Machine Learning‐Driven Nanopore Sensing for Quantitative, Label‐Free miRNA Detection

Nanopore sensors offer exceptional sensitivity for detecting single molecules, making them ideal for early disease diagnostics. In this study, we present a multiplexed nanopore-based assay that combines DNA-barcoded probes with advanced computational analysis to detect microRNAs (miRNAs) with high specificity and accuracy. Each probe selectively binds its target biomarker and induces a characteristic delay in the ionic current signal upon translocation through the nanopore. We evaluated three analytical strategies for classifying delayed versus non-delayed events: (1) moving standard deviation (MSD), (2) spectral entropy (SE), and (3) a convolutional neural network (CNN). While MSD and SE rely on manually defined thresholds and exhibit limited sensitivity, the CNN model, trained on image representations of raw current traces, achieved near-perfect classification performance across all metrics (accuracy = 0.99, precision = 0.99, recall = 0.99). Grad-CAM visualization confirmed that the CNN model focused on relevant signal regions, enhancing interpretability and generalizability. All methods produced sigmoidal concentration-response curves consistent with expected binding kinetics, and nanopore-derived delay metrics closely matched RT-qPCR validation data. All three methods were capable of distinguishing between signal classes; however, the CNN model demonstrated superior sensitivity and robustness. This work highlights the importance of data interpretation in nanopore sensing and presents a comparative framework for binary event classification. The findings pave the way for the development of machine learning-driven nanopore diagnostics capable of detecting diverse biomarker types at the single-molecule level.