Label-free biosensor based on dsDNA-templated copper nanoparticles for highly sensitive and selective detection of NAD+

We have developed a novel label-free fluorescence approach for nicotinamide adenine dinucleotide (NAD(+)) detection by coupling poly(AT-TA) dsDNA-templated fluorescent copper nanoparticles (CuNPs) as a fluorescence indicator with a dumbbell DNA probe designed for ligation. Herein, we designed a dumbbell probe which can form a self-complementary structure at both ends. In the absence of NAD(+), the extension reaction can be performed from the exposed 3'-end, and instantly opened the hybridized structure at the 5'-end, producing a chimeric poly(AT-TA) double-stranded fragment which can be used as a highly-efficient template for the formation of CuNPs, showing high fluorescence. Whereas in the presence of NAD(+), together with the E. coli DNA ligase, the 5'-phosphoryl end and the 3'-end of the DNA probe can be ligated to block the extension reaction, showing slight fluorescence. This novel NAD(+) assay is label-free, fast and easy to operate, highly sensitive and selective, which holds wide applicability as a robust analytical tool in NAD(+) related biological processes and clinical diagnosis.