An aptamer-linked assay of a target biomarker (e.g., thrombin) is facing the challenges of long-term run, complex performance, and expensive instrument, unfitting clinical diagnosis in resource-limited areas. Herein, a facile chip electrophoresis titration (ET) model was proposed for rapid, portable, and low-cost assay of thrombin via aptamer-linked magnetic nanoparticles (MNPs), redox boundary (RB), and horseradish peroxidase (HRP). In the electrophoresis titration-redox boundary (ET-RB) model, thrombin was chosen as a model biomarker, which could be captured within 15 min by MNP-aptamer 1 and HRP-aptamer 2, forming a sandwich complex of (MNP-aptamer 1)-thrombin-(HRP-aptamer 2). After MNP separation and chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) within 10 min, an ET-RB run could be completed within 5 min based on the reaction between a 3,3',5,5'-tetramethylbenzidine radical cation (TMB•+) and l-ascorbic acid in the ET channel. The systemic experiments based on the ET-RB method revealed that the sandwich complex could be formed and the thrombin content could be assayed via an ET-RB chip, demonstrating the developed model and method. In particular, the ET-RB method had the evident merits of simplicity, rapidity (less than 30 min), and low cost as well as portability and visuality, in contrast to the currently used thrombin assay. In addition, the developed method had high selectivity, sensitivity (limit of detection of 0.04 nM), and stability (intraday: 3.26%, interday: 6.07%) as well as good recovery (urine: 97-102%, serum: 94-103%). The developed model and method have potential to the development of a point-of-care testing assay in resource-constrained conditions.
Keywords: aptamer; electrophoresis titration chip; horseradish peroxidase; nanoparticles; thrombin.