| Abstract: | The development of the receptor layer of the biosensor for detecting explosive compounds is described. The covalent modification has been chosen for immobilizing E. coli nitroreductase on the gate oxide of the ion-sensitive field effect transistor (ISFET) that is comprised of silicon dioxide. The self-assembled monolayer technique has been used for immobilization. This method assumes the usage of different silanes and spacer molecules for activating the surface of SiO2. Two different immobilization strategies have been compared, one using asymmetric spacers (3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) and 4-(4-maleimidophenyl)butyric acid N-hydroxysuccinimide ester (SMPB)) and another using a symmetric glutaric dialdehyde linker both accompanied by appropriate silanes. For the first method, the dependence of functionalization efficiency on silane concentration has been studied. The sufficient density of enzyme molecules on the surface of SiO2 has been achieved at a concentration of silane of 0.0015%. The type of asymmetric linker has no influence on immobilization efficiency. The method implying glutaric dialdehyde results in higher activity of the immobilized enzyme. For this method, the immobilization procedure has been optimized. The method has been adapted for immobilization of E. coli nitroreductase inside the channel of a microfluidic system on the surface of ISFET. For this purpose, (3-aminopropyl)triethoxysilane (APTES) has been changed to the corresponding silatrane, and the concentration of the enzyme has been increased to 30 μg/mL. The optimized procedure has been successfully used to develop a biosensor for detecting explosives. |
