基于植物组织的酚电化学传感器研究
摘要
一、基于丝瓜组织的酚电化学传感器
采用无机粘土材料Laponite/壳聚糖杂化材料固定丝瓜组织匀浆,构筑了一种检测酚类物质的组织传感器。酚浓度的电化学检测采用安培法,丝瓜组织修饰电极控制的电位为-0.2V(相对于饱和甘汞电极SCE)。由于Laponite在水溶液中容易发生溶胀,可形成疏松多孔的结构,同时其具有化学惰性、较好的吸附能力和层间阳离子交换能力,使得丝瓜组织成功地固定在载体材料中。构筑丝瓜组织电极的最适参数为:Laponite/CHT的质量比为2:1,固定载体与丝瓜组织匀浆体积比为1:4,电极上组织匀浆用量为5μl。组织电极在溶液pH5.5、温度25℃、工作电位-0.2V时对儿茶酚表现了最好的电流响应。研究了该组织传感器对四种酚类化合物的响应特性,结果表明:儿茶酚、苯酚具有明显的电流响应,而间甲基苯酚和对氯苯酚几乎没有响应;固定化丝瓜组织中的多酚氧化酶对儿茶酚、苯酚的表观米氏常数KMapp分别为3.48mM、2.905 mM。
二、基于马铃薯组织的儿茶酚电化学传感器
纳米碳酸钙由于具有较大的比表面积、较高的表面能、较好的生物相容性,可作为生物分子的良好载体材料。红外光谱、扫描电镜研究表明,纳米碳酸钙/壳聚糖杂化材料能够很好地将马铃薯组织固定玻碳电极上,制成了灵敏高、选择性好的儿茶酚传感器。该组织电极对儿茶酚表现出快速、灵敏的电流响应(灵敏度为139.14 mA M-1cm-2),而对间甲基酚、对氯苯酚、苯酚的响应非常低(仅为儿茶酚响应的3%以内),从而实现了儿茶酚的选择性测定。马铃薯组织传感器测定儿茶酚线性检测范围为4.49×10-6到4×10-5M,的,检测下限为5×10-7M(当信噪比为3:1时),固定化马铃薯组织中的多酚氧化酶对儿茶酚的表观米氏常数为1.005 mM。同时,本文对组织传感器构筑和使用的最适条件进行了详细的研究。
三、基于蘑菇组织的酚传感器及抑制作用研究
多酚氧化酶在蘑菇中的含量很高,利用蘑菇组织可构筑高灵敏的酚类组织传感器。本论文以Laponite/壳聚糖杂化材料为固定载体,以蘑菇组织匀浆为敏感元,在玻碳电极上成功构筑了蘑菇组织传感器。采用红外光谱、扫描电镜对生物电极的组成、形貌以及载体与生物材料之间的相互作用进行了研究,电化学测量采用传统的三电极体系、计时安培方法。在优化的制作条件和使用条件下,该蘑菇组织传感器表现出一些优异的分析特性:高的灵敏度(对儿茶酚886.62 mA M-1cm-2),低的检测限(4.99×10-8M),高的稳定性。该组织生物传感器对四种酚类化合物均有明显的响应,其检测灵敏度大小顺序为:儿茶酚>苯酚>对氯苯酚>间甲基酚,对儿茶酚、苯酚、对氯苯酚和间甲基苯酚的表观米氏常数分别为0.2285mM、0.95881 mM、0.35616 mM和0.2485 mM。研究了苯甲酸对固定化蘑菇组织中多酚氧化酶的抑制作用,结果表明该抑制作用符合竞争性的抑制模型。
1. A novel amperometric biosensor based on sponge cucumber tissue homogenate for the determination of phenolic compounds
A composite matrix consisting of the inorganic laponite and chitosan (CHT) was used for the immobilization of sponge cucumber tissue homogenate to form a novel biosensor for the detection of catechol. Amperometric detection of catechol was evaluated by holding the tissue-homogenate electrode at– 0.2V (versus SCE). Due to the laponite’s special properties, such as high porosity due to easy swelling of laponite in aqueous solutions, chemical inertia, better adsorptivity, and good anionic exchange ability between layers, the sponge cucumber tissue homogenate was well immobilized in the composite matrix. Constructing this tissue biosensor has the optimum parameters being as follows: the mass ratio of laponite to CHT (w/w) was 2:1; the volume ratio of the immobilized carrier to tissue homogenate(v/v) ratio was 1:4; the tissue homogenate amount deposited on the electrode surface was 5μl. The tissue biosensor showed an optimum current response under the following conditions: the pH of the phosphate buffer solution was 5.5, temperature was controlled at 25℃, the applied potential was set at– 0.2 V. The results from the measurements of four phenolic compounds demonstrated: catechol and phenol have obvious current response; however, no current response was detected for m-cresol and p-chlorophenol. An apparent Michaelis constants of polyphenol oxidase in immobilized sponge cucumber tissue are 3.48mM and 2.905 mM for catechol and phenol, respectively.
2. A biosensor based on potato tissue homogenate for the determination of phenolic compounds
The calcium carbonate nanoparticles have large specific surface area, lager surface energy, and good biocompatibility, so it is a good immobilized material for immobilization of biomolecules. The experimental results of FTIR and scanning electron microscope (SEM) indicated that the potato tissue homogenate can be well immobilized on the glassy carbon electrode using laponite/chitosan composite, which formed a high sensitivity and selectivity biosensor to detect the catechol. This tissue biosensor has a fast and sensitive current response to catechol (sensitivity is 139.14 mA M-1cm-2), but low response to m-cresol, phenol, p-chlorophenol (less than 3% of catechol). Therefore, this biosensor has a good selectivity to the determination of catechol. The potato tissue biosensor provided a linear response to catechol over a concentration range from 4.49×10-6 to 4.00×10-5 with a detection limit of 5.00×10 -7 M at S/N of 3. The apparent Michaelis constant of the enzyme-catalyzed reaction for polyphenol oxidase from potato tissue homogenate is 1.005 mM. In addition, we studied the optimum conditions for the construction and use of this potato tissue biosensor in details.
3. Development of a mushroom tissue-homogenate biosensor and investigation of the inhibition effect
Due to the high content of polyphenol oxidase in mushroom, we built a highly sensitive biosensor based on the mushroom tissue. In the work, we have used the laponite/chitoson composite to immobilize the mushroom tissue homogenate on the glassy carbon electrode to fabricate a biosensor. Using the FTIR and scanning electron microscope (SEM) to characterize the composition, the morphology of the laponite/chitoson composite, and the interaction between the carrier and biomaterials. The electrochemical measurements were performed with a conventional three-electrode system and amperometric method. Under optimization of the fabrication and using conditions of the biosensor, this tissue biosensor expressed an excellent analytical performance: high sensitivity (886.62 mA M-1cm-2 for catechol), low detection limit (4.99×10-8M), and good stability. The tissue biosensor has obvious response to different phenolic compounds, the order of sensibility is as follows: catechol>phenol> p-chlorophenol >m-cresol. The apparent Michaelis constants are 0.256, 0.157, 0.043, 0.063 and 0.03mM for catechol, phenol, p-chlorophenol and m-cresol, respectively. In this report, the inhibition of polyphenol oxidase, arising from mushroom tissue homogenate immobilized in laponite/chitosan composite, was investigated using benzoic acid. The experimental result demonstrated that this inhibition is a quasi-reversible competitive type.
采用无机粘土材料Laponite/壳聚糖杂化材料固定丝瓜组织匀浆,构筑了一种检测酚类物质的组织传感器。酚浓度的电化学检测采用安培法,丝瓜组织修饰电极控制的电位为-0.2V(相对于饱和甘汞电极SCE)。由于Laponite在水溶液中容易发生溶胀,可形成疏松多孔的结构,同时其具有化学惰性、较好的吸附能力和层间阳离子交换能力,使得丝瓜组织成功地固定在载体材料中。构筑丝瓜组织电极的最适参数为:Laponite/CHT的质量比为2:1,固定载体与丝瓜组织匀浆体积比为1:4,电极上组织匀浆用量为5μl。组织电极在溶液pH5.5、温度25℃、工作电位-0.2V时对儿茶酚表现了最好的电流响应。研究了该组织传感器对四种酚类化合物的响应特性,结果表明:儿茶酚、苯酚具有明显的电流响应,而间甲基苯酚和对氯苯酚几乎没有响应;固定化丝瓜组织中的多酚氧化酶对儿茶酚、苯酚的表观米氏常数KMapp分别为3.48mM、2.905 mM。
二、基于马铃薯组织的儿茶酚电化学传感器
纳米碳酸钙由于具有较大的比表面积、较高的表面能、较好的生物相容性,可作为生物分子的良好载体材料。红外光谱、扫描电镜研究表明,纳米碳酸钙/壳聚糖杂化材料能够很好地将马铃薯组织固定玻碳电极上,制成了灵敏高、选择性好的儿茶酚传感器。该组织电极对儿茶酚表现出快速、灵敏的电流响应(灵敏度为139.14 mA M-1cm-2),而对间甲基酚、对氯苯酚、苯酚的响应非常低(仅为儿茶酚响应的3%以内),从而实现了儿茶酚的选择性测定。马铃薯组织传感器测定儿茶酚线性检测范围为4.49×10-6到4×10-5M,的,检测下限为5×10-7M(当信噪比为3:1时),固定化马铃薯组织中的多酚氧化酶对儿茶酚的表观米氏常数为1.005 mM。同时,本文对组织传感器构筑和使用的最适条件进行了详细的研究。
三、基于蘑菇组织的酚传感器及抑制作用研究
多酚氧化酶在蘑菇中的含量很高,利用蘑菇组织可构筑高灵敏的酚类组织传感器。本论文以Laponite/壳聚糖杂化材料为固定载体,以蘑菇组织匀浆为敏感元,在玻碳电极上成功构筑了蘑菇组织传感器。采用红外光谱、扫描电镜对生物电极的组成、形貌以及载体与生物材料之间的相互作用进行了研究,电化学测量采用传统的三电极体系、计时安培方法。在优化的制作条件和使用条件下,该蘑菇组织传感器表现出一些优异的分析特性:高的灵敏度(对儿茶酚886.62 mA M-1cm-2),低的检测限(4.99×10-8M),高的稳定性。该组织生物传感器对四种酚类化合物均有明显的响应,其检测灵敏度大小顺序为:儿茶酚>苯酚>对氯苯酚>间甲基酚,对儿茶酚、苯酚、对氯苯酚和间甲基苯酚的表观米氏常数分别为0.2285mM、0.95881 mM、0.35616 mM和0.2485 mM。研究了苯甲酸对固定化蘑菇组织中多酚氧化酶的抑制作用,结果表明该抑制作用符合竞争性的抑制模型。
1. A novel amperometric biosensor based on sponge cucumber tissue homogenate for the determination of phenolic compounds
A composite matrix consisting of the inorganic laponite and chitosan (CHT) was used for the immobilization of sponge cucumber tissue homogenate to form a novel biosensor for the detection of catechol. Amperometric detection of catechol was evaluated by holding the tissue-homogenate electrode at– 0.2V (versus SCE). Due to the laponite’s special properties, such as high porosity due to easy swelling of laponite in aqueous solutions, chemical inertia, better adsorptivity, and good anionic exchange ability between layers, the sponge cucumber tissue homogenate was well immobilized in the composite matrix. Constructing this tissue biosensor has the optimum parameters being as follows: the mass ratio of laponite to CHT (w/w) was 2:1; the volume ratio of the immobilized carrier to tissue homogenate(v/v) ratio was 1:4; the tissue homogenate amount deposited on the electrode surface was 5μl. The tissue biosensor showed an optimum current response under the following conditions: the pH of the phosphate buffer solution was 5.5, temperature was controlled at 25℃, the applied potential was set at– 0.2 V. The results from the measurements of four phenolic compounds demonstrated: catechol and phenol have obvious current response; however, no current response was detected for m-cresol and p-chlorophenol. An apparent Michaelis constants of polyphenol oxidase in immobilized sponge cucumber tissue are 3.48mM and 2.905 mM for catechol and phenol, respectively.
2. A biosensor based on potato tissue homogenate for the determination of phenolic compounds
The calcium carbonate nanoparticles have large specific surface area, lager surface energy, and good biocompatibility, so it is a good immobilized material for immobilization of biomolecules. The experimental results of FTIR and scanning electron microscope (SEM) indicated that the potato tissue homogenate can be well immobilized on the glassy carbon electrode using laponite/chitosan composite, which formed a high sensitivity and selectivity biosensor to detect the catechol. This tissue biosensor has a fast and sensitive current response to catechol (sensitivity is 139.14 mA M-1cm-2), but low response to m-cresol, phenol, p-chlorophenol (less than 3% of catechol). Therefore, this biosensor has a good selectivity to the determination of catechol. The potato tissue biosensor provided a linear response to catechol over a concentration range from 4.49×10-6 to 4.00×10-5 with a detection limit of 5.00×10 -7 M at S/N of 3. The apparent Michaelis constant of the enzyme-catalyzed reaction for polyphenol oxidase from potato tissue homogenate is 1.005 mM. In addition, we studied the optimum conditions for the construction and use of this potato tissue biosensor in details.
3. Development of a mushroom tissue-homogenate biosensor and investigation of the inhibition effect
Due to the high content of polyphenol oxidase in mushroom, we built a highly sensitive biosensor based on the mushroom tissue. In the work, we have used the laponite/chitoson composite to immobilize the mushroom tissue homogenate on the glassy carbon electrode to fabricate a biosensor. Using the FTIR and scanning electron microscope (SEM) to characterize the composition, the morphology of the laponite/chitoson composite, and the interaction between the carrier and biomaterials. The electrochemical measurements were performed with a conventional three-electrode system and amperometric method. Under optimization of the fabrication and using conditions of the biosensor, this tissue biosensor expressed an excellent analytical performance: high sensitivity (886.62 mA M-1cm-2 for catechol), low detection limit (4.99×10-8M), and good stability. The tissue biosensor has obvious response to different phenolic compounds, the order of sensibility is as follows: catechol>phenol> p-chlorophenol >m-cresol. The apparent Michaelis constants are 0.256, 0.157, 0.043, 0.063 and 0.03mM for catechol, phenol, p-chlorophenol and m-cresol, respectively. In this report, the inhibition of polyphenol oxidase, arising from mushroom tissue homogenate immobilized in laponite/chitosan composite, was investigated using benzoic acid. The experimental result demonstrated that this inhibition is a quasi-reversible competitive type.
引文
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