Microfluidic devices with multiple temperature zones and enhanced temperature control

Abstract

To provide a microfluidic system which combines spatial and temporal control of temperature.SOLUTION: A microfluidic system includes: an electro-wetting on dielectric (EWOD) device 36 comprising an element array configured to receive one or more liquid droplets 52; a control system configured to control actuation voltages applied to the element array to perform manipulation operations of the liquid droplets; and a plurality of thermal control elements located at different spatial locations along the EWOD device, at least one of the thermal control elements being variable in temperature with respect to time. The control system includes a thermal control unit configured to control temperatures of the thermal control elements to generate a plurality of thermal zones located at different spatial locations along the EWOD device, at least one of the thermal zones being variable in temperature with respect to time.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to droplet microfluidic devices, and more particularly to Active Matrix Electro-wetting-On-Dielectric (AM-EWOD) devices and structures and control methods for enhanced temperature control of multiple temperature zones in such devices. [Background technology]

[0002] Electrowetting on Dielectric (EWOD) is a well-known technique for manipulating droplets by application of an electric field. Active-matrix EWOD (AM-EWOD) relates to the implementation of EWOD in active-matrix arrays incorporating transistors, for example by using thin-film transistors (TFTs). As such, it is a candidate technology for digital microfluidics for lab-on-a-chip technologies. The basic principles of this technique are introduced in "Digital microfluidics: is a true lab-on-a-chip possible?, RBFair, Microfluid Nanofluid (2007) 3:245-281".

[0003] FIG. 1 shows a cross section of a portion of a conventional EWOD device. The device includes a bottom substrate 10, the top layer of which is formed of a patterned conductive material to implement a number of array element electrodes 12 (e.g., 12A and 12B in FIG. 1). The electrodes of a given array element are referred to as element electrodes 12. A droplet 14 containing a polar material (usually aqueous and / or also ionic) is constrained in a plane between the bottom substrate 10 and the top substrate 16. A suitable gap between the two substrates is implemented by a spacer 18, and a non-polar surrounding fluid 20 (e.g., oil) may be used to occupy the volume not occupied by the droplet 14. An insulator layer 22 disposed on the bottom substrate 10 separates the conductive element electrodes 12A, 12B from a first hydrophobic coating 24, on which the droplet 14 rests with a contact angle 26 represented by θ. The hydrophobic coating is formed of a hydrophobic material (usually, but not necessarily, a fluoropolymer).

[0004] On the top substrate 16 is a second hydrophobic coating 28, with which the droplet 14 may come into contact. A reference electrode 30 is disposed between the top substrate 16 and the second hydrophobic coating 28.

[0005] The contact angle θ is defined as shown in Figure 1 and is proportional to the surface tension (γ SL ), the surface tension at the interface between the liquid and the non-polar surrounding fluid (γ LG ), and the surface tension of the solid and the nonpolar surrounding fluid (γ SG ) and satisfies Young's law, expressed as follows, when no voltage is applied:

[0006]

number

[0007] During operation, the EW drive voltage (e.g., V in Figure 1) T , V0 and V 00) may be applied to different electrodes (e.g., the reference electrode 30 and each of the element electrodes 12, 12A, 12B). The resulting electrical forces effectively control the hydrophobicity of the hydrophobic coating 24. Different EW driving voltages (e.g., V0 and V 00 ), the droplet 14 can be moved in the lateral plane between the two substrates 10 and 16.

[0008] Exemplary configurations and operation of EWOD devices are described below. US6911132 (Pamula et al., issued June 28, 2005) discloses a two-dimensional EWOD array that controls the position and movement of droplets in two dimensions. US6565727 (Shenderov, issued May 20, 2003) further discloses methods for other droplet manipulations, including droplet splitting, merging, or simultaneous mixing of droplets of different materials. US7163612 (Sterling et al., issued January 16, 2007) discloses how TFT-based thin-film electronics are used to control the addressing of voltage pulses to an EWOD array, using a circuit arrangement very similar to that used in AM display technology.

[0009] The approach of US7163612 is called "Active Matrix Dielectric Electrowetting (AM-EWOD)". There are several advantages to using TFT-based thin-film electronics to control the EWOD array: Electrical drive circuitry can be integrated onto the lower substrate 10. · TFT-based thin-film electronics are well suited for AM-EWOD applications: they are cheap to manufacture, so relatively large substrate areas can be produced at relatively low cost. Conventional TFTs can be designed to operate at higher voltages than conventional CMOS transistors. This is important since many EWOD technologies require electrowetting voltages of 20V or more to be applied.

[0010] EWOD droplet manipulation devices are highly desirable platforms for the automation of chemical / biochemical reactions. Such devices may perform chemical / biochemical reactions or reaction sequences in droplets that require complex droplet temperature profiles. Different steps of the reaction may need to be performed at different temperatures. There are applications of EWOD devices that require the temperature of the sample / reagent droplets (and the products produced by combining them) to be changed to facilitate a desired chemical or biochemical reaction. Many of these reaction protocols require the droplets to be placed at multiple different temperatures at different times during the reaction sequence. Many reaction protocols require the droplets to be temperature cycled in time, and in some cases undergo many such temperature cycles. An important example of a reaction protocol that requires precise temperature control in an EWOD device over many reaction cycles is droplet-based nucleic acid amplification by polymerase chain reaction (PCR). PCR is a well-known reaction protocol for nucleic acid amplification.

[0011] One approach to handling multiple temperature requirements in an EWOD device is to provide multiple fixed temperature zones. In such an approach, a temperature control system, usually external to the EWOD device, is adjusted to control different parts or "zones" of the EWOD device at different fixed temperatures. Thus, once heated to such a temperature, the temperature remains constant in time. The temperature of the droplet can be changed by moving the droplet through the device to a location with a different temperature. This approach is particularly used for EWOD devices constructed from materials with low thermal conductivity, such as glass, because heat does not easily move longitudinally through the substrate material of the device, making it possible to achieve zones of different temperatures that are relatively close in space. Thus, each of the temperatures required for a reaction protocol must be provided in a separate temperature zone.

[0012] As an example of such an approach, US9452433B2 (Shenderov et al., issued 27 September 2016) describes a device for PCR amplification of nucleic acids with an EWOD device for droplet manipulation and for two or more reaction zones with different temperatures maintained at a specific temperature. A related patent from the same patent family, US9517469B2 (Shenderov et al., issued 13 December 2016), describes a method for PCR in which at least one reaction droplet is provided to at least two reaction zones in an electrowetting array, each reaction zone having a different temperature required for the nucleic acid amplification reaction, and electrowetting is used to move the droplet between these reaction zones. Again, the reaction zones with different temperatures are maintained at a specific temperature.

[0013] Another approach is to adjust or vary the device temperature over time. In such an approach, the temperature of the EWOD device (or in a region of the device) can be varied over time, for example the temperature of the entire device or the temperature of some substantial portion of the device. Summary of the Invention

[0014] For high sample throughput and low cost per sample, the area of ​​the EWOD device used for droplet processing should be maximized. EWOD devices are well suited to minimize sample and reagent volumes for droplet manipulation protocols, e.g., sample preparation, chemical or biochemical reactions and analyses. To maximize sample throughput and / or minimize the overall cost of running the protocol, it is desirable to make the overall area of ​​the EWOD device as small as possible, or equivalently maximize the number of droplets processed on the device (i.e., maximize the number of reactions per chip area per unit time). For droplet reaction protocols or reaction sequences with complex temperature requirements, optimizing droplet processing density is a challenge.

[0015] Both the conventional approach with multiple fixed temperature zones and the conventional approach with time-variable device temperature have significant disadvantages. Having multiple fixed temperature zones results in inefficient use of device area or inefficiency in the spatial domain. A temperature zone of one particular temperature is only required occasionally in an overall protocol step, and the remaining time, this device area is unused and essentially wasted. This disadvantage is particularly severe for protocols with many temperatures or protocols with one or more temperature steps that are used for only a short portion of the overall reaction time. Additionally, reducing the number of fixed temperature zones in this approach to improve efficiency in the spatial domain results in a non-optimized droplet temperature profile, making it difficult to achieve a balance between the number of zones and spatial efficiency.

[0016] Alternatively, having time-variable temperature devices or temperature zones provides more flexibility in the spatial domain, but results in inefficiencies in the time domain. Changing the temperature often requires that a "wait time" elapse while the temperature in the channels of the EWOD device re-equilibrates. This results in a "dead time" during which the protocol must be paused while the temperature re-equilibrates, typically for a few seconds to a few minutes. This disadvantage is especially severe for EWOD devices constructed from materials with low thermal conductivity, such as glass. However, glass or similar materials are advantageous for fabricating EWOD devices, particularly AM-EWOD devices. Thus, providing a suitable device with time-variable temperature made from the preferred materials is also difficult to achieve.

[0017] Inefficiencies in either the time or spatial domain have the overall consequence of reducing the number of droplets that can be processed on the device, i.e., reducing the number of reactions per chip area per unit time. Thus, in EWOD devices, an efficient device for carrying out reaction protocols that require precise temperature control over multiple reaction cycles has not been fully achieved.

[0018] The present invention provides enhanced temperature control in an EWOD device to optimize the temperature in the EWOD channel where droplet manipulations and reactions occur. The present invention combines spatial and temporal temperature control to provide synergistic efficiencies in space and time not achieved in previous configurations. The EWOD device control system and associated control methods minimize the number of temperature zones required and minimize the area occupied by such temperature zones. In addition, one or more different temperature zones can be varied in temperature over time. By combining spatial and temporal temperature control in an EWOD device, a temperature profile in the droplet channel of the EWOD device is generated that is optimized for the execution of a given biochemical / chemical reaction protocol or reaction sequence over many cycles. In this way, the number and size of temperature zones are optimized without compromising the reaction protocol.

[0019] The microfluidic system includes a control system and an EWOD device, and may optionally include an AM-EWOD device in particular. The control system includes an EWOD control unit having control electronics and a CPU that performs processing to control the movement of droplets on the device. The control system further includes a temperature zone control unit and at least two temperature control elements. The temperature zone control unit includes electronics and processing to control the temperature of the temperature control elements to generate different temperature control zones within the EWOD device. The temperature control elements may be arranged in thermal contact with the EWOD device, for example, either on an outer surface or inside the EWOD device so as to be in physical contact with the surface of the EWOD device. Two or more temperature control elements may be in contact with either the same surface or opposite facing surfaces of the EWOD device.

[0020] The temperature control elements can actively heat, cool, or both heat and cool the EWOD device as required or as determined by the temperature zone control unit depending on the desired reaction protocol. Heating and / or cooling can be implemented by well-known mechanisms. For example, heating can be by Joule heating or resistive heating and cooling can be by the Peltier effect, as known techniques for heating and cooling. Heating and / or cooling can be applied to the outer surface of the EWOD device, which alternately controls the temperature in the lateral plane between the two glass substrates of the EWOD device that form the EWOD channel. The EWOD channel defines the area of ​​the EWOD device where the droplet is confined, and in effect, the temperature controlled within the EWOD channel determines the temperature of the droplet placed within that channel.

[0021] The temperature control unit is configured to control the temperature control elements to generate at least two temperature zones within the channel of the EWOD device, the temperature of at least one of the temperature zones being also variable with respect to time. The temperature within the channel of the EWOD device is thus variable and controlled spatially and temporally. During a chemical / biochemical reaction protocol or sequence, droplets of a reaction mixture are moved by electrowetting between the temperature zones or are maintained in a given temperature zone. The temperature of a given droplet is thus a function of its position in the channel and the time elapsed since the start of the protocol. The combined spatial and temperature control eliminates the disadvantages of the conventional configurations and maximizes the capacity for droplet manipulation and reactions over a substantially smaller area of ​​the EWOD device compared to the conventional configurations.

[0022] By using multiple temperature zones combined with temperature control over time in one or more temperature zones, droplets can be rapidly moved between different temperatures by lateral movement through the EWOD channel of the EWOD device. In addition, the control over time of one or more zones allows different temperature values ​​to be reassigned at different points during the protocol. This means that while a variable zone is adjusted, separated by reaction steps carried out in other spatial zones, the same zone is used for different reaction steps at different times during the protocol.

[0023] As a result, inefficient use of device area common to conventional configurations with only fixed temperature zones can be avoided. When a droplet completes a reaction step in a zone at a given temperature and that temperature is no longer required for a subsequent step, the temperature of that zone is reassigned to a different value. In this way, the physical area associated with this zone is reused for other droplets at different temperatures and different times.

[0024] Similarly, the inefficient use of time of conventional time-variable configurations is eliminated in that there is no need to wait for temperature adjustment to perform the next subsequent reaction step. By efficient programming of reaction protocols into the control system, the temperature of a given zone can be varied while the droplets are "busy doing other things" in different zones, for example performing part of a droplet manipulation protocol or an incubation step in different locations of the EWOD device.

[0025] This results in a synergistic effect whereby sequenced multiple reaction droplet protocols are performed with enhanced efficiency in time and space, and more droplet processing steps are performed in a given time and in a smaller device area compared to conventional configurations. The advantages of the present invention are particularly evident for devices constructed with preferred substrate materials of low thermal conductivity, such as glass or similar materials. Low thermal conductivity means that the temperature zones of the EWOD channel are spatially closer to each other. With glass substrates, the EW electrode size is typically smaller than with other substrate materials (e.g., printed circuit boards PCBs). Thus, glass EWOD devices can operate with smaller droplets and are more spatially efficient. Efficient utilization of device area from a temperature standpoint and full realization of efficiency in an overall smaller device are achieved.

[0026] In conclusion, unexpected and enhanced results are achieved, especially with respect to EWOD devices using glass substrates, which are preferred for high quality AM-EWOD devices. Due to the costs associated with manufacturing TFT backplanes, reducing costs by reducing chip area is a synergistic advantage achieved by the present invention. AM-EWOD devices are preferably constructed with glass substrates, since glass is a standard substrate material for displays and therefore also available in AM-EWOD factories. Furthermore, AM-EWOD devices, with their very large number of array elements and high level of configurability, are particularly well suited to perform complex droplet manipulation protocols, such as PCR. Such highly complex droplet manipulation protocols often require steps at different temperature ranges, making the advantages of the present invention particularly prominent.

[0027] These and further features of the present invention will become apparent upon consideration of the following description and accompanying drawings. While the description and drawings, and in particular the embodiments of the present invention, are disclosed in detail to illustrate some of the ways in which the principles of the present invention may be employed, it should be understood that the scope of the present invention is not limited to the equivalents of the embodiments. Rather, the present invention includes all variations, modifications, and equivalents that fall within the spirit and terms of the claims appended hereto. Features described and / or shown in connection with one embodiment may be used in the same or similar manner in one or more other embodiments and / or may be used in combination with or in place of features of the other embodiments. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a cross-sectional view of a conventional EWOD device. [Diagram 2] FIG. 2 illustrates an exemplary EWOD-based microfluidic system, in accordance with an embodiment of the present invention. [Diagram 3] FIG. 3 is a schematic perspective view of an exemplary AM-EWOD device in accordance with an embodiment of the present invention. [Figure 4] FIG. 4 is a diagram illustrating a cross section through several array elements of the exemplary AM-EWOD device of FIG. [Diagram 5] 5A and 5B show circuit representations of the electrical loads appearing on the element electrodes when a droplet is present and when no droplet is present, respectively. [Figure 6] FIG. 6 is a diagram illustrating an exemplary arrangement of thin-film electronics in the exemplary AM-EWOD device of FIG. 3, in accordance with an embodiment of the present invention. [Figure 7] FIG. 7 is a diagram illustrating an exemplary arrangement of array element circuitry in accordance with an embodiment of the present invention. [Figure 8] FIG. 8 illustrates an exemplary microfluidic system according to an embodiment of the present invention that includes a temperature control element. [Figure 9]FIG. 9 is a diagram illustrating the microfluidic system of FIG. 8 illustrating an exemplary position of a droplet within an EWOD channel. [Figure 10A] FIG. 10A is a diagram illustrating the microfluidic system of FIG. 8 exhibiting various alternative configurations for the location of the temperature control element. [Figure 10B] FIG. 10B is a diagram illustrating the microfluidic system of FIG. 8 exhibiting various alternative configurations for the location of the temperature control element. [Figure 10C] FIG. 10C is a diagram illustrating the microfluidic system of FIG. 8 exhibiting various alternative configurations for the location of the temperature control element. [Figure 10D] FIG. 10D is a diagram illustrating the microfluidic system of FIG. 8 exhibiting various alternative configurations for the location of the temperature control element. [Figure 10E] FIG. 10E is a diagram illustrating the microfluidic system of FIG. 8 exhibiting various alternative configurations for the location of the temperature control element. [Figure 11] FIG. 11 is a graph showing exemplary temperature / time profiles for the first and second temperature zones and the combined temperature / time profile of a droplet as it moves by electrowetting between the two temperature zones. [Figure 12] FIG. 12 is a graphical diagram showing different protocols including temperature / time profiles of a first and second temperature zone and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones where the temperature of the temperature zones is time variable. [Figure 13] FIG. 13 is a graphical diagram showing different protocols including temperature / time profiles of a first and second temperature zone and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones where the temperature of the temperature zones is time variable. [Figure 14]FIG. 14 is a graphical diagram showing different protocols including temperature / time profiles of a first and second temperature zone and the resultant temperature / time profile of a droplet as it moves by electrowetting between two temperature zones where the temperature of the temperature zones is time variable. [Figure 15] FIG. 15 is a graphical diagram showing different protocols including temperature / time profiles of a first and second temperature zone and the resultant temperature / time profile of a droplet as it moves by electrowetting between two temperature zones where the temperature of the temperature zones is time variable. [Figure 16] FIG. 16 is a graph showing different protocols including temperature / time profiles of a first and second temperature zone and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones where the temperature of the temperature zones is time variable. [Figure 17] FIG. 17 is a graph showing different protocols including temperature / time profiles for the first and second temperature zones and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones, one time-variable temperature zone and one constant temperature zone. [Figure 18] FIG. 18 is a graph showing different protocols including temperature / time profiles for the first and second temperature zones and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones, one time-variable temperature zone and one constant temperature zone. [Figure 19] FIG. 19 is a graph showing different protocols including temperature / time profiles for the first and second temperature zones and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones, one time-variable temperature zone and one constant temperature zone. [Figure 20]FIG. 20 is a graph showing different protocols including temperature / time profiles for the first and second temperature zones and the resulting temperature / time profile of a droplet as it moves by electrowetting between two temperature zones, one time-variable temperature zone and one constant temperature zone. [Figure 21] FIG. 21 is a graphical representation showing a touchdown PCR reaction protocol according to an embodiment of the present invention. [Figure 22] FIG. 22 is a graphical representation showing the temperatures of the temperature zones for corresponding cycle numbers of the touchdown PCR reaction protocol of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Embodiments of the present invention are now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout, and it should be understood that the drawings are not necessarily to scale.

[0030] FIG. 2 illustrates an exemplary EWOD based on a microfluidic system according to an embodiment of the present invention. In the example of FIG. 2, the measurement system includes a reader 32 and a cartridge 34. The cartridge 34 can include a microfluidic device such as an EWOD or AM-EWOD 36, as well as a liquid input port to the device (not shown) and conventional electrical connections. The liquid input port can perform the function of inputting liquid into the AM-EWOD device 36 and generating droplets within the device, for example by providing it from electrowetting controlled input reservoirs. As will be described in more detail below, the microfluidic device includes an electrode array configured to receive the input droplets.

[0031] The microfluidic system may further include a control system configured to control actuation voltages applied to the electrode array of the microfluidic device to perform a manipulation operation on the droplets. For example, the reader 32 may include a control system configured as control electronics 38 and a storage device 40 for storing application software and data associated with the system. The control electronics 38 may include suitable circuitry and / or a processing device, such as a CPU, microcontroller, or microprocessor, configured to perform various control operations associated with controlling the AM-EWOD device 36.

[0032] Of these functions, the control electronics may comprise a portion of an overall control system that executes program code embodied as a control application in storage device 40 to implement aspects of the present invention. It will be clear to one skilled in the art of computer programming, and particularly in application programming for electronic control devices, how to program the control system to operate or perform the logical functions associated with the stored control application. Accordingly, for the sake of brevity, details regarding the specific programming code are omitted. Storage device 40 may comprise any non-transitory computer readable medium, such as random access memory (RAM), read only memory (ROM), erasable programmable ROM (EPROM or flash memory), or other suitable medium. Also, although code is executed by control electronics 38 in the exemplary embodiment, the functions of the control system may be executed by dedicated hardware, firmware, software, or a combination thereof without departing from the scope of the present invention.

[0033] The control system may be configured to perform some or all of the following functions: Defining appropriate timing signals to manipulate droplets on the AM-EWOD device 36 Interpreting input data indicative of sensor information measured by a sensor or sensor circuitry associated with the AM-EWOD device 36, such as calculating the position, size, center of gravity, or perimeter of a droplet on the AM-EWOD device 36. · using the calculated sensor data to define appropriate timing signals to manipulate the droplets on the AM-EWOD device 36, i.e., operate in feedback mode; · providing a graphical user interface (GUI) implementation to allow a user to program commands such as droplet manipulations (e.g., moving a droplet), analytical operations (e.g., performing an analysis), and to report results of such manipulations to the user. According to an embodiment of the present invention, and as described in more detail below, the control system may include a temperature control unit configured to control the EWOD device temperature within the EWOD channel appropriate for a given reaction protocol.

[0034] In the example of FIG. 2, an external sensor module 35 may be provided for sensing droplet properties. For example, optical sensors known in the art may be used as external sensors for sensing droplet properties. Suitable optical sensors include such as camera devices, light sensors, charge-coupled devices (CCDs) and similar image sensors. The sensor may alternatively be configured as an internal sensor circuit incorporated as part of the driver circuitry within each array element. Such sensor circuitry detects droplet properties by detecting an electrical property at the array element, such as impedance or capacitance.

[0035] The control system may provide and control, for example by control electronics 38, actuation voltages applied to the electrode array of the microfluidic device 36, e.g., required voltages and timing signals to perform droplet manipulation operations and detect droplets on the AM-EWOD device 36. The control electronics further executes application software and generates and outputs control voltages for performing droplet sensing and sensing operations. The reader 32 and cartridge 34 are electrically connected to each other during use, for example by a connecting wired cable 42, although various other methods of providing electrical communication (e.g., wireless connections) may be used as known to those skilled in the art.

[0036] 3 shows additional details of a schematic perspective view of an exemplary AM-EWOD device 36 according to an embodiment of the present invention. The AM-EWOD device 36 includes a lower substrate 44 having thin-film electronics 46 disposed thereon. The thin-film electronics 46 are arranged to drive array element electrodes 48. A plurality of array element electrodes 48 are arranged in an electrode or element array 50 having X by Y array elements, where X and Y are any integers. A droplet 52 containing a polar liquid, typically aqueous, is enclosed between the lower substrate 44 and an upper substrate 54 separated by a spacer 56, and it should also be understood that there may be multiple droplets 52.

[0037] FIG. 4 is a cross-sectional view through several array elements of the exemplary AM-EWOD device 36 of FIG. 3. In the portion of the AM-EWOD device shown in FIG. 4, the device includes a pair of array element electrodes 48A and 48B shown in cross-section that are used for the electrode or element array 50 of the AM-EWOD device 36 of FIG. 3. The device configuration is similar to the conventional configuration shown in FIG. 1, but the AM-EWOD device 36 further incorporates thin-film electronics 46 disposed on a lower substrate 44 separated from an upper substrate 54 by a spacer 56. The top layer of the lower substrate 44 (which may be considered part of the layer of thin-film electronics 46) is patterned to provide a plurality of array element electrodes 48 (e.g., specific examples of array element electrodes are 48A and 48B in FIG. 4). The term element electrode 48 hereinafter refers to both the physical electrode structure 48 associated with a particular array element and the node of the electrical circuit directly connected to this physical structure. The reference electrode 58 is disposed on the upper substrate 54 as shown in Figure 4, although alternatively the reference electrode may be disposed on the lower substrate 44 to achieve an in-plane reference electrode array. The term reference electrode 58 hereinafter refers to a physical electrode structure and / or a node in an electrical circuit directly connected to this physical structure.

[0038] Similar to the conventional structure of FIG. 1, in the AM-EWOD device 36, a non-polar fluid 60 (e.g., oil) is used to occupy the volume not occupied by the droplet 52. An insulator layer 62 is disposed on the lower substrate 44 and separates the conductive element electrodes 48A and 48B from a first hydrophobic coating 64 on which the droplet 52 rests with a contact angle 66 represented by θ. The hydrophobic coating is formed from a hydrophobic material (usually, but not necessarily, a fluoropolymer). On the upper substrate 54 is a second hydrophobic coating 68 with which the droplet 52 comes into contact. A reference electrode 58 is inserted between the upper substrate 54 and the second hydrophobic coating 68.

[0039] FIG. 5A shows a circuit representation of the electrical load 70A between the element electrode 48 and the reference electrode 58 when a droplet 52 is present. The droplet 52 is typically modeled as a resistor and a capacitor in parallel. Typically, the resistance of the droplet is relatively low (e.g., when it contains ions) and the capacitance of the droplet is relatively high (e.g., the dielectric constant of polar liquids is relatively high, e.g., .about.80 if the droplet is aqueous). In many situations, the droplet resistance is relatively small at frequencies of interest for electrowetting, and the droplet 52 effectively acts as an electrical short circuit. The hydrophobic coatings 64 and 68 have electrical properties that are modeled as a capacitor, and the insulator 62 is also modeled as a capacitor. The overall impedance between the element electrode 48 and the reference electrode 58 is approximated by a capacitor, the value of which is typically dominated by the contributions of the insulator 62 and the hydrophobic coatings 64 and 68, and may be on the order of picofarads for typical layer thicknesses and materials.

[0040] 5B is a circuit representation of the electrical load 70B between the element electrode 48 and the reference electrode 58 when no droplet is present. In this case, the droplet component is replaced by a capacitor representing the capacitance of the non-polar fluid 60 occupying the space between the upper and lower substrates. In this case, the overall impedance between the element electrode 48 and the reference electrode 58 is approximated by a capacitor whose value is dominated by the capacitance of the non-polar fluid and is small, typically on the order of femtofarads.

[0041] In effect, the electrical loads 70A / 70B function collectively as a capacitor for driving and sensing the array elements, whose value depends on whether or not a droplet 52 is present on a given element electrode 48. When a droplet is present, the capacitance is relatively high (typically on the order of picofarads), while when a droplet is absent, the capacitance is low (typically on the order of femtofarads). If a droplet only partially covers a given electrode 48, the capacitance may approximately represent the degree of coverage of the element electrode 48 by the droplet 52.

[0042] Figure 6 is a diagram illustrating an exemplary arrangement of thin-film electronics 46 in the exemplary AM-EWOD device 36 of Figure 3, in accordance with an embodiment of the present invention. The thin-film electronics 46 are located above a lower substrate 44. element That I5 Each array element 51 in the array of 0 includes an array element circuit 72 for controlling the electrode potential of a corresponding element electrode 48. Integrated row driver 74 and column driver 76 circuitry is also implemented within the thin film electronics 46 for providing control signals to the array element circuit 72. The array element circuit 72 also has sensing capability for detecting the presence or absence of a liquid droplet at the array element location. Integrated sensor row addressing 78 and column detection circuitry 80 are also further implemented within the thin film electronics for addressing and readout of the sensor circuitry in each array element.

[0043] A serial interface 82 may be provided to process a serial input data stream and facilitate programming of the required voltages to the element electrodes 48 in the array 50. A voltage supply interface 84 provides the corresponding supply voltages, the top substrate drive voltages and other necessary voltage inputs as further described herein. The number of connection wires 86 between the bottom substrate 44 and the external control electronics, power supplies and other components may be relatively small even for large array sizes. Optionally, the serial data input may be partially parallelized. For example, if two data inputs are used, the first may provide data for columns 1 to X / 2 and the second may provide data for columns (1+X / 2) to M with minimal updates to the column driver circuit 76. In this way, the rate at which data is programmed into the array may be increased, as is a standard technique used in driving circuits for liquid crystal displays.

[0044] In general, an exemplary AM-EWOD device 36 including thin-film electronics 46 may be configured as follows. The AM-EWOD device 36 includes a reference electrode 58 (optionally an in-plane reference electrode) and a plurality of individual array elements 51 on an element array 50, each array element 51 including an array element electrode 48 and an array element circuit 72, as described above. In relation, the AM-EWOD device 36 is configured to perform a method of operating the array elements to manipulate droplets on the array by controlling electrowetting voltages applied to the plurality of array elements. The applied voltages may be provided by operation of a control system shown in FIG. 2, including the control electronics 38 and applications and data stored in the storage device 40. The electrowetting voltage at each array element 51 is defined by the potential difference between the array element electrode 48 and the reference electrode 58. The method of controlling the electrowetting voltage at a given array element typically includes the steps of applying a voltage to the array element electrode 48 and applying a voltage to the reference electrode 58 by operation of the control system.

[0045] 7 is a diagram illustrating an exemplary arrangement of array element circuitry 72 present in each array element 51, according to an embodiment of the invention. The array element circuitry 72 may include actuation circuitry 88 having ENABLE, DATA and ACTUATE inputs, and outputs connected to the element electrodes 48. The array element circuitry 72 may also include droplet sensing circuitry 90 in electrical communication with the element electrodes 48. Typically, the readout of the droplet sensing circuitry 90 may be controlled by one or more addressing lines (e.g., RW) common to the elements in the same row of the array, and may have one or more outputs, e.g., OUT, common to all elements in the same column of the array.

[0046] The array element circuitry 72 typically performs the following functions: (i) Selectively actuating the element electrodes 48 by supplying a voltage to the array element electrodes. Any droplets present on the array element 51 are therefore actuated or deactuated by the electrowetting effect. (ii) Sensing the presence or absence of a droplet at a predetermined location on the array element 51. The means of sensing may be capacitive, optical, thermal or other means. Capacitive sensing is conveniently and effectively performed using impedance sensor circuitry as part of the array element circuitry.

[0047] Exemplary configurations of array element circuitry 72, including impedance sensor circuitry, are known in the art and are described in detail, for example, in the prior art section of US8653832 or in commonly assigned UK application GB1500261.1, both of which are incorporated herein by reference. These patent documents include descriptions of how droplets are actuated (by electrowetting) and how they are sensed by capacitive or impedance sensing means. Typically, the capacitive and impedance sensing is analog and may be performed simultaneously or near simultaneously at every element in the array. By processing information returned from such sensors (e.g., in application software residing in storage device 40 of reader 32), the control system described above can determine, in real time or near real time, the position, size, centroid and perimeter of each droplet present in element array 50. As referenced with respect to FIG. 2, as an alternative to the sensor circuitry, an external sensor (e.g., sensor 35), such as an optical sensor, can be provided that can be used to sense droplet properties.

[0048] The present invention provides enhanced control of temperature within the EWOD device to optimize the temperature in the EWOD channel where droplet operations and reactions occur. The present invention combines spatial and temporal temperature control to provide synergistic efficiencies in space and time not achieved in previous configurations. The EWOD device control system and associated control methods minimize the number of temperature zones required and minimize the area occupied by such temperature zones. In addition, one or more different temperature zones can be temperature variable over time. By combining spatial and temporal control of temperature in the EWOD device, a temperature profile within the droplet channel of the EWOD device is generated and optimized for the execution of a given biochemical / chemical reaction or sequence of reactions over many cycles. In this manner, a reaction protocol is efficiently executed with the number and size of temperature zones optimized.

[0049] Thus, in general terms, one aspect of the invention is a microfluidic system configured for enhanced temperature control by combining spatial and temporal temperature control. In an exemplary embodiment, the microfluidic system includes an electrowetting on dielectric (EWOD) device, the EWOD device comprising an element array configured to receive one or more droplets with a plurality of individual array elements, a control system configured to control actuation voltages applied to the element array to perform a manipulation operation on the droplets, and a plurality of temperature control elements at different spatial locations along the EWOD device, at least one temperature control element having a variable temperature with respect to time. The control system includes a temperature control unit configured to control the temperature of the plurality of temperature control elements to generate a plurality of temperature zones at different spatial locations along the EWOD device, at least one temperature zone having a variable temperature with respect to time.

[0050] Figure 8 illustrates an exemplary microfluidic system 100 according to an embodiment of the invention, including a control system 102 and an EWOD device 104 (which may be an AM-EWOD device, among other things) that defines an EWOD channel 106. Figure 9 illustrates the microfluidic system 100 of Figure 8 depicting an exemplary position of a droplet 108 within the EWOD channel 106.

[0051] Generally, the microfluidic system includes a controller system and an EWOD (or AM-EWOD) device. The controller system includes a temperature control unit configured to generate at least two temperature zones within a channel of the EWOD device, where the temperature of at least one temperature zone is dynamically (i.e., time-varying) controlled. The EWOD control unit operates to apply actuation voltages to array elements of the EWOD device to move droplets between the two temperature zones. The temperature zones are created by the temperature control elements and controlled by the temperature zone control unit.

[0052] In an exemplary embodiment, one or more of the following components are incorporated into the microfluidic system: Each of the temperature control elements is configured to heat, cool, or both heat and cool an associated temperature zone within the EWOD device. The temperature control elements are in thermal communication with either the top or bottom exterior surface of the EWOD device (either on the same surface or on opposite surfaces). If one or more temperature control elements are configured to provide heat (e.g., resistive Joule heat), the temperature zone control elements may be integrated within the EWOD device. Examples of integrated temperature control elements are described, for example, in commonly-owned U.S. application Ser. No. 13 / 092,194, filed Apr. 22, 2011, which is incorporated herein by reference. Temperature sensing elements known in the art are incorporated into one or more locations within the system components. Such temperature sensing elements may be components of the temperature control element and may include one or more external temperature sensors (e.g., thermistors) attached to the exterior surface of the EWOD device, or temperature sensors integrated into the EWOD device as described in U.S. Application Serial No. 12 / 772,245, filed May 15, 2010 by applicant (herein incorporated by reference), which may be incorporated into, for example, an array element of an AM-EWOD device, a temperature control system based on a proportional-integral-derivative (PID) control method, or into a temperature zone control unit. The microfluidic system is adapted to implement a droplet manipulation protocol that calls for droplets to be heated and / or cooled to at least two temperatures that are part of the droplet manipulation protocol.

[0053] As shown in Figures 8-9, a non-polar fluid 110 (e.g., oil) may be used to occupy the volume not occupied by the droplet 108. The EWOD device may include a first (top) substrate assembly 112 and a second (bottom) substrate assembly 114 separated by a spacer 116, which define the EWOD channel 106. To simplify illustrating the relevant configurations, the individual layers of the EWOD device components have been omitted. Thus, the first and second substrate assemblies may include the associated substrates, insulator layers, electrode layers, and associated structures that form the EWOD device, such as the various components described in connection with Figures 3-7. Figures 8 and 9 show an exemplary liquid input structure 118 for liquid input to the EWOD channel. Various configurations of input structures are known in the art, and thus, any suitable input structure may be utilized.

[0054] As mentioned above, the microfluidic system 100 further includes a control system 102. The control system 102 is configured similarly to the control system described in connection with FIG. 2 and includes control electronics for executing program code embodied as a control application embodied in a non-transitory computer-readable medium or storage device. The control system 102 may include an EWOD control unit having control electronics and a CPU processing device for controlling the movement of droplets on the EWOD device by controlling the actuation voltages applied to the array elements of the EWOD device. The control system 102 further includes a temperature zone control unit 124 and a plurality of temperature control elements. In the illustrated example, two temperature control elements 126 and 128 are shown at different spatial locations along the EWOD device. It will be understood that any number of multiple temperature control elements may be used in a given device as appropriate for a particular microfluidic operation. The temperature zone control unit 124, like the EWOD control unit 122, includes control electronics and a CPU or processing device for controlling the temperature of the temperature control elements to generate different temperature control zones within the EWOD device. The control electronics of the thermal zone control units may similarly execute program code embodied as a temperature control application embodied on a non-transitory computer readable medium or storage device within the thermal zone control units.

[0055] The temperature control elements 126 and 128 may actively heat, cool, or both heat and cool the EWOD device as required and determined by the temperature zone control unit 124 according to any desired reaction protocol. Heating and / or cooling may be implemented by any well-known mechanism. For example, heating may be by Joule heating or resistive heating, and cooling may be by the Peltier effect, as known in the art of heating and cooling. A region of the EWOD channel 106 within the EWOD device whose temperature is controlled by one of the temperature control elements is referred to herein as a temperature zone. For example, in FIG. 8 and FIG. 9, the first temperature control element 126 may operate to control the temperature of a first temperature zone 127 within the EWOD channel, and the second temperature control element 128 may operate to control the temperature of a second temperature zone 129 within the EWOD channel. Thus, the first and second temperature zones 127, 129 are located at different spatial locations along the EWOD device based on the corresponding positions of the temperature control elements. Also, any number of multiple temperature control elements may be used to control the temperature within a corresponding number of temperature zones located at different spatial locations along the EWOD device.

[0056] A droplet assumes the temperature of the temperature zone in which it is located. Due to the small size of the droplet, rapid temperature equilibration occurs between the droplet and the temperature zone. In the example of Figure 9, droplet 108 is disposed in a first temperature zone 127 and assumes the temperature of first temperature zone 127 controlled by first temperature control element 126. By application of an appropriate actuation voltage, droplet 108 is moved to a second temperature zone 129 and assumes the temperature of second temperature zone 129 controlled by second temperature control element 128.

[0057] The EWOD control unit 122 applies actuation voltages to the array elements of the EWOD device to move droplets from one temperature zone to another. The temperature zone control unit 124 and the EWOD control unit 122 are coordinated to cooperate to configure dynamically controlled temperature zones that vary the temperature in the channel of the EWOD device according to the position of the droplet in the channel. The position of the droplet in the EWOD channel may be read by a droplet position sensor integrated into the EWOD droplet manipulation device (e.g., based on sensing the droplet impedance, using the external sensor 35 of FIG. 3 or the droplet sensing circuit 90 of FIG. 7). By combining spatial and temporal temperature control in the channel of the EWOD device, the temperature profile required for the execution of a given biochemical / chemical reaction or reaction sequence is optimized, which in turn optimizes the number and size of the temperature zones. The inclusion of the droplet position sensor further enhances the system, since feedback control of the droplet position is used to determine the time at which changes to the temperature of the temperature zones are implemented.

[0058] Temperature control elements 126 and 128 are disposed in thermal contact with one of the substrate layers of the EWOD device, for example, on an outer surface of the substrate layer of the EWOD device, or internally as part of the substrate. In the examples of Figures 8 and 9, the temperature control elements are both disposed on the outer surface of the second (bottom) substrate 114.

[0059] 10A-10E show alternative arrangements for the positioning of the temperature control elements 126, 128. Two or more temperature control elements may contact the same or oppositely facing surfaces of the EWOD device, or may be disposed within or as part of one or both of the substrate assemblies in close proximity to the EWOD channel 106. In the embodiment of FIG. 10A, as an alternative example, both temperature control elements are disposed on the first (top) substrate 112. In the embodiment of FIG. 10B, the first temperature control element 126 is disposed on the second (bottom) substrate 114, and the second temperature control element 128 is disposed on the first (top) substrate 112. In the embodiment of FIG. 10C, both temperature control elements are disposed within the first (top) substrate 112. In the embodiment of FIG. 10D, the first temperature control element 126 is disposed within the second (bottom) substrate 114, and the second temperature control element 128 is disposed within the first (top) substrate 112. 10E embodiment, both temperature control elements are disposed in the second (bottom) substrate 114. Thus, any suitable location configuration of the temperature control elements relative to the EWOD channel may be used.

[0060] In this manner, heating and / or cooling is applied to one or more surfaces of substrate 112 and / or substrate 114 of the EWOD device. This in turn controls the temperature in the lateral plane between the two substrates of the EWOD device that forms the EWOD channel 106, creating distinct temperature zones, such as the exemplary temperature zones 127 and 129 in Figures 8 and 9. The EWOD channel defines an area of ​​the EWOD device in which a droplet is confined, and in effect, the temperature controlled within the EWOD channel, as described above, determines the temperature of a droplet placed within that channel.

[0061] Generally, according to an embodiment of the invention, a temperature control unit is configured to control the temperature control elements to generate a plurality of temperature zones, i.e. at least a first temperature zone and a second temperature zone, in the channel of the EWOD device, in which the temperature of at least one temperature zone is also variable with respect to time. The temperature in the channel of the EWOD device is thus variable and controlled spatially and temporally. During a chemical / biochemical reaction or sequence of reactions, droplets of the reaction mixture are electrowettingly moved between the temperature zones or maintained in a given temperature zone. The temperature of a given droplet is thus a function of both the position in the channel and the time elapsed since the start of the protocol. The combination of spatial and temporal temperature control eliminates the disadvantages of conventional configurations and maximizes the capacity for droplet manipulation and reactions over a substantially smaller area of ​​the EWOD device compared to conventional configurations.

[0062] 11-21 are graphs illustrating various protocols for controlling the temperature in the EWOD channel of an EWOD device. In general, according to an embodiment of the present invention, a microfluidic system includes a plurality of temperature control elements located at different spatial locations relative to the EWOD channel, where at least one temperature control element is controlled to be temperature-variable in time. Control of the plurality of temperature control elements results in the creation of a plurality of corresponding temperature zones in the EWOD channel, where at least one temperature zone has a temperature that is time-variable according to the change of the time-variable temperature control element. The illustrated example is based on a microfluidic system where the plurality of temperature control elements includes a first and a second temperature control element, where at least one temperature control element has a temperature that is time-variable. It will be understood that the use of two temperature control elements is a non-limiting example, and any suitable number of temperature control elements may be used as appropriate for any particular device or application. Additionally, the exact number of time-variable temperature control elements may be determined as appropriate for any particular device or application, so long as at least one temperature control element is temperature-variable in time. In other words, one or more and up to all of the temperature control elements may be time variable in temperature.

[0063] Accordingly, another aspect of the invention is a control method for performing a reaction protocol using an electrowetting on dielectric (EWOD) device that combines both spatial and temperature control within the EWOD device. In an exemplary embodiment, the control method includes the steps of receiving a droplet in an EWOD channel defined by the EWOD device, generating a first temperature zone at a first spatial location within the EWOD channel, the first temperature zone being controlled to have a first temperature, generating a second temperature zone at a second spatial location within the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature that is variable over time, making the temperature of the second temperature zone time-variable, and applying actuation voltages to an array of elements of the EWOD device to move a droplet between the first temperature zone and the second temperature zone, where the temperature of the droplet assumes the temperature of one of the first temperature zone or the second temperature zone in which the droplet is located. The temperature of the first temperature zone may be held constant during the reaction protocol or may be time-variable during the reaction protocol. The temperatures of the first temperature zone and the second temperature zone may be controlled to control the temperature of the droplets to change cyclically over multiple temperature cycles within the reaction protocol.

[0064] The meaning of the various portions of the graphs and line formats in Figures 11-21 will now be explained. In all these figures, focusing on Figure 11 as an example, the top two graph portions represent temperature as a function of time, i.e., temperature profiles, for a first temperature zone 200 (Tzone 1 or temperature zone 1) and a second temperature zone 202 (Tzone 2 for temperature zone 2). The temperature profiles for the first and second temperature zones correspond to spatial temperature zones in the EWOD channel that correspond to the positions of the temperature control elements, as described above (e.g., temperature zones 127 and 129). The third portion of the graph corresponds to the combined temperature profile (Tdroplet) of the droplet 204 as a function of both time and the spatial location of the droplet, i.e., whether the droplet is in temperature zone 1 or temperature zone 2. T1, T2, and T3 are schematic representations of different temperatures and may be any suitable temperatures for a given application or reaction protocol.

[0065] In the top graph portions of Figures 11-21, which depict the temperature profiles of the first and second temperature zones 200 and 202, the solid portions of the graphs indicate that the droplet is located in such temperature zones, and the dotted lines indicate that the droplet is not located in such temperature zones. Similarly, in the bottom graph portions, which depict the temperature profile of droplet 204, the solid portions of the graphs indicate that the droplet is located in a particular temperature zone, and the dotted lines indicate that the droplet is moving between the temperature zones. Stated differently, as will be further explained below, the graphs show how temperature changes occur as the droplet moves spatially between two temperature zones, changing the temperature within the temperature zones based on the control of a temperature control element over time.

[0066] FIG. 11 shows an example temperature / time profile for the first and second temperature zones, and a composite temperature / time profile for a droplet as it moves by electrowetting between the two temperature zones. In this particular example, the temperature of temperature zone 1 is constant over time at T1. The droplet is initially shown as being placed in temperature zone 1, and the droplet starts at a temperature of T1 in temperature zone 1. Meanwhile, the temperature of temperature zone 2 shifts from T3 to T2. The droplet is then moved from temperature zone 1 to temperature zone 2 by an appropriate actuation voltage, as shown by the dashed line in the third part of the graph, and the droplet is cooled to T2. The temperature of temperature zone 2 is then increased to T3 while the droplet is placed in temperature zone 2. The temperature of the droplet is similarly increased to T3. FIG. 11 shows how spatial and temporal temperature control are combined to reach three reaction temperatures for a droplet in an EWOD channel using only two temperature zones. The droplet begins at temperature T1 in temperature zone 1, then the droplet moves to temperature zone 2 where it experiences temperature T2 (spatial temperature control), and the droplet remains in temperature zone 2 until the temperature is increased to temperature T3 (temporal temperature control). Some or all of the droplet temperature profile may be cycled several times.

[0067] FIG. 11 provides a basic example of how spatial temperature control can be varied across two temperature zones, and how temperature can be additionally varied over time within temperature zone 2 in this example. By combining droplet temperature control via temperature zones and moving droplets between temperature zones by electrowetting, complex droplet temperature profiles can be achieved while minimizing the area of ​​the device requiring temperature control. The next figure shows a more complex protocol that still utilizes only two temperature zones when coupled to different parts or cycles of a reaction sequence for a biochemical / chemical reaction or any suitable reaction protocol. It should be understood that the various protocols of operation are presented as non-limiting examples.

[0068] Figures 12-16 are graphs showing different protocols including temperature / time profiles for the first and second temperature zones and the synthesized temperature / time profile for a droplet as it moves by electrowetting between two temperature zones, with the temperatures of both temperature zones being temporally variable.

[0069] Referring first to FIG. 12 , a droplet starts out at temperature T1 in temperature zone 1 during which time the temperature of temperature zone 2 equilibrates at temperature T2. To change the droplet temperature to temperature 2, the droplet is rapidly moved to temperature zone 2 by electrowetting. The rate at which the droplet approaches the new temperature is controlled by the speed at which the droplet is moved between the zones by the electrowetting actuation voltage. The droplet remains in temperature zone 2 while the temperature of temperature zone 2 increases to temperature T1. The droplet temperature profile follows the temperature profile for temperature zone 2 during this period. The illustrated profile is an exemplary embodiment and any temperature / time function may be applied to the droplet, and such a profile may represent a first reaction cycle (reaction cycle 1) that is part of a complex reaction sequence.

[0070] The droplet then maintains a constant temperature at temperature T1 in temperature zone 2 at the beginning of an exemplary second droplet reaction cycle (reaction cycle 2). While the droplet is in temperature zone 2, the temperature of temperature zone 1 is decreased to T2. The second reaction cycle may further include moving the droplet to temperature zone 1 by electrowetting, and the droplet follows the temperature profile applied to temperature zone 1 in the last part of the graph. This ends the second droplet temperature cycle. In this example, the droplet temperature cycles at twice the frequency of temperature zones 1 and 2. Thus, the droplet temperature cycles twice through temperature zones 1 and 2, and the rapid movement of the droplet between temperature zones 1 and 2, or temperature zones 2 and 1, creates an abrupt temperature transition between temperatures T1 and T2. Again, any temperature / time function is applied to the droplet while it remains in the variable temperature zone. By moving the droplets between two temperature zones and varying the temperature over time, efficient cycling of the droplet temperature is achieved to carry out complex reaction sequences. Four complete cycles are shown in the figure, but any suitable number of cycles may be used.

[0071] FIG. 13 shows another example of using two time-variable temperature zones to cycle the droplet temperature. Four cycles (reaction cycle 1, reaction cycle 2, reaction cycle 3 and reaction cycle 4) are again shown, but with different temperature / time profiles compared to the previous figures. Again, any suitable number of cycles may be used. The droplet starts at temperature T1 in temperature zone 1, during which time the temperature of temperature zone 2 equilibrates at temperature T2. To change the droplet temperature to temperature T2, the droplet moves to temperature zone 2 by electrowetting. In temperature zone 2, the droplet temperature increases in a stepwise manner, first to temperature T3 and then back to temperature T1, based on the controlled change in temperature of temperature zone 2 to follow the required droplet temperature / time function. While the droplet is in temperature zone 2, temperature zone 1 re-equilibrates to temperature T2. To end the second droplet temperature cycle, the droplet returns to temperature zone 1 at temperature T2, and the droplet stays in temperature zone 2 while the temperature increases stepwise to temperature T3 and back to temperature T1. In this example, the droplet temperature cycles at twice the frequency of the temperature of the temperature zones in a repeating sequence from T1 to T2 to T3. Thus, the droplet temperature cycles twice through temperature zones 1 and 2, and the rapid droplet movement between temperature zones 1 and 2 creates an abrupt temperature transition between temperatures T1 and T2.

[0072] FIG. 14 is a variation of the previous FIG. 13 and shows another example of using two time-variable temperature zones to control droplet temperature. In this example, the temperature cycles and progression are shown for comparison with FIG. 13, where the droplets undergo a series of temperature changes before entering a temperature cycle (starting with reaction cycle 1). This droplet temperature profile is useful, for example, for reverse-transcription PCR. Alternatively, the droplet temperature profile may include an initial temperature cycle before concluding with several one off temperatures.

[0073] FIG. 15 shows another example of using two time-variable temperature zones to cycle the droplet temperature. Four reaction cycles (reaction cycle 1, reaction cycle 2, reaction cycle 3 and reaction cycle 4) are again shown, but with different temperature / time profiles compared to the previous figures. Again, any suitable number of cycles may be used. The droplet starts at temperature T1 in temperature zone 1, during which time the temperature of temperature zone 2 equilibrates to temperature T2 of the reaction sequence. To change to temperature T2, the droplet moves to temperature zone 2 by electrowetting. While the droplet is in temperature zone 2 at temperature T2, temperature zone 1 re-equilibrates to temperature T3. The droplet then moves to temperature zone 1 at temperature T3, completing the first reaction cycle. During the second droplet temperature cycle, the droplet returns to temperature zone 2, which is temperature T1 of the reaction sequence, then moves to temperature zone 1 at temperature T2, and finally moves to temperature zone 2 at temperature T3. The droplet temperature cycles through the temperature zones at twice the frequency in a repeating sequence from T1 to T2, T2 to T3. The droplet temperature profile has similarities to the previous figure, but the time variation of the two temperature zones and the droplet position during temperature cycling are different to achieve comparable droplet temperature / time profiles and temperature cycling.

[0074] 16 is another example of using two time-variable temperature zones to cycle a droplet temperature. The droplet begins at temperature T1 in temperature zone 1, and abrupt transitions to temperatures T2 and T3 are enabled via movement of the droplet between temperature zones 1 and 2, in that order. Finally, the temperature of the droplet is changed back to temperature T1 by varying the temperature of temperature zone 2 while the droplet occupies the temperature zone. In this example, the temperature of temperature zone 2 is varied before or while the droplet occupies a given temperature zone.

[0075] Figures 17-20 are graphical diagrams showing different protocols, including temperature / time profiles for the first and second temperature zones and the combined temperature / time profile of a droplet as it moves by electrowetting between two temperature zones, one a time-variable temperature zone and one a constant temperature zone.

[0076] FIG. 17 can be compared to FIG. 11, except that the protocol is further illustrated as being incorporated as part of a droplet temperature cycling protocol. Two cycles are shown (Reaction Cycle 1 and Reaction Cycle 2). Again, any suitable number of cycles may be utilized. The temperature of temperature zone 1 remains constant over time at T1. The droplet is initially shown as being located in temperature zone 1, such that the droplet resides at temperature T1 in temperature zone 1. During that time period, the temperature of temperature zone 2 shifts from T3 to T2. The droplet then moves from temperature zone 1 to temperature zone 2, where the droplet cools to T2. The temperature of temperature zone 2 then increases to T3, and the temperature of the droplet increases proportionately to T3. FIG. 17 shows a step change in temperatures T2 and T3. In contrast, as shown in FIG. 18, a slower change is performed from temperature T2 to T3. A quick return to temperature T1 is achieved by moving the droplet back to temperature zone 1. In these examples, the temperature of temperature zone 1 is held constant while the droplet temperature is cycled at the same frequency as temperature zone 2.

[0077] FIG. 19 shows another example of using one time-variable temperature zone and one constant temperature zone to cycle the droplet temperature. The droplet starts at temperature T1 in temperature zone 1. To change to temperature T2, the droplet is abruptly moved to temperature zone 2 by electrowetting, and the temperature of temperature zone 1 re-equilibrates to temperature T3 while the droplet is placed in temperature zone 2. The abrupt droplet movement to temperature zone 1 brings the droplet to temperature T3. In temperature zone 1, the temperature of temperature zone 1 and the droplet increases from temperature T3 and back to temperature T1, following the required temperature / time function for the reaction of interest. The cycle may then be repeated for as many cycles as required for the reaction protocol (one additional cycle is shown in the figure for illustration). The droplet temperature cycles with the same frequency as the temperature of temperature zone 1, while the temperature in temperature zone 2 remains constant.

[0078] FIG. 20 shows another example of using one time-variable temperature zone and one constant temperature zone to cycle the droplet temperature. The droplet starts at temperature T1 in temperature zone 1. While the droplet is in temperature zone 1, the temperature of temperature zone 1 decreases from temperature T1 to temperature T2 following a temperature / time profile suitable for the reaction, and the droplet temperature follows such profile. A sudden droplet movement to temperature zone 2 brings the droplet to temperature T3 as long as the temperature of temperature zone 2 remains constant at T3. While the droplet is in temperature zone 2, the temperature of temperature zone 1 re-equilibrates to temperature T1. The droplet temperature is suddenly changed to temperature T1 by moving the droplet to temperature zone 1 by electrowetting. As described above, the cycle is repeated for the same cycles required for the reaction protocol (one additional cycle is shown in the figure for illustration). The droplet temperature cycles with the same frequency as the temperature of temperature zone 1, while the temperature of temperature zone 2 is maintained constant.

[0079] The present invention is particularly suitable for temperature control of complex reaction protocols, such as those with multiple reaction sequences that need to be carried out at different temperatures. An example of such a reaction protocol is nucleic acid amplification via PCR (polymerase chain reaction). We now present an exemplary method for PCR temperature cycling, in which droplets are moved by electrowetting between two variable temperature zones to efficiently access the three temperatures required for optimal PCR, while minimizing the area of ​​the device reserved for temperature control. Figure 15 above specifically illustrates a reaction cycle utilizing three temperatures suitable for PCR. A variation of such cycling applied to a PCR protocol is shown in more detail in Figure 21.

[0080] First, as a specific example of a PCR reaction protocol, referring to FIG. 15 showing temperature cycling suitable for PCR, the droplet starts at temperature T1 in temperature zone 1, which is controlled to have a first temperature corresponding to a dsDNA denaturation step of about 95° in temperature zone 1 for PCR. Thus, the droplet moves to temperature zone 1, and denaturation of the nucleic acid is carried out in temperature zone 1. While the droplet is located in temperature zone 1, the temperature of temperature zone 2 equilibrates to temperature T2 of the reaction sequence, which is controlled to have a second temperature corresponding to a step of annealing the primer to the ssDNA for PCR at about 60°. To change the droplet temperature to temperature T2, the droplet is suddenly moved by electrowetting to temperature zone 2, where annealing of the primer to the nucleic acid is carried out. While the droplet is present in temperature zone 2 at temperature T2, temperature zone 1 re-equilibrates to temperature T3, so as to be controlled to a third temperature corresponding to a step of DNA elongation and polymerization with an elongation temperature of about 72° for PCR. The droplet then moves to temperature zone 1 at temperature T3 where polymerization of the nucleic acid takes place. This completes the first cycle (reaction cycle 1) of the PCR reaction protocol, and while the droplet is disposed within temperature zone 1, temperature zone 2 re-equilibrates back to T1. During the next run of the second droplet temperature cycle, the droplet moves back to temperature zone 2 at temperature T1 of the reaction sequence, then to temperature zone 1 at temperature T2, and finally to zone 2 at temperature T3, and so on. Although reaction cycles 1, 2, 3, and 4 are shown in FIG. 15 for illustrative purposes, a typical PCR may include about 40 or more cycles as may be appropriate for a given PCR protocol.

[0081] Thus, multiple PCR cycles may be additionally performed by repeating the PCR cycles described above until the nucleic acid amplification protocol is fully completed. The droplet temperature cycles at twice the frequency of the temperature of the temperature zones, and the rate of approach to each new droplet temperature is determined by the speed of droplet movement via electrowetting. The area of ​​the device reserved for temperature management is limited to two temperature zones, and the method of combining droplet movement and temperature change of the temperature zones allows three temperatures for efficient PCR to be achieved by requiring only two spatial areas.

[0082] FIG. 21 is a graphical diagram illustrating in more detail a variation of the PCR reaction protocol of FIG. 15, referred to in the art as touchdown PCR. In touchdown PCR, the PCR protocol described above is modified such that for the first approximately 14 cycles of PCR, the primer annealing temperature T2 varies by 1-2°C per cycle from an initial value of approximately 10°C, which is above the DNA / primer melting temperature. These different T2 temperatures are labeled T2a, T2b, etc. in FIG. 21, and by cycle 15 the primer annealing temperature is at T2 (e.g., approximately 60°C, as described above). The primer annealing temperature remains at T2 for subsequent cycles, such as cycles 15 and 16 in FIG. 21, with no further change in annealing temperature. This is illustrated in FIG. 22, which shows how the temperature T2 varies over the first 14 cycles and then remains at a constant temperature for the remainder of the protocol (e.g., up to cycle 40 or more). Thus, even with adjustment of primer annealing temperatures over a large number of cycles, the area of ​​the device reserved for temperature control is still restricted to two spatial temperature zones, allowing for enhanced touchdown PCR over a minimal device area.

[0083] Returning to structural diagrams 8-10E, any number of reaction protocols can be programmed into the control system 102. In particular, the programmed reaction protocols can be provided to the EWOD control unit 122 and stored in the memory of the EWOD control unit, or can be transmitted to the EWOD control unit 122 as program code from some external source for execution. Such programmed reaction protocols cause the EWOD control unit 122 to send control signals to the temperature zone control unit 124, which in turn generates temperature control signals to control the temperatures of the temperature control elements 126, 128 to generate temperature zones 127 and 129 within the EWOD channel 106. Additionally, the EWOD control unit 122 can further generate voltage control signals to apply actuation voltages to move droplets between temperature zones according to the reaction protocol, and to perform any additional associated droplet manipulation operations. In this manner, various reaction protocols can be preprogrammed into the EWOD device control system and then executed as needed when a desired protocol is being executed.

[0084] The use of multiple temperature zones at different spatial locations combined with temperature control over time within one or more temperature zones achieves a number of advantages over conventional configurations. In general terms, droplets can be rapidly transitioned between different temperatures by lateral movement through the EWOD channel of the EWOD device. Furthermore, temperature control of one or more zones allows different spatial zones to be reassigned to different temperature values ​​at different points during a protocol. This means that the same zones are used for different reaction steps at different times during the protocol, while a variable zone is being adjusted, separated by reaction steps carried out in other spatial zones.

[0085] As a result, the synergistic effect of combining spatial and temporal temperature control eliminates the inefficient use of device area common to conventional configurations with only fixed temperature zones. When a droplet completes a reaction step at a given temperature in a zone and that temperature is not required for a subsequent step, the temperature of that zone may be reassigned to a different value. The physical area associated with that zone may thus be reused for alternative droplet operations at different temperatures and different times. Furthermore, the inefficient use of time in conventional time-variable configurations is eliminated in that there is no need to wait for temperature adjustments to perform subsequent reaction steps. By efficiently programming the reaction protocol into the control system, the temperature of a given zone may be variable when the droplet is "busy doing other things" in a different zone, e.g., performing part of a droplet operation protocol or an incubation step in a different location of the EWOD device.

[0086] As a result, a synergistic effect is achieved, whereby droplet protocols of multiple sequential reactions are performed with enhanced efficiency in both time and space, such that many droplet processing steps are performed in a smaller device area at a given time compared to conventional configurations. The advantages of the present invention are particularly pronounced for devices constructed from preferred substrate materials with low thermal conductivity, such as materials such as glass. Low thermal conductivity means that the multiple temperature zones of the EWOD channel are spatially close to each other. When using glass substrates, the EW electrode size is typically smaller than when using other substrate materials (e.g., printed circuit boards PCBs). Thus, glass EWOD devices operate with smaller droplets and are more spatially efficient. From a thermal perspective, efficient use of device area and therefore full realization of efficiency in an overall smaller device is achieved.

[0087] As a result, enhanced results beyond expectations are achieved, especially in relation to EWOD devices using glass substrates, which are favorable for high quality AM-EWOD devices. Cost reduction by reducing chip area is a synergistic advantage achieved by the present invention, due to the costs associated with manufacturing TFT backplanes. AM-EWOD devices are preferably constructed with glass substrates, since glass is a standard substrate material for displays and therefore an available substrate material in AM-EWOD manufacturing plants. Furthermore, AM-EWOD devices have a very large number of array elements and a high level of configurability, making them particularly well suited to perform complex droplet manipulation protocols, such as PCR, including touchdown PCR. Very complex droplet manipulation protocols often require steps at a range of different temperatures, which particularly promotes the advantages of the present invention.

[0088] Additional advantages of the various protocols of the present invention include one or more of the following: Three or more temperature zones are realized in the footprint of two physical temperature zones in the system, where space is reserved for increased throughput or multiple parallel operations. An optimal temperature profile for droplets undergoing biochemical / chemical reactions of a reaction sequence may be achieved by combining droplet movement between temperature zones (of different temperatures) and temperature fluctuations of the temperature zones when occupied by the droplets. This allows the required droplet temperature profile to be optimized in the smallest space with the most favorable temperature approach rate (°C / s).

[0089] The temperature of a given temperature zone can re-equilibrate during the time the droplets are in another temperature zone, which will be at the optimal temperature for the next step of the chemical / biochemical reaction or reaction sequence when the droplets arrive via EWOD from the other temperature zone, which saves time and increases throughput.

[0090] The droplet temperature profile may be cycled by repeated temperature cycling through the temperature zones. The frequency of temperature zone cycling may be the same as or different from the droplet temperature cycling. In addition, device area utilization may be optimized for repeated reaction temperature cycling. EWOD droplet manipulation along a one-dimensional linear path "there and back" may access three or more different reaction temperatures without looped droplet movement, i.e., no two-dimensional droplet path is required, which provides simplified manipulation and space savings.

[0091] The temperature of a temperature zone may be varied before, during or after the movement of the droplet through a given temperature zone. Rapid droplet temperature changes are made possible by the fast electrowetting motion between temperature zones. The droplet is small and therefore quickly reaches thermal equilibrium with its surroundings.

[0092] Considering PCR specifically, for optimal PCR, the reaction mixture must be cycled typically 35 to 45 times through three discrete temperatures, e.g., 95°C to denature the double standard DNA, 55-60°C to anneal the primers to the ssDNA, and 70-75°C for optimal extension of the new DNA strand. The device and method for PCR on EWOD of the present invention provides optimal reaction temperature profiles and minimized temperature control space requirements. Furthermore, for touchdown PCR, defining the droplet temperature profile for this procedure is greatly facilitated by combining droplet temperature control via variable temperature zones and the method of moving droplets between temperature zones by electrowetting. This process favors the formation of amplicons from primer / DNA template pairs with the highest complementarity and reduces the formation of non-specific products.

[0093] Thus, one aspect of the invention can be described as a microfluidic system configured for enhanced temperature control by combining spatial and temporal temperature control. In an exemplary embodiment, the microfluidic system comprises an electrowetting on dielectric (EWOD) device comprising an array of elements configured to receive one or more droplets, an array of elements comprising a plurality of individual array elements, a control system configured to control actuation voltages applied to the array of elements to perform a manipulation operation on the droplets, and a plurality of temperature control elements disposed at different spatial locations along the EWOD device, at least one of the temperature control elements being temperature variable with respect to time. The control system includes a temperature control unit configured to control the temperature of the plurality of temperature control elements to generate a plurality of temperature zones disposed at different spatial locations along the EWOD device, at least one of the temperature zones being temperature variable with respect to time by time-varying the temperature of at least one of the temperature control elements having a time-varying temperature. The microfluidic system may include one or more of the following features, individually or in combination:

[0094] In an exemplary embodiment of the microfluidic system, the temperature control unit is configured to control the temperature of at least one temperature zone whose temperature is variable with respect to time depending on the position of the droplet in the EWOD channel of the EWOD device.

[0095] In an exemplary embodiment of the microfluidic system, the plurality of temperature control elements are variable in temperature with respect to time, and the temperature control unit is configured to control the plurality of variable temperature control elements to vary the temperature of the plurality of temperature zones with respect to time.

[0096] In an exemplary embodiment of the microfluidic system, the temperature control unit is configured to control at least one temperature control element to maintain at least one temperature zone at a constant temperature.

[0097] In an exemplary embodiment of a microfluidic system, the EWOD device includes a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and a plurality of temperature control elements are disposed on an outer surface of one or both of the substrate assemblies.

[0098] In an exemplary embodiment of a microfluidic system, the EWOD device includes a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and a plurality of temperature control elements are disposed within one or both of the substrate assemblies.

[0099] In an exemplary embodiment of the microfluidic system, the plurality of temperature control elements comprises temperature control elements controlled by a temperature control unit that performs either heating or cooling.

[0100] In an exemplary embodiment of the microfluidic system, the plurality of temperature control elements comprises temperature control elements controlled by a temperature control unit that performs both heating and cooling.

[0101] In an exemplary embodiment of the microfluidic system, the temperature control elements include Joule heating elements, resistive heating elements, and / or Peltier effect elements.

[0102] In an exemplary embodiment of the microfluidic system, the microfluidic system further comprises a droplet sensor for sensing the position of a droplet within the EWOD channel of the EWOD device.

[0103] Another aspect of the invention is a method of controlling a reaction protocol using an electrowetting on dielectric (EWOD) device with enhanced temperature control by combining spatial and temporal temperature control. In an exemplary embodiment, the method of controlling comprises the steps of receiving a droplet in an EWOD channel defined by an EWOD device, generating a first temperature zone at a first spatial location in the EWOD channel, the first temperature zone being controlled to have a first temperature, generating a second temperature zone at a second spatial location in the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature that is time-variable, time-varying the temperature of the second temperature zone, and applying an actuation voltage to an array of elements of the EWOD device to move the droplet between the first temperature zone and the second temperature zone, wherein the temperature of the droplet is taken as the temperature of one of the first temperature zone or the second temperature zone in which the droplet is located. The method of controlling may include one or more of the following features, individually or in combination.

[0104] In an exemplary implementation of the control method, the temperature of the second temperature zone is time-variable depending on the position of the droplet within the EWOD channel.

[0105] In an exemplary embodiment of the control method, the temperature of the second temperature zone is variable when the droplet is located within the first temperature zone.

[0106] In an exemplary embodiment of the control method, the temperature of the second temperature zone is variable when the droplet is located in the second temperature zone.

[0107] In an exemplary embodiment of the control method, the temperature of the first temperature zone is kept constant during the reaction protocol.

[0108] In an exemplary embodiment of the control method, the control method further includes time-varying the temperature of the first temperature zone during the reaction protocol.

[0109] In an exemplary embodiment of the control method, the temperatures of the first temperature zone and the second temperature zone are controlled to control the temperature of the droplets to be cyclically variable over multiple droplet temperature cycles within the reaction protocol.

[0110] In an exemplary embodiment of the control method, the temperatures of the first and second temperature zones are controlled to control the temperature of the droplet to undergo a series of temperature changes before performing a temperature cycle.

[0111] Another aspect of the invention is a method of performing nucleic acid amplification via polymerase chain reaction (PCR) using an electrowetting on dielectric (EWOD) device with enhanced temperature control by combining spatial and temporal temperature control.

[0112] In an exemplary embodiment, a PCR method comprises receiving a droplet in an EWOD channel defined by an EWOD device, the droplet comprising a nucleic acid, and performing a PCR cycle, the PCR cycle comprising: generating a first temperature zone at a first spatial location in the EWOD channel, the first temperature zone being controlled to have a first temperature for performing a denaturing step, moving the droplet to the first temperature zone, denaturing the nucleic acid in the first temperature zone, generating a second temperature zone at a second spatial location in the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature for performing a primer annealing step, moving the droplet to the second temperature zone, annealing the primer of the nucleic acid in the second temperature zone, re-equilibrating the temperature of the first temperature zone to a third temperature for performing nucleic acid polymerization while the droplet is in the second temperature zone, moving the droplet to the first temperature zone, and polymerizing the nucleic acid in the first temperature zone. The PCR method further includes performing multiple PCR cycles by repeating the PCR cycles until the nucleic acid amplification is complete.

[0113] In an exemplary embodiment, the PCR method is a touchdown PCR method, further comprising adjusting the primer annealing temperature within a second temperature zone between 1-2°C starting from 10°C higher than the primer melting temperature of each PCR cycle for a first portion of the plurality of PCR cycles, and maintaining the primer annealing temperature within the second temperature zone constant in each PCR cycle for a second portion of the plurality of PCR cycles following the first portion of the PCR cycles.

[0114] Although the present invention has been shown and described with respect to one or more embodiments, equivalent substitutes and modifications may occur to those skilled in the art upon reading and understanding the present specification and the accompanying drawings. In particular, with respect to the various functions performed by the above-described elements (components, assemblies, devices, compositions, etc.), the terms used to describe such elements (including "means") are intended to correspond to any element that performs the particular function of the described element (i.e., functionally equivalent), unless otherwise indicated, even if it is not structurally equivalent to the disclosed structures that perform that function in one or more exemplary embodiments of the present invention shown herein. Furthermore, although a particular feature of the present invention is described with respect to only one or more of the examples, such feature may be combined with one or more other features of other embodiments as may be desirable or advantageous for a given or particular application. [Industrial Applicability]

[0115] The described embodiments can be used to provide enhanced AM-EWOD devices. The AM-EWOD devices can form part of a lab-on-a-chip system. As such, the devices can be used to manipulate, react, and sense chemical, biochemical, or physiological materials. Applications include healthcare diagnostic testing, materials testing, chemical or biochemical materials synthesis, proteomics, and research tools in the life sciences and forensics. The invention as originally claimed in the present application is set forth below. 〔1〕 In a microfluidic system, An electrowetting on dielectric (EWOD) device comprising an array of elements configured to receive one or more droplets, the array of elements comprising a plurality of individual array elements; a control system configured to control actuation voltages applied to the array of elements to perform a manipulation operation on the droplet; a plurality of temperature control elements disposed at different spatial locations along the EWOD device, at least one of the temperature control elements being variable in temperature with respect to time; Equipped with wherein the control system includes a temperature control unit configured to control temperatures of a plurality of temperature control elements to generate a plurality of temperature zones arranged at different spatial locations along the EWOD device, and at least one of the temperature zones is temperature variable with respect to time by time-varying the temperature of the at least one of the temperature control elements having a time-varying temperature. Microfluidic systems. 〔2〕 The microfluidic system of [1], wherein the temperature control unit is configured to control the temperature in at least one temperature zone, the temperature being variable with respect to time, depending on the position of a droplet in an EWOD channel of the EWOD device. 〔3〕 The microfluidic system of claim 1 or 2, wherein a plurality of temperature control elements have a variable temperature with respect to time, and the temperature control unit is configured to control the plurality of variable temperature control elements to vary the temperatures of a plurality of temperature zones with respect to time. 〔4〕 The microfluidic system of any one of claims 1 to 3, wherein the temperature control unit is configured to control at least one temperature control element to maintain at least one temperature zone at a constant temperature. 〔5〕 A microfluidic system as described in any one of [1] to [4], wherein the EWOD device comprises a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and the plurality of temperature control elements are disposed on an outer surface of one or both of the first and second substrate assemblies. 〔6〕 A microfluidic system as described in any one of [1] to [4], wherein the EWOD device comprises a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and the plurality of temperature control elements are disposed within one or both of the first and second substrate assemblies. 〔7〕 The microfluidic system according to any one of claims 1 to 6, wherein the plurality of temperature control elements include temperature control elements controlled by the temperature control unit to perform heating or cooling. 〔8〕 A microfluidic system described in any one of [1] to [7], wherein the plurality of temperature control elements include temperature control elements controlled by the temperature control unit to perform both heating and cooling. 〔9〕 The microfluidic system of any one of claims 1 to 8, wherein the temperature control element comprises a Joule heating element, a resistive heating element, and / or a Peltier effect element. 〔10〕 A microfluidic system described in any one of [1] to [9], comprising a droplet sensor for detecting the position of the droplet within the EWOD channel of the EWOD device. 〔11〕 1. A control method for performing a reaction protocol using an electrowetting on dielectric (EWOD) device, the control method comprising: receiving a droplet within an EWOD channel defined by the EWOD device; generating a first temperature zone at a first spatial location within the EWOD channel, the first temperature zone being controlled to have a first temperature; generating a second temperature zone at a second spatial location within the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature that is variable with respect to time; Varying the second temperature of the second temperature zone over time; and applying an actuation voltage to an array of elements of the EWOD device to move the droplet between the first temperature zone and the second temperature zone, wherein the temperature of the droplet is taken to be the temperature of one of the first temperature zone or the second temperature zone in which the droplet resides; and a control method comprising the steps of: 〔12〕 The control method described in

[11] , wherein the second temperature of the second temperature zone is time-variable depending on the position of the droplet in the EWOD channel. 〔13〕 The control method of claim 11 or 12, wherein the second temperature of the second temperature zone is variable when the droplet is located in the first temperature zone. 〔14〕 The control method according to any one of claims 11 to 13, wherein the second temperature of the second temperature zone is variable when the droplet is located in the second temperature zone. 〔15〕 The method of any one of claims 11 to 14, wherein the first temperature of the first temperature zone is kept constant during the reaction protocol. 〔16〕 The method of any one of claims 11 to 14, further comprising time-varying the first temperature of the first temperature zone during the reaction protocol. 〔17〕 The control method of any one of

[11] to

[16] , wherein the temperatures of the first temperature zone and the second temperature zone are controlled to control the temperature of the droplet to be cyclically variable over multiple droplet temperature cycles within a reaction protocol. 〔18〕 The control method of

[17] , wherein the temperatures of the first temperature zone and the second temperature zone are controlled to control the temperature of the droplet to undergo a series of temperature changes before performing a temperature cycle. 〔19〕 1. A method for performing nucleic acid amplification via polymerase chain reaction (PCR) using an electrowetting on dielectric (EWOD) device, the PCR method comprising: receiving a droplet within an EWOD channel defined by the EWOD device, the droplet comprising nucleic acid; performing a PCR cycle, generating a first temperature zone at a first spatial location within the EWOD channel, the first temperature zone being controlled to have a first temperature for performing a denaturation step; and moving the droplet to the first temperature zone; organizing the nucleic acid within the first temperature zone; generating a second temperature zone at a second spatial location in the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature for performing a step of annealing a primer; and moving the droplet to the second temperature zone; annealing a primer to the nucleic acid in the second temperature zone; re-equilibrating the temperature of the first temperature zone to a third temperature to carry out nucleic acid polymerization while the droplet is in the second temperature zone, and moving the droplet to the first temperature zone; and polymerizing the nucleic acid in the first temperature zone; performing a PCR cycle; and performing a plurality of PCR cycles by repeating the PCR cycles until the nucleic acid amplification is complete; A method comprising the steps of: 〔20〕 adjusting the primer annealing temperature within a second temperature zone between 1-2°C starting from 10°C above the primer melting temperature of each PCR cycle for a first portion of the plurality of PCR cycles; and maintaining the temperature of primer annealing constant within the second temperature zone within each PCR cycle for a second portion of a plurality of PCR cycles subsequent to the first portion of the PCR cycles; The method of claim 19, further comprising:

[0116] Reference symbol list 10 - Bottom Board 12 - Element electrode 12A - Element electrode 12B - Element electrode 14 - Droplet 16 - Top Board 18 - Spacer 20 - Non-polar ambient fluid 22 - Insulator layer 24 - First hydrophobic coating 26 - contact angle 28 - Second hydrophobic coating 30 - reference electrode 32 - Leader 34 - Cartridge 35 - External Sensor Module 36 - EWOD or AM-EWOD device 38 - Control electronics 40 - Storage Devices 42 - Cable for connecting wiring 44 - Bottom board 46 - Thin film electronic circuit 48 - Element electrode 48A - Element electrode 48B - Element electrode 50 - Electrode or element array 52 - Droplet 54 - Top board 56 - Spacer 58 - Reference electrode 60 - nonpolar fluid 62 - Insulator layer 64 - First hydrophobic coating 66 - contact angle 68 - Second hydrophobic coating 70A - Electrical load when droplets are present 70B - Electrical load when no droplets are present 72 - Array element circuit 74 - Integrated row driver 76 - Column Driver 78 - Integrated Sensor Row Addressing 80 - Column detection circuit 82 - Serial Interface 84 - Voltage supply interface 86 - number of connecting wires 88 - Actuation Circuit 90 - Droplet or Impedance Sensing Circuit 100 - Exemplary Microfluidic Systems 102 - Control Systems 104 - EWOD device 106 - EWOD Channel 108 - Droplet 110 - Nonpolar fluid 112 - 1st (top) board assembly 114 - Second (lower) board assembly 116 - Spacer 118 - Liquid input structure 122 - EWOD control unit 124 - Temperature Zone Control Unit 126 - First temperature control element 127 - First Temperature Zone 128 - Second temperature control element 129 - Second Temperature Zone 200 - Temperature profile for the first temperature zone 202 - Temperature profile for the second temperature zone 204 - Droplet Temperature Profile

Claims

1. In a microfluidic system, An electrowetting on dielectric (EWOD) device comprising an array of elements configured to receive one or more droplets, said array of elements comprising a plurality of individual array elements; a control system configured to control actuation voltages applied to the array of elements to perform a manipulation operation on the droplet; a plurality of temperature control elements arranged at different spatial locations along the EWOD device, at least one of said temperature control elements being variable in temperature with respect to time; Equipped with wherein the control system includes a temperature control unit configured to control temperatures of a plurality of temperature control elements to generate a plurality of temperature zones arranged at different spatial locations along an EWOD device, the temperature control unit configured to control temperatures of the plurality of temperature control elements to achieve a predetermined droplet temperature profile by combining spatial temperature control, which varies the temperature of the plurality of temperature zones, and temporal temperature control, which varies the temperature in at least one temperature zone with respect to time, according to a reaction protocol programmed into the control system, wherein the spatial temperature control comprises moving the droplet between temperature zones by electrowetting, and the temporal temperature control comprises applying an arbitrary temperature / time function to the droplet while it remains in the at least one temperature zone. Microfluidic systems.

2. The microfluidic system of claim 1 , wherein the temperature control unit is configured to control the temperature in at least one temperature zone having a variable temperature with respect to time depending on the position of a droplet in an EWOD channel of the EWOD device.

3. 3. The microfluidic system of claim 1, wherein a plurality of temperature control elements are variable in temperature with respect to time, and the temperature control unit is configured to control the plurality of temperature control elements to vary the temperatures of a plurality of temperature zones with respect to time.

4. 4. The microfluidic system of claim 1, wherein the temperature control unit is configured to control at least one temperature control element to maintain at least one temperature zone at a constant temperature.

5. 5. The microfluidic system of claim 1, wherein the EWOD device comprises a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and the plurality of temperature control elements are disposed on an outer surface of one or both of the first and second substrate assemblies.

6. 5. The microfluidic system of claim 1, wherein the EWOD device comprises a first substrate assembly and a second substrate assembly defining an EWOD channel for receiving one or more droplets, and the plurality of temperature control elements are disposed within one or both of the first and second substrate assemblies.

7. 7. The microfluidic system of claim 1, wherein the plurality of temperature control elements comprises temperature control elements controlled by the temperature control unit to perform heating or cooling.

8. 8. The microfluidic system of claim 1, wherein the plurality of temperature control elements comprises temperature control elements controlled by the temperature control unit to perform both heating and cooling.

9. 9. The microfluidic system of claim 1, wherein the temperature control elements comprise Joule heating elements, resistive heating elements, and / or Peltier effect elements.

10. 10. The microfluidic system of claim 1, comprising a droplet sensor for detecting the position of the droplet in an EWOD channel of the EWOD device.

11. 1. A temperature control method for executing a pre-programmed reaction protocol using an electrowetting on dielectric (EWOD) device, the temperature control method comprising: receiving a droplet within an EWOD channel defined by said EWOD device; generating a first temperature zone at a first spatial location within the EWOD channel, the first temperature zone being controlled to have a first temperature; generating a second temperature zone at a second spatial location within the EWOD channel different from the first spatial location, the second temperature zone being controlled to have a second temperature that is variable with respect to time, wherein the first temperature zone and the second temperature zone are controlled by the preprogrammed reaction protocol to achieve a predetermined droplet temperature profile by a combination of spatial temperature control that varies the temperature of the first temperature zone and the temperature of the second temperature zone and temporal temperature control that varies the temperature in at least one of the first temperature zone and the second temperature zone with respect to time; wherein the spatial temperature control comprises moving the droplet between temperature zones by electrowetting, and the temporal temperature control comprises applying an arbitrary temperature / time function to the droplet while it remains in the at least one temperature zone, and applying an actuation voltage to an array of elements of the EWOD device to move the droplet between the first temperature zone and the second temperature zone, wherein the temperature of the droplet is taken to be the temperature of one of the first temperature zone or the second temperature zone in which the droplet resides; A temperature control method comprising the steps of:

12. The temperature control method of claim 11 , wherein the second temperature of the second temperature zone is time-variable depending on the position of the droplet within the EWOD channel.

13. 13. The temperature control method of claim 11 or 12, wherein the second temperature of the second temperature zone is variable when the droplet is located in the first temperature zone.

14. 14. The method of claim 11, wherein the second temperature of the second temperature zone is variable when the droplet is located in the second temperature zone.

15. 15. The method of claim 11, wherein the first temperature of the first temperature zone is kept constant during the reaction protocol.

16. 15. The method of claim 11, further comprising time-varying the first temperature of the first temperature zone during the reaction protocol.

17. 17. The temperature control method of claim 11, wherein the temperatures of the first temperature zone and the second temperature zone are controlled to control the temperature of the droplet to be cyclically variable over multiple droplet temperature cycles within a reaction protocol.

18. 20. The temperature control method of claim 17, wherein temperatures of the first temperature zone and the second temperature zone are controlled to control the temperature of the droplet to undergo a series of temperature changes before performing a temperature cycle.

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