Wu Li-Chen, Cheng Chien-Ming
Biochemistry Laboratory, Department of Applied Chemistry, National Chi-Nan University, Puli, Nantou 545, Taiwan.
Anal Biochem. 2005 Nov 15;346(2):234-40. doi: 10.1016/j.ab.2005.08.031. Epub 2005 Sep 19.
A flow-injection enzymatic analytical system was developed for determination of glycerol and triacylglycerol based on enzymatic reactions in capillary followed by electrochemical detection. The hydrogen peroxide produced from the enzyme reaction was monitored by a platinum-based electrochemical probe. Different immobilization strategies on silica support were studied. The best and most effective configuration found for the measurement of glycerol and triacylglycerols in this system was the tandem connection of a lipase column and a silica-fused capillary column coimmobilized with glycerokinase (GK) and glycerol-3-phosphate oxidase (GPO). Lipase helps the breakdown of triacylglycerol to yield free fatty acids and glycerol, while glycerokinase catalyzes the adenosine-5-triphosphate-dependent phosphorylation of glycerol to yield alpha-glycerol phosphate, which can subsequently be oxidized by 3-glycerol phosphate oxidase to produce hydrogen peroxide. Response-surface methodology (RSM) was applied to optimize the proposed system for glycerol. Experiment settings were designed by central composite design to investigate the combined effects of pH, flow rate, reaction temperature, and ATP concentration on collected signals. The fitted model, per RSM, showed that the optimum conditions of the system are 2 mM ATP in 0.1 M carbonate buffer (pH 11.0), flow rate of 0.18 mL/min, temperature of 35 degrees C, 20 microL of sample injection, and applied voltage of 0.650 V. The proposed biosensing system using lipase, GK, and GPO exhibited a flow-injection analysis peak response of 2.5 min and a detection limit of 5 x 10(-5) M glycerol (S/N = 3) with acceptable reproducibility (CV < 4.30%). It also had linear working ranges from 10(-4) to 10(-2) M for glycerol and from 10(-3) to 10(-2) M for triacylglycerol. The capillary enzyme reactor was stable up to 2 months in continuous operation, and it was possible to analyze up to 15 samples per hour. The present biosensing system holds promise for on-line detection of triacylglycerol in serum and glycerol content in fermented products.
基于毛细管中的酶促反应并结合电化学检测，开发了一种流动注射酶分析系统，用于测定甘油和三酰甘油。酶促反应产生的过氧化氢由铂基电化学探针监测。研究了在二氧化硅载体上的不同固定化策略。在该系统中，用于测定甘油和三酰甘油的最佳且最有效的配置是脂肪酶柱与共固定有甘油激酶（GK）和甘油-3-磷酸氧化酶（GPO）的二氧化硅熔融毛细管柱串联连接。脂肪酶有助于三酰甘油分解产生游离脂肪酸和甘油，而甘油激酶催化甘油在三磷酸腺苷依赖下磷酸化生成α-甘油磷酸，随后α-甘油磷酸可被3-甘油磷酸氧化酶氧化产生过氧化氢。应用响应面方法（RSM）对所提出的甘油分析系统进行优化。通过中心复合设计来设计实验设置，以研究pH、流速、反应温度和ATP浓度对采集信号的综合影响。根据RSM拟合的模型表明，该系统的最佳条件为：在0.1 M碳酸盐缓冲液（pH 11.0）中加入2 mM ATP，流速为0.18 mL/min，温度为35℃，进样量为20 μL，施加电压为0.650 V。所提出的使用脂肪酶、GK和GPO的生物传感系统在流动注射分析中呈现出2.5分钟的峰响应，甘油的检测限为5×10⁻⁵ M（信噪比 = 3），具有可接受的重现性（变异系数 < 4.30%）。它对甘油的线性工作范围为10⁻⁴至10⁻² M，对三酰甘油的线性工作范围为10⁻³至10⁻² M。毛细管酶反应器在连续运行中稳定长达2个月，每小时可分析多达15个样品。本生物传感系统有望用于在线检测血清中的三酰甘油和发酵产品中的甘油含量。
