WO2017024297A1 - Multiplexed detection on microfluidic analytical devices - Google Patents

Abstract

A three-dimensional microfluidic device is described, including: a plurality of porous, hydrophilic cellulosic layers each comprising one or more hydrophilic regions and/or hydrophilic channels embedded in the porous, hydrophilic cellulosic layer; wherein the hydrophilic channel is fluidically connected to the hydrophilic region; the hydrophilic regions comprise a sample deposition zone, one or more assay zones in fluidic communication with the sample deposition zone; and a buffer deposition zone; and the assay zone comprises one or more reagents embedded therein selected from the group consisting of antigens and antibodies; and a valve layer comprising a valve switchable from a first position where the buffer zone and the assay zone are not in fluidic communication, to a second position where the buffer zone and the assay zone are in fluidic communication.

Description

MULTIPLEXED DETECTION ON MICROFLUIDIC ANALYTICAL DEVICES

RELATED APPLICATION

[0001] This application claims the priority and benefits to U.S. Provisional Application No. 62/201,902, filed August 6, 2015, the entire content of which is expressly incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] The present invention was made with United States government support under Grant No. HDTRA1-14-C-0037 and HDTRA1-14-R-0012 awarded by the Defense Threat

Reduction Agency (DTRA). The United States government may have certain rights in this invention.

TECHNICAL FIELD

[0003] This technology relates generally to analytic devices. In particular, this invention relates to microfluidic devices.

BACKGROUND

[0004] Analytical tests, commonly used in chemical and biochemical analysis, have been designed to be performed in well-equipped centralized laboratories; therefore, they are often inadequate for point-of-care (POC) applications in low-income countries. Laboratory-based tests (e.g., ELISA assays and fluorescent immunoassays) require: i) expensive and nonportable equipment, ii) specialized facilities, and iii) trained personnel to execute lengthy experimental procedures (i.e., on the order of hours) and dispose the hazardous wastes that are produced. These resources are scarce in laboratories and clinics in low-income countries, therefore, these tests are typically not available there. Low-cost analytical devices, such as immunochromatographic lateral-flow tests and lab-on-a-chip (LOC) systems, are better suited for POC applications and many of them are commercially available. Their price, however, is often too high to allow, for example, large-scale screening of a population for a disease in low-income countries. The multiplex capabilities of these devices are also limited, and a single device can typically detect only one analyte/disease. [0005] Microfluidic paper-based analytical devices (μ-PADs) have been introduced as a new type of low-cost devices that require small amount of samples (i.e., few μΐ^) and are suitable for POC setting. The primary material used in the fabrication of μ-PADs is chromatography paper. Hydrophobic barriers patterned into each layer of paper define a two-dimensional network of hydrophilic, microfluidic channels. Aqueous samples wick along these predefined channels by capillary action to the sensing area and the detection of analytes is usually performed using optical, colorimetric, luminometric, and electrochemical readouts. Simple μ-PADs to perform multistep assays require the user to add the sample and most of the necessary reagents manually. Three-dimensional (3D), fully-integrated μ-PADs designed to minimize the complexity of user interface have also been developed. In these devices, the user is only required to add the sample and a buffer solution to the entry ports of the device to run the complete assay. Using fully-integrated 3D μ-PADs, our group achieved the multiplex detection of metabolites (i.e., glucose and proteins) in artificial urine samples, and enzymes (i.e., alkaline phosphatase and aspartate aminotransferase) and total protein in whole-blood samples. More recently, Schonhorn et al. developed a 3D μ-PAD for the determination of human chorionic gonadotropin (hCG) in urine samples. Schonhorn, et al, Lab Chip, 2014, 14, 4653-4658. The analytical assays, in all of these cases, are based on one-step reactions with color readout.

[0006] There remains a need for new and efficient microfluidic devices for quick, multi-step essays with minimal user interface.

SUMMARY

[0007] Described herein is a new class of three-dimensional (3D) microfluidic devices (e.g., μ-PADs) that can perform multi-step, indirect immunoassays, with minimal user interface, and in short period of time (e.g., less than 15 minutes). The devices are capable of detecting multiple target analytes from a single sample, and they optionally have all necessary reagents pre-stored inside them and are easy-to-use. The end user simply has to add a few drops of sample and optionally buffer at the entry ports of the device to run the assay. Optionally, the device can be designed such that the user can open the device to read the results. The design of the devices is optionally modular, and can be easily changed for the detection of single or multiple analytes, depending on the needs of the analyses.

[0008] In one aspect, a three-dimensional microfluidic device is described, including: a plurality of porous, hydrophilic cellulosic layers each including one or more hydrophilic regions and/or hydrophilic channels embedded in the porous, hydrophilic cellulosic layer; wherein the hydrophilic channel is fluidically connected to the hydrophilic region; the hydrophilic regions include a sample deposition zone, one or more assay zones in fluidic communication with the sample deposition zone; and a buffer deposition zone; and the assay zone includes one or more reagents embedded therein selected from the group consisting of antigens and antibodies; and a valve layer including a valve switchable from a first position where the buffer zone and the assay zone are not in fluidic communication, to a second position where the buffer deposition zone and the assay zone are in fluidic communication.

[0009] In any one of the embodiments described herein, the plurality of cellulosic layers include a first porous, hydrophilic cellulosic layer including a first hydrophilic region in fluidic communication with the buffer deposition zone and a second porous, hydrophilic cellulosic layer including a second hydrophilic region in fluidic communication with the assay zone; and the valve layer is disposed in between the first and second porous, hydrophilic cellulosic layers and the valve aligns with at least portions of the first and second hydrophilic regions.

[0010] In any one of the embodiments described herein, the valve includes a cavity within the valve layer wherein in the first position, the first and second hydrophilic regions are separated by the cavity and not in contact; and in the second position, the first and second hydrophilic regions are pressed into the cavity to contact each other.

[0011] In any one of the embodiments described herein, the valve layer is a double-sided adhesive.

[0012] In any one of the embodiments described herein, the cellulosic layer includes a material selected from the group consisting of nitrocellulose acetate, cellulose acetate, cellulosic paper, filter paper, tissue paper, writing paper, printing paper, blotting paper, chromatography paper, paper towel, and cloth.

[0013] In any one of the embodiments described herein, the porous, hydrophilic cellulosic layer includes a hydrophobic barrier substantially permeating the thickness of the cellulosic layer and defining the hydrophilic regions and/or hydrophilic channels. [0014] In any one of the embodiments described herein, the hydrophobic barrier includes wax.

[0015] In any one of the embodiments described herein, the hydrophilic regions from two adjacent cellulosic layers are aligned vertically and in contact with each other to enable vertical fluidic flow.

[0016] In any one of the embodiments described herein, the hydrophilic channels connecting the hydrophilic regions within the same cellulosic layer enables horizontal fluidic flow.

[0017] In any one of the embodiments described herein, the device includes one, two, three, four, five, six, or more assay zones.

[0018] In any one of the embodiments described herein, the reagent is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0019] In any one of the embodiments described herein, the reagent is an antigen for a disease-specific antibody of interest in a sample.

[0020] In any one of the embodiments described herein, the reagent is the antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0021] In any one of the embodiments described herein, the hydrophilic regions further includes a storage region embedding one or more antigens or antibodies and in fluidic communication with the buffer zone.

[0022] In any one of the embodiments described herein, the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0023] In any one of the embodiments described herein, the storage region is embedded with a secondary, reporter antibody specific for a disease-specific antibody of interest in a sample.

[0024] In any one of the embodiments described herein, the storage region is embedded with a secondary, reporter antibody specific for the antibody of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0025] In any one of the embodiments described herein, the reporter antibody is conjugated with metal nanoparticles. [0026] In any one of the embodiments described herein, the reporter antibody is conjugated with gold nanoparticles.

[0027] In any one of the embodiments described herein, the device further includes a splitting layer for splitting the sample into a plurality of hydrophilic regions each in fluidic communication with one of the assay regions.

[0028] In any one of the embodiments described herein, the device further includes a blotting layer to promote efficient fluidic flow.

[0029] In any one of the embodiments described herein, the blotting layer includes a blotting paper.

[0030] In any one of the embodiments described herein, the device further includes a docking layer configured to align the cellulosic layer including the assay zones with an adjacent cellulosic layer and to enable removing the assay zones from the device for observation.

[0031] In any one of the embodiments described herein, the device further includes a filtration membrane.

[0032] In any one of the embodiments described herein, the filtration membrane is a plasma separation membrane in fluid communication with the sample deposition zone and configured to filter the sample.

[0033] In any one of the embodiments described herein, the plasma separation membrane is selected from the group consisting of plasma separation membranes.

[0034] In any one of the embodiments described herein, the device further includes an additional buffer deposition zone; and an additional valve switchable from a first position where the additional buffer zone and the assay zone are not in fluidic communication, to a second position where the additional buffer zone and the assay zone are in fluidic

communication.

[0035] In any one of the embodiments described herein, the device further includes an additional storage zone embedding an additional reporter antibody and in fluidic

communication with the additional buffer zone. [0036] In another aspect, a method of detecting a disease-specific antibody or antigen in a sample is described, including: providing the device of any one of the embodiments described herein; depositing a biological fluidic sample into the sample deposition zone; allowing the fluidic sample to flow into the one or more assay zones; depositing a buffer into the buffer deposition zone; and activating the valve to switch from the first position to the second position.

[0037] In any one of the embodiments described herein, the sample is a blood sample, a plasma sample, or a urine sample.

[0038] In any one of the embodiments described herein, the sample includes the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0039] In any one of the embodiments described herein, the method further includes the sample.

[0040] In any one of the embodiments described herein, the method further includes the fluid in the sample into two, three, four, five, six, or more assay zones.

[0041] In any one of the embodiments described herein, the reagent is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0042] In any one of the embodiments described herein, the sample includes a disease- specific antibody and the reagent is an antigen for the disease-specific antibody.

[0043] In any one of the embodiments described herein, the method further includes allowing the antigen to bind to the disease-specific antibody in the sample.

[0044] In any one of the embodiments described herein, the hydrophilic regions further includes a storage region embedding one or more antigens or antibodies and in fluidic communication with the buffer zone. [0045] In any one of the embodiments described herein, the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

[0046] In any one of the embodiments described herein, the hydrophilic regions further includes a storage region embedding a reporter antibody specific for a disease-specific antibody of interest in a sample.

[0047] In any one of the embodiments described herein, the reporter antibody is conjugated with metal nanoparticles.

[0048] In any one of the embodiments described herein, the reporter antibody is conjugated with gold nanoparticles.

[0049] In any one of the embodiments described herein, the method further includes allowing the reporter antibody bind to the disease-specific antibody.

[0050] In any one of the embodiments described herein, the buffer includes a reporter antibody specific for the disease-specific antibody.

[0051] In any one of the embodiments described herein, the reporter antibody is conjugated with metal nanoparticles.

[0052] In any one of the embodiments described herein, the reporter antibody is conjugated with gold nanoparticles.

[0053] In any one of the embodiments described herein, the method further includes allowing the reporter antibody bind to the disease-specific antibody.

[0054] In any one of the embodiments described herein, the method further includes observing the assay zone for color indicative of the gold nanoparticles.

[0055] It is contemplated that any embodiment disclosed herein may be properly combined with any other embodiment disclosed herein. The combination of any two or more embodiments disclosed herein is expressly contemplated.

[0056] These and other aspects and embodiments of the disclosure are illustrated and described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The invention is described with reference to the following figures, which are presented for the purpose of illustration only and are not intended to be limiting.

[0058] In the Drawings:

[0059] Figures 1A-1E illustrates images of a 3D microfluidic device (e.g., μ-PAD device), in closed state (Figures 1 A-1B) and opened state (Figures 1C-1E). The images include top view of a device showing the relative size dimensions (Figure 1 A), an angle view showing the thickness (d=17 mm) of a device (Figure IB), and top views of open devices revealing the yellow sensing layers with either two (Figure 1C), four (Figure ID) or six (Figure IE) white capture zones.

[0060] Figure 2 illustrates an exploded-view schematic of the device described herein, indicating the layers of the four-plex 3D microfluidic device (e.g., μ-PAD) and their specific function in the device. Layers 1 and 13 are made of cellulose acetate; layers 2-3, 5-8, and 11 are made of chromatography paper; layer 4 and 10 are made of double side adhesive; layer 9 is made of nitrocellulose; and layer 12 is made of blotting paper.

[0061] Figure 3 shows the schematic of the flow of the solutions inside a 4-plex device. The colors are representative for specific: yellow line for plasma flow; white line for buffer flow, and red line for Ab2-AuNPs flow.

[0062] Figure 4 shows a schematic of the chemical reactions occurring on the sensing area.

[0063] Figures 5A-5H shows images of actual device capture zones, after performing indirect immunoassays for HIV, syphilis (i.e., denoted as Syph), Hepatitis B (i.e., denoted as HBV), and Hepatitis C (i.e., denoted as HCV), in our 2-plex (A -C), 4-plex (D-F), and 6-plex (G-H) μ-PADs. Specifically, Figures 5A-5B indicate assays for HBV; Figure 5C indicates assays for HCV; Figures 5D-5E indicate assays for HIV and Syph; Figures 5G-5H indicates assays for HIV, Syph, HCV and HBV.

[0064] Figures 6A-6C show exploded-view schematics, indicating the layers of the 2-plex device (Figure 6A), 4-plex device (Figure 6B), and 6-plex device (Figure 6C) (e.g., μ-PADs).

[0065] Figure 7 shows the schematic of the preparation of the capture zones. [0066] Figures 8A-8C show the schematic of the flow of the solutions inside a 2-plex device (Figure 8A), 4-plex device (Figure 8B), and 6-plex device (Figure 8C). The colors are representative for specific: yellow line for plasma flow; white line for buffer flow, and red line for Ab2-Au Ps.

[0067] Figure 9 shows images of the multiplex microfluidic paper-based analytical device described herein used for the detection of more than one antibodies, e.g., anti-Brucella IgG and IgM antibodies. The images include a top view of the device showing the relative size dimensions (left) and an angle view showing the thickness of the device (right).

[0068] Figure 10 shows an exploded-view schematic of the multiplex microfluidic paper- based analytical device used for the detection of anti-Brucella IgG and IgM antibodies in bovine serum. Layers 2-3, 5-9 and 12 are made of wax-patterned (green and blue colors) chromatography paper; layers 1 and 14 are made of cellulose acetate; layers 4 and 11 are made of double-sided tape; layer 10 is made of waxed-patterned (yellow color) nitrocellulose; and layer 13 is made of blotting paper.

[0069] Figures 11A-11B show schematic representation of the sensing layer and indirect immunoassay performed with the multiplex microfluidic paper-based analytical device.

Figure 11A is a top view of the sensing layer: the sensing layer is divided into four sensing zones for the detection of anti-Brucella IgG (left) and IgM (right) antibodies and includes the positive control (PC) for both detections. Figure 11B is a side view of the sensing layer: the two steps indirect immunoassay and the reading of the test are done on the sensing layer of the device.

[0070] Figures 12A-12B show images of the sensing layer after performing the indirect immunoassay for the detection of anti-Bovine IgG antibodies. Results for negative serum (Figure 12A) and positive serum (Figure 12B) are showed.

[0071] Figure 13 shows a device described herein including plasma separation membrane. The plasma membrane is placed between a layer of tape and cellulose acetate. The top of the membrane is exposed for sample delivery.

[0072] Figures 14A-14D show plasma separation membrane results for Whatman™ plasma separation membranes. Figure 14A shows the result for GF/DVA at sample volumes (μί): 50, 40, 35, 30, 27, 25, 20, and 15. Figure 14B shows the result for MF1 at sample volumes (HL): 35, 30, 25, 20, 17, 15, 10, and 8. Figure 14C shows LF1 at sample volumes (μΐ,): 30, 25, 20, 15, 13, 10, 8, and 5. Figure 14D shows Fusion 5 at sample volumes (μί): 35, 30, 25, 20, 17, 15, 10 and 8. The back scan was flipped horizontally to facilitate image comparisons.

DETAILED DESCRIPTION

[0073] In one aspect, a three-dimensional microfluidic device is described, including: a plurality of porous, hydrophilic cellulosic layers each including one or more hydrophilic regions and/or hydrophilic channels embedded in the porous, hydrophilic cellulosic layer; wherein the hydrophilic channel is fluidically connected to the hydrophilic region; the hydrophilic regions include a sample deposition zone, one or more assay zones in fluidic communication with the sample deposition zone; and a buffer deposition zone; and the assay zone includes one or more reagents embedded therein selected from the group consisting of antigens and antibodies; and a valve layer including a valve switchable from a first position where the buffer deposition zone and the assay zone are not in fluidic communication, to a second position where the buffer zone and the assay zone are in fluidic communication.

[0074] In some embodiments, the plurality of cellulosic layers includes a first porous, hydrophilic cellulosic layer including a first hydrophilic region in fluidic communication with the buffer deposition zone and a second porous, hydrophilic cellulosic layer including a second hydrophilic region in fluidic communication with the assay zone; and the valve layer is disposed in between the first and second porous, hydrophilic cellulosic layers and the valve aligns with at least portions of the first and second hydrophilic regions. In these

embodiments, the multiple layers may be stacked to form the microfluidic device and the valve aligns vertically with at least portions of the first and second hydrophilic regions.

[0075] Any valves known in the art for controlling fluid flow can be used. For example, and without any limitation, cantilever push valve, multi-layer push valve, magnetic valve, and fluidic diode valve can be used. The valve is used herein to control the right reaction sequence for a multi-step immunoassay. In some embodiments, the valve is at the first position so that the buffer fluid does not reach the assay zone, and the sample fluid is allowed to reach the assay zone, e.g., to react with any embedded reagents in the one or more assay zones. Subsequently, the valve is switched to the second positon so that any added buffer, optionally including a secondary antigen or antibody or carrying/dissolving a secondary antigen or antibody from a storage zone, will flow to the assay zone after the sample fluid has already reached the assay zone (and optionally reacted with the reagents embedded in the assay zone).

[0076] In some embodiments, the valve includes a cavity within the valve layer wherein in the first position, the first and second hydrophilic regions are separated by the cavity and not in contact; and in the second position, the first and/or second hydrophilic regions are pressed into the cavity to contact each other. In some specific embodiments, the valve layer is a double-sided adhesive, e.g., a double-sided tape.

[0077] Exemplary microfluidic devices described herein are shown in Figures 1 A-1E. A new class of 3D devices, e.g., microfluidic paper-based analytical devices (μ-PADs), is described, that can perform multi-step, indirect immunoassays, with minimal user interface, and in short time, e.g., less than 15 minutes. The devices are capable of detecting multiple target analytes from a single sample. They optionally have all necessary reagents pre-stored inside them, and are easy-to-use. In some embodiments, the end user simply has to add a few drops of sample and buffer at the entry ports of the device to run the assay, and then open the device to read the results. See, Figures 1C-1E. Figures 1A-1E illustrate images of a 3D microfluidic device (e.g., μ-PAD device), in closed state (Figures 1 A-1B) and open state (Figures 1C-E). The images include top view of a device showing the relative size dimensions (Figure 1 A), an angle view showing the thickness (e.g., d=17 mm) of a device (Figure IB), and top views of open devices revealing the yellow sensing layers with either two (Figure 1C), four (Figure ID) or six (Figure IE) white capture/assay zones.

[0078] In certain embodiments, the design of the devices is modular, and can be easily changed for the detection of single or multiple analytes, depending on the needs of the analyses. In non-limiting embodiments, three different types of devices are described: i) a device with two capture zones (2-plex device), ii) a device with four capture zones (4-plex device), and iii) a device with six capture zones (6-plex device) (Figures 1 A-1E). In some embodiments, these devices enable indirect immunoassays with visual readout. The final detection may be based on the visual readout of color indicator, e.g., gold nanoparticles, bound to target analytes on the capture zones of the devices - for the detection of antigens or antibodies, e.g., antibodies against HIV virus (anti-HIV), Treponema pallidum (anti- Treponema pallidum), Hepatitis C virus (anti-HCV), Hepatitis A virus (anti-HAV), or anti- Brucella IgG and IgM antibodies. [0079] The microfluidic device disclosed herein is now described in more details with reference to Figure 2. Figure 2 shows a three-dimensional microfluidic device described herein. The device includes a plurality of porous, hydrophilic cellulosic layers, e.g., layer 2 (entry layer), layer 3 (storage layer), layer 5 (junction layer), layers 6-8 (splitting layers I, II, and III), layer 9 (sensing layer), layer 11 (washing layer). Each of these cellulosic layer may independently include a material selected from the group consisting of nitrocellulose acetate, cellulose acetate, cellulosic paper, filter paper, tissue paper, writing paper, printing paper, blotting paper, chromatography paper, paper towel, and cloth. Each of these cellulosic layers include a hydrophobic barrier (e.g., shown as the dark shade labeled as 219 in the junction layer 5) substantially permeating the thickness of the cellulosic layer and defining one or more hydrophilic regions (e.g., regions 203, 204, 205, 209, 211, 212, 213, 214, 216, 217 and 218) and channels fluidically connected to the hydrophilic regions (channel 215 connecting regions 213 and 214; channel 210 connecting regions 209 and 211). In certain embodiments, the plurality of porous, hydrophilic cellulosic layers each including a hydrophobic barrier substantially permeating the thickness of the cellulosic layer and defining one or more hydrophilic regions and/or channels fluidically connected to the hydrophilic regions. In certain embodiments, the hydrophobic barrier includes polymers, e.g., polymerized photoresist. In certain embodiments, the hydrophobic barrier includes wax. The hydrophilic regions includes a sample deposition zone 203, one or more assay zones 217 in fluidic communication with the sample deposition zone 203; and a buffer deposition zone 204.

[0080] In this non-limiting example of the device described in Figure 2, the device includes 13 different layers, 10 layers are made of cellulose-based materials (i.e., chromatography paper, nitrocellulose, blotting paper, printing paper, and cellulose acetate), 2 layers are made of thin polymeric films (i.e., adhesive (layers 4 and 10), and 1 layer is made of cellulose- based material and thin polymeric film (i.e., cellulose acetate and blood separation membrane (layer 1)). The cellulose-based layers are patterned using wax printing, to form hydrophilic channels, and backed with a patterned layer of double-sided adhesive, thus joining each layer to the next to generate an integrated 3D device. Double-sided adhesive layer 4 is also used to create a cavity valve 207; the valve is an empty gap formed by aligning cavity 207 and vertically-overlapping hydrophilic regions 206 and 209 in adjacent layers. [0081] Each layer of a device has a specific function, as indicated in Figure 2. For example, the "top layer" layer 1 serves as an entry port for the device including sample entry point 201 and buffer entry point 202. Layer 1 may optionally include a filtration membrane 220 to filter the biological sample of interest, e.g., blood, using a blood separation membrane. The filtration membrane 220 may be a plasma separation membrane in fluid communication with the sample deposition zone and configured to filter the sample. In certain embodiments, the plasma separation membrane is selected from the group consisting of Whatman™ GF/DVA, MF1, LF1 and Fusion 5 plasma separation membranes. In certain embodiments, the plasma separation membrane is selected from the group consisting of Whatman™ MF1 and LF1 plasma separation membranes. In certain embodiments, the plasma separation membrane is selected to filter off red blood cells and/or to prevent hemolysis.

[0082] The entry layer (layer 2) is a patterned cellulosic layer including a sample deposition zone 203 and a buffer deposition zone 204. The zones 203 and 204 are aligned vertically and in contact with hydrophilic regions 205 and 206 of storage layer 3, respectively, to enable vertical fluid flow. The hydrophilic region 206 may optionally be a storage region embedding one or more antigens or antibodies and in fluidic communication with the buffer zone 204. In certain embodiments, the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In certain embodiments, the storage region is embedded with a secondary, reporter antibody specific for a disease-specific primary antibody of interest in a sample. In certain embodiments, the storage region is embedded with a secondary, reporter antibody specific for the antibody of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In certain embodiments, the reporter antibody is conjugated with metal nanoparticles. In certain embodiments, the reporter antibody is conjugated with gold nanoparticles.

[0083] The cavity valve layer (layer 4) controls the fluidic flow to allow the required sequential mixing of necessary reagents, and is important in multi-step immunoassays. Any valves known in the art for controlling fluid flow can be used. In the non-limiting example shown in Figure 2, the valve layer is a double-sided tape 208 with a hole 207 in the middle. The valve layer 4 is disposed between a first porous, hydrophilic cellulosic layer (layer 3) including a first hydrophilic region (region 206) in fluidic communication with the buffer deposition zone 204 and a second porous, hydrophilic cellulosic layer (layer 5) including a second hydrophilic region 209 in fluidic communication with the assay zone 217. The valve 207 aligns with at least portions of the first and second hydrophilic regions 206 and 209. In certain embodiments, the valve is a cavity valve 207 within the valve layer wherein before the cavity valve 207 is compressed (i.e., in the first position), the first and second hydrophilic regions 206 and 209 are separated by the cavity 207 and not in contact. As a result, the buffer deposition zone 204 and the assay zone 217 are not in the fluidic communication. When the cavity is pressed, e.g., one may apply a force through opening 202 at the cavity to compress the cavity, the first and/or second hydrophilic regions 206 and 209 are pressed into the cavity to contact each other (the second position of the valve) to enable fluidic communication therebetween. As a result, when the valve is at the second position, e.g., pressed position, the buffer deposition zone 204 will be in fluidic contact with hydrophilic zone 209 and hydrophilic zone 211 through hydrophilic channel 210, and eventually in fluidic communication with the assay zone 217.

[0084] Note that the tape 208 (layer 4) is about half of size of adjacent layers 3 and 5, so that the double side tape 208 is not placed in between hydrophilic regions 205 and 211. As a result, regions 205 and 211 are in contact and in vertical fluidic communication to ensure that sample deposition zone 203 and downstream assay region 217 are in fluidic contact.

[0085] The splitting layers I, II, III (layers 6-8) split a single fluidic sample into two, four, or six independently optimized channels, depending on the type of the device (i.e., 2-plex, 4- plex, and 6-plex). Shown in Figure 2 is a 4-plex microfluidic device. The sensing layer (layer 9) is where the immunoassay occurs. The sensing layer may include one, two, three, four, five, six, or more assay zones. In certain embodiments, the assay zone includes one or more reagents embedded therein. In certain embodiments, the reagent is selected from the group consisting of antigens and antibodies. Non-limiting examples of the reagents include the antibody and antigen of HIV, HAV, HBV, Treponema pallidum, syphilis and Brucella. (e.g., Brucella-IgM and Bruce lla-IgG). In certain embodiments, the assay zone includes the same reagent in each assay zone for repeated assay. In other embodiments, the assay zone includes different reagents in each assay zone for a plurality of different assays using a single sample.

[0086] In certain specific embodiments, the reagent embedded in the assay zone is an antigen for a disease-specific antibody of interest in a sample. For instance, the reagent may be the antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In these

embodiments, the sample is deposited into the sample entry point 201 and flow through hydrophilic regions 203, 205, 211, 212, 213, hydrophilic channel 215, hydrophilic regions 214, 216, and eventually reach assay zone 217 to react with the embedded reagent, e.g., antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In these specific embodiments, the capture antigens may be immobilized in the capture/assay zones of the capture/sensing layer 9 that is made of nitrocellulose.

[0087] In certain specific embodiments, after the first step of the immunoassay is complete, i.e., binding of the embedded antigen in assay zone 217 with a disease-specific antibody in a sample, a buffer is introduced by a user through the entry point 202 into buffer deposition zone 204. In certain embodiments, the buffer may include desired antigens or antibodies for the second step of the immunoassay reaction. In other embodiments, the hydrophilic regions further includes a storage region, e.g., 206, embedding one or more antigens or antibodies and in fluidic communication with the buffer zone. In certain embodiments, the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In certain specific embodiments, the storage region is embedded with a secondary, reporter antibody specific for a disease-specific antibody of interest in a sample. Non-limiting examples of the secondary, reporter antibodies include reported antibodies specific for the antibody of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In certain embodiments, the reporter antibody is conjugated with metal nanoparticles, e.g., gold nanoparticles. In certain embodiments, reporter antibodies conjugated gold nanoparticles are stored in the storage layer 3 that is made of chromatographic paper; sucrose is used to preserve and stabilize the dry antibodies. After the buffer is added, the valve 207 is activated, e.g., switched to the second position, to enable fluidic communication between the buffer deposition zone and the assay zone, so that the reagent in the buffer solution, e.g., a reporter antibody specific for a disease-specific primary antibody, will mix with the buffer and flow from hydrophilic region 206 to region 209, and through hydrophilic channel 210 to hydrophilic region 211, then through hydrophilic region 212, 213, hydrophilic channel 215, hydrophilic regions 214, 216, and eventually reach assay zone 217. Once reaching the assay zone 217, the reporter antibody may bind to any primary, disease-specific antibody bond to the antigen embedded in the assay zone. In certain embodiments, the reporter antibody is conjugated with a metal particle with color, e.g., gold. After the unbounded reporter antibody is washing away, the remaining gold/reddish color will indicate the presence of the disease-specific antibody, and hence, the disease state of a patient. [0088] Note that the buffer may also be added before or at the same time as the sample is added to the sample deposition zone. As long as the valve is not activate, i.e., remaining in the first position, the buffer will have no fluidic contact with the assay zone, which ensures the desired two-step immunoassay reaction sequence.

[0089] In certain embodiments, the device further includes a docking layer 10 configured to align the cellulosic layer 9 including the assay zones 217 with an adjacent cellulosic layer, e.g., layer 8, and to enable removing the assay zones from the device for observation. The dock layer aligns the assay layer within the device and facilitates the opening of the device to read the results. In certain embodiments, the dock layer 10 is made of one-sided adhesive so its upper non-sticky side is easily separated from the above layer.

[0090] In certain embodiments, the device further includes a washing layer, layer 11, having hydrophilic regions 218 in contact with assay zone 217 to protect the sensing layer from back flow. In certain embodiments, the device further includes a blotting layer (layer 12) acts as a reservoir to wick-away excess fluid and to assure continual flow. In certain embodiments, the device further includes a backing layer (layer 13) to protect the device and the user and serves as a support for the device. In the described device shown in Figure 2, all necessary reagents are optionally stored within the device layers. The function of each layer is described in Figure 2.

[0091] As described in one or more embodiments herein, the fluidic flow inside a device is predetermined by: i) lateral-flow channels (e.g., channel 210) defined by wax barriers, and ii) vertical-flow paths defined by holes that are integrated into tape located on the backside of each layer and enabled by aligned hydrophilic regions from adjacent layers (e.g., regions 203 and 205). This combination of fluid flow regimes introduces specific capabilities to the devices. For instance, the lateral-flow channels used in the "splitting layer" makes multiplexing possible; intersecting channels within the "splitting layer" split a single sample into two, four, or six aliquots that can be analyzed separately. The vertical-flow channels enable the compartmentalization of the flow path into different regions, where all the necessary reagents can be stored; the reagents can be stored independently in different layers. It also allows short microfluidic path lengths, small volume requirements, and therefore rapid assays. [0092] Capillary forces move fluid from the point of entry situated at the top of the device, e.g., region 203, to the sensing area, e.g., assay zone 217, and beyond, e.g., region 218 and blotting layer 12, without the need for external pumps. The sample and buffer required to perform the assay dissolve and, if required by the assay, react with the reagents stored in the layers of the device. In certain embodiments, a cavity valve, e.g., valve 207, is used as a tool to control fluidic flow inside the devices. To open the valve and let the fluid flow through, the user needs to press the cavity valve. The cavity valve layer allows the performance of multistep indirect assays. Thus, in certain specific embodiments, the primary antibodies (Ab-ls) in a sample first reacts with the capture antigens (Ag)

immobilized on the assay zone (e.g., zone 217) before reacting with labeled secondary antibodies (Ab-2s; optionally stored in storage zone 206) to complete the assay. The flow- control valves allow the selective addition of the patients' sample (potentially containing the Ab-ls) and the buffer (containing the Ab-2s) to the capture zones in the right order.

[0093] In some embodiments, the device described herein further include one or more additional buffer deposition zones; and one or more additional valves each corresponding to one of the additional buffer deposition zones and each switchable from a first position where its corresponding additional buffer zone and the assay zone are not in fluidic communication, to a second position where its corresponding additional buffer zone and the assay zone are in fluidic communication. In some embodiments, the device further includes one or more additional storage zones each embedding an additional reporter antibody. Thus, in these embodiments, the device includes two or more different buffer zones/storage zones capable of allowing different antigens/antibodies, e.g., different reporter antibodies, to react with the primary antibodies bounded at the assay zone. In some embodiments, each of the buffer deposition zones optionally corresponds to and is in fluidic communication with a specific storage zone, and is fluidically controlled by a specific valve to enable fluidic communication with a corresponding assay zone. As a result, a plurality of secondary/reporter antibodies can be used to react with a single disease-specific primary antibody at distinct assay zones. This design can be used, e.g., for the detection of multiple subtypes of a primary disease- specific antibody by using a plurality of reporter antibodies each specific for a subtype of the primary antibody.

Method of Analyzing a Sample [0094] In another aspect, a method of detecting a disease-specific antibody or antigen in a sample is described, including: providing the device of any one of the embodiments disclosed herein; depositing a biological fluidic sample into the sample deposition zone; allowing the fluidic sample to flow into the one or more assay zones; depositing a buffer into the buffer deposition zone; and activating the valve to switch from the first position to the second position. In some embodiments, the sample is a blood sample, a plasma sample, or a urine sample. In some embodiments, the sample comprises the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In some embodiments, the method further includes filtering the sample. In some embodiments, the method further includes splitting the fluid in the sample into two, three, four, five, six, or more assay zones. In these embodiments, the assay zone comprises one or more reagents, e.g., antigens and antibodies, embedded therein. Non-limiting examples of the reagent is selected from the group consisting of antigens and antibodies of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella. In certain embodiments, the method further comprises observing the assay zone for color indicative of the gold particles.

[0095] In some embodiments, the device is modular and can be easily changed depending on the needs of the analyses. In a non-limiting embodiments, three different types of devices are developed (exploded views of these devices are shown in Figures 6A-6C). The design of each device differs, beginning in the splitting layer and continuing in the layers below, where the number of channels determines the number of target analytes being tested. The design of these devices is similarly as the device shown in Figure 2. The first configuration, termed duplex (2-plex; Figure 6 A), has two capture zones 601 to detect antibodies against one disease and one of the two zones incorporates a positive control zone. See, e.g., Figure 1C. We used this configuration to detect anti-HIV, anti-Treponema pallidum, anti-HCV, and anti-HAV antibodies in human serum. The second configuration, termed quadraplex (4- plex; Figure 6B), has four capture zones 602 to detect antibodies against two diseases and incorporate a positive and a negative control zone. See, e.g., Figure ID. We used this configuration to detect, in a single sample of human serum, anti-HIV and anti-Treponema pallidum antibodies. The third configuration, termed sixplex (6-plex; Figure 6C), has six capture zones 603 to detect antibodies against four diseases and incorporate a single positive and a negative control zone. See, e.g., Figure IE. We used this design to detect, in a single sample of human serum, anti-HIV, anti-Treponema pallidum, anti-HBV, and anti-HCV antibodies. A detailed sample and buffer flow for the devices described in Figures 6A, 6B, and 6C are illustrated in Figures 8A, 8B, and 8C, respectively.

[0096] In some embodiments, the method of using the device disclosed herein is described in more detail with reference to Figure 3. In certain embodiments, the method includes: i) the user places a drop of sample (10 μΐ_^ of blood or serum; labeled as " 1") and buffer (30 uL of Phosphate Buffered Saline, PBS; labeled as "2") into the sample port (indicated by "S" on the entry layer of the device). The sample wicks through the blood separation membrane 301— in blood samples, the red blood cells need to be removed by filtration as they can interfere with the interpretation of the results— and the cell-free sample follows the channel into the splitting layer 302. In the splitting layer 302, the sample is redirected through a predetermined number of channels to the capture zones 303. At that point, only the target primary antibodies bind to the immobilized capture antigens, while the remaining sample flows through - into the blotting layer; ii) the user adds buffer (50 μΐ_^ of PBS; labeled as "3") to the buffer port (as indicated by "B" on the entry layer of the device). The buffer wicks through the first three layer of the device including sample layer 304 and the storage layer 305 and releases the pre-stored secondary antibodies conjugated with gold nanoparticles (Ab- 2s-Au Ps) stored in storage zone 306. The user also physically compresses the buffer port 307 with a tip (e.g., pipette tip), thus activating the cavity valve 308 and allowing the fluid (i.e., buffer solution containing Ab-2s-Au Ps) to wick through the device and to continue along their designated path to the capture zones 303. On the capture zones, the Ab-2s- Au Ps bind to the target primary antibodies (Ab-ls) present, completing the assay; iii) the user adds more buffer (100 μΐ_^ of PBS) to the buffer port 307 in order to wash any remaining reagents from the channels; iv) the user opens the device by removing the layers above the "dock layer" 309 (the splitting layer 302 and the dock layer 309 can be separated) to reveal the sensing layer 310 and read the results on the capture zones 303 devoted to the detection of each disease. The ease of reading of the results is an important advantage of the design of these devices. A white capture zone indicates a negative result (i.e., no immobilized antibodies) and a reddish capture zone or a capture zone that have red traces indicates a positive result (i.e., Ab-ls and Au P conjugated Ab-2s are immobilized). The capture zones devoted to positive controls (indicated by "PC" on the sensing layer of each device) should be reddish, indicating that the fluid has wicked through the device up to the capture zones, and the μ-PAD had successfully performed the assay. The capture zones devoted to negative controls (indicated by "NC" on the sensing layer of the device) should be white, indicating that there is not non-specific binding of Ab-2s-Au Ps to the capture zone and, in case that the sample is blood, the red blood cells have not been hemolyzed. If the positive control capture zone is white or the negative control capture zone is reddish, these indicate the μ-PAD device did not work properly, and the results cannot be interpreted.

[0097] In some embodiments, methods of embedding the antigens into the assay zone are described with reference to Figure 7. The cellulosic layers are first patterned using the hydrophobic barrier, e.g., wax, to define one or more assay zones. Antigens are then added and bound to the hydrophilic assay zones. Finally, the free active sites are blocked by blocking agent.

[0098] In some embodiments, a schematic indicating the sequence of reactions occurring at the capture zones is shown in Figure 4. As shown in Figure 4, capture antigens are embedded in the assay zones of the device. Samples containing primary antibodies flow through the assay zone to allow the binding of the primary antibodies with the embedded antigen. Subsequently, reporter antibodies bound to a gold nanoparticle flow through the assay zone and, where the primary antibodies are present and bound with the antigen, will bind to the primary antibodies and present a reddish color for a user to observe.

Microfluidic Devices for detecting HIV, Syphilis, Hepatitis B and Hepatitis C.

[0099] In some embodiments, the performance of these devices for the diagnosis of four infectious diseases: HIV, Syphilis, Hepatitis B and Hepatitis C are tested. Patients are often tested for these diseases in parallel, as all these diseases are sexually transmitted.

Challenges in the development of devices to detect these diseases arose from the availability and composition of the commercially-available samples from infected humans; untreated serum samples from infected humans are infectious and require special handling. We used serum-based serological control samples from humans that contain anti-HIV, anti-Treponema pallidum, anti-HB V, and anti-HCV antibodies; the samples are not infectious as the viruses and bacteria have been inactivated by heat. The fact that these serological control samples are not pre-concentrated or purified, verifies that our devices can detect antibodies against this set of diseases in physiologically-relevant concentrations. The devices could also test blood samples as they incorporate a blood separation membrane to remove the red blood cells. [0100] In some embodiments, the devices are designed to provide qualitative results (yes/no), because the detection of this set of diseases (i.e., HIV, Syphilis, Hepatitis B and Hepatitis C) is based on the presence/absence of specific antibodies against the pathogenic agent (i.e., virus, bacteria). In some embodiments, this type of devices is contemplated to provide semi-quantitative results by comparing the color of the sensing area with a calibrated color bar or by using a scanner or a cell-phone camera to digitize the results and express them in terms of concentration levels (e.g., low, middle, high). Figures 5A-5H shows images of the "sensing layer" of devices that were used for the singleplex or multiplex detection of anti- HIV, anti-Treponema pallidum, anti-HBV, and anti-HCV antibodies. In all cases, the devices have worked properly as the "PC" zones have reddish spots and the "NC" zones are white. The devices tested with serum samples obtained from non-infected individuals (negative samples) exhibit capture zones, devoted to the detection of each specific type of antibodies, which are white (Figure 5A). The devices tested with serum samples that contain anti-HIV, anti-Treponema pallidum, anti-HBV, and anti-HCV antibodies (positive samples) exhibit capture zones, devoted to the detection of each specific type of antibodies, which have reddish spots (Figures 5B-H). Specifically, Figures 5A-5B indicate assays for HBV; Figure 5C indicates assays for HCV; Figures 5D-5E indicate assays for HIV and Syph; Figures 5G- 5H indicates assays for HIV, Syph, HCV and HBV.

[0101] Without wishing to be bound to any particular theory, it is believed that the devices are suitable for use at the point-of-care because i) they are fully-functional, ii) they can detect disease-specific antibodies in physiologically-relevant concentrations, and ii) the user can interpret the results, by looking at the "sensing layer" of a device, and determine if the test sample is positive or negative by eye. The user can also take a picture of the "sensing layer" to analyze the images locally (either manually or using a custom software) and/or send them to a central location for remote analysis, interpretation, and logging.

[0102] In some embodiments, the devices described herein have ten characteristics that make them attractive candidates for further development for POC diagnosis in resource-limited settings: i) Low-cost. The bill of materials per device is less than $2. ii) Equipment-free. These devices do not require any instrumentation for liquid handling or signal detection, iii) Easy-to-use. The user can add only two liquids (i.e., a sample and a buffer solution), and open the device to read the results with naked eye. iv) Rapid analysis. The test is completed in less than 15 minutes, iv) Multiplex-capable. These devices can detect multiple target analytes present in a single sample, v) Minimal sample size. These devices require a single drop of blood (~ 20 μΕ). vi) Sensitive. They can detect antibodies at concentrations relevant to clinical assays, without needing of an amplification step, vii) Specific. The devices are capable of selective detection of target analytes with minimal background signal, viii) Fieldable. They can be easily delivered (e.g., even by mail) and used in a wide range of locations, ix) Web accessible. Results can be rapidly transferred to the web by imaging with a cell-phone or other devices, and stored in a global database.

Diagnostic feedback from analysis of the data is also straightforward, x) Disposable. The devices can be disposed easily by incineration. The characteristics of these devices exceed the ASSURED criteria established for POC devices for use in resource-limited settings, as defined by the World Health Organization.

Fully Multiplexed IgG And IgM Immunoassay

[0103] In some embodiments, a multiplex microfluidic paper-based analytical device (μ- PAD) is designed to detect both IgG and IgM anti-Brucella antibodies via indirect immunoassay. The device can detect both isotypes from a single sample and has all the necessary reagents pre-stored inside to perform the assay (Figure 9). It is simple to use since the end user has to add only a few drops of sample and buffer at the entry ports of the device to run the assay, and then open the device to read the results (Figure 10). The assay takes less than 15 minutes to perform. The design of the devices is modular, and can be easily changed for the detection of antibodies from different populations, depending on the needs of the analysis. The reading of the test is based on the visual detection of gold nanoparticles bound to target analytes on the capture zones of the device. Using this method, commercially available antibodies against Brucella IgG (anti-Bovine IgG) in purified bovine serum were detected.

[0104] The device is described in more detail with reference to Figure 10. Each layer from the multiplex μ-PAD designed for the detection of Brucella has a specific function. The top layer (Figure 10, layer 1) is the first layer of the device and has three cavities to deposit assay fluid. Below is the entry layer (Figure 10, layer 2) that serves as entry point for the device microfluidic channels. It includes three entry regions/channels where the patient's sample (Figure 10, layer 1, channel "S"), and the buffer (Figure 10, layer 1, 2 channels "B"), are dropped. Below the sample layer is the spitting and storage layer (Figure 10, layer 3); it also hosts three regions/channels. The sample channel is free of reagent and is used to split the patient's sample and bring it the lower layer. The second and third channels contain the red gold nanoparticles (Au Ps) conjugated with isotype-specific secondary antibodies required to perform the indirect immunoassay. The second channel 703 (bottom left of the layer) stores the AuNPs conjugated with the anti-bovine IgG secondary antibodies and the third channel 704 (bottom right of the layer) stores the AuNPs conjugates with the antibovine IgM secondary antibodies. During the test, the buffer goes through this third layer to dilute the conjugated AuNPs and bring them to the lower layer. Below the channels from this layer are two flow-control valves 701. These valves allow the successive addition of the patient's sample and buffer into the sensing layer of the device. They are empty gaps created by three layers of patterned double-sided tapes placed between the splitting and storage layer and the junction layers (Figure 10, layer 5-6). To open the valves and let the fluid flow, the user needs to press the area above the gaps so that layers 3 and 5 are in contact. During the assay, the patient's sample first flows vertically through layers 3 and 5 and reaches the junction layers 5 and 6. Once the valves are opened, the buffer then flows vertically through layers 3 and 4 before reaching the junction layers and flowing toward the splitting layers (Figure 10, layer 7-9). The splitting layers divide the liquid into four lateral-flow branches. This layer 7 has hydrophilic wells 705 that receives the liquid from the sixth layer and directs it to the four lateral-flow branches connected to the sensing layer (Figure 10, layer 10). Layer 10 is where the indirect immunoassay takes place. It is divided into four hydrophilic sensing areas 702 where the antigens needed for the assay are immobilized and the results are read. Below each of these sensing areas are channels connected to the washing layer (Figure 10, layer 12). This layer is to wash the sensing areas and to separate it from the blotting layer (Figure 10, layer 13) so no back flow can contaminate the sensing layer. Finally, the last layer of the device is the backing layer (Figure 10, layer 14). It protects both the user and the device in addition to increasing the rigidity of the device.

[0105] The design of the microfluidic channels inside the multiplex μ-PAD allows for the separate detection of the two anti-Brucella antibody isotypes, IgG and IgM. In the splitting and storage layer 3 of the device, the sample is split into two distinct channels that are connected to the two distinct detection areas of the sensing layer (Figure 11 A). The left area of the sensing layer is for the detection of anti-Brucella IgG antibody (labeled IgG) and the right area is for the detection of the anti-Brucella IgM antibody (labeled IgM). Both areas include a sensing zone for the detection of the antibody (top channel) and a sensing zone for the positive control (PC, bottom channel). [0106] The indirect immunoassay is performed in two simple steps to detect both anti- Brucella antibody IgG and IgM isotypes (Figure 1 IB). The user first adds a drop (about 20 μΐ,) of blood into the sample port of the multiplex μ-PAD. The blood wicks through the device using the sample channels to reach the sensing areas with the pre-immobilized capture antigen (Figure 1 IB, step 1). At that point, only the target primary antibodies (Ab-ls) present in the blood bind to the capture antigens, while the remaining sample flows through the lower layers. A drop of buffer is then added to the buffer channels of the device. The buffer dissolves and releases the pre-stored secondary antibodies conjugated with red gold nanoparticles (Ab-2s-Au Ps) situated in the layer 3 of the device before reaching the sensing area. The anti-bovine IgG conjugated Au Ps reach the left area of the device dedicated to the detection of anti-Brucella IgG and the anti-bovine IgM conjugated AuNPs reach the right area of the device dedicated to the detection of anti-Brucella IgM. The red conjugated AuNPs in the buffer then bind to the target primary antibodies (Ab-ls) (Figure 1 IB, step 2), completing the assay. To read the result, the user opens the device to reveal the sensing layer with the capture zones devoted to the detection of each isotypes. A white capture zone without immobilized antibodies indicates a negative result, and a red capture zone with immobilized antibodies and red gold nanoparticles indicates a positive result.

[0107] Commercially available and pre-purified IgG antibodies against Brucella in bovine serum were detected (Figures 12A-12B). The assays run with the positive serum containing the antibodies show sensing zones with red color indicating the detection of the anti-Brucella IgG antibodies (Figure 12B, top sensing zones). As for the assays run with negative serum that is not infected, the sensing zones are white indicating that no anti-Brucella IgG antibodies are detected (Figure 12A, top sensing zones). The positive controls (PC) from all these assays show a red color indicating that the flow channels from the device worked properly and the negative controls (NC) show a white sensing zone indicating that no nonspecific binding occurred.

[0108] The multiplex μ-PAD for the detection of anti-Brucella is a better device compared with other point-of-care analytical devices due to its capability to detect both anti-Brucella IgG and IgM antibody within the same sample. The multiplex μ-PAD is also affordable, transportable, and easy to use and to dispose. This makes the μ-PAD an ideal tool to screen animal quickly and efficiently in a wide range of healthcare settings.

Implementation Of Plasma Separation To Paper-Based Devices [0109] To determine an appropriate and fully-functional plasma separation membrane for the paper-based, multiplexed immunoassay for testing of whole blood samples, we compared four different glass fiber membranes commercially available from Whatman™ (GF/DVA, MFl, LFl, and Fusion 5) and identified the membrane that is most appropriate for our multiplexed immunoassay.

[0110] We have incorporated a plasma separation membrane into the fully functional microfluidic paper-based analytical device (μ-PAD) that allows for antibody detection using whole blood samples (Figure 13). The plasma membrane 1301, placed between the first cellulose acetate layer and the double-sided tape layer, is designed to trap red blood cells allowing only blood plasma to pass through the membrane and into the hydrophilic channels of the μ-PAD, e.g., the hydrophilic regions 1302 in the wax-printed cellulose layer, which provides the capability of multi-target detection from a single sample with minimal user input and sample preparation. This device designed with an integrated plasma separation membrane eliminates the sample preparation step to remove red blood cells prior to running the assay.

[0111] Using a model for the top layer of the fully multiplexed immunoassay, we tested four commercially available plasma separation membranes (Whatman™ GF/DVA, MFl, LFl, and Fusion 5). We found that MFl and LFl membranes were suited to the device. Each membrane has an appropriate blood volume related to the membrane absorbance. Appropriate volumes for the MFl membrane ranged from 10 to 17 μΐ_^, and appropriate volumes for the LFl membrane ranged from 10 to 20 μΐ_^. Blood volume is controlled to prevent hemolysis and red blood cell overflow. Volumes that were too large resulted in the paper channel filling with red blood cells from spill over or hemolysis (Figures 14A-14D).

[0112] Although the terms, first, second, third, etc., may be used herein to describe various elements, these elements are not to be limited by these terms. These terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary

embodiments. Spatially relative terms, such as "above," "below," "left," "right," "in front," "behind," and the like, may be used herein for ease of description to describe the relationship of one element to another element, as illustrated in the figures. It will be understood that the spatially relative terms, as well as the illustrated configurations, are intended to encompass different orientations of the apparatus in use or operation in addition to the orientations described herein and depicted in the figures. For example, if the apparatus in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term, "above," may encompass both an orientation of above and below. The apparatus may be otherwise oriented (e.g., rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Further still, in this disclosure, when an element is referred to as being "on," "connected to," "coupled to," "in contact with," etc., another element, it may be directly on, connected to, coupled to, or in contact with the other element or intervening elements may be present unless otherwise specified.

[0113] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting of exemplary embodiments. As used herein, singular forms, such as "a" and "an," are intended to include the plural forms as well, unless the context indicates otherwise.

[0114] It will be appreciated that while a particular sequence of steps has been shown and described for purposes of explanation, the sequence may be varied in certain respects, or the steps may be combined, while still obtaining the desired configuration. Additionally, modifications to the disclosed embodiment and the invention as claimed are possible and within the scope of this disclosed invention.

Claims

Claims

1. A three-dimensional microfluidic device comprising: a plurality of porous, hydrophilic cellulosic layers each comprising one or more hydrophilic regions and/or hydrophilic channels embedded in the porous, hydrophilic cellulosic layer; wherein the hydrophilic channel is fluidically connected to the hydrophilic region; the hydrophilic regions comprise a sample deposition zone, one or more assay zones in fluidic communication with the sample deposition zone; and a buffer deposition zone; and the assay zone comprises one or more reagents embedded therein selected from the group consisting of antigens and antibodies; and a valve layer comprising a valve switchable from a first position where the buffer zone and the assay zone are not in fluidic communication, to a second position where the buffer deposition zone and the assay zone are in fluidic communication.

2. The device of claim 1, wherein the plurality of cellulosic layers comprise a first porous, hydrophilic cellulosic layer comprising a first hydrophilic region in fluidic communication with the buffer deposition zone and a second porous, hydrophilic cellulosic layer comprising a second hydrophilic region in fluidic communication with the assay zone; and the valve layer is disposed in between the first and second porous, hydrophilic cellulosic layers and the valve aligns with at least portions of the first and second hydrophilic regions.

3. The device of claim 2, wherein the valve comprises a cavity within the valve layer wherein in the first position, the first and second hydrophilic regions are separated by the cavity and not in contact; and in the second position, the first and second hydrophilic regions are pressed into the cavity to contact each other.

4. The device of any one of claims 1-3, wherein the valve layer is a double-sided adhesive.

5. The device of any one of the preceding claims, wherein the cellulosic layer comprises a material selected from the group consisting of nitrocellulose acetate, cellulose acetate, cellulosic paper, filter paper, tissue paper, writing paper, printing paper, blotting paper, chromatography paper, paper towel, and cloth.

6. The device of any one of the preceding claims, wherein the porous, hydrophilic cellulosic layer comprises a hydrophobic barrier substantially permeating the thickness of the cellulosic layer and defining the hydrophilic regions and/or hydrophilic channels.

7. The device of claim 6, wherein the hydrophobic barrier comprises wax.

8. The device of any one of the preceding claims, wherein the hydrophilic regions from two adjacent cellulosic layers are aligned vertically and in contact with each other to enable vertical fluidic flow.

9. The device of any one of the preceding claims, wherein the hydrophilic channels connecting the hydrophilic regions within the same cellulosic layer enables horizontal fluidic flow.

10. The device of any one of the preceding claims, wherein the device comprises one, two, three, four, five, six, or more assay zones.

11. The device of any one of the preceding claims, wherein the reagent is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

12. The device of claim 11, wherein the reagent is an antigen for a disease-specific antibody of interest in a sample.

13. The device of claim 11, wherein the reagent is the antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

14. The device of any one of the preceding claims, wherein the hydrophilic regions further comprises a storage region embedding one or more antigens or antibodies and in fluidic communication with the buffer zone.

15. The device of claim 14, wherein the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

16. The device of claim 14, wherein the storage region is embedded with a secondary, reporter antibody specific for a disease-specific antibody of interest in a sample.

17. The device of claim 14, wherein the storage region is embedded with a secondary, reporter antibody specific for the antibody of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

18. The device of claim 16 or 17, wherein the reporter antibody is conjugated with metal nanoparticles.

19. The device of claim 16 or 17, wherein the reporter antibody is conjugated with gold nanoparticles.

20. The device of any one of the preceding claims, further comprising a splitting layer for splitting the sample into a plurality of hydrophilic regions each in fluidic communication with one of the assay regions.

21. The device of any one of the preceding claims, further comprising a blotting layer to promote efficient fluidic flow.

22. The device of claim 21, wherein the blotting layer comprises a blotting paper.

23. The device of any one of the preceding claims, further comprising a docking layer configured to align the cellulosic layer comprising the assay zones with an adjacent cellulosic layer and to enable removing the assay zones from the device for observation.

24. The device of any one of the preceding claims, further comprising a filtration membrane.

25. The device of claim 24, wherein the filtration membrane is a plasma separation membrane in fluid communication with the sample deposition zone and configured to filter the sample.

26. The device of claim 25, wherein the plasma separation membrane is selected from the group consisting of plasma separation membranes.

27. The device of any one of the preceding claims, further comprising an additional buffer deposition zone; and an additional valve switchable from a first position where the additional buffer zone and the assay zone are not in fluidic communication, to a second position where the additional buffer zone and the assay zone are in fluidic

communication.

28. The device of claim 27, further comprising an additional storage zone embedding an additional reporter antibody and in fluidic communication with the additional buffer zone.

29. A method of detecting a disease-specific antibody or antigen in a sample, comprising: providing the device of any one of the preceding claims; depositing a biological fluidic sample into the sample deposition zone; allowing the fluidic sample to flow into the one or more assay zones; depositing a buffer into the buffer deposition zone; and activating the valve to switch from the first position to the second position.

30. The method of claim 29, wherein the sample is a blood sample, a plasma sample, or a urine sample.

31. The method of claim 29, wherein the sample comprises the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

32. The method of claim 29, further comprising filtering the sample.

33. The method of any one of claims 29-32, further comprising splitting the fluid in the sample into two, three, four, five, six, or more assay zones.

34. The method of any one of claims 29-33, wherein the reagent is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

35. The method of any one of claims 29-34, wherein the sample comprises a disease-specific antibody and the reagent is an antigen for the disease-specific antibody.

36. The method of claim 35, further comprising allowing the antigen to bind to the disease-specific antibody in the sample.

37. The method of claim 29, wherein the hydrophilic regions further comprises a storage region embedding one or more antigens or antibodies and in fluidic communication with the buffer zone.

38. The method of claim 37, wherein the antibody or antigen is the antibody or antigen of HIV, HAV, HBV, Treponema pallidum, syphilis or Brucella.

39. The method of any one of claims 29-38, wherein the hydrophilic regions further comprises a storage region embedding a reporter antibody specific for a disease- specific antibody of interest in a sample.

40. The method of claim 39, wherein the reporter antibody is conjugated with metal nanoparticles.

41. The method of claim 40, wherein the reporter antibody is conjugated with gold nanoparticles.

42. The method of any one of claims 39-41, further comprising allowing the reporter antibody bind to the disease-specific antibody.

43. The method of claim 35, wherein the buffer comprises a reporter antibody specific for the disease-specific antibody.

44. The method of claim 43, wherein the reporter antibody is conjugated with metal nanoparticles.

45. The method of claim 44, wherein the reporter antibody is conjugated with gold nanoparticles.

46. The method of claim 45, further comprising allowing the reporter antibody bind to the disease-specific antibody.

47. The method of claim 42 or 46, further comprising observing the assay zone for color indicative of the gold nanoparticles.