A Supramolecular Indicator System for Detecting Urea Based on the Cellulose-Nanodiamond Composite Material and Urease

Abstract

A supramolecular indicator system (cellulose-nanodiamonds-urease) for reusable biochemical detection of urea has been fabricated using sequential immobilization of the components. Modified nanodiamonds (MNDs) were covalently immobilized onto DEAE cellulose granules via the nucleophilic addition reaction. At DEAE cellulose: MND ratio of 4:1 (w/w), up to 110 μg of nanoparticles bound onto 1 mg of the polymer during the addition reaction. Urease was immobilized by covalent conjugation onto the polymer-MND composite with the benzoquinone-activated surface. In comparative experiments, the enzyme was immobilized onto initial polymer granules via nonspecific adsorption and covalent conjugation. However, when these indicator systems were repeatedly used to detect the analyte, the enzyme was considerably inactivated, and that was evidenced by a decrease in the colored product yield. At the same time, the enzyme covalently bound onto the DEAE cellulose-MND composite showed higher functional efficacy and enabled more stable yields of the colored product in repeated urea assays. Comparative experiments with the indicator systems repeatedly used to detect urea at 37 °C demonstrated that the enzyme covalently conjugated onto the DEAE cellulose-MND composite showed greater thermo stability, and its activity was reduced at a much slower rate than the activity of the enzyme covalently bound to the polymer. The data obtained in the present study offer the prospect of designing a new type of reusable indicator assay systems (polymer carrier-nanodiamond-biomarker supramolecular systems) for biomedical analytical applications.