A Tungsten-Oxide Nanozyme-Type Redox Mediator for Enzyme-Free Colorimetric Glucose Determination in Human Serum

A fully enzyme-free, low-cost spectrophotometric method is reported for the determination of glucose in human serum. The method couples the intrinsic redox reactivity of an in-house-synthesized mixed-valence tungsten-oxide (WOₓ) nanozyme which functions here as a regenerable surface redox mediator (electron shuttle) rather than as a substrate-specific enzyme mimic, to the classic, highly selective Cu(I)-2,2′-biquinoline (cuproine) chromogenic reaction. Glucose reduces the mixed-valence tungsten bronze at its surface; the reduced tungsten centers in turn reduce Cu(II) to Cu(I), which is captured by 2,2′-biquinoline as the intensely colored bis-chelate and concentrated within a cetyltrimethylammonium bromide (CTAB) micellar pseudo-phase, giving a metal-to-ligand charge-transfer band at 550 nm. The redox-mediation mechanism is supported by X-ray photoelectron spectroscopy (XPS), cyclic voltammetry, Arrhenius kinetics, and control experiments. Under optimized conditions the method is linear over 3.2–34.5 mmol L−1 (R2 = 0.9962), with an apparent molar absorptivity of 4.51 × 10³ L mol−1 cm−1, a limit of detection of 0.66 mmol L−1, and intra- and inter-day precision (relative standard deviation, RSD) of ≤2.1% and ≤3.5%, respectively. Because detection rests on non-enzymatic redox chemistry, selectivity was examined against individually added endogenous serum constituents including reducing sugars, ascorbate, urate, and thiols, and in aggregate through the 120-sample method comparison; fructose and ascorbate were identified as the principal chemical interferences and are discussed explicitly. Applied to 120 human serum samples in parallel with a hexokinase reference method, the method showed close agreement (Passing-Bablok slope 0.997, Bland–Altman mean bias −0.17 mmol L−1). The reagents are stable at room temperature and no enzyme is required; the method does, however, require a thermostatted incubation at 45 °C, and its practical niche relative to established enzymatic and point-of-care approaches is discussed candidly.