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

Abstract

Circulating tumour DNA (ctDNA) is widely used in liquid biopsies due to having a presence in the blood that is typically in proportion to the stage of the cancer and because it may present a quick and practical method of capturing tumour heterogeneity. This paper outlines a simple electrochemical technique adapted towards point-of-care cancer detection and treatment monitoring from biofluids using a label-free detection strategy. The mutations used for analysis were the KRAS G12D and G13D mutations, which are both important in the initiation, progression and drug resistance of many human cancers, leading to a high mortality rate. A low-cost DNA sensor was developed to specifically investigate these common circulating tumour markers. Initially, we report on some developments made in carbon surface pre-treatment and the electrochemical detection scheme which ensure the most sensitive measurement technique is employed. Following pre-treatment of the sensor to ensure homogeneity, DNA probes developed specifically for detection of the KRAS G12D and G13D mutations were immobilized onto low-cost screen printed carbon electrodes using diazonium chemistry and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide coupling. Prior to electrochemical detection, the sensor was functionalised with target DNA amplified by standard and specialist PCR methodologies (6.3% increase). Assay development steps and DNA detection experiments were performed using standard voltammetry techniques. Sensitivity (as low as 0.58 ng/μL) and specificity (>300%) was achieved by detecting mutant KRAS G13D PCR amplicons against a background of wild-type KRAS DNA from the representative cancer sample and 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 receive results from the system was 3.5 h with appreciable scope for optimisation, thus far comparing favourably to the UK National Health Service biopsy service where patients can wait for weeks for biopsy results.

Detection of the p53 Gene Mutation Using an Ultra-sensitive and Highly Selective Electrochemical DNA Biosensor

“…It is attributed to the contribution of the increasing molecular material of dsDNA (double-stranded DNA) in the electrode surface and the formation of a more compact adlayer which physically blocks access of the [Fe(CN)6] 3-/4ions to the surface of the electrode. Also, the increase of negative charge during DNA hybridization may be due to the double-stranded formation that creates more electrostatic repulsion force between the redox probe and the electrode surface, as reported in previous literature [28,32,33]. Using Dox (Fig.…”

Rapid Multiplex Strip Test for the Detection of Circulating Tumor DNA Mutations for Liquid Biopsy Applications

“…In this approach, signal enhancement was accomplished via DNA-induced target recycling. This biosensor had an LOD of 0.033 fM, >1 h analysis time with quite good specificity, and about 4 h fabrication time, but it lacked multiplicity and universality [ 39 , 40 ] RNase HII-aided target recycling was also performed in another SERS assay to detect single-stranded ctDNA in blood samples. T-rich DNA sequences produced by the RNAse were captured on the sensor’s surface.…”

Reverse Electrochemical Sensing of FLT3-ITD Mutations in Acute Myeloid Leukemia Using Gold Sputtered ZnO-Nanorod Configured DNA Biosensors

“…A key advantage in coupling DNA amplifications steps with sensor-based mutation detec-tions is the possibility of developing multiplex sensor devices. Since mutations leading to the prognosis of AML progression is not limited to signal-pathway genes such as FLT3 only (e.g., KRAS, NRAS, KIT, PTPN11, and NF1), the proposed sensor system can be designed as a multiplexed panel of DNA probes targeting various DNA sequence mutations in a single platform, leading to concomitant identification of multiple mutations that can yield an entire gene mutation profile for AML patients [55,56].…”