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

Abstract

It is well-known that two major issues, preventing improved outcomes from cancer are late diagnosis and the evolution of drug resistance during chemotherapy, therefore technologies that address these issues can have a transformative effect on healthcare workflows. In this work we present a simple, low-cost DNA biosensor that was developed specifically to detect mutations in a key oncogene (KRAS). The sensor employed was a screen-printed array of carbon electrodes, used to perform parallel measurements of DNA hybridisation. A DNA amplification reaction was developed with primers for mutant and wild type KRAS sequences which amplified target sequences from representative clinical samples to detectable levels in as few as twenty cycles. High levels of sensitivity were demonstrated alongside a clear exemplar of assay specificity by showing the mutant KRAS sequence was detectable against a significant background of wild type DNA following amplification and hybridisation on the sensor surface. The time to result was found to be 3.5 h with considerable potential for optimisation through assay integration. This quick and versatile biosensor has the potential to be deployed in a low-cost, point-of-care test where patients can be screened either for early diagnosis purposes or monitoring of response to therapy.

Keywords: DNA biosensors; KRAS; electrochemical; liquid biopsy.

Figure 1

Image of screen printed electrode array employed (8 × working electrodes with common Ag reference and carbon counter electrodes along with schematic showing DNA functionalisation and DNA target binding.

Figure 2

Copies of KRASG12D mutant and KRAS wild type detected in total DNA isolated from SK-UT-1 cells using ddPCR (BioRad QX200).

Figure 3

CV results showing hybridisation selectivity between a (a) wild type probe (b) mutant probe and a representative sample containing PCR amplified KRAS G12D mutant allele with a total KRASG12D ssDNA concentration of 0.854 ng/µL.

Figure 4

CV results showing repeated cycling of (a) KRAS G12D probe functionalised electrodes (b) KRAS G12D amplicons over 8-chip multielectrodes using the same current and voltage inputs.

Figure 5

EIS responses for clean, probe modified and target bound electrodes following 25 PCR cycles (a) and 30 PCR cycles (b,c) CV results showing the correlation between oxidative and reductive peak currents and PCR cycle number.

Figure 6

(a) CV results showing DNA hybridisation target controls using (i) G12D mutant probe in ultrapure water (ii) G12D mutant probe in human plasma (iii) G12D mutant probe in representative sample containing KRAS G12D mutant amplicons in plasma (b) CV results (% signal change) showing G12D amplicons concentration dose response.