A highly sensitive electrochemical magneto-genosensing assay for the specific detection of a single nucleotide variation in the KRAS oncogene in human plasma

Abstract

Liquid biopsy is a non-invasive and efficient technique for the detection of tumor biomarkers in biological fluids, which currently represents a new frontier in theranostics and precision medicine. Among mutations of the KRAS oncogene, p.G12D single nucleotide variation in the KRAS gene plays a central role in the early diagnosis and therapeutic treatment of colorectal cancer. In this context, we developed a highly sensitive magneto-genosensing assay based on PNA capture probes immobilized on magnetic microbeads. To detect the mutation of the KRAS oncogene, two capture probe sequences recognizing wild-type and p.G12D tumor DNA were used in association with a DNA signaling probe allowing for electrochemical detection using an enzyme conjugate. The assay conditions were optimized using a 32 full-factorial design, obtaining an outstanding specificity, evidenced by a remarkably lower (>95%) signal of the singly-mismatched- compared to that of fully-complementary-DNA. Ultra-high sensitivity was achieved in 10-fold diluted human plasma reaching detection limits of 818 fM for the p.G12D and 1.8 pM for the wild-type targets. The genosensing assay was tested on genomic tumor DNA samples provided by IRCCS-Regina Elena National Cancer Institute and finally integrated into a smart portable multichannel potentiostat capable of performing up to four simultaneous acquisitions. The developed magneto-genosensing assay demonstrated portability, simplicity, and high sensitivity, showing good potential as theranostic tool for personalized medicine in oncology.