Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types
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 te...
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| Format: | Article |
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MDPI AG
2021-02-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/11/2/42 |
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| author | Bukola Attoye Matthew J. Baker Fiona Thomson Chantevy Pou Damion K. Corrigan |
| author_facet | Bukola Attoye Matthew J. Baker Fiona Thomson Chantevy Pou Damion K. Corrigan |
| author_sort | Bukola Attoye |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-09T05:36:27Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T05:36:27Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-42033722bfba4c4e938cf34ac6fcf5852023-12-03T12:28:27ZengMDPI AGBiosensors2079-63742021-02-011124210.3390/bios11020042Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour TypesBukola Attoye0Matthew J. Baker1Fiona Thomson2Chantevy Pou3Damion K. Corrigan4Department of Biomedical Engineering, University of Strathclyde, 40 George Street, Glasgow G1 1QE, UKTechnology and Innovation Centre, Department of Pure and Applied Chemistry, University of Strathclyde, 99 George Street, Glasgow G1 1RD, UKWolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UKWolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow G61 1QH, UKDepartment of Biomedical Engineering, University of Strathclyde, 40 George Street, Glasgow G1 1QE, UKCirculating 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.https://www.mdpi.com/2079-6374/11/2/42electrochemicalDNA biosensorsKRASliquid biopsycancer point-of-care diagnostic tests |
| spellingShingle | Bukola Attoye Matthew J. Baker Fiona Thomson Chantevy Pou Damion K. Corrigan Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types Biosensors electrochemical DNA biosensors KRAS liquid biopsy cancer point-of-care diagnostic tests |
| title | Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types |
| title_full | Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types |
| title_fullStr | Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types |
| title_full_unstemmed | Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types |
| title_short | Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types |
| title_sort | optimisation of an electrochemical dna sensor for measuring kras g12d and g13d point mutations in different tumour types |
| topic | electrochemical DNA biosensors KRAS liquid biopsy cancer point-of-care diagnostic tests |
| url | https://www.mdpi.com/2079-6374/11/2/42 |
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