Folding vertical-flow electrochemical paper-based device for quantum dot-linked immunosorbent assay

In this work, we report the design and fabrication of a fully integrated, foldable electrochemical paper-based analytical device (ePAD) for quantum dot (QD)-linked immunosorbent assays using carcinoembryonic antigen (CEA) as a model analyte. The device comprises a circular assay zone positioned adjacent to a circular three-electrode electrochemical cell incorporating a bismuth precursor-modified working electrode as the transducer. A sandwich-type immunoassay is performed in the assay zone employing a reporter antibody functionalized with PbS QDs. Following immunocomplex formation, the assay zone is folded onto the adjacent electrochemical cell. The immunocomplex is then acidically dissolved, releasing Pb(II) ions that are vertically eluted into the electrochemical cell and subsequently quantified by anodic stripping voltammetry. The proposed platform leverages a combination of various concepts and technologies, including: (i) the use of paper as an analytical substrate; (ii) the “pen-on-paper” fabrication strategy; (iii) a folding configuration enabling vertical elution; (iv) low-cost and simple in-house preparation of the reporter antibody-PbS QDs conjugate; (v) a bismuth precursor-modified working electrode; and (vi) the favorable electrochemical properties of the PbS QDs. The platform enables ultrasensitive determination of CEA, achieving a limit of detection (LOD) of 3 pg mL −1 and a coefficient of variation of 12.9%. Recovery studies in serum samples yielded values ranging from 86% to 97%, demonstrating satisfactory analytical performance in complex matrices.