Selective Surface-Confinement and Elegant Sensing of Vitamin B12 on Hydroxyl-Functionalized Screen-Printed Carbon Electrode

Vitamin B12 (Cyanocobalamin; B12) is an essential micronutrient involved in cellular metabolism and neurological function, and its deficiency is associated with diabetes, hypertension, and cancer. However, trace-level detection of B12 in real samples is challenging due to its low abundance, structural complexity, and matrix interference, while conventional chromatographic methods remain costly and time-consuming. In this work, we present a simple, surface-confined electrochemical sensing strategy for B12 using a hydrogen peroxide (H₂O₂)-assisted electrochemically activated screen-printed carbon electrode (SPCE*) in 0.1 M H₂SO₄ (SPCE*@B12). The activation process generates hydroxyl-rich (>C-OH) oxygenated functional groups on the carbon surface, which enable covalent attachment of B12 through an ether linkage (C-O-C) formation with the hydroxyl moieties of its ribose component within 60 s of exposure time. This stable covalent immobilization enhances surface confinement and markedly improves electron-transfer kinetics at the B12-Co2+/+ sites. Comprehensive physicochemical and spectroscopic characterization using HRTEM, FESEM, FTIR, Raman spectroscopy, XPS, and UPLC confirmed successful functionalization and robust confinement of B12 on the SPCE* surface. The resulting sensor supports single-drop analysis (1-3 μL) with high sensitivity and selectivity, exhibiting a linear electrochemical response over the nanomolar concentration range (50-140 ng mL⁻¹) and a detection limit of 2.49 ng mL⁻¹ within 2 min of the voltammetric run, comparable to that achieved by UPLC, the gold-standard analytical method. Method validation using eight real samples, including beef liver (#1), quail egg (#2), beef meat (#3), chicken egg (#4), yogurt (#5), curd (#6), egg white (#7), and milk (#8), demonstrated excellent agreement with UPLC quantification, with relative errors below 5%. Overall, this rapid, low-volume electrochemical platform enables ultrasensitive B12 detection for routine nutritional and food-quality analysis.