电化学生物传感器的研制及应用
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摘要
本文对电化学生物传感器的类型、制备方法以及在分析化学中的应用作了较为详细的评述。在此基础上,采用多种电极材料,对生物材料的固定化方法进行了一系列探索,构筑了多种性能良好的生物传感器,并对其应用进行了研究。各项研究工作简述如下:
1.葡萄糖氧化酶生物传感器的制备与应用研究
1.1聚中性红修饰玻碳电极研制葡萄糖生物传感器
以中性红为电子传递介质,电聚合于Nafion修饰玻碳电极表面,以戊二醛作交联剂固定葡萄糖氧化酶,最后覆盖一层Nafion膜防止酶流失,构建一种新型葡萄糖生物传感器。详细探讨了传感器的电化学性能及其对葡萄糖的最佳响应条件,结果表明,30℃时,传感器在pH 7.0的PBS中线性响应范围为1.0×10~(-5)～5.0×10~(-3) mol·L-1。该传感器制作简单、性能优良,有潜在应用前景。
1.2基于静电吸附碳纳米管和壳聚糖固定葡萄糖氧化酶生物传感器的研究本文以Nafion-聚中性红修饰电极为基底,自组装多壁碳纳米管和壳聚糖之后,再固定上葡萄糖氧化酶,制成葡萄糖氧化酶生物传感器。实验表明,该传感器在30℃的PBS (pH 7.0)中对葡萄糖的线性响应范围为5.0×10~(-6)～2.0×10~(-3) mol/L,线性相关系数为0.9948,检出限为1.0×10~(-6) mol/L,达到95 %稳态电流所用的时间<10 s,于4℃环境保存30 d后峰电流值约为原来的84.3 %,且具有较低的工作电位,能有效地消除抗坏血酸等的干扰。
2.辣根过氧化物酶生物传感器的制备与应用研究
2.1明胶固定辣根过氧化物酶制备H2O2传感器
用明胶将辣根过氧化物酶(HRP)固定于多壁碳纳米管(MWNT)和茜素红(AR)修饰的玻碳(GC)电极上,制成HRP生物传感器(HRP/AR/MWNT/GC),然后在3 %戊二醛(GA)中进行交联改性,以克服明胶膜易溶胀的缺点并提高膜的稳定性。同时详细探讨了该传感器对H2O2的响应性能,并优化了实验条件。结果表明,该传感器对H2O2的线性响应范围为5.0×10~(-6)～1.0×10~(-3) mol/L,线性相关系数为0.9932,检出限为1.0×10-7 mol/L,且放于4℃环境30 d后,峰电流值约为原来的72.1 %。该传感器响应快速,灵敏度高,且具有良好的重现性、稳定性及较长的使用寿命,具有潜在的应用价值。
2.2茜素红修饰玻碳电极研制辣根过氧化物酶生物传感器
以多壁碳纳米管(MWNT)修饰玻碳电极为基底,自组装一层带负电荷的电子媒介体茜素红(AR),再通过分子间静电作用力吸附固定辣根过氧化物酶(HRP),制成辣根过氧化物酶生物传感器(HRP/AR/MWNT/GC)。探讨了媒介体组装时间、pH、温度、工作电位对电极响应的影响。结果表明,该传感器对H2O2具有良好的催化作用,还原峰电流与H2O2的浓度在1.0×10~(-6)～1.0×10~(-3) mol/L范围内呈现良好的线性关系:ip=139.4811+1.0537c,R = 0.9953,检出限为2.0×10-8 mol/L。该传感器响应快速,灵敏度高,且具有良好的重现性、稳定性及较长的使用寿命,具有潜在的应用价值。
3.细胞色素c在纳米杂化膜修饰玻碳电极上的直接电化学
以多壁碳纳米管(MWNT)修饰玻碳(GC)电极为基底,自组装金纳米粒子(AuNPs)及L-半胱氨酸(L-Cys)研制杂化膜修饰电极(L-Cys/AuNPs/MWNT/GC)。实验表明,该膜修饰电极在PBS(pH 7.0)中对细胞色素c(Cyt c)的直接电子转移反应具有良好的电催化行为,于0.121 V(vs. Ag/AgCl)附近有一对可逆的氧化还原峰,峰电流与浓度在4.037×10~(-5)～3.230×10~(-4) mol/L范围内呈现良好的线性关系,线性相关系数R=0.9957,检出限是2.423×10~(-5) mol/L。该传感器响应快速,性能优良。
This article described in detail the types, preparation methods and applications of electrochemical biosensors in the field of analytical chemistry. On the basis of different electrode material and lots of researches on immobilization of bio-material, some novel biosensors was fabricated. Electrochemical behaviors and their applications of the resulting modified electrodes were studied. The main research work was as follows: 1. Preparation of Glucose Oxidase Biosensors and Applications
1.1 Glucose Biosensor Based on Poly(neutral red) Modified Glassy Carbon Electrode
A novel glucoseoxidase biosensor was fabricated through crosslinking method. Neutral red, which was used as the electron mediator, electropolymerized on the Nafion modified glassy carbon electrode, forming NR/Nafion/GC electrode. Then glucose oxidase was immobilized on NR/Nafion/GC electrode by a cross-linker Glutaraldehyde. Lastly, additional Nafion membrane prevent the glucose oxidase dissipating from Glucose Biosensor. The biosensor kept a linear relationship with the concentration of glucose in the range of 1.0×10~(-5)～5.0×10~(-3) mol·L-1 in pH 7.0 PBS at 30℃.
1.2 Glucose Oxidase Biosensor Based on Carbon Nanotubes And Chitosan Through Electrostatic Adsorption
A novel glucose biosensor was fabricated by means of self-assembled technique to immobilize Glucose Oxidase (GOD). Neutral Red was electorpolymerized on glassy carbon electrode (GCE) modified with Nafion to form a positively charged surface, then Multi-walled Carbon Nanotubes (MWNT) and Chitosan (Cs) was linked by electrostatic adsorption step by step, finally GOD was electrostaticly absorbed in Cs film to prepare a glucose biosensor. The experiments showed a linear range to concentration of glucose was 5.0×10~(-6)～2.0×10~(-3) mol/L with a detection limit of 1.0×10~(-6) mol/L in PBS(pH 7.0) at 30℃, and response time was less than 10 s. The biosensor retained 84.3% of its original peak current after thirty days. Moreover, the biosensor exhibited good reproducibility, high selectivity and anti-interference ability to ascorbic acid, etc.
2. Preparation of Horseradish Peroxidase Biosensors and Applications
2.1 Hydrogen Peroxide Biosensor Based on Gelatin Immobilizing Horseradish Peroxidase
A novel Hydrogen Peroxide biosensor was obtained by successfully immobilizing horseradish peroxidase (HRP) on a glassy carbon (GC) electrode, which was modified with multi-walled carbon nanotubes (MWNT) and alizarin red(AR). Then the biosensor was immerged in 3% glutaraldehyde (GA) in order to overcome the swelling and improve the stability of the gelatin membrane. The experimental condition was optimized, and the electrochemistry behavior of the HRP/AR/MWNT/GC biosensor in PBS (pH 7.0) and H2O2 were discussed. The biosensor displayed rapid response and expanded linear response range from 5.0×10~(-6) to 1.0×10~(-3) mol/L (R=0.9932) with detection limit of 1.0×10-7 mol/L in PBS (pH 7.0), as well as acceptable preparation reproducibility and excellent stability.
2.2 Horseradish Peroxidase Biosensor Based on Alizarin Red Modified Glassy Carbon Electrode
A novel biosensor was fabricated by multi-walled carbon nanotubes (MWNT) modified glassy carbon (GC) electrode self-assembling Alizarin red (AR) coated with negative charge and staticly adsorbing with horseradish peroxidase (HRP) to fabricate amperometric biosensor (HRP/AR/MWNT/GC). The experimental condition was optimized, and the sensor showed a good catalytic behavior to H2O2. The linear response of the sensor to H2O2 is in the range of 1.0×10~(-6)～1.0×10~(-3) mol/L with a correlation coefficient of 0.9953. The detection limit of the biosensor was 2.0×10-8 mol/L. Experiments showed that the sensor response rapidly with high sensitivity and good reproducibility, stability and longer service life, which possessed potential application value.
3. Direct Electrochemistry of Cytochrome c at Nanohybrid Film Modified Electrode
Being novel favorite promoters, L-cysteine (L-Cys), gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWNT), because of particular multi-absorbing sites, was immobilized on glassy carbon (GC) electrode surface by layer-by-layer self-assembly technique to form a robust and effective nanohybrid film. The film modified electrode (L-Cys/AuNPs/MWNT/GC) can promote the direct electron change reaction of cytochrome c (Cyt c). Experiments showed a pair of well-defined and nearly reversible peaks of Cyt c at about 0.121 V (vs.Ag/AgCl) was obtained. The sensor responded rapidly to Cyt c in the linear range from 4.037×10~(-5) to 3.230×10~(-4) mol/L with detection limit of 2.423×10~(-5) mol/L in PBS(pH 7.0) .
1.葡萄糖氧化酶生物传感器的制备与应用研究
1.1聚中性红修饰玻碳电极研制葡萄糖生物传感器
以中性红为电子传递介质,电聚合于Nafion修饰玻碳电极表面,以戊二醛作交联剂固定葡萄糖氧化酶,最后覆盖一层Nafion膜防止酶流失,构建一种新型葡萄糖生物传感器。详细探讨了传感器的电化学性能及其对葡萄糖的最佳响应条件,结果表明,30℃时,传感器在pH 7.0的PBS中线性响应范围为1.0×10~(-5)～5.0×10~(-3) mol·L-1。该传感器制作简单、性能优良,有潜在应用前景。
1.2基于静电吸附碳纳米管和壳聚糖固定葡萄糖氧化酶生物传感器的研究本文以Nafion-聚中性红修饰电极为基底,自组装多壁碳纳米管和壳聚糖之后,再固定上葡萄糖氧化酶,制成葡萄糖氧化酶生物传感器。实验表明,该传感器在30℃的PBS (pH 7.0)中对葡萄糖的线性响应范围为5.0×10~(-6)～2.0×10~(-3) mol/L,线性相关系数为0.9948,检出限为1.0×10~(-6) mol/L,达到95 %稳态电流所用的时间<10 s,于4℃环境保存30 d后峰电流值约为原来的84.3 %,且具有较低的工作电位,能有效地消除抗坏血酸等的干扰。
2.辣根过氧化物酶生物传感器的制备与应用研究
2.1明胶固定辣根过氧化物酶制备H2O2传感器
用明胶将辣根过氧化物酶(HRP)固定于多壁碳纳米管(MWNT)和茜素红(AR)修饰的玻碳(GC)电极上,制成HRP生物传感器(HRP/AR/MWNT/GC),然后在3 %戊二醛(GA)中进行交联改性,以克服明胶膜易溶胀的缺点并提高膜的稳定性。同时详细探讨了该传感器对H2O2的响应性能,并优化了实验条件。结果表明,该传感器对H2O2的线性响应范围为5.0×10~(-6)～1.0×10~(-3) mol/L,线性相关系数为0.9932,检出限为1.0×10-7 mol/L,且放于4℃环境30 d后,峰电流值约为原来的72.1 %。该传感器响应快速,灵敏度高,且具有良好的重现性、稳定性及较长的使用寿命,具有潜在的应用价值。
2.2茜素红修饰玻碳电极研制辣根过氧化物酶生物传感器
以多壁碳纳米管(MWNT)修饰玻碳电极为基底,自组装一层带负电荷的电子媒介体茜素红(AR),再通过分子间静电作用力吸附固定辣根过氧化物酶(HRP),制成辣根过氧化物酶生物传感器(HRP/AR/MWNT/GC)。探讨了媒介体组装时间、pH、温度、工作电位对电极响应的影响。结果表明,该传感器对H2O2具有良好的催化作用,还原峰电流与H2O2的浓度在1.0×10~(-6)～1.0×10~(-3) mol/L范围内呈现良好的线性关系:ip=139.4811+1.0537c,R = 0.9953,检出限为2.0×10-8 mol/L。该传感器响应快速,灵敏度高,且具有良好的重现性、稳定性及较长的使用寿命,具有潜在的应用价值。
3.细胞色素c在纳米杂化膜修饰玻碳电极上的直接电化学
以多壁碳纳米管(MWNT)修饰玻碳(GC)电极为基底,自组装金纳米粒子(AuNPs)及L-半胱氨酸(L-Cys)研制杂化膜修饰电极(L-Cys/AuNPs/MWNT/GC)。实验表明,该膜修饰电极在PBS(pH 7.0)中对细胞色素c(Cyt c)的直接电子转移反应具有良好的电催化行为,于0.121 V(vs. Ag/AgCl)附近有一对可逆的氧化还原峰,峰电流与浓度在4.037×10~(-5)～3.230×10~(-4) mol/L范围内呈现良好的线性关系,线性相关系数R=0.9957,检出限是2.423×10~(-5) mol/L。该传感器响应快速,性能优良。
This article described in detail the types, preparation methods and applications of electrochemical biosensors in the field of analytical chemistry. On the basis of different electrode material and lots of researches on immobilization of bio-material, some novel biosensors was fabricated. Electrochemical behaviors and their applications of the resulting modified electrodes were studied. The main research work was as follows: 1. Preparation of Glucose Oxidase Biosensors and Applications
1.1 Glucose Biosensor Based on Poly(neutral red) Modified Glassy Carbon Electrode
A novel glucoseoxidase biosensor was fabricated through crosslinking method. Neutral red, which was used as the electron mediator, electropolymerized on the Nafion modified glassy carbon electrode, forming NR/Nafion/GC electrode. Then glucose oxidase was immobilized on NR/Nafion/GC electrode by a cross-linker Glutaraldehyde. Lastly, additional Nafion membrane prevent the glucose oxidase dissipating from Glucose Biosensor. The biosensor kept a linear relationship with the concentration of glucose in the range of 1.0×10~(-5)～5.0×10~(-3) mol·L-1 in pH 7.0 PBS at 30℃.
1.2 Glucose Oxidase Biosensor Based on Carbon Nanotubes And Chitosan Through Electrostatic Adsorption
A novel glucose biosensor was fabricated by means of self-assembled technique to immobilize Glucose Oxidase (GOD). Neutral Red was electorpolymerized on glassy carbon electrode (GCE) modified with Nafion to form a positively charged surface, then Multi-walled Carbon Nanotubes (MWNT) and Chitosan (Cs) was linked by electrostatic adsorption step by step, finally GOD was electrostaticly absorbed in Cs film to prepare a glucose biosensor. The experiments showed a linear range to concentration of glucose was 5.0×10~(-6)～2.0×10~(-3) mol/L with a detection limit of 1.0×10~(-6) mol/L in PBS(pH 7.0) at 30℃, and response time was less than 10 s. The biosensor retained 84.3% of its original peak current after thirty days. Moreover, the biosensor exhibited good reproducibility, high selectivity and anti-interference ability to ascorbic acid, etc.
2. Preparation of Horseradish Peroxidase Biosensors and Applications
2.1 Hydrogen Peroxide Biosensor Based on Gelatin Immobilizing Horseradish Peroxidase
A novel Hydrogen Peroxide biosensor was obtained by successfully immobilizing horseradish peroxidase (HRP) on a glassy carbon (GC) electrode, which was modified with multi-walled carbon nanotubes (MWNT) and alizarin red(AR). Then the biosensor was immerged in 3% glutaraldehyde (GA) in order to overcome the swelling and improve the stability of the gelatin membrane. The experimental condition was optimized, and the electrochemistry behavior of the HRP/AR/MWNT/GC biosensor in PBS (pH 7.0) and H2O2 were discussed. The biosensor displayed rapid response and expanded linear response range from 5.0×10~(-6) to 1.0×10~(-3) mol/L (R=0.9932) with detection limit of 1.0×10-7 mol/L in PBS (pH 7.0), as well as acceptable preparation reproducibility and excellent stability.
2.2 Horseradish Peroxidase Biosensor Based on Alizarin Red Modified Glassy Carbon Electrode
A novel biosensor was fabricated by multi-walled carbon nanotubes (MWNT) modified glassy carbon (GC) electrode self-assembling Alizarin red (AR) coated with negative charge and staticly adsorbing with horseradish peroxidase (HRP) to fabricate amperometric biosensor (HRP/AR/MWNT/GC). The experimental condition was optimized, and the sensor showed a good catalytic behavior to H2O2. The linear response of the sensor to H2O2 is in the range of 1.0×10~(-6)～1.0×10~(-3) mol/L with a correlation coefficient of 0.9953. The detection limit of the biosensor was 2.0×10-8 mol/L. Experiments showed that the sensor response rapidly with high sensitivity and good reproducibility, stability and longer service life, which possessed potential application value.
3. Direct Electrochemistry of Cytochrome c at Nanohybrid Film Modified Electrode
Being novel favorite promoters, L-cysteine (L-Cys), gold nanoparticles (AuNPs) and multi-walled carbon nanotubes (MWNT), because of particular multi-absorbing sites, was immobilized on glassy carbon (GC) electrode surface by layer-by-layer self-assembly technique to form a robust and effective nanohybrid film. The film modified electrode (L-Cys/AuNPs/MWNT/GC) can promote the direct electron change reaction of cytochrome c (Cyt c). Experiments showed a pair of well-defined and nearly reversible peaks of Cyt c at about 0.121 V (vs.Ag/AgCl) was obtained. The sensor responded rapidly to Cyt c in the linear range from 4.037×10~(-5) to 3.230×10~(-4) mol/L with detection limit of 2.423×10~(-5) mol/L in PBS(pH 7.0) .
引文
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