A novel ligase chain reaction-based electrochemical biosensing strategy for highly sensitive point mutation detection from human whole blood

• Published in: Talanta (Oxford) 2020-08, Vol.216, p.120966-120966, Article 120966
• Main Authors: Liu, Zhou-Jie, Yang, Liang-Yong, Wei, Qing-Xia, Ye, Chen-Liu, Xu, Xiong-Wei, Zhong, Guang-Xian, Zheng, Yan-Jie, Chen, Jin-Yuan, Lin, Xin-Hua, Liu, Ai-Lin
• Format: Article
• Language: English
• Subjects: Biosensing Techniques; Clinical sample; CYP2C192 allele; Cytochrome P-450 CYP2C19 - blood; Cytochrome P-450 CYP2C19 - genetics; Electrochemical biosensor; Electrochemical Techniques; Humans; Ligase Chain Reaction; Point Mutation
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2020.120966

Challenged by the detection of trace amounts of mutants and disturbance from endogenous substances in clinical samples, herein, we present a novel electrochemical biosensor based on ligase chain reaction (eLCR) via the thermostable ligase with high mutation recognizing ability. The lengthened double-stranded DNAs exponentially generated via LCR were uniformly distributed on a bovine serum albumin-modified gold electrode, in which the phosphate buffer was tactfully added to remove adsorbed uninterested-probes, and thereafter the amperometry current was collected for the specific binding of streptavidin-poly-HRP and subsequent catalysis in the 3, 3′, 5, 5′-tetramethylbenzidine substrate that contained hydrogen peroxide. It found that, under optimized conditions, the proposed biosensor exhibited a high selectivity of mutant targets from the 104-fold excess of co-existent wild targets within a detection limit of 0.5 fM. Impressively, without the involvement of pre-PCR, the homozygous mutants were specifically distinguished from the wild genotype of CYP2C19*2 allele in human whole blood samples. Therefore, the proposed eLCR, due to its advantages in simple primer design, operational ease and ease of miniaturization, has demonstrated its considerable potential for point-of-care testing in the diagnosis of point mutation-related diseases and personalized medicine. [Display omitted] •A new eLCR coupling the dsDNA-bovine serum albumin platform and HRP-H2O2-TMB system.•Effective removal of unwanted DNA probes' adsorption via phosphate buffer regulation.•High selectivity of mutants from the co-existent excessive wild targets (104-fold).•Successful discrimination of CYP2C19*2 allele in clinical samples without pre-PCR.