**4. Conclusions**

We were able to successfully produce a simple DNA sensor requiring no labelling processes or external indicators using a multi-carbon electrode. An electrochemical detection scheme involving a DNA hybridisation technique and screen-printed carbon electrodes were developed and shown through a series of comparative measurements to be sensitive and specific for the KRAS G12D and G13D mutations. The DNA modified sensors demonstrated superior performance to electrochemical pre-treatment with acetate buffer containing NaCl and characterisation using ferri-ferrocyanide buffer. Improved sensor sensitivity was achieved by designing a PCR reaction capable of amplifying either mutant KRAS G13D or wild-type KRAS through primer choice from representative patient samples. Cyclic voltammetry and Square Wave measurements were very sensitive for charactering the surface of the modified SPCEs and Differential Pulse voltammetry measurements provided the desired response and indicated detection was possible from samples containing as few as 0.58 ng/μ<sup>L</sup> concentrations of amplicons. In addition, the response was found to be consistent with previously observed results, i.e., large signal decreases being evident upon amplification of the mutant allele, offering the promise of quantitation of mutant sequences from clinical samples. Both non-complementary DNA probes and wild-type DNA amplification reaction was successfully used as control. These results increase the prospect of simple, rapid and cost-effective measurement of nucleic acid tumour markers from blood and other body fluids. The current time to result of the electrochemical sensor was 3.5 h, providing notable scope for optimisation. It is essential to note that the sensor being developed can be potentially used for both early detection of cancer and monitoring the response to cancer treatment.

**Author Contributions:** Conceptualization, D.K.C.; methodology, B.A.; validation, D.K.C.; formal analysis, B.A.; investigation, B.A.; resources, F.T. and C.P.; data curation, B.A.; writing—original draft preparation, B.A.; writing—review and editing, B.A., D.K.C., C.P. and F.T.; supervision, D.K.C., and M.J.B.; project administration, B.A. and D.K.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** The lead author was supported by EPSRC (grant reference number EP/L015595/1). F.T. and C.P. were supported by CRUK and ECMC core funding.

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of the University of Strathclyde (protocol code UEC17/83—17/04/2018).

**Informed Consent Statement:** Not applicable. The HCT116 and SK-UT-1 cell lines were obtained from American Type Culture Collection organization (ATCC, catalogue #CCL-247 TM and HTB-114TM).

**Data Availability Statement:** Data is contained within this article and additional data can be found in preceding article https://doi.org/10.3390/IECB2020-07067.

**Acknowledgments:** Special gratitude goes to CDT Medical Devices at the University of Strathclyde through which the gran<sup>t</sup> was received.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
