An electrochemical biosensor for the detection of microRNA-31 as a potential oral cancer biomarker

Abstract

Oral cancer presents substantial challenges to global health due to its elevated mortality rates. Approximately 90% of these malignancies are oral squamous cell carcinoma (OSCC). A significant contributor to the prevalence of oral cancer is the difficulty in detecting cancerous biomarkers, further exacerbated by socioeconomic disadvantages and late-stage diagnoses. Given the critical nature of oral cancer, the early detection of biomarkers is essential for reducing mortality rates. This study investigates the application of microRNA-31 (miRNA-31) as a biomarker for the electrochemical detection of oral cancer, recognizing the considerable potential that microRNAs have demonstrated in cancer screening and diagnosis. The methodology employed includes the use of a glassy carbon electrode modified with graphene and a molecular tethering agent designed to enhance sensitivity and specificity. The biosensor exhibited a limit of detection of 10^{- 11} M (70 pg/mL or 6.022 × 10⁶ copies/µL) in buffer and 10^{- 10} M (700 pg/mL or 6.022 × 10⁷ copies/µL) in diluted serum for the complementary target miRNA-31 using the Six Sigma method. The efficacy of this biosensor was further validated through specificity studies utilizing a non-complementary miRNA in both buffer and human serum samples. The electrochemical biosensor displayed exceptional performance and high sensitivity in detecting miRNA-31, confirming its role as an innovative sensor for the non-invasive diagnosis of oral cancer. Furthermore, the proposed biosensor demonstrates several advantages over current methodologies, including reduced detection time, and cost-effective reagents.

Keywords: Graphene; Impedance; Nanomaterials; Nucleic acid; Serum; ssDNA.

Fig. 1

The schematic representation of the fabrication principles for the developed biosensor

Fig. 2

Nyquist plots of different modified electrode surfaces performed in 0.1 M KCl with 5.0 mM [Fe (CN)₆]^3−/4−. The inset shows the equivalent circuit used for fitting

Fig. 3

Electrochemical impedance response for various optimization steps on the developed glassy carbon electrode. Graphene loading on the bare electrode and its effect in DRCT (A). DNA concentration and its effect in DRCT (B). RNA hybridization time and its impact in DRCT (C). RNA hybridization temperature and its effect in DRCT (D)

Fig. 4

The sensitivity calibration curve is based on the relationship between ΔR_CT (%) and the logarithmic concentration of miRNA-31 in buffer. The inset shows the full calibration curve of the concentration range

Fig. 5

The sensitivity calibration curve is based on the relationship between ΔR_CT (%) and the logarithmic concentration of miRNA-31 in diluted serum

Fig. 6

Specificity studies of the target miRNA-31 and non-complementary miRNA-25 in buffer and diluted serum. The asterisks indicate statistical significance (* p < 0.05, and *** p < 0.001 using a two-tailed test)

Fig. 7

Time stability studies of the developed biosensor with 10⁻⁷ M miRNA-31. The asterisk indicates statistical significance (* p < 0.05 using a two-tailed test)