In Situ Single‐Particle LSPR Biosensor for Amplification‐Free Detection of miR‐451 via Dark‐Field Scattering Spectroscopy

Colorectal cancer (CRC) is frequently diagnosed at advanced stages due to the asymptomatic nature of early disease and the semi-invasive nature of colonoscopy. Developing effective noninvasive methods for CRC-related biomarker detection is crucial for reducing CRC mortality. Notably, miR-451 has been reported as a CRC-related biomarker candidate. We present an in situ single-particle spectroscopic assay for amplification-free quantitative detection of miR-451 using gold nanoparticles (AuNPs). Functionalized substrates with immobilized AuNPs enable real-time monitoring of localized surface plasmon resonance (LSPR) peak shifts at the single-particle level. By statistically analyzing the proportion of AuNPs exhibiting LSPR shifts, we achieved quantitative detection of miR-451. This in situ microfluidic configuration allows spectral analysis of the same nanoparticles before and after detection, eliminating spatial sampling errors. Specificity is conferred by a stem-loop DNA probe on the AuNP surface via strand displacement. The main advance lies in in situ tracking of the same immobilized nanoparticles with statistical single-particle readout. This strategy demonstrates a detection limit of 58 fm and excellent specificity against other miRNAs, with proof-of-concept feasibility in spiked FBS samples.