Development of nonenzymatic glucose sensors with high reproducibility and stability is an urgent need to reduce cost of regular diabetic monitoring. Here, we have fabricated ruthenium dioxide–poly(vinyl chloride)–Nafion (RuO
2–PVC–Nafion) composite for direct glucose sensing in sodium hydroxide and phosphate buffer nonenzymatically for the first time. The restricted activity of the RuO
2–PVC film electrode in alkaline pH is extended to neutral pH using Nafion as an outer membrane, which reduces the distance between Ru active sites by bridging effect and improves the electrode stability. The catalytic rate, measured in terms of change of RuO
2 resistance, is similar irrespective of the medium for the high temperature annealed RuO
2 (700 °C), whereas the low temperature annealed RuO
2 (300 °C) is highly sensitive for the change in the pH of the solution. This is revealed by observing large Michaelis–Menten kinetic constant
K M for the RuO
2 (700 °C) than the low temperature annealed RuO
2 (300 °C) due to effective increase in the catalytic active sites similar to oxygen evolution reaction. Contrast to this, the buffer solution does not influence significantly the apparent
K M observed for RuO
2 (300 °C) and has greater impact on the high temperature 500 and 700 °C annealed RuO
2 samples. Cyclic voltammetry, chrono amperommetry, and electrochemical impedance spectroscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction techniques are used for characterization of the sensor behavior. The RuO
2–PVC–Nafion senses glucose selectively in the presence of potential interferences like fructose, galactose, mannose, sucrose, starch, uric acid, ascorbic acid, dopamine, and catechol in NaOH and phosphate buffer. Glucose sensing in the blood serum of the diabetic and nondiabetic patients is made. The results suggest that the RuO
2–PVC–Nafion is a promising candidate for the development of nonenzymatic glucose sensors.
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