A trifunctional split dumbbell probe coupled with ligation-triggered isothermal rolling circle amplification for label-free and sensitive detection of nicotinamide adenine dinucleotide.

The nicotinamide adenine dinucleotide (NAD+) is an important small biomolecule that participates in a variety of physiological functions, and it has been regarded as a potential biomarker for disease diagnosis and a promising target for disease treatment. The conventional methods for NAD+ assay often suffer from complicated procedures, expensive labeling, poor selectivity, and unsatisfactory sensitivity. Herein, we develop a label-free and sensitive method for NAD+ assay based on the integration of a trifunctional split dumbbell probe with ligation-triggered isothermal rolling circle amplification (RCA). We design a trifunctional split dumbbell probe that can act as a probe for NAD+ recognition, a template for RCA reaction, and a substrate for SYBR Green I binding. In the presence of target NAD+, it can serve as a cofactor to active E. coli DNA ligase which subsequently catalyzes the ligation of split dumbbell probe to form a circular template for RCA reaction, generating numerous dumbbell probe amplicons which can be easily and label-free monitored by using SYBR Green I as the fluorescent indicator. Due to the high fidelity of NAD+-dependent ligation and high amplification efficiency of RCA amplification, this method exhibits high sensitivity with a detection limit of 85.6 fM and good selectivity with the capability of discriminating target NAD+ from its analogs. Moreover, this method can be applied for accurate and sensitive detection of NAD+ in complex biological samples and cancer cells, holding great potential in NAD+-related biological researches and clinical diagnosis.