An Interface-Regulated Electrochemical Biosensing Platform Based on the Cascade Amplification of Primer Exchange Reaction and CRISPR/Cas12a for Noninvasive Bladder Cancer Diagnosis

Early screening and timely treatment of bladder cancer are crucial to improving patient survival rates. However, the lack of sensitive and noninvasive diagnostic approaches remains a major challenge. Herein, we developed an electrochemical biosensor for urinary miRNA-182 in bladder cancer detection based on the primer exchange reaction (PER) and CRISPR/Cas12a one-pot cascade reaction. Specifically, the presence of the target miRNA-182 initiated the PER reaction to generate multiple CRISPR/Cas12a activation sequences, which subsequently activated the trans-cleavage activity of CRISPR/Cas12a and induced the cleavage of methylene-blue-labeled signal probes (SP-MB). The uncleaved SP-MB was captured by the complementary sequence (CS) immobilized on the electrode surface, generating an electrochemical response signal that decreased with increasing target concentration, thereby enabling the quantitative detection of miRNA-182. Notably, the detection performance of biosensors constructed on different DNA nanostructure-modified interfaces (S, H, TDN, and TDR) containing CS was systematically compared to elucidate the regulatory mechanism of the biosensing interface. The optimized interface significantly improved the dynamic detection range, with a low detection limit of 1.60 pM. This study not only provides a promising noninvasive diagnostic strategy for bladder cancer but also provides fundamental insights into the rational design of DNA nanostructure-based biosensing interfaces for high-performance electrochemical biosensors.