Apparatus and Method for Sample Analysis

Abstract

A digital microfluidic and analyte detection device includes a first substrate and a second substrate. The first substrate and the second substrate are aligned generally parallel to each other in a side view and a gap is defined therebetween. At least one of the first substrate and the second substrate has an electrode array configured to generate electrokinetic forces to push at least one droplet within the gap along at least one of the first substrate and the second substrate. The electrode array has a plurality of electrodes that define an electrode array region in a plan view. At least one of the first substrate and the second substrate has a well array that defines a well array region in a plan view. The well array region is defined within the electrode array region and overlaps a portion of each of the plurality of electrodes. The well array region overlaps less than 75% of the electrode array region in a plan view.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application 62 / 856,563, filed on June 3, 2019, which is incorporated herein by reference in its entirety. Background of the Invention

[0003] Field of the disclosed subject matter

[0004] The disclosed subject matter relates to devices, systems, and methods for sample analysis, such as in integrated devices for performing analyte analysis.

[0005] Description of the related art

[0006] Analytical devices typically need to process a sample (e.g., a biological fluid) to prepare and analyze discrete volumes of the sample. Digital microfluidics allows for the processing of discrete volumes of fluid, including the electrokinetic movement, mixing, and splitting of fluid droplets disposed in a gap between two surfaces, at least one of which includes an electrode array coated with a hydrophobic material and / or a dielectric material.

[0007] Such devices and systems are particularly beneficial in integrated devices for performing analyte analysis. Generally, digital microfluidics can be used to introduce a fluid (e.g., a sample or a reagent) into one or more wells for analysis. However, the presence of wells can affect the surface properties of the device and the behavior of droplets moving across the device. For example, compared to the surface of the surrounding electrode array without wells, the area of the device with wells can exert an increased surface tension or drag on the droplets. The increased surface tension or drag can prevent the effective loading of fluid into the wells or pores because the fluid droplets tend to bypass the wells and the associated increased surface tension or become stuck or pinned at the top of the well region.

[0008] Accordingly, there is still a need to improve such devices and systems. Positioning, sizing, and orienting the wells relative to the device electrodes can minimize the effects of the different surface properties created by the wells and facilitate the effective loading of fluid into the wells.

[0009] Overview

[0010] The objects and advantages of the disclosed subject matter will be set forth in the description which follows, and will become apparent from the description, as well as will be learned by practice of the disclosed subject matter. The additional advantages of the disclosed subject matter will be realized and obtained by means of the methods and systems particularly pointed out in the written description and claims hereof as well as from the appended drawings.

[0011] To achieve these and other advantages and in accordance with the purpose of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter includes a digital microfluidics and analyte detection device. The device generally includes a first substrate and a second substrate, the first substrate and the second substrate being aligned generally parallel to each other in a side view and defining a gap therebetween. At least one of the first substrate and the second substrate has an electrode array configured to generate electrokinetic forces to push at least one droplet within the gap along at least one of the first substrate and the second substrate. The electrode array has a plurality of electrodes defining an electrode array region in a plan view. At least one of the first substrate and the second substrate has a well array defining a well array region in a plan view. The well array region is defined within the electrode array region and overlaps a portion of each of the plurality of electrodes. The well array region overlaps less than 75% of the electrode array region in a plan view.

[0012] Each of the plurality of electrodes may overlap less than 25% of the well array region. The electrode array may include a first electrode having a first electrode region in a plan view and a second electrode adjacent to the first electrode and having a second electrode region in a plan view. The first electrode region and the second electrode region may together define a generally parallelogram shape having a longitudinal axis passing therethrough in a plan view. The well array region may have a generally rectangular shape having a diagonal axis passing between diagonals therethrough. The diagonal axis of the well array region may be generally parallel or collinear with the longitudinal axis of the first electrode region and the second electrode region. A first portion of the well array region may overlap the first electrode region and a second portion of the well array region may overlap the second electrode region. The first portion may have dimensions substantially equal to those of the second portion.

[0013] The electrode array may further include a third electrode having a third electrode region in a plan view and a fourth electrode having a fourth electrode region in a plan view. The first electrode, the second electrode, the third electrode, and the fourth electrode may be serially connected to define a path along which a droplet may engage the well array. Additionally or alternatively, the first electrode, the second electrode, the third electrode, and the fourth electrode may together define a generally square shape having a diagonal axis passing therethrough. The diagonal axis may be disposed at an angle of about 45 degrees relative to the diagonal axis defined by the well array. The well array region may overlap portions of the first electrode region, the second electrode region, the third electrode region, and the fourth electrode region that are substantially similar in size to each other.

[0014] The electrode array can be configured to push at least one droplet along a path, with at least a portion of the at least one droplet making fluid contact with at least one well of the well array. Additionally or alternatively, the electrode array can be configured to continuously push droplets around a peripheral portion of the well array. The electrode array can also be configured to push the at least one droplet along a path from the first electrode to the second electrode, and at least a portion of the peripheral edge of the well array can be positioned at an angle of 0 degrees to 55 degrees relative to the path.

[0015] The well array can include a plurality of femtoliter wells, and each femtoliter well can be configured to accommodate a single bead. The device can include at least one of a magnet and an electromagnet proximate to the plurality of wells. At least one of the first substrate and the second substrate can include at least one of PET, PMMA, COP, COC, PC, and glass. Additionally, both the electrode array and the well array can be defined in one of the first substrate or the second substrate.

[0016] According to another aspect of the disclosed subject matter, an analyte detection module for performing analyte detection is provided. The analyte detection module generally includes a substrate having a first layer and a second layer. The first layer includes an electrode array configured to generate an electrokinetic force to push at least one droplet along the surface of the substrate. The electrode array has a plurality of electrodes that define an electrode array region in a plan view. The second layer has a well array that defines a well array region in a plan view. The well array region is defined within the electrode array region and overlaps a portion of each of the plurality of electrodes. The well array region overlaps less than 75% of the electrode array region in a plan view.

[0017] According to another aspect of the disclosed subject matter, a method of loading droplets into a well array of an analyte detection module is provided. The method includes introducing a mother droplet into a gap defined between a first substrate and a second substrate. At least one of the first substrate and the second substrate has an electrode array defined therein that has an electrode array region in a plan view. At least one of the first substrate and the second substrate has a well array that defines a well array region in a plan view, the well array region being defined within the electrode array region and overlapping less than 75% of the electrode array region in a plan view. The method further includes continuously pushing the mother droplet around a peripheral portion of the well array by generating an electrokinetic force on the mother droplet by the electrode array to set at least a portion of the mother droplet into fluid contact with at least one well in the well array. The method further includes filling the at least one well in the well array with at least one daughter droplet released from the mother droplet.

[0018] The mother liquor droplets can be pushed around the peripheral portion of the well array for a plurality of consecutive cycles within a range of 5 - 20 consecutive cycles.

[0019] It should be understood that the foregoing general description and the following detailed description are both exemplary and are intended to provide further explanation of the disclosed subject matter as claimed.

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to illustrate and provide a further understanding of the disclosed subject matter. Together with the description, the drawings serve to explain the principles of the disclosed subject matter. Brief description of the drawings

[0022] Figure 1A is a schematic side view of an exemplary analyte detection module of an integrated digital microfluidics and analyte detection device according to the disclosed subject matter.

[0023] Figure 1B is a schematic side view of another exemplary analyte detection module of an integrated digital microfluidics and analyte detection device according to the disclosed subject matter.

[0024] Figure 2 is a schematic plan view of an exemplary embodiment of an integrated digital microfluidics and analyte detection device according to the disclosed subject matter.

[0025] Figure 3 is Figure 1A a partial schematic plan view of the analyte detection module of

[0026] Figure 4A is Figure 1A a partial schematic plan view of the analyte detection module of

[0027] Figure 4B is a partial schematic plan view of another exemplary analyte detection module according to the disclosed subject matter.

[0028] Figure 5 is Figure 1A a schematic side view of the analyte detection module in which droplets are disposed.

[0029] Figure 6 is Figure 1A a schematic partial side view of the analyte detection module in which droplets containing particles or beads are disposed on the well array.

[0030] Figure 7 is a schematic side view of another exemplary analyte detection module according to the disclosed subject matter.

[0031] Description

[0032] Reference will now be made in detail to various exemplary embodiments of the disclosed subject matter, which are illustrated in the accompanying drawings. The structure and corresponding methods of operation and methods of use of the disclosed subject matter will be described in conjunction with a detailed description of the system.

[0033] The systems, devices, and methods described herein relate to sample analysis including in integrated digital microfluidic and analyte detection devices. As used interchangeably herein, "digital microfluidics (DMF)", "digital microfluidic module (DMF module)", or "digital microfluidic device (DMF device)" refers to a module or device that utilizes digital or droplet-based microfluidic technology to process discrete small volumes of liquid in the form of droplets. Digital microfluidics utilizes the principles of emulsion science to generate fluid-fluid dispersions (e.g., water-in-oil emulsions) in channels, and thus can produce monodisperse droplets or bubbles or with very low polydispersity. Digital microfluidics is based on the microscopic manipulation of discontinuous fluid droplets within a reconfigurable network. By combining the basic operations of droplet formation, translocation, splitting, and merging, complex instructions can be programmed.

[0034] Digital microfluidics operates on discrete volumes of fluid, which can be processed by binary electrical signals. By using discrete unit volume droplets, microfluidic operations can be defined as a set of repeated basic operations, e.g., moving one unit of fluid one unit of distance. Droplets can be formed using the surface tension properties of the liquid. The actuation of the droplets is based on the presence of an electrostatic force generated by an electrode placed below the bottom surface on which the droplet lies. Different types of electrostatic forces can be used to control the shape and movement of the droplets. One technique that can be used to generate the above electrostatic force is based on dielectrophoresis, which relies on the dielectric constant difference between the droplet and the surrounding medium and can utilize a high-frequency AC electric field. Another technique that can be used to generate the above electrostatic force is based on electrowetting, which relies on the dependence of the surface tension between the droplet present on the surface and the surface on the electric field applied to the surface.

[0035] As used herein, "sample", "test sample", or "biological sample" refers to a fluid sample that contains or is suspected of containing the analyte under consideration. The sample can be derived from any suitable source. As embodied herein, the sample can include a liquid, a flowing particulate solid, or a fluid suspension of solid particles. As embodied herein, the sample can be processed prior to the analysis described herein. For example, the sample can be separated or purified from its source prior to analysis; however, as embodied herein, an unprocessed sample containing the analyte can be analyzed directly. The source of the analyte molecule can be synthetic (e.g., produced in a laboratory), environmental (e.g., air, soil, fluid samples such as water supply, etc.), animal (e.g., mammal, reptile, amphibian, or insect), plant, or any combination thereof. By way of example and not limitation, as embodied herein, the source of the analyte is human body substances (e.g., body fluids, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus, tears, lymph fluid, amniotic fluid, interstitial fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, feces, tissue, organs, etc.). The tissue can include, but is not limited to, skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin, etc. The sample can be a liquid sample or a liquid extract of a solid sample. In some cases, the source of the sample can be an organ or tissue, such as a biopsy sample, which can be solubilized by tissue decomposition or cell lysis.

[0036] As embodied herein and as further described herein, an integrated digital microfluidics and analyte detection device can have two modules: a sample preparation module and an analyte detection module. As embodied herein, the sample preparation module and the analyte detection module are separate or separate and adjacent. As embodied herein, the sample preparation module and the analyte detection module are co-located, mixed, or cross. The sample preparation module can include a plurality of electrodes for moving, combining, diluting, mixing, separating droplets of samples and reagents. The analyte detection module (or "detection module") can include an array of wells in which signals related to the analyte are detected. As embodied herein, the detection module can also include a plurality of electrodes for moving droplets of the prepared sample to the array of wells. As embodied herein, the detection module can include an array of wells in a first substrate (e.g., an upper substrate) that is disposed above a second substrate (e.g., a lower substrate) spaced apart by a gap. In this way, the array of wells is in an inverted orientation. As embodied herein, the detection module can include an array of wells in a second substrate (e.g., a lower substrate) that is disposed below a first substrate (e.g., an upper substrate) spaced apart by a gap. As embodied herein, the first substrate and the second substrate are in a facing arrangement. One or more electrodes present in the first substrate and / or the second substrate can be used to push (e.g., by electroactuation) the droplets to the array of wells. As embodied herein, the array of wells, including the area between the wells, can be hydrophobic. Alternatively, the plurality of electrodes can be limited to the sample preparation module and other means can be used to push droplets of the prepared sample (and / or droplets of immiscible fluids) to the detection module.

[0037] Droplet-based microfluidics refers to the generation and actuation (e.g., moving, merging, splitting, etc.) of droplets via active or passive forces. Examples of active forces include, but are not limited to, electric fields. Exemplary active force techniques include electrowetting, dielectrophoresis, optoelectrowetting, electrode-mediated, electric field-mediated, electrostatic actuation, etc. or combinations thereof. For example, and as further described herein, the device may actuate droplets across the upper surface of the first layer (or the upper surface of the second layer, when present) in the gap via droplet-based microfluidics such as electrowetting or via a combination of electrowetting and a continuous fluid flow of droplets. Alternatively, the device may include microchannels to convey droplets from a sample preparation module to a detection module. As a further alternative, the device may rely on actuating droplets across the surface of a hydrophobic layer in a gap via droplet-based microfluidics. Electrowetting may involve changing the wetting characteristics of a surface by applying an electric field to the surface and affecting the surface tension between a droplet present on the surface and the surface. A continuous fluid flow may be used to move droplets via an external pressure source, such as an external mechanical pump or an integrated mechanical micropump, or a combination of capillary forces and electrokinetics. Examples of passive forces include, but are not limited to, T-junctions and flow focusing methods. Other examples of passive forces include using a denser immiscible liquid, such as a heavy oil fluid, which may be coupled to a droplet on a first substrate surface and move the droplet across the surface. The denser immiscible liquid may be any liquid that is denser than water and does not mix with water to a significant extent. For example, the immiscible liquid may be hydrocarbons, halogenated hydrocarbons, polar oils, non-polar oils, fluorinated oils, chloroform, dichloromethane, tetrahydrofuran, 1-hexanol, etc.

[0038] According to aspects of the disclosed subject matter, a digital microfluidics and analyte detection device is provided. The device generally includes a first substrate and a second substrate, the first substrate and the second substrate being generally parallel and aligned with each other in a side view and defining a gap therebetween. At least one of the first substrate and the second substrate has an electrode array configured to generate electrokinetic forces to push at least one droplet within the gap along at least one of the first substrate and the second substrate. The electrode array has a plurality of electrodes defining an electrode array region in a plan view. At least one of the first substrate and the second substrate has a well array defining a well array region in a plan view. The well array region is defined within the electrode array region and overlaps a portion of each of the plurality of electrodes. The well array region overlaps less than 75% of the electrode array region in a plan view.

[0039] The accompanying drawings, in which like reference numerals refer to identical or functionally similar elements throughout the views, are used to further illustrate various embodiments in accordance with the disclosed subject matter and to explain all of the various principles and advantages. For purposes of explanation and illustration and not limitation, an exemplary embodiment of a device for sample analysis included in an integrated device for analyte analysis in accordance with the disclosed subject matter is shown in Figures 1A - 7 as follows.

[0040] Figure 1A An exemplary analyte detection module of an integrated digital microfluidic and analyte detection device 10 is illustrated. Device 10 includes an analyte detection module that includes a first substrate 11 and a second substrate 12, where the second substrate 12 is generally aligned parallel to the first substrate with a gap 13 therebetween. As embodied herein, the second substrate 12 may be positioned above the first substrate 11, or alternatively, the second substrate 12 may be positioned below the first substrate 11. That is, it is to be appreciated that the terms “first” and “second” are interchangeable and are used herein only as reference points. As Figure 1A shown, the second substrate 12 may be coextensive with the first substrate 11. Alternatively, the first substrate 11 and the second substrate 12 may have different lengths.

[0041] At least one of the first substrate 11 and the second substrate 12 includes an electrode array defined therein. By way of example and not limitation, and as embodied herein, the first substrate 11 may include a plurality of electrodes positioned on an upper surface of the first substrate 11 to define an electrode array. The electrode array, such as, by way of example and not limitation, the electrode arrays 200 or 400 shown in Figures 3 - 4B and further discussed herein, is configured to generate electrokinetic forces to push at least one droplet along at least one of the first substrate 11 and the second substrate 12, as further discussed herein. Although a plurality of electrodes 17 are depicted in the first substrate 11, the device in accordance with the disclosed subject matter may have electrodes in the first substrate 11, the second substrate 12, or both the first and second substrates.

[0042] Still referring to Figure 1A , device 10 may include a first portion 15 onto which droplets, such as sample droplets, reagent droplets, etc., may be introduced onto at least one of the first substrate 11 and the second substrate 12. Device 10 may include a second portion 16 toward which droplets may be pushed. The first portion 15 may also be referred to as a sample preparation module, and the second portion 16 may be referred to as an analyte detection module. For example, a liquid may be introduced into the gap 13 via a droplet actuator (not shown). Alternatively, the liquid may enter the gap via a fluid inlet, interface, or channel. As further discussed herein, for example with respect to Figure 6, the apparatus 10 may include chambers for containing samples, wash buffers, binding members, enzyme substrates, waste liquids, etc. Analytical reagents may be contained in an external reservoir that is part of the integrated device, where a predetermined volume can be pushed from the reservoir to the device surface when needed for a specific analysis step. Additionally, the analytical reagents may be deposited on the device in the form of dry, printed, or lyophilized reagents, where they can be stored for an extended period without losing activity. Such dry, printed, or lyophilized reagents may be rehydrated before or during analyte analysis.

[0043] Further reference Figure 1A , a dielectric / hydrophobic material layer 18 may be disposed on the upper surface of the first substrate. By way of example and not limitation, and as embodied herein, Teflon may be used as both the dielectric material and the hydrophobic material. However, as further described herein, any suitable material having dielectric and hydrophobic properties may be used. The layer 18 may coat a plurality of electrodes 17 in the electrode array. Alternatively, and as shown in the exemplary device depicted in Figure 1B , a dielectric material layer 38 may be disposed on the upper surface of the first substrate and coat a plurality of electrodes 17 of the electrode array. A hydrophobic material layer 34 may cover the dielectric layer 38. In this manner, any suitable combination of materials having dielectric and hydrophobic properties may be used to form the layer 38 and the layer 34, respectively, as further described herein.

[0044] At least one of the first substrate 11 and the second substrate 12 has a well array 19. By way of example and not limitation, and with reference to Figure 1A , the well array 19 may be located in the layer 18 of the first substrate 11 in the second portion 16 of the device. With reference to Figure 1B , the well array 19 may alternatively be located in the layer 34. Although reference is made herein to the well array 19 in the first substrate 11, the well array 19 may be located on the first substrate 11, the second substrate 12, or both the first and second substrates. As embodied herein, the plurality of electrodes 17 and the well array 19 may be defined in the same one of the first substrate or the second substrate. Alternatively, the plurality of electrodes 17 and the well array 19 may be defined in different substrates.

[0045] The first substrate and the second substrate can be made of flexible materials, such as paper (with inkjet-printed electrodes) or polymers, such as PET, PMMA, COP, COC, and PC. Alternatively, the first substrate and the second substrate can be made of non-flexible materials, such as printed circuit boards, plastics, or glass. For purposes of illustration and not limitation, as embodied herein, one or both of the substrates can be made of a single sheet, which can undergo subsequent processing to generate multiple electrodes. For example, one or more sets of multiple electrodes can be fabricated on a substrate, which can be cut to form multiple substrates covered with multiple electrodes. For example, the electrodes can be bonded to the surface of the conductive layer via a general adhesive or solder.

[0046] The electrodes can be composed of metals, metal mixtures or alloys, metal-semiconductor mixtures or alloys, or conductive polymers. Some examples of metal electrodes include copper, gold, indium, tin, indium tin oxide, and aluminum. For example, the dielectric layer comprises an insulating material having low electrical conductivity or capable of maintaining an electrostatic field. For example, the dielectric layer can be made of porcelain (such as ceramics), polymers, or plastics. The hydrophobic layer can be made of materials having hydrophobic properties, such as Teflon and general fluorocarbons. In another example, the hydrophobic material can be a fluorinated surfactant (e.g., FluoroPel). In embodiments including a hydrophilic layer deposited on the dielectric layer, the hydrophilic layer can be a layer of glass, quartz, silica, metal hydroxide, or mica.

[0047] The multiple electrodes can include a certain number of electrodes per unit area of the first substrate, and this number can increase or decrease based on the size of the electrodes and the presence or absence of interdigitated electrodes. The electrodes can be fabricated using a variety of processes, including lithography of the electrodes, atomic layer deposition, laser scribing or etching, laser ablation, flexographic printing, and inkjet printing. For example, but not limited to, a specific mask pattern can be applied to a conductive layer disposed on the upper surface of the first substrate, and then the exposed conductive layer can be laser ablated to generate multiple electrodes on the first substrate.

[0048] Figure 2is a plan view of an exemplary embodiment of an integrated digital microfluidics and analyte detection device according to the disclosed subject matter. The digital microfluidics module is depicted as having a plurality of electrodes forming an electrode array 1049, which are operatively connected to a plurality of reagent reservoirs 1051, which can be used to generate droplets to be delivered to the well array 1054. For example, one or more reservoirs 1051 can contain reagents or samples. Different reagents can be present in different reservoirs. Contact pads 1053 for connecting the electrode array 1049 to a power source (not shown) are also depicted in the microfluidics module 1050. Traces connecting the electrode array 1049 to the contact pads are not depicted. The electrode array 1049 can deliver one or more droplets, such as but not limited to buffer droplets or droplets containing buffer and / or labels (such as but not limited to, cleaved labels or dissociated aptamers), to the well array 1054.

[0049] For example and as embodied herein, an electric potential generated by the plurality of electrodes drives a droplet formed on the upper surface of a first layer (or second layer, when present) covering the plurality of electrodes across the surface of the digital microfluidics device to be received by the well array. In this manner, each electrode can independently drive a droplet across the surface of the digital microfluidics device.

[0050] Now referring to Figure 3 , the electrode array has an electrode array region in a plan view. For example but not limited to, and as embodied herein, the electrode array 200 can have a substantially rectangular shape including four sides 211, 212, 213, and 214 in a plan view, and the electrode array region is defined within the perimeter of the region defined by the electrode array. As further discussed herein, the electrode array 200 can include a first electrode 201, a second electrode 202, a third electrode 203, and a fourth electrode 204, each electrode defining a region of the electrode array region. Although the electrode array 200 is shown as having a substantially square shape, electrode arrays within the scope of the disclosed subject matter can have any shape. For example but not limited to, referring to the electrode array 400 shown in Figure 4B , the electrode array can have a substantially rectangular shape. Additionally or as a further alternative, the electrode array can define a non-linear perimeter, such as but not limited to, if the electrodes forming the electrode array cross each other and / or cross other electrodes formed on a substrate.

[0051] Referring again to Figure 3, the well array 19 has a well array region in a plan view. By way of example and not limitation, and as embodied herein, the well array 19 may have a substantially rectangular shape in a plan view, having four sides 191, 192, 193, and 194 that form the perimeter of the well array region. The well array 19 is disposed within the electrode array 200 and has a well array region defined within the electrode array region in a plan view. Thus, for purposes of illustration and not limitation, as embodied herein, the well array region overlaps less than 75% of the electrode array region in a plan view. That is, by way of example and not limitation as shown, the well array region defined by the sides 191, 192, 193, and 194 of the well array 19 overlaps less than 75% of the electrode array region defined by the sides 211, 212, 213, and 214 of the electrode array 200 in a plan view. For purposes of illustration and not limitation, as embodied herein, the well array region may overlap less than 75% of each electrode in the electrode array. By way of example and not limitation, as Figure 3 shown, the well array region formed by each of the sides 191, 192, 193, and 194 may overlap less than 75% of each of the first electrode 201, the second electrode 202, the third electrode 203, and the fourth electrode 204, respectively.

[0052] According to another aspect of the disclosed subject matter, an analyte detection module for performing analyte detection is provided. The analyte detection module generally includes a substrate having a first layer and a second layer. The first layer includes an electrode array configured to generate electrokinetic forces to push at least one droplet along the surface of the substrate. The electrode array has a plurality of electrodes that define an electrode array region in a plan view. The second layer has a well array that defines a well array region in a plan view. The well array region is defined within the electrode array region and overlaps a portion of each of the plurality of electrodes. The well array region overlaps less than 75% of the electrode array region in a plan view.

[0053] According to another aspect of the disclosed subject matter, a method of loading droplets into a well array of an analyte detection module is provided. The method includes introducing a mother droplet into a gap defined between a first substrate and a second substrate. At least one of the first substrate and the second substrate has an electrode array defined therein, the electrode array having an electrode array region in a plan view. At least one of the first substrate and the second substrate has a well array defining a well array region in the plan view, the well array region being defined within the electrode array region and overlapping the electrode array region by less than 75% in the plan view. The method further includes generating electrokinetic forces on the mother droplet through the electrode array to continuously push the mother droplet around a peripheral portion of the well array to bring at least a portion of the mother droplet into fluid contact with at least one well in the well array. The method further includes filling the at least one well in the well array with at least one daughter droplet released from the mother droplet.

[0054] Now referring to Figure 4A , as embodied herein, the electrode array 200 can include a first electrode 201 and a second electrode 202. The first electrode 201 can have a first electrode region in a plan view and the second electrode 202 can have a second electrode region in a plan view. For purposes of illustration and not limitation, and the first electrode region and the second electrode region can jointly define a generally parallelogram shape having a longitudinal axis passing therethrough, and as embodied herein, the shape can be a generally rectangular shape having a longitudinal axis passing therethrough. For purposes of illustration only and not limitation, the longitudinal axis 301 of the first electrode region and the second electrode region is depicted in dashed lines in Figure 4A . Additionally, the well array 19 can define a well array region having a generally rectangular shape with a diagonal axis passing between diagonals in the plan view. For purposes of illustration only, the diagonal axis 302 of the well array 19 is also depicted in dashed lines in Figure 4A . The diagonal axis 302 of the well array 19 can be generally parallel or collinear with the longitudinal axis 301 of the first electrode region and the second electrode region. Additionally or alternatively, and as further embodied herein, in the plan view, a first portion 304a of the well array 19 can overlap the first electrode 201, and a second portion 304b of the well array 19 can overlap the second electrode 202. The first portion 304a of the well array 19 can have dimensions generally equal to those of the second portion 304b of the well array 19.

[0055] Further referring to Figure 4A, the electrode array 200 may further include a third electrode 203 and a fourth electrode 204. As embodied herein, the first electrode 201, the second electrode 202, the third electrode 203, and the fourth electrode 204 may define a substantially square shape having a diagonal axis passing therethrough. For illustrative purposes only and not by way of limitation, the diagonal axis 306 is depicted as a dashed line in Figure 4A . The diagonal axis 306 of the electrode array 200 may be disposed at an angle of about 45 degrees with respect to the diagonal axis 302 of the region of the well array 19. As embodied herein, the well array 19 may partially overlap with a portion having dimensions substantially similar to those of each of the regions of the first electrode 201, the second electrode 202, the third electrode 203, and the fourth electrode 204.

[0056] Still referring to Figure 4A , the first electrode 201, the second electrode 202, the third electrode 203, and the fourth electrode 204 may serially define a path 305 along which at least one droplet (not depicted) may be pushed by the electrokinetic force of the electrode array. When pushing at least one droplet along the path 305, at least a portion of the droplet may engage at least a portion of the well array 19. For example, the electrode array 200 may be configured to push a droplet along the path 305 such that at least a portion of the droplet makes fluid contact with at least one well in the well array 19. Additionally or alternatively, the electrode array 200 may be configured to push a droplet from the first electrode 201 to the second electrode 202 along the path 305. As embodied herein, at least a portion of the peripheral edge of the well array 19 may be at an angle of 0 degrees to 55 degrees with respect to the path 305 and may be at an angle of about 45 degrees with respect to the path 305. As embodied herein, the peripheral edge of the well array 19 may be defined by four sides 191, 192, 193, and 194.

[0057] Referring to Figure 4B , an electrode array 400 having six electrodes is depicted. The first electrode 401, the second electrode 402, the third electrode 403, the fourth electrode 404, the fifth electrode 405, and the sixth electrode 406 may define a path 415 along which at least one droplet (not depicted) may be pushed by the electrokinetic force of the electrode array. The electrode array 400 and the well array 419 may have any features or combinations of features of the electrode arrays and well arrays described herein. For example, and as embodied herein, when pushing at least one droplet (not shown) along the path 415, at least a portion of the droplet may engage at least a portion of the well array 419.

[0058] Moving or pushing a droplet along a path to bring at least a portion of the droplet into fluid contact with at least one well in the well array 19, which can be performed to load the droplet into the well array. According to the disclosed subject matter, an electrokinetic force can be generated by the electrode array 200 to continuously push a mother droplet around the peripheral portion of the well array 19, and at least one well in the well array 19 can be filled with at least one daughter droplet released from the mother droplet. The mother droplet can be continuously cycled and pushed to the peripheral portion of the well array. As embodied herein, the mother droplet can be continuously pushed around the peripheral portion of the well array 19 along path 305. For example but not limited to, the droplet can be pushed around the peripheral portion of the well array for 5 - 20 continuous cycles.

[0059] For purposes of illustration and not limitation, Figure 5 is a schematic side view of another exemplary integrated digital microfluidic and analyte detection device 100, in which a droplet 180 is pushed in a slit 170. As embodied herein, the droplet 180 can contain a plurality of beads or particles 190. The arrow indicates the direction of movement of the droplet from a first portion 115 to a second portion 130 including the well array 160. Although beads or particles are illustrated herein, the droplet can contain analyte molecules in place of or in addition to the solid support. For purposes of illustration and not limitation, exemplary droplet configurations and contents are described in U.S. Patent Application Publication 2018 / 0095067, which is incorporated herein by reference in its entirety.

[0060] In addition, or as an alternative, in addition to moving aqueous fluids, immiscible fluids, such as organic - based immiscible fluids, can also be pushed by dielectrophoresis. Droplet actuation can be related to the dipole moment and dielectric constant, which are interrelated, and also to the conductivity. As embodied herein, the immiscible liquid can have a molecular dipole moment greater than about 0.9 D, a dielectric constant greater than about 3, and / or a conductivity greater than about 10 -9 S m -1 . Examples of using immiscible liquids in the analyte analysis assays disclosed herein include assisting the movement of aqueous droplets, displacing the aqueous fluid located above the wells, displacing un - deposited beads / particles / analyte molecules from the wells prior to optical interrogation of the wells, sealing the wells, etc. Some examples of organic - based immiscible fluids that can be moved in the devices disclosed herein include 1 - hexanol, dichloromethane, dibromomethane, THF, and chloroform. Organic - based oils that meet such criteria are also movable under similar conditions. As embodied herein, using immiscible fluid droplets, the slits / spacings in the device can be filled with air.

[0061] Figure 6 is Figure 5Schematic partial side view of the device, where the droplet 180 containing the beads or particles 190 is partially positioned above the well array 160. As discussed above with reference to Figure 4A the exemplary embodiments, the droplet 180 can be continuously pushed along a path, where at least a portion of the droplet is in fluid contact with at least one well in the well array 160. Continuously moving the droplet along the path while maintaining fluid contact with at least one well in the well array 160 can facilitate the deposition of the particles or beads 190 into the well array 160. The wells 160 can be sized to accommodate one bead or particle 190 per well, or alternatively, can be sized to accommodate multiple beads or particles 190 per well. Although beads or particles are depicted herein, droplets containing any other contents (such as, but not limited to, analyte molecules) can also be moved as described herein. The wells 160 can also be sized to accommodate one analyte molecule per well, or alternatively can be sized to accommodate multiple analyte molecules per well.

[0062] As shown for illustrative purposes only and not for limitation, reference Figure 7 , the beads or particles 190 can be magnetic, and a force can be applied to the beads or particles 190 using a magnet or an electromagnet 825, which can facilitate loading the beads or particles 190 into the wells 160. For illustrative purposes and not for limitation, exemplary techniques for loading beads, particles, or other droplet contents into wells are described in U.S. Patent Application Publication 2018 / 0095067, which is incorporated herein by reference in its entirety.

[0063] As embodied herein, the fluid sample can be diluted before analysis. For example, in embodiments where the source of the analyte molecules is human body fluid (such as, blood, serum), the fluid can be diluted with a suitable solvent (such as, a buffer, such as PBS buffer). The fluid sample can be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more before use.

[0064] As embodied herein, the sample can undergo pre-analytical processing. The pre-analytical processing can provide additional functions, such as non-specific protein removal and / or efficient and inexpensive mixing functions. General methods for pre-analytical processing can include using electrokinetic capture, AC electrokinetics, surface acoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, or other preconcentration techniques known in the art. As embodied herein, the fluid sample can be concentrated before analysis. For example, in embodiments where the source of the analyte molecules is human body fluid (such as, blood, serum), the fluid can be concentrated by precipitation, evaporation, filtration, centrifugation, or a combination thereof. The fluid sample can be concentrated about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold, or more before use.

[0065] As embodied herein, the analyte is not amplified prior to measurement of the analyte (e.g., the copy number of the analyte is not increased). For example, when the analyte is DNA or RNA, the analyte is not replicated to increase the copy number of the analyte. As embodied herein, the analyte is a protein or a small molecule.

[0066] As used herein, the terms "one or more droplets" and "one or more fluid droplets" are used interchangeably and refer to discrete volumes of liquid that are generally spherical in shape and are bounded on at least one side by a well or substrate of a microfluidic device. In the context of droplets, generally spherical means a shape such as a sphere, a partially flattened sphere, e.g., a disk shape, a slug shape, a truncated sphere, an ellipse, a hemisphere, or an ovoid. The volume of the droplets in the devices disclosed herein can range from about 10 μl to about 5 pL, e.g., 10 μl - 1 pL, 7.5 μl - 10 pL, 5 μl - 1 nL, 2.5 μl - 10 nL, or 1 μl - 100 nL, e.g., 10 μl, 5 μl, 1 μl, 800 nL, 500 nL, or less.

[0067] For example, a well array includes a plurality of individual wells. The well array can include a plurality of wells, and the number of wells can range from 10 to 10 9 wells / mm 2 . As embodied herein, an array of about 100,000 to 500,000 wells (e.g., femtoliter wells) can be fabricated that coat an area of about 12 mm 2 . Each well can measure about 4.2 μm wide X 3.2 μm deep (volume of about 50 femtoliters) and may be capable of accommodating a single bead / particle (about 3 μm in diameter). At this density, the femtoliter wells are spaced apart from each other by about 7.4 μm. For example, the well array can be fabricated to have individual wells with diameters from 10 nm to 10,000 nm.

[0068] Placing a single bead, particle, analyte molecule, or other suitable content in a well can permit a digital or an analog readout. For example, for a small number of positive wells (<~70% positive), Poisson statistics can be used to quantify the analyte concentration in digital format; for a large number of positive wells (>~70%), the relative intensity of the wells carrying the signal is compared to the signal intensity generated by a single bead, particle, or analyte molecule, respectively, and used to generate an analog signal. The digital signal can be used for lower analyte concentrations, while the analog signal can be used for higher analyte concentrations. Digital and analog quantification can be used in combination, which can expand the linear dynamic range. As used herein, a "positive well" is a well having a signal associated with the presence of a bead / particle / analyte molecule that is above a threshold. As used herein, a "negative well" is a well having no signal associated with the presence of a bead, particle, or analyte molecule. As embodied herein, the signal from a negative well can be at a background level, e.g., below the threshold.

[0069] The wells can be any of a variety of shapes, such as cylindrical with a flat bottom, cylindrical with a round bottom, cube, cuboid, frustum of a cone, inverted frustum of a cone, or cone. As embodied herein, the wells can include sidewalls, which can be oriented to facilitate the receipt and retention of microbeads or microparticles present in a droplet that has been pushed above the well array. For example, the wells can include a first sidewall and a second sidewall, where the first sidewall can be opposite the second sidewall. For example, and as embodied herein, the first sidewall is oriented at an obtuse angle relative to the bottom of the well, and the second sidewall is oriented at an acute angle relative to the bottom of the well. The movement of the droplet can be in a direction parallel to the bottom of the well and from the first sidewall to the second sidewall.

[0070] For example, the well array can be fabricated by one or more of molding, pressing, heating, or laser or a combination thereof. For example, nanoimprint / nanosphere lithography can be used to fabricate the well array. Other fabrication methods known in the art can also be used. The integrated device for performing analyte analysis and its various components can be formed, for example but not limited to, using the materials and techniques described in U.S. Patent Application Publication 2018 / 0095067, which is hereby incorporated by reference in its entirety.

[0071] The systems, devices, and methods described herein have demonstrated desirable performance characteristics not achievable by conventional DMF analyte detection devices. The well array region can impose increased surface tension or drag on a droplet compared to the surrounding electrode array region. In conventional devices, the increased surface tension or drag in the well array region would prevent fluid from being effectively loaded into the wells or pores because fluid droplets tend to bypass the well array region and associated elevated surface tension or become stuck or pinned at the top of the well region. Positioning, sizing, and orienting the well array relative to the device electrodes can minimize the effects of different substrate surface characteristics in the well array region and facilitate effective loading of fluid into the wells.

[0072] For example, sizing the well array region in a plan view to overlap less than 75% of the electrode array region can reduce the amount of droplets covering the well array region and allow more droplets to remain above the electrode array. Such a configuration can reduce the tendency for droplets to become pinned at the top of the well array region and improve droplet entry onto the well array. Orienting the well array relative to the electrode array can similarly improve fluid loading into the wells. For example, aligning the diagonal axis of the well array region substantially parallel or collinear with the longitudinal axes of the first and second electrode regions can reduce the amount of droplets covering the well array region and allow more droplets to remain above the electrode array.

[0073] Continuously pushing a droplet around the peripheral portion of the well array can similarly facilitate effective loading of fluid into the wells within the well array in cases where at least a portion of the droplet is in fluid contact with at least one well of the well array. Sizing the well array region relative to the electrode array region and additionally or alternatively orienting the well array relative to the electrode array can minimize the pinning effect and allow the droplet to be continuously pushed around the periphery of the well array without getting stuck on the well array. For example, such a configuration can allow the droplet to continuously cycle around the well array 20 or more times.

[0074] For purposes of illustration and not limitation, data are provided to demonstrate various operational characteristics achieved by the systems, devices, and methods described herein. Table 1 describes the results of bead loading analysis using a method for loading droplets into a well array according to the disclosed subject matter.

[0075] Table 1.

[0076]

[0077] A plurality of wells are arranged such that the peripheral edge of the well array is at an angle of about 45 degrees with respect to the droplet movement path. The plurality of wells includes 32K wells with a pitch of 11 µm. Referring to Table 1, the "Wells" column represents the number of wells in the plurality of wells. The "Filled Wells" column represents the number of wells loaded with beads after continuously pushing a mother droplet containing beads suspended therein in contact with at least one well in the well array, with at least a portion of the mother droplet in fluid contact with the well array. The droplet pushed around the well array contains 100K beads. The "Filling Percentage" column of Table 1 describes the percentage of the total wells in the well array filled with beads. As described in Table 1, a bead loading efficiency of about 92% to about 99% is achieved using the method of loading droplets into the well array according to the disclosed subject matter. Similar results were observed using 70K beads.

[0078] In accordance with other aspects of the disclosed subject matter, the analyte detection module of the digital microfluidic and analyte detection device described herein can be combined with, for example but not limited to, a sample preparation module as described in U.S. Patent Application Publication 2018 / 0095067, which is incorporated herein by reference in its entirety.

[0079] As embodied herein, the sample preparation module can be used to perform steps of an immunoassay. Any immunoassay format can be used to generate a detectable signal that indicates the presence of the analyte of interest in the sample and is proportional to the amount of the analyte in the sample.

[0080] As embodied herein and further described herein, the detection module includes a well array that is optically interrogated to measure a signal related to the amount of analyte present in the sample. The well array can have a sub-femtoliter volume, femtoliter volume, sub-nanoliter volume, nanoliter volume, sub-microliter volume, or microliter volume. For example, the well array can be a femtoliter well array, nanoliter well array, or microliter well array. As embodied herein, the wells in the array can all have substantially the same volume. The well array can have a volume of up to 100 µl, such as about 0.1 femtoliter, 1 femtoliter, 10 femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50 microliter, or 100 microliter.

[0081] As embodied herein and further described herein, both the sample preparation module and the detection module can be present on a single substrate, and both the sample preparation module and the detection module can include a plurality of electrodes for moving droplets. As embodied herein, such a device can include a first substrate and a second substrate, wherein the second substrate is positioned above the first substrate and separated from the first substrate by a gap. The first substrate can include a first portion (e.g., a proximal portion) where the sample preparation module is located, where droplets are introduced into the device, and a second portion (e.g., a distal portion) towards which the droplets are pushed, where the detection module is located. As used herein, "proximal" with respect to "distal" and "first" with respect to "second" are relative terms and are interchangeable with each other.

[0082] The height of the gap between the first substrate and the second substrate can be up to 1 mm, such as 0.1 µm, 0.5 µm, 1 µm, 5 µm, 10 µm, 20 µm, 50 µm, 100 µm, 140 µm, 200 µm, 300 µm, 400 µm, 500 µm, 1 µm - 500 µm, 100 µm - 200 µm, etc. The volume range of the droplets generated and pushed in the device described herein can be from about 10 µl to about 5 pL, such as 10 µl–1 pL, 7.5 µl–10 pL, 5 µl–1 nL, 2.5 µl–10 nL or 1 µl–100 nL, 800 - 200 nL, 10 nL - 0.5 µl, for example, 10 µl, 1 µl, 800 nL, 100 nL, 10 nL, 1 nL, 0.5 nL, 10 pL or less.

[0083] As embodied herein, the first portion and the second portion are separate or separate and adjacent. As embodied herein, the first portion and the second portion are co-located, mixed or crossed. The first substrate can include a plurality of electrodes covering the upper surface of the first substrate and extending from the first portion to the second portion. The first substrate can include a layer disposed on the upper surface of the first substrate, covering the plurality of electrodes and extending from the first portion to the second portion. The first layer can be made of a dielectric and hydrophobic material. Examples of dielectric and hydrophobic materials include polytetrafluoroethylene materials (e.g., Teflon®) or fluorosurfactants (e.g., FluoroPel TM). The first layer can be deposited in a manner that provides a substantially flat surface. The well array can be positioned in the second portion of the first substrate, covering a portion of the plurality of electrodes and forming a detection module. The well array can be positioned within the first layer. As embodied herein, before or after fabricating the well array in the first layer, a hydrophilic layer can be disposed above the first layer in the second portion of the first substrate to provide a well array with a hydrophilic surface. The gap / spacing between the first substrate and the second substrate can be filled with air or an immiscible fluid. As embodied herein, the gap / spacing between the first substrate and the second substrate can be filled with air.

[0084] As embodied herein, both the sample preparation module and the detection module can be fabricated using a single substrate, but the plurality of electrodes for moving droplets can only be present in the sample preparation module only. As embodied herein, the first substrate can include a plurality of electrodes covering the upper surface of the first substrate at the first portion of the first substrate, wherein the plurality of electrodes do not extend to the second portion of the first substrate. As embodied herein, the plurality of electrodes are only positioned in the first portion. As described herein, a first dielectric / hydrophobic material layer can be disposed on the upper surface of the first substrate and can encapsulate the plurality of electrodes. As embodied herein, the first layer can be disposed only above the first portion of the first substrate. Alternatively, the first layer can be disposed above the upper surface of the first substrate over the first portion as well as the second portion. The well array can be positioned in the first layer in the second portion of the first substrate, forming a detection module that does not include the plurality of electrodes present beneath the well array.

[0085] As embodied herein, the second substrate can extend over the first portion and the second portion of the first substrate. As embodied herein, the second substrate can be substantially transparent, at least in the region covering the well array. Alternatively, the second substrate can be disposed in a spaced-apart manner above the first portion of the first substrate and cannot be disposed above the second portion of the first substrate. Thus, as embodied herein, the second substrate can be present in the sample preparation module but not in the detection module.

[0086] As embodied herein, the second substrate can include a conductive layer that forms an electrode. The conductive layer can be disposed on the lower surface of the second substrate. As described herein, the conductive layer can be encapsulated by a first layer made of a dielectric / hydrophobic material. As embodied herein, the conductive layer can be encapsulated by a dielectric layer. The dielectric layer can be encapsulated by a hydrophobic layer. The conductive layer and any one or more of the layers encapsulating it can be disposed across the lower surface of the second substrate or can be present only on the first portion of the second substrate. As embodied herein, the second substrate can extend over the first portion and the second portion of the first substrate. As embodied herein, the second substrate and any layer disposed thereon (e.g., the conductive layer, the dielectric layer, etc.) can be substantially transparent, at least in the region covering the well array.

[0087] As embodied herein, the plurality of electrodes on the first substrate can be configured as coplanar electrodes and the second substrate can be configured to be electrode-free. The electrodes present in the first layer and / or the second layer can be made of a substantially transparent material such as indium tin oxide, fluorine-doped tin oxide (FTO), doped zinc oxide, etc.

[0088] As embodied herein, the sample preparation module and the detection module can be fabricated on a single substrate. Alternatively, the sample preparation module and the detection module can be fabricated on separate substrates, which can then be joined to form an integrated microfluidic and analyte detection device. As embodied herein, the first substrate and the second substrate can be spaced apart using spacers that can be positioned between the substrates. The devices described herein can be flat and can have any shape, such as rectangular or square, rectangular or square with rounded corners, circular, triangular, etc.

[0089] Although the disclosed subject matter is described herein in accordance with certain preferred embodiments, those skilled in the art will recognize that various modifications and improvements can be made to the disclosed subject matter without departing from its scope. Additionally, although the various features of one embodiment of the disclosed subject matter may be discussed herein or shown in the drawings of this embodiment and not shown in other embodiments, it is apparent that the various features of one embodiment can be combined with one or more features of another embodiment or features from multiple embodiments.

[0090] In addition to the specific embodiments claimed below, the disclosed subject matter also relates to other embodiments having any other possible combinations of the dependent features claimed below and those features disclosed above. Thus, the specific features recited in the dependent claims and disclosed above can be combined with each other in other ways within the scope of the disclosed subject matter such that the disclosed subject matter should be considered to particularly relate to other embodiments having any other possible combinations. Accordingly, the foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to the embodiments disclosed.

[0091] It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and systems of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Accordingly, the disclosed subject matter is intended to include modifications and variations within the scope of the appended claims and their equivalents.

Claims

1. A digital microfluidics and analyte detection device, comprising: a first substrate and a second substrate, the first substrate and the second substrate being aligned parallel to each other in a side view and defining a gap therebetween; the second substrate having an electrode array configured to generate an electrokinetic force to push at least one droplet within the gap along one of the first substrate and the second substrate, the electrode array having a plurality of electrodes defining an electrode array region in a plan view; and the second substrate having a well array defining a well array region in a plan view, wherein at least a portion of the at least one droplet is in fluid contact with at least one well of the well array, the well array region being defined within the electrode array region and overlapping at least a portion of each of the plurality of electrodes, wherein the well array region overlaps less than 75% of the electrode array region in a plan view; wherein the well array includes a plurality of femtoliter wells, each femtoliter well being configured to accommodate a single bead.

2. The device according to claim 1, wherein each of the plurality of electrodes overlaps less than 25% of the well array region.

3. The device according to claim 1, wherein the electrode array comprises: a first electrode having a first electrode region in a plan view, and a second electrode adjacent to the first electrode and having a second electrode region in a plan view, the first electrode region and the second electrode region together defining a parallelogram shape having a longitudinal axis passing therethrough in a plan view.

4. The device according to claim 3, wherein the well array region has a rectangular shape having a diagonal axis passing between diagonals thereof, the diagonal axis of the well array region being parallel or collinear with the longitudinal axis of the first electrode region and the second electrode region.

5. The device according to claim 3, wherein a first portion of the well array region overlaps the first electrode region, and a second portion of the well array region overlaps the second electrode region, the first portion having the same size as the second portion.

6. The device according to claim 3, the electrode array further comprising a third electrode having a third electrode region in a plan view and a fourth electrode having a fourth electrode region in a plan view, wherein the first electrode, the second electrode, the third electrode, and the fourth electrode are connected in series to define a path along which the droplet engages the well array.

7. The device according to claim 6, wherein the first electrode, the second electrode, the third electrode, and the fourth electrode together define a square shape having a diagonal axis passing therethrough, the diagonal axis being disposed at a 45-degree angle with respect to the diagonal axis defined by the well array.

8. The device according to claim 6, wherein the well array region overlaps a portion of each of the first electrode region, the second electrode region, the third electrode region, and the fourth electrode region having the same size.

9. The device according to claim 6, wherein the electrode array is configured to push the at least one droplet along the path.

10. The device according to claim 6, wherein the electrode array is configured to continuously push the at least one droplet around a peripheral portion of the well array.

11. The device according to claim 3, wherein the electrode array is configured to push the at least one droplet along a path from the first electrode to the second electrode, and wherein at least a portion of a peripheral edge of the well array is at an angle of 0 degrees to 55 degrees relative to the path.

12. The device according to claim 1, further comprising at least one of a magnet and an electromagnet proximate to the plurality of wells.

13. The device according to claim 1, wherein at least one of the first substrate or the second substrate comprises at least one of PET, PMMA, COP, COC, PC, and glass.

14. The device according to claim 1, wherein both the electrode array and the well array are defined in one of the first substrate or the second substrate.

15. An analyte detection module for performing analyte detection, comprising: a substrate having: a first layer, the first layer including an electrode array configured to generate an electrokinetic force to push at least one droplet along a surface of the substrate, the electrode array having a plurality of electrodes that define an electrode array region in a plan view; and a second layer, the second layer having a well array that defines a well array region in a plan view, wherein at least a portion of the at least one droplet is in fluid contact with at least one well of the well array, the well array region being defined within the electrode array region and overlapping at least a portion of each of the plurality of electrodes, wherein the well array region overlaps less than 75% of the electrode array region in a plan view; wherein the well array includes a plurality of femtoliter wells, each femtoliter well being configured to accommodate a single bead.

16. The analyte detection module according to claim 15, wherein each of the plurality of electrodes overlaps less than 25% of the well array region.

17. The analyte detection module according to claim 15, wherein the electrode array comprises: a first electrode having a first electrode region in a plan view, and a second electrode adjacent to the first electrode and having a second electrode region in a plan view, the first electrode region and the second electrode region together defining a parallelogram shape having a longitudinal axis passing therethrough in a plan view.

18. The analyte detection module according to claim 17, wherein the well array region has a rectangular shape having a diagonal axis passing between diagonals therethrough, the diagonal axis of the well array region being parallel or collinear with the longitudinal axis of the first electrode region and the second electrode region.

19. The analyte detection module according to claim 17, the electrode array further comprising a third electrode having a third electrode region in a plan view and a fourth electrode having a fourth electrode region in a plan view, wherein the first electrode, the second electrode, the third electrode, and the fourth electrode are serially defined to form a path, and the droplet engages with the well array along the path.

20. The analyte detection module according to claim 19, wherein the electrode array is configured to continuously push the at least one droplet around a peripheral portion of the well array.

21. A method of loading droplets into a well array of an analyte detection module, comprising: introducing a mother droplet into a gap defined between a first substrate and a second substrate in a side view, the second substrate having an electrode array having a plurality of electrodes defining an electrode array region in a plan view, and the second substrate having a well array defining a well array region in a plan view, the well array region being defined within the electrode array region and overlapping a portion of each of the plurality of electrodes, wherein the well array region overlaps less than 75% of the electrode array region in the plan view; generating an electrokinetic force on the mother droplet by the electrode array to continuously push the mother droplet around a peripheral portion of the well array to bring at least a portion of the mother droplet into fluid contact with at least one well in the well array; and filling the at least one well in the well array with at least one daughter droplet released from the mother droplet; wherein the well array includes a plurality of femtoliter wells, each femtoliter well being configured to accommodate a single bead.

22. The method according to claim 21, wherein the mother droplet is pushed around the peripheral portion of the well array for a plurality of consecutive cycles within a range of 5 - 20 consecutive cycles.

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