Study On Amperometric Nitrate Reductase And Horseradish Peroxidase Biosensors Based On Organic-inorganic Hybrid Material

Nitrate and phenolic compounds, as main pollutants, are threatening human health and our living environment. The fast, accurate detection of these two pollutants is of great significance. With the merits of prompt analysis, high sensitivity and low cost, biosensors have been widely used in enviromental monitoring, clinical diagnosis, food analysis and military medicine.In this dissertation, with nanometer technology we immobilized nitrate reductase and horseradish peroxidase enzymes respectively by sol-gel derived organic-inorganic hybrid material to fabricate various nitrate reductase and horseradish peroxidase biosensors. The detailed work of this thesis has been set out as follows:By dialysis and optimizing the parameters of hybrid material, an amperometric nitrate reductase biosensor with good performance and stability had been prepared. The electroanalytical properties of optimized biosensor at -0.712V(vs SCE) were measured: linear response range, sensitivity and detection limit were 0.01 0.4mmol/L (R²=0.9928), 6.866nA/(μmol/L), 3.37×10^-6mol/L, respectively. It reached a steady-state response to the added nitrate in 6.7s. After 30 days'interval use, the response current dropped to 87.6% of its initial value.To enhance the sensitivity and stability of the nitrate reductase biosensor, single-wall carbon nanotubes (SWNTs) were used and two enzyme electrodes were fabricated by different methods: First, nitrate reductase was embedded on SWNTs modified electrode by hybrid material; Second, SWNTs were added to hybrid material directly. The results showed that response currents of the two electrodes were both significantly enhanced and the sensitivity were 8.806 A/(μmol/L),11.361 nA/(μmol/L), respectively. Applied potential and response time had changed slightly whereas the second electrode had better stability and reproducibility.An amperometric horseradish peroxidase biosensor which can determine phenolic compounds (such as hydroquinone, phenol, catechol, chlorophenol, cresol) had been fabricated by immobilizing horseradish peroxidase enzymes on the surface of Au electrode with hybrid material. The electroanalytical properties of optimized biosensor at -0.20V(vs SCE) were measured: linear response range, sensitivity and detection limit were 1⁷0μmol/L (R2=0.9996), 93nA/(μmol/L), 0.023μmol/L, respectively. It reached a steady-state response to the added hydroquinone in 2.4s. After 30 days'interval use, the response current dropped to 91.6% of its initial value.Two types of horseradish peroxidase biosensors based on SWNTs and Prussian Blue modified electrodes had been fabricated which can both reduced operating potential and eliminated the interferences of electroactive substances (such as ascorbic acid and uric acid) effectively. Response time were 4.6s and 9.9s, respectively.