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Glucose concentration is an important parameter in biomedicine since glucose is involved in many metabolic pathways in organisms. Many methods for glucose detection have been developed for use in various applications, particularly in the field of healthcare in diabetics. In this study, ratiometric fluorescent glucose-sensing membranes were fabricated based on the oxygen levels consumed in the glucose oxidation reaction under the catalysis of glucose oxidase (GOD). The oxygen concentration was measured through the fluorescence quenching effect of an oxygen-sensitive fluorescent dye like platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) by oxygen molecules. Coumarin 6 (C6) was used as a reference dye in the ratiometric fluorescence measurements. The glucose-sensing membrane consisted of two layers: The first layer was the oxygen-sensing membrane containing polystyrene particles (PS) doped with PtP and C6 (e.g., PS@C6^PtP) in a sol–gel matrix of aminopropyltrimethoxysilane and glycidoxypropyltrimethoxysilane (GA). The second layer was made by immobilizing GOD onto one of three supporting polymers over the first layer. These glucose-sensing membranes were characterized in terms of their response, reversibility, interferences, and stability. They showed a wide detection range to glucose concentration in the range of 0.1 to 10 mM, but high sensitivity with a linear detection range of 0.1 to 2 mM glucose. This stable and sensitive ratiometric fluorescent glucose biosensor provides a reliable way to determine low glucose concentrations in blood serum by measuring tear glucose.

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe 3 O 4 , via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K m and V max ) were determined by Michaelis-Menten enzyme kinetics. The K m values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring. © KSBB

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