Clark氧电极生物传感器的研究及分析应用
摘要
生物传感器是一类特殊的化学传感器,它以生物活性单元(如酶、抗体、核酸、细胞、组织等)作为生物敏感元件,对被测目标物具有高度选择性的检测器。它通过各种物理、化学型信号转换器捕捉目标物与敏感元件之间的反应,然后将反应的程度用离散或连续的电信号表达出来,从而得出被测物的浓度。与传统的分析方法相比,生物传感器这种新的检测手段具有如下优点:(1)选择性好;(2)响应快;(3)成本低;(4)可连续在线监测。Clark氧传感器具有灵敏度高、线性范围宽、仪器简单、分析速度快等优点,将酶或细菌生物活性单元与Clark氧传感器相结合,在生化分析等领域具有广泛的应用前景。
本文以生物相容性好的高分子物质如鸡蛋膜、壳聚糖和聚乙烯醇作为载体材料,以半乳糖氧化酶、乙醇氧化酶和甲烷氧化细菌为分子识别元件,用适当的交联剂分别固定在载体基质上,并与Clark氧电极(电化学换能器)相结合,制备简单、快速、灵敏的几种基于耗氧反应的酶和细菌的生物传感器。通过优化实验条件,分别建立了测定半乳糖、乙醇和甲烷的分析方法,并将其应用于实际样品中半乳糖、乙醇的分析测定。
第一章:主要综述了生物传感器的特点、研究现状及其应用。
第二章:以戊二醛为交联剂,将半乳糖氧化酶共价交联固定于鸡蛋壳膜载体上,基于半乳糖氧化酶能催化半乳糖的氧化反应,利用Clark氧电极构建了半乳糖生物传感器,通过氧浓度的变化间接测定半乳糖,并对实验条件、电极的响应特性、可能存在的干扰物进行了考察。实验结果表明,用于半乳糖测定的最佳实验条件为:底液磷酸缓冲溶液浓度为74 mM,pH=7.0,实验温度为室温。该传感器的平均响应时间为100秒,半乳糖浓度在1.0×10~(-4)—8.5×10~(-3)mol/L呈良好的线性关系(r~2=0.9984),以信噪比的3倍计算传感器的检出限为5.0×10~(-5)mol/L,相对标准偏差为3.74%(n=7),连续使用3个月后,响应值降为初始值的83.6%。实际样品中可能存在的干扰物对测定无干扰,并进行了回收实验。结果表明,此种传感器具有选择性高,重现性好,操作简便,干扰少,检测速度快,使用寿命长等优点,对于食品和临床的半乳糖检测具有重要的意义。
第三章:在氧气存在下,乙醇氧化酶可催化氧化乙醇生成乙醛和过氧化氢,氧浓度的变化可引起Clark氧电极电流信号的改变。基于上述原理,以壳聚糖为交联剂,将乙醇氧化酶固定于新鲜鸡蛋壳膜上,将固定有酶的鸡蛋膜紧贴于Clark氧电极表面设计出一种简便、快速、灵敏和稳定性高的乙醇生物传感器。并对实验条件、电极的响应特性、可能存在的干扰物及实际样品进行了考察。实验结果表明,酶的固定化及样品测定的最佳实验条件为:壳聚糖浓度为0.50%(w/v),乙醇氧化酶的最佳用量为1.0 mg,底液磷酸缓冲溶液浓度为25 mM,pH=7.4,实验温度为室温。该传感器的平均响应时间为1分钟,乙醇浓度在6.0×10~(-5)-8.0×10~(-4)mol/L呈良好的线性关系(r~2=0.9988),该传感器的检出限为3.0×10~(-5)mol/L,RSD为3.20%(n=11),连续使用3个月后,响应值降为初始值的86.6%。在干扰测定中只有甲醇、正丙醇、正丁醇表现出一定形式的干扰,实际样品中其它可能存在的干扰物对测定无干扰。并对酒样品中乙醇含量进行了测定和回收实验,结果满意。
第四章:甲烷氧化细菌能在室温下催化氧化甲烷,并在催化过程中消耗氧。采用聚乙烯醇将甲烷氧化细菌固定化,并与Clark氧电极结合设计出一种简便,快速,灵敏的生物传感系统。并对实验条件、传感系统的响应特性等进行了考察。实验结果表明,细菌的固定化及甲烷测定的最佳实验条件为:聚乙烯醇浓度为0.50%(w/v),细菌的最佳用量和比率分别为40 mg和1∶1。底液磷酸缓冲溶液的pH=7.0,实验温度为室温。建立了甲烷测定的生物传感新方法。该方法的平均响应时间为100秒,甲烷浓度在1%-5%(v/v)呈良好的线性关系(r~2=0.9912),RSD为3.10%(n=8),检测限(S/N=3)为0.3%(v/v)。连续使用1个月后,响应值降为初始值的50.0%。
第五章:结合在不同高分子材料上酶与细菌的固定化包括在蛋壳膜上用戊二醛固定半乳糖氧化酶、壳聚糖固定乙醇氧化酶、以及聚乙烯醇固定甲烷氧化细菌,初步探讨了固定化机理。
第六章:合成了一种对甲烷具有选择性吸附的传感膜材料(穴番A)。以香草醛为原料,在乙醇溶液中和1,2-二溴乙烷反应生成1,2-二(4-甲酰-2-甲氧基苯氧基)乙烷,再依次对其进行硼氢化和质子化,三步合成笼状超分子主体化合物穴番-A。产物通过硅胶柱分离纯化后用核磁光谱对其进行了分析鉴定,并且用荧光和紫外光谱对它的发光性能进行了研究。
The biosensor is a special chemical sensor.It uses the biologically sensitive components as active units(such as enzyme,antibody,nucleic acid,cell, tissue) and promises highly selectively to analyte.The analyte was sensed through various kinds of physical-chemical signal transducer.The analytical methods based on biosensor are characterized by high selectivity,easy realization for on-line continuous monitoring,fast response and low cost. This paper mainly concerns the application of enzyme or cell-based Clark oxygen electrode biosensors in the field of bio-chemical analysis.The paper consists of six chapters.
Chapter 1:Biosensors are analytical devices composed of a recognition element of biological origin and a physico-chemical transducer.The biological element is capable of sensing the presence,activity or concentration of a chemical analyte in solution.The conception,basal constitution,the types,work principle and the merit of the biosensor and the development of application of the biosensor in the fields of environment monitoring,ferment,food analysis,bio-medicine and the military field.
Chapter 2:A galactose biosensor using an enzyme-immobilized eggshell and the Clark oxygen electrode for galactose determination has been fabricated. Galactose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent.The galactose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a Clark oxygen electrode.When galactose passed through sensor, galactose was oxidized by dissoloving oxygen under catalysis of galactose oxidase present in the eggshell membrane,then produced hydrogen peroxide and the depleted dissolved oxygen.The decrease in the oxygen level was monitored by Clark oxygen electrode.The effect of pH,phosphate buffer concentration and temperature on the response of the biosensor has been studied in detail.Common matrix interferents did not give significant interference.The results showed response time of biosensor is 100 s,the linear range of is 1.0×10~(-4)—8.5×10~(-3)mol/L galactose(r~2=0.9984) with a detection limit of 50μM(S/N =3),RSD=3.74%for 5.0 mM galactose(n=7). It can retain 83.6%of initial enzyme activity after 3 months when stored in a pH 7.0 phosphate.In brief,the biosensor has displayed a faster response to the galactose,good reproducibility,selectivity,and stability for long-term use. The results showed that the eggshell membrane technology holds a great promise for the fabrication of a biosensor as well as practical.
Chapter 3:A biosensor for determination of ethanol has been fabricated by employing an alcohol oxidase immobilized onto an eggshell membrane with chitosan as a cross-linking agent and a Clark oxygen electrode as the transducer.The detection scheme was based on the enzymatic reactions of ethane leading to the depletion of the dissolved oxygen level in the base solution and then the decrease in the oxygen level was monitored and related to the ethanol concentration.The effects of enzyme loading,chitosan concentration,pH,and temperature on the response of the biosensor were investigated.Storage and repeatability of the biosensor were also studied in detail.The analytical range of the biosensor was from 6.0×10~(-5)-8.0×10~(-4) mol/L ethanol with a detection limit of 30μM(S/N=3) and 1 min response time.In the optimization studies of the biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30%(w/v), respectively.The phosphate buffer(pH 7.4,25 mM) and room temperature (20-25℃) were chosen as the optimum working conditions.In the characterization studies of the biosensor some parameters such as interference effects,operational and storage stability were studied in detail. The biosensor was also tested withvarious wine samples.There were no significant differences between the values obtained from the proposed biosensor and gas chromatographic methods.Furthermore,the recovery tests for ethanol were performed by adding various known amounts of ethanol to the beer and liquor samples.The amounts of added ethanol were then evaluated by the proposed ethanol biosensor.The recovery tests demonstrate that the proposed biosensor offers an excellent,accurate and precise method for the determination of ethanol in real samples.
Chapter 4:A microbial biosensing system for the measurement of methane has been developed using an immobilized mixed culture of Pseudomonas aeruginosa and Klebsiella sp.together with Clark oxygen(O_2) electrode. When methane was applied to the biosensing system,methane was oxidized by dissoloving oxygen under catalysis of methane-oxidizing bacteria present in the immobilized small ball,then the depleted dissolved oxygen.The dissolved O_2 content decreased until a steady-state was reached and was monitored by Clark oxygen electrode.The biosensing system response depends linearly on methane concentration between 1.0 and 5.0%v/v with a detection limit of 0.3%v/v(S/N=3) and a 100-s response time.Phosphate buffer(pH 7.0,25 mM) and room temperature(20-25℃) were chosen as the optimum working conditions.Some parameters including pH,temperature, operational and storage stability were studied in detail for characterization of the biosenisng system.Satisfactory repeatability was confirmed with a relative standard deviation of 3.1%obtained by subjecting the microbial biosensing system to 3.0%v/v methane in pH 7.0 phosphate buffer(25 mM) eight times.Our proposed microbial biosensing system exhibits fast response to methane with good reproducibility.The most promising features of our system are cost-effective,simple sensing design and ease of operation.This microbial biosensing system using co-immobilized Pseudomonas aeruginosa and Klebsiella sp appears very attractive for rapid determination of methane.
Chapter 5:On the base of previous experiments,the mechanisms of immobilization of enzyme andbacteria were brought forward.
Chapter 6:A novel material of sensing membrane-synthesis and characterization of Cryptophane A
Cryptophane-A was synthesized from vanillin using a three-step method,it was purified by elution through a silica gel column and characterized by nuclear magnetic resonance(NMR) spectroscopy.Cryptophanes consist of two rigid cone-shaped units(cyclotriveratrylene) linked together by three bridges.It can complex with neutral molecules and plays an important part in molecular probe and gas sensor field,so the fluorescence and UV spectra of it were studied in the article.
本文以生物相容性好的高分子物质如鸡蛋膜、壳聚糖和聚乙烯醇作为载体材料,以半乳糖氧化酶、乙醇氧化酶和甲烷氧化细菌为分子识别元件,用适当的交联剂分别固定在载体基质上,并与Clark氧电极(电化学换能器)相结合,制备简单、快速、灵敏的几种基于耗氧反应的酶和细菌的生物传感器。通过优化实验条件,分别建立了测定半乳糖、乙醇和甲烷的分析方法,并将其应用于实际样品中半乳糖、乙醇的分析测定。
第一章:主要综述了生物传感器的特点、研究现状及其应用。
第二章:以戊二醛为交联剂,将半乳糖氧化酶共价交联固定于鸡蛋壳膜载体上,基于半乳糖氧化酶能催化半乳糖的氧化反应,利用Clark氧电极构建了半乳糖生物传感器,通过氧浓度的变化间接测定半乳糖,并对实验条件、电极的响应特性、可能存在的干扰物进行了考察。实验结果表明,用于半乳糖测定的最佳实验条件为:底液磷酸缓冲溶液浓度为74 mM,pH=7.0,实验温度为室温。该传感器的平均响应时间为100秒,半乳糖浓度在1.0×10~(-4)—8.5×10~(-3)mol/L呈良好的线性关系(r~2=0.9984),以信噪比的3倍计算传感器的检出限为5.0×10~(-5)mol/L,相对标准偏差为3.74%(n=7),连续使用3个月后,响应值降为初始值的83.6%。实际样品中可能存在的干扰物对测定无干扰,并进行了回收实验。结果表明,此种传感器具有选择性高,重现性好,操作简便,干扰少,检测速度快,使用寿命长等优点,对于食品和临床的半乳糖检测具有重要的意义。
第三章:在氧气存在下,乙醇氧化酶可催化氧化乙醇生成乙醛和过氧化氢,氧浓度的变化可引起Clark氧电极电流信号的改变。基于上述原理,以壳聚糖为交联剂,将乙醇氧化酶固定于新鲜鸡蛋壳膜上,将固定有酶的鸡蛋膜紧贴于Clark氧电极表面设计出一种简便、快速、灵敏和稳定性高的乙醇生物传感器。并对实验条件、电极的响应特性、可能存在的干扰物及实际样品进行了考察。实验结果表明,酶的固定化及样品测定的最佳实验条件为:壳聚糖浓度为0.50%(w/v),乙醇氧化酶的最佳用量为1.0 mg,底液磷酸缓冲溶液浓度为25 mM,pH=7.4,实验温度为室温。该传感器的平均响应时间为1分钟,乙醇浓度在6.0×10~(-5)-8.0×10~(-4)mol/L呈良好的线性关系(r~2=0.9988),该传感器的检出限为3.0×10~(-5)mol/L,RSD为3.20%(n=11),连续使用3个月后,响应值降为初始值的86.6%。在干扰测定中只有甲醇、正丙醇、正丁醇表现出一定形式的干扰,实际样品中其它可能存在的干扰物对测定无干扰。并对酒样品中乙醇含量进行了测定和回收实验,结果满意。
第四章:甲烷氧化细菌能在室温下催化氧化甲烷,并在催化过程中消耗氧。采用聚乙烯醇将甲烷氧化细菌固定化,并与Clark氧电极结合设计出一种简便,快速,灵敏的生物传感系统。并对实验条件、传感系统的响应特性等进行了考察。实验结果表明,细菌的固定化及甲烷测定的最佳实验条件为:聚乙烯醇浓度为0.50%(w/v),细菌的最佳用量和比率分别为40 mg和1∶1。底液磷酸缓冲溶液的pH=7.0,实验温度为室温。建立了甲烷测定的生物传感新方法。该方法的平均响应时间为100秒,甲烷浓度在1%-5%(v/v)呈良好的线性关系(r~2=0.9912),RSD为3.10%(n=8),检测限(S/N=3)为0.3%(v/v)。连续使用1个月后,响应值降为初始值的50.0%。
第五章:结合在不同高分子材料上酶与细菌的固定化包括在蛋壳膜上用戊二醛固定半乳糖氧化酶、壳聚糖固定乙醇氧化酶、以及聚乙烯醇固定甲烷氧化细菌,初步探讨了固定化机理。
第六章:合成了一种对甲烷具有选择性吸附的传感膜材料(穴番A)。以香草醛为原料,在乙醇溶液中和1,2-二溴乙烷反应生成1,2-二(4-甲酰-2-甲氧基苯氧基)乙烷,再依次对其进行硼氢化和质子化,三步合成笼状超分子主体化合物穴番-A。产物通过硅胶柱分离纯化后用核磁光谱对其进行了分析鉴定,并且用荧光和紫外光谱对它的发光性能进行了研究。
The biosensor is a special chemical sensor.It uses the biologically sensitive components as active units(such as enzyme,antibody,nucleic acid,cell, tissue) and promises highly selectively to analyte.The analyte was sensed through various kinds of physical-chemical signal transducer.The analytical methods based on biosensor are characterized by high selectivity,easy realization for on-line continuous monitoring,fast response and low cost. This paper mainly concerns the application of enzyme or cell-based Clark oxygen electrode biosensors in the field of bio-chemical analysis.The paper consists of six chapters.
Chapter 1:Biosensors are analytical devices composed of a recognition element of biological origin and a physico-chemical transducer.The biological element is capable of sensing the presence,activity or concentration of a chemical analyte in solution.The conception,basal constitution,the types,work principle and the merit of the biosensor and the development of application of the biosensor in the fields of environment monitoring,ferment,food analysis,bio-medicine and the military field.
Chapter 2:A galactose biosensor using an enzyme-immobilized eggshell and the Clark oxygen electrode for galactose determination has been fabricated. Galactose oxidase was covalently immobilized on an eggshell membrane with glutaraldehyde as a cross-linking agent.The galactose biosensor was fabricated by positioning the enzyme-immobilized eggshell membrane on the surface of a Clark oxygen electrode.When galactose passed through sensor, galactose was oxidized by dissoloving oxygen under catalysis of galactose oxidase present in the eggshell membrane,then produced hydrogen peroxide and the depleted dissolved oxygen.The decrease in the oxygen level was monitored by Clark oxygen electrode.The effect of pH,phosphate buffer concentration and temperature on the response of the biosensor has been studied in detail.Common matrix interferents did not give significant interference.The results showed response time of biosensor is 100 s,the linear range of is 1.0×10~(-4)—8.5×10~(-3)mol/L galactose(r~2=0.9984) with a detection limit of 50μM(S/N =3),RSD=3.74%for 5.0 mM galactose(n=7). It can retain 83.6%of initial enzyme activity after 3 months when stored in a pH 7.0 phosphate.In brief,the biosensor has displayed a faster response to the galactose,good reproducibility,selectivity,and stability for long-term use. The results showed that the eggshell membrane technology holds a great promise for the fabrication of a biosensor as well as practical.
Chapter 3:A biosensor for determination of ethanol has been fabricated by employing an alcohol oxidase immobilized onto an eggshell membrane with chitosan as a cross-linking agent and a Clark oxygen electrode as the transducer.The detection scheme was based on the enzymatic reactions of ethane leading to the depletion of the dissolved oxygen level in the base solution and then the decrease in the oxygen level was monitored and related to the ethanol concentration.The effects of enzyme loading,chitosan concentration,pH,and temperature on the response of the biosensor were investigated.Storage and repeatability of the biosensor were also studied in detail.The analytical range of the biosensor was from 6.0×10~(-5)-8.0×10~(-4) mol/L ethanol with a detection limit of 30μM(S/N=3) and 1 min response time.In the optimization studies of the biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30%(w/v), respectively.The phosphate buffer(pH 7.4,25 mM) and room temperature (20-25℃) were chosen as the optimum working conditions.In the characterization studies of the biosensor some parameters such as interference effects,operational and storage stability were studied in detail. The biosensor was also tested withvarious wine samples.There were no significant differences between the values obtained from the proposed biosensor and gas chromatographic methods.Furthermore,the recovery tests for ethanol were performed by adding various known amounts of ethanol to the beer and liquor samples.The amounts of added ethanol were then evaluated by the proposed ethanol biosensor.The recovery tests demonstrate that the proposed biosensor offers an excellent,accurate and precise method for the determination of ethanol in real samples.
Chapter 4:A microbial biosensing system for the measurement of methane has been developed using an immobilized mixed culture of Pseudomonas aeruginosa and Klebsiella sp.together with Clark oxygen(O_2) electrode. When methane was applied to the biosensing system,methane was oxidized by dissoloving oxygen under catalysis of methane-oxidizing bacteria present in the immobilized small ball,then the depleted dissolved oxygen.The dissolved O_2 content decreased until a steady-state was reached and was monitored by Clark oxygen electrode.The biosensing system response depends linearly on methane concentration between 1.0 and 5.0%v/v with a detection limit of 0.3%v/v(S/N=3) and a 100-s response time.Phosphate buffer(pH 7.0,25 mM) and room temperature(20-25℃) were chosen as the optimum working conditions.Some parameters including pH,temperature, operational and storage stability were studied in detail for characterization of the biosenisng system.Satisfactory repeatability was confirmed with a relative standard deviation of 3.1%obtained by subjecting the microbial biosensing system to 3.0%v/v methane in pH 7.0 phosphate buffer(25 mM) eight times.Our proposed microbial biosensing system exhibits fast response to methane with good reproducibility.The most promising features of our system are cost-effective,simple sensing design and ease of operation.This microbial biosensing system using co-immobilized Pseudomonas aeruginosa and Klebsiella sp appears very attractive for rapid determination of methane.
Chapter 5:On the base of previous experiments,the mechanisms of immobilization of enzyme andbacteria were brought forward.
Chapter 6:A novel material of sensing membrane-synthesis and characterization of Cryptophane A
Cryptophane-A was synthesized from vanillin using a three-step method,it was purified by elution through a silica gel column and characterized by nuclear magnetic resonance(NMR) spectroscopy.Cryptophanes consist of two rigid cone-shaped units(cyclotriveratrylene) linked together by three bridges.It can complex with neutral molecules and plays an important part in molecular probe and gas sensor field,so the fluorescence and UV spectra of it were studied in the article.
引文
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