Apparatuses and methods for manipulating droplets on a printed circuit board

Description

RELATED DECLARATIONS

[0001] This patent application is related to U.S. Patent Application Serial No. 10 / 253,342, filed September 24, 2002, and U.S. Provisional Patent Application Serial No. 60 / 648,051, filed January 28, 2005.

TECHNICAL FIELD

[0002] The present patent application relates generally to devices and methods which enable micromanipulation of droplets to be carried out on a substrate of a printed circuit board (PCB). More specifically, the subject of the described patent is a device and method enabling the production and operation of microfluidic systems based on droplets and operating on the basis of conventional PCB technology, where the droplets are moved by applying electrical potentials to electrodes formed on the PCB. The patent also relates to the use of a solder mask as an electrode insulator to manipulate the droplets, and techniques to adapt other traditional layers and materials present on a printed circuit board for use in droplet-based microfluidics.

BASICS

[0003] The microfluidic technique is a rapidly growing field of fluid testing in sub-microliter volumes. Microfluidic devices are increasingly used and recognized in many areas of biology, chemistry, medicine, environmental monitoring, development of new drugs and consumer electronics. The miniaturization of conventional equipment, particularly analytical equipment, is expected to provide numerous benefits, including reduced consumption (and cost) of reagents and samples, increased throughput and automation, reduced analysis duration, and the creation of more reliable, inexpensive and portable instrumentation. As devices of this type become more and more functions, they fully

EP 1 859 330B1 integrated miniaturized Total Medical Diagnostic (pTAS) systems or labs-on-a-chip chips are becoming a reality and are gaining in importance.

[0004] The lab-on-a-chip microcircuit is a new paradigm that aims to miniaturize and integrate the fluid manipulation technique within an integrated circuit. A lab-on-a-chip microcircuit, if it is to be a truly self-contained unit, should enable dosing, transport, mixing of liquids, their incubation, detection or separation and waste disposal. Lab-on-a-chip microfluidic systems can be broadly divided into continuous flow and discrete flow systems. The name of the continuous flow systems explains the principle of their operation, while in the discrete flow systems the liquid is divided into droplets. A common limitation of continuous flow systems is the need to physically restrict fluid transport over fixed channels, while droplet-based systems (i.e., discrete flow) may either be restricted by physically existing flow channels or operate on flat systems without channels. The fluid transport mechanisms generally used in continuous flow systems may be pressure controlled by external pumps or electrokinetically controlled using high voltages. Continuous flow systems may require complex duct systems and large auxiliaries such as external valves or power supplies. As a different approach to channel systems, the unidirectional flow of fluid in the channels is forced by centrifugal forces. The paradigm of continuous flow microfluidic systems has clear limitations in terms of versatility, which makes it difficult to achieve a high degree of integration of various functions and controls in these systems.

[0005] Discrete flow or liquid droplet microfluidic systems are constantly evolving towards meeting the expectations of the lab-on-a-chip microchip concept for carrying out all steps of the analysis, including sampling, preparation, processing, including transport. , mixing and incubation, detection and disposal of waste. These stages are

ΕΡ 1 859 330Β1 designed to be integrated on an integrated circuit, without the use of significant external auxiliary circuits. Recently, in discrete-flow systems, several droplet manipulation methods have been developed based on the use of soft lithography, multiphase hydrodynamic flows, continuous electro-wetting, dielectric electro-wetting (EWOD), electrophoresis, electrostatics, and surface acoustic waves. Some of these techniques manipulate droplets or nuggets in physically defined channels, while others allow manipulation of the droplets on planar surfaces without any physically defined channels. Channelless droplet approaches are called digital microfluidic as the liquid is broken down into discrete droplets and manipulated by software.

The analytical protocols in droplet systems are very similar to lab scale biochemical analysis protocols, which are also generally performed on discrete volumes of liquid. Therefore, recognized protocols can easily be adapted to the format required by digital microfluidic technology. Some of the distinguishing features of digital microfluidic systems include: ease of reconfiguration (droplet operations and trajectories are selected from a software control panel, allowing users to instantly create any combination of microfluidic operations); The ability to program operation also provides design flexibility as a single general purpose microfluidic processor chip can be redesigned and reprogrammed for a variety of applications; conditional steps can be implemented as each operation of the microfluidic system can be carried out under the direct control of the computer, thus allowing maximum flexibility of the operation; omni-directional droplet transport, as channels only exist in a virtual sense and can be instantly reconfigured by software; small droplet volumes (<1 µl); work only in the electronic system, without use

ΕΡ 1,859 330Β1 external pumps or valves; simultaneous and independent control of multiple droplets; channelless operation (no pre-priming required).

[0007] Many modern lab-on-a-chip microchip technologies (including both continuous and discrete flow devices) are relatively inflexible and designed to perform only a single determination or a small set of very similar determinations. Due to the rigid configuration of the currently used microfluidic circuits, a new chip design is required for each new application, which makes developing new applications expensive. In addition, many of these devices are manufactured using expensive microfabrication techniques derived from the semiconductor chip field. As a result, applications of microfluidic systems are developing relatively slowly due to the cost and effort required to develop new systems for specific applications. While serial production makes microfabricated chips cheap to mass produce, developing new chips can be disproportionately costly and time-consuming due to the high cost of prototyping and the long processing cycle time associated with standard semiconductor microfabrication techniques. In order to broaden the range of applications and enhance the impact of microfluidic technology on medicine, drug discovery, environmental and food monitoring, and other areas, including consumer electronics, there has long been a need for both a microfluidic approach that will enable the creation of easier to configure, flexible integrated circuits, as well as techniques allowing for cheaper and faster development and production of these systems.

[0008] Over the past few years, advances have been made using different approaches to the microfluidic technique based on the manipulation of individual nano-sized droplets by directly controlling them with electric forces. 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

ΕΡ 1,859,330Β1 studies belong to the team of Pamuła et al. (And are commonly assigned to the assignee of this patent).

[0009] US-A-2004/0055891 also discloses a droplet manipulation device comprising an electrode with a one-sided structure in which all conductive elements are limited to a single surface on which the droplets are manipulated and a second, additional surface parallel to the droplet. the first can be placed to hold droplets to be handled. Droplet manipulation uses techniques based on the electro-wetting effect in which electrodes placed on or built into a first surface are sequentially connected and de-energized in a controlled manner.

[0010] Paik et al, in a paper entitled Thermal Effects on droplet transport in digital microfluidics with applications to chip cooling, Ninth International Conference on Thermal and Thermochemical Phenomena in electronic systems, 2004 (ITHERM Ό4), Las Vegas, NV, USA, January 1-4, 2004, Piscataway, NJ, USA, IEEE, 2004, pages 649-654, describe a cooling technique that uses electro-wetting manipulation of discrete liquid droplets with a volume of the order of nanoliters, immersed in oil and placed over the electrode system, and present the results of research on the influence of temperature-dependent system parameters on droplet transport.

[0011] These various techniques offer numerous advantages in implementing the digital microfluidic paradigm as described above, however, the currently used manufacturing techniques for these microfluidic systems are still dependent on quite complex and expensive manufacturing techniques. These microfluidic integrated circuits are currently produced by microfabrication processes using costly manufacturing steps based on semiconductor processing techniques routinely used in the electronic integrated circuit manufacturing industry. In addition to the higher cost of semiconductor manufacturing techniques,

EP 1 859 330B1 semiconductor production lines are not readily available and typically do not offer production or prototype cycle times of the order of 24 hours.

[0012] Microfluidic integrated circuits are typically fabricated using custom-made processes based on traditional semiconductor microfabrication procedures. The systems are manufactured on a glass substrate in a series of repeated steps of depositing layers and etching patterns therein using standard photolithography techniques. Typically, two metal layers (one for electrodes, the other for cabling) are required, plus two or three insulating layers and layers to define the gap between the bottom and top plates. Due to the high cost of producing a photoresist and producing the ICs themselves, a single prototype production cycle for up to 100 chips can cost as much as $ 10,000 and take up to 3 months, depending on the number of photolithographic layers required. Moreover, since the process flow is not standardized, the yields of the resulting systems can be very low in the first few fabrication attempts.

[0013] The cost and time required for prototyping are a serious obstacle to the development and optimization of microfluidic devices using droplets. In addition, it is believed that the high cost of the systems and the inability to quickly adapt to customer requirements or improve device design will limit the commercial prospects for this versatile technology. In short, in order to accelerate the development of these devices and their acceptance by users, faster, more reliable and cheaper manufacturing technology is required. Because microfluidic designs are relatively large in size and seldom require the use of frontier semiconductor manufacturing techniques, the use of cheaper, lower resolution batch production methods should be considered.

[0014] In particular, printed circuit board (PCB) technology offers many possibilities and materials similar to the microfabrication of traditional semiconductor circuits, but at a much lower cost.

ΕΡ 1,859,330Β1 resolutions. The conductor and insulator layers are deposited on top of each other and subsequently photo-etched to form complex multi-level structures. In the field of digital microfluidic chip manufacturing, PCB technology is considered to offer an excellent compromise in terms of resolution, availability, cost and ease of manufacture. In addition, it is believed that an additional benefit of using PCBs as a substrate is that the devices can be easily and cheaply integrated into electronic sensor, analyzer and control systems. [0015] Typically, copper track width and track spacing in PCB manufacturing are measured in mils (1 mil = 25.4 µm), which is orders of magnitude larger than the submicron size typically obtained in semiconductor fabrication. Usually, the production of PCBs does not require the costly ultra-clean environment that is required for the production of semiconductor integrated circuits. Printed boards are also generally made of a reinforced plastic, e.g., epoxy resin with glass fiber, TEFLON®, polyimide, etc., while the substrate for the production of microfluidic systems produced on semiconductor micro-fabrication lines is silicon or glass. Also, instead of a mask positioning device for semiconductor devices, the positioning can be done manually in the manufacture of printed circuit boards. Cheap masks made in the form of transparencies or MYLAR sheets are used in place of the costly chromium-on-glass photolithographic masks used in the production of semiconductor systems. In the manufacture of printed circuit boards, through holes are drilled mechanically or with a laser and then electroplated, while in the case of semiconductor systems, vacuum etching and vapor deposition are used, which requires processing in a vacuum. Multilayer connections are usually achieved by joining together individual, independently etched wafers, while in the production of semiconductor systems, a single substrate is used and multiple layers are formed or individual wafers are joined. Generally speaking, these are the fundamental differences between PCB manufacturing processes and circuit manufacturing processes

EP 1 859 330B1 semiconductor manufacturing processes, even though the most modern PCB manufacturing processes tend to adapt certain processes in the field of semiconductor fabrication (e.g. vacuum evaporation).

[0016] In today's highly competitive commercial environment, it is imperative that products be marketed quickly and cost-effectively, particularly in industries such as consumer electronics and medical diagnostics. The subject of this patent application is the use of printed circuit board (PCB) manufacturing techniques that are widely available, reliable, cheap and well proven. Manufacturing easy-to-reconfigure microfluidic platforms using reliable, readily available, and low-cost manufacturing technology will enable wider uptake and accelerate lab-on-a-chip microchip device development and acceptance in many potential biomedical and other applications.

[0017] The attractiveness of PCB technology as an inexpensive, well-established, flexible and readily available manufacturing process for the development of microfluidic systems has already been recognized by researchers working in the field of more traditional continuous flow microfluidic systems. For example, researchers have previously demonstrated a number of continuous-flow microfluidic systems based on PCB technology, including a bubble detector, a pH control system, a micropump and a capacitive pressure sensor. PCB systems for the manipulation and dielectrophoresis of single cells have also recently been described, using a hybrid approach using printed circuit boards to integrate silicon-based microfluidic systems. However, there remains a long felt need for cheap, flexible and easily reconfigurable systems for handling droplets in discrete flow.

SUMMARY

[0018] The present invention is defined by independent claim No. 1.

EP 1 859 330B1

[0019] The devices and methods described herein provide a new way of using a substrate produced by a standard printed circuit board (PCB) manufacturing process to manipulate droplets in digital microfluidic systems. This unconventional application of well-known PCB manufacturing processes includes a variety of new aspects, including: (1) the use of copper traces and contact fields on PCBs as electrodes for manipulating droplets where droplet-like liquids are directed (i.e. transported) on the outer surface of the PCB substrate by electrical forces (as opposed to placing electronics on the board and directing the flow of electronic signals); (2) the use of the solder mask material as a dielectric electrode material acting as an insulator when manipulating the droplets with induced electric fields (as opposed to using a solder mask, as its name implies, to protect the copper traces from solder access); (3) using a photoformable liquid or dry solder mask to create physical structures that hold the liquids; (4) using through holes in the PCB to electrically connect the electrodes to drive the droplets with each other or with the pads; (5) use of solder mask filled ports inside the electrodes on the PCB to allow the electrodes to be tightly packed for droplet control; (6) filling the through holes inside the electrodes with conductive epoxy to allow the electrodes to be packed tightly without losing their conductive surface; (7) filling the through holes inside the electrodes with an optically clear epoxy resin to allow optical measurements to be made through the droplet; (8) applying copper traces adjacent to the electrodes on the same PCB surface to provide a common reference potential (in a coplanar configuration); (9) the use of copper embedded in the control electrodes and placed on the dielectric layer as reference electrodes; (10) using holes drilled in the printed circuit board to create a streaming interface enabling

ΕΡ 1 859 330Β1 liquid injection and discharge to / from PCB and (11) use of copper tracks as heating elements.

[0020] Thus, in accordance with the present invention, there is provided a droplet manipulation device, the device comprising:

(a) a printed circuit board substrate including a first side surface and a second side surface;

(b) an array of electrodes disposed on the surface of the first side of the substrate;

(c) a dielectric layer disposed on the surface of the first side of the substrate and formed to cover the electrodes; and (d) an electrode selector for dynamically creating an activation sequence of the electrode array whereby a droplet disposed on the surface of the first side of the substrate is electrically manipulated, the printed circuit board substrate comprising a series of openings extending from the first side of the substrate surface to the second side of the substrate surface, with each electrode hole is aligned with one of a series of holes in the substrate, creating a set of through holes in the device.

[0021] In another embodiment, the droplet manipulation device comprises a printed circuit board substrate including a first side surface and a second side surface. The control electrode array is disposed on the surface of the first substrate side, and the array of one or more reference elements connectable to a common reference potential is arranged at least substantially coplanar with respect to the steering electrode array. A layer of dielectric material is disposed on the surface of the first side of the substrate and formed to cover the control electrodes. The system is also provided with an electrode selector for dynamically creating an activation sequence of the electrode array whereby a droplet placed on the surface of the first side of the substrate is electrically manipulated.

[0022] In yet another embodiment, the droplet manipulation device comprises a printed circuit board substrate including a first surface.

ΕΡ 1 859 330Β1 side and the surface of the other side. The control electrode array is disposed on the surface of the first side of the substrate, an elongate reference element is also formed which is substantially parallel to the surface of the first side of the substrate and is spaced therefrom, thus defining a space between the reference element and the surface of the first side of the substrate, wherein this distance is sufficient to accommodate a droplet placed in this space. A layer of dielectric material is disposed on the surface of the first side of the substrate and formed to cover the control electrodes. The system is also provided with an electrode selector for dynamically creating an activation sequence of the electrode array whereby a droplet placed on the surface of the first side of the substrate is electrically manipulated.

[0023] In a further embodiment, the droplet manipulation device comprises a printed circuit board substrate having a first side surface and a second side surface, a control electrode array disposed on the first side surface of the printed circuit board substrate, and a dielectric material layer disposed on the first side surface of the circuit board substrate. printed and formed to cover the control electrodes. The apparatus also includes a second printed circuit board substrate including the first side surface and the second side surfaces, the second printed circuit board substrate being substantially parallel to the first printed circuit board substrate and spaced thereto to form a space between the second side surface of the second board substrate. printed circuits and the surface of the first side of the first printed circuit board, the distance is sufficient to accommodate the droplet disposed in the space. The control electrode array and the one or more reference elements are disposed on the surface of the second side of the second printed circuit board substrate. The device also includes an electrode selector for dynamically creating an activation sequence of the electrode array whereby a droplet disposed between the first side surface of the first printed circuit board substrate and the second printed circuit board substrate second side surface is electrically manipulated.

ΕΡ 1 859 330Β1

[0024] Methods for moving the droplet are also envisaged in connection with the device of the present invention.

[0025] In one instance, the method of moving the droplet comprises the step of placing the droplet on a substrate surface of the printed circuit board. The surface comprises an array of electrodes and a droplet is initially placed on the first of these electrodes adjacent the second electrode separated from the first electrode by the first gap. The method still includes the step of connecting the first electrode to a first voltage and the second electrode to a second voltage which is different from the first voltage so that the droplet moves towards the second electrode.

[0026] Another aspect of the method for moving the droplet comprises the step of placing the droplet on a substrate surface of a printed circuit board. The surface comprises an array of control electrodes and an at least substantially coplanar array of one or more reference elements, a droplet being placed on the first of the control electrodes. The method still includes the step of energizing the first control electrode to move the droplet from the first control electrode to the second control electrode.

[0027] In another embodiment, the method for moving the droplet comprises the step of placing the droplet between the substrate surface of the printed circuit board and an elongate reference member substantially parallel to and spaced from the surface of the printed circuit board. A surface of a printed circuit board contains an array of control electrodes and a droplet is placed on the first of these electrodes. The method still includes the step of energizing the first control electrode to move the droplet from the first control electrode to the second control electrode.

[0028] In yet another embodiment, the method of moving the droplet comprises the step of placing the droplet between the substrate surface of the first printed circuit board and the substrate surface of the second printed circuit board substantially parallel to and spaced from the surface of the first printed circuit board. The surface of the first printed circuit board includes

The control electrode array and the droplet are placed on the first control electrode and the surface of the second printed circuit board includes the control electrode array and the array of one or more reference elements. The method still includes the step of energizing the first control electrode to move the droplet from the first control electrode to the second control electrode.

[0029] In another case, the method of moving a droplet on the array of control electrodes on the surface of a printed circuit board without physically distinguished reference elements requires placing a droplet on the first of the control electrodes and moving it towards the second or third control electrode. The method then requires that the second and third control electrodes are connected to a suitable voltage such that the droplet will be directed towards the second or third electrode due to the non-uniformity of the electric field between them. In this case, the droplet may not move abruptly, i.e., from one electrode to the adjacent electrode, but will move continuously in a non-uniform electric field gradient towards or away from the target electrode due to a phenomenon known as dielectrophoresis.

[0030] Methods of combining two or more droplets together into a single droplet and dividing the droplet into two or more droplets are also provided.

[0031] In one instance, the method of combining two or more droplets into a single droplet comprises the step of arranging first and second droplets on a substrate of a printed circuit board. The surface includes an electrode array of at least three electrodes, including a first outer electrode, a center electrode adjacent the first outer electrode, and a second outer electrode adjacent to the central electrode. The first droplet is placed on the first outer electrode and is adjacent to the center electrode, while the second droplet is placed on the second outer electrode and also adjacent to the center electrode. The method also includes selecting one of the three electrodes as a target electrode and selecting two or more of the

There are three electrodes for activation or deactivation, depending on the selection of the target electrode. The method then includes the step of activating or deactivating selected electrodes to move the first or second droplets towards the remaining droplet or to move the first and second droplets towards each other whereby the first and second droplets combine to form a single, connected droplet on the target electrode. .

[0032] In another case, the method of dividing the droplet into two or more droplets comprises the step of arranging the starting droplet on a substrate of the printed circuit board. The surface comprises an electrode array, the electrode array including at least three electrodes including a first outer electrode, a center electrode adjacent the first outer electrode, and a second outer electrode adjacent the middle electrode. The exit blob is initially located on at least one of the three electrodes and is adjacent to at least one of the other electrodes. The method also includes the step of connecting each of the three electrodes to a first voltage to align the output droplet across the three electrodes. The method then involves connecting the center electrode to a second voltage different from the first voltage to divide the output droplet into first and second result droplets, a first droplet being formed on the first outer electrode and a second droplet being formed on the second outer electrode.

[0033] In yet another case, the method of dividing the droplet into two or more droplets comprises the step of arranging the starting droplet on a substrate of the printed circuit board. The surface includes an electrode array of at least three electrodes, including a first outer electrode, a center electrode adjacent the first outer electrode, and a second outer electrode adjacent to the central electrode. The exit blob is initially located on at least one of the three electrodes and partially overlaps at least one of the other three electrodes. The method then includes the step of connecting the center electrode to a first voltage to align the exit droplet such that the exit droplet at least partially overlaps the three electrodes. It still involves connecting the center electrode to

ΕΡ 1 859 330Β1 of the second voltage and at least one of the first and second outer electrodes to a third voltage, the second and third voltages being different from the first voltage, to divide the output droplet into a first and second result droplet, the first droplet being formed on the first outer electrode and a second droplet is formed on the second outer electrode.

[0034] It is therefore an object of the present invention to create and operate a device for the production and operation of droplet-based microfluidic systems, based on conventional printed circuit board (PCB) technology, where the droplets are moved by applying electrical potentials to electrodes formed on the PCB. .

[0035] The subject matter of the invention described herein has been defined above, while other objects related in whole or in part to the present invention will become apparent from the following description with reference to the accompanying drawings described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

Figure 1A is a top view and Figures 1B-1D are side views of an embodiment of the described invention showing a tightly coplanar arrangement on a printed circuit board with electrode through holes filled or unfilled.

Figure 2A is a top view and Figure 2B is a side view of an embodiment of the present invention showing a substantially coplanar arrangement on a printed circuit board with electrode through holes filled or unfilled.

Figure 3A is a top view and Figure 3B is a side view of an embodiment of the present invention showing an embedded coplanar arrangement on a printed circuit board with electrode through holes filled or unfilled;

ΕΡ 1 859 330Β1

Figure 4Α is a top view and Figure 4B is a side view of an embodiment of the present invention showing a parallel board array or a two-plane array on printed circuit boards with filled or unfilled electrode through holes.

Figure 5 is a perspective view of a droplet positioned on a dense electrode array with through holes on a printed circuit board for droplet manipulation in accordance with the present invention (liquid reservoirs not shown);

Figure 6 is a photograph showing a front view of an integrated circuit on a printed circuit board used to test droplet transport efficiency with different shapes and sizes of drive electrodes in accordance with the present invention;

Figures 7A-7D are photographs of different shapes of electrodes in accordance with the subject of the present invention.

Figures 8A-8B are photographs of an embodiment of the present invention showing, respectively, a front view and a rear view of a circuit structure on a printed circuit board realizing a three-phase droplet transporter and other droplet dispensing, storage, and mixing structures;

Figures 9A-9B are photographs of another embodiment of the present invention showing, respectively, a front view and a rear view of a circuit structure on a printed circuit board realizing the three-phase droplet transporter and other structures for dispensing, storing and mixing droplets;

Figure 10 is a graph depicting droplet transport characteristics (frequency versus threshold voltage) for various 1.5mm electrode shapes, in accordance with the present invention;

Figure 11 is a graph showing droplet transport characteristics (frequency versus threshold voltage) for various 1.0 mm electrode shapes, in accordance with the present invention;

ΕΡ 1 859 330Β1

Figure 12 is a graph showing the stability of the droplet transport voltage over time, in accordance with the present invention;

Figure 13 is a graph showing the minimum voltage required for droplet transport at a given switching frequency, in accordance with the present invention; and

Figures 14A-14D are photographs of time-shifted images demonstrating droplet transport and assembly in accordance with the present invention.

DETAILED DESCRIPTION

[0037] A printed circuit board (PCB), sometimes also called a printed wiring board, is a substrate used to interconnect electronic components with conductive fields and tracks formed on the substrate. Typically, PCBs are made by applying a layer of copper over the entire surface of the substrate, sometimes on both sides (a process called making a blank PCB), and then removing unwanted copper (e.g. by acid etching) after applying a temporary mask, leaving only the desired copper traces. Electrical connections (passages) between the opposite sides of the substrate can be created by drilling holes in the substrate, mechanically or by laser, and metallizing the inside of the holes to ensure continuous electrical connection between both sides. Multi-layer tiles can also be formed, obtained by bonding together separately processed substrates. The electrode traces in the copper layer are typically obtained by etching copper from a blank PCB in a subtractive process, however some plants use semi-additive or fully additive processes where the copper is deposited onto a substrate by electroplating or other techniques.

[0038] As discussed above, the digital microfluidic technique is a microfluidic approach in which discrete liquid droplets are manipulated by electrical forces on a substrate containing the electrode array. IN

ΕΡ 1,859,330Β1 of the commonly used configuration, the droplets are sandwiched between two parallel plates with the upper plate physically holding the droplets and the lower plate containing an array of individually addressable driving or driving electrodes (or elements) that are electrically mutually insulated. Typically, one or more reference electrodes (or elements) are also required to control the electric potential of the droplet. The reference electrodes can be placed on the same substrate as the control electrodes (coplanar) or on the opposite plate (biplanar). The space between the two plates surrounding the droplet generally remains open and can be filled with air or an immiscible liquid to prevent evaporation. Examples of immiscible liquids that can be used with water droplets include silicone oil, fluorosilicone oil, or hydrocarbon oils. When the reference electrodes and control electrodes are placed on the same substrate, the opposing plate is not part of the electrical circuit, but merely serves as a physically containment cover for liquids and may not be necessary for the correct operation of the instrument.

[0039] The movement of the droplets is achieved by applying a potential difference between the reference electrode and one or more control electrodes. The applied potential difference may be direct (DC) or alternating (AC) voltage, and the reference electrodes need not be physically different from the control electrodes. A droplet adjacent to an activated control electrode will be attracted to that electrode and will begin to move towards it. The electrodes may be sequentially activated according to a user-defined pattern (possibly using an electrode selector) to move the droplets along any path defined by adjacent electrodes. In addition to displacement, thanks to the appropriate design of the control electrodes and the use of appropriate patterns of their activation, other operations can be performed, including combining, dividing, deforming and dispensing droplets.

[0040] The digital micro fluidic processor essentially consists of an array of control electrodes with one or more reference electrodes. A complete chip can contain many other types of structures, including channels, liquid tanks, top plates, sensors, inlets, outlets, etc. The electrode system requires a network

ΕΡ 1 859 330Β1 of interconnection enabling electrical connection of specific electrodes and connection of electrodes with contact fields in order to connect external circuits. Previously, digital microfluidic integrated circuits were fabricated on a glass or silicon substrate using thin deposition and photolithography techniques borrowed from the field of semiconductor fabrication. Multi-level electrical connection systems replacing cabling were created by depositing and shaping successive layers of conductors and insulators on a single output substrate. The present invention now relates to a device and method enabling the advantageous construction of digital myrofluidic processors by a standard PCB manufacturing process, as opposed to traditional glass or silicon based processes.

[0041] The object of the present invention takes advantage of the ease with which multiple layers of conductors can be produced in a PCB manufacturing process as opposed to a glass or silicon based process. The reason for this is essentially the fact that in PCB manufacturing processes, the metal layers are produced on discrete substrates that are joined by a lamination operation at the end of the process, rather than produced sequentially on a single substrate layer.

[0042] The conductive path patterns are applied to the PCB substrate by subtractive electroplating, panel electroplating, pattern coating, or additive electroplating. In general, two or more wiring layers are required to perform complex droplet manipulation operations, necessitating the use of multi-layer wafers. Multilayer tiles are assembled by joining together several double-sided tiles or by sequentially applying several layers to tiles that do not require mechanical drilling (e.g., through holes are chemically etched or laser cut and then coated with a metal layer in non-electroplating processes). By definition, double-sided plates have conductive tracks on both sides of the plates, they can be further classified into plates without metallization of through holes and plates with metallization of these holes. Shallow

ΕΡ 1 859 330Β1 with metallized through holes can be further classified into plating metallized through hole plates and infill metallized through hole plates. In the case of plated through-hole metallization, the holes are metallized by copper cladding (e.g. by electroplating, plating without electric current, or a combination of both), and in the case of metallization of through holes by filling, the holes can be filled with a conductive paste such as copper, silver or conductive epoxy resin, etc.

[0043] In digital micro fluidic integrated circuits, through holes are drilled through the center of the control electrodes on one side of the multi-layer wafer to make electrical connections to the other side of the wafer. The drop trace is defined by the surface of the driving electrode. In order to obtain small droplet volumes, the surface area of ​​the control electrodes should be minimized. Since the through holes are drilled through the control electrodes, it is important to minimize the diameter of the through holes included in the electrode region diameter / track width. For this reason, through-holes play an important role in determining the minimum droplet volume obtainable in PCB systems. The PCB manufacturing industry aims to reduce the size of the through holes for another reason, namely to avoid blocking the paths of the paths and to maximize the PCB area available for the traces. Many processes use small through holes cut with an excimer laser. There are a number of varieties of multilayer processes used in the PCB manufacturing industry, including, but not limited to, Surface Laminar Circuits (SLC) in which through holes are formed by a photochemical process, DYCOstrate where through holes are simultaneously etched with dry plasma, Film Redistribution Layer (FRL), where the outermost dielectric layer is photosensitive, while the inner layers form a regular multi-layer plate, Conductive Adhesive Bonded Flex (Z-Link); Built-up Structure System (IBSS) where the dielectric is photosensitive; Sequential Bonding Cores / Any-Layer Inner Via-hole (ALIVH), where to drill

ΕΡ 1 859 330Β1 through holes, CO2 laser is used, and the holes are then filled with silver paste; Carrier Formed Circuits, where individual circuits are prepared on stainless steel supports and then laminated with FR-4 material; Roli Sheet Buildup, where one-side epoxy coated films are then laminated by hot rolling and pressure application; and Sheet Buildup, which is similar to the Roli Sheet Buildup process but laminates to double-sided or multi-layer circuits. In one embodiment, using a multilayer wafer (Z-Link) for the production of microfluidic systems, multilayer flexible wafers consisting of copper foils coated with a polyimide layer can be laminated and then placed on a rigid wafer to form a multilayer wafer. In this case, the holes in the individual flexible layers can be punched, laser-drilled or plasma-drilled. The openings connecting the different layers can then be filled with conductive adhesive.

GENERAL DESCRIPTION OF EXECUTION

[0044] With reference to Figures 1A-1D, 2A-2B, 3A-3B, 4A-4B, and 5, the requirements to be met in order to adapt PCB substrates for droplet manipulation will now be discussed in more detail. As will be discussed in more detail below, Figures 1A-1D refer to a digital microfluidic chip 10 on a PCB having a closely coplanar arrangement on the PCB with filled or unfilled electrode through holes; Figures 2A-2B refer to a digital microfluidic PCB integrated circuit 20 having an essentially coplanar arrangement on the PCB with filled or unfilled electrode through holes; Figures 3A-3B refer to a digital microfluidic PCB chip 30 having an embedded coplanar on the PCB with filled or unfilled electrode through holes; Figures 4A-4B relate to a digital microfluidic chip 40 on a PCB having a parallel or biplanar wafer arrangement on the PCB, filled or

EP 1 859 330B1 unfilled through holes in the electrodes; and Figure 5 depicts a droplet positioned on a dense electrode array with through holes in the PCB for droplet manipulation. Figure 5 shows in general terms the concept of the present invention where liquid samples are digitized into single, discrete droplets D, which can then be independently dispensed, transported, incubated, detected or reacted with other droplets (digital micro-fluidic approach).

[0045] In each of the embodiments shown in Figures 1A-1D, 2A-2B, 3A-3B and 4A-4B, as will be individually described in more detail below, there is a PCB substrate 12 having an upper surface 13 of the first side and a lower surface 14 second page. Control electrodes 16 (or elements), such as copper track electrodes, may be placed on the top surface 13 of PCB 12, and reference electrodes (or elements) 18, such as a copper track or parallel plate reference electrode, may be placed in a variety of configurations to allow manipulation of the droplets. A solder mask such as a photoformable liquid (LPI) solder mask is used as an outer layer in traditional PCB manufacturing processes to protect the copper traces from etching or galvanization or, finally, from the effects of solder when arranging electronic components on the board. However, in an application for controlling the movement of droplets in accordance with the present invention, this outer layer is an insulator 22 which serves to isolate the droplets from the potentials applied to the control electrodes and reference electrodes 16, 18. The control electrodes 16 are completely insulated by the insulator 22, preferably being LPI solder mask or other type of temporary solder mask containing a dielectric. Complete insulation means that the control electrode 16 is dielectric coated on all sides, including the edges. Insulator 22 (LPI solder mask) is applied using conventional processes including but not limited to curtain coating, spin coating, spray coating, or screen printing. In the event that a reference electrode 18 is desired, some of the copper may remain exposed and

ΕΡ 1 859 330Β1 uninsulated to ensure that the droplets are in direct contact with the reference potential. The exposed portion lies as close to the control electrodes 16 as possible in PCB manufacturing processes, this is determined by the resolving power with which the copper structures are formed and the resolving power with which the solder mask is applied and positioned relative to the copper layer. . The exposed portion of the reference electrode 18 can obtain an optional conductive surface finish, which is typically silver, gold, or non-electrolytic nickel plating and gold plating (EN IG process).

Base material

[0046] As discussed above, the electrostatic microfluidic devices of the present invention include a substrate plate 12 which can be made of almost any material commonly used in PCB production. These materials include, but are not limited to, FR-2, FR-4, FR-5, polyimide, Kapton, Rogers, Duroid, BT, cyanate esters, and ploitetrafluoroethylene (PTFE). Rigid, rigid flexible, or flexible plates may be used as substrate materials for the manufacture of these devices.

Forming the electrodes

[0047] An outer copper conductive layer of the PCB is formed to form the drive electrodes required to allow manipulation of the droplets with electric fields. The control electrodes 16 can take a wide variety of shapes and forms depending on the particular application. For example, square-shaped electrode arrays, circular-shaped electrode arrays, hexagonal-shaped electrode arrays, star-shaped and other electrode shapes, interlaced or comb-shaped, and elongated electrodes can be used. The reference electrodes 18 may also be formed in the same conductive layer or layer

ΕΡ 1 859 330Β1 separate on the same substrate (coplanar) or on a separate substrate (biplanar).

In one embodiment, as shown in Figures 1A-1D, reference electrodes 18 may be formed in the same copper conductive layer as the control electrodes 16 and the insulator 22 is removed from above a portion of the reference electrodes 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, reference electrodes 18 may be positioned next to or between control electrodes 16.

[0049] In another embodiment, as shown in Figures 2A-2B, the reference elements 18 may be formed as a discrete conductive layer disposed directly on the insulator 22. The conductive layer may be a thin metal foil deposited by a vacuum process, a non-electrolytic plating, electroplating process. or by other means to form corresponding reference elements 18. The reference elements 18 may have a variety of shapes and forms and may be located either directly above and / or next to the controls 16 (i.e. the reference elements 18 need not be precisely aligned with the controls 16). In one arrangement, the references 18 may form a lattice or network of conductive lines positioned above the controls 16. In this arrangement, the reference elements 18 can shield electrically control electrodes 16 at those points where they overlap, so that the mutual overlap should ideally be minimized in the step of sizing and positioning the reference elements 18 relative to the control elements 16. In another system, the step of the reference grid is chosen smaller than the step of the electrode network, but not by an integer factor. This arrangement of a separate conductive network can be accomplished by using additive PCB metallization processes in which metal is deposited on the insulator 22, or alternatively in a subtractive process in which reference elements 18 and control elements 16 are formed on opposite sides of a thin flexible substrate. In the latter case, the flexible base of the circuits plays the role of insulation between the control elements 16a

The flexible system can then be laminated and bonded to a rigid substrate providing mechanical rigidity and allowing electrical interconnections between the electrodes to be made.

[0050] In a further embodiment, shown in Figures 3A-3B, the reference elements 18 may be arranged in an embedded coplanar with control elements 16. In such an arrangement, the through-holes 25 together with the metal shell 26 may function as reference elements 18 in areas not covered by the insulator. 22. There may also be other ports 24 with a metal shell 26 in the system, coated with insulator 22 and functioning as described below.

[0051] In another embodiment, shown in Figs. 4A-4B, the reference elements 18 may be placed on a separate substrate having the form of a parallel plate. Typically, the substrate containing the control electrodes 16 and the substrate containing the reference elements 18 are positioned opposite to each other and separated by a space G, which contains the liquid, thereby forming a multilayer structure. An additional embodiment with parallel plates may include two opposing surfaces which are themselves electrostatic microfluidic systems on the PCB (the top plate may be a PCB having a first side 13 'top and a second side 14' bottom surface) and have control elements 16 on both surfaces. and reference elements 18 on at least one surface.

[0052] Since very little current is sufficient to charge the drive electrodes 16 and manipulate the droplets with electric fields, the conductive material constituting the electrodes may have a much greater resistance than acceptable materials in typical PCB applications. Therefore, many types of materials in addition to copper can be used in this case. They also include materials normally considered unsuitable for creating conductive areas and paths on PCBs. Likewise, the conductive layer may be much thinner than is commonly used for PCBs. Ideally, the conductive layer should be as thin as possible in order to minimize the topography of the conductive elements which must then be covered with an insulating layer. Additionally, minimizing the thickness of the conductor

EP 1 859 330B1 increases the planarity of the PCB surface which is desirable for consistent and reliable manipulation of the droplets on that surface. Conductor thickness may be minimized by using a substrate stock material with a minimum conductor thickness (e.g.,% ounce or 5 pm copper film) or by adding a polishing or etching step to reduce the conductor thickness prior to depositing the insulator layer.

Connections between the electrodes and through holes

[0053] The conductive tracks on the PCB substrate 12 are used to make electrical connections to the control electrodes 16 and reference elements 18. Each control electrode 16 or reference element 18 may be connected to one or more control electrodes 16 or reference elements 18, other components. placed on the same PCB substrate 12 or to areas that allow external connections. In one embodiment, the external connection areas are arranged along the edges of the PCB and the PCB is adapted for use in slot 28 of the edge card connector (see Figures 8A-8B). In another embodiment, an array of conductive areas is disposed on the surface of the PCB, and contact with these areas is provided by spring loaded pins, test clips, or an anisotropic conductive material strip 29 (see Figure 9A). In yet another embodiment, the pin head, socket connector, or other discrete electronic component is connected to the PCB to facilitate connections to the external circuit.

[0054] As shown in Figures 1A-1D, 2A-2B, 3A-3B and 4A-4B, the electrical connections between the various conductive layers of the substrate 12 can be made by methods known in the PCB art in which the holes or through holes 24 are drilled into the substrate 12 from the side of both conductive regions (upper surface 13 and lower surface 14) on each side of the substrate 12 at the point where the electrical connection is to be made. While the through holes 24 are shown as circles in the figures, it is evident that

They can be of any shape, such as square, oval, etc., that can be formed into the substrate material 12. The interior of the opening 24 may also be metallized by non-electrolytic plating, electroplating, or other methods to form plating 26 (metallization of the inside of the through hole), so that a continuous electrical connection is formed between the opposite sides of the plate at the location of the through hole. As mentioned above, instead of metallizing the interior of the through hole, conductive pastes (metallization by filling the through hole) can be used to ensure the continuity of the electrical connection.

[0055] Several different approaches are available for establishing the electrical connections between electrodes and traces. In one, a wire or track is from the electrode on the same side of the PCB, then, if necessary, a track may be led to the other side of the substrate through a through hole remote from the electrode. In another approach, ports are made through the electrodes. In this case, it may be necessary to fill or cover the hole to prevent liquid from flowing out or evaporating through the hole. Through hole 24 may be closed with a metal coating by non-electrolytic or electroplating technology, and may be filled using various techniques and materials (conductive epoxy, non-conductive epoxy, clear epoxy or any other material). After the through holes are filled with any of these filler materials, the surface of the PCB can be copper plated using non-electrolytic technology or electroplating to completely close the opening for droplets moving across the surface.

In one approach, the hole to be drilled has a diameter so small that an insulator embedded in a liquid form, such as a conventional solder mask, cannot penetrate it due to viscous or surface tension phenomena, or it can be made large enough so that the solder mask can penetrate it to form a through hole 24 filled with the solder mask '(see Figure 1B). Alternatively, an additional process step can be used by filling

There are openings with epoxy or similar material prior to embedding the insulator, thus forming a through hole 24 filled with epoxy resin (see Figure 1C) or a through hole 24 'filled with clear epoxy resin (see Figure 1D). Another approach is to use a dry insulation layer that covers the opening, effectively covering and sealing the surface of the system. A possible disadvantage of some of these approaches is that they result in a non-conductive region within the conductive electrode which reduces the area of ​​the electrode that can be used to generate an electric field. To overcome this problem, several techniques are available for producing conductive fillings, including the use of conductive epoxy resins to fill the hole and the use of electroplating or electroplating technology to create a conductive surface covering the non-conductive filler material. Another solution is to electroplate the hole so that it is completely filled with metal. This approach may require a planarization step to remove excess metal deposited on the surface of the substrate by over-electroplating. The planarization and control of the thickness of the conductor layer on the substrate surface can in this case be simplified by the use of an island electroplating process in which an additional amount of metal is deposited only in the area surrounding the through hole. The resulting islets can then be removed by polishing the PCB surface. In this method, quite significant amounts of metal can be deposited inside the holes without increasing the final thickness of the metal layer on the PCB surface.

Electrode insulation

[0057] Still referring to Figures 1A-1D, 2A-2B, 3A-3B, and 4A-4B, the control electrodes 16 are typically electrically insulated by an insulator 22 to prevent direct electrical current from flowing between the electrodes and a conductive liquid when applied to it. constant potential (DC) control electrodes. It should be noted that in order to manipulate the droplets with induced

ΕΡ 1,859,330Β1 electric fields, alternating potentials (AC) can also be applied to the control electrodes. While any dielectric material can be used, traditional PCB manufacturing processes typically employ a solder mask to protect the copper traces on the PCB and expose the copper only where electronic components will later be soldered. The most direct approach to insulating control electrodes 16 is to use the solder mask material (or other dielectric) as the electrical insulator 22. Both liquid solder masks and dry film masks are suitable for use as electrode insulators 22. Photoformable solder masks are generally preferred as they can be easily molded to provide electrical access to the reference elements 18 or the contact fields beneath the insulator layer 22.

[0058] Solder masks are available in two varieties: photoformable liquid masks (LPI) and dry film solder masks (DFSS). LPI masks are not conformal. DFSS materials provide near vertical sidewalls and are described for use in making electroplating molds, sealing liquid channels, and as masks for cleaning microchannels by powder blowing. However, DFSS materials have not been used to form liquid reservoirs or as sealing materials to form supports or seals between two parallel plates as envisaged in the present invention.

[0059] In some applications, it may be found that there are no solder mask materials with the desired combination of thermal, mechanical, electrical or optical properties. In such cases, the solder mask materials may be substituted for or combined with other types of insulating materials. For example, spin coating materials such as polyimide, dip coating, spin coating or sputtering or brush application materials such as TEFLON® AF or Cytopp vacuum sputtered or sputter coated materials can be used on PCB substrates. such as silicon dioxide, and polymers such as poly (para-xylylene) (Parylene).

EP 1 859 330B1

[0060] As an alternative material to the solder mask for forming the insulators 22, thin layers of parylene deposited by a vapor deposition process may be used. Parylene is the technical name for the family of poly (para-xylylene) polymers, it includes pariylene C, D and N. The term parislene as used herein refers to any composition of poly (para-xylylene) or a mixture of such compounds. The main advantage of Parylene is that it can be deposited as an even layer with a thickness much less than solder masks, both LPI and DFSS. In PCB manufacturing processes, LPI masks can be deposited as 0.5 mil layers (1 mil = 25.4 µm), while Parylene can be deposited as a pore-free layer as low as 0.5 µm. Such a thin insulator layer reduces the potential required to move the droplet. In some applications, the dielectric should be etched to expose the copper electrodes. Parylene can be etched by reactive ion etching, plasma firing, chemical etching or laser ablation. Alternatively, the Parylene may be selectively deposited by masking areas to remain exposed with tape (e.g., 3M® Mask Plus II Water Soluble Wave Solder Tape No. 5414, which is used to mask the gold contacts on the PCB during wave soldering). Other representative examples of materials that could be used as dielectrics include silicones, polyurethanes, acrylics, and other dielectric materials applied by spin coating or deposited.

[0061] In general, it is desirable to minimize the thickness of insulator 22 in order to reduce the voltage required to induce fluid movement.

Support layers

[0062] It is also envisioned that additional layers of solder mask material may be deposited and formed on the PCB surface to create physical structures such as cavities and channels (not shown in the figures) used to collect and direct the liquid flow.

ΕΡ 1 859 330Β1

ADDITIONAL PROCESSES

A combination of subtractive and additive processes

[0063] In a further embodiment of the manufacture of the PCB droplet manipulation systems of the present invention, a combination of subtractive and additive processes may be used. Subtractive processes can be used to produce a multilayer wafer that defines the waveform and all electrode connections that control the movement of the droplet. A formable dielectric layer can then be applied to such a plate. Through holes can be made in this dielectric by means of laser drilling or a photolithography process using masks. In one embodiment, LPI material may be used as the dielectric. The electrode area exposed in the aperture may optionally be plated to form flush with the dielectric surface. At this point, an additive process can be used to form all the electrodes, using non-electrolytic copper deposition for this purpose, as smaller track spacing can be achieved with this process.

Post-processing

[0064] The finishing of the device may involve a combination of standard PCB manufacturing processes and custom processes. For example, in one step, a hydrophobic coating may be applied to the finished PCB to facilitate droplet transport. In addition, the use of a solder mask as a dielectric may be undesirable for some applications, in which case uninsulated PCBs may be coated with special materials not found in standard PCB manufacturing processes. However, even in such cases, the use of PCBs as the starting substrate and PCB manufacturing processes to form conductive paths still retains many, if not most, of the advantages of a fully compatible PCB manufacturing process.

[0065] In one embodiment, all the conductive tracks required for the electrical connections may be formed on the multi-layer.

ΕΡ 1 859 330Β1

PCB. Some or all of the outer copper layers can then be removed by polishing or chemical etching. Such a PCB, containing all the connections required to manipulate the droplets, can then serve as a substrate for downstream processes to form control and reference electrodes with smaller track spacing. Control electrodes can be formed using techniques used in semiconductor fabrication, including thin film deposition and photolithography, to achieve tight spacing between tracks.

Coating of coplanar reference elements

[0066] In an embodiment in which the reference electrodes 18 are formed in the same layer as the control electrodes 16 (see, for example, Figures 1A-1D), a distinct recess may appear in the LPI solder mask as the mask only covers the electrodes. control, leaving the reference electrodes exposed. This recess may compromise operational reliability as the droplet may not be in contact with the reference element. In this case, the reference electrodes may be additionally coated so that the surface of the reference element forms flush with the LPI solder mask (situation not shown in the figures). This coating step may be carried out prior to surface finishing with copper or nickel.

Reference electrodes on the outer surface

[0067] In one embodiment, after forming the copper electrodes as described above, the LPI coating may be used as a dielectric interlayer, and then another copper layer may be formed over the LPI layer to form the reference electrodes. The dielectric can also be a thin (2 mils or less) prepreg plate in a typical multilayer PCB design, or it can be a flexible plate with copper structures embedded in it to serve as reference electrodes on the outermost

ΕΡ 1 859 330Β1 layer. The copper layer directly beneath this outer copper layer then has structures forming the control electrodes.

Embedding of electronic circuits and detectors on PCBs

[0068] In a further embodiment of the invention, it is envisaged that the PCB of the system object of the present invention may also include electronic components disposed in areas which are not used for liquid manipulation. Electronic components may include microcontrollers, relays, high voltage multipliers, voltage converters (DC-DC for boosting voltage, DC-AC, AC-DC, etc.), electro-optical components such as LEDs, photodiodes, photomultiplier (PMT), heating elements, thermistors, resistance temperature detectors (RTDs) and electrodes for electrochemical measurements. Copper traces can also be used to measure droplet impedance. Resistive heating elements are realized in the form of meandering copper tracks, and their resistance characteristics will depend on the dimensions of these tracks. In one embodiment, a PCB comprising an optical detector, such as a photomultiplier or a photodiode, can be used as a parallel board to form a sandwich structure with the PCB containing the droplet manipulation system. In another embodiment, the gold-plated electrodes obtained from standard PCB manufacturing processes can be used for electrochemical measurements.

Openings constituting the entrance and exit of the jet system

[0069] Mechanically tapped holes are typically used to attach the PCB to another surface. It is further envisioned that in the PCB microfluidic systems of the present invention, these openings may be used as microfluidic system input / output ports for introducing and removing liquid on and off the PCB substrate surface. It is further envisaged that these openings are capable of engaging a fluid source including, but not limited to, flexible tubing, syringes, pipettes, glass capillaries, intravenous tubing, or microdialysis probe. Fluid flow in

ΕΡ 1,859,330Β1 of these tubes may be forced by pressure or other factors. The continuous flow of liquid in the tubes can be connected to the PCB through said openings, this flow can be divided into discrete droplets either directly or via an intermediate reservoir formed on the PCB.

For example, in one embodiment, metallized orifices (see, for example, holes 32 in Figures 9A-9B) may be positioned adjacent the control electrodes for use as fluid input / output ports, bringing liquid to the surface of the electrodes, or discharging liquid. it from this surface. In another embodiment, non-metallized holes (see, for example, holes 34 in Figures 9A-9B) may be positioned for introducing and draining liquids and may be connected to a channel etched in a solder mask that then leads to a reservoir (not shown in the figure). The reservoir may have electrodes for dispensing, for example by employing electric field-mediated droplet dispensing techniques. In yet another embodiment, the metallized holes in the jet system entrance / exit may be dielectric coated and have additional concentric ring electrodes around the holes. In this case, the droplets may be dispensed radially from the opening by pressing the liquid through the opening and then using the electric field to dispense the droplets onto the electrodes. In an additional embodiment, the openings can be used to drain liquid into a waste container or other container outside of the integrated circuit by collecting droplets in the region of the opening and allowing them to drip by gravity into a container positioned below the opening.

Extracting droplets out of the plane from through holes

[0071] Generally, the droplets placed in the device of the present invention are manipulated within a horizontal plane in a sandwich structure formed by one or both boards containing PCB arrays. In another embodiment, holes drilled in the PCB may be used to extract droplets from the sandwich structure in a vertical plane. Droplets

ΕΡ 1 859 330Β1 can be extracted through the holes in many different ways. One method uses the pressure difference between a droplet bounded by a sandwich structure and a large opening, the droplets could be passively forced through an opening larger than the droplet diameter by simply placing a droplet under the opening. The droplets could also be extracted by electric forces where an additional plate would be added to the layered structure and the droplets would be drawn from one layered structure to another newly formed structure by applying an appropriate electric potential. Here, in order to simplify the droplet extraction process, a layer structure may be formed between the PCB coplanar substrate and another substrate containing electrodes. While the two plates are planar-parallel, the droplets will only touch the coplanar PCB substrate and will travel vertically to the second substrate only when an electric potential is applied to this substrate, which will pull the droplet out of the plane by electrostatic interactions. The droplets can also be moved vertically by gravity after they are extruded onto another plate. Applications for such vertical droplet displacement include squeezing DNA or proteins. Extraction of droplets through such openings can also be used to increase the path length in absorbance measurements and to transport liquid to another layer structure to allow transport within another layer.

Synthesis and biochemical analysis

[0072] A number of biochemical reactions can be performed by manipulating liquids on the PCB substrate as described herein. As described herein, the present invention provides an apparatus for detecting a target analyte in sample solutions by optical and electrical detection means. The sample solution may contain any number of components, but not limited to, body fluids (including, but not limited to, blood, sweat, tears, urine, plasma, blood serum, lymph, saliva,

ΕΡ 1 859 330Β1 anus and vagina, semen derived from essentially any organism, with mammalian samples being preferred, particularly human samples preferred; food and environmental samples (including, but not limited to, air, agricultural, water, and soil samples); samples of biological weapons related agents; research samples; purified samples such as purified genomic DNA, RNA, proteins, cells etc .; raw samples (bacteria, viruses, fungi, etc.). The types of analysis that can be performed on the PCB substrate described herein include enzymatic analysis, DNA amplification by isothermal or thermocycling, immunity testing, including layered and homogeneous systems, and cell count-based analyzes using optical and electrical detection methods. Analytes that can be measured in biological samples include metabolites, electrolytes, gases, hormones, cytokines, peptides, DNA and RNA.

[0073] In one of the processes using the device of the present invention, a physiological sample of human origin can be introduced into a PCB reservoir. The reservoir may be formed by a dry film solder mask. The sample can then be dispensed in the form of droplets which will be mixed with the appropriate reagent droplets placed on the PCB or incorporated into the PCB. The course of some enzyme assays can be optically monitored (e.g. by measuring absorbance, by reflectometry, fluorescence or luminescence). In the case of absorbance measurement, the through-holes can be filled with an optically transparent material so that light passes through a droplet positioned on one of the openings allowing the absorbance to be measured.

[0074] In other processes, biochemical samples may also be synthesized on a PCB substrate using the droplet manipulation technique described herein. For example, on a PCB, a whole series of protein-containing droplets can be dispensed from a reservoir and mixed with various reagents, and then incubated to automatically find the crystallization conditions for a given protein.

ΕΡ 1 859 330Β1

Transport between the side walls

[0075] In a further process, copper tracks with a thickness of the same order as the droplet height may be used such that the droplet is contained between tracks lying on the same substrate and covered with an insulator. The droplet is moved by electric fields applied mainly in the plane of the substrate, not perpendicular to it. Unlike the coplanar system, where the droplet rests on coplanar control and reference electrodes, and in a parallel-plate system where the droplet is sandwiched between control electrodes on a substrate and a common reference electrode on a second, parallel substrate, in this structure the droplet is sandwiched between the coplanar electrodes: control and reference.

SPECIFIC DESIGN

[0076] While the general embodiments of the present invention have been discussed above, a more specific embodiment of making an apparatus for manipulating micro-volume liquid samples, the apparatus comprising a printed circuit board substrate, will now be discussed.

[0077] In a preferred embodiment, the FR-4 substrate is laminated on both sides with a% ounce (~ 9pm) of copper foil. 8 mil through holes are drilled into the substrate. These holes are then plated with copper by electroplating and filled with a solder mask or epoxy resin. It is preferable to coat the holes with copper to a thickness of about 5 microns by an island electroplating process in which only the through holes are galvanized while the remainder of the plate is covered with a mask. The islands are mechanically planarized and then the through holes are filled with a solder mask or non-conductive epoxy. Upon completion of the processing of the holes, a plating less than 5 µm thick is applied. In the event that unfilled through holes are required, a further drilling step may be performed to obtain the unfilled through holes which are electroplated if necessary. On

At this stage, a designed pattern of electrodes with a minimum distance between the 2 mil tracks is transferred to the copper coating, the pattern is etched through the mask. An approximately 0.5 mil thick LPI solder mask is formed. Finally, by laminating and forming the dry film solder mask, physical structures (cavities or channels) are formed that retain the liquid and serve as material retainers. In other embodiments, the retention layer may be obtained by applying one or more layers of a LPI solder mask, or by applying by lamination followed by etching the copper foil.

EXPERIMENTAL RESEARCH AND RESULTS

[0078] Experiments have been carried out in which the double layer single board electric field drop manipulator structure described herein was presented to a PCB manufacturer for commercial electronics and tested upon completion. The design consisted of various shaped control electrodes for transporting and mixing liquid droplets, and specialized shaped electrodes for dispensing droplets from a larger volume of liquid. The electrodes were connected to the contact areas by conductive tracks formed in the same copper layer on the PCB substrate. Where necessary, tracks were led to the other side of the wafer using conventional ports located remote from the control electrodes. Several different layouts and several different wiring diagrams were tested.

[0079] Some arrays contained multiple copies of a single linear array of electrodes where corresponding electrodes in each copy of the array were connected to the same electrical signal - thus multiple identical circuits could be controlled simultaneously. Other systems included an electrode bus, "a transporter structure, in which every fourth electrode in the linear array of control electrodes was connected to the same control signal.

ΕΡ 1 859 330Β1

The use of such a structure makes it possible to control any long transport route using a predetermined number of control signals. Multiple droplets can be placed on or removed from a bus and transported synchronously. The contact areas were arranged along one side of the PCB and were designed to be connected either with a standard edge connector or using a standard SOIC test clamp.

[0080] Figures 6, 7, 8A-8B and 9A-9B illustrate several examples of integrated circuits manufactured for experimental purposes. Figure 6 shows the front side of the PCB with the system used to test the transport efficiency of droplets with different electrode shapes (round 16a, square 16b, low curvature star 16c, higher curvature star 16d) (see Figure 7) and different drive electrode sizes (results are discussed in discussion). based on Figures 10-12 below). The integrated circuit shown in Figure 6 includes 16 different electrode arrays in a linear array. Figures 8A and 8B show front and rear views, respectively, of the structure of the system forming the three-phase droplet transporter and other structures for dispensing droplets from a reservoir included in the chip for storing and mixing the droplets. Through holes 24 are used to route electrical signals from the rear side of the PCB to the control electrodes on the front side, the electrical contact providing an edge connector socket 28 located along one side of the PCB. Figures 9A and 9B show front and rear views, respectively, of the structure of the system forming the three-phase droplet transporter and other structures for dispensing droplets from the input / output port 32, for storing and mixing the droplets. The through holes 24 are used to route electrical signals from the rear side of the PCB to the control electrodes on the front side, the electrical contact providing an array of conductive areas connected using the SOIC test terminal 29.

[0081] The designed drive electrode array has a pitch of 1.0 mm or 1.5 mm and a nominal 2 mil spacing between adjacent electrodes. The substrate material was FR-4 with a% ounce copper film. The copper remained

EP 1 859 330B1 is formed to form control electrodes, tracks and contact areas. The nominal minimum widths / distances from the process used could be 2 mils / 2 mils, this being the spacing between adjacent electrodes and the paths between the electrodes and the contact areas. A CARAPACE® EMP 110 brand of photoformable liquid solder mask (available from Electra Polymers & Chemicals, Ltd.) was used as the electrode insulator. The nominal thickness of the solder mask insulator was 0.6 mils. After receiving the PCB from the manufacturer, a thin hydrophobic coating of TEFLON® AF material was applied to the upper surface of the system. TEFLON® AF was applied to the PCB surface by a spin coating process with a 1% solution in FC-75 solvent at 3000 rpm. for 20 seconds followed by a 30 minute cure at 150 ° C.

[0082] The PCBs were assembled in a sandwich configuration with an indium tin oxide coated glass top plate. The top plate was also coated with a thin layer of TEFLON® AF so that all internal surfaces in contact with the droplet were hydrophobic. The conductive indtin oxide layer on the top plate was used as a reference electrode. The PCB and the top board were separated by a slot about 0.8 mm wide. One or more electrolyte droplets (0.1 M KCI) were injected into the sandwich structure and deposited on a control electrode. The droplet volume was sufficient to cover a single electrode and was approximately 2.2 µl for 1.5 mm pitch electrodes and 1.1 µΙ for 1 mm electrodes. The remaining volume of the space between the two plates was filled with air or a low viscosity silicone oil (1 cSt).

[0083] Referring to Figures 6, 7 and 10-12, droplet transport studies were performed by sequential activation of drive electrodes as described in US Patent No. 6,911,132 and U.S. Patent Application Publication No. 2004/0058450, both by Pamula et al. Using a PCB similar to that shown in Figure 6, tests were conducted for 4 different electrode shapes (round 16a, square 16b, star-shaped, low curvature 16c,

EP 1 859 330B1 stellate with higher curvature 16d) (see Figure 7) for each of the two electrode sizes (1.0 mm and 1.5 mm pitch).

[0084] For each electrode size and shape, the maximum rate at which droplets could be transported between adjacent electrodes as a function of the applied voltage was determined as shown in Figures 10 and 11. The droplets could be successfully transported at voltages below 40V (for size electrodes 1.0 mm), and the transport speed increased with increasing voltage above the threshold. Higher voltages were required to move the droplets than those previously reported for other systems, as in this case a greater thickness solder mask insulator was used. For example, the solder mask insulation is approximately 16 times thicker than the insulation used in previously manufactured microcircuits, therefore four (4) times the voltage is required, which is in line with the formula for electrostatic energy (1 / 2CV²) for this transport mechanism.

[0085] As expected, above the initial threshold voltage, the transport speed, and consequently the maximum speed at which the droplet could be moved, increased with increasing voltage. The range of the tested voltages was 0 - 200 V for 1.5 mm electrodes and 0 - 100 V for 1.0 mm electrodes, and the droplet transport rate was observed up to 24 Hz. The curves obtained as a result of the tests showed the expected general shape - the higher the applied voltage, the higher the possible transport frequency. However, the curves for the 1.5 mm electrodes (Figure 10) were not very smooth and showed a clear influence of the shape of the electrodes. In contrast, the curves for the electrodes of 1.0 mm (Figure 11) are quite predictable and show little dependence on the shape of the electrodes. In addition, a scaling effect was observed, as the threshold voltage for the electrodes of 1.0 mm was, at the same frequencies, 10-20 V lower than for the 1.5 mm electrodes.

[0086] As shown in the graphical representation in Figure 12, further tests were performed to determine the stability of the droplet transport process over time. In a program-controlled cycle, the droplets circulated in an arrangement of four 1.5 mm square electrodes with the minimum voltage required

ΕΡ 1,859 330Β1 for transport holding at 4 Hz or 8 Hz switching frequency. The voltage necessary to maintain transport continuity was tested and regulated at five-minute intervals. Tests conducted for an hour or more have shown a general trend of increasing the required voltage over time, possibly due to deterioration of the insulator properties over time and contamination of its surface. However, in each case over 20,000 droplet transport cycles were performed over the course of the experiment.

[0087] With reference to the graph shown in Figure 13, tests were also performed to determine the minimum voltage required to induce the droplet transport phenomenon at a given switching frequency. Digital microfluidic systems of both open design (i.e., coplanar, no top plate) and constrained (i.e. biplanar, top plate) designs were used on PCBs (see Figures 1B and 4B, respectively). Electrodes (1.5 x 1.5 mm²) were formed into copper reaching a final thickness of ~ 25 µm. A 150 µm diameter through hole is drilled in each electrode to provide an electrical connection to the back of the plate. Earthing bars were formed along all of the control electrodes to ensure the continuity of the droplet connection to the ground, and a photoformable liquid (LPI) solder mask (~ 17 pm) was applied to act as an insulator, exposing only the rails. The only step in the final processing of the system was to apply a TEFLON® AF coating with a brush to make the surface hydrophobic. Droplets of a polarizable, conductive liquid (1M KCI) were transported in both open (coplanar) and confined (biplanar) systems. In the open system, each droplet had a volume of 6 µl, a small drop (2 µl) of silicone oil was added to it which surrounded the droplet. In the case of the limited system, the volume of individual droplets was 2.5 µl, and for ease of transport, the entire chip was filled with silicone oil. [0088] For both systems, the minimum droplet displacement voltages required for efficient droplet transport at frequencies from 1 to 32 Hz were measured. As shown graphically in Figure 13, the operating voltages for the droplets in a confined (biplanar) and open (coplanar) pattern

EP 1 859 330B1 varied between 140-260V and 125-220V, respectively, depending on the switching frequency used. This seems to suggest that the movement of the droplets is facilitated in the absence of a limiting top plate, possibly due to a lower drag force acting on the droplet in an unrestricted configuration. Droplet electrolysis, usually caused by inadequate coating of the surface with an insulator, was not observed when a LPI solder mask was used as the insulator, up to the maximum tested voltage of 350 V. However, electrostatic charging was observed above the voltage of 300 V.

[0089] In Figs. 14A-14D, individual sequences of time-shifted images are shown, illustrating a top view of droplet transport and mixing. Figures 14A-14B show droplet transport and mixing, respectively, for droplets bounded by an upper plate (600 µm) (biplanar arrangement). Figures 14C14D show droplet transport and mixing, respectively, for droplets in an open (coplanar) system. Mixing was performed at a switching frequency of 8 Hz and was completed in 5 seconds for 2 2.5 µM restricted droplets and 1.8 seconds for two 6 µM droplets in the open system. Thus, the mixing rate (volume per unit time) observed in an open (coplanar) system is almost seven times greater than in a confined (biplanar) system. This improvement in mixing efficiency can be attributed to an increase in circulation in a larger droplet, as previously it has been shown that circulation deteriorates as the droplet shrinks.

LITERATURE

[0090] The literature listed below is intended to supplement or clarify, or provide a basis or lecture on, methodology, techniques and / or processes used.

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V. Srinivasan, VK Pamuła, and RB Fair, An integrated digital microfluidic lab-on-a-chip for clinical diagnostics on human physiological fluids, Lab Chip, 4 (4), 310, (2004)

B. Berge, and J. Peseux, Variable focal lens controlled by an external voltage: An application of electrowetting, The European Physical Journal E, vol. 3, p. 159 (2000).

MWJ Prins, WJJ Welters, and JW Weekamp, ​​Fluid control in multichannel structures by electrocapillary pressure, Science, vol. 291, pp. 277-280 (2001).

T. Merkel, L. Pagel, and HW Glock, Electric fields in fluidic channels and sensor applications with capacitance, Sensors and Actuators A, vol. 80, pp. 1-7 (2000).

A. Wego, and L. Pagel, Aself-filling micropump based on PCB technology, Sensors and Actuators A, vol. 88, pp. 220-226 (2001).

C. Laritz, and L. Pagel, A microfluidic pH-regulation system based on printed Circuit board technology, Sensors and Actuators A, vol. 84, pp. 230-235 (2000).

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A. Wego, S. Richter, L. Pagel, Fluidic microsystems based on printed Circuit board technology, Journal of Micromechanics and Microengineering, vol. 11, no. 5, pp. 528-531 (Sept 2001).

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

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C. Laritz and L. Pagel, A microfluidic pH-regulation system based on printed Circuit board technology, Sensors and Actuators A-Physical, vol. 84, no. 3, pp. 230-235 (Sept 1,2000).

T. 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, pp. 98-105 (Oct. 12, 1999).

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G. 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, pp. 121-125 (Sept. 1997).

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JA Schwartz, JV Vykoukal and PRC Gascoyne, Droplet-based chemistry on a programmable micro-chip, Lab on a Chip, vol. 4, no. 1, pp. 11-17 (2004). Y. Tan, JS Fisher, Al 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, pp. 292-298 (2004).

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JD Tice, AD Lyon, and RF Ismagilov, Effects of viscosity on droplet formation and mixing in microfluidic channels, Analytica Chimica Acta, vol. 507, pp. 73-77 (2004).

T. Merkel, L Pagel, H. Glock, Electric fields in fluidic channels and sensor applications with capacitance, Sensors and Actuators, vol. 80, pp. 1-7 (2000). G. 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, pp. 317-325 (2003).

L. Altomare, M. Borgatti, G. Medoro, M. Manaresi, M. Tartagni, R. Guerrieri and R. Gambari, Levitation and Movement of Human Tumor Cells Using a Printed Circuit Board Device Based on Software-Controlled Dielectrophoresis, Biotechnology and Bioengineering, vol. 82, pp. 474-479 (2003).

[0091] It is understood that various details relating to the subject matter of the present invention may be changed without departing from the scope of the present invention. Moreover, the above description has been provided for purposes of illustration only, and is not intended to limit the scope.

ΕΡ 1 859 330Β1