Development of electrochemical DNA detection methods to measure circulating tumour DNA for enhanced diagnosis and monitoring cancer

Liquid biopsies are becoming an increasingly important potential replacement for existing biopsy procedures which can be invasive, painful and compromised by tumour heterogeneity. This paper reports a simple electrochemical approach tailored towards point of care cancer detection and treatment monitoring from biofluids using a label free detection strategy. The mutations under test were the KRAS G12D and KRAS G13D mutations which are both important in the development and progression of many human cancers and whose presence correlates with poor outcomes. These common circulating tumour markers were investigated in clinical samples and amplified by standard and specialist PCR methodologies for subsequent electrochemical detection.
Following pre-treatment of the sensor to give a clean surface, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low cost carbon electrodes using diazonium chemistry and EDC/NHS coupling. Following functionalisation of the sensor it was possible to sensitively and specifically detect mutant KRAS G12D and G13D PCR product from cancer patients against a background of wild type KRAS DNA from the representative sample. Our findings give rise to the basis of a simple and very low cost system for measuring ctDNA biomarkers in patient samples. The current time to result of the system is 4.5 hours with considerable scope for optimisation and already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result.
The paper will report the technical developments we have made in the production of clean carbon surfaces for functionlisation, show the assay performance data for KRAS G12D and G13D with a range of PCR systems and demonstrate the potential for measuring response to treatment offered by the system.