基于丝网印刷技术的苹果酸脱氢酶电极设计
发布时间：2019-02-15 13:17
【摘要】：L-苹果酸是普遍存在于动物、植物及微生物体内的一种重要羧酸,在细胞内参与多种生命活动,在食品、医药以及化工等领域都有重要的应用。对于L-苹果酸的定量检测,目前主要包括色谱法、光谱法、酶法等,这些方法虽然能够满足测定精确度的要求,但一般步骤繁琐、成本较高。苹果酸脱氢酶(MDH)在存在其辅酶NAD(P)+时可在生理条件下催化苹果酸的转化及NAD(P)H的生成。利用对NAD(P)H敏感的电化学传感器对其进行检测,可以实现对L-苹果酸的定性定量分析。根据这一原理可以通过将苹果酸脱氢酶固定于NADH敏感电极表面形成电流型酶电极的途径解决L-苹果酸的定量检测问题。本文将苹果酸脱氢酶同壳聚糖、戊二醛制成固定化酶制剂,并将其整合至由碳纳米管修饰的电化学基底电极表面构成酶电极,得到对L-苹果酸敏感的酶生物传感器:(1)利用鱼精蛋白(Pr)与多壁碳纳米管(CNT)形成稳定的复合物,以此复合物对丝网印刷电极(SPE)的工作表面进行修饰,获得碳纳米管功能化的丝网印刷电极Pr/CNT/SPE。通过表面微观结构表征发现,鱼精蛋白的应用使碳纳米管修饰电极表面形成了稳定、致密的微观结构,显著提高了修饰电极对NADH的电催化性能。修饰电极对NADH在2-1500μmol/L的范围内存在线性响应,回归方程i(μA)=-1.8764c(mmol/L)-0.0516,R2=0.984。响应时间为6 s,灵敏度为1.8764μA·L/mmol/Lol,检测限为0.05μmol/L(S/N=3)。实验还确定了最佳的Pr/CNT质量分数,确定Pr/CNT质量分数为0.5作为制备Pr/CNT/SPE的最佳方案。(2)以Pr/CNT/SPE作为基底电极构建对L-苹果酸灵敏的电化学酶电极。以戊二醛(GDI)对壳聚糖(CHIT)进行改性处理,获得表面保留有自由醛基的酶固定化载体。将MDH同此固定化载体以合适的比例混合可以得到固定化酶CHIT-GDI-MDH。以此固定化酶修饰Pr/CNT/SPE,并以核微孔膜防止固定化酶的泄漏,即得完整的苹果酸脱氢酶电极CHIT-GDI-MDH/Pr/CNT/SPE。该酶电极对5-300μmol/L内的L-苹果酸存在线性响应,其可用以定量检测的回归方程为i(μA)=-0.69c(mmol/L)-0.0024(R2=0.995)。响应时间为6-8 s,检测灵敏度为0.69μA·L/mmol;检测限为0.03μmol/L(S/N=3)。实验还对酶电极的抗干扰性能进行了评估,发现酶电极具备一定的抗干扰性能,适合用于实际样本中L-苹果酸的分析检测。(3)酶电极的制备方法及检测条件优化。选取CHIT-GDI:MDH比例、pH、工作电位以及测试温度4个因素作为实验变量,首先通过单因素实验分析了其各自对酶电极传感性能的影响并确定出其各自的最佳水平。随后,再以4个因素进行正交试验,进一步寻找最优方案。结果显示,CHIT-GDI-MDH/Pr/CNT/SPE的最佳制备方法及工作条件为:以每30U MDH同100μL CHIT-GDI混合制备CHIT-GDI-MDH,选用pH为7.5的缓冲液,检测温度45℃,检测电位0.36V。该酶酶电极性能稳定,测试结果可靠,可用于实际样本中L-苹果酸的分析测试。其制作过程简单,使用方便,适于工业化生产。
[Abstract]:L-malic acid is an important carboxylic acid in animals, plants and microbes. It is involved in many life activities in cells and has important applications in the fields of food, medicine and chemical industry. The quantitative determination of L-malic acid mainly includes chromatography, spectrometric method and enzymatic method. Although these methods can meet the requirements of accuracy, the general steps are tedious and the cost is high. Malate dehydrogenase (MDH) can catalyze the transformation of malic acid and the formation of NAD (P) H under physiological conditions in the presence of its coenzyme NAD (P). The qualitative and quantitative analysis of L- malic acid can be realized by using electrochemical sensor sensitive to NAD (P) H. According to this principle, the problem of quantitative detection of L-malic acid can be solved by immobilization of malate dehydrogenase on the surface of NADH sensitive electrode to form current-type enzyme electrode. In this paper, malate dehydrogenase and glutaraldehyde were prepared into immobilized enzyme preparation and integrated into the surface of electrochemical substrate electrode modified by carbon nanotubes to form enzyme electrode. An enzyme biosensor sensitive to L-malic acid was obtained. (1) A stable complex of protamine (Pr) and multiwalled carbon nanotube (CNT) was obtained to modify the working surface of screen printing electrode (SPE). Preparation of carbon Nanotube functionalized screen Printing electrode Pr/CNT/SPE. It was found that the application of protamine made the surface of carbon nanotube modified electrode form stable and compact microstructure, and the electrocatalytic performance of modified electrode for NADH was improved significantly. The modified electrode has a linear response to NADH in the range of 2-1500 渭 mol/L. The regression equation I (渭 A) = -1.8764c (mmol/L) -0.0516C R2O 0.984. The response time is 6 s, the sensitivity is 1.8764 渭 A / mol / L ol / L and the detection limit is 0. 05 渭 mol/L (S/N=3). The experiment also determined the best Pr/CNT mass fraction, The best method for the preparation of Pr/CNT/SPE was determined as the mass fraction of Pr/CNT was 0.5. (2) the electrochemical enzyme electrode sensitive to L-malic acid was constructed by using Pr/CNT/SPE as the substrate electrode. Chitosan (CHIT) was modified with glutaraldehyde (GDI) to obtain the immobilized carrier with free aldehyde group on the surface. The immobilized enzyme CHIT-GDI-MDH. can be obtained by mixing MDH with the immobilized carrier in an appropriate proportion. The Pr/CNT/SPE, was modified by immobilized enzyme and the leakage of immobilized enzyme was prevented by nuclear microporous membrane. The complete malate dehydrogenase electrode CHIT-GDI-MDH/Pr/CNT/SPE. was obtained. The enzyme electrode has a linear response to L-malic acid in 5-300 渭 mol/L. The regression equation for quantitative detection is I (渭 A) = -0.69c (mmol/L) -0.0024 (R2O0.995). The response time was 6-8 s, the detection sensitivity was 0.69 渭 A / L / mmol and the detection limit was 0.03 渭 mol/L (S/N=3). The anti-interference performance of the enzyme electrode was also evaluated. It was found that the enzyme electrode had certain anti-interference performance and was suitable for the analysis and detection of L- malic acid in practical samples. (3) the preparation method and detection conditions of enzyme electrode were optimized. Four factors, CHIT-GDI:MDH ratio, pH, working potential and test temperature, were selected as experimental variables. Firstly, the effects of CHIT-GDI:MDH ratio, pH, working potential and test temperature on the sensing performance of enzyme electrode were analyzed by single factor experiments and their optimal levels were determined. Then, the orthogonal experiment was carried out with four factors to find the best scheme. The results showed that the optimal preparation method and working conditions of CHIT-GDI-MDH/Pr/CNT/SPE were as follows: CHIT-GDI-MDH, was prepared by mixing every 30 U MDH with 100 渭 L CHIT-GDI in buffer solution of pH 7.5, detection temperature was 45 鈩，
本文编号：2423399
[Abstract]:L-malic acid is an important carboxylic acid in animals, plants and microbes. It is involved in many life activities in cells and has important applications in the fields of food, medicine and chemical industry. The quantitative determination of L-malic acid mainly includes chromatography, spectrometric method and enzymatic method. Although these methods can meet the requirements of accuracy, the general steps are tedious and the cost is high. Malate dehydrogenase (MDH) can catalyze the transformation of malic acid and the formation of NAD (P) H under physiological conditions in the presence of its coenzyme NAD (P). The qualitative and quantitative analysis of L- malic acid can be realized by using electrochemical sensor sensitive to NAD (P) H. According to this principle, the problem of quantitative detection of L-malic acid can be solved by immobilization of malate dehydrogenase on the surface of NADH sensitive electrode to form current-type enzyme electrode. In this paper, malate dehydrogenase and glutaraldehyde were prepared into immobilized enzyme preparation and integrated into the surface of electrochemical substrate electrode modified by carbon nanotubes to form enzyme electrode. An enzyme biosensor sensitive to L-malic acid was obtained. (1) A stable complex of protamine (Pr) and multiwalled carbon nanotube (CNT) was obtained to modify the working surface of screen printing electrode (SPE). Preparation of carbon Nanotube functionalized screen Printing electrode Pr/CNT/SPE. It was found that the application of protamine made the surface of carbon nanotube modified electrode form stable and compact microstructure, and the electrocatalytic performance of modified electrode for NADH was improved significantly. The modified electrode has a linear response to NADH in the range of 2-1500 渭 mol/L. The regression equation I (渭 A) = -1.8764c (mmol/L) -0.0516C R2O 0.984. The response time is 6 s, the sensitivity is 1.8764 渭 A / mol / L ol / L and the detection limit is 0. 05 渭 mol/L (S/N=3). The experiment also determined the best Pr/CNT mass fraction, The best method for the preparation of Pr/CNT/SPE was determined as the mass fraction of Pr/CNT was 0.5. (2) the electrochemical enzyme electrode sensitive to L-malic acid was constructed by using Pr/CNT/SPE as the substrate electrode. Chitosan (CHIT) was modified with glutaraldehyde (GDI) to obtain the immobilized carrier with free aldehyde group on the surface. The immobilized enzyme CHIT-GDI-MDH. can be obtained by mixing MDH with the immobilized carrier in an appropriate proportion. The Pr/CNT/SPE, was modified by immobilized enzyme and the leakage of immobilized enzyme was prevented by nuclear microporous membrane. The complete malate dehydrogenase electrode CHIT-GDI-MDH/Pr/CNT/SPE. was obtained. The enzyme electrode has a linear response to L-malic acid in 5-300 渭 mol/L. The regression equation for quantitative detection is I (渭 A) = -0.69c (mmol/L) -0.0024 (R2O0.995). The response time was 6-8 s, the detection sensitivity was 0.69 渭 A / L / mmol and the detection limit was 0.03 渭 mol/L (S/N=3). The anti-interference performance of the enzyme electrode was also evaluated. It was found that the enzyme electrode had certain anti-interference performance and was suitable for the analysis and detection of L- malic acid in practical samples. (3) the preparation method and detection conditions of enzyme electrode were optimized. Four factors, CHIT-GDI:MDH ratio, pH, working potential and test temperature, were selected as experimental variables. Firstly, the effects of CHIT-GDI:MDH ratio, pH, working potential and test temperature on the sensing performance of enzyme electrode were analyzed by single factor experiments and their optimal levels were determined. Then, the orthogonal experiment was carried out with four factors to find the best scheme. The results showed that the optimal preparation method and working conditions of CHIT-GDI-MDH/Pr/CNT/SPE were as follows: CHIT-GDI-MDH, was prepared by mixing every 30 U MDH with 100 渭 L CHIT-GDI in buffer solution of pH 7.5, detection temperature was 45 鈩，
本文编号：2423399
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