Paper‐Based ELISA

Abstract

# These authors contributed equally to this work.1 This paper describes enzyme-linked immunosorbent assays (ELISA) performed in a 96-microzone plate made out of paper (paper-based ELISA, or P-ELISA). ELISA is widely used in biochemical analyses, including immunoassays, food industry assays for food allergens, and toxicological assays. These assays are typically carried out in microtiter plates or small vials.1,2 ELISA combines the specificity of antibodies and the high-turnover catalysis by enzymes, to provide specificity and sensitivity. 1,2 We have recently described a 96-microzone paper plate-fabricated by patterning hydrophobic polymer in hydrophilic paper-as a platform for biochemical analysis. 3,4 Although microfluidic paper-based analytical devices (µPADs) were designed primarily to provide analytical capability at low cost in developing countries, [5][6][7] we expect that they will also be useful in applications such as point-of-care clinical analysis, military field operations, and others where high throughput, low volumes of sample, low cost, and robustness are important. 6,7 These devices have so far been prototyped using analyses of simple metabolites: glucose, protein, and certain enzymes. 8-10 P-ELISA combines the sensitivity and specificity of ELISA with the convenience, low cost and ease-of-use of paper-based platforms.Porous membranes, including nitrocellulose and filter paper, have been used for decades in dot-immunobinding assays (DIA). [8][9][10][11][12][13] Though DIAs are the simplest form of immunoassays on paper, they typically require one piece of nitrocellulose for each assay, the pieces of nitrocellulose have to be processed individually in Petri dishes, and the assays take several hours to complete. 9 Quantitative DIAs have been reported, 14 but DIAs are typically qualitative, and provide only "yes/no" results. 15 Traditional ELISA, usually performed in 96-well plates (fabricated by injection molding in plastic), is quantitative 2 and well-suited for high-throughput assays, but each assay requires large volumes (~20-200 µL) of analyte and reagents, the incubation and blocking steps are long (≥1 h per step, because the reagents must diffuse to the surface of the wells), and the results are quantified using a plate reader, typically an ~$20,000 instrument. 9,16Paper microzone plates for ELISA can have the same layout as plastic 96-wellplates, but each test zone requires only ~3 µL of sample, and the results can be measured using a desktop scanner, typically a ~$100 instrument. In addition, an entire P-ELISA can be completed in less than one hour. The ease of fabrication of paper microzone plates also opens opportunities for a wide range of non-standard formats, and customized connections to carry reagents between zones. To evaluate the feasibility of P-ELISA, and the potential advantages and disadvantages of P-ELISA and 96-well-plate-based ELISA,we developed a three-step procedure that i) immobilizes targeted antigens and then incubates them with their primary antibodies on a 96-microzo...

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