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. 2016 Jul 26:6:30474.
doi: 10.1038/srep30474.

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers

Sharma T Sanjay  1 , Maowei Dou  1 , Jianjun Sun  2 , XiuJun Li  1   2   3
Affiliations

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers

Sharma T Sanjay et al. Sci Rep. .

Abstract

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings.

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Figures

Figure 1
Figure 1. Chip design of the PMMA/paper hybrid microfluidic microplate.
(a) 3D schematic of the exploded view of the hybrid device. (b) Cross-section view of the chip. The chip consists of three PMMA layers. The top layer (I) for fluid delivery consists of inlet reservoir ‘1’ and fluid distribution channel ‘3’. The middle layer (II) for incubation consists of 56 unique funnel-shaped microwells, with upper microwell ‘4’ and lower microwell ‘6’, with paper disks ‘5’ placed in between. The lowermost layer (III) for fluid removal consists of an outlet channel ‘7’, which leads to a common outlet reservoir ‘8’. (c) 3D exploded view of the funnel-shaped microwell. (d) Photograph of the actual assembled device with water and different colored dyes in alternate columns.
Figure 2
Figure 2
Rapid immobilization of antibodies (a) and effectiveness of blocking buffer (b) for ELISA on the hybrid device. (a) Fluorescence image of Cy3-labeled IgG immobilized on the hybrid device. Different concentrations of IgG includes 100 μg/mL, PBS, 50 μg/mL, PBS, 25 μg/mL, PBS, 12.5 μg/mL, and PBS, respectively, from left to right. (b) The mean fluorescence intensity of 20 μg/mL of Cy-3 IgG immobilized on the hybrid device with and without the blocking buffer.
Figure 3
Figure 3. Optimization of the incubation time for BCIP/NBT.
The graph shows the average brightness value of the enzymatically-developed colour as measured by ImageJ for different IgG concentrations against the incubation time.
Figure 4
Figure 4. Rapid detection of IgG by on-chip ELISA on a hybrid microfluidic microplate.
(a) Partial scanned image of the microfluidic microplate with different IgG concentrations by an office scanner: (1) 100 μg/mL, (2) 10 μg/mL, (3) 1 μg/mL, (4) 100 ng/mL, (5) 10 ng/mL, (6) 1 ng/mL, (7) 0.1 ng/mL and (8) 0 ng/mL. (b) Sigmoidal plot of the corrected brightness of microwells versus different IgG concentrations. The inset shows the calibration curve of IgG where the range of linearity lies between 1 ng/mL to 1 × 104 ng/mL.
Figure 5
Figure 5. Detection of HBsAg by ELISA on a hybrid microfluidic microplate.
(a) Partial scanned image of the microfluidic microplate with different HBsAg concentrations by an office scanner: (1) 340 μg/mL, (2) 34 μg/mL, (3) 3.4 μg/mL, (4) 340 ng/mL, (5) 34 ng/mL, (6) 3.4 ng/mL, (7) 0.34 ng/mL, and (8) 0 ng/mL. (b) Sigmoidal curve of the corrected brightness of HBsAg over a concentration range from 3.4 × 102 pg/mL to 3.4 × 108  pg/mL. The upper inset is the schematic of the colorimetric ELISA for detection of HBsAg, where a primary antibody (rabbit anti-HBsAg) and an ALP-conjugated secondary antibody (goat anti-rabbit IgG) are used together to form a sandwich-type immunoassay. The lower inset shows the calibration curve of HBsAg where the range of linearity lies between 0.34 ng/mL to 3.4 × 104  ng/mL.
Figure 6
Figure 6. Schematic illustration of the approach of enzymatic immunoassay on the paper/PMMA hybrid device, comprising six steps: (1) Immobilizing antibody on paper, (2) Blocking, (3) Washing, (4) Binding of enzyme conjugated antibody, (5) Washing, and (6) Enzymatic production of insoluble NBT diformazan.
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