Thermometric Sensing of Peroxide in Organic Media. Application To Monitor the Stability of RBP-Retinol-HRP Complex

摘要

The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid—HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlied pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detec-don of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5—7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a relinoic acid—HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP—retinoic acid—HIRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.