Dual Protein Corona-Mediated Target Recognition System for Visual Detection and Single-Molecule Counting of Nucleic...

Rapid, highly sensitive, and specific nucleic acid detection plays a crucial role in advancing point-of-care (POC) diagnostics for pathogens and viruses, cancer monitoring, and optimizing clinical treatments. Herein, leveraging the precise recognition ability of CRISPR/dCas9 and the powerful localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs), we report the design of a dual protein corona-mediated detection platform to simultaneously fulfill rapid POC testing and single-molecule counting of nucleic acids in a one-pot and one-step manner. This system uses guide RNA as a molecular bridge to anchor dCas9 protein onto AuNPs, forming artificial protein coronas. Upon recognizing a target, the interaction between the two protein coronas on the same nucleic acid molecule triggers cross-linked aggregation of AuNPs. Then, a target as low as 100 aM can be visually detected within 30 min, making the platform particularly well-suited for rapid POC application and the screening of emerging epidemics. Additionally, the superior LSPR properties of AuNPs increase the light-scattering signal generated during target-induced aggregation, enabling the visualization of the aggregated AuNPs as diffraction-limited spots under confocal microscopy. By counting these spots, the platform achieves unprecedented detection sensitivity, identifying a target as low as 1 aM, which is equivalent to just 6 molecules in a 10 μL system, demonstrating single-molecule detection capability. This dual protein corona-mediated detection system offers exceptional promise for large-scale screening of pathogenic viruses and the early detection of cancer, particularly in applications requiring ultrahigh sensitivity at the single-molecule level.