Detection of p53 DNA using commercially available personal glucose meters based on rolling circle amplification coupled with nicking enzyme signal amplification

Abstract

The development of cost-effective methods for early detection and identification of prognostic markers still remains a significant challenge to improve diagnosis and reduce the mortality of cancer. Herein, on the basis of rolling circle amplification (RCA) coupled with nicking endonuclease-assisted signal amplification (NESA), a simple, sensitive and portable biosensor was developed for the determination of p53 DNA by using the personal glucose meter (PGM) as readout. Initially, biotin-modified hairpin probe (HP) was immobilized onto streptavidin-coated magnetic beads (MBs). The target DNA hybridized with the loop region of the HP, which triggered target recycling process and produced the complementary sequences for the padlock probes. Next, the liberated complementary sequences hybridized with the padlock probes to form a circular template, inducing the subsequent RCA reaction and replicating a long tandem repeated sequences. Then, numerous DNA-invertase conjugation were tagged on the resulted RCA products on the surface of MBs. The DNA-invertase efficiently catalyzed the hydrolysis of sucrose to generate abundant glucose, leading to an amplified response of glucometer. By virtue of the multiple signal amplification strategy, the proposed biosensor toward p53 DNA could achieve a low detection limit of 0.36 pM with a linear calibration range from 0.5 to 10 pM and exhibited excellent sequence selectivity. In addition, the resulting biosensor was also applied to detect the p53 DNA sequence in spiked human serum samples with satisfactory results, which possessed enormous potential to be applied in clinical diagnostics and biomedical research.