Preparation and characterization of sensing membranes for the detection of glucose, lactate and tyramine in microtiter plates

In this work, sensing membranes for the detection of glucose, lactate and tyramine were successfully prepared by immobilizing enzymes and fluorophore on sol–gels. The membranes were fabricated on the bottom of the wells in a microtiter plate. Glucose oxidase (GOD), lactate oxidase (LOD) and tyramine oxidase (TOD) were immobilized on individual sol–gels or a mixture of different sol–gels (3-glycidoxypropyl-trimethoxysilane (GPTMS), methyl-triethoxysilane (MTES), aminopropyl-trimethoxysilane (APTMS)). The oxidation of the analytes specifically catalyzed by the enzymes resulted in the reduction of the oxygen concentration, which changed the fluorescence intensity (FI) of the oxygen sensitive ruthenium complex acting as the transducer. The linear calibration graphs were in the ranges of 0.0–5.0 g/l for glucose, 0.0–9.0 mg/l for lactate and 0.0–100 mg/l for tyramine. The values of the detection limit were found to be 0.10–0.52 g/l for glucose, 7.77 mg/l for lactate and 6.30–8.73 mg/l for tyramine. The covalent binding between the epoxy and amine groups of the sol–gels and enzymes, respectively, prevented the enzymes from being washed out and preserved the high stability of the sensing membranes. The different ratios of silanes in the sol–gels, which were used as the supporting matrix for the immobilization of the enzymes led to different responses of the sensing membranes to various concentrations of glucose, lactate and tyramine. The kinetic parameters of the enzymatic reactions, and the stability and other parameters for the sensing membranes were also investigated.