CRISPR-dCas13a-based electrochemical biosensor for sensitive detection of miR-331-5p as a potential biomarker of Parkinson's disease.

Abstract

This study aimed to develop a CRISPR-dCas13a-based electrochemical biosensor as a proof-of-concept analytical platform for the sensitive and selective detection of miR-331-5p, which has been reported to be associated with Parkinson's disease. The biosensor was prepared by sequentially forming a self-assembled monolayer with 11-mercaptoundecanoic acid (MUA) on a gold electrode, functionalizing the surface with a generation-4 polyamidoamine (PAMAM-G4) dendrimer, immobilizing dCas13a, and assembling the target-specific sgRNA recognition complex. The electrode-modification steps were evaluated using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The impedance response increased linearly with miR-331-5p concentration over the experimentally evaluated range of 5-400 pM. The limit of detection and limit of quantification were 7.82 and 23.71 pM, respectively. Responses to the non-target miRNAs miR-146a-5p, miR-19 b-3p, miR-24-3p, and miR-7-5p were substantially lower than the response to miR-331-5p. Triplicate spike-recovery measurements in healthy human serum and storage-stability studies supported the analytical applicability and reproducibility of the platform. These findings demonstrate a CRISPR-dCas13a/EIS-based proof-of-concept analytical platform for miR-331-5p detection. Validation in well-characterized clinical cohorts will be required before any diagnostic application can be established.