CRISPR-mediated 3D nanostructured surface plasmon resonance imaging biosensor for highly sensitive microRNA detection

MicroRNA(miRNA) has emerged as a particularly promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, current detection methods for miRNA remain challenges in terms of sensitivity and pre-amplification steps. This study proposes a novel surface plasmon resonance imaging (SPRi) biosensor that combines tetrahedral DNA nanostructures (TDN)-based surface organization with CRISPR-Cas13a-mediated molecular recognition, enabling amplification-free, sensitive, and specific detection of AD-related miRNA-137. The biotin-labeled RNA reporters are anchored to TDN framework on the gold film surface, ensuring the orientation of the probe. In the presence of target miRNA-137, the CRISPR-Cas13a system activates, cleaves these reporters and prevents subsequent binding of streptavidin-HRP, thereby inhibiting precipitate formation from the enzymatic reaction. Under optimal condition, this sensor, integrated with the self-developed highly sensitive intensity-modulated SPRi system, displays a good linear response to miRNA-137 ranging from 0.5 pM to 500 nM with a detection limit of 212 fM. The platform demonstrates excellent specificity of discriminating single-base mutation, and reliable analysis capability in clinical plasma samples. Notably, our sensor confirms significantly reduced miRNA-137 levels in AD patients compared to healthy people. This innovative assay provides a powerful tool for the detection of AD-related miRNAs, promising to advance early diagnosis research for AD.