阅读说明：本技术 数字微流体凝集测定 (Digital microfluidic agglutination assay ) 是由 A·斯卡拉沃诺斯 J·L·拉曼纳 A·R·惠勒 于 2020-05-04 设计创作，主要内容包括：本公开提供一种用于在“双板”DMF装置格式上执行凝集测定的方法。将含有感兴趣分析物(颗粒、细胞等)的液滴加载到所述DMF装置中,并且使其与溶液相或干燥的凝集抗体或抗原混合。所述凝集剂结合到它们在所述样品液滴中的互补靶标(例如,抗体或抗原),从而导致形成不溶性聚集物。DMF装置上的主动混合减少了反应时间并且增强了凝集效果。由于凝集的样品夹在所述DMF装置上的两个板之间,因此可直截了当地通过肉眼或经由数码相机查看结果。(The present disclosure provides a method for performing agglutination assays on a "two-plate" DMF device format. The droplets containing the analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with solution phase or dry agglutinated antibody or antigen. The agglutinating agent binds to their complementary target (e.g., antibody or antigen) in the sample droplet, resulting in the formation of insoluble aggregates. Active mixing on a DMF device reduces reaction time and enhances agglutination effect. The results can be viewed directly by eye or via a digital camera, since the agglutinated sample is sandwiched between two plates on the DMF device.)

1. A method of characterizing a sample using an agglutination assay to determine the presence or absence of a preselected analyte, comprising the steps of:

providing a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive 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 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; and

visually characterizing any agglutinates formed as a result of the presence of the preselected analyte in the fluid sample.

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 as a result of the presence of the preselected analyte in the fluid sample, the method comprises: determining an amount of agglutination of the analyte by the agglutinating agent using image analysis of the fluid sample.

4. The method of claim 1, 2, or 3, further comprising a surfactant mixed in the fluid sample containing the analyte, or the agglutinating agent, or both.

5. The method of any one of claims 1 to 3, further comprising a surfactant in a pre-dried form, the method further comprising: coating one or more drive electrodes in a predetermined spot or across the entire array of drive electrodes with the surfactant in the pre-dried form such that when the fluid sample is contacted with the pre-dried surfactant, the fluid sample dissolves.

6. The method of claim 4 or 5, wherein the surfactant is one of an ionic surfactant and a non-ionic surfactant.

7. The method of claim 6, wherein the ionic surfactant is selected from the group consisting of sodium lauryl sulfate, sodium stearate, cetrimide and sodium lauryl sulfate.

8. The method of claim 6 or 7, wherein the non-ionic surfactant is selected from the group consisting of alkylphenol hydroxypolyethylene, polysorbate, poloxamine, poloxamer and sorbitan esters.

9. The method of any one of claims 1 to 8, wherein the agglutinating agent is a liquid agglutinating agent loaded and metered to a preselected drive electrode.

10. The method of any one of claims 1 to 9, further comprising the steps of: actively mixing the agglutinating agent with the fluid sample using electrowetting on the DMF device.

11. The method of any one of claims 1 to 10, wherein the agglutinating agent comprises one or more of a chemical agglutinating agent and a biological agglutinating agent.

12. The method of claim 11, wherein the chemical agglutinating 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) hydrochloride, cross-linked 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); cross-linked polyvinylpyrrolidone; polyvinylpyrrolidone, poly (melamine-co-formaldehyde); partial methylation; brominating hexylenediamine; 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 of claim 11, wherein the biological agglutinating agent is selected from the group consisting of proteins, antibodies, viruses and antigens, DNA, RNA, and DNA or RNA-based aptamers.

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

15. The method of claim 13, wherein the antibody comprises anti-a, anti-B, and anti-D.

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

17. The method of any one of claims 1 to 16, wherein the agglutinating agent comprises a particle coated with the agglutinating agent.

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

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

20. The method of claim 17, wherein the analyte for which detection is made is an antibody, and wherein the particles are coated with an antigen or other agent capable of capturing the antibody of interest.

21. The method according to any one of claims 1 to 20, for the agglutination of a suspension of polymer particles.

22. A method according to any one of claims 1 to 20 for agglutination of a suspension of red blood cells.

23. The method according to any one of claims 1 to 20, wherein said fluid is blood comprising at least red blood cells.

24. The method of claim 23, wherein the agglutinating agent is a chemical agglutinating agent for agglutinating red blood cells for determining hematocrit levels.

25. A two-plate electrowetting DMF device, comprising:

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 drive electrodes; and

a surface on the first or second plate having a surfactant in pre-dried form coating the surface in preselected locations, another surface on the first or second plate having an agglutinating agent in pre-dried form coating the other surface in preselected locations.

26. A DMF device according to claim 25, further comprising a microprocessor connected to a power source and the plurality of actuation electrodes and programmed with instructions for powering the actuation electrodes in a preselected pattern for moving over the electrodes droplets of a fluid sample being investigated for the presence of a pre-analyte located therein and an agglutinating agent.

27. The DMF device of claim 25 or 26, wherein the surface coated by the surfactant and the surface coated with the agglutinating agent are different or the same.

28. A DMF apparatus according to claim 25, 26 or 27, comprising: a camera positioned such that its field of view encompasses the DMF device, and wherein the image is analyzed for determining an amount of agglutination of the analyte caused by the agglutinating agent using image analysis of the fluid sample.

29. The method of claim 3, wherein the step of determining the amount of agglutination of the analyte by the agglutinating agent using image analysis of the agglutinating agent is performed using an image analysis algorithm programmed to determine the amount of agglutination in the agglutinated product using all or part of the droplet agglutination algorithm.

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

31. A kit, comprising:

a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes;

a microprocessor connected to a power source and the plurality of drive electrodes and programmed with instructions for powering the drive electrodes in a pre-selected pattern for moving the droplets of the fluid sample and the agglutinating agent over the electrodes; and

a surfactant for placement on one of the two plates; and

an agglutinating agent for placement on one of the two plates;

a camera positioned such that its field of view encompasses the DMF device; and

an image analysis algorithm for visually characterizing any agglutinates formed as a result of the presence of the preselected analyte in the fluid sample, comprising using image analysis of the fluid sample to determine an amount of agglutination of the analyte by the agglutinating agent.

Technical Field

The present disclosure relates to a method for performing agglutination assays using a "two-plate" Digital Microfluidic (DMF) device format. The droplets containing the analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with the liquid phase or dry agglutinated antibody or antigen.

Background

Agglutination assays are commonly used to detect the presence of an analyte in a sample; typical applications include infectious disease and pathogen detection, and 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 lumps or aggregates that are visible to the naked eye. Thus, agglutination assays have unique advantages over other techniques for such assays (which rely on instrumental measurement of photon or electrical energy) -the reading of assay results is very straightforward.

In conventional agglutination assays, antibody or antigen coated particles are combined with a sample, manually mixed, and the presence of aggregates is determined by visual inspection. Significant drawbacks of standard techniques include the need for manual mixing, the potential for errors in interpreting assay readings, and low throughput. Agglutination assays in microfluidic devices have recently been developed in an effort to address these shortcomings. For example, Castro et al¹A microfluidic method for agglutination assays is described that relies on hydrodynamic mixing in conjunction 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,²Light scattering,³Fluorescence⁴And an optical microscope.^5,6These detection methods are useful for method development and optimization, but they require auxiliary detectors which (ultimately) negate the main advantage of agglutinationAbility to read test results without complex detection schemes.

Another challenge affecting previously reported microfluidic-based agglutination assays is the requirement that the sample must be diluted prior to introduction into the microfluidic device, which adds to the complexity of the assay.⁵An ideal method would be to accept the untreated sample, which would be compatible with non-expert use. In addition, manipulation of agglutinates in narrow channels may cause channel blockage, which may lead to device failure and reliability problems. Finally, as an alternative to conventional microfluidics, there are many "paper microfluidics" formats (e.g., Yoon and You) that are flow-based⁷) Examples of agglutination assay methods that are performed, but these methods typically have low throughput and/or require manual washing steps. A new approach relying on Digital Microfluidics (DMF) is presented here, which overcomes the limitations of the above-mentioned techniques.

DMF is a liquid handling technique that uses electrostatic forces to manipulate liquid droplets of picoliters to microliter sizes. The most powerful format of DMF is the "two-plate" configuration, where the droplet is sandwiched between the top and bottom counter-electrode plates, exposing an array of insulated electrodes. When operating in a two-plate format, droplets can be dispensed, split, combined and mixed, making DMF very suitable for sample processing,⁸Immunoassay method^9-11And chemical reactions.¹²DMF differs from traditional microfluidics in a number of ways, including versatility (versatile device architecture can be applied to a variety of applications), absolute control of the position of reagents (liquid or dry) without moving parts, and operation without the opportunity for channel blockage in open geometry.

The present inventors are aware of two previous reports of agglutination assays performed on a DMF apparatus. One method uses back-light scattering detection for latex immunoagglutination assays,⁷and another method uses functionalized gold nanoparticles to agglutinate biomarkers of interest, detected in conjunction with a micro-isolation procedure.¹³It should be noted that none of these techniques are shown in the "standard" format of digital microfluidics. That is, the first method⁷Is not usedElectric field to manipulate the droplet (instead, using wires controlled in XYZ stages to mechanically push/pull the droplet around the surface), and a second method¹³So-called "single-plate" DMF is used (in contrast to the more powerful "double-plate" DMF format used in the methods disclosed herein). These "non-standard" DMF formats⁷ _, ¹³Useful for proof of concept, but we consider them almost certainly incompatible with the type of sample-in-answer-out (sample-in-answer-out) system that has become a universal plate for dual-plate digital microfluidics.¹¹

The term Digital Microfluidics (DMF) has been widely used to describe liquid droplet manipulation systems. Several fluid actuators have been reported, such as using chemistry¹⁴Or a thermal gradient,¹⁵A magnet,¹⁶Sound wave,¹⁷Machine with a movable working part⁷And an electrical method.¹³Since the present invention relies on the use of electrostatic liquid handling forces, commonly referred to as electrowetting on media (EWOD), only a comparison between single and dual plate DMF EWOD devices will be concerned and all other devices (non-EWOD) will not be discussed further since they use other liquid handling techniques not relevant to the present invention.

DMF EWOD systems generally fall into two main categories; single-plate DMF and double-plate DMF. There are several reasons and significant technical challenges to switch from a single-plate DMF apparatus to a double-plate DMF apparatus. The term single-plate DMF apparatus is used to describe an open system in which droplets are freely located on a horizontal solid substrate, while the term double-plate DMF apparatus is used to describe a cover system in which droplets are confined between two plates. Both types of devices require sufficient ground to operate; single-plate devices require external leads that are in direct contact with the droplets or electrodes that lie in the same plane as the actuation electrodes, whereas in dual-plate devices, the ground is located in the top plate.

In addition to the number of plates, the two types of DMF devices also differ greatly in their ability to perform droplet operations. In a two-plate system, droplet movement is easier. In addition, the splitting and distribution of droplets is almost a unique option for a dual plate system. In contrast, when thorough mixing, evaporation (for species concentration) and direct contact with liquid is requiredIn the case of droplets, a single plate device is preferred because droplets are readily available. Single board devices typically operate at much higher voltages and much lower frequencies, which require different hardware instrumentation than dual board systems. Thus, although there have been previous reports on digital microfluidics for agglutination assays,^7,13it is not clear how to transition agglutination from these devices to the two-plate DMF system, as there are numerous technical differences between the two types of devices that need to be optimised and determined in order to perform agglutination assays on the two-plate DMF devices reported here.

Furthermore, even from the standpoint of agglutination assays, any of the previous reports are not ideal-backscatter techniques⁷Require auxiliary instrumentation for analysis, and are based on nanoparticle technology¹³Is slow (as the user must wait for each sample to evaporate before analysis) and requires custom, expensive, nanoparticle-based reagents. It should be noted that both of these approaches were reported more than a decade ago, with no subsequent publication, indicating that population perception was slow (or not).

Disclosure of Invention

The present disclosure provides a novel method for performing agglutination assays on a "two-plate" Digital Microfluidic (DMF) device format. The droplets containing the analyte of interest (particles, cells, etc.) are loaded into the DMF device and mixed with solution phase or dry agglutinated antibody or antigen. The agglutinating agent binds to their complementary target (e.g., antibody or antigen) in the sample droplet, resulting in the formation of insoluble aggregates. Active mixing on DMF reduced reaction time and enhanced agglutination effect. The results can be viewed directly by eye or via a digital camera, since the agglutinated sample is sandwiched between two plates on the DMF device.

Accordingly, the present disclosure provides a method of characterizing a sample containing an analyte using a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes, the method comprising the steps of:

loading an agglutinating agent on the DMF device;

loading a fluid sample containing the analyte on the DMF device; and

contacting the fluid sample with the agglutinating agent using electrowetting

To cause agglutination of the analyte, thereby producing an agglutinate.

The method may further comprise the steps of: characterizing the amount of agglutination of the analyte by the agglutinating agent.

The fluid loaded on the DMF apparatus may contain a surfactant.

The surfactant may be in a pre-dried form, and the method may further comprise: coating one or more drive electrodes or across device surfaces with the surfactant in pre-dried form in predetermined points such that when the fluid sample is contacted with the pre-dried surfactant, the fluid sample dissolves and the surfactant may be present in the fluid in an amount of at least 0.01 wt%. The procedure for drying and reconstituting reagents on the DMF apparatus may be according to US2014/0141409A 1(Foley et al)¹⁸) The method described in (1) is performed.

The surfactant may be one of an ionic surfactant and a nonionic surfactant. The ionic surfactant may be selected from the group consisting of sodium lauryl sulfate, sodium stearate, cetrimide and sodium lauryl sulfate. Nonionic surfactants include, but are not limited to, alkylphenol hydroxypolyethylenes (e.g., Triton X RTM), polysorbates (e.g., Tween RTM), poloxamines (e.g., Tetronic RTM), poloxamers (e.g., Pluronic RTM), and sorbitan esters. The poloxamer may include

The agglutinating agent can be a liquid that is loaded and metered to a preselected drive electrode.

The agglutinating agent can be in a pre-dried form, and the method can further comprise: coating one or more actuation electrodes with the pre-dried form of the agglutinating agent in predetermined spots on the surface of the DMF device such thatSuch that when a fluid is contacted with the pre-dried agglutinating agent, the pre-dried agglutinating agent dissolves. The procedure for drying and reconstituting reagents on the DMF apparatus may be according to US2014/0141409A 1(Foley et al)¹⁸) The method described in (1) is performed.

The method may further comprise the steps of: actively mixing the agglutinating agent with the fluid sample using electrowetting on the DMF device.

The agglutinating agent can include any one or combination of substances capable of producing an agglutinate. Examples of substances include chemical agglutinating agents and biological agglutinating agents. For agglutination of red blood cells, the chemical agglutinating agent may be selected from the group consisting of: poly-L-lysine hydrobromide, poly (dimethyldiallylammonium) chloride: (A)RTM、RTM、RTM), poly-L-arginine hydrochloride, poly-L-histidine, poly (4-vinylpyridine) hydrochloride, crosslinked poly (4-vinylpyridine), methyl chloride quaternary salts, 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); cross-linked polyvinylpyrrolidone; polyvinylpyrrolidone, poly (melamine-co-formaldehyde); partial methylation; brominating hexylenediamine; poly (glutamic acid, lysine) 1:4 hydrobromide; poly (lysine, alanine) 3:1 hydrobromide; poly (lysine, alanine) 2:1 hydrobromide; poly-L-lysine succinylation; poly (lysine, alanine) 1:1 hydrobromide; and poly (lysine, tryptophan) 1:4 hydrobromide.

The chemical agglutinating agent may be poly (dimethyldiallylammonium) chloride.

The biological agglutinating agent may be selected from the group consisting of proteins, antibodies, viruses and antigens, DNA, RNA, and DNA or RNA based aptamers.

The protein may comprise a lectin capable of reversibly binding to the carbohydrate structure. The antibodies may include anti-A, anti-B and anti-D.

The virus may comprise an influenza virus.

The step of characterizing the amount of agglutination of the analyte by the agglutinating agent may comprise visual characterization. This visual characterization can be performed by a human observing the DMF apparatus to estimate the amount of aggregation. Alternatively, the step of visually characterizing is performed using a camera. The camera may be a webcam, a cell phone camera, a digital camera (including a digital single lens reflex DSLR), a video camera, a monitoring camera, a point-and-shoot camera, a camera with a CCD detector, a camera with a CMOS detector, a monochrome camera, a color camera.

The particles may be coated with the agglutinating agent. These may include any one or combination of polymer particles (e.g., latex), gold, silver, nanoparticles, and microparticles. The polymer particles may comprise latex.

The analyte against which detection is being made may be an antibody, and wherein the particles may be coated with an antigen or other agent capable of capturing the antibody of interest.

The analyte against which detection is being made may be an antigen, and wherein the particles may be coated with an antibody or other agent capable of capturing the antigen of interest.

The analyte against which detection is being made may be a bacterium, and wherein the particles may be coated with an antibody or other agent capable of capturing the bacterium of interest.

The analyte against which detection is being made may be a virus, and wherein the particles may be coated with an antibody or other agent capable of capturing the virus of interest.

The method is useful for the agglomeration of suspensions of polymer particles.

The method may be used for the agglutination of suspensions of nanoparticles.

The method may be used for agglutination of a suspension of red blood cells.

The fluid may be a blood sample containing at least red blood cells.

The fluid may contain a virus suspension for detecting viruses using agglutination of red blood cells or particles.

The method may be used for agglutination of a suspension of white blood cells.

The fluid may be a serum sample or a plasma sample containing at least white blood cells.

The method can be used for agglutination of suspensions of any other type of eukaryotic cells.

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

The cells may be red blood cells.

The agglutinating agent, when used with red blood cells, can be used to determine hematocrit levels.

The invention provides a double-plate electrowetting DMF device, which comprises:

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 drive electrodes; and

a surface on the first or second plate having a surfactant in pre-dried form coating the surface or coating the entire plate in preselected locations, another surface on the first or second plate having an agglutinating agent in pre-dried form coating the other surface in preselected locations.

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

The present disclosure provides a kit, comprising:

a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes;

a surfactant for placement on one of the two plates; and

an agglutinating agent for placement on one of the two plates.

The agglutinating agent can be selected for agglutination of red blood cells.

A further understanding of the functional and advantageous aspects of the present disclosure may be realized by reference to the following detailed description and drawings.

Drawings

Implementations will now be described, by way of example only, with reference to the accompanying drawings, in which:

fig. 1 shows in three (3) plots the DMF device and associated steps of an agglutination assay performed on the DMF device, wherein the leftmost plot labeled (i) shows loading of one or more samples containing an analyte of interest into the DMF device containing an agglutinating agent, the middle plot labeled (ii) shows metering the samples into subsamples, each subsample is then mixed with the agglutinating agent for a predetermined period of time, and the rightmost plot labeled (iii) shows agglutination being observed visually or by a camera on the DMF device.

Figure 2 is a graph depicting the results of a DMF agglutination assay for blood typing. A whole blood sample is loaded onto the device, after which it is metered into four sub-droplets. Each sub-droplet was mixed with separate droplets containing anti-a (left), anti-B (left two), anti-a/anti-B blend (right two), and anti-D (right) antibodies on a DMF apparatus. After 2 minutes of mixing, the results can be determined visually. The specific samples here formed aggregates with anti-a, anti-AB and anti-d (rh), which indicated the a + type.

Figure 3 is a graph depicting the results of a bead-based DMF agglutination assay. A first sample of bacterial lysate 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 latex beads coated with an antibody non-specific to PBP2 (left second). The first sample formed weak aggregates with latex beads coated with PBP2 antibody and no aggregates with latex beads coated with antibodies non-specific for PBP2, indicating that the bacteria were susceptible to methicillin. Similarly, a second sample of bacterial lysate was loaded into the device, which was then metered into a pair of sub-droplets, which were mixed with droplets containing latex beads coated with antibodies to PBP2 (right two) and droplets coated with antibodies non-specific to PBP2 (right). The second sample formed a strong aggregate with latex beads coated with antibodies specific to PBP2 and did not form an aggregate with latex beads coated with antibodies non-specific to PBP2, indicating that the bacteria were methicillin resistant. In both cases, after loading the samples, the process was automated, resulting in results after about 2 minutes of mixing.

Fig. 4 is a diagram illustrating the workflow of automated image analysis for determining the output of a DMF agglutination assay. A) An image collected from a digital camera showing the steps of capturing an isolation of the ROI (region of interest) from the initial image. (i) An image of the device is captured at an angle to reduce reflections. (ii) Perspective correction is performed. (iii) The portion of the image featuring the center of the device is isolated. (iv) In the isolated image, a region of interest (ROI) is identified for each droplet, and (v) each ROI is masked and stored as a separate image for analysis. B) The analyzed image (left) and data (right) for each ROI are shown. The pixel intensity variation for each ROI indicates the degree of agglutination.

FIG. 5 is a graph depicting the results of a DMF agglutination assay for determining hematocrit levels. On the left side four droplets are shown, with different hematocrit levels (ratio of red blood cell volume to total blood volume) -20% (upper), 40% (upper two), 60% (lower two), 80% (lower). The droplets are mixed with a chemical agglutinating agent, resulting in non-specific agglutination of the red blood cells. The higher the hematocrit level, the larger the point of coagulation will be. The hematocrit level may be estimated by the naked eye or determined using a digital camera. The output of the processed image captured using the digital camera is shown on the right. The pixel intensity difference can be used to determine the hematocrit level of the sample.

Fig. 6 is a diagram with three graphs depicting the results of a DMF agglutination assay for donor compatibility testing. A whole blood sample is loaded onto the device and then metered into four sub-droplets, as shown in the left-most drawing. Each sub-droplet is then mixed with a separate droplet containing plasma from the intended donor on a DMF apparatus, as shown in the middle panel. After 5 minutes of mixing, the results can be determined visually. The particular sample here formed an aggregate with the first two samples D1, D2 on the left (in the right-hand panel), indicating that these donor samples were incompatible with the recipient's blood, and the other two samples D3, D4 (on the right) did not show any signs of agglutination, indicating that these donor samples were compatible with the recipient's blood.

FIG. 7 is a graph depicting the results of a DMF agglutination assay for determining hematocrit levels with sorted pixel intensity versus number of pixels. On the left side of the vertical axis, five droplets are shown, having different, artificially defined hematocrit levels between 20% and 60% -60% (upper), 50% (upper two), 40% (upper three), 30% (lower two), 20% (lower). The droplets are mixed with a chemical agglutinating agent, resulting in non-specific agglutination of the red blood cells. The higher the hematocrit level, the larger the point of coagulation will be. Hematocrit levels were determined using image analysis. The output of the processed image captured using the digital camera is shown on the right. The integral of the pixel intensity for each sample is used to determine the hematocrit level of the sample.

FIG. 8 is a graph of hematocrit fraction versus hematocrit (%), which depicts a calibration curve generated from the image shown in FIG. 7. The markers are experimental data and the error bars indicate ± 1 standard deviation of n ═ 3 experiments under each condition. Fitting a dashed second order polynomial to the data, where R²＝0.9990。

FIG. 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-DMF on-DMF Droplet Agglutination Assessment (DAAD) method (right). For this set of 12 samples, the comparison between the gold standard and the DMF-DAAD method yielded p ≧ 0.5045.

Fig. 10A to 10D are a series of graphs depicting a comparison of performance between agglutination detection algorithms for detecting agglutination in 344 sample images. Known data include 225 positive samples and 119 negative samples as defined by the gold standard method. Negative is a sample that does not show any sign of agglutination and positive is a sample that shows any sign of agglutination.

Fig. 10A shows the performance of the histogram method.

Fig. 10A left hand graph is a plot of agglutination score versus gold standard results for sample images generated by the histogram method. Within the graph, black and gray represent the number of correct/incorrect evaluations using the threshold T (10 a.u.).

The right hand graph of fig. 10A is a graph of a Receiver Operating Characteristic (ROC) curve, which shows the true positive rate of the method versus the false positive rate of the method. The dotted line in the ROC curve represents the result of random guessing (coin toss). The area under the curve (AUC) of the method was 0.981.

Fig. 10B shows the performance of the standard deviation method.

FIG. 10B is a graph of the agglutination fraction of sample images produced by the standard deviation method versus gold standard results on the left hand side. Within the graph, black and gray represent the number of correct/incorrect evaluations using a threshold T (0.13 a.u.).

Fig. 10B, right hand graph, is a graph of Receiver Operating Characteristic (ROC) curves showing true positive rate of the method versus false positive rate of the method. The dotted line in the ROC curve represents the result of random guessing (coin toss). The area under the curve (AUC) of the method was 0.9990.

Fig. 10C shows the performance of the variance method.

FIG. 10C left hand side graph is a plot of agglutination score versus gold standard results for sample images generated by the variance method. Within the graph, black and gray represent the number of correct/incorrect evaluations using the threshold T (1 a.u.).

The right hand graph of fig. 10C is a graph of a Receiver Operating Characteristic (ROC) curve, showing the true positive rate of the method versus the false positive rate of the method. The dotted line in the ROC curve represents the result of random guessing (coin toss). The area under the curve (AUC) of the method was 0.9997.

Fig. 10D shows the performance of the droplet agglutination evaluation on DMF (DAAD) method.

Fig. 10D left hand side graph is a plot of aggregation fraction versus gold standard results for sample images produced by the DAAD method. Within the graph, black and gray represent the number of correct/incorrect evaluations using a threshold T (0.152 a.u.).

Fig. 10D, right hand graph, is a graph of Receiver Operating Characteristic (ROC) curves showing true positive rate of the method versus false positive rate of the method. The dotted line in the ROC curve represents the result of random guessing (coin toss). The area under the curve (AUC) of the method was 1.000.

Detailed Description

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

As used herein, the terms "comprises" and "comprising" are to be interpreted as inclusive and open-ended, and not exclusive. In particular, the terms "comprises" and "comprising," and variations thereof, as used in the specification and claims, are meant to encompass the specified features, steps or components. The terms are not to be interpreted to exclude the presence of other features, steps or components.

As used herein, the term "exemplary" means "serving as an example, instance, or illustration," and should not be construed as preferred or advantageous over other configurations disclosed herein.

As used herein, the terms "about" and "approximately," when used in conjunction with a particle size range, mixture composition, or other physical characteristic or feature, are intended to encompass slight variations that may be present in the upper and lower limits of the size range, so as not to exclude embodiments in which, on average, most of the sizes are satisfied, but statistical sizes may be present outside this region. It is not intended that embodiments such as these be excluded from the present disclosure. The terms "about" and "approximately" mean plus or minus 25% or less, unless otherwise specified.

It will be understood that, unless otherwise specified, any given range or group is simply meant to be a recitation of each member of the range or group, and every possible subrange or sub-group contained therein, and similarly referring to any subrange or sub-group therein. Unless otherwise indicated, the disclosure refers to and explicitly encompasses each and every specific member and sub-range or sub-group combination.

As used herein, the term "approximately," when used in conjunction with a quantity or parameter, refers to a range spanning from about one tenth to ten times the quantity or parameter recited.

As used herein, "agglutination" refers to a process in which clumps of cells or inert particles are formed due to interactions between specific antibodies and antigen components or due to other chemicals that can induce the same clumping effect.

Agglutination is defined as the formation of a clump of cells or inert particles by antibodies specific for surface antigenic components (direct agglutination) or for antigenic components adsorbed or chemically coupled to red blood cells or inert particles (passive red blood cell agglutination and passive agglutination, respectively).¹⁹Erythrocytes are also agglutinated by non-antibody substances such as vegetable proteins, viruses, heavy metal salts, inorganic glial acids and bases, and basic proteins (protamine, histones). Agglutination inhibition or inhibition of hemagglutination refers to the inhibition of these reactions by soluble antigens that react with the binding sites of antibodies and thereby prevent their binding and agglutination to the particles.

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

The agglutination assay differs from other assays, for example, it differs from assays such as US2017/0056887(Hadwen et al)²⁰) The coagulation assay disclosed in (1):

agglutination is the process of particle (solid/semi-solid or cell) agglomeration. There are many instances of agglutination. For example, hemagglutination is the aggregation of red blood cells, and leukocyte agglutination is the aggregation of white blood cells.

Coagulation, on the other hand, is the process by which a liquid changes to a solid/semi-solid state. An example of coagulation is the process of blood coagulation, in which blood changes from a liquid to a gel, thereby forming a blood clot. Blood coagulation is similar to the process of gelation. Three main steps of coagulation; i) platelet plug formation, ii) an endogenous or exogenous pathway, and iii) a common pathway.

The main difference between agglutination and coagulation is: agglutination is the process of particle aggregation, whereas coagulation is the process of forming a shaped blood clot. Many particles are capable of agglutinating, while only blood is capable of clotting.

Agglutination is due to antigen-antibody reactions, whereas coagulation is due to activation of multiple plasma factors.

As used herein, the phrase "agglutinating agent" refers to any substance used in agglutination assays that can result in the production of particle aggregates (agglutinates).

As used herein, the phrase "chemical agglutinating agent" refers to a substance used in agglutination assays that will produce the same effect as particle aggregation, but does not rely on antigen-antibody reactions, but on other processes that will break down particle suspensions and force the particles to collapse into each other and form an agglutinate.

Fig. 1 illustrates a dual (2) plate digital microfluidic device for use in the present disclosure for generating agglutination in a liquid analyte sample being tested for the presence or absence of a particular analyte. The main steps of using the DMF apparatus are depicted in three (3) panels from the leftmost panel to the rightmost panel. In a DMF apparatus, the steps of performing an agglutination assay on a DMF apparatus begin with: (ii) the left-most panel labeled (i) showing loading of one or more samples containing the analyte of interest into a DMF device containing an agglutinating agent; (iii) the middle panel labeled (ii) showing the metering of the sample into subsamples, then mixing each subsample with an agglutinating agent for a predetermined period of time; and the right-most panel labeled (iii), which shows agglutination observed either visually or by camera on a DMF apparatus.

Thus, broadly speaking, the present disclosure provides a method of characterizing a sample containing an analyte using a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes. The dual plate configuration of the DMF apparatus allows for dispensing, splitting, and merging of droplets. In single plate DMF devices, higher forces/voltages are required to shunt and distribute the droplets-even above the dielectric breakdown of the dielectric. In addition, a two-plate DMF device is ideal for imaging droplets and the contents of droplets because most of the droplets appear as flat areas, whereas in a single-plate DMF device, the curvature of the droplets does not allow for the same ease of imaging. Furthermore, in a single plate DMF device, the area of the droplet exposed to the air is larger, resulting in faster evaporation of the droplet, which may interfere with the assay.

The method comprises the following steps: a fluid sample (which may contain a surfactant) containing the analyte for which the test is to be performed and an agglutinating agent (which may contain a surfactant) are loaded onto a preselected number of actuation electrodes of a DMF device, after which the fluid sample is contacted with the agglutinating agent using electrowetting to agglutinate the analyte to produce an agglutinate, and then the amount of agglutination of the analyte by the agglutinating agent is characterized.

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

In one embodiment, the surfactant is in a pre-dried form, and when the surfactant is in the pre-dried form, the method further comprises: one or more of the actuation electrodes is coated with a surfactant in a pre-dried form in a predetermined spot or across the entire device surface such that when the fluid sample comes into contact with the pre-dried surfactant, the fluid sample dissolves such that the surfactant is present in the fluid in an amount of at least 0.01% by weight. In another embodiment, the agglutinating agent is a liquid agglutinating agent that is loaded and metered to a preselected drive electrode.

In a preferred embodiment, the method comprises the steps of: the use of electrowetting on a DMF device actively mixes the agglutinating agent with the fluid, which advantageously accelerates the agglutination process in the presence of the analyte for which the test is being performed.

The surfactant may be an ionic surfactant or a non-ionic surfactant, depending on the analyte for which the test is being performed. Ionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium stearate, cetrimide and sodium lauryl sulfate. Nonionic surfactants include, but are not limited to, alkylphenol hydroxypolyethylenes (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 depends on the type of agglutination assay to be performed and the type of sample being analysed. Surfactants for specific assays and sample types should be screened to determine surfactant compatibility.

For example, when testing blood to determine blood type, nonionic surfactants are preferred, as will be discussed in the examples below. Nonionic surfactants are used with blood to maintain an isotonic environment of the cells, thereby preventing cell lysis.

The agglutinating agent can include any one or combination of substances capable of producing an agglutinate. Examples of substances include chemical agglutinating agents and biological agglutinating agents. For agglutination of red blood cells, the chemical agglutinating agent may 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) hydrochloride, crosslinked poly (4-vinylpyridine), methyl chloride quaternary salts, poly (4-vinylpyridine-co-styrene); poly (4-vinylpyridine poly (hydrogen fluoride)); poly (4-vinylpyridine-p-toluene)Sulfonate salts); poly (4-vinylpyridine-tribromide); poly (4-vinylpyrrolidone-co-2-dimethylaminoethyl methacrylate); cross-linked polyvinylpyrrolidone; polyvinylpyrrolidone, poly (melamine-co-formaldehyde); partial methylation; brominating hexylenediamine; 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.

Chemical agglutinating agents are used to cause agglutination of any red blood cells, so they can be used as a positive control for blood agglutination assays, and can also be used to agglutinate red blood cells to determine hematocrit levels, as will be discussed in the examples below.

A biological agglutinating agent is any biological substance of any nature that is capable of producing an agglutinate. For agglutination of red blood cells, examples include proteins such as lectins (proteins capable of reversibly binding 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 agglutinating agents are used to determine the presence or absence of red blood cells or a particular analyte of interest in 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, influenza viruses are used to determine the amount of antibodies present in plasma against the virus and to determine the level of immunity of a patient sample.

The step of characterizing the amount of agglutination of the analyte by the agglutinating agent is preferably by visual/optical characterization. Other reported characterization methods include the use of electrochemical (e.g., impedance spectroscopy), absorbance, and turbidity techniques. However, implementation of these techniques requires additional hardware equipment and several modifications to the DMF apparatus, so that the present process using visual characterization by operator visual inspection of the results of the agglutination reaction or using a camera is quite advantageous, as no additional modifications to the system are required.

Visual characterization can be performed by a human visually observing the DMF apparatus to estimate the amount of aggregation. Alternatively, the step of visually characterizing is performed using a camera. Non-limiting examples of cameras that may be used include webcams, cell phone cameras, digital cameras (including digital single lens reflex DSLR), video cameras, surveillance cameras, point-and-shoot cameras, cameras with CCD detectors, cameras with CMOS detectors, monochrome cameras, black and white cameras, color cameras. For visual/optical characterization involving cameras, a droplet agglutination assessment on DMF (DAAD) was developed. DAAD is an image analysis algorithm for automated detection of agglutinates in droplets on a DMF device. The DAAD algorithms may be stored in a microprocessor associated with the camera, or they may be stored on a microprocessor connected to the DMF power supply controlling the drive electrodes of the DMF device, or may be stored on a remote computer. The DAAD algorithm may be executed by a microprocessor or computer.

In one embodiment, the agglutinating agent comprises particles coated with the agglutinating agent. 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 agglutinating agent. For example, to detect an antigen, the particles are coated with an antibody or other agent capable of capturing the antigen of interest. In the case of detecting antibodies, the particles should be coated with an antigen or other agent capable of capturing the antibody of interest.

The method is useful for the agglomeration of suspensions of polymer particles. Non-limiting examples include coated latex particles for rubella antibody detection,²¹Latex particles coated with antibodies for the detection of any virus,²²Latex particles coated with streptolysin O,²³Latex particles coated with antibodies for C-reactive protein detection,²⁴Coated latex particles for identifying staphylococcus aureus,²⁵And coated latex particles for identifying any type of bacteria.

The methods disclosed herein can be used for the agglutination of nanoparticle suspensions. Non-limiting examples include nanoparticles coated with antibodies for detecting antigens and nanoparticles coated with antigens for detecting specific antibodies.

The method may be used for agglutination of a suspension of red blood cells to determine the blood type of a patient, as will be discussed in the examples below. 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 solution (e.g., Phosphate Buffered Saline (PBS)), a solution containing PBS and serum albumin (e.g., human serum albumin, bovine serum albumin).

However, it will be appreciated that other types of blood samples can be characterized using this method, including whole blood (white and red blood cells, platelets and plasma), and also including diluted blood (a portion of which is taken and mixed with other things, to name a few examples), suspensions of white blood cells, serum and plasma.

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

Other types of fluid samples can be characterized using this method, including viral 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 may be mixed with agglutinating agents for virus detection.

Other types of fluid samples, including suspensions of any other type of eukaryotic cell, can be characterized using this method. The agglutinating agent can be any substance capable of causing cell agglutination.

Examples

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

Example 1

The first embodiment shown in fig. 2 is a blood typing assay that uses blood agglutinating antibodies to cause agglutination of red blood cells. A panel 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) -was used to determine ABO and rhesus (Rh) blood groups. In the embodiment shown in fig. 2, the agglutinating agent is loaded into the device in solution; a similar method is also shown, where the agglutination reagent is preloaded onto the device as a dry spot that dissolves upon exposure to the sample or another reagent (e.g., a lysis buffer). The complete assay takes only a few minutes and is fully automated.

In embodiments using predried reagents, e.g., Foley et al¹⁸Recombination is performed as described in US2014/0141409a 1.

The assay relies on a phenomenon known as hemagglutination. According to the national library of medicine, hemagglutination (or hemagglutination) is defined as "the aggregation of erythrocytes by lectins, including antibodies, lectins and viral proteins".²⁶Traditionally, hemagglutination assays have been used to detect variations or polymorphisms in surface markers (antigens) found on red blood cell membranes to classify blood by category (blood type). There are currently 339 certified blood group antigens, of which 297 belong to one of the 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.

The ABO system is particularly unique in that it is the only blood group system in which antigens are not present on the surface of red blood cells, and reverse complementary antibodies (reciprocal antibodies) are consistently and predictably found as soluble entities in plasma. ABO antigens are often called histocompatibility antigens because their widespread distribution means that they can also be used as histocompatibility antigens in general. Most importantly, the widespread distribution of anti-a and anti-B makes transfusions of different blood types catastrophic, as Hemolytic Transfusion Reactions (HTR) can lead to hyperacute rejection of incompatible kidney, liver and heart transplants. Likewise, Rh antigens are commonly used to prevent fetal and neonatal Hemolytic Disease (HDFN).^27,28

The assays described in this example were performed in blood samples; the relevant tests can be performed in serum, which is commonly referred to as retrotyping. In retrotyping, the patient's serum is mixed with red blood cells (e.g., a cells and B cells of ABO) having known surface antigens, and observation of hemagglutination indicates the presence or absence of the corresponding antibody.²⁹In either format (positive or "negative"), there is a strong incentive to develop novel, rapid and easy to perform blood typing assays, as evidenced by the global blood typing market which is projected to reach $ 25 billion by 2022.³⁰

Example 2

Another application of the new platform is the use of the system for donor-recipient cross-matching of blood, a critical operation that must be performed quickly on-site in a (time-precious) high-risk environment such as an emergency room or wound care laboratory. In particular, the first step in plasma donation in such an environment is to determine the type of acceptor based on the ABO/Rh system so that the type of donor can be identified (e.g., B + donor for B + acceptor). But this level of selectivity is not sufficient because there are many other subtypes that may lead to incompatibility that are not captured by the ABO/Rh system, so that a second step (usually performed at the patient's bedside, just before transfusion) is usually performed, in which plasma from a potential donor is tested directly for agglutination with the patient's blood. An example of a simulated cross-matching test performed by DMF hemagglutination and analyzed by DAAD is shown in fig. 6. In this example, two of the four potential donors were found to be compatible with the potential acceptor.

Example 3

Agglutination of red blood cells can be used to determine the hematocrit of a blood sample. FIG. 5 is a graph showing the results of a DMF agglutination assay for determining hematocrit levels. On the left side of fig. 5, droplets are shown, which have different hematocrit levels (ratio of red blood cell volume to total blood volume) -20% (upper), 40% (upper two), 60% (lower two), 80% (lower). The droplets are mixed with a chemical agglutinating agent, resulting in non-specific agglutination of the red blood cells. The higher the hematocrit level, the larger the point of coagulation will be. The hematocrit level may be estimated by the naked eye or determined using a digital camera. The output of the processed image captured using the digital camera is shown on the right. The pixel intensity difference can be used to determine the hematocrit level of the sample.

Especially for hematocrit determination, the integral of the pixel intensity found (inclusively) between a small fraction of the total number of pixels is defined as the 'hematocrit fraction'. FIG. 7 in an initial experiment, the hematocrit fraction from a training set of diluted blood samples with artificially defined hematocrit levels between 20% and 60% was found and plotted as a function of hematocrit level and with a second order polynomial: y-0.0249 x2+1.092x + 107.3. The hematocrit fraction of each droplet was compared to the calibration curve to determine the predicted% hematocrit (fig. 8).

FIG. 7 shows the results of a DMF agglutination assay for determining hematocrit levels. On the left side of fig. 7 five droplets are shown, which have different hematocrit levels (ratio of red blood cell volume to total blood volume) -60% (upper), 50% (upper two), 40% (upper three), 30% (lower two), 20% (lower). Similar to fig. 5, the droplets are mixed with a chemical agglutinating agent, resulting in non-specific agglutination of the red blood cells. The higher the hematocrit level, the larger the point of coagulation will be.

FIG. 8 shows a calibration curve of hematocrit fraction as a function of known hematocrit levels. The markers are experimental data and the error bars indicate ± 1 standard deviation of n ═ 3 experiments under each condition. Fitting a dashed second order polynomial to the data, where R²＝0.9990。

FIG. 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 yielded p ≧ 0.5045.

Example 4

A fourth embodiment of the present disclosure, shown in FIG. 3, is a Latex Immunoagglutination Assay (LIA) which uses a suspension of latex particlesTo detect the analyte of interest. In the absence of analyte, the beads are suspended as individual units and the suspension appears "smooth" (i.e., no heterogeneous clumps), whereas in the presence of analyte, the particles aggregate, forming a macroscopic heterogeneous aggregate. These assays have a wide range of utility, such as detecting monovalent and multivalent antigens, proteins, drugs, steroid hormones, and even microorganisms.³¹LIA is commonly used by clinicians for influenza detection,³²And antibiotic susceptibility testing.³³For the latter, there is great interest in being able to distinguish between strains of bacteria that are or are not resistant to antibiotics to determine which therapy to prescribe. For example, the main cause of infection in hospitals is methicillin-resistant Staphylococcus aureus³⁴(MRSA); clearly, prescribing methicillin to patients infected with MRSA is time and resource consuming. 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 close-up in fig. 3.

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

Visual determination of agglutination results

The simplest detection mode of an agglutination assay is that the user observes with the naked eye; this method is applicable to the embodiments already put into practice as described above. But also agglutinatesIt is suitable for automation by image processing. There have been several reports of automated detection of hemagglutination in fluid channels, but they rely on ancillary equipment (e.g., microscope, slide, or slide, or slide, or slide, or slide, or,⁵A waveguide,³⁵Etc.) and complex post-processing procedures. For example, Huet et al⁵Artificial networks were trained in MATLAB to detect the progress of agglutination, but the algorithms are not general and a new training set is required for each new imaging setup.

In contrast, the present method as depicted in fig. 4A and 4B is straightforward and applicable to any system with a digital camera. For optical characterization involving a camera, droplet agglutination on DMF (DAAD) was performed in 8 steps. The first six image pre-processing steps (i-vi) were identical for blood typing (fig. 2), latex agglutination assay (fig. 3), hematocrit analysis (fig. 5) and donor compatibility test (fig. 6).

Fig. 4A-I show step I) in which a camera is used to collect images of the device. The camera is positioned at an angle relative to a plane perpendicular to the DMF apparatus. Images are typically captured at the maximum resolution of each camera (although lower resolution images may also be processed).

Fig. 4A-ii show step ii, in which the image is perspective corrected by defining four coordinates in the source image and four reference coordinates. A 3 x 3 matrix is calculated based on each set of coordinates (image-reference pair pairs) and then the same matrix is applied to the source image to obtain a perspective corrected image.

Fig. 4A-iii show step iii) in which the center of the DMF device is automatically located by detecting known device features, and isolating this region of the image for further processing.

Fig. 4A-iv illustrate step iv, where drops are detected by identifying contours and combining adjacent contours to form a rectangular region of interest (ROI) for each drop that defines a mask to extract an image.

Fig. 4A-v show step v, where the ROI image corresponding to each drop is masked, isolated and converted from RGB to grayscale.

FIG. 4B-left side 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], then sorted by pixel value from lowest to highest.

Fig. 4B-right side shows step vii, where the slope of the pixel intensity in this gradient is then used as an indication of the degree of agglutination (procedure used in blood typing, donor compatibility test, step viii). For example, in fig. 4B, a steep slope of the pixel intensity of A, D (Rh) and a, B blends indicates agglutination, while a flat slope of B indicates no agglutination. A similar image processing method was developed to automate the detection of latex bead agglutination (fig. 3), highlighting the flexibility of this method compared to previous reports.⁵

Within the visual characterization, other analysis methods may be performed. Three alternative agglutination detection algorithms were tested and compared to DAAD: histogram method, standard deviation method and variance method. In the histogram method, DAAD substeps (i) - (v) are performed to isolate each ROI image. For each image, a histogram is generated based on the number of pixels per pixel intensity value. The histogram is smoothed with a moving average filter (window ═ 10 bins) and in the smoothed data set, the main peak is identified by finding the local maximum by comparing the pixel intensity with the neighboring values.

The average pixel intensity of the main peak in the smoothed histogram is defined as the threshold T. Finally, the agglutination fraction 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 intensity greater than T. In the standard deviation method (adapted from previous reports)³⁶) In which DAAD substeps (i) - (vi) are performed, after which the array is (again) normalized to the range [0, 1%]And the standard deviation σ of the pixel intensity is defined as the agglutination fraction. In the variance method (adapted from previous reports)^5,6,37) DAAD substeps (i) - (v) are performed to isolate each ROI image. Computing each pixel relative to its neighbors using a 3 x 3 matrixAnd determining the local variance of the imageThe mean variance of all pixels in the image.

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

In summary, the inventors report a 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 examples. The first embodiment is a blood typing hemagglutination assay-the first embodiment known to be performed on a double plate DMF device. This method has proven to be compatible with the use of solution phase or dry agglutinated antibodies, and the antibodies can be mixed with undiluted whole blood, and the results can be determined visually within minutes. As an extension of the blood typing assay, a donor compatibility test may be performed to indicate the correct donor for the recipient patient when the blood sample is mixed with the intended donor sample (second embodiment). In addition, it is shown that the above assay is performed using hemagglutination, which uses a chemical reagent to determine the hematocrit of the sample (third embodiment). In a fourth embodiment, the DMF method for performing a Latex Immunoagglutination Assay (LIA) is performed. In this example, the test is shown for antibiotic susceptibility; it is contemplated that any LIA should be compatible. Finally, imaging-based readings are reported using a custom but generalizable algorithm for interpreting the results of DMF agglutination assays. When considered together, the user may load the sample, press a button, and receive the results within minutes.

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

Table 1: comparison between other agglutination methods using digital microfluidic platforms and the methods disclosed herein.

The inventors are aware of literature reports demonstrating agglutination assays using digital microfluidics, but they are not relevant to the present invention.^7,13No previous reports have used a two-plate electrowetting device to perform agglutination assays. In contrast, different types of assays have been shown on a two-plate DMF apparatus: coagulation assay²⁰And plasma separation using lectins³⁸However, none of the above is relevant to the present invention. In addition, detection of agglutination is performed by the naked eye or using DAAD (our unique detection algorithm that detects agglutination in images captured with a digital camera). The method does rely on the use of an absorbance module to determine agglutination, as previously reported³⁹And the algorithm does not use any previously reported methods to detect agglutination because they rely on expensive imaging equipment (microscope settings or high-end DSLR cameras)^7,13And is highly dependent on the imaging conditions (brightness, contrast, white balance, etc.). The performance of some other previously reported methods was compared to DAAD, and it has been shown herein that the present DAAD outperforms all of these methods.

In general, in one aspect, the disclosure provides a method of characterizing a sample containing an analyte using a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes. The method comprises the following steps: loading a fluid sample containing an analyte and a surfactant with an agglutinating agent onto the DMF device; and contacting the fluid sample with an agglutinating agent using electrowetting to cause the analyte to agglutinate with the agglutinating agent.

In another aspect, the present disclosure provides a two-plate electrowetting DMF device, comprising: 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 drive electrodes; and a surface on the first or second plate having a surfactant in pre-dried form coating the surface in preselected locations, another surface on the first or second plate having an agglutinating agent in pre-dried form coating the other surface in preselected locations.

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

The present disclosure also provides a kit comprising: a two-plate electrowetting digital microfluidic Device (DMF) having a plurality of drive electrodes; a surfactant for placement on one of the two plates; and an agglutinating agent for placement on one of the two plates.

The specific embodiments described above have been shown by way of example, and it should be understood that these embodiments may be susceptible to various modifications and alternative forms. It should be further understood that the claims are not intended to be limited to the particular forms disclosed, but rather to cover all modifications, equivalents, and alternatives falling within the spirit and scope of this disclosure.

Reference to the literature

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