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.