Department of Laboratory Medicine, the Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, People's Republic of China.
Department of ophthalmology, the Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210004, People's Republic of China.
Mikrochim Acta. 2019 Aug 31;186(9):662. doi: 10.1007/s00604-019-3780-3.
An electrochemical aptamer-based assay is described for the determination of CFP-10 which is an early secretary biomarker of Mycobacterium tuberculosis. CFP-10 is specifically captured by its aptamer and then induces a DNA cross-linking click reaction, the release of CFP-10, and an amplification cycle of repeated CFP-10 release. This mechanism (with dual amplification via DNA click and target release cycle) causes more and more CFP-10 Apt strands on the electrode surface to expose their 5' overhang and to hybridize with the DNA complexes linked to the gold nanoparticles (AuNPs). Consequently, large amounts of AuNPs, each loaded with a number of quadruplex DNA motifs, can be bound on the electrode surface and remarkably enhance the signal. Under optimal conditions, the method has a detection limit as low as 10 pg.mL of CFP-10. The method was successfully applied to the diagnosis of M. tuberculosis in sputum. Graphical abstract Schematic representation of an electrochemical CFP-10 (10-kDa culture filtrate protein) assay using click DNA cycling in combination with gold nanoparticles loaded with quadruplex DNA motifs. Click chemistry reaction between Dibenzocyclooctyne (DBCO)-DNA and azido-DNA can liberate the CFP-10 antigen for the next cycle, which can be viewed as the first amplification step. G-quadruplex-based DNAzyme is formed due to the guanine-rich sequences of DNA S1, which can be viewed as the second amplification step.
描述了一种基于电化学适体的测定方法，用于测定 CFP-10，这是结核分枝杆菌的早期分泌生物标志物。CFP-10 被其适体特异性捕获，然后诱导 DNA 交联点击反应、CFP-10 的释放和重复 CFP-10 释放的扩增循环。这种机制（通过 DNA 点击和目标释放循环进行双重扩增）导致电极表面上越来越多的 CFP-10 Apt 链暴露其 5'突出端，并与连接到金纳米粒子（AuNPs）的 DNA 复合物杂交。因此，可以将大量 AuNPs 结合到电极表面上，每个 AuNPs 都负载有多个四链体 DNA 基序，从而显著增强信号。在最佳条件下，该方法的检测限低至 10pg.mL 的 CFP-10。该方法成功应用于痰液中结核分枝杆菌的诊断。电化学 CFP-10（10-kDa 培养滤液蛋白）测定的示意图，使用点击 DNA 循环结合负载四链体 DNA 基序的金纳米粒子。Dibenzocyclooctyne (DBCO)-DNA 和叠氮-DNA 之间的点击化学反应可以释放 CFP-10 抗原以进行下一个循环，这可以看作是第一个扩增步骤。由于 DNA S1 中富含鸟嘌呤的序列，形成了基于 G-四链体的 DNA 酶，这可以看作是第二个扩增步骤。
