3D vertical-flow paper-based device for simultaneous detection of multiple cancer biomarkers by fluorescent immunoassay

A novel three-dimensional (3D) vertical-flow paper-based device (3VPD), integrated with a sandwich-type fluorescent immunoassay, was developed for multiplexed detection of cancer biomarkers simultaneously based on the principle of origami. The developed 3VPD, composed of a sample layer, a splitting layer, and a test layer, could be fabricated simply by wax-printing on paper at a large scale. Herein, a vertical-flow immunoassay was introduced into the device, in which fluorescein isothiocyanate labeled antibodies were coated on the splitting layer and mouse monoclonal capture antibodies were functionalized on the test layer. The proposed 3VPD showed high sensitivity and selectivity for detecting three cancer biomarkers, carcinoembryonic antigen (CEA), a-fetoprotein (AFP), and cancer antigen 199 (CA199) with detection limits of 0.03 ng/mL, 0.05 ng/mL, and 0.09 U/mL in human serum, respectively. The results obtained in human serum were compatible with the reference values which were acquired from parallel single-analyte. The developed 3VPD provides a new strategy to develop an inexpensive and sensitive approach for simultaneous detection of multiple analytes and comprehensive clinical diagnosis by a single test.