Ferrocene-labeled and purification-free electrochemical biosensor based on ligase chain reaction for ultrasensitive single nucleotide polymorphism detection
Single nucleotide polymorphisms (SNPs) are crucial during the early diagnosis of a given disease as well as in evaluating their response to certain drugs. Thus, this study sought the development of ferrocene (Fc)-labeled electrochemical biosensor for SNP detection. This proposed system involves the ligation of four short probes (e.g., A, B, A′, and B′, where B′ is labeled with an Fc-tag) in the presence of target DNA via ligase chain reaction (LCR), resulting in the formation of Fc-tagged duplex AB-A′B′ in 2 n . Subsequently, immobilization of the Fc-tagged duplex AB-A′B′ on a single-stranded DNA capture probe (SC-DNA)-carboxyl multi-wall carbon nanotube (MWCNT-COOH) modified glassy carbon electrode (GCE) was accomplished through hybridization. Owing to the specificity of hybridization, and the use of Fc as electrochemical probe for detection of duplex AB-A′B′, such strategy realized directly analysis of LCR products without the need for purification . By taking advantage of the thermal stability and high-discrimination ability of HiFi Taq DNA ligase for single-base differences, the specificity of hybridization, the EGFR T790 M mutant DNA (MT-DNA) biosensor was developed to offer a low limit of detection (0.75 aM), a high discrimination of single-base mismatches [as low as 0.01% (molar fraction)], a wide linear range of more than 7 orders of magnitude (1 aM–10 pM), and the recovery rates (95.3%–107.8%) from human serum samples. Thus, the biosensor under development was found to be economical, highly-sensitive, and exceptionally selective for detection of SNPs, and as well as extending the versatile applications of LCR to offer great potential for diagnosis and individual clinical regimens.
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