Aza-Arenophilic Gels as Supports for the Reversible Immobilization of Enzymes—Bioanalytical Applications

Abstract

A method is described for the simple, reversible immobilization of enzymes to an aza-arenophilic support. The support is prepared by reacting Sepharose CL-4B first with 3,5-dichloro-2,4,6-trifluoropyridine and 4-dimethylaminopyridine followed with 2-mercaptoethanol. The enzymes were non-covalently adsorbed using a phosphate buffer (0.5M, pH 7.4) and readily desorbed using sodium acetate (0.1M, pH 2.9). The same solid support could be used many times to reversibly bind enzymes. Urease bioreactors prepared by this method were incorporated into a flow injection manifold and used for the quantitation of urea. Calibration curves were found to be linear up to 120 mgdL⁻¹ urea with a detection limit of.0.08mgdL⁻¹. Recovery yields varied between 98 – 103%. The within day RSD was <1.9% and the day to day RSD was < 2.8 for a 20mgdL⁻¹ solution. There was no detectable decrease in bioreactor activity over a period of 34 days. Up to 18 urea samples can be analysed manually per hour.

Key Words:

• reversible immobilization
• aza-arenophilic solid support
• flow injection analysis
• urea quantitation