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.
Highlights
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]
This work presents a KRAS G12D and G13D DNA oligonucleotide probe modified sensor array that can accurately detect mutant KRAS amplicons and forms the basis of a system for the accurate detection of circulating tumour DNA (ctDNA) in patient samples and monitoring of response during treatment. This was achieved by amplifying mutant DNA isolated from a human cancer cell line recovered from clinical samples, using electrochemical techniques and Screen Printed Carbon electrodes (SPCEs) to detect a clinically relevant mutation, comparing the signal change from DNA hybridisation experiments involving amplified KRAS mutant samples and amplified wild-type KRAS samples, varying concentration of amplified products to determine concentration effects and establishing a limit of detection for the DNA amplification reaction
Electrografting using in situ generated diazonium cations is important for modifying the surface of the SPCE by allowing the formation of covalent bonds between the carbon surface and organic films [39,40,41]
Summary
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]. This work presents a KRAS G12D and G13D DNA oligonucleotide probe modified sensor array that can accurately detect mutant KRAS amplicons and forms the basis of a system for the accurate detection of ctDNA in patient samples and monitoring of response during treatment This was achieved by amplifying mutant DNA isolated from a human cancer cell line recovered from clinical samples, using electrochemical techniques and SPCEs to detect a clinically relevant mutation, comparing the signal change from DNA hybridisation experiments involving amplified KRAS mutant samples and amplified wild-type KRAS samples, varying concentration of amplified products to determine concentration effects and establishing a limit of detection for the DNA amplification reaction. Employing eight eight working working electrodes electrodes with with a common common Ag reference and carbon counter electrodes printed electrode array employing along with a schematic showing modification steps and DNA functionalisation
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