Electrochemical DNA Detection Methods to Measure Circulating Tumour DNA for Enhanced Diagnosis and Monitoring of Cancer

Abstract

Liquid biopsies are becoming increasingly important as a 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 G13D mutations, which are both important in the development and progression of many human cancers and which have a presence that 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 present 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 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide coupling. Following the functionalisation of the sensor, it was possible to sensitively and specifically detect a mutant KRAS G13D PCR product against a background of wild-type KRAS DNA from the representative cancer 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 was 3.5 h with considerable scope for optimisation, and it already compares favourably to the UK National Health Service biopsy service where patients can wait weeks for their result. This paper reports the technical developments we made in the production of consistent carbon surfaces for functionalisation, assay performance data for KRAS G13D and detection of PCR amplicons under ambient conditions.

Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types

“…It has been observed that these effects can be reversed when micro-or nanoscale electrodes are employed [20,46], but for this study, the electrodes used were comfortably on the macro scale (diameter = 2.95 mm). For nanomolar (>10 nM) and micromolar concentrations, an increase in the peak current following hybridisation was consistently observed (and has also been observed in other data from our lab involving SPCEs) [1] for carbon electrodes which is likely explained by the high surface density of hybridised DNA amplicons changing the interfacial properties of the electrode and, therefore, altering the electrochemical response. The underlying physical mechanism of this effect is actively under investigation.…”

“…In recent years, analytical electrochemistry has emerged as a powerful tool for the rapid in-vitro analysis of biological analytes for the early detection of certain diseases, such as cancer [ 1 ]. Cancer is a genetic disease by nature, caused by mutations in certain genes thereby resulting in cellular malfunction [ 2 ].…”

“…Considering the choice of steps and ease of use of the assay being developed, the system can be very easily automated and integrated into a final device capable of fast and seamless clinical measurements. The presented work builds on a recent publication [ 1 ] showing the possible detection of KRAS G12D mutations, by developing understanding or surface pre-treatment steps (essential to realising a reproducible analytical technique) and by introducing the detection of the KRAS G13D mutation which expands the assay towards a multi-marker assay and permits the analysis of more tumour types.…”

“…builds on a recent publication [1] showing the possible detection of KRAS G12D mutations, by developing understanding or surface pre-treatment steps (essential to realising a reproducible analytical technique) and by introducing the detection of the KRAS G13D mutation which expands the assay towards a multi-marker assay and permits the analysis of more tumour types.…”