An amplification-free and in situ detection system for miRNA-451 with single-nanoparticle LSPR biosensing on a multi-channel microfluidic chip

Early screening for colorectal cancer (CRC) is crucial for reducing its mortality. This study developed a amplification-free, highly sensitive sensing system based on localized surface plasmon resonance (LSPR) technology integrated with a multi-channel microfluidic chip platform for the in situ detection of the key CRC biomarker microRNA-451 (miRNA-451) using gold nanoparticles (AuNPs), achieving a limit of detection (LOD) of 19.2 fM. The core of this system is a multi-channel microfluidic chip serving as the detection substrate, where single-stranded DNA (ssDNA) probes are immobilized on the AuNPs surfaces to construct the sensor. In the presence of the target miRNA-451, it hybridizes specifically with two types of ssDNA probes, inducing the formation of AuNPs dimers and consequently causing a red shift in their LSPR scattering spectra. Unlike conventional methods, this study employed a precise single-nanoparticle tracking strategy, quantifying the target by statistically analyzing the proportion of AuNPs exhibiting significant spectral red shifts within the same field of view (FOV). This approach effectively mitigates spatial sampling errors arising from FOV inconsistencies. Furthermore, the system decouples the "hybridization" and "detection" functions, significantly enhancing detection efficiency and accuracy. This work provides a robust technical platform for the amplification-free and highly sensitive detection of miRNA-451.