Method for kernel extraction and amplification by using biological field programmable gate array

Abstract

Disclosed herein is a method for performing kernel extraction and amplification by using a biological field programmable gate array (bio-field programmable gate array), and a method for performing kernel extraction and amplification by using a biological field programmable gate array (bio-field programmable gate array). The method comprises arranging a hybrid droplet comprising one or more target nucleic acids adsorbed to a magnetic bead on a microelectrode array, the microelectrode array comprising microelectrodes operable to form one or more actuation patterns. The method comprises extracting the one or more target nucleic acids from the mixed droplet by attracting the one or more target nucleic acids using a magnetic force generated by one or more coils below the microelectrode, and switching one or more of the microelectrodes to correspond to a treatment actuation pattern to move the hybrid droplet to a treatment position. The method includes combining the one or more target nucleic acids and an amplified droplet to form a pre-amplified droplet, heating the pre-amplified droplet under a programmed temperature planning scheme to generate an amplified droplet, and visualizing the amplified droplet.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 337,478, filed on May 2, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to molecular biology techniques, and more particularly to molecular biology techniques involving the manipulation of nucleic acids. Background of the Invention

[0005] Molecular biology techniques, such as polymerase chain reaction (PCR) and rapid diagnostic tests (RDT), are useful in testing for a variety of analytes associated with disease states. Molecular biology techniques involve the detection and analysis of nucleic acids (e.g., DNA and RNA). Various techniques are available to amplify and detect specific regions of nucleic acids, allowing identification of pathogens and genetic mutations. Rapid bedside (point-of-care) testing slows disease spread and reduces health care costs. Therefore, there is a need for such molecular biology techniques that allow rapid detection and analysis of target nucleic acids (including nucleic acids associated with infectious diseases, cancers or other genetic changes). SUMMARY OF THE INVENTION

[0007] In a first aspect, a method for nuclear extraction and amplification using a bio-field programmable gate array includes arranging a hybrid droplet at a first position on a microelectrode array, wherein the hybrid droplet includes one or more target nucleic acids, a lysis buffer, and magnetic beads, wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and the microelectrode array includes a plurality of microelectrodes arranged in an array and operable to form one or more actuated patterns, each microelectrode including: a heater under the microelectrode, and a coil under the microelectrode. The method further includes extracting the one or more target nucleic acids from the hybrid droplet by: using a magnetic force generated by the coil under the first position to attract the one or more target nucleic acids adsorbed to the magnetic beads, and switching one or more of the plurality of microelectrodes to correspond to a disposal actuation pattern to move the hybrid droplet to a disposal position on the microelectrode array. The method includes combining the one or more target nucleic acids and an amplifying droplet to form a pre-amplifying droplet at the first location, heating the pre-amplifying droplet using a heater below the first location under a programmed temperature schedule to generate an amplified droplet, and visualizing the amplified droplet.

[0008] In a second aspect, a non-transitory computer readable medium for performing nuclear extraction and amplification using a biological field programmable gate array comprises logic, which when executed by a computing device causes the computing device to perform at least the following steps: detecting a mixed droplet at a first position on a microelectrode array, wherein the mixed droplet comprises one or more target nucleic acids, a lysis buffer, and magnetic beads, wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and the microelectrode array comprises a plurality of microelectrodes arranged in an array and operable to form one or more actuation patterns, each microelectrode comprising: a heater below the microelectrode, and a wire below the microelectrode; coil; extracting the one or more target nucleic acids from the mixing droplet by: using a magnetic force generated by a coil below the first position to attract the one or more target nucleic acids adsorbed to the magnetic beads, and switching one or more of the plurality of microelectrodes corresponding to a disposal actuation pattern to move the mixing droplet to a disposal position on the microelectrode array; merging the one or more target nucleic acids and amplification droplets to form pre-amplification droplets at the first position; heating the pre-amplification droplets using a heater below the first position under a programmed temperature planning scheme to generate amplified droplets; and visualizing the amplified droplets.

[0009] These and additional features provided by the embodiments described herein will be more fully understood in view of the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The embodiments illustrated in the drawings are illustrative and exemplary in nature and are not intended to limit the subject matter defined by the claims. The following detailed description of the illustrative embodiments may be understood when read in conjunction with the following drawings, in which like structures are designated by like reference numerals, and in which:

[0012] Figure 1 schematically depicts an exemplary biological field programmable gate array of the present disclosure, according to one or more embodiments shown and described herein;

[0013] Figure 2 schematically depicts exemplary non-limiting components of a biological field programmable gate array of the present disclosure, according to one or more embodiments shown and described herein;

[0014] Figure 3 schematically depicts an actuation pattern on a microelectrode array of the present disclosure, according to one or more embodiments shown and described herein;

[0015] Figure 4Aschematically depicts a droplet on a microelectrode array and corresponding detected positions of the droplet illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0016] Figure 4B schematically depicts a droplet (with an actuation pattern) on a microelectrode array and corresponding detected positions of the droplet illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0017] Figure 4C schematically depicts a droplet moving on a microelectrode array (driven by an actuation pattern) and a corresponding detected position of the droplet after the movement, illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0018] Figure 5A schematically depicts two droplets on a microelectrode array and corresponding detected positions of the droplets illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0019] Figure 5B schematically depicts two droplets on a microelectrode array with an actuation pattern therebetween and corresponding detected positions of the droplets illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0020] Figure 5C schematically depicts two droplets merging on a microelectrode array (driven by an actuation pattern) and corresponding detected positions of the merged droplets after merging, illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0021] Fig. 6A schematically depicts a droplet on a microelectrode array and corresponding detected positions of the droplet illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0022] Figure 6B schematically depicts a droplet on a microelectrode array illustrated at a graphical user interface of the present disclosure having two actuation patterns at both sides of the droplet and the corresponding detected position of the droplet, according to one or more embodiments shown and described herein;

[0023] Figure 6Cschematically depicting the splitting of a droplet into two split droplets on a microelectrode array via the two actuation patterns and corresponding detected positions of the split droplets after splitting, as illustrated at a graphical user interface of the present disclosure, according to one or more embodiments shown and described herein;

[0024] Figure 7 Schematically depicts a process of mixing droplets on a microelectrode array by activating an actuation pattern in a cyclic sequence of the present disclosure, according to one or more embodiments shown and described herein;

[0025] Figure 8 Schematically depicts a process of merging and mixing sample droplets and reagent droplets of the present disclosure, according to one or more embodiments shown and described herein;

[0026] Fig. 9 Schematically depicts a process of extracting target nucleic acid from a mixing droplet of the present disclosure, according to one or more embodiments shown and described herein;

[0027] Fig.10 A flow chart illustrating illustrative steps of the present disclosure for nuclear extraction and amplification by using a biological field programmable gate array, according to one or more embodiments shown and described herein;

[0028] Fig.11 Illustrated is a photograph of a bio-FPGA chip of the present disclosure next to a penny, an illustrative example of a portable device incorporating the bio-FPGA chip, and a photograph of a cartridge housing the bio-FPGA chip, according to one or more embodiments shown and described herein;

[0029] Fig.12 illustrates an example of thermal cycling using a bio-FPGA chip of the present disclosure, according to one or more embodiments shown and described herein; and

[0030] Fig.13 Illustrated are exemplary LAMP results of amplified nucleic acids following processing using a bio-FPGA chip of the present disclosure, according to one or more embodiments shown and described herein. DETAILED DESCRIPTION OF THE INVENTION

[0032] Covid-19 testing is useful in the fight against the SARS-CoV-2 virus. Diagnostic tests, such as polymerase chain reaction (PCR), are useful in determining whether a person may be infected by SARS-CoV-2. PCR tests can detect RNA specific to the virus and can detect the virus within a few days of infection, even in those who do not have symptoms. When completed in a laboratory, such tests may take several days, a week, or possibly longer to return results. The embodiments disclosed herein relate to methods for nucleic acid extraction, amplification, and analysis and portable, programmable, handheld biological field programmable gate arrays (bio-FPGAs). The bio-FPGA disclosed herein performs several tasks in parallel, including sample processing, extraction, labeling, targeting, molecular biomarker detection (with high specificity and sensitivity), and both real-time quantitative PCR (qPCR) and digital PCR (dPCR). The bio-FPGA is a microscale liquid handling technology that generates multiple droplets for manipulating microfluidic operations in the air, moving, splitting, and merging the droplets. Each droplet acts as an independent reactor, which enables multiple parallel biological and chemical reactions that require solvent exchange. The bio-FPGA according to the present disclosure reduces reagent and energy consumption, speeds up analysis, and enables bedside diagnostic applications. In addition to Covid-19 testing, the bio-FPGA and methods of use disclosed herein are useful in detecting and diagnosing other infectious diseases (including other viruses, bacteria, parasites, and other infectious agents), and detecting other analytes of interest that may be missed by other types of tests, such as genetic changes or cancer cells.

[0033] In some embodiments, the bio-FPGA is manufactured using a standard 0.35 μm complementary metal oxide semiconductor (CMOS) process and has the listed functions. The bio-FPGA moves droplets (samples or reagents) with a diameter of 200-500 μm to a target area at a speed of up to 1000 μm / s through the electrowetting (ElectroWetting on Dielectric; EWOD) force on the dielectric. The position and volume of the target droplet can be manipulated by using one or more actuation patterns of a microelectrode dot array (MEDA). The actuation pattern can move the target droplet from one position to another. The actuation pattern can control a large number of target droplets at the same time and automatically run the set program step by step. The droplet can be moved toward or away from the area where the pattern is actuated.

[0034] In some embodiments, the bio-FPGA can split droplets symmetrically (1:1) or asymmetrically (1:n, n=2, 3, ..., etc.). With respect to the droplets disclosed herein, splitting refers to the process in which a liquid droplet flows in two opposite directions at equal or unequal rates and forms two sister droplets. In order to split, a pattern with two actuation areas will be set in the programming MEDA. The two areas move the droplet in opposite directions at the same time to create sister droplets. Both symmetric and asymmetric splitting can be achieved, based on specific parameters of the actuation pattern, such as whether the actuation pattern has equal or unequal sizes on the bio-FPGA.

[0035] In some embodiments, the bio-FPGA can move two individual droplets along a prescribed path to mix together. To perform the merging of droplets, a pattern with two actuation regions can be set in the programming MEDA. The two regions move the droplets toward each other until the droplets merge together. Both symmetric and asymmetric merging can be achieved, based on the characteristics of the droplets, such as whether the droplets are of equal or unequal size.

[0036] In some embodiments, the bio-FPGA can dilute droplets by merging and asymmetric or symmetric splitting, so that high dilution factors can be achieved. For dilution, a pattern with two or more actuation areas can be set in programming MEDA. Asymmetric or symmetric merging and splitting can be used for dilution.

[0037] In some embodiments, the bio-FPGA may include at least two different sensing methods, including capacitive and optical sensing. Capacitive sensing can be used to confirm sample volume and position. Optical sensing can measure the concentration and quantity of target molecules. The capacitive sensing circuit under each microelectrode may include a capacitive sensor for the volume and position of the target droplet. Each microelectrode presents "1" (if there is a sample) or "0" (if there is no sample). Optionally, the sensing result can include a 2D binary array, which includes detailed information on volume and position. Optical sensors can detect the fluorescence of labeled reagents, such as in PCR tests or real-time monitoring. Such optical sensors are also useful for visualizing isothermal amplifications such as loop-mediated isothermal amplification (LAMP).

[0038] In some embodiments, the bio-FPGA can perform DNA or RNA extraction for amplification via a thermal control plate on the chip. Solid phase extraction can also be performed on the chip. Magnetic beads can be collected by electromagnetic electrodes on the chip.

[0039] In some embodiments, the bio-FPGA can perform a PCR assay, where on-chip thermal control is used to manipulate the temperature profile to promote amplification of the target DNA, as defined in the biological protocol for the PCR test. In addition, the bio-FPGA can perform in-situ cycling real-time quantification of amplicons. The target temperature for the PCR step can be manipulated by the on-chip temperature profile. The actuation pattern can be set in the programming MEDA. The target temperature can be controlled via a given pattern, i.e., different geometric patterns can be used to achieve different set temperatures.

[0040] In some embodiments, the bio-FPGA can perform immunolabeling. The bio-FPGA can move, mix, and incubate cells, tissues, or organoids in droplets with labeled antibody solutions. The bio-FPGA can decant and wash target samples through a series of movements, splitting, and / or merging of droplets on the chip. During such a process, a droplet can be considered as a reactor, and multiple droplets can be considered as independent reactors. Actuation patterns can be set in programming MEDA to achieve decantation and washing via movement, merging, and / or splitting of sample droplets and other droplets.

[0041] In some embodiments, the bio-FPGA can manipulate both samples and reagents to avoid contamination during testing procedures. The bio-FPGA can process multiple samples simultaneously to verify results and enhance testing accuracy.

[0042] The methods and devices disclosed herein are suitable for the detection of a wide variety of analytes (including but not limited to DNA and RNA). In some embodiments, the target analyte may indicate the presence of a specific virus, bacteria, fungus, parasite, or other target cell. For example, in some embodiments, the methods and devices disclosed herein detect target nucleic acids indicating the following viruses: coronaviruses, such as SARS-CoV-1, SARS-CoV-2, and MERS; arboviruses, such as dengue virus, chikungunya virus, Zika virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus; influenza virus; human immunodeficiency virus; and other target viruses. In some embodiments, the target analyte is a genetic mutation in a target nucleic acid.

[0043] Various embodiments of the bio-FPGA method, system, and apparatus are described in more detail herein. Whenever possible, the same reference numerals will be used throughout the drawings to refer to the same or like parts.

[0044] Unless otherwise expressly stated, it is in no way intended that any method described herein be construed as requiring that its steps be performed in a particular order, and requiring a particular orientation when using any apparatus. Thus, when a method claim does not actually recite an order to be followed by its steps, or any apparatus claim does not actually recite an order or orientation for individual components, or does not specifically state in the claims or specification that the steps will be limited to a particular order, or does not recite a particular order or orientation for components of the apparatus, it is in no way intended that an order or orientation be inferred in any respect. This applies to any possible non-express basis for interpretation, including logical issues relating to arrangement of steps, operational flow, order of components, or orientation of components; clear meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0045] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a component includes aspects having two or more such components unless the context clearly dictates otherwise.

[0046] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (e.g., reaction conditions), and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0047] As used herein, the term "about" when referring to a value or amount of mass, weight, time, volume, concentration, or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the expressly stated amount as such variations are suitable for practicing the disclosed methods.

[0048] For the purpose of defining the present technology, the transitional phrase "consisting of" may be introduced in the claims as a closed leading term that limits the scope of the claim to the recited components or steps and any naturally occurring impurities. For the purpose of defining the present technology, the transitional phrase "consisting essentially of" may be introduced in the claims to limit the scope of one or more claims to the recited elements, components, materials, or method steps and any unrecorded elements, components, materials, or method steps that do not materially affect the novel features of the claimed subject matter. The transitional phrases "consisting of" and "consisting essentially of" may be interpreted as a subset of open transitional phrases (e.g., "comprising" and "including"), such that any use of an open phrase to introduce a description of a series of elements, components, materials, or steps should be interpreted as also disclosing the description of the series of elements, components, materials, or steps by using the closed terms "consisting of" and "consisting essentially of". For example, a description of a composition "comprising" components A, B, and C should be interpreted as also disclosing a composition "consisting of components A, B, and C" as well as a composition "consisting essentially of components A, B, and C." Any numerical value recited in this application may be considered to include open embodiments consistent with the transitional phrases "comprising" or "including," as well as closed or partially closed embodiments consistent with the transitional phrases "consisting of" and "consisting essentially of."

[0049] It should be understood that each maximum numerical limit given throughout this specification includes each lower numerical limit, as if such lower numerical limits were explicitly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were explicitly written herein. Each numerical range given throughout this specification will include each narrower numerical range that falls within such wider numerical range, as if such narrower numerical ranges were explicitly written herein.

[0050] Turning to the attached figure, Figure 1 Schematically depicts an exemplary biological field programmable gate array (bio-FPGA) of the present disclosure. The bio-FPGA 100 includes a microelectrode array 101 fabricated on a substrate 120. The microelectrode array 101 may include a plurality of microelectrodes 122 on a surface of the microelectrode array 101. Figure 1As illustrated in , the microelectrodes 122 can be arranged in a grid array. In the grid array, the microelectrodes 122 are arranged in a two-dimensional grid pattern. The shape of the microelectrode 122 can be cubic, rectangular, circular (oval), or any shape suitable for the microelectrode array 101. The microelectrode 122 can have a diameter of several microns to hundreds of microns, depending on the circuit under the microelectrode 122. The interval between each microelectrode 122 can have a distance from several microns to hundreds of microns. The diameter of the microelectrode 122 and the selection of the gap between the microelectrodes 122 can depend on the desired spatial resolution, signal-to-noise ratio and the cost of the device. The microelectrode array 101 can also include a linear array, a spiral array, a random array or other suitable arrays. Each microelectrode 122 is connected to the control and detection circuit.

[0051] The substrate 120 may further be integrated with a heater 132, a coil 142, and a capacitive sensor 209. In some embodiments, each microelectrode 122 may have a heater 132, a temperature sensor 208, a coil 142, and a capacitive sensor 209 below the microelectrode 122. Each microelectrode 122, heater 132, coil 142, and capacitive sensor 209 are connected to a control and detection circuit built into the substrate 120. The control and detection circuit may be connected to the input / output hardware 205 of the controller 201 through the pins and wires 115 of the substrate 120. The optical sensor 210 may also be connected to the controller 201 through the input / output hardware 205.

[0052] The controller 201 may include various modules for detecting the surface of the microelectrode array. For example, the controller may use an optical sensor 210 to detect the fluorescence of the surface of the microelectrode array 101. The controller 201 may use a temperature sensor 208 to detect the temperature of the droplets on the surface of the microelectrode array 101. The controller 201 may use one or more capacitive sensors 209 and optical sensors 210 to detect the position and size of the droplets on the surface of the microelectrode array 101.

[0053] The controller 201 may include various modules that control the components of the microelectrode array. For example, the controller 201 may apply an electric field to the microelectrode 122 to switch it to an on-state microelectrode 332, where the on-state microelectrode 332 exhibits a positive or negative potential. Similarly, the controller 201 may switch the microelectrode 122 to an "off" state, where the microelectrode 122 exhibits a zero potential. The controller 201 may control the circuitry built into the substrate 120 to turn on the heater 132 to generate heat and turn on the coil 142 to generate a magnetic field, or turn off the heater 132 from generating heat and turn off the coil 142 from generating a magnetic field.

[0054] See also Figure 2 , depicting exemplary, non-limiting components of the bio-FPGA. The bio-FPGA 100 may include a controller 201. The controller 201 may include various modules. For example, the controller 201 may include an actuation pattern module 222, a temperature control module 232, a magnetic control module 242, a visualization module 252, and a positioning module 262. The controller 201 may further include various components, such as a memory 202, a processor 204, an input / output hardware 205, a network interface hardware 206, a data storage component 207, and a local interface 203. The controller 201 may include a temperature sensor 208, a capacitive sensor 209, and / or an optical sensor 210.

[0055] The controller 201 can be any device or component combination including a processor 204 and a memory 202 (e.g., a non-transitory computer-readable memory). The processor 204 can be any device capable of executing a machine-readable instruction set stored in the non-transitory computer-readable memory. Therefore, the processor 204 can be an electrical controller, an integrated circuit, a microchip, a computer, or any other computing device. The processor 204 may include any processing component configured to receive and execute programming instructions (e.g., from a data storage component 207 and / or a memory component 202). The instructions may be in the form of a machine-readable instruction set stored in the data storage component 207 and / or the memory component 202. The processor 204 is communicatively coupled to other components of the controller 201 via a local interface 203. Therefore, the local interface 203 can enable any number of processors 204 to be communicatively coupled to each other, and allow components coupled to the local interface 203 to operate in a distributed computing environment. The local interface 203 can be implemented as a bus or other interface to facilitate communication between components of the controller 201. In some embodiments, each of the components can operate as a node that can send and / or receive data. Although in Figure 1 The embodiment depicted in includes a single processor 204 , but other embodiments may include more than one processor 204 .

[0056] The memory 202 (e.g., a non-transitory computer-readable memory component) may include RAM, ROM, flash memory, a hard disk, or any non-transitory memory device capable of storing machine-readable instructions so that the machine-readable instructions can be accessed and executed by the processor 204. The machine-readable instruction set may include logic or algorithms written in a programming language of any generation (e.g., 1GL, 2GL, 3GL, 4GL, or 5GL) (e.g., a machine language that can be directly executed by the processor 204, or an assembly language, object-oriented programming (OOP), a scripting language, microcode, etc., which can be compiled or assembled into machine-readable instructions and stored in the memory 202). Alternatively, the machine-readable instruction set may be written in a hardware description language (HDL) (e.g., logic implemented via a field-programmable gate array (FPGA) configuration or an application-specific integrated circuit (ASIC) or its equivalent). Thus, the functionality described herein may be implemented in any conventional computer programming language, as pre-programmed hardware elements, or as a combination of hardware and software components. For example, the memory component 202 may be a machine-readable memory (which may also be referred to as a non-transitory processor-readable memory or medium) storing instructions that, when executed by the processor 204, cause the processor 204 to perform the methods or control schemes described herein. Although in Figure 1 The embodiment depicted in includes a single non-transitory computer readable memory 202, but other embodiments may include more than one memory module. The memory is used to store the actuation pattern module 222, the temperature control module 232, the magnetic control module 242, the visualization module 252, and the positioning module 262 during operation. During operation, each of the actuation pattern module 222, the temperature control module 232, the magnetic control module 242, the visualization module 252, and the positioning module 262 can be in the form of an operating system, application modules, and other program modules. Such program modules can include, but are not limited to, routines, subroutines, programs, objects, components, and data structures for performing specific tasks or executing specific abstract data types according to the present disclosure, as will be described below.

[0057] Input / output hardware 205 may include a monitor, keyboard, mouse, printer, camera, microphone, speakers, and / or other devices for receiving, sending, and / or presenting data.

[0058] Network interface hardware 206 may include any wired or wireless networking hardware, such as a modem, a LAN port, a Wi-Fi card, a WiMax card, mobile communications hardware, and / or other hardware for communicating with other networks and / or devices.

[0059] The data storage unit 207 stores the collected visualization data, the data generated by the sensor, and the data for operating the microelectrodes, heaters, and coils. The actuation pattern module 222, the temperature control module 232, the magnetic control module 242, the visualization module 252, and the positioning module 262 may also be stored in the data storage unit 207 during or after operation.

[0060] See also Figure 3 , depicts an actuation pattern on a microelectrode array of the present disclosure. An actuation pattern is a group of microelectrodes in an "on" state, where an electric field is applied to the actuation pattern to manipulate droplets in the vicinity of the actuation pattern. Actuation patterns can include a variety of configurations, such as parallel electrodes, annular electrode arrays, serpentine electrodes, interdigitated electrodes, and asymmetric electrode patterns. The choice of an actuation pattern depends on the specific application and the desired droplet movement. For example, parallel electrodes are typically used for linear movement, while annular electrode arrays are often used to mix or separate droplets. In Figure 3 , the actuation pattern 322 consists of applying an electric field to a 3 x 3 array of on-state microelectrodes 332. The choice of positive or negative electric field can depend on the characteristics of the droplet to be manipulated. For example, if the droplet is positively charged, then the negative electric field will attract it, while the positive electric field will repel it. Conversely, if the droplet is negatively charged, then the positive electric field will attract it, while the negative electric field will repel it. In addition to the charge of the droplet, the choice of positive or negative electric field also depends on the electrode configuration and the desired manipulation method. For example, in a parallel electrode configuration, applying a positive electric field to one electrode and a negative electric field to another electrode can create an electric field gradient that moves the droplet in a certain direction. In some embodiments, the droplet actuation capability can depend on the leakage current and the avalanche breakdown voltage.

[0061] Figures 4A-4C Depicts the process of using the bio-FPGA to move droplets on a microelectrode array. In some embodiments, the bio-FPGA can first be used to control sensors (e.g., Figure 1 Capacitive sensor 209 or optical sensor 210 in the positioning module 262 (for example, in Figure 2) to detect the position of the droplet after it is placed on the microelectrode array. The bio-FPGA then determines the target position and the path between the first position of the droplet and the target position. The bio-FPGA can use the actuation pattern module 222 to program a series of actuation patterns to manipulate the movement of the droplet to the target position. During this process, the bio-FPGA can use the positioning module 262 (e.g., in Figure 2 ) and associated sensors to continuously monitor the position and movement of the droplets. The bio-FPGA uses an actuation pattern module 222 (e.g., Figure 2 ) to compare the position of the droplet with its programmed manipulation in order to adjust the actuation pattern to move the droplet as desired.

[0062] See also Figure 4A , depicting droplets 410 on a microelectrode array 401 and corresponding detected positions 403 of the droplets illustrated on a graphical user interface 402. The droplets 410 may be sample droplets, reagent droplets, mixing droplets, pre-amplification droplets, or amplified droplets. After the droplets 410 are arranged on the microelectrode array 401, the bio-FPGA may detect the position of the droplets 410 using an optical sensor 210 or a capacitive sensor 209 below the microelectrode 122. For example, the capacitive sensor 209 may detect the dielectric properties of the material on the electrode. The bio-FPGA may map the dielectric properties of the material on the microelectrode array 401 (e.g., the permittivity of the droplets, air, and other media), and determine the size and position of the droplets 410. The bio-FPGA may include a graphical user interface 402 and display the positions of the droplets in the form of a grid array that simulates the configuration of the microelectrode array 401, with each cell 442 representing the microelectrode 122 at that position. As shown in Figure 4A As illustrated in , the graphical user interface 402 may display the microelectrode area covered by the droplet as shaded cells 420 .

[0063] See also Figure 4B , depicts a droplet 410 (at its first position) on a microelectrode array 401 illustrated at a graphical user interface, an actuation pattern 332 representing a target position, and a corresponding detected position of the droplet. After the bio-FPGA determines the target position for moving the droplet 410, an actuation pattern can be created near the droplet that includes an on-state microelectrode 332. The position of the droplet 410 can be displayed on the graphical user interface 402.

[0064] See also Figure 4C, depicts a droplet 412 (driven by an actuation pattern) on a microelectrode array 401 and a corresponding detected position of the droplet after movement, illustrated at a graphical user interface. An electric field is applied to the droplet 410 after the actuation pattern. The droplet 410 moves from a first position 410a to a target position 410b, driven by an electrowetting on dielectric (EWOD) force. The EWOD force is proportional to the square of the applied voltage on the microelectrode 122. After the droplet 412 moves to the target position, the graphical user interface may display its detected position 403 in the form of a shaded cell 420.

[0065] Figures 5A-5C Depicts the process of using the bio-FPGA to merge two droplets into one droplet on a microelectrode array. In some embodiments, the bio-FPGA can be used to control sensors (e.g., Figure 1 Capacitive sensor 209 or optical sensor 210 in the positioning module 262 (for example, in Figure 2 ) to detect the position of the droplet, determine the target position for the droplet to be merged, and one or more paths between the position of the droplet and the target position. In some embodiments, only one droplet is moved and the target position is the position of another droplet. The bio-FPGA can use the actuation pattern module 222 to program a series of actuation patterns to manipulate the droplet to move to the target position. The bio-FPGA can estimate the size of the merged droplet and program the actuation pattern at the target position based on the estimated size of the merged droplet. During this process, the bio-FPGA can use the positioning module 262 and the sensor to continuously monitor the position and movement of the droplet. The bio-FPGA uses the actuation pattern module 222 (for example, in Figure 2 ) to compare the position of the droplet with its programmed manipulation in order to adjust the actuation pattern to move the droplet as desired.

[0066] See also Figure 5A , depicting two droplets 510 and 512 on a microelectrode array 501 and corresponding detected positions 503 and 504 of the droplets illustrated on a graphical user interface 502. The droplets 510 and 512 may be sample droplets, reagent droplets, mixing droplets, pre-amplification droplets, or amplified droplets. The bio-FPGA may detect the positions of the droplets 510 and 512 using a capacitive sensor 209 under the microelectrode 122 or using an optical sensor 210. The bio-FPGA may display the positions 503 and 504 of the droplets as shaded cells 420 at the graphical user interface 502.

[0067] See also Figure 5B, depicting an actuation pattern on a microelectrode array 501 (including an on-state microelectrode 332 between two droplets 510 and 512) and corresponding detected positions of the droplets, illustrated at a graphical user interface 502. The actuation pattern includes a 4x4 array of on-state microelectrodes 332 at least partially located between the droplet 510 and the droplet 512. When the actuation pattern occurs, an EWOD force is applied to the two droplets 510 and 512, so that the droplets are attracted to the position of the actuation pattern. The positions of the droplets 510 and 512 can be displayed on the graphical user interface 502, wherein cells 442 represent each corresponding microelectrode 122 in the microelectrode array 501, and shaded cells 420 represent positions 503, 504 of the droplets 510 and 512. In the initial stage of merging, the two droplets 510 and 512 are still at their first positions, as indicated by Figure 5B This is represented by the shaded cell 420 .

[0068] See also Figure 5C , depicts a merged droplet 514 formed by merging two droplets 510 and 512 from first locations 510a and 512a on a microelectrode array 501, an actuation pattern, and a corresponding detected position of the merged droplet after merging, illustrated at a graphical user interface 502. The 4 x 4 array actuation pattern drives the two droplets 510 and 512 to merge at the location of the actuation pattern via an EWOD force to form the merged droplet 514. After the droplets 510 and 512 merge at the location of the actuation pattern, the graphical user interface 502 may display the position 505 of the merged droplet 514 as a shaded cell 420.

[0069] Figures 6A-6C Depicted is the process of using the bio-FPGA to split a droplet into two sister droplets on a microelectrode array. In some embodiments, the bio-FPGA can be used to control sensors (e.g., Figure 1 Capacitive sensor 209 or optical sensor 210 in the positioning module 262 (for example, in Figure 2 220 (illustrated in FIG. 221 ), detects the position of the first droplet, determines two or more target positions for the split sister droplets to be moved, and a path between the position of the first droplet and the target positions. The split can be symmetrical (the split droplets exhibit substantially equal volumes) or asymmetrical (the split droplets exhibit unequal volumes). The bio-FPGA can use the actuation pattern module 222 (e.g., in Figure 2) to program a series of actuation patterns to manipulate the split droplets to move to the target location. The bio-FPGA can estimate the size of the split droplets and program the actuation patterns at the target location based on the estimated size of the split droplets. During this process, the bio-FPGA can use the positioning module 262 (e.g., in Figure 2 ) and sensors (e.g., capacitive sensor 209 and optical sensor 210) to continuously monitor the position and movement of the droplets. The bio-FPGA uses an actuation pattern module 222 (e.g., Figure 2 ) to compare the position of the droplet with its programmed manipulation in order to adjust the actuation pattern to move the droplet as desired.

[0070] See also Fig. 6A , depicting a droplet 610 on a microelectrode array 601 and a corresponding detected position 603 of the droplet illustrated at a graphical user interface 602. The droplet 610 may be a sample droplet, a reagent droplet, a mixing droplet, a pre-amplification droplet, or an amplified droplet. The bio-FPGA may detect the position of the droplet 610 using a capacitive sensor 209 under the microelectrode 122 or using an optical sensor 210. The bio-FPGA may display the position 603 of the droplet 610 as a shaded cell 420 at the graphical user interface 602.

[0071] See also Figure 6B , depicting a droplet 610 on a microelectrode array 601 (with two actuation patterns at both sides of the droplet 610) and the corresponding detected position of the droplet as illustrated at a graphical user interface 602. The actuation pattern includes a 2x 2 array of two on-state microelectrodes 332 at least partially located on the left and right sides of the droplet 610, although other actuation patterns are possible and contemplated. When the actuation pattern occurs, the EWOD force applied to the droplet 610 causes the droplet to be attracted to the two target positions of the actuation pattern. The position of the droplet 610 can be displayed on the graphical user interface 602, where cells 442 represent each microelectrode 122 in the microelectrode array 601, and shaded cells 420 represent the position of the droplet 610. In the initial stage of merging, the droplet 610 is still at its first position, as represented by the detected position 603 composed of shaded cells 420.

[0072] See also Figure 6C, depicting two sister droplets 612 and 614 split from a droplet 610 located at a first position 610a on a microelectrode array 601 by the two actuation patterns illustrated at a graphical user interface 602, and corresponding detected positions 604 and 605 of the split droplets 612 and 614 after the splitting. The two 2x2 array actuation patterns are arranged at least partially on the left and right sides of the droplet 610, where the electric field (as a result of the voltage applied to the on-state microelectrode 332) causes the surface tension of the droplet 610 to change through the EWOD force and causes the droplet 610 to split into two smaller split droplets 612 and 614.

[0073] It may be noted that during the process of splitting, the droplet 610 can change its shape and the bio-FPGA can control the shape and size of the split droplet. For example, during the process of splitting, the bio-FPGA can control the shape of the droplet in the following manner. For droplets having spherical and hemispherical shapes, after the bio-FPGA applies a voltage to the on-state microelectrode 332 of the actuation pattern, the surface tension of the droplet changes, and the droplet can deform or elongate, depending on the direction of the applied force. Taking into account the surface properties of the microelectrode array 601 and the viscosity of the liquid in the droplet 610, the bio-FPGA can further control the amplitude and duration of the applied voltage to split the droplet 610 into two or more smaller split droplets (e.g., split droplets 612 and 614). In some cases, a droplet can undergo a series of shape changes before it splits, thereby forming an intermediate shape, such as a dumbbell or teardrop shape. The ability to control these intermediate shapes can be useful in some applications, such as in droplet-based microfluidics, where it may be desirable to precisely control the shape and size of the droplets to perform specific chemical and biological assays. The resulting droplet shape can be spherical, rectangular, dumbbell-shaped (two droplets connected by a thin, elongated neck), or other shapes. The shape of the split droplet can be useful for droplets used as reaction vessels or carriers for chemical or biological assays, where the size and shape of the droplet can affect the efficiency and accuracy of the ongoing reaction. The user can use the bio-FPGA to manipulate the shape of the droplet to achieve the desired experimental results.

[0074] The bio-FPGA can use capacitive sensors and optical sensors to monitor the shape of the droplet during breakup, thereby using the monitored shape and position of the droplet as feedback to further adjust the actuation pattern and applied voltage on the on-state microelectrode 332. After the droplets 612 and 614 are broken up and reach the desired target position, the graphical user interface 502 can display the positions 604 and 605 of the broken-off droplets 612 and 614 in the form of shaded cells 420, as shown in FIG. Figure 6C As shown in .

[0075] See also Figure 7 , depicting the process of mixing droplets 710 on microelectrode array 701 by activating on-state microelectrodes 332 in a cyclic sequence. In some embodiments, to control or accelerate the reaction rate in the droplet, the bio-FPGA can use actuation pattern module 222 (e.g., Figure 2 ) to mix the droplets 710. The bio-FPGA can use sensors such as capacitive sensors or optical sensors to determine the position of the droplets 710, and use microelectrodes 122 under the droplets to create an electric field pattern that drives the droplets to move and induces mixing. The specific actuation pattern of the microelectrode array 701 can vary depending on the desired mixing protocol. In one embodiment, the mixing protocol can involve applying a series of voltages in a sequential manner to move the merged droplets back and forth, thereby mixing the composition in the merged droplets. Alternatively, in another embodiment, a more complex mixing protocol can involve applying a more complex electric field pattern to create a more intricate flow pattern within the droplets. For example, in Figure 7 In the embodiment of the present invention, a series of voltages are programmed in a sequential manner so as to be applied to the on-state microelectrode 332 in a clockwise manner. The on-state microelectrode 332 is activated to switch to the "on" state, in an orderly manner from microelectrode 1 to microelectrode 12. When an electric field is applied to the next on-state microelectrode 332, the previous on-state microelectrode 332 switches to the "off" state. Therefore, the liquid phase in the droplet is driven to move in a clockwise manner. The bio-FPGA can control the speed of mixing by changing the duration of the on and off states of the microelectrode. Similar strategies are also used for counterclockwise or other mixing processes. In some embodiments, it is desired that the mixing step promotes effective mixing of reagents and ensures that the reaction occurs uniformly throughout the droplet. In the present invention, such cyclic mixing can be any actuation pattern that produces turbulence.

[0076] In addition to the ability to move, merge, split, and mix droplets, the bio-FPGA can also perform dilution, washing, and decanting of droplets. For example, the bio-FPGA can merge sample droplets with droplets of water or other solvents to dilute the sample droplets. Dilution is the process of reducing the concentration of sample droplets by adding solvents or other liquids. The bio-FPGA can merge sample droplets with droplets of water or another solvent to dilute the sample droplets. The bio-FPGA can control the volume and concentration of droplets to achieve precise dilution levels, allowing accurate and reproducible experiments.

[0077] The bio-FPGA may include other components to trap and immobilize a selected composition in a droplet, and separate the composition from the droplet by moving the droplet to another location other than the trapped location. Washing and decanting involve separating unwanted components or contaminants from the target composition. The bio-FPGA may trap or capture and immobilize a selected composition in a droplet, and move the droplet to another location other than the trapped location, thereby separating the composition from the droplet. For example, the device may trap and immobilize DNA or RNA molecules in a droplet, and then move the droplet to a different location, thereby leaving behind unwanted proteins or other contaminants. The bio-FPGA may also mix the trapped composition with droplets of a solvent or reagent to wash or decant the composition, thereby further purifying the target composition. For example, the device may mix the trapped DNA molecules with droplets of a wash buffer to remove any remaining contaminants.

[0078] See also Figure 8 , depicting an exemplary process of merging and mixing sample droplets and reagent droplets. At time 801, sample droplets 810 and reagent droplets 812 are arranged on a microelectrode array including a plurality of microelectrodes 122. The bio-FPGA can detect the position and size of the droplets, and determine the actuation pattern and any necessary movement routes for the two droplets 810 and 812 to be merged. At time 802, the bio-FPGA applies a calculated actuation pattern, including a 4x 4 array of on-state microelectrodes 332 arranged at least partially between the sample droplet 810 and the reagent droplet 812, so that the two droplets are merged into a single droplet, which is represented by the merged droplet 814. At the moment of 803, the bio-FPGA implements mixing measures on the merged droplets by applying a series of voltages in a sequential clockwise manner. The sample droplet can contain cells, tissues, blood or other fluids with a desired target nucleic acid (e.g., DNA or RNA). The reagent droplet can include magnetic beads and a lysis buffer reagent. The reagent droplets can also include a fluorescent reagent, such as a fluorophore, and a quencher.

[0079] See also Fig. 9 , depicting the process of extracting target nucleic acid from a hybrid microdroplet. At time 901, the bio-FPGA can detect the position and size of a hybrid microdroplet 910 on a microelectrode array composed of microelectrodes 122. The bio-FPGA can use a magnetic control module 242 (e.g., Figure 2 ) to control the coil 142 to generate a magnetic field. For example, the bio-FPGA can turn on the coil 142 below the microelectrode below the mixing droplet 910. The coil 142 is integrated on the substrate 120 (e.g., Figure 1 ). The bio-FPGA can control the circuit to pass current through the coil to generate a magnetic field. The magnetic field can interact with the magnetic beads in the hybrid droplet 910 and trap and fix the magnetic beads in the hybrid droplet 910 at a first position. The hybrid droplet can be lysed to release the nucleic acid, and the magnetic beads can be coated with special molecules that allow adsorption of the target nucleic acid. Therefore, the magnetic field generated locally under the hybrid droplet can trap and fix both the magnetic beads and the nucleic acid adsorbed to the magnetic beads.

[0080] At time 902, the bio-FPGA can generate an actuation pattern at a position different from the coil 142 that generates the magnetic field. The actuation pattern (including, for example, a 4x 4 array of microelectrodes in the on state) can move the mixed droplet to the position of the actuation pattern 903, thereby leaving the trapped magnetic beads and the nucleic acid adsorbed to the magnetic beads at the original droplet position (e.g., the first position 910a). In this way, the nucleic acid is separated from the droplet, and the fluid and other reagents are removed.

[0081] Fig.10 The diagram illustrates a flow chart of illustrative steps for performing nuclear extraction and amplification by using a biological field programmable gate array. At block 1001, the method for performing nuclear extraction and amplification by using a biological field programmable gate array may include arranging sample droplets containing target nucleic acids on the microelectrode array and arranging reagent droplets including magnetic beads and lysis buffer on the microelectrode array. The microelectrode array may include microelectrodes operable to form one or more actuation patterns, a heater under each microelectrode, and a coil under each microelectrode. The magnetic beads may be iron oxide, iron or cobalt, and the magnetic beads may be coated with silicon dioxide, amino groups, carboxyl groups, etc.

[0082] At block 1002, the method for performing nuclear extraction and amplification using a biological field programmable gate array may include merging the sample droplet and the reagent droplet using a merging actuation pattern to form a merged droplet, and mixing the merged droplet by applying a bias voltage to a microelectrode under the merged droplet in a cyclic sequence to form a mixed droplet. In some embodiments, the merging process may be performed in conjunction with Figures 5A-5C In other embodiments, the mixing process can be similar to that described in Figure 7 and 8 The process is similar to that depicted in. Other blending, merging and mixing processes and patterns are contemplated and possible.

[0083] At block 1003, the method for nuclear extraction and amplification using a biological field programmable gate array may include arranging a mixing droplet at a first position on a microelectrode array. The mixing droplet may be prepared on the microelectrode array as in blocks 1001 and 1002 or as in Figures 5A-5C , 7 and 8. The hybrid droplets can be prepared in advance and transferred to the microelectrode array. The hybrid droplets can include target nucleic acids, lysis buffer and magnetic beads. The hybrid droplets can further include antibodies or other suitable marker molecules for marking target nucleic acids. Such marking allows multiple biological activity tests to be processed simultaneously at the same time on the same microelectrode array. The hybrid droplets can include different reagents to decompose or destroy cells, thereby releasing nucleic acids such as DNA and RNA from cells and tissues. For example, in some embodiments, the reagents can include one or more of the following: sodium dodecyl sulfate, triton X-100, guanidine hydrochloride, proteinase K, lysozyme, etc.

[0084] The method may further include heating the hybrid droplet at the lysis temperature using one or more heaters disposed below the microelectrodes. The bio-FPGA may use a temperature control module 232 (e.g., Figure 2The temperature of the mixed droplet is controlled by a temperature of 20°C (as illustrated in Figure 1). The cleavage reaction is carried out at a cleavage temperature that is high enough to destroy the cell membrane or cell wall, but not so high as to degrade or denature the DNA or RNA being extracted. For example, in a thermal-based cleavage method comprising heating to 95-100°C, the temperature is high enough to denature proteins and destroy cell membranes, but care must be taken not to overheat the sample and degrade the nucleic acid. On the other hand, in the case of enzymatic cleavage with proteinase K digestion or lysozyme treatment, the cleavage temperature is lower, such as 37-65°C, to ensure that the enzyme activity is optimal and the nucleic acid is not degraded. In some embodiments, the cleavage temperature is the optimal temperature for the cleavage reaction, depending on the specific cleavage method and the type of cell or microorganism to be lysed. The cleavage temperature is selected for the best application to ensure the optimal yield and quality of the nucleic acid. The nucleic acid can be released after cell lysis and decantation of the lysis buffer.

[0085] At block 1004, the method for nuclear extraction and amplification using a biological field programmable gate array may include extracting the target nucleic acid from the hybrid droplet. The method may include using a magnetic force generated by a coil below the first position to attract the target nucleic acid absorbed to the magnetic beads, and switching one or more of the plurality of microelectrodes to correspond to a disposal actuation pattern to move the hybrid droplet to a disposal position on the microelectrode array. The process of trapping and fixing the nucleic acid adsorbed on the magnetic beads may be similar to that in Fig. 9 The process is similar to that described in .

[0086] After separating the mixed droplets and the magnetic beads, the bio-FPGA can purify the nucleic acids adsorbed to the magnetic beads. In some embodiments, the bio-FPGA can perform a washing step by adding or moving droplets of a wash buffer to a position on the array (e.g., a first position of a droplet). The bio-FPGA can wash the trapped magnetic beads by gently moving the droplets back and forth on the magnetic beads using the actuation pattern of the microelectrode array. The washing step can help remove any non-specifically bound materials, such as proteins or cell debris, which may have been captured with the nucleic acids. Fig. 9 Similar to the measures implemented in separating the mixed droplets and the magnetic beads as illustrated in , after washing, the droplets are moved to a disposal position, leaving behind the washed nucleic acids adsorbed to the magnetic beads. The washing step can be performed multiple times to achieve the desired conditions for further molecular biological assays (e.g., PCR or other rapid diagnostic tests (RDT)).

[0087] After washing, the bio-FPGA can elute the nucleic acid from the magnetic beads. The bio-FPGA can be eluted by adding droplets of elution buffer to the first location where the magnetic beads are trapped, and gently moving the droplets of elution buffer back and forth on the beads using a microelectrode array. The elution buffer helps release the nucleic acid from the beads, which can then be collected in the eluted droplets. In this way, the nucleic acid is extracted and purified for further processing, such as amplification. In some embodiments, the extracted nucleic acid can be used for detection and visualization without amplification.

[0088] At block 1005, the method for nuclear extraction and amplification using a biological field programmable gate array may include combining the target nucleic acid and an amplification droplet using a pre-amplification actuation pattern to form a pre-amplification droplet.

[0089] The amplification droplet can include, for example, primers, nucleotides, polymerases and buffers. Polymerases can be enzymes that make new chains of DNA or RNA. Primers can be short oligonucleotides that provide starting points for DNA or RNA synthesis. For example, a user can determine the region of nucleic acid to be copied or amplified, and design primers to separate and amplify the desired sequence. The buffer used in the amplification droplet can include additional components to optimize droplet formation and amplification. In some embodiments, the buffer can include dNTPs, MgCl2 and buffer salts to maintain pH and ionic strength of the reaction, and can include additional components to optimize droplet formation and amplification.

[0090] The amplification droplet may further include a fluorescent probe to detect the amplified product in each droplet. A fluorescent probe is a molecule that absorbs light with a specific wavelength and emits light with a different wavelength. The fluorescent probe may be designed to anneal to a specific region of a target nucleic acid sequence and may be labeled with a fluorescent reporter molecule at one end and a quencher at the other end. When the fluorescent probe is intact, the fluorescence of the reporter molecule may be quenched by the quencher molecule. During the extension phase of the PCR reaction, the probe degrades and releases the fluorescent reporter molecule, thereby allowing its fluorescence to be detected.

[0091] At block 1006, the method for nuclear extraction and amplification using a biological field programmable gate array may include heating the pre-amplification droplet using a heater below the first location under a programmed temperature schedule to generate an amplified droplet.

[0092] After the target nucleic acid and the amplification droplet are mixed to form the pre-amplification droplet, the pre-amplification droplet can be heated using a heater under a programmed temperature schedule to generate amplified droplets. The programmed temperature schedule can be based on a loop-mediated isothermal amplification (LAMP) process or a PCR cycling process.

[0093] The target nucleic acid can be amplified using PCR or dye-based real-time PCR (qPCR). During amplification, the fluorescent probe can anneal to the target sequence and can be cleaved by the polymerase, thereby separating the fluorescent reporter molecule from the quencher molecule. The bio-FPGA can use a temperature control module 232 (e.g., Figure 2 ) to control the heater in heating the pre-amplification droplets to the desired temperature.

[0094] During PCR cycling, the bio-FPGA can use a heater to heat the preamplification droplet to a series of temperature changes that enable the following aspects: denaturation of the DNA or RNA template, annealing of the primer to the template, and primer extension to generate new copies of the target sequence. For example, the PCR cycling process can include a denaturation step by heating the preamplification droplet to a high temperature (e.g., 94-98°C) to separate or denature the double-stranded DNA or RNA template into a single-stranded molecule. The single-stranded molecule is the template for the next step. The PCR cycling process can include an annealing step. During annealing, the preamplification droplet is then cooled to a lower temperature (e.g., 50-65°C) to enable the primer to anneal to the target. The primer can be a short DNA or RNA sequence that is complementary to the region flanking the target sequence. The PCR cycling process can include an extension step to increase the temperature (e.g., 72°C) to allow the polymerase to extend the primer, wherein a new copy of the target sequence is synthesized using the single-stranded template as a template. The PCR cycling process can repeat the denaturation, annealing, and extension steps multiple times, wherein each cycle results in a doubling of the target sequence (e.g., as in Fig.12 As illustrated in , thermal cycling can be provided in which heating and cooling are performed between 95° C. and 55° C. at 20 second intervals, although other temperatures and time intervals are possible and contemplated). After multiple cycles, a large number of copies of the target sequence are generated in the reaction mixture. The bio-FPGA can determine the number of cycles for amplification based on the starting concentration of the template DNA or RNA and the desired level of amplification.

[0095] The PCR cycling process can be modified by changing the temperature and duration of each step and the number of cycles performed to optimize the amplification of the target sequence. In addition, variations of PCR, such as reverse transcription PCR (RT-PCR) and nested PCR, can involve additional steps or modifications to the cycling process.

[0096] During loop-mediated isothermal amplification (LAMP), the bio-FPGA can use a heater to raise the pre-amplification droplets to the desired reaction temperature running at a constant temperature (e.g., 60-65°C). The bio-FPGA can use 4-6 primers of LAMP to recognize 6-8 different regions of the target nucleic acid. For example, a strand-displacing DNA or RNA polymerase can initiate synthesis, and 2 of the primers form a loop structure to facilitate subsequent rounds of amplification. The products and byproducts of the reaction during the LAMP process can be seen by eye (e.g., in Fig.13 ) or visualized by other suitable means (e.g., a camera).

[0097] At block 1007, the method for nuclear extraction and amplification using a biological field programmable gate array may include visualizing the amplified droplet.

[0098] The bio-FPGA may use a visualization module 252 (e.g., Figure 2 ) to visualize the amplified droplets, for example using an optical sensor 210 (e.g., Figure 1 ). The amplifying droplet may include a fluorescent probe and a quencher. When the fluorescent probe is degraded, the fluorescent reporter molecule may no longer be quenched and emit fluorescence. The fluorescent signal may be detected by an optical sensor 210 in real time during qPCR or after amplification in end-point PCR. The fluorescent signal may indicate the presence or absence of the target sequence in the sample. For example, Fig.13 As illustrated in , the optical sensor can detect positive sample droplets at a higher light intensity, and detect negative sample droplets at a lower light intensity.

[0099] Fig.11 A photograph of an illustrative PCR chip of the present disclosure adjacent to a penny, an illustrative example of a bio-FPGA chip integrated portable device, and a photograph of a kit containing the bio-FPGA chip are illustrated. The PCR chip may be approximately 3.8 x 5.3 mm 2, with a volume of less than about 1 μl. The PCR chip can be made of SiO2 or Si3N4. The package of the PCR chip is a chip on board, so that the integrated circuit (e.g., microprocessor) is attached (tethered, directly bonded) to the printed circuit board and covered with epoxy resin. In some embodiments, a portable device can be used as a controller for the bio-FPGA. In a particular embodiment, the portable device can be a handheld mobile phone, a tablet or other such device with an Android or other operating system and a display (to display a graphical user interface). In some embodiments, the display can be a touch screen of various sizes (e.g., ranging in size from a typical mobile phone to a page-sized tablet or larger). In some embodiments, the device has a resin (e.g., Bakelite) (polyoxybenzyl glycol anhydride)) shell, an automated dropper, and an optical design detector.

[0100] Fig.12 An example of thermal cycling using a bio-FPGA chip is illustrated. A thermal cycle with 20 second intervals between 95°C and 55°C is depicted. Advantageously, the bio-FPGA can control a heater to heat at a small local area with a fast ramp rate. It should be noted that the bio-FPGA in the present disclosure can control a heater to heat locally through a microelectrode array.

[0101] Fig.13 The illustrated loop-mediated isothermal amplification (LAMP) results of the nucleic acid amplified after processing using the bio-FPGA chip are illustrated. When primers are added during the amplification process, the heat treatment during the LAMP process can quench the bond between the target nucleic acid and the fluorescent molecule, so that the released fluorescent reporter molecules are detected by the bio-FPGA using an optical sensor. In this case, the positive droplet contains the target nucleotide, and the negative droplet does not contain the target nucleic acid, and therefore only the positive droplet shows a high light intensity. Suitable optical sensors for detecting fluorescence include cameras, etc.

[0102] Although particular embodiments have been illustrated and described herein, it should be understood that various other changes and modifications may be made without departing from the spirit and scope of the claimed subject matter. In addition, although various aspects of the claimed subject matter have been described herein, such aspects need not be used in combination. Therefore, it is intended that the appended claims cover all such changes and modifications within the scope of the claimed subject matter.

[0103] Further aspects of the embodiments described herein are provided by the subject matter of the following numbered clauses.

[0104] 1. A method for nuclear extraction and amplification by using a biological field programmable gate array, the method comprising:

[0105] Arranging a mixing type microdroplet at a first position on a microelectrode array, wherein the mixing type microdroplet comprises one or more target nucleic acids, a lysis buffer and magnetic beads, wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and the microelectrode array comprises a plurality of microelectrodes arranged in an array and operable to form one or more actuation patterns, each microelectrode comprising: a heater under the microelectrode, and a coil under the microelectrode;

[0106] extracting the one or more target nucleic acids from the hybrid droplet by: using a magnetic force generated by a coil below the first position to attract the one or more target nucleic acids absorbed to the magnetic beads, and switching one or more of the plurality of microelectrodes to correspond to a treatment actuation pattern to move the hybrid droplet to a treatment position on the microelectrode array;

[0107] combining the one or more target nucleic acids and the amplification droplet to form a pre-amplification droplet at the first location;

[0108] heating the pre-amplification droplet using a heater below the first position under a programmed temperature schedule to generate an amplified droplet; and

[0109] The amplified droplets are visualized.

[0110] 2. A method according to clause 1, wherein the mixed droplets are formed by: arranging sample droplets containing the one or more target nucleic acids on the microelectrode array; arranging reagent droplets containing the magnetic beads and the lysis buffer on the microelectrode array; using a merging actuation pattern to merge the sample droplets and the reagent droplets to form merged droplets; and mixing the merged droplets by applying a bias voltage to the microelectrodes below the merged droplets in a cyclic sequence to form mixed droplets.

[0111] 3. A method according to any preceding clause, wherein after arranging the hybrid droplet, the method further comprises heating the hybrid droplet at a lysis temperature using a heater below the first position.

[0112] 4. A method according to any of the preceding clauses, wherein before merging the one or more target nucleic acids and the amplification droplets, the method further comprises decanting the mixed droplets by: using one or more decanting actuation patterns to merge and split the one or more target nucleic acids and the one or more decanting droplets.

[0113] 5. A method according to any of the preceding clauses, wherein after extracting the one or more target nucleic acids from the mixed droplets, the method further comprises releasing the one or more target nucleic acids from the magnetic beads by adding droplets of elution buffer to the one or more target nucleic acids absorbed to the magnetic beads.

[0114] 6. A method according to any preceding clause, wherein when the amplifying droplets comprise a fluorescent and a quenching agent, an optical sensor is used to visualize the amplified droplets.

[0115] 7. A method according to any of the preceding clauses, wherein the programmed temperature planning scheme is based on a loop-mediated isothermal amplification process or a polymerase chain reaction cycling process.

[0116] 8. The method according to any preceding clause, wherein the mixing droplet further comprises an antibody to label the mixing droplet.

[0117] 9. A method according to any preceding clause, wherein the magnetic beads comprise iron oxide, iron or cobalt, and wherein the magnetic beads are coated with silica, amino groups or carboxyl groups.

[0118] 10. A method according to any preceding clause, wherein the amplifying droplets comprise one or more of primers, nucleotides, a polymerase and a buffer.

[0119] 11. A method according to any preceding clause, wherein the microelectrode array further comprises a capacitive sensor beneath each microelectrode, wherein the capacitive sensor detects the volume and position of the droplet.

[0120] 12. A non-transitory computer readable medium for performing nuclear extraction and amplification by using a biological field programmable gate array, the medium comprising logic which, when executed by a computing device, causes the computing device to perform at least the following steps:

[0121] Detecting a hybrid droplet at a first position on a microelectrode array using a capacitive sensor or an optical sensor, wherein the hybrid droplet comprises one or more target nucleic acids, a lysis buffer, and magnetic beads, wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and the microelectrode array comprises a plurality of microelectrodes arranged in an array and operable to form one or more actuation patterns, each microelectrode comprising: a heater under the microelectrode, a coil under the microelectrode, and a capacitive sensor under the microelectrode;

[0122] extracting the one or more target nucleic acids from the hybrid droplet by: using a magnetic force generated by a coil below the first position to attract the one or more target nucleic acids absorbed to the magnetic beads, and switching one or more of the plurality of microelectrodes to correspond to a treatment actuation pattern to move the hybrid droplet to a treatment position on the microelectrode array;

[0123] combining the one or more target nucleic acids and the amplification droplets to form pre-amplification droplets;

[0124] heating the pre-amplification droplet using a heater below the first position under a programmed temperature schedule to generate an amplified droplet; and

[0125] The amplified droplets are visualized.

[0126] 13. A medium according to claim 12, wherein the mixed droplets are formed by: detecting sample droplets containing the one or more target nucleic acids on the microelectrode array; detecting reagent droplets containing the magnetic beads and the lysis buffer on the microelectrode array; merging the sample droplets and the reagent droplets using a merging actuation pattern to form merged droplets; and mixing the merged droplets by applying a bias voltage to the microelectrodes below the merged droplets in a cyclic sequence to form mixed droplets.

[0127] 14. The medium of clause 12 or clause 13, wherein after detecting the hybrid droplet, the method further comprises heating the hybrid droplet at a lysis temperature using a heater below the first location.

[0128] 15. A medium according to any one of clauses 12-14, wherein before merging the one or more target nucleic acids and the amplification droplets, the method further comprises decanting the mixed droplets by using one or more decanting actuation patterns to merge and split the one or more target nucleic acids and the one or more decanting droplets.

[0129] 16. A medium according to any one of clauses 12-15, wherein after extracting the one or more target nucleic acids from the mixed droplets, the method further comprises releasing the one or more target nucleic acids from the magnetic beads by adding droplets of elution buffer to the one or more target nucleic acids absorbed to the magnetic beads.

[0130] 17. The medium of any one of clauses 12-16, wherein when the amplifying droplets comprise a fluorescent and a quencher, the optical sensor is used to visualize the amplified droplets.

[0131] 18. A medium according to any one of clauses 12-17, wherein the programmed temperature planning scheme is based on a loop-mediated isothermal amplification process or a polymerase chain reaction cycling process.

[0132] 19. A medium according to any one of clauses 12-18, wherein the amplifying droplets comprise one or more of primers, nucleotides, a polymerase and a buffer.

[0133] 20. The medium according to any of clauses 12-19, wherein the capacitive sensor or the optical sensor detects the volume and position of a droplet.

[0134] It will be apparent to those skilled in the art that various modifications and variations may be made to the embodiments described herein without departing from the scope of the claimed subject matter. Therefore, it is intended that this specification covers modifications and variations of the various embodiments described herein, provided that such modifications and variations are within the scope of the appended claims and their equivalents.

Claims

1. A method for nuclear extraction and amplification by using a biological field programmable gate array, the method comprising: A mixing droplet is arranged at a first position on a microelectrode array, wherein The mixed droplets contain one or more target nucleic acids, a lysis buffer and magnetic beads. wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and The microelectrode array comprises a plurality of microelectrodes arranged in an array and operable to form one or more actuation patterns, each microelectrode comprising: a heater under the microelectrode, and a coil under the microelectrode; The one or more target nucleic acids are extracted from the mixed droplets by: attracting the one or more target nucleic acids absorbed to the magnetic beads using a magnetic force generated by a coil below the first position, and switching one or more of the plurality of microelectrodes corresponding to a disposition actuation pattern to move the mixing droplet to a disposition position on the microelectrode array; merging the one or more target nucleic acids and the amplification droplet to form a pre-amplification droplet at the first position; heating the pre-amplification droplet using a heater below the first position under a programmed temperature schedule to generate an amplified droplet; and The amplified droplets are visualized.

2. The method according to claim 1, wherein the hybrid droplets are formed by: disposing sample droplets comprising the one or more target nucleic acids on the microelectrode array; Arranging reagent droplets comprising the magnetic beads and the lysis buffer on the microelectrode array; merging the sample droplet and the reagent droplet using a merging actuation pattern to form a merged droplet; and The merged droplets are mixed by applying bias voltages on microelectrodes beneath the merged droplets in a cyclic sequence to form mixed droplets. 3 . The method of claim 1 , wherein after disposing the hybrid droplet, the method further comprises heating the hybrid droplet at a lysis temperature using a heater below the first position.

4. The method of claim 1, wherein before merging the one or more target nucleic acids and the amplification droplets, the method further comprises decanting the mixed droplets by: using one or more decanting actuation patterns to merge and split the one or more target nucleic acids and the one or more decanting droplets.

5. The method according to claim 1, wherein after extracting the one or more target nucleic acids from the mixed droplets, the method further comprises releasing the one or more target nucleic acids from the magnetic beads by adding droplets of elution buffer to the one or more target nucleic acids absorbed to the magnetic beads.

6. The method of claim 1, wherein when the amplifying droplets comprise a fluorescent and quenching agent, an optical sensor is used to visualize the amplified droplets.

7. The method according to claim 1, wherein the programmed temperature planning scheme is based on a loop-mediated isothermal amplification process or a polymerase chain reaction cycling process. The method according to claim 1 , wherein the mixing droplet further comprises an antibody to label the mixing droplet.

9. The method of claim 1, wherein the magnetic beads comprise iron oxide, iron, or cobalt, and wherein the magnetic beads are coated with silica, amino groups, or carboxyl groups.

10. The method of claim 1, wherein the amplifying droplets comprise one or more of primers, nucleotides, a polymerase, and a buffer.

11. The method of claim 1, wherein the microelectrode array further comprises a capacitive sensor beneath each microelectrode, wherein the capacitive sensor detects the volume and position of the droplet.

12. A non-transitory computer readable medium for performing nuclear extraction and amplification by using a biological field programmable gate array, the medium comprising logic which, when executed by a computing device, causes the computing device to perform at least the following steps: A capacitive sensor or an optical sensor is used to detect a mixed droplet at a first location on the microelectrode array, wherein The mixed droplets contain one or more target nucleic acids, a lysis buffer and magnetic beads. wherein the one or more target nucleic acids are adsorbed to the magnetic beads, and The microelectrode array comprises a plurality of microelectrodes arranged in an array and operable to form one or more actuation patterns, each microelectrode comprising: a heater under the microelectrode, a coil under the microelectrode, and a capacitive sensor under the microelectrode; The one or more target nucleic acids are extracted from the mixed droplets by: attracting the one or more target nucleic acids absorbed to the magnetic beads using a magnetic force generated by a coil below the first position, and Switching one or more of the plurality of microelectrodes in response to a disposition actuation pattern to move the mixing droplet to a disposition position on the microelectrode array; merging the one or more target nucleic acids and the amplification droplet to form a pre-amplification droplet; heating the pre-amplification droplet using a heater below the first position under a programmed temperature schedule to generate an amplified droplet; and The amplified droplets are visualized.

13. The medium of claim 12, wherein the hybrid droplets are formed by: detecting sample droplets comprising the one or more target nucleic acids on the microelectrode array; detecting a reagent droplet comprising the magnetic beads and the lysis buffer on the microelectrode array; merging the sample droplet and the reagent droplet using a merging actuation pattern to form a merged droplet; and The merged droplets are mixed by applying bias voltages on microelectrodes beneath the merged droplets in a cyclic sequence to form mixed droplets.

14. The medium of claim 12, wherein after detecting the hybrid droplet, the method further comprises heating the hybrid droplet at a lysis temperature using a heater below the first location.

15. The medium of claim 12, wherein prior to merging the one or more target nucleic acids and the amplification droplets, the method further comprises decanting the mixing droplets by using one or more decanting actuation patterns to merge and split the one or more target nucleic acids and the one or more decanting droplets.

16. The medium of claim 12, wherein after extracting the one or more target nucleic acids from the mixed droplets, the method further comprises releasing the one or more target nucleic acids from the magnetic beads by adding droplets of elution buffer to the one or more target nucleic acids absorbed to the magnetic beads.

17. The medium of claim 12, wherein when the amplifying droplets comprise a fluorescent and a quencher, the optical sensor is used to visualize the amplified droplets.

18. The medium of claim 12, wherein the programmed temperature planning scheme is based on a loop-mediated isothermal amplification process or a polymerase chain reaction cycling process.

19. The medium of claim 12, wherein the amplifying droplets comprise one or more of primers, nucleotides, a polymerase, and a buffer.

20. The medium of claim 12, wherein the capacitive sensor or the optical sensor detects the volume and position of a droplet.

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