Nicotinamide adenine dinucleotide detection based on silver nanoclusters stabilized by a dumbbell-shaped probe

We have developed a novel method for detecting nicotinamide adenine dinucleotide (NAD⁺) based on fluorescent silver nanoclusters (AgNCs) stabilized by a dumbbell-shaped DNA template containing two cytosine-loops joined in a dsDNA stem. The design involves two types of components: a dumbbell-shaped DNA template and three enzymes. In the presence of NAD⁺ as a cofactor, Escherichia coli DNA ligase (E.coli DNA ligase) catalyzes template ligation to generate a sealed (no terminal nucleotides) dumbbell-shaped structure, preventing digestion by exonuclease III (Exo III) and exonuclease I (Exo I). The loop regions of the intact template serve as sites for the deposition of highly fluorescent AgNCs. In the absence of NAD⁺, the ligation reaction does not occur, and the unsealed dumbbell-shaped template is digested into mononucleotides via cooperation of Exo III and Exo I. The destruction of the DNA template results in the agglomeration of AgNCs into silver nanoparticles with low fluorescence. The fluorescence enhancement depends on the ligation and digestion of the DNA template, allowing quantitative detection of NAD⁺ in the range of 0.5 nM-5000 nM with a detection limit of ∼0.25 nM.