Digital microfluidic agglutination assay

Abstract

The present disclosure provides a method for performing an agglutination assay on a “dual-plate” DMF device format. Droplets containing an analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with a solution-phase or dried agglutination antibody or antigen. The agglutinants bind to their complementary targets (e.g., antibody or antigen) in the sample droplets, resulting in the formation of insoluble aggregates. Active mixing on the DMF device reduces the reaction time and enhances the agglutination effect. Since the agglutinated sample is sandwiched between two plates on the DMF device, the results can be straightforwardly viewed by the naked eye or via a digital camera.

Description

Technical Field

[0001] The present disclosure relates to a method for performing an agglutination assay using a "dual-plate" digital microfluidic (DMF) device format. Droplets containing an analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with a liquid-phase or dried agglutination antibody or antigen. Background Art

[0002] Agglutination assays are commonly used to detect the presence of an analyte in a sample; typical applications include infectious disease and pathogen detection, as well as blood typing for donor compatibility. Agglutination assays rely on the interaction of an antibody or antigen with an analyte of interest; the result of this interaction is the formation of large, insoluble clumps or aggregates that are visible to the naked eye. Thus, agglutination assays have a unique advantage over other techniques used for such assays, which rely on instrumental measurements of photon energy or electrical energy—the results of the assay are very straightforward to read.

[0003] In traditional agglutination assays, particles coated with an antibody or antigen are combined with a sample, manually mixed, and the presence of aggregates is determined by visual inspection. The obvious disadvantages of standard techniques include the need for manual mixing, the possibility of misinterpreting assay readings, and low throughput. Agglutination assays in microfluidic devices have recently been developed in an effort to address these disadvantages. For example, Castro et al. 1 describe a microfluidic method for agglutination assays that relies on hydrodynamic mixing combined with imaging-based detection, which is reported to be useful for limiting user input and minimizing assay variability. Other microfluidic implementations of agglutination assays rely on flow cytometry, 2 light scattering, 3 fluorescence 4 and optical microscopy. 5,6 These detection methods are useful for method development and optimization, but they require auxiliary detectors that ultimately negate the main advantage of agglutination—the ability to read test results without a complex detection scheme.

[0004] Another challenge affecting previously reported microfluidic-based agglutination assays is the requirement that the sample must be diluted before introduction into the microfluidic device, which increases the complexity of the assay. 5 An ideal method would be to accept an untreated sample, which would be compatible with non-expert use. Additionally, manipulating agglutinates in narrow channels can cause channel blockage, which can lead to device failure and reliability issues. Finally, as an alternative to conventional microfluidics, there are many "paper microfluidics" formats based on flow measurement (e.g., Yoon and You 7)Examples of agglutination assay methods implemented, but these methods typically have low throughput and / or require manual washing steps. A new method relying on digital microfluidics (DMF) is introduced here, which overcomes the limitations of the above-mentioned technologies.

[0005] DMF is a liquid handling technology that uses electrostatic forces to manipulate liquid droplets ranging in size from picoliters to microliters. The most powerful format of DMF is the "dual-plate" configuration, where the droplets are sandwiched between a top electrode plate and a bottom electrode plate, thus exposing an insulating electrode array. When operated in the dual-plate format, droplets can be dispensed, split, merged, and mixed, making DMF very suitable for sample handling, 8 immunoassay 9-11 and chemical reactions. 12 DMF differs from traditional microfluidics in many ways, including versatility (a general-purpose device architecture can be applied to multiple applications), absolute control over the position of (liquid or dry) reagents without moving parts, and operation in an open geometry with no chance of channel clogging.

[0006] The inventors are aware of two previous reports of agglutination assays implemented on DMF devices. One method uses backscatter detection for latex immunoagglutination assays, 7 and another method uses functionalized gold nanoparticles to agglutinate biomarkers of interest, combined with a microseparation procedure for detection. 13 It should be noted that neither of these techniques has been demonstrated in the "standard" format of digital microfluidics. That is, the first method 7 does not use an electric field to manipulate droplets (instead, wires using XYZ stage control mechanically push / pull droplets around the surface), and the second method 13 uses a so-called "single-plate" DMF (in contrast to the more powerful "dual-plate" DMF format used in the method disclosed herein). These "non-standard" DMF formats 7 , 13 are useful for proof-of-concept, but we believe they are almost certainly incompatible with the type of fully integrated sample-in-answer-out system that has become the common board for dual-plate digital microfluidics. 11

[0007] The term digital microfluidics (DMF) has been widely used to describe liquid droplet manipulation systems. Several fluid actuators have been reported, such as those using chemical 14 or thermal gradients, 15 magnets, 16 acoustic waves, 17 mechanical 7 and electrical methods. 13Since the present invention relies on the use of electrohydrodynamic forces, commonly known as electrowetting-on-dielectric (EWOD), only the comparison between single-plate and double-plate DMF EWOD devices will be focused on, and all other devices (non-EWOD) will not be further discussed because they use other liquid manipulation techniques that are not relevant to the present invention.

[0008] DMF EWOD systems are generally divided into two main categories; single-plate DMF and double-plate DMF. There are several reasons and significant technical challenges in moving from single-plate DMF devices to double-plate DMF devices. The term single-plate DMF device is used to describe an open system in which droplets are freely located on a horizontal solid substrate, while the term double-plate DMF device is used to describe an enclosed system in which droplets are confined between two plates. Both types of devices require sufficient grounding for operation; single-plate devices require an external wire in direct contact with the droplet or an electrode in the same plane as the actuating electrode, while in double-plate devices, the grounding is located within the top plate.

[0009] In addition to the number of plates, these two types of DMF devices also differ significantly in their ability to perform droplet operations. In a double-plate system, droplet movement is easier. Additionally, droplet splitting and dispensing are almost exclusive options for double-plate systems. In contrast, when sufficient mixing, evaporation (for species concentration), and direct contact with liquid droplets are required, single-plate devices are preferred because the droplets are readily available. Single-plate devices typically operate at much higher voltages and much lower frequencies, which requires different hardware instrumentation than double-plate systems. Therefore, although there have been previous reports on digital microfluidics for agglutination assays, 7,13 it is not clear how to transition agglutination from these devices to a double-plate DMF system because there are numerous technical differences between the two types of devices that need to be optimized and determined in order to perform agglutination assays on the double-plate DMF devices reported here.

[0010] Furthermore, even from the perspective of agglutination assays, neither of the previous reports was ideal - the backscattering technique 7 requires auxiliary instrumentation for analysis, and the nanoparticle-based technique 13 is slow (because the user has to wait for each sample to evaporate and then perform the analysis) and requires custom, expensive, nanoparticle-based reagents. It should be noted that both of these methods were reported over a decade ago with no subsequent publications, indicating slow (or no) uptake by the community. SUMMARY OF THE INVENTION

[0011] The present disclosure provides a novel method for performing an agglutination assay in a "dual-plate" digital microfluidic (DMF) device format. Droplets containing an analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with a solution-phase or dried agglutinating antibody or antigen. The agglutinants bind to their complementary targets (e.g., antibody or antigen) in the sample droplets, resulting in the formation of insoluble aggregates. Active mixing on the DMF reduces the reaction time and enhances the agglutination effect. Since the agglutinated sample is sandwiched between two plates on the DMF device, the results can be straightforwardly viewed by the naked eye or via a digital camera.

[0012] Accordingly, the present disclosure provides a method for characterizing a sample containing an analyte using a dual-plate electrowetting digital microfluidic (DMF) device having a plurality of drive electrodes, the method comprising the steps of:

[0013] Loading an agglutinant onto the DMF device;

[0014] Loading a fluid sample containing the analyte onto the DMF device; and

[0015] Using electrowetting to bring the fluid sample into contact with the agglutinant

[0016] To agglutinate the analyte, thereby producing agglutinates.

[0017] The method may further comprise the step of characterizing the amount of agglutination of the analyte caused by the agglutinant.

[0018] The fluid loaded onto the DMF device may contain a surfactant.

[0019] The surfactant may be in a pre-dried form, and the method may further comprise: coating one or more drive electrodes or across the device surface at a predetermined point with the pre-dried form of the surfactant such that when the fluid sample contacts the pre-dried surfactant, the fluid sample dissolves and the surfactant is present in the fluid in an amount of at least 0.01 wt%. The process of drying and reconstituting reagents on the DMF device may be performed according to the method described in US2014 / 0141409 A1 (Foley et al. 18 )

[0020] The surfactant can be one of an ionic surfactant and a non-ionic surfactant. The ionic surfactant is optionally selected from the group consisting of sodium dodecyl sulfate, sodium stearate, cetrimonium bromide, cetrimonium chloride, and sodium lauryl sulfate. The non-ionic surfactant includes, but is not limited to, alkylphenol hydroxy polyethylene (e.g., Triton X RTM), polysorbate (e.g., Tween RTM), poloxamine (e.g., Tetronic RTM), poloxamer (e.g., Pluronic RTM), and sorbitan ester. The poloxamer may include

[0021] The aggregating agent can be a liquid loaded and metered into a preselected drive electrode.

[0022] The aggregating agent can be in a pre-dried form, and the method may further include: coating one or more drive electrodes at a predetermined point on the surface of the DMF device with the pre-dried form of the aggregating agent such that when the fluid contacts the pre-dried aggregating agent, the pre-dried aggregating agent dissolves. The process of drying and reconstituting reagents on the DMF device can be performed according to the method described in US 2014 / 0141409 A1 (Foley et al. 18 )

[0023] The method may further include the step of actively mixing the aggregating agent with the fluid sample using electrowetting on the DMF device.

[0024] The aggregating agent can include any one or a combination of substances capable of producing aggregates. Examples of substances include chemical aggregating agents and biological aggregating agents. For the aggregation of red blood cells, the chemical aggregating agent is optionally selected from the group consisting of poly-L-lysine hydrobromide, poly(dimethyldiallylammonium) chloride( RTM, RTM, RTM), poly-L-arginine hydrochloride, poly-L-histidine, poly(4-vinylpyridine), poly(4-vinylpyridine) hydrochloride, crosslinked poly(4-vinylpyridine), methyl chloride quaternary salt, poly(4-vinylpyridine-co-polystyrene); poly(4-vinylpyridine poly(hydrogen fluoride)); poly(4-vinylpyridine-p-toluenesulfonate); poly(4-vinylpyridine-tribromide); poly(4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); crosslinked polyvinylpyrrolidone; polyvinylpyrrolidone, poly(melamine-co-formaldehyde); partially methylated; hexamethonium bromide; poly(glutamic acid, lysine) 1:4 hydrobromide; poly(lysine, alanine) 3:1 hydrobromide; poly(lysine, alanine) 2:1 hydrobromide; poly-L-lysine succinylated; poly(lysine, alanine) 1:1 hydrobromide; and poly(lysine, tryptophan) 1:4 hydrobromide.

[0025] The chemical flocculant may be poly(dimethyldiallylammonium) chloride.

[0026] The biological flocculant may be selected from the group consisting of proteins, antibodies, viruses and antigens, DNA, RNA, and DNA- or RNA-based aptamers.

[0027] The protein may comprise a lectin capable of reversibly binding to a saccharide structure. The antibodies may include anti-A, anti-B, and anti-D.

[0028] The virus may include influenza virus.

[0029] The step of characterizing the amount of aggregation of the analyte caused by the flocculant may include visual characterization. This visual characterization may be performed by a person observing the DMF device to estimate the amount of aggregation. Alternatively, the step of performing visual characterization using a camera. The camera may be a webcam, a mobile phone camera, a digital camera (including a digital single-lens reflex camera DSLR), a video camera, a surveillance camera, a point-and-shoot camera, a camera with a CCD detector, a camera with a CMOS detector, a monochrome camera, a black-and-white camera, or a color camera.

[0030] The particles may be coated with the flocculant. These may include any one or a combination of polymer particles (e.g., latex), gold, silver, nanoparticles, and microparticles. The polymer particles may include latex.

[0031] The analyte being detected may be an antibody, and wherein the particles may be coated with an antigen or other agent capable of capturing the antibody of interest.

[0032] The analyte being detected may be an antigen, and wherein the particles may be coated with an antibody or other agent capable of capturing the antigen of interest.

[0033] The analyte being detected can be bacteria, and the particles can be coated with antibodies or other agents capable of capturing the bacteria of interest.

[0034] The analyte being detected can be a virus, and the particles can be coated with antibodies or other agents capable of capturing the virus of interest.

[0035] The method can be used for the agglutination of suspensions of polymer particles.

[0036] The method can be used for the agglutination of suspensions of nanoparticles.

[0037] The method can be used for the agglutination of suspensions of red blood cells.

[0038] The fluid can be a blood sample containing at least red blood cells.

[0039] The fluid can contain a virus suspension for detecting the virus using the agglutination of red blood cells or particles.

[0040] The method can be used for the agglutination of suspensions of white blood cells.

[0041] The fluid can be a serum sample or a plasma sample containing at least white blood cells.

[0042] The method can be used for the agglutination of suspensions of any other type of eukaryotic cell.

[0043] The agglutinating agent can be any substance capable of causing cell agglutination.

[0044] The cell can be a red blood cell.

[0045] The agglutinating agent can be used to determine the hematocrit level when used with red blood cells.

[0046] The present invention provides a double-plate electrowetting DMF device, which includes:

[0047] A first electrode plate, a second electrode plate spaced apart from the first electrode plate, one of the first electrode plate and the second electrode plate having a plurality of driving electrodes; and

[0048] A surface on the first plate or the second plate, another surface on the first plate or the second plate, the surface having a surfactant in a pre-dried form coated on the surface or coating the entire plate, and the another surface having an agglutinating agent in a pre-dried form coated on the another surface at a preselected position.

[0049] The surface coated with the surfactant and the surface coated with the agglutinating agent are different or the same.

[0050] The present disclosure provides a kit, which includes:

[0051] A double-plate electrowetting digital microfluidic device (DMF), the DMF having a plurality of driving electrodes;

[0052] A surfactant, the surfactant being for placement on one of the two plates; and

[0053] A coagulant, the coagulant being for placement on one of the two plates.

[0054] The coagulant can be selected for the agglutination of red blood cells.

[0055] A further understanding of the functions and advantageous aspects of the present disclosure can be achieved by referring to the following detailed description and the drawings. Description of the Drawings

[0056] Embodiments will now be described by way of example only with reference to the drawings, in which:

[0057] Figure 1 The DMF device and the associated steps of an agglutination assay performed on the DMF device are shown in three (3) figures, where the leftmost figure labeled (i) shows loading one or more samples containing an analyte of interest into the DMF device containing a coagulant, the middle figure labeled (ii) shows metering the sample into subsamples and then mixing each subsample with the coagulant for a predetermined period of time, and the rightmost figure labeled (iii) shows observing agglutination visually or by camera on the DMF device.

[0058] Figure 2 is a figure depicting the results of a DMF agglutination assay for blood group typing. A whole blood sample is loaded onto the device, after which it is metered into four sub-droplets. Each sub-droplet is mixed on the DMF device with separate droplets containing anti-A (left), anti-B (second from left), anti-A / anti-B blend (second from right), and anti-D (right) antibodies. After mixing for 2 minutes, the results can be determined by the naked eye. The specific sample here forms agglutinates with anti-A, anti-AB, and anti-D (Rh), indicating type A+.

[0059] Figure 3A schematic diagram depicting the results of a bead-based DMF agglutination assay. A first bacterial lysate sample was loaded into the device and then metered into a pair of daughter droplets, which were mixed with droplets containing latex beads coated with PBP2 antibody (left) or droplets containing latex beads coated with an antibody non-specific to PBP2 (second from left). The first sample formed weak aggregates with the latex beads coated with PBP2 antibody and did not form aggregates with the latex beads coated with an antibody non-specific to PBP2, indicating that the bacteria were susceptible to methicillin. Similarly, a second bacterial lysate sample was loaded into the device and then metered into a pair of daughter droplets, which were mixed with droplets containing latex beads coated with PBP2 antibody (second from right) and droplets containing latex beads coated with an antibody non-specific to PBP2 (right). The second sample formed strong aggregates with the latex beads coated with PBP2 antibody and did not form aggregates with the latex beads coated with an antibody non-specific to PBP2, indicating that the bacteria were methicillin-resistant. In both cases, after loading the sample, the process was automated such that results were obtained after approximately 2 minutes of mixing.

[0060] Figure 4 A schematic diagram showing the workflow of automated image analysis for determining the output of a DMF agglutination assay. A) Images collected from a digital camera showing the steps from initial image capture to isolation of the ROI (region of interest). (i) The image of the device was captured at an angle to reduce reflection. (ii) Perspective correction was performed. (iii) The image portion featuring the center of the device was isolated. (iv) In the isolated image, the region of interest (ROI) was identified for each droplet, and (v) each ROI was masked and stored as a separate image for analysis. B) Images (left) and data (right) showing the analysis of each ROI. The change in pixel intensity of each ROI indicates the degree of agglutination.

[0061] Figure 5 A schematic diagram depicting the results of a DMF agglutination assay for determining hematocrit levels. Four droplets with different hematocrit levels (ratio of red blood cell volume to total blood volume) - 20% (top), 40% (second from top), 60% (second from bottom), 80% (bottom) - are shown on the left. The droplets were mixed with a chemical agglutinating agent, resulting in non-specific agglutination of red blood cells. The higher the hematocrit level, the greater the agglutination point. The hematocrit level can be estimated by the naked eye or determined using a digital camera. The output of the processed image captured using a digital camera is shown on the right. The pixel intensity difference can be used to determine the hematocrit level of the sample.

[0062] Figure 6It is a schema with three figures, which depicts the results of a DMF agglutination assay for donor compatibility testing. A whole blood sample is loaded onto the device, and then it is metered into four sub-droplets, as shown in the leftmost figure. Then each sub-droplet is mixed with a separation droplet containing plasma from the intended blood donor on the DMF device, as shown in the middle figure. After 5 minutes of mixing, the results can be determined by the naked eye. Here, the specific sample forms agglutinates with the first two samples D1 and D2 on the left (in the right-hand figure), which indicates that these donor samples are incompatible with the recipient's blood, and the other two samples D3 and D4 (on the right) show no signs of agglutination, which indicates that these donor samples are compatible with the recipient's blood.

[0063] Figure 7 It is a graph of sorted pixel intensity versus number of pixels that depicts the results of a DMF agglutination assay for determining hematocrit levels. On the left side of the vertical axis, five droplets are shown, which have different, artificially defined hematocrit levels between 20% and 60% - 60% (top), 50% (second from top), 40% (third from top), 30% (second from bottom), 20% (bottom). The droplets are mixed with a chemical agglutinating agent, which results in non-specific agglutination of red blood cells. The higher the hematocrit level, the larger the agglutination point will be. Image analysis is used to determine the hematocrit level. The output of the processed image captured using a digital camera is shown on the right. The integral of the pixel intensity of each sample is used to determine the hematocrit level of the sample.

[0064] Figure 8 It is a graph of hematocrit fraction versus hematocrit (%) that depicts the calibration curve generated from the Figure 7 image shown. The markers are experimental data, and the error bars represent ±1 standard deviation for n = 3 experiments under each condition. The dashed second-order polynomial is fitted to the data, where R 2 = 0.9990.

[0065] Figure 9 It shows a bar graph of hematocrit measurements collected from 12 finger prick whole blood samples from volunteers using the gold standard (left) and the DMF-DMF upper droplet agglutination assessment (DAAD) method (right). For this set of 12 samples, the comparison between the gold standard and the DMF-DAAD method yields p ≥ 0.5045.

[0066] Figure 10 A to Figure 10 D are a series of graphs that depict the performance comparison between agglutination detection algorithms for detecting agglutination in 344 sample images. The known data includes 225 positive samples and 119 negative samples defined by the gold standard method. Negative is a sample that does not show any signs of agglutination, and positive is a sample that shows any signs of agglutination.

[0067] Figure 10 A shows the performance of the histogram method.

[0068] Figure 10 The left - hand graph is a plot of the agglutination fraction of the sample images produced by the histogram method against the gold - standard results. Inside the graph, black and grey represent the number of correct / incorrect evaluations using the threshold T (10 a.u.).

[0069] Figure 10 The right - hand graph is a plot of the receiver operating characteristic (ROC) curve, which shows the true - positive rate of the method against the false - positive rate of the method. The dashed line in the ROC curve represents the result of random guessing (tossing a coin). The area under the curve (AUC) of the method is 0.981.

[0070] Figure 10 B shows the performance of the standard - deviation method.

[0071] Figure 10 The left - hand graph is a plot of the agglutination fraction of the sample images produced by the standard - deviation method against the gold - standard results. Inside the graph, black and grey represent the number of correct / incorrect evaluations using the threshold T (0.13 a.u.).

[0072] Figure 10 The right - hand graph is a plot of the receiver operating characteristic (ROC) curve, which shows the true - positive rate of the method against the false - positive rate of the method. The dashed line in the ROC curve represents the result of random guessing (tossing a coin). The area under the curve (AUC) of the method is 0.9990.

[0073] Figure 10 C shows the performance of the variance method.

[0074] Figure 10 The left - hand graph is a plot of the agglutination fraction of the sample images produced by the variance method against the gold - standard results. Inside the graph, black and grey represent the number of correct / incorrect evaluations using the threshold T (1 a.u.).

[0075] Figure 10 The right - hand graph is a plot of the receiver operating characteristic (ROC) curve, which shows the true - positive rate of the method against the false - positive rate of the method. The dashed line in the ROC curve represents the result of random guessing (tossing a coin). The area under the curve (AUC) of the method is 0.9997.

[0076] Figure 10 D shows the performance of the droplet agglutination assessment on DMF (DAAD) method.

[0077] Figure 10The left - hand side D figure is a graph of the agglutination fraction of the sample image generated by the DAAD method against the gold - standard results. Inside the graph, black and gray represent the number of correct / incorrect evaluations using the threshold T (0.152 a.u.).

[0078] Figure 10 The right - hand side D figure is a graph of the receiver operating characteristic (ROC) curve, which shows the true - positive rate of the method against the false - positive rate of the method. The dashed line in the ROC curve represents the result of random guessing (coin - flipping). The area under the curve (AUC) of the method is 1.000. Detailed Description

[0079] Various embodiments and aspects of the present disclosure will be described with reference to the details discussed below. The following description and drawings are illustrative of the present disclosure and should not be construed as limiting the present disclosure. Numerous specific details are described to provide a thorough understanding of the various embodiments of the present disclosure. However, in some instances, well - known or conventional details are not described in order to provide a concise discussion of the embodiments of the present disclosure.

[0080] As used herein, the terms “comprising” and “including” should be interpreted as inclusive and open - ended, rather than exclusive. Specifically, when used in the specification and claims, the terms “comprising” and “including” and their variants mean including the specified features, steps, or components. These terms should not be construed as excluding the presence of other features, steps, or components.

[0081] As used herein, the term “exemplary” means “serving as an example, instance, or illustration” and should not be construed as being preferred or advantageous over other configurations disclosed herein.

[0082] As used herein, the terms “about” and “approximately”, when used in connection with a particle - size range, mixture composition, or other physical property or characteristic, are intended to cover minor variations that may exist in the upper and lower limits of the size range, so as not to exclude embodiments in which, on average, most sizes are met but statistical sizes may lie outside this region. Embodiments such as these are not intended to be excluded from the present disclosure. Unless otherwise specified, the terms “about” and “approximately” mean plus or minus 25% or less.

[0083] It should be understood that, unless otherwise specified, any specified range or group is a convenient way of referring to each member of the separately - mentioned range or group, as well as every possible sub - range or sub - group contained therein, and similarly for any sub - range or sub - group thereof. Unless otherwise specified, the present disclosure contemplates and expressly includes every specific member and combination of sub - ranges or sub - groups.

[0084] As used herein, the term "approximate", when used in connection with a quantity or parameter, means a range spanning from about one-tenth to ten times the stated quantity or parameter.

[0085] As used herein, "agglutination" refers to the process in which clumps of cells or inert particles are formed due to the interaction between specific antibodies and antigenic components or due to other chemical substances that can induce the same clumping effect.

[0086] Agglutination is defined as the formation of clumps of cells or inert particles by antibodies specific for surface antigenic components (direct agglutination) or for antigenic components adsorbed or chemically conjugated to red blood cells or inert particles (passive hemagglutination and passive agglutination, respectively). 19 Red blood cells are also agglutinated by non-antibody substances such as plant proteins, viruses, heavy metal salts, inorganic colloidal acids and bases, and basic proteins (protamine, histone). Agglutination inhibition or hemagglutination inhibition refers to the inhibition of these reactions by soluble antigens that react with the binding sites of the antibodies and thereby prevent their binding to and agglutination of the particles.

[0087] As used herein, the phrase "agglutination assay" refers to a research procedure that uses the agglutination process to qualitatively evaluate and quantitatively measure the presence, amount, and functional activity of a target entity (analyte).

[0088] Agglutination assays are different from other assays. For example, they differ from coagulation assays such as those disclosed in US2017 / 0056887 (Hadwen et al. 20 ) as follows:

[0089] Agglutination is the process of clumping of particles (solid / semi-solid or cells). There are many examples of agglutination. For example, hemagglutination is the aggregation of red blood cells, and leukagglutination is the aggregation of white blood cells.

[0090] On the other hand, coagulation is the process by which a liquid changes to a solid / semi-solid state. An example of coagulation is the process of blood clotting, in which blood changes from a liquid to a gel, forming a blood clot. Blood clotting is similar to the gelation process. Coagulation has three main steps: i) platelet plug formation, ii) the intrinsic or extrinsic pathway, and iii) the common pathway.

[0091] The main difference between agglutination and coagulation is that: agglutination is the process of aggregation of particles, while coagulation is the process of forming a defined blood clot. Many particles can agglutinate, while only blood can coagulate.

[0092] Agglutination is due to antigen-antibody reactions, while coagulation is due to the activation of multiple plasma factors.

[0093] As used herein, the phrase "agglutinating agent" refers to any substance used in an agglutination assay that causes the formation of particle aggregates (agglutinates).

[0094] As used herein, the phrase "chemical agglutinating agent" refers to a substance used in an agglutination assay that will produce the same effect as particle aggregation, but does not rely on an antigen-antibody reaction, but rather on other processes that will disrupt the particle suspension and force the particles to collapse onto each other and form agglutinates.

[0095] Figure 1 Shown is a dual (2)-plate digital microfluidic device for use in the present disclosure, which is used to produce agglutination in a liquid analyte sample being tested for the presence or absence of a specific analyte. The main steps of using the DMF device are depicted in three (3) figures from the leftmost figure to the rightmost figure. In the DMF device, the steps of performing an agglutination assay on the DMF device begin with: the leftmost figure labeled (i), which shows loading one or more samples containing the analyte of interest into the DMF device containing the agglutinating agent; the middle figure labeled (ii), which shows metering the samples into subsamples and then mixing each subsample with the agglutinating agent for a predetermined period of time; and the rightmost figure labeled (iii), which shows observing agglutination visually or via a camera on the DMF device.

[0096] Thus, broadly speaking, the present disclosure provides a method of characterizing a sample containing an analyte using a dual-plate electrowetting digital microfluidic device (DMF) having a plurality of driving electrodes. The dual-plate configuration of the DMF device allows for droplet dispensing, splitting, and merging. In a single-plate DMF device, higher forces / voltages are required to split and dispense droplets—even higher than the dielectric breakdown of the dielectric. Additionally, the dual-plate DMF device is ideal for imaging droplets and the contents of droplets because most of the droplet presents as a flat area, while in a single-plate DMF device, the curvature of the droplet does not allow for the same ease of imaging. Further, in a single-plate DMF device, the droplet is exposed to a greater area of air, resulting in faster droplet evaporation, which can interfere with the assay.

[0097] The method includes: loading a fluid sample (which may contain a surfactant) containing the analyte being tested and an agglutinating agent (which may contain a surfactant) onto a preselected number of driving electrodes of the DMF device, then using electrowetting to bring the fluid sample into contact with the agglutinating agent to cause agglutination of the analyte to produce agglutinates, and then characterizing the amount of agglutination of the analyte caused by the agglutinating agent.

[0098] In one embodiment, the fluid sample and / or the agglutinating agent may contain a surfactant. The surfactant can be used to reduce non-specific binding of the analyte to the top or bottom plate of the DMF device. The surfactant can also be used to improve the movement of the fluid sample or the agglutinating agent on the DMF device.

[0099] In one embodiment, the surfactant is in a pre-dried form, and when the surfactant is in a pre-dried form, the method further comprises: coating one or more drive electrodes at a predetermined point or across the entire device surface with the surfactant in pre-dried form such that when the fluid sample contacts the pre-dried surfactant, the fluid sample dissolves and the surfactant is present in the fluid in an amount of at least 0.01% by weight. In another embodiment, the aggregating agent is a liquid aggregating agent loaded and metered to a preselected drive electrode.

[0100] In a preferred embodiment, the method comprises the step of actively mixing the aggregating agent with the fluid using electrowetting on a DMF device, which advantageously accelerates the aggregation process in the presence of an analyte being tested for.

[0101] Depending on the analyte being tested for, the surfactant can be an ionic surfactant or a non-ionic surfactant. Ionic surfactants include, but are not limited to, sodium dodecyl sulfate, sodium stearate, cetrimonium bromide, cetrimonium chloride, and sodium lauryl sulfate. Non-ionic surfactants include, but are not limited to, alkylphenol ethoxylates (e.g., Triton X RTM), polysorbates (e.g., Tween RTM), poloxamines (e.g., Tetronic RTM), poloxamers (e.g., Pluronic RTM), and sorbitan esters. The choice of ionic or non-ionic surfactant to be used is based on the type of aggregation assay to be performed and the type of sample being analyzed. The surfactant to be used for a specific assay and sample type should be screened to determine surfactant compatibility.

[0102] For example, when testing blood to determine blood type, a non-ionic surfactant is preferred, as will be discussed in the following examples. Non-ionic surfactants are used with blood to maintain an isotonic environment for the cells, thus preventing cell lysis.

[0103] The aggregating agent can include any one or combination of substances capable of producing aggregates. Examples of substances include chemical aggregating agents and biological aggregating agents. For the aggregation of red blood cells, chemical aggregating agents can be selected from substances such as: polymeric cations including, but not limited to: poly-L-lysine hydrobromide, poly(dimethyldiallylammonium) chloride (e.g., RTM, RTM, RTM), poly-L-arginine hydrochloride, poly-L-histidine, poly(4-vinylpyridine), poly(4-vinylpyridine) hydrochloride, crosslinked poly(4-vinylpyridine), methyl chloride quaternary salt, poly(4-vinylpyridine-co-styrene); poly(4-vinylpyridine poly(hydrogen fluoride)); poly(4-vinylpyridine-p-toluenesulfonate); poly(4-vinylpyridine-tribromide); poly(4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); crosslinked polyvinylpyrrolidone; polyvinylpyrrolidone, poly(melamine-co-formaldehyde); partially methylated; hexamethonium bromide; poly(glutamic acid, lysine) 1:4 hydrobromide; poly(lysine, alanine) 3:1 hydrobromide; poly(lysine, alanine) 2:1 hydrobromide; succinylated poly-L-lysine; poly(lysine, alanine) 1:1 hydrobromide; and poly(lysine, tryptophan) 1:4 hydrobromide. The most preferred polymeric cation is poly(dimethyldiallylammonium) chloride.

[0104] Chemical agglutinants are used to cause the agglutination of any red blood cells, so they can be used as positive controls for blood agglutination assays and can also be used to agglutinate red blood cells to determine the hematocrit level, as will be discussed in the following examples.

[0105] Biological agglutinants are any biologically active substances capable of producing aggregates. For the agglutination of red blood cells, examples include proteins such as lectins (proteins capable of reversibly binding to carbohydrate structures) and antibodies (e.g., anti-A, anti-B, anti-D), viruses (e.g., influenza virus), antigens, DNA, RNA, and DNA- or RNA-based aptamers. Biological agglutinants are used to determine the presence or absence of a specific analyte of interest in red blood cells or a sample. For example, the antibody anti-A is used to detect the presence or absence of antigen A on the surface of red blood cells. As another example, the influenza virus is used to determine the amount of antibodies against the virus present in plasma and to determine the immune level of a patient sample.

[0106] The step of characterizing the amount of agglutination of the analyte caused by the agglutinant is preferably by visual / optical characterization. Other reported characterization methods include using electrochemistry (e.g., impedance spectroscopy), absorbance, and turbidity techniques. However, the implementation of these techniques requires additional hardware equipment and several modifications to the DMF device, making the visual characterization by visual inspection of the results of the agglutination reaction by the operator or the present process using a camera quite advantageous as no additional modifications to the system are required.

[0107] Visual characterization can be performed by visually observing the DMF device by a person to estimate the amount of agglutination. Alternatively, the steps of visual characterization are performed using a camera. Non-limiting examples of cameras that can be used include webcams, mobile phone cameras, digital cameras (including digital single-lens reflex cameras DSLR), video cameras, surveillance cameras, point-and-shoot cameras, cameras with CCD detectors, cameras with CMOS detectors, monochrome cameras, black and white cameras, and color cameras. For performing visual / optical characterization involving a camera, Droplet Agglutination Assessment on DMF (DAAD) has been developed. DAAD is an image analysis algorithm for automatically detecting agglutinates in droplets on a DMF device. The DAAD algorithm can be stored in a microprocessor associated with the camera, or they can be stored on a microprocessor of a DMF power supply that is connected to drive electrodes controlling the DMF device, or can be stored on a remote computer. The DAAD algorithm can be executed by a microprocessor or a computer.

[0108] In one embodiment, the agglutinant includes particles coated with an agglutinant. Non-limiting examples include polymers (e.g., latex), gold, silver, nanoparticles, and microparticles. Depending on the analyte of interest, the particles are coated with an agglutinant. For example, for detecting an antigen, the particles are coated with an antibody or other agent capable of capturing the antigen of interest. In the case of detecting an antibody, the particles should be coated with an antigen or other agent capable of capturing the antibody of interest.

[0109] The method can be used for the agglutination of suspensions of polymer particles. Non-limiting examples include latex particles coated for rubella antibody detection, 21 latex particles coated with an antibody for detecting any virus, 22 latex particles coated with streptolysin O, 23 latex particles coated with an antibody for C-reactive protein detection, 24 latex particles coated for identifying Staphylococcus aureus, 25 and latex particles coated for identifying any type of bacteria.

[0110] The methods disclosed herein can be used for the agglutination of nanoparticle suspensions. Non-limiting examples include nanoparticles coated with an antibody for detecting an antigen and nanoparticles coated with an antigen for detecting a specific antibody.

[0111] The method can be used for the agglutination of suspensions of red blood cells to determine a patient's blood type, as will be discussed in the following examples. In this application, the fluid is a blood sample containing at least only red blood cells. These blood cells are mixed with a liquid diluent such as, but not limited to, plasma, isotonic buffer solutions (e.g., phosphate buffered saline (PBS)), solutions containing PBS and serum albumin (e.g., human serum albumin, bovine serum albumin).

[0112] However, it will be appreciated that this method can be used to characterize other types of blood samples, including whole blood (white blood cells and red blood cells, platelets, and plasma), and can also include diluted blood (taking a portion of whole blood and mixing it with something else, for some examples), suspensions of white blood cells, serum, and plasma.

[0113] The method can be used for the agglutination of suspensions of red blood cells to determine the hematocrit level, as will be discussed in the following examples. In this application, the fluid is a blood sample containing at least red blood cells. These blood cells can be mixed with a liquid diluent. In this example, any agglutinating agent can be used to determine the hematocrit level.

[0114] This method can be used to characterize other types of fluid samples, including virus suspensions in liquid diluents, such as but not limited to whole blood, serum, plasma, isotonic buffer solutions (e.g., PBS), solutions containing PBS and serum albumin (e.g., human serum albumin, bovine serum albumin), nasal mucus, nasopharyngeal mucus, urine, and saliva. These fluid samples can be mixed with an agglutinating agent for virus detection.

[0115] This method can be used to characterize other types of fluid samples, including suspensions of any other type of eukaryotic cells. The agglutinating agent can be any substance capable of causing cell agglutination.

[0116] Examples

[0117] Non-limiting and exemplary embodiments of the methods disclosed herein will now be discussed, but it will be appreciated that the present disclosure is not limited to these embodiments.

[0118] Example 1

[0119] Figure 2 The first example shown is a blood typing assay that uses blood agglutination antibodies to cause red blood cell agglutination. A set of 3 antibodies (monoclonal or polyclonal) - specific for antigens A (anti-A), B (anti-B), and RhD (anti-D) and a blend of A and B (anti-A,B) - are used to determine the ABO and rhesus (Rh) blood types. In Figure 2 the example shown, the agglutination reagent is loaded into the device in solution form; a similar method is also shown, where the agglutination reagent is pre-loaded onto the device as a dry spot that dissolves when exposed to the sample or another reagent (e.g., a dissolution buffer). The complete assay takes only a few minutes and is fully automated.

[0120] In the example using pre-dried reagents, as described by Foley et al. 18 reconstitution is performed as described in US2014 / 0141409A1.

[0121] The determination relies on a phenomenon called hemagglutination. According to the U.S. National Library of Medicine, hemagglutination (or haemagglutination) is defined as "the aggregation of red blood cells by lectins including antibodies, lectins, and viral proteins". 26 Traditionally, hemagglutination assays have been used to detect variations or polymorphisms of surface markers (antigens) found on the red blood cell membrane to classify blood into categories (blood types). There are currently 339 certified blood group antigens, 297 of which belong to one of 33 blood group systems. Among these blood group systems, ABO and Rh are the most well-known systems because of their importance in transfusion medicine.

[0122] The ABO system specifically is unique because it is the only blood group system in which the antigens are not present on the surface of red blood cells; instead, the reciprocal antibodies are consistently and predictably found as soluble entities in the plasma. ABO antigens are often called tissue blood group antigens because their widespread distribution means they are also commonly used as histocompatibility antigens. Most importantly, the widespread distribution of anti-A and anti-B makes transfusion of incompatible blood types catastrophic because hemolytic transfusion reactions (HTRs) can cause hyperacute rejection of incompatible kidney, liver, and heart grafts. Similarly, Rh antigens are commonly used to prevent hemolytic disease of the fetus and newborn (HDFN). 27,28

[0123] The determination described in this example is performed on a blood sample; the related test can be carried out in serum, which is commonly called reverse typing. In reverse typing, the patient's serum is mixed with red blood cells having known surface antigens (e.g., A cells and B cells for ABO), and the observation of hemagglutination indicates the presence or absence of the corresponding antibodies. 29 In either format (forward or "reverse"), there is a strong motivation to develop novel, rapid, and easy-to-perform blood typing assays, as evidenced by the global blood typing market, which is expected to reach $2.5 billion by 2022. 30

[0124] Example 2

[0125] Another application of the new platform is the use of the system for blood donor-recipient cross-matching, a critical operation that must be performed rapidly on-site in high-risk environments (where time is of the essence) such as the emergency room or trauma care laboratory. Specifically, the first step in plasma donation in such an environment is to determine the recipient's type according to the ABO / Rh system in order to be able to identify the donor's type (e.g., a B+ donor for a B+ recipient). However, this level of selectivity is insufficient because there are many other subtypes that are not captured by the ABO / Rh system and that can cause incompatibility, such that a second step is typically performed (usually at the patient's bedside, just prior to transfusion), in which plasma from potential donors is directly tested for agglutination with the patient's blood. Figure 6 An example of a simulated cross-matching test performed by DMF hemagglutination and analyzed by DAAD is shown. In this example, two out of four potential donors were found to be compatible with the potential recipient.

[0126] Example 3

[0127] Agglutination of red blood cells can be used to determine the hematocrit of a blood sample. Figure 5 is a schema showing the results of a DMF agglutination assay for determining hematocrit levels. On the Figure 5 left side are shown droplets that have different hematocrit levels (ratio of red blood cell volume to total blood volume) - 20% (top), 40% (second from top), 60% (second from bottom), 80% (bottom). The droplets are mixed with a chemical agglutinating agent, which results in non-specific agglutination of red blood cells. The higher the hematocrit level, the larger the agglutination spot will be. The hematocrit level can be estimated by the naked eye or determined using a digital camera. On the right side is shown the output of an image captured using a digital camera after processing. Pixel intensity differences can be used to determine the hematocrit level of the sample.

[0128] Specifically for hematocrit determination, the integral of pixel intensity found between (inclusive) a small fraction of the total number of pixels is defined as the 'hematocrit fraction'. Figure 7 In an initial experiment, the hematocrit fractions of a training set of diluted blood samples with artificially defined hematocrit levels between 20% and 60% were found and plotted as a function of the hematocrit level and fit to a second-order polynomial: y = -0.0249x2 + 1.092x + 107.3. The hematocrit fraction of each droplet was compared to the calibration curve to determine the predicted % hematocrit ( Figure 8 ).

[0129] Figure 7 shows the results of a DMF agglutination assay for determining hematocrit levels. On the Figure 7Five droplets are shown on the left side thereof, which have different hematocrit levels (ratio of red blood cell volume to total blood volume) – 60% (top), 50% (second from top), 40% (third from top), 30% (second from bottom), 20% (bottom). Similar to Figure 5 , the droplets are mixed with a chemical aggregating agent, which results in non-specific aggregation of red blood cells. The higher the hematocrit level, the larger the aggregation points will be.

[0130] Figure 8 shows a calibration curve of hematocrit fraction as a function of known hematocrit levels. The markers are experimental data, and the error bars represent ±1 standard deviation for n = 3 experiments under each condition. The dashed second-order polynomial is fitted to the data, where R 2 = 0.9990.

[0131] Figure 9 shows a bar graph of hematocrit measurements collected from 12 finger prick whole blood samples from volunteers using the gold standard (left) and the DMF-DAAD method (right). For this set of 12 samples, the comparison between the gold standard and the DMF-DAAD method yields p ≥ 0.5045.

[0132] Example 4

[0133] Figure 3 The fourth example of the present disclosure shown is a latex immunoagglutination assay (LIA), which uses a suspension of latex particles to detect an analyte of interest. In the absence of the analyte, the beads are suspended as individual units and the suspension looks "smooth" (i.e., no heterogeneous clumps), while in the presence of the analyte, the particles aggregate, forming visible heterogeneous aggregates to the naked eye. These assays have wide utility, such as detecting monovalent and multivalent antigens, proteins, drugs, steroid hormones, and even microorganisms. 31 LIA is commonly used by clinicians for influenza detection, 32 and antibiotic susceptibility testing. 33 For the latter, there is great interest in being able to distinguish between strains of bacteria that are resistant or susceptible to antibiotics to determine which therapy to prescribe. For example, the main cause of infection in hospitals is methicillin-resistant Staphylococcus aureus 34 (MRSA); obviously, prescribing methicillin to a patient infected with MRSA wastes time and resources. As a proof of principle, a latex bead-based agglutination assay on DMF was developed for detecting methicillin resistance and susceptibility in strains of bacteria, as Figure 3 featured.

[0134] In this example, a susceptible strain of bacteria (first sample - a pair of droplets on the left) and a resistant strain (second sample - a pair of droplets on the right) are mixed with latex beads coated with penicillin-binding protein 2 (PBP2) antibody (monoclonal or polyclonal) (left droplet in each pair). Each sample is also mixed with latex beads coated with an antibody non-specific to PBP2 as a negative control (right droplet in each pair). The results indicate that the first sample shows weak agglutination, indicating susceptibility (suggesting that patients infected with these bacteria may be treatable with methicillin), and the second sample shows strong agglutination (indicating that patients infected with these bacteria should receive alternative treatment). In summary, the DMF-based assay allows for the rapid detection of three states that can be easily identified by the naked eye: no agglutination (for the negative control), weak agglutination for antibiotic-susceptible bacteria, and strong agglutination for antibiotic-resistant bacteria).

[0135] Visual determination of agglutination results

[0136] The simplest detection mode of the agglutination assay is for the user to observe with the naked eye; this method is applicable to the examples already put into practice above. However, agglutination is also suitable for automation through image processing. There have been several reports of automated detection of hemagglutination in fluid channels, but they rely on auxiliary equipment (e.g., microscopes, 5 waveguides, 35 etc.) and complex post-processing procedures. For example, Huet et al. 5 trained an artificial network in MATLAB to detect the progress of agglutination, but the algorithm is not general, and each new imaging setup requires a new training set.

[0137] In contrast, the method depicted in Figure 4 A and Figure 4 B is straightforward and applicable to any system with a digital camera. To perform the optical characterization involving the camera, the droplet agglutination assessment on DMF (DAAD) is carried out in 8 steps. For blood typing ( Figure 2 ), latex agglutination assay ( Figure 3 ), hematocrit analysis ( Figure 5 ), and donor compatibility testing ( Figure 6 ), the first six image preprocessing steps (i-vi) are the same.

[0138] Figure 4 A-i shows step i), where a camera is used to collect an image of the device. The camera is positioned at an angle relative to the plane perpendicular to the DMF device. Images are typically captured at the maximum resolution of each camera (but lower resolution images can also be processed).

[0139] Figure 4A-ii shows step ii, where the image is perspective-corrected by defining four coordinates in the source image and four reference coordinates. A 3×3 matrix is calculated based on each set of coordinates (image-reference correspondence pair), and then the same matrix is applied to the source image to obtain the perspective-corrected image.

[0140] Figure 4 A-iii shows step iii), where the center of the DMF device is automatically located by detecting known device features, and this region of the image is isolated for further processing.

[0141] Figure 4 A-iv shows step iv, where droplets are detected by identifying contours and combining adjacent contours to form a rectangular region of interest (ROI) for each droplet to define a mask for extracting the image.

[0142] Figure 4 A-v shows step v, where the ROI images corresponding to each droplet are masked, isolated, and converted from RGB to grayscale.

[0143] Figure 4 B - Left shows step vi, where each isolated image is flattened into a one-dimensional array and normalized so that the pixel intensities cover the entire 8-bit range [0 - 255], and then sorted from lowest to highest pixel value.

[0144] Figure 4 B - Right shows step vii, where then the slope of the pixel intensities in this gradient is used as an indication of the degree of agglutination (a process used in blood typing, donor compatibility testing, step viii). For example, in Figure 4 B, a steep slope of the pixel intensities for A, D(Rh), and A,B blends indicates agglutination, while a flat slope for B indicates no agglutination. A similar image processing method was developed to automate the detection of latex bead agglutination ( Figure 3 ), highlighting the flexibility of this method compared to previously reported ones. 5

[0145] Within the visual characterization, other analytical methods can be performed. Three alternative agglutination detection algorithms were tested and compared with DAAD: the histogram method, the standard deviation method, and the variance method. In the histogram method, DAAD sub-steps (i)–(v) were performed to isolate each ROI image. For each image, a histogram was generated based on the number of pixels for each pixel intensity value. The histogram was smoothed with a moving average filter (window = 10 bins), and in the smoothed dataset, the main peak was identified by finding the local maximum by comparing pixel intensities with adjacent values.

[0146] The average pixel intensity of the main peak in the smoothed histogram was defined as the threshold T. Finally, the agglutination score was defined as 100 × (S>T / S), where S is the number of pixels in the ROI image and S>T is the number of pixels with an intensity greater than T. The standard deviation method (adapted from a previous report) was used to calculate the agglutination score. 36 ), DAAD substeps (i)–(vi) were performed, after which the array was (again) normalized to the range [0,1] and the standard deviation σ of the pixel intensities was defined as the agglutination score. 5,6,37 ), perform DAAD sub-steps (i)–(v) to isolate each ROI image. Use a 3×3 matrix to calculate the relative position of each pixel to its neighboring pixels The local variance of the image The average variance of all pixels in .

[0147] The agglutination fraction is defined as A series of 86 samples (344 ROIs) were evaluated by DAAD and the three alternative methods. The "optimal" agglutination thresholds (for highest true positive rate and lowest false positive rate) for the alternative methods were found to be 10 a.u., 0.13 a.u., and 1 a.u. for the histogram method, the standard deviation method, and the variance method, respectively. Figure 10 ).

[0148] In summary, the inventors report the first dual-plate digital microfluidic system and method capable of performing agglutination assays. The inventors have demonstrated four non-limiting and exemplary embodiments of the present invention in the above four embodiments. The first embodiment is a blood typing hemagglutination assay - the first embodiment known to be implemented on a dual-plate DMF device. This method has been shown to be compatible with the use of solution-phase or dried agglutination antibodies, which can be mixed with undiluted whole blood and the results can be determined by the naked eye within minutes. As an extension of the blood typing assay, when the blood sample is mixed with the expected donor sample, a donor compatibility test can be performed to indicate the correct donor for the recipient patient (second embodiment). In addition, the above assay is demonstrated using hemagglutination of red blood cells, which uses chemical reagents to determine the hematocrit of the sample (third embodiment). In the fourth embodiment, a DMF method for performing a latex immunoagglutination assay (LIA) is implemented. In this example, the test is demonstrated for antibiotic susceptibility; but it is expected that any LIA should be compatible. Finally, an imaging-based readout is reported using a custom but generalizable algorithm for interpreting the results of the DMF agglutination assay. When considered together, a user can load a sample, push a button, and receive results within minutes.

[0149] Table 1 below summarizes some of the significant differences between the new method reported here and two previous DMF agglutination methods reported in the literature.

[0150]

[0151] Table 1: Comparison between other agglutination methods using a digital microfluidic platform and the method disclosed herein.

[0152] The inventors are aware of literature reports showing agglutination assays using digital microfluidics, but they are all irrelevant to the present invention. 7,13 None of the previously reported methods used a dual-plate electrowetting device to perform an agglutination assay. In contrast, different types of assays have been demonstrated on a dual-plate DMF device: coagulation assays 20 and plasma separation using lectins 38 , but none of the above are relevant to the present invention. Additionally, the detection of agglutination is performed by the naked eye or using DAAD (our unique detection algorithm that detects agglutination in images captured using a digital camera). This method does rely on using an absorbance module to determine agglutination, as previously reported 39 , and the algorithm does not use any previously reported method to detect agglutination because these methods rely on expensive imaging equipment (microscope setups or high-end DSLR cameras) 7,13 and are highly dependent on imaging conditions (brightness, contrast, white balance, etc.). The performance of some other previously reported methods was compared with DAAD, and it has been shown herein that the present DAAD outperforms all of these methods.

[0153] Broadly speaking, on the one hand, the present disclosure provides a method for characterizing a sample containing an analyte using a dual-plate electrowetting digital microfluidic device (DMF) having a plurality of driving electrodes. The method includes the steps of loading a fluid sample containing an analyte and a surfactant and an agglutinating agent onto the DMF device; and using electrowetting to bring the fluid sample into contact with the agglutinating agent to cause agglutination of the analyte with the agglutinating agent.

[0154] On the other hand, the present disclosure provides a dual-plate electrowetting DMF device, which includes: a first plate, a second plate spaced apart from the first plate, one of the first plate and the second plate having a plurality of driving electrodes; and a surface on the first plate or the second plate, another surface on the first plate or the second plate, the surface having a surfactant in a pre-dried form coated on the surface at a preselected position, the another surface having an agglutinating agent in a pre-dried form coated on the another surface at a preselected position.

[0155] The surface coated with the surfactant and the surface coated with the agglutinating agent are different or the same.

[0156] The present disclosure also provides a kit, which includes: a dual-plate electrowetting digital microfluidic device (DMF), the DMF having a plurality of driving electrodes; a surfactant for placement on one of the two plates; and an agglutinating agent for placement on one of the two plates.

[0157] The above specific embodiments have been shown by way of example, and it should be understood that these embodiments may readily have various modifications and alternative forms. It should also be understood that the claims are not intended to be limited to the specific forms disclosed, but are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

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Claims

1. A method of characterizing a sample using an agglutination assay to determine the presence or absence of a preselected analyte, wherein The following steps are involved: Providing a double-plate electrowetting digital microfluidic device DMF having a plurality of driving electrodes; loading a fluid sample containing the analyte and an agglutinating agent capable of causing agglutination onto a separation driving electrode of the DMF device; contacting a droplet of the fluid sample with the agglutinating agent using electrowetting to agglutinate any of the analytes present in the sample with the agglutinating agent to produce an agglutinate; Using electrowetting to bring droplets including the condensate to a plurality of corresponding locations on the DMF device; and Any agglutinates formed due to the presence of the preselected analyte in the fluid sample are visually characterized by: collecting images of the DMF device using a camera, including a plurality of locations on the DMF device; identifying, for each droplet, a corresponding region of interest, the region of interest comprising a corresponding position among the plurality of positions; converting the region of interest into a normalized and sorted array of pixel intensities for each droplet; as well as The degree of agglutination of each region of interest is determined based on the slope of the gradient of the sorted pixel intensity array. 2 . The method of claim 1 , wherein the step of visually characterizing the agglutinate is performed by a user observing the agglutinate or by using a camera.

3. The method of claim 2, wherein when using a camera to visually characterize any agglutinates formed due to the presence of the preselected analyte in the fluid sample, the method include: Image analysis of the fluid sample is used to determine the amount of agglutination of the analyte caused by the agglutinating agent.

4. The method of claim 1, further comprising a surfactant mixed in the fluid sample containing the analyte, or the agglutinating agent, or both.

5. The method according to claim 1, further comprising a surfactant in a pre-dried form, the method further comprising: include: One or more drive electrodes are coated with the pre-dried form of the surfactant in predetermined spots or across the entire array of drive electrodes such that when the fluid sample comes into contact with the pre-dried surfactant, the fluid sample dissolves.

6. The method according to claim 4 or 5, wherein the surfactant is one of an ionic surfactant and a nonionic surfactant.

7. The method according to claim 6, wherein the ionic surfactant is selected from the group consisting of sodium lauryl sulfate, sodium stearate, cetrimonium bromide, cetrimonium chloride and sodium lauryl sulfate.

8. The method of claim 6, wherein the nonionic surfactant is selected from the group consisting of alkylphenol hydroxypolyethylene, polysorbate, poloxamine, poloxamer and sorbitan ester.

9. The method of any one of claims 1 to 5, wherein the agglutinating agent is a liquid agglutinating agent that is loaded and metered to a preselected drive electrode.

10. The method according to any one of claims 1 to 5, further comprising: The following steps are involved: The agglutinating agent was actively mixed with the fluid sample using electrowetting on the DMF device.

11. The method according to any one of claims 1 to 5, wherein the aggregating agent comprises one or more chemical aggregating agents and biological aggregating agents.

12. The method according to claim 11, wherein the chemical aggregating agent is selected from the group consisting of: poly-L-lysine hydrobromide, poly(dimethyldiallylammonium) chloride, poly-L-arginine hydrochloride, poly-L-histidine, poly(4-vinylpyridine), poly(4-vinylpyridine) hydrochloride, crosslinked poly(4-vinylpyridine), methyl chloride quaternary salt, poly(4-vinylpyridine-co-styrene); poly(4-vinylpyridine poly(hydrogen fluoride)); poly(4-vinylpyridine-p-toluenesulfonate); poly(4-vinylpyridine-tribromide); poly(4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); crosslinked polyvinylpyrrolidone; polyvinylpyrrolidone, poly(melamine-co-formaldehyde); partially methylated; hexamethonium bromide; poly(glutamic acid, lysine) 1:4 hydrobromide; poly(lysine, alanine) 3:1 hydrobromide; poly(lysine, alanine) 2:1 hydrobromide; succinylated poly-L-lysine; poly(lysine, alanine) 1:1 hydrobromide; poly(lysine, tryptophan) 1:4 hydrobromide; and poly(dimethyldiallylammonium) chloride.

13. The method according to claim 11, wherein the biological aggregating agent is selected from the group consisting of proteins, antibodies, viruses and antigens, DNA, RNA, and DNA- or RNA-based aptamers.

14. The method according to claim 13, wherein the protein comprises a lectin capable of reversibly binding to a carbohydrate structure.

15. The method according to claim 13, wherein the antibody comprises anti-A, anti-B, and anti-D.

16. The method according to claim 13, wherein the virus comprises an influenza virus.

17. The method according to any one of claims 1 to 5, wherein the aggregating agent comprises particles coated with the aggregating agent.

18. The method according to claim 17, wherein the particles comprise any one or a combination of polymer particles, gold, silver, nanoparticles, and microparticles.

19. The method according to claim 18, wherein the polymer particles are latex particles.

20. The method according to claim 17, wherein the analyte to be detected is an antibody, and wherein the particles are coated with an antigen or other agent capable of capturing the antibody.

21. The method according to any one of claims 1 to 5, which is used for the aggregation of a suspension of polymer particles.

22. The method according to any one of claims 1 to 5, which is used for the aggregation of a suspension of red blood cells.

23. The method according to any one of claims 1 to 5, wherein the fluid is blood containing at least red blood cells.

24. The method according to claim 23, wherein the aggregating agent is a chemical aggregating agent for aggregating red blood cells for determining the hematocrit level.

25. The method according to claim 3, wherein the step of using image analysis of the agglomerates to determine the amount of agglutination of the analyte caused by the aggregating agent is performed using an image analysis algorithm, the image analysis algorithm being programmed to determine the amount of agglutination in the agglutination product using all or part of a droplet agglutination algorithm.

26. The method according to claim 3, wherein the algorithm is stored in a microprocessor associated with the camera, or in a microprocessor of a DMF power supply connected to the drive electrodes controlling the DMF device, or the algorithm is stored on a remote computer and is programmed to be executed by the microprocessor or the computer.

27. A dual-plate electrowetting DMF device for use in the method according to any one of claims 1-26, the dual-plate electrowetting DMF device comprising: a first plate and a second plate spaced apart from the first plate, one of the first plate and the second plate having a plurality of drive electrodes; a surface on the first plate or the second plate, another surface on the first plate or the second plate, the surface having a surfactant in a pre-dried form coated on the surface at a preselected position, the another surface having an aggregating agent in a pre-dried form coated on the another surface at a preselected position; and a microprocessor that executes instructions to implement the method according to any one of claims 1 to 26.

28. The DMF device according to claim 27, the microprocessor being connected to a power supply and the plurality of drive electrodes and programmed with instructions for powering the drive electrodes in a preselected pattern to move droplets of a fluid sample and an aggregating agent over the electrodes to study the presence of a pre-analyte located therein.

29. The DMF device according to claim 27 or 28, wherein the surface coated with the surfactant and the surface coated with the aggregating agent are different or the same.

30. The DMF device according to claim 27 or 28, which comprises: a camera positioned such that its field of view encompasses the DMF device, and wherein the image is analyzed to determine the amount of agglutination of the analyte caused by the aggregating agent using image analysis of the fluid sample.

31. A kit for use in the method according to any one of claims 1-26, the kit comprising: a dual-plate electrowetting digital microfluidic device DMF having a plurality of drive electrodes; a microprocessor connected to a power supply and the plurality of drive electrodes and programmed with instructions for powering the drive electrodes in a preselected pattern to move droplets of the fluid sample and the aggregating agent; and a surfactant for placement on one of the dual plates; and an aggregating agent for placement on one of the dual plates; a camera positioned such that its field of view encompasses the DMF device; and An image analysis algorithm, the image analysis algorithm being used to visually characterize any aggregates formed due to the presence of the preselected analyte in the fluid sample, including using image analysis of the fluid sample to determine the amount of aggregation of the analyte caused by the aggregating agent.

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