无试剂二氧化钛凝胶酶传感器检测苹果酸对映异构体研究

Studies on the Determination of Malate Enantimers by Reagentless Sensor with Enzyme Encapsulated in Titania Sol-gel Ion Matrix

研制了基于TiO2凝胶的D型和L型苹果酸脱氢酶(D-MDH、L-MDH)安培电化学传感器,并用它检测果汁饮料中D-苹果酸、L-苹果酸含量。利用气相沉积法在玻碳电极表面形成的TiO2凝胶膜,为固定化的MDH提供了一个生物兼容性的微环境。在溶解氧存在的条件下,苹果酸被凝胶膜中的MDH催化氧化,其氧化产物在电位55mV下有电流响应,利用此响应值对苹果酸进行测定,测定时不需要向体系中添加媒介体。D-MDH/TiO2、L-MDH/TiO2两电极对苹果酸表现出快速的响应(响应时间小于10s)。传感器对苹果酸的测定具有较宽的线性范围(1×10-6～2×10-4mol/L)和较低的检测限(0.5×10-6mol/L)。由计算得到的固定化L-MDH和D-MDH的表观米氏常数(KMapp)分别为0.29mmol/L和0.31mmol/L。两电极具有较高的稳定性。本法快速、灵敏度高、选择性强,适合于测定果汁饮料中的苹果酸对映异构体。

Two novel amperometric D-, L-malate sensor was constructed by immobilizing D-, L-malate dehydrate enzyme （D-MDH and L-MDH） in a titania sol-gel matrix. The method for determination of content of D-, L＇-malate was constructed. This matrix provided a microenvironment for retaining the native structure and bioactivity of the entrapped enzyme by vapor deposition method. Malate could be oxidized by dissolving oxygen in presence of immobilized MDH to form a detectable product which was determined at 55 mY without any mediator. The L-malate sensor exhibited a fast response （less than 10 seconds）. The linear range for phenol determination was from 1.0×10^-5 to 2×10^-3 mol/L with a detection limit of 0.5×10^-6 mol/L. The apparent Michaelis-Menten constant of the encapsulated tyrosinase was calculated to be 0.29 mmol/L for L-MDH and 0.31mmol/L for D-MDH. The stability of the biosensors were evaluated. The results suggested that the two sensors are suitable for the determination of malate enantimers in fruit juices quickly with high sensitivity and selectivity.