Developing a Low-Cost, Simple-to-Use Electrochemical Sensor for the Detection of Circulating Tumour DNA in Human Fluids

Attoye, Bukola; Pou, Chantévy; Blair, Ewen O.; Rinaldi, Christopher A.; Thomson, Fiona; Baker, Matthew J.; Corrigan, Damion K.

doi:10.3390/bios10110156

Cited by 15 publications

(8 citation statements)

References 27 publications

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“…Therefore, we expected a reduction in the peak current when specific DNA hybridisation had taken place, and this was found to be the case for lower concentrations of DNA (pico-to-low nanomolar concentrations) as shown in Figure 2B,C. 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) [31] 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.…”

Section: Dna Sensor Hybridisation Specificitysupporting

confidence: 82%

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

Attoye

Baker

Pou

et al. 2020

The 1st International Electronic Conference on Biosensors

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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.

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Section: Dna Sensor Hybridisation Specificitysupporting

confidence: 82%

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

Attoye

Baker

Pou

et al. 2020

The 1st International Electronic Conference on Biosensors

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“…Further optimisation can be made using isothermal amplification which can cut down the number of thermal cycles and, in turn, decrease the overall amplification time from 1 h to 30 min [ 53 ]. As our previously published work shows that we were able to detect KRAS amplicons in plasma to mimic a ‘clinical sample’ [ 54 ], near future work will explore the detection of non-specified clinical samples containing different KRAS mutations and mutations in other genes involved in cancer, e.g., P53 and BRCA1. Analysing multiple mutations simultaneously in a given sample without prior knowledge of the alterations using multiplex techniques and direct detection of ctDNA from cancer patient samples will support the future direction of PoC clinical testing.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Capture DNA probes were immobilized onto the electrode’s surface, while the hybridization of the target was performed through ruthenium redox mediator and cyclic voltametric measurements. The fabrication time of the sensor is about 1 h, the analysis time is 3.5 h, while the LOD is 4 copies/ng for the single and 0.58 ng/μL for the multiplex format [ 37 , 38 ]. An urchin-like gold nanocrystal-multiple graphene aerogel was also exploited for ctDNA analysis.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2022

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In the era of personalized medicine, molecular profiling of patient tumors has become the standard practice, especially for patients with advanced disease. Activating point mutations of the KRAS proto-oncogene are clinically relevant for many types of cancer, including colorectal cancer (CRC). While several approaches have been developed for tumor genotyping, liquid biopsy has been gaining much attention in the clinical setting. Analysis of circulating tumor DNA for genetic alterations has been challenging, and many methodologies with both advantages and disadvantages have been developed. We here developed a gold nanoparticle-based rapid strip test that has been applied for the first time for the multiplex detection of KRAS mutations in circulating tumor DNA (ctDNA) of CRC patients. The method involved ctDNA isolation, PCR-amplification of the KRAS gene, multiplex primer extension (PEXT) reaction, and detection with a multiplex strip test. We have optimized the efficiency and specificity of the multiplex strip test in synthetic DNA targets, in colorectal cancer cell lines, in tissue samples, and in blood-derived ctDNA from patients with advanced colorectal cancer. The proposed strip test achieved rapid and easy multiplex detection (normal allele and three major single-point mutations) of the clinically relevant KRAS mutations in ctDNA in blood samples of CRC patients with high specificity and repeatability. This multiplex strip test represents a minimally invasive, rapid, low-cost, and promising diagnostic tool for the detection of clinically relevant mutations in cancer patients.

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Developing a Low-Cost, Simple-to-Use Electrochemical Sensor for the Detection of Circulating Tumour DNA in Human Fluids

Cited by 15 publications

References 27 publications

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

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

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

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

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