SENSITIVE ELISA FOR DISEASE DIAGNOSIS ON SURFACE MODIFIED POLY(METHYL METHACRYLATE) (PMMA) MICROFLUIDIC MICROPLATES

Abstract

Certain embodiments are directed to an ultrasensitive poly(methyl methacrylate) (PMMA) ELISA microfluidic microplate, where the protein is covalently bound to a poly-lysine modified or carboxylated PMMA surface.

Claims

1. An ultrasensitive microfluidic microtiter plate comprising a plurality of microwells having a poly-lysine modified or carboxylated poly(methyl methacrylate) (PMMA) capture surface, wherein the plate has a limit of detection for IgG of 200 pg/mL.

2. The microfluidic plate of claim 1, further comprising a capture protein covalently bound to the PMMA capture surface.

3. The microfluidic plate of claim 2, wherein the capture protein is covalently bound through a poly-lysine modification or carboxylation modification.

4. The microtiter plate of claim 2, wherein the capture protein is an antibody or antibody fragment.

5. The microtiter plate of claim 1, wherein the plate comprises 8 or more microwells.

6. The microtiter plate of claim 5, wherein the plate comprises 96 to 800 microwells.

7. The microtiter plate of claim 5, wherein each microwell is about 0.001 to 3 mm in diameter and 0.01 to 4 mm in depth.

8. The microtiter plate of claim 1, wherein the microwells have a flat or rounded floor.

9. The microtiter plate of claim 1, wherein the microwells are funnel shaped, having an upper diameter of 0.1 to 5 mm and a lower diameter of 0.001 to 1 mm.

10. The microtiter plate of claim 1, wherein the microwells are arranged in an array.

11. The microtiter plate of claim 10, wherein the array is a radial array or columnar array.

12. An enzyme linked immunosorbent assay (ELISA) kit comprising the microtiter plate of claim 1.

13. The kit of claim 12, further comprising a capture agent.

14. The kit of claim 13, wherein the kit comprises a plurality of capture agents.

15. The kit of claim 13, wherein the capture agent is coupled to the microtiter plate.

16. The kit of claim 13, wherein the capture agent is provided in a container separate from the microtiter plate.

17. The kit of claim 13, wherein the capture agent is an antibody.

18. The kit of claim 13, further comprising detection reagents and wash reagents.

19. A method for detecting an analyte in a sample comprising: contacting an microtiter plate of claim 1, comprising a capture agent covalently bound to the microtiter plate, wherein the capture agent binds a target analyte, with a sample suspected of comprising the target analyte; and detecting the presence of a signal.

20. The method of claim 19, wherein the signal is detected by direct visualization or by or an office scanner.

Description

DESCRIPTION OF THE DRAWINGS

[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0022] FIG. 1. Photograph of the actual device with black background.

[0023] FIG. 2A-2B. Schematic of the covalent modification of PMMA. A. poly-lysine modification; B. Carboxylation.

[0024] FIG. 3A-3C. FTIR analysis of surface modified PMMA. A. poly-lysine modification, method 1 (poly-lysine in Dimethyl sulfoxide (DMSO)); B. poly-lysine modification, method 2 (poly-lysine in Sodium hydroxide (NaOH) treated PMMA); C. Carboxylation.

[0025] FIG. 4A-4B. Fluorescence intensity of surface modified PMMA. A. poly-lysine modification; B. Carboxylation.

[0026] FIG. 5A-5B. Detection of IgG on poly-lysine modified PMMA (#1 method). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations from left to right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of IgG over a concentration range of 10.sup.2 pg/mL to 10.sup.8 pg/mL.

[0027] FIG. 6A-6B. Detection of IgG on poly-lysine modified PMMA (#2 method). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations from left to right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of IgG over a concentration range of 10.sup.2 pg/mL to 10.sup.8 pg/mL.

[0028] FIG. 7A-7B. Detection of HBsAg on poly-lysine modified PMMA (#1 method). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations, from left to right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4 μg/mL, 34 μg/mL, and 340 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of ELISA of HBsAg over a concentration range of 34×10.sup.1 pg/mL to 34×10.sup.7 pg/mL.

[0029] FIG. 8A-8B. Detection of HBsAg on poly-lysine modified PMMA (#2 method). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations, from left to right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4 μg/mL, 34 μg/mL, and 340 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of ELISA of HBsAg over a concentration range of 34×10.sup.1 pg/mL to 34×10.sup.7 pg/mL.

[0030] FIG. 9A-9B. Detection of HBcAg on surface modified PMMA (poly-lysine). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations from left to right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of HBcAg over a concentration range of 10.sup.2 pg/mL to 10.sup.7 pg/mL.

[0031] FIG. 10A-10B. Detection of IgG on surface modified PMMA (carboxylation). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations from left to right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, 10 μg/mL, and 100 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of IgG over a concentration range of 10.sup.2 pg/mL to 10.sup.8 pg/mL.

[0032] FIG. 11A-11B. Detection of HBsAg on surface modified PMMA (carboxylation). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations, from left to right: blank, 0.34 ng/mL, 3.4 ng/mL, 34 ng/mL, 340 ng/mL, 3.4 μg/mL, 34 μg/mL, and 340 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of ELISA of HBsAg over a concentration range of 34×10.sup.1 pg/mL to 34×10.sup.7 pg/mL.

[0033] FIG. 12A-12B. Detection of HBcAg on surface modified PMMA (carboxylation). (A) Scanned image of enzymatic converted substrate in different columns of the chip with concentrations from left to right: blank, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL, respectively. (B) Sigmoidal curve of the corrected brightness of HBcAg over a concentration range of 10.sup.2 pg/mL to 10.sup.7 pg/mL.

[0034] FIG. 13A-13B. Multiplex assay in surface modified PMMA (poly-lysine). Scanned image of the enzyme-catalyzed substrate, (A) and bar plot of corrected brightness of the scanned image (B) for detection of HBsAg and HBcAg. From left to right: immobilized probe, none (1), HBsAg (2) and (3), HBcAg (4) and (5), and HBsAg+HBcAg (6), (7), and (8), respectively. Test: From left to right, solution containing, anti-HBsAg and anti-HBcAg (1) and (6), HBsAg (2), (4), and (7), and HBcAg (3), (5), and (8). “a” and “b” shows that the data are significantly different from each other at p=0.05.

[0035] FIG. 14A-14B. Multiplex assay in surface modified PMMA (carboxylation). Scanned image of the enzyme-catalyzed substrate, (A) and bar plot of corrected brightness of the scanned image (B) for detection of HBsAg and HBcAg. From left to right: immobilized probe, none (1), HBsAg (2) and (3), HBcAg (4) and (5), and HBsAg+HBcAg (6), (7), and (8), respectively. Test: From left to right, solution containing, anti-HBsAg and anti-HBcAg (1) and (6), HBsAg (2), (4), and (7), and HBcAg (3), (5), and (8). “a” and “b” shows that the data are significantly different from each other at p=0.05.

[0036] FIG. 15A-15B. Anti-interference test for the detection of HBsAg in surface modified PMMA (poly-lysine). Corrected brightness of the scanned image of ELISA as measured by ImageJ (A) and scanned image of the chip (B) for the detection of HBsAg. From left to right: detection of 0 ng/mL of HBsAg in the solution containing 1 μg/mL HBcAg (1), 100 ng/mL CEA (2), 250 μg/mL BSA (3), and 10 ng/mL PSA (4), respectively and 200 ng/mL of HBsAg in 1 μg/mL HBcAg (5), 100 ng/mL CEA+10 ng/mL PSA (6), 250 μg/mL BSA (7), and PBS (8), respectively. “a” and “b” shows that the data are significantly different from each other at p=0.05.

[0037] FIG. 16A-16B. Anti-interference test for the detection of HBsAg in surface modified PMMA (carboxylation). Corrected brightness of the scanned image of ELISA as measured by ImageJ (A) and scanned image of the chip (B) for the detection of HBsAg. From left to right: detection of 0 ng/mL of HBsAg in the solution containing 1 μg/mL HBcAg (1), 100 ng/mL CEA (2), 250 μg/mL BSA (3), and 10 ng/mL PSA (4), respectively and 200 ng/mL of HBsAg in 1 μg/mL HBcAg (5), 100 ng/mL CEA+10 ng/mL PSA (6), 250 μg/mL BSA (7), and PBS (8), respectively. “a” and “b” shows that the data are significantly different from each other at p=0.05.

DESCRIPTION

[0038] Infectious diseases, cancer, and other diseases are often diagnosed by immunoassay. Enzyme Linked ImmunoSorbent Assay (ELISA), one of the most commonly and widely used laboratory immunoassay methods in medical diagnostic and research applications, detects proteins based on their binding to immobilized antibodies or antigens. Even though, most ELISAs today are performed in 96-well plates and are well suited for high-throughput assays, highly complicated and specialized instruments have to be utilized to automate the assay, including robotic pipetters, plate washers, and spectrophotometers. These traditional quantitative immunoassay experiments take several hours to complete because of the hour-long incubation. Likewise, other critical issue is the consumption of large volumes of precious samples and reagents, and must be performed in a laboratory setting, which is not suitable for point-of-care detection. The progress in the field of microfluidics and MEMS technology could provide an ELISA system for real time detection, multiplexing, and reducing sample usage, for economical and high throughput diagnosis (Herrmann, (2006) Lab on a Chip, 6, 555-560).

[0039] In certain aspects a microtiter plate is made from poly (methyl methacrylate) (PMMA). PMMA has various advantages over other expensive substrates. It is more rigid and less fragile, disposable, and easy to fabricate using techniques such as hot embossing or CO.sub.2 laser ablation. Also, it does not require the longer fabrication and incubation time required for PDMS. In certain aspects PMMA is modified with polylysine or carboxylation so there is covalent binding of protein to the PMMA surface, increasing its specificity and sensitivity. Certain aspects are directed to multiplex detection of different biomarkers, for example Hepatitis B (Hepatitis B Surface antigen (HBsAg) and Hepatitis B core Antigen (HBcAg)). A microfluidics chip is created for sensitive and specific multiplex disease detection using modified PMMA, which can be both used in remote setting without laboratory facility, and also in developed countries with sophisticated instruments like microplate reader.

[0040] Hepatitis B virus (HBV) is used as a representative disease/biomarker for proof of concept studies. HBV infection is a major cause of chronic hepatic damage and of hepatocellular carcinomas worldwide (Lai et al., The Lancet, 2003, 362:2089-94). HBsAg, a qualitative serological biomarker for a developing HBV infection, can diagnose acute and chronic hepatitis B virus (Rodella et al., Journal of clinical virology, 2006, 37:206-12; Jaroszewicz et al., Journal of hepatology, 2010, 52:514-22; Ben Slama et al., Gastroentérologie Clinique et Biologique, 2010, 34:S112-S118). Also, the titer of serum HBsAg indicates the level of infection and severity of disease (Ben Slama et al., Gastroentérologie Clinique et Biologique, 2010, 34:S112-S118; Ganem and Prince, New England Journal of Medicine, 2004, 350:1118-29).

[0041] In one embodiment a microfluidic chip is prepared from poly-lysine modified or carboxylated PMMA, which is a less expensive replacement of microplate that can be used to read data by microtiter plate or scanner for low resource setting. It is economical; does not require trained personnel or considerable volumes of biological samples. Similarly, all the reagent delivery and washing steps can be integrated into the device so that it does not require robust method of reagent delivery into each well manually. In one aspect a funnel shaped PMMA has been created by laser ablation of PMMA. In certain embodiments the ELISA takes place on the upper surface of funnel, which is poly-lysine modified or carboxylated.

[0042] Certain aspects of the invention are directed to immunoassays, immunoassay devices, and immunoassay kits for detecting one or more target analyte. Immunoassays generally involve contacting with a sample directly or indirectly with a capture agent. In certain aspects the capture agent can be a polypeptide. In other aspects the capture agent can be directly or indirectly linked to a solid support, i.e., modified PMMA as described herein. Specific binding of a capture agent with a target analyte from the sample can then be detected. In certain aspects the capture agent is an antibody and the antibody can be detected by an antibody detecting polypeptide, e.g., a secondary antibody. In certain aspects the capture agent is covalently linked to the solid support, i.e., the poly-lysine modified or carboxylated PMMA microtiter plate described herein.

[0043] In a particular aspect, an immunoassay may be carried in one or more of the following steps: (i) a poly-lysine modified or carboxylated PMMA support is coated with a capture polypeptide or agent (e.g., an antibody), (ii) the support is washed and then blocked with a blocking buffer, (iii) the support is washed and a detection reagent (e.g., a secondary antibody) is added, (iv) the support can be washed and the appropriate detection reagents or methodology added or performed; and (v) the support is examined or assessed for any detectable signal.

[0044] In certain aspects a label is capable of generating a measurable signal when it is contacted with the appropriate substrate or stimulus (e.g., light comprising an appropriate wavelength of electromagnetic radiation). In some embodiments, the detection reagent is conjugated to a label selected from horseradish peroxidase (HRP), I.sup.125, alkaline phosphatase, fluorescein isothiocyanate (FITC), tetramethyl rhodamine isothiocyanate (TRITC), green fluorescent protein (GFP), allophycocyanin, phycocyanin, phycoerythrin, and phycoerythrocyanin. In some embodiments, the detection reagent is not labeled, and can be detected, for example, with a secondary antibody, that is optionally labeled.

[0045] Other embodiments are directed to an assay kit comprising a poly-lysine modified or carboxylated PMMA support. In one particular aspect, the kit may contain an antibody capture polypeptide, a capture antibody, and/or an antibody detection polypeptide. The kits of the invention may further contain at least one of following reagents for carrying out the immunoassay such as blocking buffer, stopping reagents, a label substrate, and washing solutions, for example.

[0046] According to other embodiments, an assay device used for detecting target analytes may include a poly-lysine modified or carboxylated PMMA support coated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more capture agents (e.g., capture polypeptide or an antibody). The immunoassay device of the invention may include a capture agent linked to the poly-lysine modified or carboxylated PMMA substrate.

[0047] In certain aspects the poly-lysine modified or carboxylated PMMA substrate can be part of a microfluidic chip. For example, the chip used in this study was designed in Adobe Illustrator CS5 and micro-machined using Laser cutter (Epilog Zing 16, Golden, Colo.). In certain aspects the wells in the PMMA layer are designed according to the dimension of standard 384 well plate. In one example, as seen from FIG. 1, the X-axis and Y-axis offset is 4.5 mm (d), which, is the length between the centers of 2 wells. The position of the first well corresponds to A1 of microtiter plate, with the center of first well. A1 row offset of the chip is 8.99 mm (a), similar to microtiter plate. In addition, A1 column offset of 12.13 mm (b) is same as microtiter plate. The diameter of each well is 3.6 mm.

Covalent Modification of PMMA

[0048] FIG. 2A illustrates one method for covalent modification of PMMA. First PMMA is sonicated for 10 minutes in 50% aqueous 2-propanol solution. PMMA is dried. After drying the dried PMMA is immersed in poly-lysine (e.g., 0.2%, 0.1%, or 0.05% poly-lysine solutions) in DMSO for 20 minutes at room temperature. PMMA is then rinsed with 2-Propanol. Finally, PMMA is immersed in Glutaraldehyde solution (1% w/v) at room temperature for 30 minutes. Once the PMMA is poly-lysine modified a protein is added so that there is covalent binding of protein to the PMMA surface.

[0049] In an alternatively method, PMMA is immersed in 1N NaOH solution at 55° C. for 30 minutes. Then PMMA is immerged in a poly-lysine solution (e.g., 0.2%, 0.1%, or 0.05%) at pH 7 at room temperature for 1 hour. Finally, PMMA is immersed in Glutaraldehyde solution (1% w/v) at room temperature for 30 minutes. Once the PMMA is poly-lysine modified, protein is added so that there is covalent binding of protein to the PMMA surface.

[0050] In yet another method of modification PMMA can be carboxylated (see FIG. 2B). Carboxylation methods include sonication of PMMA for 10 minutes in 50% aqueous 2-propanol solution. PMMA is dried. PMMA is then submerged in 3M sulfuric acid at 60 degrees for 20 minutes. After that it is rinsed first with water, then with 2-propanol and dried. Then EDC/NHS solution (0.35M EDC+0.1M NHS) is added and incubated for 15-20 minutes. Once the PMMA is modified, protein is added so that there is covalent binding of protein to the PMMA surface.

FTIR Analysis of Modified PMMA Surface

[0051] From FT-IR spectrum of the poly-lysine modified PMMA, it can be seen that there is a distinct absorption band from 1,150 cm.sup.−1 to 1,250 cm.sup.−1, which can be attributed to the C—O—C stretching vibration. The two bands at 1,387 cm.sup.−1 and 750 cm.sup.−1 can be attributed to the α-methyl group vibrations. The band at 986 cm.sup.−1 is the characteristic absorption vibration of PMMA, together with the bands at 1,063 cm.sup.−1 and 841 cm.sup.−1. The band at 1,723 cm.sup.−1 shows the presence of the acrylate carboxyl group. The band at 1,435 cm.sup.−1 can be attributed to the bending vibration of the C—H bonds of the —CH.sub.3 group. The two bands at 2,995 cm.sup.−1 and 2,951 cm.sup.−1 can be assigned to the C—H bond stretching vibrations of the —CH.sub.3 and —CH.sub.2— groups, respectively.

[0052] Poly-lysine modification method 1 (FIG. 3A). Some major changes can be observed in the spectrum of PMMA once it was modified. The presence of Amide I (1652 cm.sup.−1), Amide II (1533 cm.sup.−1) and C—N stretch after the treatment of PMMA with polylysine shows that the PMMA is aminated by Polylysine. The aminated PMMA was further treated with glutaraldehyde as can be seen from C═O (1637 cm.sup.−1) and C—N (1442 cm.sup.−1) stretch vibration to covalently bind the antibody to the PMMA surface. The inventors see strong absorption for Amide I (1646 cm.sup.−1) and Amide II (1555 cm.sup.−1) after the addition of an antibody, which proves the covalent binding of the antibody to the modified PMMA surface.

[0053] Poly-lysine modification method 2 (FIG. 3B). Some major changes can be observed in the spectrum of PMMA once it was treated with NaOH to get hydroxyl group followed by treatment with polylysine to get aminated PMMA. The presence of Amide I (1617 cm.sup.−1), Amide II (1534 cm.sup.−1) and C—N (1435 cm.sup.−1) stretch after the treatment of PMMA with polylysine shows that the PMMA is aminated by Polylysine. Also, we can see CH.sub.2 stretching mode of vibration at 2933 cm.sup.−1 and V1 proton mode band of peptide at 3239 cm.sup.−1. The aminated PMMA was further treated with glutaraldehyde as can be seen from C═O (1637 cm.sup.−1) and C—N (1440 cm.sup.−1) stretch vibration to covalently bind the antibody to the PMMA surface. The inventors can see strong absorption for Amide I (1643 cm.sup.−1) and Amide II (1541 cm.sup.−1) after the addition of an antibody, which proves the covalent binding of the antibody to the modified PMMA surface.

[0054] FT-IR of carboxylated PMMA (FIG. 3C). From FT-IR spectrum of the PMMA, it can be seen that there is a distinct absorption band from 1,150 cm.sup.−1 to 1,250 cm.sup.−1, which can be attributed to the C—O—C stretching vibration. The two bands at 1,387 cm.sup.−1 and 750 cm.sup.−1 can be attributed to the α-methyl group vibrations. The band at 986 cm.sup.−1 is the characteristic absorption vibration of PMMA, together with the bands at 1,063 cm.sup.−1 and 841 cm.sup.−1. The band at 1,723 cm.sup.−1 shows the presence of the acrylate carboxyl group. The band at 1,435 cm.sup.−1 can be attributed to the bending vibration of the C—H bonds of the —CH.sub.3 group. The two bands at 2,995 cm.sup.−1 and 2,951 cm.sup.−1 can be assigned to the C—H bond stretching vibrations of the —CH.sub.3 and —CH.sub.2— groups, respectively.

[0055] Some major changes can be observed in the spectrum of PMMA once it was modified. Treatment of PMMA with H.sub.2SO.sub.4 creates a carboxyl functional group in the PMMA surface. The splitted C═O stretch vibration for the acrylated (1724 cm.sup.−1) and non-acrylated (1699 cm.sup.−1) carboxyl group proves the formation of carboxyl functional group at the surface of PMMA. Then, the well-known EDC/NHS method was used to treat the carboxylated PMMA to get the amine-reactive sulfo-NHS ester, which can be proved by the S═O (1347 cm.sup.−1) assymetric stretch, S—O (853 cm.sup.−1) stretch and S═O (1152 cm.sup.−1) symmetric stretch. A strong absorption for Amide I (1558 cm.sup.−1) and Amide II (1646 cm.sup.−1) can be seen after the addition of the antibody, which proves the covalent binding of the antibody to the modified PMMA surface.

[0056] Once the PMMA was modified, 20 μg/mL of Cy-3 IgG was added to the PMMA surface and incubated for 20 minutes and washed with PBST for three times. Fluorescence intensity was measured before and after washing with PBST (FIG. 4A-4B). The covalent modification method with highest antibody binding will be used in subsequent experiment for multiplex infectious diseases detection.

[0057] Detection of IgG. For IgG detection assay, the primary Antibody, IgG (0.1 ng/mL-100 μg/mL in 10 mM, pH 8.0 PBS) was pipetted to the chip. After the chip was incubated with primary antibody for 20 minutes, the unreacted PMMA surface was blocked with Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05% Tween 20) for another 20 minutes. After that, it was washed with washing buffer, PBST (10 mM, pH 7.4 PBS+0.05% Tween 20). Following washing, anti-rabbit IgG-Alkaline phosphatase (6 μg/mL) was added. It was then incubated for another 7 minutes. Then, the final wash was done with washing buffer for three times. Finally, the substrate for the alkaline phosphatase, i.e., BCIP/NBT (Nitroblue tetrazolium+5-bromo, 4-chloro, 3-indoyl phosphate) was added. NBT is used with the alkaline phosphatase substrate BCIP in western blotting and immunohistological staining and immunoassay procedures. These substrate systems produce an insoluble NBT diformazan end product that is blue to purple in color and can be observed visually. After 10 minutes, the PMMA was scanned with scanner and the brightness value was measured by using the software ImageJ.

[0058] The limit of detection (LOD) is defined as the concentration value that generates a signal three standard deviation above the blank value. The calibration curve of IgG on surface modified PMMA (#1 method) was linear over the range of 10.sup.2 pg/mL to 10.sup.6 pg/mL with a regression curve of y=9.72 log (x)+98.95 (r.sup.2=0.98). The LOD of IgG on surface modified PMMA (#1 method) was found to be 200 pg/mL. (FIG. 5A-5B)

[0059] The calibration curve of IgG on surface modified PMMA (#2 method) was linear over the range of 10.sup.2 pg/mL to 10.sup.7 pg/mL with a regression curve of y=12.26 log (x)+42.49 (r.sup.2=0.98). The LOD of IgG on surface modified PMMA (#2 method) was found to be 140 pg/mL. (FIG. 6A-6B)

[0060] The limit of detection (LOD) is defined as the concentration value that generates a signal three standard deviation above the blank value. The calibration curve of IgG on surface modified PMMA was linear over the range of 10.sup.2 pg/mL to 10.sup.8 pg/mL with a regression curve of y=17.86 log (x)+9.57 (r.sup.2=0.98). The LOD of IgG on surface modified PMMA was found to be 190 pg/mL. (FIG. 10A-10B)

[0061] HBsAg Detection assay. Different concentration of HBsAg (0.34 ng/mL-340 μg/mL in 10 mM, pH 8.0 PBS) was introduced to the wells in the modified PMMA surface. After the PMMA was incubated with antigen for 20 minutes, the unreacted PMMA surface was blocked with Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05% Tween 20) for another 20 minutes. After that, primary antibody i.e., anti-HBsAg was added and incubated for 20 minutes. It was washed once with washing buffer, PBST (10 mM, pH 7.4 PBS+0.05% Tween 20). Following washing, alkaline phosphatase labelled secondary antibody (6 μg/mL) was added. It was again incubated for another 7 minutes. Then, the final wash was done with washing buffer for three times. Finally, the substrate for the alkaline phosphatase, i.e., BCIP/NBT (Nitroblue tetrazolium+5-bromo, 4-chloro, 3-indoyl phosphate) was added. After 10 minutes, the PMMA was scanned with scanner and the brightness value was measured using the software ImageJ.

[0062] The calibration curve of HBsAg on poly-lysine modified PMMA (#1 method) was linear over the range of 34×10.sup.1 pg/mL to 34×10.sup.6 pg/mL with a regression curve of y=13.13 log (x)+75.1 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA (#1 method) was found to be 180 pg/mL. (FIG. 7A-7B)

[0063] The calibration curve of HBsAg on poly-lysine modified PMMA (#2 method) was linear over the range of 34×10.sup.1 pg/mL to 34×10.sup.6 pg/mL with a regression curve of y=16.46 log (x)+74.9 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA (#2 method) was found to be 160 pg/mL. (FIG. 8A-8B)

[0064] The calibration curve of HBsAg on carboxylated PMMA was linear over the range of 34×10.sup.1 pg/mL to 34×10.sup.6 pg/mL with a regression curve of y=11.05 log (x)+91.76 (r.sup.2=0.98). The LOD of HBsAg on surface modified PMMA was found to be 360 pg/mL. (FIG. 11A-11B)

[0065] Detection of HBcAg. Different concentration of HBcAg (0.1 ng/mL-10 μg/mL in 10 mM, pH 8.0 PBS) was introduced to the wells in the modified PMMA surface. After the PMMA was incubated with Antigen for 20 minutes, the unreacted PMMA surface was blocked with Bovine Serum Albumin (4.5% BSA w/v in PBS+0.05% Tween 20) for another 20 minutes. After that, primary antibody i.e., anti-HBsAg was added and incubated for 20 minutes. It was washed once with washing buffer, PBST (10 mM, pH 7.4 PBS+0.05% Tween 20). Following washing, alkaline phosphatase labelled secondary antibody (6 μg/mL) was added. It was again incubated for another 7 minutes. Then, the final wash was done with washing buffer for three times. Finally, the substrate for the alkaline phosphatase, i.e., BCIP/NBT (Nitroblue tetrazolium+5-bromo, 4-chloro, 3-indoyl phosphate) was added. After 10 minutes, the PMMA was scanned with scanner and the brightness value was measured using the software ImageJ.

[0066] The calibration curve of HBcAg on surface modified PMMA was linear over the range of 10.sup.2 pg/mL to 10.sup.7 pg/mL with a regression curve of y=14.07 log (x)+29.64 (r.sup.2=0.98). The LOD of HBcAg on surface modified PMMA was found to be 380 pg/mL. (FIG. 12A-12B)

[0067] Multiplex Detection. The surface modified PMMA was used for simultaneous colorimetric detection of HBsAg and HBcAg. As shown in the diagram first column is negative control without any antigen, hence no color development. Second and third columns are for the detection of HBsAg while fourth and fifth columns are for the detection of HBcAg. Third and fourth columns do not develop color, as they do not have the respective antibody against the antigen but second and fifth columns develop color as they have their respective antibody. Sixth, seventh and eight columns have both the antigen i.e. HBsAg and HBcAg. All of them develop color as they have their respective antibody or the mixture of both the antibody. (FIG. 13A-13B and FIG. 14A-14B)

[0068] Anti-interference Test. The detection assay needs to have a high anti-interference capability to screen various infectious diseases as the serum contains complex ingredients consisting of hundreds of different proteins with a wide range of concentration that may interfere the detection of target proteins. Anti-interference experiments were performed in the various columns of the modified PMMA. The experiment shows the detection of HBsAg 200 ng/mL with and without various interfering proteins (1 μg/mL HBcAg, 100 ng/mL carcinoembryonic antigen (CEA), 250 μg/mL BSA, and 10 ng/mL prostate specific antigen (PSA)). As shown in the diagram, first four columns do not contain HBsAg while the last four columns contains 200 ng/mL of HBsAg with various concentration of interfering proteins. In the absence of HBsAg, there is no development of color. Furthermore, the color intensity for the detection of 200 ng/mL of HBsAg in the presence of different interfering protein was similar to the detection of 200 ng/mL of HBsAg without the interfering protein. It demonstrates that even 1,250 times concentrated interfering proteins could not influence the specific detection of HBsAg. (FIG. 15A-15B and FIG. 16A-16B).