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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(SiO2). 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. 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.

AFM studies of slides of  silicon dioxide(SiO2) at each stage of the process of immobilization using glutaric dialdehyde showed interesting results. First, it is worth noting that the roughness of the films of APTES obtained using the proposed technique (Ra = 0.169 nm) differs slightly from the roughness of the initial surface of silicon (on average, the roughness parameter for a silicon plate is ~0.12 nm).The results of the AFM studies of the morphology of the obtained films of the enzyme showed that even minimum values of used concentrations of MPTES were sufficient for the formation of a dense film of the enzyme on the surface of SiO2.

Silatranes compare favorably with silanes by the ability to form uniform films on the surface of  silicon dioxide(SiO2) in an aqueous solution. The activity of immobilized nitroreductase in the case of the use of silatrane was the same as in the case of silane. The concentration of the enzyme is another important aspect during the immobilization in the microchannel. Because of the notable decrease in the ratio of the volume of the reaction mixture to the area. That the the developed procedure has been successfully implemented during the creation of a bio-sensor for the detection of explosives with silicon dioxide(SiO2).


 

 

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