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.