Electron Transfer Mechanism–Mediated Host–Guest Nanoswitch Powered Amplification-Free CRISPR/Cas12a–Electrochemiluminescence Bioassay for Alzheimer’s Disease Diagnosis

Developing a high-performance amplification-free electrochemiluminescence (ECL) assay platform that operates at a low trigger potential is a promising strategy for broadening the applications of ECL sensing. In this work, we present a host-guest interaction-mediated split-type CRISPR/Cas12a-ECL assay platform by using the highly sensitive host-guest recognition between the β-cyclodextrin-functionalized gold nanoclusters (β-CD-AuNCs) probe and methylene blue (MB) system as a proof of concept. Efficient ECL quenching of β-CD-AuNCs by MB is achieved via an electron transfer mechanism based on host-guest recognition between them. By integrating the high-specific recognition and cleavage activity of the CRISPR/Cas technology, the high quantum yield, and low trigger potential β-CD-AuNCs-based ECL probes, together with the highly sensitive and selective host-guest recognition-based split-type assay design, a novel "trinity" detection platform has been successfully constructed. Using Amyloid-β oligomers (AβOs), a key biomarker for Alzheimer's disease (AD) diagnosis and therapy, as the analyte, this amplification-free CRISPR/Cas-ECL biosensing platform enables ultrasensitive and accurate detection of AβO without requiring additional signal amplification strategies. The proposed sensing platform exhibits a linear detection range from 1.0 × 10^-8 to 1.0 × 10^-1 μg/mL for AβO detection, with a detection limit as low as 0.2 fg/mL (S/N = 3). This sensitivity approaches single-molecule levels and is 3-4 orders of magnitude lower than that of traditional ELISA. Furthermore, owing to its outstanding performance including high specificity, excellent selectivity, superior sensitivity, and strong anti-interference capability, the platform demonstrates remarkable detection performance in monitoring AβO in clinical AD blood samples, showing a good Pearson's correlation between the method and ELISA results. This work provides a powerful tool for clinical diagnosis and paves the way for therapeutic development, while also offering a rational design strategy for next-generation ECL biosensing platforms.