The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Analyst. 2023 Feb 13;148(4):799-805. doi: 10.1039/d2an02006h.
The development of sensitive adenosine triphosphate (ATP) sensors is imperative due to the tight relationship between the physiological conditions and ATP levels . Herein, a fluorescent aptasensor for ATP is presented, which adopts a strategy that combines a split aptamer and a DNAzyme/walker with terminal deoxynucleotidyl transferase (TDT)-assisted formation of DNA-AgNCs to realize fluorescence detection of ATP. A multifunctional oligonucleotide sequence is rationally designed, which integrates a split aptamer, a DNAzyme and a DNA walker. Both multifunctional oligonucleotide and its substrate strand are connected to the surface of FeO@Au nanoparticles Au-S bonds. The existence of ATP can induce the formation of the complete aptamer, and then activate the DNAzyme to circularly cleave the substrate strand, leaving 2',3'-cyclophosphate at the 3'end of the strand. This blocks the polymerization of dCTP to form poly(C) even in the presence of TDT and dCTP, due to the lack of free 3'-OH. In contrast, when ATP is absent, the DNAzyme/walker cannot work and then TDT catalyzes the formation of poly(C) at the free 3'-OH of the substrate strand, which is subsequently utilized as the template to prepare DNA-AgNCs. The fluorescence response derived from AgNCs thus reflects the ATP concentration. Under the optimum conditions, the aptasensor shows a linear response range from 5 nM to 10 000 nM, with a detection limit of 0.27 nM. The level of ATP in human serum can be effectively measured by the aptasensor with good recovery, indicating its application potential in medical samples.
由于生理状态与 ATP 水平之间存在紧密关系，因此开发灵敏的三磷酸腺苷 (ATP) 传感器至关重要。本文提出了一种用于 ATP 的荧光适体传感器，它采用了一种结合了分裂适体和 DNA 酶/行走者与末端脱氧核苷酸转移酶 (TDT) 辅助形成 DNA-AgNCs 的策略，实现了对 ATP 的荧光检测。合理设计了一种多功能寡核苷酸序列，它集成了分裂适体、DNA 酶和 DNA 行走者。多功能寡核苷酸及其底物链都通过 Au-S 键连接到 FeO@Au 纳米粒子的表面。ATP 的存在可以诱导完整适体的形成，然后激活 DNA 酶循环切割底物链，使链的 3'端留下 2'，3'-环磷酸。这阻止了 dCTP 的聚合以形成 poly(C)，即使在存在 TDT 和 dCTP 的情况下，也由于缺少游离 3'-OH。相比之下，当没有 ATP 时，DNA 酶/行走者无法工作，然后 TDT 催化游离 3'-OH 上的 poly(C)形成，随后将其用作模板来制备 DNA-AgNCs。源自 AgNCs 的荧光响应因此反映了 ATP 浓度。在最佳条件下，适体传感器的线性响应范围为 5 nM 至 10000 nM，检测限为 0.27 nM。该适体传感器可有效测量人血清中的 ATP 水平，回收率良好，表明其在医学样本中的应用潜力。
