抗坏血酸生物传感器的研究及其分析应用
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摘要
第一章：简要概述生物传感器的工作原理、分类、特点及应用,并对酶生物传感器的研究进展、生物活性组分的固定化方法及抗坏血酸的检测方法作了综述。
第二章：采用包埋法将抗坏血酸氧化酶(A.O)固定于壳聚糖与鸡蛋壳膜之间制得壳聚糖/抗坏血酸氧化酶/鸡蛋壳膜,将其与溶解氧电极偶联制得抗坏血酸生物传感器,对影响传感器响应性能的溶液pH、温度与缓冲溶液浓度进行了优化。磷酸缓冲溶液的pH 5.0,浓度为100mmol/L,实验温度为室温,此生物传感器的响应性能良好。该生物传感器的响应时间为100s,抗坏血酸浓度在0.07-0.77mmol/L呈现良好的线性关系,检出限为16μmol/L, RSD=4.05%(n=10).间断使用90天后,响应值变为初始值的85.0%。
第三章：采用包埋-交联法,以戊二醛做交联剂、壳聚糖为包埋剂,制备壳聚糖/抗坏血酸氧化酶/戊二醛/鸡蛋膜,并与氧电极偶联制成抗坏血酸生物传感器。对溶液温度、pH、戊二醛浓度、酶固定量等因素对传感器的影响进行了探索。磷酸缓冲溶液的pH 5.0,戊二醛浓度为15%,酶固定量为6 Units为传感器的最佳实验条件。抗坏血酸浓度在0.02～0.82mmol/L间呈良好的线性关系,检测限为12μmol/L,间断使用90天后,响应值变为初始值的87.6%。
第四章：通过包埋-交联法将抗坏血酸氧化酶固定于猪膀胱膜上,将固定有酶的膀胱膜紧贴于氧电极表面制得抗坏血酸生物传感器,并考察了影响传感器响应的缓冲溶液pH、浓度、戊二醛浓度、酶固定量等因素。在优化实验条件下,该传感器的响应时间为80s,抗坏血酸浓度在0.01～0.88 mmol/L之间呈现良好线性关系,检出限为, RSD=3.10%(n=20)。而且,此传感器具有良好的使用寿命和贮藏稳定性。实际样品中可能存在的干扰物质对抗坏血酸的测定无干扰。该传感器应用于维生素C饮品中抗坏血酸含量的测定,结果与试剂盒所测结果基本吻合。此外,进行了回收率实验,回收率为97.1-104%。结果表明：膀胱膜比一般天然膜材料透气性和韧性更好,制备出的传感器具有响应速度快、灵敏度高、线性范围宽、检测限低、重现性好、检测结果准确、使用寿命长等优点,为食品药品中抗坏血酸的检测提供了一种准确、可靠的新方法。
第五章：基于金属卟啉的化学稳定性、良好的生物和电催化活性,制备金属铁卟啉纳米粒子,并将其应用于抗坏血酸生物传感器的研制,制得以金属铁卟啉纳米粒子为电子媒介体的抗坏血酸生物传感器。对实验条件的优化进行了初步探索。
Chapter 1:A biosensor, a analytical device composed of a recognition element of biological origin and a physico-chemical transducer, becomes one of the important research fields of analytical chemistry. The biological element is capable of sensing the presence, activity or concentration of a chemical analyte in solution. The conception, basal constitution, working principles, types, characteristic and application of biosensors were summarized. In addition, research status of enzyme biosensor and detecting methods of L-ascorbic acid were reviewed.
Chapter 2:A L-ascorbic acid biosensor using a chitosan/ascorbate oxide/ eggshell membrane as a biological molecular recognition component and a dissolved oxygen electrode as a transducer has been fabricated. The ascorbate oxidase was entrapped between chitosan and eggshell membrane. The effect of phosphate buffer concentration, pH and temperature on the response of the L-ascorbic acid biosensor has been studied in detail. The resulting sensor exhibited a fast response of 100 s, high sensitivity and good storage stability. The linear response is 0.07-0.77 mmol/L of L-ascorbic acid concentration with a detection limit of 16μmol/L
Chapter 3:A L-ascorbic acid biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for L-ascorbic acid determination has been fabricated. Ascorbate oxidase was covalently immobilized on an eggshell membrane by chitosan with glutaraldehyde as a cross-linking agent. In the optimization studies of the biosensor, the most suitable enzyme and glutaraldehyde concentration were found to be 6 units and 15%(w/v), respectively. The phosphate buffer of pH 5.0 was chosen as the optimum working condition. The biosensor response has a linear range of L-ascorbic acid concentration between 0.02 mmol/L and 0.82 mmol/L with a detection limit of 12μmol/L (S/N=3). Compare with the membrane without glutaraldehyde, enzyme of the proposed biosensor was immobilized firmly。In the characterization studies of the glucose biosensor, some parameters such as interference effects and storage stability were investigated in detail.
Chapter 4:A L-ascorbic acid biosensing assay was developed using the enzyme-immobilized pig bladder membrane and dissolved oxygen electrode. The ascorbate oxidase immobilized with chitosan was bound to bladder membrane by glutaraldehyde as a crosslinking agent. The phosphate buffer (100 mM, pH=5.0), glutaraldehyde concentration (15%), and 0.035 mg enzyme loading were established as the optimum test conditions. This biosensor exhibited a response time of 80 s, a generous linear range of 0.010 mM to 0.88 mM with a detection limit of 10μM, repeatbility (3.1%, n=20), recoveries (98.7-102%), and good stability with a shelf-life of more than 3 months. The reproducibility of fabrication of the biosensors was investigated by using three different membranes (R.S.D.=3.0%). The comparison of the analytical performance between the published and the proposed biosensor methods was made. This biosensor has been applied to determine the L-ascorbic acid content in real samples, and the results were agreement with those obtained by a commercial colorimetric ascorbic acid assay kit.
Chapter 5:Porphyrins are recognized as perfect host for molecular recognition owing to their diversity, rigid molecular structures, the position and direction of the substitutes on the porphyrin ring being controllable, Metalloporphyrin is a prototype of metal hexacyanoferrates with remarkable electro-, catalytic-, photo-and biochemical properties. In this paper, a type of iron (Ⅲ)-porphyrin (FeTPP) nanoparticles was prepared and applied for the fabrication of L-ascorbic acid biosensor. The effects of experimental conditions on biosensors'response have been investigated.
第二章：采用包埋法将抗坏血酸氧化酶(A.O)固定于壳聚糖与鸡蛋壳膜之间制得壳聚糖/抗坏血酸氧化酶/鸡蛋壳膜,将其与溶解氧电极偶联制得抗坏血酸生物传感器,对影响传感器响应性能的溶液pH、温度与缓冲溶液浓度进行了优化。磷酸缓冲溶液的pH 5.0,浓度为100mmol/L,实验温度为室温,此生物传感器的响应性能良好。该生物传感器的响应时间为100s,抗坏血酸浓度在0.07-0.77mmol/L呈现良好的线性关系,检出限为16μmol/L, RSD=4.05%(n=10).间断使用90天后,响应值变为初始值的85.0%。
第三章：采用包埋-交联法,以戊二醛做交联剂、壳聚糖为包埋剂,制备壳聚糖/抗坏血酸氧化酶/戊二醛/鸡蛋膜,并与氧电极偶联制成抗坏血酸生物传感器。对溶液温度、pH、戊二醛浓度、酶固定量等因素对传感器的影响进行了探索。磷酸缓冲溶液的pH 5.0,戊二醛浓度为15%,酶固定量为6 Units为传感器的最佳实验条件。抗坏血酸浓度在0.02～0.82mmol/L间呈良好的线性关系,检测限为12μmol/L,间断使用90天后,响应值变为初始值的87.6%。
第四章：通过包埋-交联法将抗坏血酸氧化酶固定于猪膀胱膜上,将固定有酶的膀胱膜紧贴于氧电极表面制得抗坏血酸生物传感器,并考察了影响传感器响应的缓冲溶液pH、浓度、戊二醛浓度、酶固定量等因素。在优化实验条件下,该传感器的响应时间为80s,抗坏血酸浓度在0.01～0.88 mmol/L之间呈现良好线性关系,检出限为, RSD=3.10%(n=20)。而且,此传感器具有良好的使用寿命和贮藏稳定性。实际样品中可能存在的干扰物质对抗坏血酸的测定无干扰。该传感器应用于维生素C饮品中抗坏血酸含量的测定,结果与试剂盒所测结果基本吻合。此外,进行了回收率实验,回收率为97.1-104%。结果表明：膀胱膜比一般天然膜材料透气性和韧性更好,制备出的传感器具有响应速度快、灵敏度高、线性范围宽、检测限低、重现性好、检测结果准确、使用寿命长等优点,为食品药品中抗坏血酸的检测提供了一种准确、可靠的新方法。
第五章：基于金属卟啉的化学稳定性、良好的生物和电催化活性,制备金属铁卟啉纳米粒子,并将其应用于抗坏血酸生物传感器的研制,制得以金属铁卟啉纳米粒子为电子媒介体的抗坏血酸生物传感器。对实验条件的优化进行了初步探索。
Chapter 1:A biosensor, a analytical device composed of a recognition element of biological origin and a physico-chemical transducer, becomes one of the important research fields of analytical chemistry. The biological element is capable of sensing the presence, activity or concentration of a chemical analyte in solution. The conception, basal constitution, working principles, types, characteristic and application of biosensors were summarized. In addition, research status of enzyme biosensor and detecting methods of L-ascorbic acid were reviewed.
Chapter 2:A L-ascorbic acid biosensor using a chitosan/ascorbate oxide/ eggshell membrane as a biological molecular recognition component and a dissolved oxygen electrode as a transducer has been fabricated. The ascorbate oxidase was entrapped between chitosan and eggshell membrane. The effect of phosphate buffer concentration, pH and temperature on the response of the L-ascorbic acid biosensor has been studied in detail. The resulting sensor exhibited a fast response of 100 s, high sensitivity and good storage stability. The linear response is 0.07-0.77 mmol/L of L-ascorbic acid concentration with a detection limit of 16μmol/L
Chapter 3:A L-ascorbic acid biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for L-ascorbic acid determination has been fabricated. Ascorbate oxidase was covalently immobilized on an eggshell membrane by chitosan with glutaraldehyde as a cross-linking agent. In the optimization studies of the biosensor, the most suitable enzyme and glutaraldehyde concentration were found to be 6 units and 15%(w/v), respectively. The phosphate buffer of pH 5.0 was chosen as the optimum working condition. The biosensor response has a linear range of L-ascorbic acid concentration between 0.02 mmol/L and 0.82 mmol/L with a detection limit of 12μmol/L (S/N=3). Compare with the membrane without glutaraldehyde, enzyme of the proposed biosensor was immobilized firmly。In the characterization studies of the glucose biosensor, some parameters such as interference effects and storage stability were investigated in detail.
Chapter 4:A L-ascorbic acid biosensing assay was developed using the enzyme-immobilized pig bladder membrane and dissolved oxygen electrode. The ascorbate oxidase immobilized with chitosan was bound to bladder membrane by glutaraldehyde as a crosslinking agent. The phosphate buffer (100 mM, pH=5.0), glutaraldehyde concentration (15%), and 0.035 mg enzyme loading were established as the optimum test conditions. This biosensor exhibited a response time of 80 s, a generous linear range of 0.010 mM to 0.88 mM with a detection limit of 10μM, repeatbility (3.1%, n=20), recoveries (98.7-102%), and good stability with a shelf-life of more than 3 months. The reproducibility of fabrication of the biosensors was investigated by using three different membranes (R.S.D.=3.0%). The comparison of the analytical performance between the published and the proposed biosensor methods was made. This biosensor has been applied to determine the L-ascorbic acid content in real samples, and the results were agreement with those obtained by a commercial colorimetric ascorbic acid assay kit.
Chapter 5:Porphyrins are recognized as perfect host for molecular recognition owing to their diversity, rigid molecular structures, the position and direction of the substitutes on the porphyrin ring being controllable, Metalloporphyrin is a prototype of metal hexacyanoferrates with remarkable electro-, catalytic-, photo-and biochemical properties. In this paper, a type of iron (Ⅲ)-porphyrin (FeTPP) nanoparticles was prepared and applied for the fabrication of L-ascorbic acid biosensor. The effects of experimental conditions on biosensors'response have been investigated.
引文
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