A Hyperbranched Rolling Circle Amplification Assay Using Aptamer-Hemin/G-Quadruplex Recognition for the Detection of Creatine Kinase-MB.

Creatine kinase-myocardial band (CK-MB) remains an important biomarker for the early diagnosis and clinical management of myocardial infarction. Here, we report a sensitive colorimetric assay for CK-MB based on a conformation-locked aptamer guanine-quadruplex (G-quadruplex) system integrated with magnetic nanoparticle capture and hyperbranched rolling circle amplification (HRCA). Target binding induces conformational unlocking of the aptamer, initiating HRCA-mediated signal amplification and generating a concentration-dependent colorimetric response in the presence of a chromogenic substrate. A compact 3D-printed imaging module, combined with a Python-based processing pipeline, was used to extract grayscale intensity values from assay images for quantitative analysis. Using only 25 μL of sample, the assay achieved limits of detection (LOD) of 0.268 ng/mL in aqueous samples and 0.328 ng/mL in blood-spiked samples, and limits of quantification (LOQ) of 0.89 ng/mL and 1.09 ng/mL, respectively, with a linear dynamic range of 0.5-100 ng/mL. Grayscale intensity was inversely proportional to CK-MB concentration, enabling quantitative detection across clinically relevant levels. The HRCA amplification process and target-induced activation were further validated by TapeStation gel electrophoresis, confirming the formation of amplified DNA nanostructures. This platform provides a low-volume, amplification-based strategy for CK-MB detection that is compatible with simple optical readout and has strong potential for point-of-care applications.