Highly sensitive electrochemical analysis of telomerase activity based on magnetic bead separation and exonuclease III-aided target recycling amplification

Abstract

Telomerase is considered a pivotal biomarker for early cancer diagnosis and a valuable therapeutic target. However, the current methods to detect telomerase activity have some limitations. Herein, we propose a homogeneous electrochemical strategy to develop a simple, rapid, and highly sensitive assay to detect human telomerase activity from crude cancer cell extracts. Our strategy is based on magnetic bead separation and exonuclease III-aided target recycling amplification. The complementary probes can hybridize with the extended telomeric repeats, which allows exonuclease III to recognize and digest the latter once the hybrid product is separated with magnetic beads. The released complementary probes can hybridize with and open multiple methylene blue (MB)-labeled hairpin (HP) DNA probes, allowing exonuclease III to digest the duplex. Then, the opened hairpin couples with the captured mononucleotides on the surface of the gold electrode. By taking advantage of the exonuclease III-aided target recycling strategy, the present assay enables the detection of telomerase activity at a single-cell level. Furthermore, the assay is carried out in a homogeneous solution achieved by magnetic purification, which removes the interferents present in crude lysates and avoids false negatives, thus, providing a powerful platform to detect telomerase activity in samples of early-stage cancer.