A Reusable Paper-in-Polymer-Pond (PiPP) Hybrid Microfluidic Microplate for Multiplexed Ultrasensitive Detection of Cancer Biomarkers

Abstract

Conventional affinity-based colorimetric enzyme-linked immunosorbent assay (ELISA) is one of the most widely used methods for the detection of biomarkers. However, rapid point-of-care (POC) detection of multiple cancer biomarkers by conventional ELISA is limited by long incubation time, large reagent volume, and costly instrumentations along with low sensitivity due to the nature of colorimetric methods. Herein, we have developed a reusable and cost-effective paper-in-polymer-pond (PiPP) hybrid microfluidic microplate for ultrasensitive and high throughput multiplexed detection of disease biomarkers within an hour without using specialized instruments. A piece of pre-patterned chromatography paper placed in the polymer PMMA pond facilitates rapid protein immobilization to avoid intricate surface modifications of polymer and can be changed with a fresh paper layer to reuse the device. Reagents can be simply delivered from the top PMMA layer to multiple microwells in the middle PMMA layer via flow-through microwells and reagent delivery, thereby increasing the efficiency of washing and avoiding repeated manual pipetting or costly robots. Quantitative colorimetric analysis was achieved by calculating the brightness of images scanned by an office scanner or a smartphone camera. Sandwich type immunoassay was performed in the PiPP hybrid device after the optimization of multiple conditions. Limits of detection of 0.32 ng/mL for carcinoembryonic antigen (CEA) and 0.20 ng/mL for Prostate Specific Antigen (PSA) were obtained, which were at least 10 folds better than those of commercial ELISA kits. We envisage that this simple but versatile hybrid device can have broad applications in various bioassays in resource-limited settings.