T7 Endonuclease I-mediated voltammetric detection of KRAS mutation coupled with horseradish peroxidase for signal amplification.

Rapid and selective sensing of KRAS gene mutation which plays a crucial role in the development of colorectal, pancreatic, and lung cancers is of great significance in the early diagnosis of cancers. In the current study, we developed a simple electrochemical biosensor by differential pulse voltammetry technique for the specific detection of KRAS mutation that uses the mismatch-specific cleavage activity of T7-Endonuclease I (T7EI) coupled with horseradish peroxidase (HRP) to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) substrate in the presence of hydrogen peroxide (H2O2). In addition, we synthesized the nanocomposite composed of multi-walled carbon nanotube/chitosan-ionic liquid/gold nanoparticles (MWCNT/Chit-IL/AuNPs) on screen-printed carbon electrode surface to increase the electrode surface area and electrochemical signal. In principle, T7E1 enzyme recognized and cleaved the mismatched site formed by the presence of KRAS gene mutation, removing 5'-biotin of capture probes and subsequently reducing the differential pulse voltammetry signal compared to wild-type KRAS gene. With this proposed strategy, a limit of detection of 11.89 fM was achieved with a broad linear relationship from 100 fM to 1 µM and discriminated 0.1% of mutant genes from the wild-type target genes. This confirms that the developed biosensor is a potential platform for the detection of mutations in early disease diagnosis.