Electrochemical impedance spectroscopy (EIS) is a highly useful approach for the label free measurement of DNA hybridisation at functionalised electrode surfaces. Since label free detection relies upon a change in the electrochemical signal arising directly from the presence of target oligonucleotide or DNA/RNA sequences it is necessary to improve understanding of the conditions which produce a stable baseline value, promote optimal hybridisation of complementary sequences and can reduce non-specific binding effects. This study investigates both artificial DNA oligonucleotide sequences and clinical samples of MRSA genomic DNA, initially demonstrating that the use of tris(2-carboxyethyl)phosphine (TCEP) during probe layer formation improves both the initial baseline signal reproducibility and also the magnitude of the response upon hybridisation with a complementary target Having demonstrated enhanced performance from TCEP modified electrodes, the assay is then used to detect clinical samples of MRSA. It is shown that improved performance is achieved both in terms of signal magnitude and discrimination against negative controls. Finally, formamide is added to the EIS measurement buffer and it is demonstrated that EIS measurement is possible in the presence of high formamide concentrations and that non-specific binding is also reduced under such conditions. The importance of these findings lies in the design of future electrochemical assays for nucleic acid biomarkers which are capable of functioning under ambient conditions but still have discriminatory power. A diagnostic device which does not have to operate at elevated temperatures will lead to increased simplicity and substantial battery and time savings which will further the widespread realisation of portable clinical diagnostic devices. (C) 2017 Elsevier B.V. All rights reserved.