基于有机—无机杂化材料的硝酸还原酶和辣根过氧化物酶电极的研究
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摘要
硝酸盐和酚类化合物是水体中重要的污染物质,威胁着人们的身体健康和生存环境。因此,对这两类污染物的快速准确测定具有重大的意义。生物传感器具有分析速度快、灵敏度高、成本低等特点,已经在环境监测、临床医学、食品分析、军事医学等领域得到了广泛应用。
本论文使用基于溶胶-凝胶技术的有机-无机杂化材料,采用包埋方法并结合纳米技术分别制备了硝酸还原酶和辣根过氧化物酶两类电极,以检测水中的硝酸盐和酚类污染物。具体研究内容如下:
利用了透析、调整固定化参数和杂化材料各组分比例等方法对原有的硝酸还原酶电极进行改进,制备出性能更好、稳定性更强的硝酸还原酶电极。酶电极的响应电流与硝酸根浓度在0.01～0.4mmol/L范围内呈线性关系(R~2=0.9928),检测灵敏度为6.866nA/(μmol/L),检测限为3.37×10~(-6)mol/L。工作电位为-0.712V,响应时间仅为6.7s。一个月后,响应电流为初始值的87.6%。
由于碳纳米管具有众多独特的性质,对于提高生物传感器检测的灵敏度和稳定性具有重大意义。因此基于单壁碳纳米管制成了两种酶电极:一种是将硝酸还原酶包埋在杂化材料里,在单壁碳纳米修饰过的金电极表面形成酶膜制成酶电极;另一种是将硝酸还原酶包埋在掺杂有SWNTs的杂化材料里制成硝酸还原酶电极。实验结果表明两种电极的响应电流都大幅增加,检测灵敏度分别为8.806nA/(μmol/L)和11.361nA/(μmol/L)。响应时间和工作电位变化不大,而第二种方法制备的酶电极稳定性更强。
将辣根过氧化物酶直接包埋在杂化材料里,在金电极表面形成酶膜,制备出能够检测水中多种酚类化合物的生物传感器。酶电极的响应电流与对苯二酚浓度在1～70μmol/L范围内呈线性关系(R2=0.9996),检测灵敏度为93nA/(μmol/L),检测限为0.023μmol/L。工作电位为-0.20V,响应时间仅为2.4s。一个月后,响应电流为初始值的91.6%。
分别使用单壁碳纳米管和普鲁士蓝对辣根过氧化物酶电极进行修饰,有效地降低了工作电位(分别降至-0.05V和-0.03V),增强了酶电极的抗干扰性。两种电极的响应时间分别为4.6s和9.9s。
Nitrate and phenolic compounds, as main pollutants, are threatening human health and our living environment. The fast, accurate detection of these two pollutants is of great significance. With the merits of prompt analysis, high sensitivity and low cost, biosensors have been widely used in enviromental monitoring, clinical diagnosis, food analysis and military medicine.
In this dissertation, with nanometer technology we immobilized nitrate reductase and horseradish peroxidase enzymes respectively by sol-gel derived organic-inorganic hybrid material to fabricate various nitrate reductase and horseradish peroxidase biosensors. The detailed work of this thesis has been set out as follows:
By dialysis and optimizing the parameters of hybrid material, an amperometric nitrate reductase biosensor with good performance and stability had been prepared. The electroanalytical properties of optimized biosensor at -0.712V(vs SCE) were measured: linear response range, sensitivity and detection limit were 0.01~ 0.4mmol/L (R~2=0.9928), 6.866nA/(μmol/L), 3.37×10~(-6)mol/L, respectively. It reached a steady-state response to the added nitrate in 6.7s. After 30 days’interval use, the response current dropped to 87.6% of its initial value.
To enhance the sensitivity and stability of the nitrate reductase biosensor, single-wall carbon nanotubes (SWNTs) were used and two enzyme electrodes were fabricated by different methods: First, nitrate reductase was embedded on SWNTs modified electrode by hybrid material; Second, SWNTs were added to hybrid material directly. The results showed that response currents of the two electrodes were both significantly enhanced and the sensitivity were 8.806 A/(μmol/L),11.361 nA/(μmol/L), respectively. Applied potential and response time had changed slightly whereas the second electrode had better stability and reproducibility.
An amperometric horseradish peroxidase biosensor which can determine phenolic compounds (such as hydroquinone, phenol, catechol, chlorophenol, cresol) had been fabricated by immobilizing horseradish peroxidase enzymes on the surface of Au electrode with hybrid material. The electroanalytical properties of optimized biosensor at -0.20V(vs SCE) were measured: linear response range, sensitivity and detection limit were 1~70μmol/L (R2=0.9996), 93nA/(μmol/L), 0.023μmol/L, respectively. It reached a steady-state response to the added hydroquinone in 2.4s. After 30 days’interval use, the response current dropped to 91.6% of its initial value.
Two types of horseradish peroxidase biosensors based on SWNTs and Prussian Blue modified electrodes had been fabricated which can both reduced operating potential and eliminated the interferences of electroactive substances (such as ascorbic acid and uric acid) effectively. Response time were 4.6s and 9.9s, respectively.
本论文使用基于溶胶-凝胶技术的有机-无机杂化材料,采用包埋方法并结合纳米技术分别制备了硝酸还原酶和辣根过氧化物酶两类电极,以检测水中的硝酸盐和酚类污染物。具体研究内容如下:
利用了透析、调整固定化参数和杂化材料各组分比例等方法对原有的硝酸还原酶电极进行改进,制备出性能更好、稳定性更强的硝酸还原酶电极。酶电极的响应电流与硝酸根浓度在0.01～0.4mmol/L范围内呈线性关系(R~2=0.9928),检测灵敏度为6.866nA/(μmol/L),检测限为3.37×10~(-6)mol/L。工作电位为-0.712V,响应时间仅为6.7s。一个月后,响应电流为初始值的87.6%。
由于碳纳米管具有众多独特的性质,对于提高生物传感器检测的灵敏度和稳定性具有重大意义。因此基于单壁碳纳米管制成了两种酶电极:一种是将硝酸还原酶包埋在杂化材料里,在单壁碳纳米修饰过的金电极表面形成酶膜制成酶电极;另一种是将硝酸还原酶包埋在掺杂有SWNTs的杂化材料里制成硝酸还原酶电极。实验结果表明两种电极的响应电流都大幅增加,检测灵敏度分别为8.806nA/(μmol/L)和11.361nA/(μmol/L)。响应时间和工作电位变化不大,而第二种方法制备的酶电极稳定性更强。
将辣根过氧化物酶直接包埋在杂化材料里,在金电极表面形成酶膜,制备出能够检测水中多种酚类化合物的生物传感器。酶电极的响应电流与对苯二酚浓度在1～70μmol/L范围内呈线性关系(R2=0.9996),检测灵敏度为93nA/(μmol/L),检测限为0.023μmol/L。工作电位为-0.20V,响应时间仅为2.4s。一个月后,响应电流为初始值的91.6%。
分别使用单壁碳纳米管和普鲁士蓝对辣根过氧化物酶电极进行修饰,有效地降低了工作电位(分别降至-0.05V和-0.03V),增强了酶电极的抗干扰性。两种电极的响应时间分别为4.6s和9.9s。
Nitrate and phenolic compounds, as main pollutants, are threatening human health and our living environment. The fast, accurate detection of these two pollutants is of great significance. With the merits of prompt analysis, high sensitivity and low cost, biosensors have been widely used in enviromental monitoring, clinical diagnosis, food analysis and military medicine.
In this dissertation, with nanometer technology we immobilized nitrate reductase and horseradish peroxidase enzymes respectively by sol-gel derived organic-inorganic hybrid material to fabricate various nitrate reductase and horseradish peroxidase biosensors. The detailed work of this thesis has been set out as follows:
By dialysis and optimizing the parameters of hybrid material, an amperometric nitrate reductase biosensor with good performance and stability had been prepared. The electroanalytical properties of optimized biosensor at -0.712V(vs SCE) were measured: linear response range, sensitivity and detection limit were 0.01~ 0.4mmol/L (R~2=0.9928), 6.866nA/(μmol/L), 3.37×10~(-6)mol/L, respectively. It reached a steady-state response to the added nitrate in 6.7s. After 30 days’interval use, the response current dropped to 87.6% of its initial value.
To enhance the sensitivity and stability of the nitrate reductase biosensor, single-wall carbon nanotubes (SWNTs) were used and two enzyme electrodes were fabricated by different methods: First, nitrate reductase was embedded on SWNTs modified electrode by hybrid material; Second, SWNTs were added to hybrid material directly. The results showed that response currents of the two electrodes were both significantly enhanced and the sensitivity were 8.806 A/(μmol/L),11.361 nA/(μmol/L), respectively. Applied potential and response time had changed slightly whereas the second electrode had better stability and reproducibility.
An amperometric horseradish peroxidase biosensor which can determine phenolic compounds (such as hydroquinone, phenol, catechol, chlorophenol, cresol) had been fabricated by immobilizing horseradish peroxidase enzymes on the surface of Au electrode with hybrid material. The electroanalytical properties of optimized biosensor at -0.20V(vs SCE) were measured: linear response range, sensitivity and detection limit were 1~70μmol/L (R2=0.9996), 93nA/(μmol/L), 0.023μmol/L, respectively. It reached a steady-state response to the added hydroquinone in 2.4s. After 30 days’interval use, the response current dropped to 91.6% of its initial value.
Two types of horseradish peroxidase biosensors based on SWNTs and Prussian Blue modified electrodes had been fabricated which can both reduced operating potential and eliminated the interferences of electroactive substances (such as ascorbic acid and uric acid) effectively. Response time were 4.6s and 9.9s, respectively.
引文
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