FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Background constrains analytical sensitivity: surveying larger sensor areas samples more analyte molecules but also accumulates false positives, limiting gains in detection performance. Here we introduce FINDER―Fluorogenic INstantaneous Digital Enumeration and Recognition―a single-molecule platform that combines kinetic fingerprinting with fluorogenic transient probes for rapid molecular classification under near-zero-background conditions. By suppressing both solution and surface-associated background at micromolar probe concentrations, FINDER classifies individual molecules within seconds-scale observation windows per field of view. This regime allows sensitivity to scale with surveyed sensor area, enabling amplification-free quantification of the miRNA cancer biomarker hsa-miR-16 with an 11 aM detection limit. FINDER further generalizes to HPV16 DNA biomarker detection, two-color RNA/DNA co-profiling, and rapid discrimination of clinically relevant EGFR single-nucleotide variants using multidimensional kinetic filtering. Rapid per-field classification permits tens of fields to be surveyed within minutes. By converting kinetic specificity into area-scalable sensitivity, FINDER enables semi-automated attomolar biomarker counting without amplification in practical workflows.