Encoded DNA Nanoswitch Array for Multiplexed MicroRNA Profiling via Nanopore Readout

DNA nanoswitches are programmable molecular devices that convert target recognition events into highly specific and controllable conformational changes. Nevertheless, their application in multiplexed analysis of low abundance miRNAs remains limited, and methodological breakthroughs are urgently needed for analysis in complex clinical samples. Here, we have seamlessly integrated rolling circle amplification (RCA), cloverleaf structured DNA nanoswitch arrays, and glass nanopore detection technology to construct a high throughput analysis platform, which is expected to be applied in the diagnostic research of acute myocardial infarction (AMI). The design leverages RCA to convert target miRNAs into stable DNA reporters followed by enzymatic cleavage, which enables sensitive molecular detection and effectively alleviates nanopore clogging. The cloverleaf design enhances signal amplitude by 70.77% over conventional dumbbell structures. In clinical plasma samples, the platform accurately differentiated AMI patients from healthy controls by profiling a panel of five potential AMI characteristic miRNAs. A reference free ternary encoding (RFTC) strategy further enables a 12 carrier DNA nanoswitch array capable of simultaneously detecting up to 60 targets. By combining label free DNA nanoswitch arrays with glass nanopore sensing, this work establishes a robust methodological framework for multiplex nucleic acid analysis. The platform also exhibits promising potential for analyzing circulating miRNAs and screening acute myocardial infarction related biomarkers, offering a facile modular strategy for flexible detection of diverse molecular targets in future clinical translational research.