DNA-Templated Click Ligation Chain Reaction Catalyzed by Heterogeneous CuO for Enzyme-Free Amplification and Ultrasensitive Detection of Nucleic Acids.

Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via NA-emplated lick igation hain eaction (DT-CLCR) catalyzed by a eterogeneous anocatalyst made of CuO (hnCuO). This hnCuO-DT-CLCR method is built on two cross-amplifying hnCuO-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCuO-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCuO-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCuO-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCuO-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.