**1. Introduction**

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]. Cancer is a genetic disease by nature, caused by mutations in certain genes thereby resulting in cellular malfunction [2]. Imaging tests can sometimes be inconclusive

**Citation:** Attoye, B.; Baker, M.J.; Thomson, F.; Pou, C.; Corrigan, D.K.Optimisation of an Electrochemical DNA Sensor for Measuring KRAS G12D and G13D Point Mutations in Different Tumour Types. *Biosensors* **2021**, *11*, 42. https://doi.org/ 10.3390/bios11020042

Received: 19 December 2020 Accepted: 1 February 2021 Published: 5 February 2021

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and broadly do not provide information on the stage or type of cancer, so further biopsy is needed [3]. Serious medical risks and related metastasis may ensue from gathering multiple biopsies from different regions of a primary tumour [4].

The period at which a tumour shows clinical symptoms usually corresponds with the later stages of progression (e.g., Phases III and IV), when the cancer is metastatic or unresectable, causing surgery and therapy to be less effective. In addition, surgical biopsy procedures are not possible or recommended for some patients; therefore, liquid biopsies that are able to detect the presence of tumour DNA hold promise as a noninvasive alternative.

Most body fluids, including blood, contain tumour biomarkers and short fragments of cell-free DNA (cfDNA) that can be detectable as shown in Figure 1A below. In cancer patients, a fraction of cfDNA called circulating tumour DNA (ctDNA) can be found, which emerges from tumours and may feature the same mutations and genetic modifications as those present in the primary tumour [5]. While circulating tumour cells (CTCs) that have been shed into the vasculature of a primary tumour are also transported around the body in circulation, they are present at quantities of around 10 cells/mL of blood, suggesting that only very low concentrations are present in clinical samples. In contrast to ctDNA, CTCs are rare in peripheral blood and are difficult to separate from other cells, increasing the credibility for the use of ctDNA in liquid biopsy applications. The mechanism of ctDNA release from tumour cells is poorly understood [6], however it is thought to be released in small quantities following apoptosis or necrosis. ctDNA typically comprises 0.01–1% of the circulating free DNA in blood [7] and it is important to note that this can be shed as both double stranded and single stranded DNA [8]. At present, ctDNA can be detected in blood and other body fluids like lymph, urine and stool [9]. Due to the small fraction of ctDNA concealed by large background levels of wild-type cell-free DNA, sensitive amplification reactions such as polymerase chain reaction (PCR) will need to be implemented to achieve detection and discrimination above wild type signals. A point of care (PoC) measurement of circulating tumour DNA (ctDNA) may offer a non-invasive strategy for evaluating response to treatment, monitoring disease recurrence, capturing tumour heterogeneity and gaining insights into a tumour's mutational profile [9,10].

Single nucleotide variations (SNV) in the Kirsten rat sarcoma viral oncogene homolog, commonly abbreviated 'KRAS' are present across many human tumour types with KRAS G12D and G13D being specific variants observed. KRAS is a member of the RAS family of proteins which are a part of at least six signalling pathways in a healthy human cell and is the most commonly mutated protein across many human tumour types [11]. KRAS mutations take place in approximately 90% of pancreatic cancers [12], 30% of lung cancers [13], 60% of thyroid cancers and 43% of colorectal cancers [14]. KRAS activated mutations drive cancer initiation, progression and drug resistance, directly leading to nearly a million deaths per year. SNVs have been used as biomarkers for predicting disease risk [15,16], and its combination with liquid biopsies will create innovations in biomarker detection that will enhance clinical outcomes for patients at all cancer stages [17].

From a PoC viewpoint, Electrochemical DNA biosensors represent an exciting approach in the detection of clinically important biomarkers due to their rapidity and simplicity [18–20]. Electrochemical biosensors are used to directly convert a biological binding event to an electronic signal [21]. A range of electrode materials and electrochemical measurement approaches have been employed for sensitive measurements [8,22–24]. The possibilities of electrochemical biosensors, once matured as a technology to provide efficient clinical workflows, is vast. In electrochemical DNA biosensing, a change in signal is obtained when recognition and hybridisation of two opposing strands of DNA occur as a result of their base-pair complementarity. A double stranded DNA sequence with tumourspecific mutations can indicate the diagnosis of a specific cancer [24]. As the concentration of ctDNA is directly proportional to the tumour grade, attaining high sensitivity for the DNA sensor is key for the early detection of disease, developing tailored therapies and monitoring therapy efficiency.

Given the continued need for the miniaturization of advanced electronics, the area of screen printing techniques has been adapted for electronic circuit fabrication. Screen printed electrodes (SPEs) are evolving as they are easy to use and can be produced on a large scale. SPEs are also very practical as they are disposable and low cost when manufactured in large volumes. SPEs are usually composed of working electrodes made of conductive inks like carbon, platinum, gold or silver. Although, carbon with organic solvents, binding pastes and some additives that provide functional characteristics are contained in conductive inks found in Screen Printed Carbon electrodes (SPCEs), they can be modified in order for their electrochemical properties to be improved [24]. Carbon electrodes are also chemically inert, specifically at negative potential ranges in all media, making them particularly suitable electrode sensors for electroanalytical chemistry, providing an advantage over metal electrodes [25]. SPCEs are simple, sensitive, cost-effective (~£2 each) and disposable, making them preferable for rapid electrochemical analyses and suitable as electrodes for characterizing the processes implemented herein, specifically for the detection of ctDNA.

The SPCE sensor shown in this study was developed by characterising the surface of the electrode chip to determine the treatments and buffers with optimal sensitivity. In order to make the surface of the SPCE as homogenous as possible, it is important that they are pretreated. These pre-treatments remove any binder residues left on the carbon surface after the curing process [26–29], with well-established electrochemical oxidative pre-treatments not only showing removal of binder residues left on the surface of SPCE after curing but also improvement of carbon surface sensitivity [30]. In this study, two common buffers are compared for pre-treatment: NaOH and NaCl. Up until now, few studies have been done on electrochemically pre-treating and characterising the surface of activated screen printed carbon electrodes [28–30]. We make the choice of a characterisation redox buffer after observing the effect of surface chemistry in relation to electron transfer rates using an inner-sphere redox mediator (Ferri-ferrocyanide) and an outer-sphere redox mediator (ruthenium hexaminechloride). Further voltammetric characterisation is performed to reveal DNA hybridisation effects and thus mutation detection in both potassium ferriferrocyanide (1 mM Fe(CN)6 3−/4− in 0.1× PBS) and ruthenium hexaminechloride (1 mM Ru(NH3)6Cl3 in 0.1× PBS) solutions.

This work presents a KRAS G12D and G13D DNA oligonucleotide probe modified sensor array that can accurately detect mutant KRAS amplicons and therefore 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. Cyclic Voltammetry (CV), Square Wave Voltammetry (SWV) and Differential Pulse Voltammetry (DPV) are routinely used electrochemical measurement techniques that supply information on electron transfer reaction kinetics of any combined chemical reaction [31]. In these techniques, a potential waveform is applied to the working electrode (WE). The peak current obtained is directly influenced by hybridisation between target and immobilised probe DNA strands [32]. In this study, DPV, SWV and CV were used depending on whether electrodes needed to be cleaned, electrografted, or characterised during sensor measurement. Considering the choice of steps and ease of use of the assay being developed, the system can be very easily automated and integrated into a final device capable of fast and seamless clinical measurements. The presented work builds on a recent publication [1] showing the possible detection of KRAS G12D mutations, by developing understanding or surface pre-treatment steps (essential to realising a reproducible analytical technique) and by introducing the detection of the KRAS G13D mutation which expands the assay towards a multi-marker assay and permits the analysis of more tumour types.

**Figure 1.** (**A**) Schematic showing circulating tumour DNA (ctDNA) retrieval and analysis [33–35] (**B**) Image of a screenprinted electrode array employing eight working electrodes with a common Ag reference and carbon counter electrodes along with a schematic showing modification steps and DNA functionalisation.

#### **2. Materials and Methods**
