Homogeneous Electrochemical Strategy for Human Telomerase Activity Assay at Single-Cell Level Based on T7 Exonuclease-Aided Target Recycling Amplification

Abstract

As an important biomarker for early cancer diagnostics and a valuable therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Herein, a homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification is proposed for a simple, rapid, and highly sensitive assay of human telomerase activity from crude cancer cell extracts. In this strategy, a 5' methylene blue (MB)-labeled hairpin (HP) probe is designed, which can hybridize with the telomerase reaction products to initiate the subsequent digestion by T7 exonuclease, and a large amount of MB-labeled mononucleotides are released to result in the significantly amplified electrochemical signal. By taking advantage of the high amplification efficiency of T7-aided target recycling, the present assay enables the detection of telomerase activity at the single-cell level, which is superior or comparable to that of the reported literature. Furthermore, the assay was carried out in a homogeneous solution without complex modification or immobilization procedures, which has the merits of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensors. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.

Sensing telomerase: From in vitro detection to in vivo imaging

“…This assay can sensitively measure telomerase activity with a detection limit of as low as 10 HeLa cells per mL. Recently, a series of electrochemical methods have been reported for telomerase assay using Methylene Blue 42 and hexaammineruthenium( iii ) chloride 43 as the reporters. In addition, metal–organic frameworks have been used in the development of electrochemical methods for telomerase assays.…”

Advances in the detection of telomerase activity using isothermal amplification

“…In addition to the above several assays, ferrocenylnaphthalene diimide as a tetraplex DNA-specific binder 42, horseradish peroxidase-conjugated avidin (avidin-HRP) for the electrochemical reduction of 3,3',5,5-tetramethylbenzidine (TMB) 43 and T7 exonuclease-aided 5'-methylene blue (MB) release 44 affected by telomerase elongation using isothermal amplification are also worthy of attention.…”

Highly sensitive homogeneous electrochemical aptasensor for antibiotic residues detection based on dual recycling amplification strategy

“…In the absence of AMP, HP can resist the cleavage by T7 exonuclease, and the electrostatic repellence between HP and the negatively charged ITO electrode prevents MB attached to HP from approaching the electrode surface so almost no electrochemical response is observed on the sensing electrode (Liu et al 2013c;Liu et al 2015;Zhang et al 2015). Once in the presence of the target AMP, the aptamer sequence region in HP specifically recognizes AMP to form the target-aptamer complex (Song et al 2012a), leading to the exposure of the primer recognition fragment (in skyblue) originally caged in HP.…”