet holes for placing liquid withdrawals on the surface of the electrode. In another embodiment, non-metallic perforations (see, for example, perforations 34 in Figures 9A-9B) may be provided for fluidic inlet and outlet and may be connected to a channel etched into the welding mask, which then leads to a deposit (not shown). This reservoir may have electrodes for dispensing, such as using droplet dispensing techniques mediated by electric field. In yet another embodiment, metallic perforations provided for fluidic inlet / outlet can be covered by a dielectric and also have concentric electrode rings around the perforation. In this case, the droplets can be dispensed radially out of the hole by pressurizing the liquid through the hole and then using an electric field to dispense the droplets onto the electrodes. In a further embodiment, perforations can be used to expel liquid into a waste container or any other container outside the chip by collecting the droplets throughout the area and allowing the drops to fall by gravity into a container placed under the hole.

p00115Removing droplets out of the plane from the traffic lagoons

p00116Generally, droplets moved in apparatus of the present subject are handled in a horizontal plane in a sandwich structure with one or both of the plates comprising PCB. In a further embodiment, the holes drilled in a PCB could be used to extract droplets from the sandwich structure in a vertical plane. Droplets can be extracted through holes in various ways. In a method that exploits the pressure difference between a droplet confined in a sandwich structure and a large hole, the droplets could be passively pushed through a hole with a diameter greater than the radius of the droplet simply by placing it below the hole. The droplets could also be extracted by electrical means where another plate is added to the sandwich structure and the droplets can be dragged out of a sandwich structure to the newly formed sandwich structure by applying an electrical potential. In this case, to simplify the extraction process, a sandwich structure can be formed between a coplanar PCB substrate and other electrode substrate. Although both of these plates form a parallel plate arrangement, the droplets will only touch the coplanar PCB substrate and move vertically on the other substrate when an electrical potential is applied on the other substrate to electrostatically drag the droplet out of the plane . The droplet could also move vertically with gravity to be stamped on another plate. Applications for said vertical droplet pulse include DNA or protein stamping applications. Droplets extracted from said holes can also be used to increase the path length for absorbance measurements and for transport to another sandwich structure to allow transport to another layer.

p00117Biochemical synthesis and analysis

p00118A series of biochemical reactions can be performed by manipulating liquids on PCB substrates as disclosed herein. As disclosed herein, the present subject provides an apparatus for detecting a target analyte in sample solutions by means of electrical and optical detection means. The sample solution may comprise any number of elements, including, but not limited to, body fluids (including, but not limited to, blood, sweat, tears, urine, plasma, serum, lymph, saliva, anal and vaginal secretions, semen of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred); food and environmental samples (including, but not limited to, air, agricultural, water and soil samples); samples of biological warfare agents; research samples; purified samples, such as purified genomic DNA, RNA, proteins, cells, etc .; and raw samples (bacteria, viruses, fungi, etc.). The types of assays that can be performed on the PCB substrate as disclosed herein include enzymatic assays, isothermally amplifying DNA.

p00119or by thermocycling, immunoassays, including sandwich and homogeneous arrangements, and cell-based assays with optical and electrical detection means. Analytes measured in physiological samples include metabolites, electrolytes, gases, proteins, hormones, cytokines, peptides, DNA and RNA.

p00120In a process using the apparatus of the invention, a human physiological sample can be introduced into a reservoir in the PCB. The deposit could be defined by the dry film welding mask. The sample can then be dispensed into droplets that will be mixed with the appropriate reagent droplets provided on the PCB or introduced onto the PCB. Some of the enzymatic assays can then be monitored optically (for example, by absorbance, reflectometry, fluorescence and luminescence). In the case of absorbance, transit gaps can be filled with an optically transparent material so that light can pass through a droplet located over one of these transit gaps for absorbance measurements.

p00121In another process, biochemical samples can also be synthesized on a PCB substrate using droplet manipulation techniques described herein. For example, on the PCB, a series of protein droplets can be dispensed from a reservoir and mixed with different reagents and incubated to automatically discover conditions to crystallize a protein.

p00122Lateral wall transport

p00123In a further process, copper tracks with thicknesses of the same order as the height of the droplet can be used, so that the droplet is contained between the tracks arranged on the same substrate and covered by an insulator. The droplet is driven by electric fields applied mainly in the plane of the substrate instead of perpendicular to it. Unlike the coplanar arrangement, where the droplet sits on the coplanar drive and reference electrodes and the parallel plate arrangement, in which the droplet is sandwiched between the driving electrodes on a substrate and a common reference electrode on a substrate In parallel, in this structure a droplet is sandwiched between the coplanar drive and reference electrodes.

SPECIFIC REALIZATION

p00125Although general embodiments of the present invention have been described hereinbefore, more specific embodiments of manufacturing an apparatus for handling samples and micro-volume liquid in which the apparatus comprises a printed circuit board substrate will be described below.

p00126In a preferred embodiment, a FR-substrate is laminated with a ¼ oz copper sheet (ѝ9 m) on both sides. 8 thousand transit gaps are drilled through the substrate. These transit gaps are then electroplated with copper and filled with a welding mask or an epoxy. Preferably, transit gaps they are buttoned to a thickness of approximately 5 m where the traffic lagoons are specifically plated while the rest of the plate is covered by a mask. The buttons are mechanically planarized and then the transit gaps are filled with welding mask or a non-conductive epoxy. After processing the traffic gaps, a smooth plating stage is performed at a thickness of less than 5 m. If unfilled traffic gaps are required, another drilling stage can be performed to obtain unified holes and plating is performed if necessary. In this phase, the designed electrode pattern is transferred to copper with a minimum separation of 2 mils tubes by recording it through a mask. The LPI is modeled and coated to a thickness of approximately 0.5 mils. Finally, a dry film welding mask is laminated and modeled to form the physical structures (for example, wells and / or channels) to contain liquids and also to serve as separation material. In other embodiments, the separation layer can also be obtained using one or more LPI welding mask coatings or by laminating and etching a copper sheet.

EXPERIMENTAL TESTS AND RESULTS

p00128Experiments were conducted in which a single-layer two-plate design for a droplet manipulator mediated by an electric field as disclosed herein was submitted to a commercially available electronic PCB manufacturer and tested. The design consisted of a series of different forms of control electrode for the transport and mixing of liquid droplets as well as specialized electrode forms for dispensing droplets from a larger liquid volume. The electrodes were connected to contact welding points by conductive tracks modeled in the same copper layer on the surface of the PCB. Where necessary, the tracks were routed between the two sides of the board using conventional tracks at remote locations of the control electrodes. Several different chip designs and interconnection schemes were tested.

p00129Some chips contained multiple copies of a single linear series of electrodes where the corresponding elements in each copy of the series were connected to the same electrical signal - therefore multiple identical series could be controlled simultaneously. Other chips contained an electrode "bus" or conveyor structure where each fourth electrode in a contiguous line of control electrodes was connected to the same control signal. The use of said structure allows arbitrarily long transport paths to be controlled using a fixed number of control signals. Multiple droplets can be diverted on or off the bus and transported synchronously. The contact solder points were arranged along the side of the PCB and were designed to contact them using a conventional card edge connector or a conventional SOIC test clip.

p00130Figures 6, 7, 8A-8B and 9A-9B illustrate several examples of chips manufactured for experimental purposes. Figure 6 illustrates the front of a PCB chip used to test the transport performance of droplets of different shapes (circular 16a, square 16b, star with a small curvature 16c, star with a large curvature 16d) (see Figure 7) and sizes of the control electrodes (results described in reference to Figures 10-12 below). The chip illustrated in Figure 6 contains 16 different series of linear electrodes. Figures 8A and 8B are front and rear views of a chip design featuring a three-phase droplet conveyor as well as other structures for dispensing from a deposit in the chip, storage and mixing of droplets. The tracks 24 are used to route the electrical signals from the back of the PCB to the control electrodes on the front and the electrical contact is established through the card edge connector socket 28 located along one side of the PCB Figures 9A and 9B are front and rear views of another chip design featuring a three-phase droplet conveyor as well as other structures for dispensing from a fluidic inlet / outlet port 32, storage and mixing of droplets. The tracks 24 are used to route the electrical signals from the back of the PCB to the control electrodes on the front and the electrical contact is established by a series of welding points designed to make contact with them using a test clip SOIC 29.

p00131The series of control electrodes were designed with a spacing of 1.0 mm or 1.5 mm and a nominal separation of 2 thousand between adjacent electrodes. The substrate material was FR-4 with a copper coating of ¼ oz. Copper was modeled to form control electrodes, tracks and contact solder points. The width of the tubes / nominal minimum separation of the process used was 2 thousand / 2 thousand, which was the separation used between adjacent electrodes as well as the track width between the control electrodes and the contact welding points. A liquid photosensitive welding mask material, CARAPACE® EMP 110 (available from Electra Polymers &amp; Chemicals, Ltd.) was used as an electrode insulator. The nominal thickness of the welding mask insulator was 0.6 thousand. After the PCBs were received from the manufacturer, a thin hydrophobic coating of TEFLON® AF was applied to the top surface of the chip. TEFLON® AF was applied by centrifugal coating of a 1% solution in FC-75 at 3000 rpm for 20 seconds on the surface of the PCB, followed by a 30 minute cure at 150 ° C.

p00132The PCBs were assembled like a sandwich with a glass top plate coated with indium-tin oxide. The top plate was also coated with a thin layer of TEFLON® AF so that all interior surfaces in contact with the droplet were hydrophobic. The conductive indium tin oxide film on the top plate was used as the reference electrode. The PCB and the top plate were separated by a gap of approximately 0.8 mm. One or more electrolyte droplets (0.1 M KCI) were injected into the sandwich structure and deposited on a control electrode. The volume of the droplets was sufficient to cover a single electrode and it was approximately 2.2 l for the 1.5 mm spacing electrodes and 1.1 l for the 1 mm electrodes. The remaining volume between the two plates was filled with air or low viscosity silicone oil (1 cSt.). Referring to Figures 6, 7 and 10-12, tests on the transport of droplets were performed by sequential activation of the control electrodes as described in US Patent No. 6,911,132 and the Publication of the Application for US Patent No. 2004/0058450, both of Pamula et al., Using a PCB similar to that shown in Figure 6, tests were performed on 4 different electrode shapes (circular 16a, square 16b, star with small curvature 16c, star with greater curvature 16d) (see Figure 7) in each of the electrode sizes (1.0 mm and 1.5 mm spacing).

p00133For each electrode size and shape, the maximum speed at which the droplets could be transported between adjacent control electrodes was determined based on the applied voltage, as shown in Figures 10 and 11. The droplets were successfully transported at voltages. less than 40 V (for an electrode size of 1.0 mm) with the transport speed increasing with the voltage beyond that threshold. Higher voltages were required for droplet impulse than those described above in other systems due to the use of thicker welding mask insulator. For example, the isolation of the welding mask is approximately 16 times thicker than the insulation used with previous microfabricated devices and, therefore, approximately four (4) times the voltage is required due to the dependence of electrostatic energy (1 / 2 CV2) of the transport mechanism.

p00134As expected, beyond the initial threshold voltage, the transport speed and, consequently, the maximum speed at which the droplet could be exchanged increased with the voltage. The voltage range tested was around 0 - 200 V for 1.5 mm electrodes and 0 - 100 V for 1.0 mm electrodes, and droplet transport rates of up to 24 Hz were observed. The resulting test curves showed a general expected form - the higher the applied voltage, the greater the possible transfer frequency. However, the curves for the 1.5 mm electrode (figure 10) were not too smooth and there seems to be a significant effect on the shape of the electrode. As an alternative, the curves for the 1.0 mm electrodes (figure 11) are quite predictable and do not show a great dependence on the shape of the electrode. In addition, there was a scaling effect where the threshold voltages of the 1.0 mm electrodes were 10-20 V lower than the 1.5 mm electrodes at corresponding frequencies.

p00135As shown schematically in Figure 12, an additional test was performed to determine the stability of droplet transport over time. A droplet was programmably cycled through four 1.5 mm square electrodes to the minimum voltage required to maintain the transport at a switching frequency of 4 Hz or 8 Hz. At five minute intervals the minimum voltage for Continuous transport was tested and adjusted. Tests that were performed for an hour or more demonstrate a general trend of increasing voltage requirement over time that is presumably due to the degradation of the insulator and contamination of the insulator surface. However, in each case more than 20,000 droplet transport cycles were performed during the experiment.

p00136Referring to the graph shown in Figure 13, tests were also performed to determine the minimum voltage requirements for the transport of droplets at a given switching frequency. Digital microfluidic chips were used for both an open structure (i.e., co-planar without a top plate) and confined (i.e., bi-planar with a top plate) on a PCB (see Figures 1B and 4B, respectively). Electrodes (1.5 x 1.5 mm2) were modeled in copper at a final thickness of ~ 25 m. 150 m transit gaps were drilled in each electrode to provide electrical contacts with the back of the plate. Grounding rails were modeled next to all the driving electrodes to provide a continuous ground connection to the droplets, and a liquid photosensitive welding (LPI) mask (~ 17 m) was modeled to act as an insulator, leaving exposed Only the rails. As the only post-processing stage, TEFLON® AF was coated by brushing to make the surface hydrophobic. Droplets of a polarizable and conductive liquid (1 M KCI) were transported in both open (co-planar) and confined (bi-planar) systems. For the open system, each Droplet had a volume of 6 L and a small drop of silicone oil (2 L) was added and seemed to surround the droplet. For the confined system, the volume of each droplet was 2.5 l, and the entire chip was filled with silicone oil to facilitate transport.

p00137The minimum movement voltages required to successfully transport droplets were measured for each system at switching frequencies ranging between 1 and 32 Hz. As shown in Figure 13, the operating voltages for droplets in the confined systems (bi -planar) and open (co-planar) varied between 140-260V and 125-220V, respectively, depending on the switching frequency of the droplets. This seems to suggest that droplet driven is facilitated by the absence of an upper confinement plate, possibly due to the reduced drag experienced by the unconfined drop. The electrolysis of the droplets, typically due to inadequate insulation coverage, was not observed using an LPI welding mask as an insulator up to the maximum tested voltage of 350 V. The insulation load, however, was experienced beyond 300 V .

p00138Referring to Figures 14A-14D, top views of various image sequences are shown at time intervals demonstrating the transport and mixing of droplets. Figures 14A-14B represent the transport and mixing of droplets, respectively, for droplets confined by an upper plate (600 m) (bi-planar). Figures 14C-14D represent the transport and mixing of droplets, respectively, for droplets in an open (co-planar) system. Mixing was performed at a switching frequency of 8 Hz and was completed in 5 seconds for two “confined” droplets of 2.5 l, and in 1.8 seconds for two 6 l droplets in an “open” system. Therefore, the mixing rates (volume per unit of time) observed in the open (co-planar) system are almost seven times higher than in the confined (bi-planar) system. This improved mixing can be attributed to the greater circulation experienced in the thicker droplet, since the circulation has been shown to worsen previously as the droplets become thinner.

REFERENCES

p00140The references listed below supplement, explain, provide background for or teach methodology, techniques and / or processes.

<dl><dt>MG</dt><dd> Pollack, RB Fair, and AD Shenderov, “Electrowetting-based actuation of liquid droplets for microfluidic actuation”, Appl. Phvs Lett., Vol. 77, pages 1725-1726 (2000).</dd></dl>

<dl><dt>V.</dt><dd> Srinivasan, VK Pamula and RB Fair, "An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids", Lab Chip, 4 (4), 310, (2004) </dd></dl>

<dl><dt>B.</dt><dd> Berge and J. Peseux, “Variable focal lens controlled by an external voltage: An application of electrowetting”, The </dd></dl>

p00141European Physical Journal E, vol. 3, page 159 (2000).

<dl><dt>MWJ </dt><dd>Prins, WJJ Welters and JW Weekamp, ​​"Fluid control in multichannel structures by electrocapillary pressure", Science, vol. 291, pages 277-280 (2001).</dd></dl>

<dl><dt>T. </dt><dd>Merkel, L. Pagel and HW Glock, “Electric fields in fluidic channels and sensor applications with capacitance”, </dd></dl>

p00142Sensors and Actuators A, vol. 80, pages 1-7 (2000).

p00143A. Wego, and L. Pagel, "A self-filling micropump based on PCB technology", Sensors and Actuators A, vol. 88, pages 220-226 (2001).

p00144C. Laritz and L. Pagel, "A microfluidic pH-regulation system based on printed circuit board technology", Sensors and Actuators A, vol. 84, pages 230-235 (2000).

p00145Li et al., "Development of PDMS-based Microfluidic Device for Cell-based Assays", Chemical Journal of Chinese Universities, vol. 25, no. 1, pages 4-6 (2004).

p00146A. Wego, S. Richter, L. Pagel, "Fluidic microsystems based on printed circuit board technology", Journal of Micromechanics and Microengineering, vol. 11, no. 5, pages 528-531 (September 2001).

p00147A. Wego, HW Glock, L. Pagel and S. Richter, “Investigations on thermo-pneumatic volume actuators based on PCB technology”, Sensors and Actuators A-Physical, vol. 93, no. 2, pages 95-102 (September 30, 2001).

p00148A. Wego and L. Pagel, "A self-filling micropump based on PCB technology", Sensors and Actuators A-Physical, vol. 88, no. 3, pages 220-226 (March 5, 2001).

p00149C. Laritz and L. Pagel, "A microfluidic pH-regulation system based on printed circuit board technology", Sensors and Actuators A-Physical, vol. 84, no. 3, pages 230-235 (September 1, 2000).

p00150T. Merkel, M. Graeber, and L. Pagel, "A new technology for fluidic microsystems based on PCB technology", Sensors and Actuators A-Physical, vol. 77, no. 2, pages 98-105 (October 12, 1999).

p00151NT Nguyen and XY Huang, “Miniature valveless pumps based on printed circuit board technique”, Sensors and

p00152Actuators A-Physical, vol. 88, no. 2, pages 104-111 (February 15, 2001).

<dl><dt>CW </dt><dd>Li, CN Cheung, J. Yang, CH Tzang, and MS Yang, “PDMS-based microfluidic device with multi-height structures manufactured by single-step photolithography using printed circuit board as masters”, Analyst, vol. 128, no. 9, pages 1137-1142 (2003).</dd></dl>

<dl><dt>TO.</dt><dd> P. Sudarsan and VM Ugaz, "Printed circuit technology for fabrication of plastic-based microfluidic devices", Analytical Chemistry vol. 76, no. 11, pages 3229-3235 (June 1, 2004).</dd></dl>

<dl><dt>G. </dt><dd>Jobst, I. Moser, P. Svasek, M. Varahram, Z. Trajanoski, P. Wach, P. Kotanko, F. Skrabal, and G. Urban, “Mass producible miniaturized flow through a device with a biosensor array”, Sensors and Actuators B-Chemical, vol. 43, pages 121-125 (September 1997).</dd></dl>

p00153MA Unger, HP Chou, T. Thorsen, A. Scherer, SR Quake, "Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography", Science, vol. 288, pages 113-116 (2000).

p00154AR Wheeler, H. Moon, CA Bird, RR Loo, CJ Kim, JA Loo, RL Garrell, “Digital microfluidics with in-line sample purification for proteomics analyses with MALDI-MS”, Analytical Chemistry, vol. 77, no. 2, pages 534-40 (2005).

<dl><dt>JA</dt><dd> Schwartz, JV Vykoukal and PRC Gascoyne, "Droplet-based chemistry on a programmable micro-chip", Lab on a Chip, vol. 4, no. 1, pages 11-17 (2004).</dd></dl>

<dl><dt>AND. </dt><dd>Tan, JS Fisher, AI Lee, V. Cristini and AP Lee, "Design of microfluidic channel geometries for the control of droplet volume, chemical concentration, and sorting", Lab on a Chip, vol. 4, no. 4, pages 292-298 (2004).</dd></dl>

p00155JD Tice, AD Lyon and RF Ismagilov, “Effects of viscosity on droplet formation and mixing in microfluidic channels”,

p00156Analytica Chimica Acta, vol. 507, pages 73-77 (2004).

p00157T. Merkel, L Pagel, H. Glock, “Electric fields in fluidic channels and sensor applications with capacitance”, Sensors and Actuators, vol. 80, pages 1-7 (2000).

p00158G. Medoro, N. Manaresi, A. Leonardi, L. Altomare, M. Tartagni and R. Guerrieri, "A Lab-on-a-Chip for Cell Detection and Manipulation", IEEE Sensors Journal, vol. 3, pages 317-325 (2003).

p00159L. Altomare, M. Borgatti, G. Medoro, M. Manaresi, M. Tartagni, R. Guerrieri and R. Gambari, “Levitation and

p00160Movement of Human Tumor Cells Using a Printed Circuit Board Device Based on Software-Controlled Dielectrophoresis ”, Biotechnology and Bioengineering, vol. 82, pages 474-479 (2003).

p00161It will be understood that various details of the matter disclosed in this document may be changed without departing from the scope of the matter disclosed in this document. In addition, the above description is for illustration purposes only and not for limitation purposes.