Construction Of Bi-enzyme Modified Electrode And Its Application In The Detection Of Ethyl Carbamate

Ethyl carbamate(EC) is known as a genotoxic carcinogen that widely exists in fermented food and alcoholic beverages, and is therefore a public health concern. As a consequence, rapid and sensitive detection of EC and effective control of EC content attract wide research interests. Based on high specificity to substrate, enzyme biosensors provide a simple, rapid, sensitive and low-cost mean to detect harmful comounds and improve food safety.Urethanase can decompose EC to equal molar mass of ethanol, CO2 and NH4+, the later can act as substrate of glutamate dehydrogenase(GLDH) to reduce α-ketoglutarate. In this process, equal molar mass of NADH was oxidized. With its maximum absorption wavelength at 340 nm and oxidation current on the surface of electrode, NADH can be detected, which can be further used to quantify the concentration of EC. Based on these principles, a high-sensitive spectrophotometric method for EC determination was firstly established through glutamate dehydrogenase/urethanase cascade reaction with the corresponding change in NADH concerntration. Next, a bi-enzyme electrode was fabricated through immobilizing urethanase and glutamate dehydrogenase on the surface of pyrolytic graphite electrode(PGE). According to the change of current response produced by NADH after reaction, the concentration of EC was determined. The main research contents of this thesis include the following two aspects:(1) Urethanase and GLDH were added into buffer solution containing α-ketoglutarate and NADH for the establishment of cascade reaction system. 5 min after the addition of EC, the change in the absorbance at 340 nm was determined. A relationship between the change of absorbance and the concentration of EC was established. This spectrophotometric method can detect EC in concentration range of 0.3-50 μmol·L-1 with the low limit detection of 9.28 nmol·L-1. The accuracy and precision of the method were verified using mimic Chinese rice wine samples. The recovery of EC in samples was 95.54%-100.01%, and the relative standard deviation(RSD) was 1.634%-4.611%, indicating high accuracy and satisfactory precision of the method.(2) A sol/gel method was developed to immobilize urethanase and GLDH on the surface of PGE. After the addition of different concentration of EC, NADH consumed which can be indicated by the current change during the reaction. Then the concentration of EC can be detected. The detection range of EC is within 0.5-50 μmol·L-1 with the low limit detection of 5.26 nmol·L-1.The accuracy and precision of the detection were verified in synthetic yellow rice wine. The recovery of EC in samples was 100.19%-103.79%, RSD was 2.86%-7.14%, indicating a good accuracy and high precision. Reproducibility, repeatability, stability and interferents-tolerance of the method were also investigated and satisfactory results were obtained.