Intercalating methylene blue in molecular beacon for sensitive detection of salivary TNF-α towards early diagnosis of oral cancer

Abstract

Oral cancer is the sixth most common cancer globally, and pro-inflammatory cytokines are associated with oral cancer development and progression. Thus, monitoring salivary pro-inflammatory cytokines is essential for the early diagnosis of oral cancer. Electrochemical molecular beacons are popular in the design of biosensors for the real-time monitoring of analytes. Labelling redox probes on molecular beacons reporting electrochemical signals is critical to enhance their sensitivity. Herein, we designed an electrochemical biosensor, denoted as screen-printed carbon electrode (SPCE)-graphene oxidize-aptamer conjugated with methylene blue (MB) (SPCE-GO-aptamer (MB)), by intercalating methylene blue (i.e., the redox probe) in the hairpin structure of a molecular beacon having specific affinity to TNF-α on a graphene oxide (GO)-modified glass screen-printed carbon electrode for the quantitative detection of TNF-α. For comparison, another sensing interface termed SPCE-GO-aptamer-MB, on which methylene blue was covalently attached to the end of the molecular beacon on a GO-modified SPCE was employed for the quantitative detection of TNF-α. Techniques including electrochemistry, XPS, and SEM were used to characterize the surface of the functionalized sensor. Because a larger amount of MB could be intercalated in the hairpin of the molecular beacon, a relatively larger MB signal was observed for SPCE-GO-aptamer (MB) than that on SPCE-GO-aptamer-MB when both biosensing interfaces were exposed to the same concentration of TNF-α. The linear range of SPCE-GO-aptamer (MB) and SPCE-GO-aptamer-MB for the detection of TNF-α was 1–400 pg mL⁻¹ with a detection limit of 1 pg mL⁻¹ and 10–300 pg mL⁻¹ with a detection limit of 10 pg mL⁻¹, respectively. The SPCE-GO-aptamer(MB) sensing interface was validated by detecting TNF-α in saliva with a desirable performance, which promises a non-invasive method for monitoring the dynamic cytokine concentration in oral cancer.