A novel polymer-based nitrocellulose platform for implementing a multiplexed microfluidic paper-based enzyme-linked immunosorbent assay

Nitrocellulose (NC) membranes, as porous paper-like substrates with high protein-binding capabilities, are very popular in the field of point-of-care immunoassays. However, generating robust hydrophobic structures in NC membranes to fabricate microfluidic paper-based analytical devices (mu PADs) remains a great challenge. At present, the main method relies on an expensive wax printer. In addition, NC membranes very easy to adhere during the printing process due to electrostatic adsorption. Herein, we developed a facile, fast and low-cost strategy to fabricate mu PADs in NC membranes by screen-printing polyurethane acrylate (PUA) as a barrier material for defining flow channels and reaction zones. Moreover, hydrophobic barriers based on UV-curable PUA can resist various surfactant solutions and organic solvents that are generally used in immunoassays and biochemical reactions. To validate the feasibility of this PUA-based NC membrane for immunoassays in point-of-care testing (POCT), we further designed and assembled a rotational paper-based analytical device for implementing a multiplexed enzyme-linked immunosorbent assay (ELISA) in a simple manner. Using the proposed device under the optimal conditions, alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) could be identified, with limits of detection of 136 pg/mL and 174 pg/mL, respectively, which are below the threshold values of these two cancer biomarkers for clinical diagnosis. We believe that this reliable device provides a promising platform for the diagnosis of disease based on ELISA or other related bioassays in limited settings or remote regions.

Automated summary

Nitrocellulose (NC) membranes are widely used in point-of-care immunoassays for their high protein-binding capabilities. However, creating hydrophobic structures in NC membranes for microfluidic paper-based analytical devices (muPADs) is challenging. In this study, we developed a simple and low-cost method using screen-printed polyurethane acrylate (PUA) to fabricate muPADs in NC membranes, and demonstrated the feasibility of using PUA-based NC membranes for immunoassays. A rotational paper-based analytical device was also designed and assembled for multiplexed enzyme-linked immunosorbent assay (ELISA) with promising results.