Integrated Immunoassays on Paper/Polymer Hybrid Microfluidic Devices for Low-Cost Detection of Disease Biomarkers
Infectious diseases and cancers have been the major cause of global death and disability causing a significant impact on global health and economies. Enzyme-linked immunosorbent assay (ELISA) is one of the most widely used laboratory diagnostic methods for infectious diseases and cancer. ELISA detects proteins based on their binding to immobilized antibodies or antigen. Even though most ELISAs performed today in 96-well plates are well suited for high throughput assays, performing ELISA in low-resource settings is limited by several factors, such as long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we have developed multiple simple, miniaturized poly(methyl methacrylate) (PMMA) and paper/PMMA hybrid microfluidic devices for the detection of infectious diseases and cancer biomarkers in low-resource settings. At first, we developed a surface modified PMMA microfluidic microplate, where the protein was covalently bound either to the carboxylated PMMA surface using carbodiimide chemistry or to the amine dense PMMA surface functionalized with polylysine. Immobilization efficiency of proteins on the surface of the modified PMMA was remarkably increased due to the covalent binding of the protein, thereby improving the sensitivity of ELISA and decreasing the background noise. The surface modified PMMA microplate, where the protein can be covalently immobilized within 20 min was used for the ultrasensitive multiplex detection of various biomarkers including Immunoglobulin G (IgG), Hepatitis B surface antigen (HBsAg), and Hepatitis B core antigen (HBcAg) with limits of detection (LODs) of 200 pg/mL, 180 pg/mL, and 300 pg/mL, respectively. The results of the assay can be viewed by the naked eye or scanned through a simple desktop scanner for quantitative analysis within 90 min as compared to 18 hours in traditional microplates. The surface modified microplate was found to be at least 10-fold more sensitive than traditional microplates with much less reagent consumption. To avoid the complicated surface modification steps, we also developed a novel paper/PMMA hybrid microfluidic microplate using a porous, 3D paper in flow-through microwells. The use of low-cost chromatography paper in the funnel-shaped microwells facilitated the rapid immobilization of protein within 10 min as compared to overnight incubation in traditional microplates. In addition, it also helped in the efficient washing, decreasing the background noise. The top reagent delivery channels can transfer reagents to multiple microwells, avoiding repeated manual pipetting or the use of costly robots. Results of these colorimetric ELISA could be observed by the naked eye within an hour. LODs of 1.3 ng/mL and 1.6 ng/mL for IgG and HBsAg were achieved without any specialized equipment, which was comparable to commercially used microplate ELISA. Likewise, for the device to be used in high-end laboratories and hospitals, the hybrid device was redesigned to make it compatible with traditional microplate readers. Chemiluminescence ELISA of HBsAg, HBcAg, and Hepatitis C virus core antigen (HCVcAg) was performed in the hybrid device with LODs of 50 pg/mL, 35 pg/mL, and 10 pg/mL, respectively, using commercial microplate reader to read the device. Our hybrid device was found to be 100-fold more sensitive than 96-well commercial microplate, even with the reduced amount of sample (5 µL as compared to 50 µL) and assay time (1 hr compared to 18 hr). To further increase the sensitivity of the device and to measure low-concentration analytes, we developed a reusable, cost-effective, and eco-friendly PMMA/paper hybrid plug-and-play (PnP) device for analyte enrichment and detection. The sample flowed back and forth through the low-cost 3D paper substrate within PMMA channels, thereby, enriching the amount of analyte adsorbed and dramatically decreasing the incubation time. After the enrichment assay, the paper substrate can be replaced so that the device can be reused. LODs, 10-fold better than commercial microplate reader and a wide linear range of five and six orders of magnitude was obtained for IgG and HBsAg, respectively. Finally, we demonstrated the broader application of our microfluidic approach by developing paper in PMMA pond hybrid microfluidic device for simultaneous detection of cancer biomarkers including prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA). The porous 3D paper kept in between the flow-through reservoir and pond, helped in rapid immobilization of protein and efficient washing, thus increasing the sensitivity and decreasing the noise. Sandwich type immunoassay was performed accordingly in the hybrid device, where the reagent is delivered automatically to the paper-substrate through the reagent delivery channel. LOD of 0.32 ng/mL for CEA and 0.20 ng/mL for PSA was obtained, which is sensitive enough to detect clinical cut off value of 5 ng/mL and 4 ng/mL for CEA and PSA, respectively. We envisage that these simple polymer/paper hybrid microfluidic microplates can be used in both underdeveloped and developed countries for low-cost, sensitive, and high-throughput bioassays of infectious diseases, cancer biomarkers, and other bio-molecules.
Sharma Timilsina, Sanjay, "Integrated Immunoassays on Paper/Polymer Hybrid Microfluidic Devices for Low-Cost Detection of Disease Biomarkers" (2018). ETD Collection for University of Texas, El Paso. AAI10815068.