Label-free multiplexed protein quantification in clinical samples using encodable hydrogel barcode and low-aspect-ratio micropore

Abstract

Pore-based sensing technique is an attractive protein detection platform enabling high-throughput label-free assay with portable equipment. However, conventional pore-based sensors suffer from low multiplexing capability due to complicated molecular encoding and limited robustness because of susceptibility to external factors. In this study, we present a micropore sensing platform for protein detection using geometrically encoded hydrogel barcodes. By incubating antibody-functionalized hydrogel barcodes with target proteins and translocating the hydrogel barcodes through the pore, the target concentration can be quantified by measuring an ionic current in real time. Simultaneously, the various geometries of the hydrogel barcodes allow the decoding of up to 64 analytes by measuring changes in the electrical signals representing each code. By optimization of the assay, three types of breast cancer biomarkers (MUC1, CEA, HER2) are detected with robust sensitivity comparable to ELISA. Multiplexed detection of breast cancer markers is conducted to demonstrate a high specificity and an acceptable recovery rate. To validate the clinical applicability, we differentiate of biomarker expression in plasma samples between breast cancer patients and healthy controls using micropore sensor. This pioneering approach in multiplexed protein detection offers a label-free, robust, portable, and efficient diagnostic tool for diverse clinical applications. © 2025 Elsevier B.V.