Target-Induced Shielding Layer at the Dual-Affinity Probe Interface for Rapid Colorimetric Detection of Cardiac Biomarkers

Cardiac troponin I (cTnI) is a key biomarker for acute myocardial infarction (AMI); however, the available detection methods are limited by slow responses, complex steps, and costly instrumentation, restricting their suitability for rapid or point-of-care testing. Herein, we report a rapid and sensitive colorimetric assay for the detection of cTnI based on a nanoparticle shielding layer and its interfacial interactions with an enzyme. To achieve high sensitivity, we employed a dual-affinity silver nanoparticle (AgNP) probe coated with two highly specific affinity molecules, i.e., aptamer (Apt) and antibody (Ab), offering effective detection of the target molecule. The affinity layer coating was inherently imperfect, leaving exposed regions at the interface of the AgNP probe to inhibit the enzyme (urease) by Ag-SH interaction. Upon introduction of cTnI, the probe selectively binds to it, and a target-induced shielding layer is formed that prevents the interaction of the enzyme with the AgNP surface, rendering the enzyme active in solution. Consequently, the active enzyme generates a colored endpoint for visual detection in <15 min with a detection limit of 0.088 ng/mL. We assessed the assay performance in a complex matrix to evaluate the stringency. The working of the assay was studied using microscale thermophoresis (MST), which showed complete masking of the urease-particle interaction in the presence of cTnI. Unlike surface-based methods, such as ELISA, our solution-phase assay eliminates immobilization and blocking steps and achieves enhanced sensitivity, representing a paradigm shift in biomarker detection.