College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, 250014, China.
College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan, 250014, China.
Talanta. 2021 Mar 1;224:121962. doi: 10.1016/j.talanta.2020.121962. Epub 2020 Dec 3.
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.
烟酰胺腺嘌呤二核苷酸（NAD）是一种重要的小分子生物物质，参与多种生理功能，它已被视为疾病诊断的潜在生物标志物和疾病治疗的有前途的靶点。NAD 测定的常规方法通常存在复杂的程序、昂贵的标记、较差的选择性和不理想的灵敏度。在此，我们开发了一种基于多功能分裂哑铃探针与连接触发等温滚环扩增（RCA）集成的无标记和灵敏的 NAD 测定方法。我们设计了一种多功能分裂哑铃探针，它可以用作 NAD 识别的探针、RCA 反应的模板和 SYBR Green I 结合的底物。在存在靶 NAD 的情况下，它可以作为大肠杆菌 DNA 连接酶的辅因子，随后催化分裂哑铃探针的连接，形成 RCA 反应的圆形模板，产生大量哑铃探针扩增子，可以使用 SYBR Green I 作为荧光指示剂轻松且无标记地监测。由于 NAD 依赖性连接的高保真度和 RCA 扩增的高效率，该方法具有 85.6 fM 的检测限，具有较高的灵敏度和良好的选择性，能够区分靶 NAD 与其类似物。此外，该方法可用于复杂生物样品和癌细胞中 NAD 的准确灵敏检测，在 NAD 相关的生物学研究和临床诊断中具有很大的潜力。
