Structure-switching G-quadruplex: An efficient CRISPR/Cas12a signal reporter for label-free colorimetric biosensing.

G-quadruplex is widely used as a signal reporter for colorimetric biosensor construction. However, the effectiveness of CRISPR/Cas12a in trans-cleaving G-quadruplexes is significantly influenced by their resistance to nuclease, resulting in a weak colorimetric signal response. Herein, a structure-switching G-quadruplex regulated by transducer DNA is used as a signal reporter to construct CRISPR/Cas12a-based biosensors. The transducer DNA lacks a stable secondary structure, enabling efficient cleavage by CRISPR/Cas12a, which subsequently affects the catalytic activity of the G-quadruplex/hemin DNAzyme. We used microRNAs (miRNAs) and ATP as model targets to develop a label-free colorimetric detection platform. By optimizing the DNA sequences and reaction conditions, the biosensors exhibit excellent detection selectivity and sensitivity. The reliability of the proposed method was validated by its consistency with RT-qPCR for miRNAs detection and a commercial chemiluminescence kit for ATP assay, demonstrating its potential in clinical diagnosis and bioanalytical studies. The assay is concise and cost-effective because it does not require DNA labeling, magnetic separation, or enzymatic DNA amplification. Our design strategy avoids the use of G-quadruplex as a cleavage substrate for CRISPR/Cas12a while ensuring an efficient response of the G-quadruplex/hemin DNAzyme to CRISPR/Cas12a system, addressing the issue of G-quadruplex resistance to CRISPR/Cas12a nuclease activity.