An amplification-free dual-readout biosensor integrating colorimetry and single-particle counting for ultrasensitive miRNA detection in esophageal cancer.

Esophageal cancer (EC) remains highly lethal partly because many patients are diagnosed at advanced stages, and current diagnostic pathways rely on invasive endoscopy and centralized molecular testing. Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers, yet amplification-based assays can be workflow-intensive and introduce protocol-dependent bias. Here we report an amplification-free, dual-readout gold nanoparticle (AuNP) biosensor for hsa-miR-320b analysis in plasma RNA extracts. Target miR-320b bridges a biotinylated capture probe on streptavidin magnetic beads and a thiolated detection probe on 40-nm AuNPs to form sandwich complexes. A shared magnetic-separation workflow enables two sequential readouts: a colorimetric verification based on AuNP depletion in the supernatant (LOD 0.085 pM) and a digital release-to-count (R2C) mode, in which RNase H selectively cleaves the RNA strand within the DNA/RNA heteroduplex to release AuNP-DNA labels for dark-field single-particle counting (LOD 5.3 fM) without nucleic-acid amplification. The assay demonstrates single-base mismatch discrimination, short-term storage stability, and spike recoveries of 89.8 -92.9% in plasma RNA extracts. In a cohort of 60 clinical plasma samples (20 EC, 20 benign lesions, 20 healthy controls), the R2C digital readout showed an EC-associated elevation of plasma miR-320b relative to benign lesions and healthy controls, with trends consistent with reverse transcription quantitative PCR (RT-qPCR) on the same extracts. This work provides a practical route to amplification-free digital miRNA quantification using countable plasmonic labels and is readily extended to other circulating targets.