新型生物分子固定技术用于构建电化学免疫传感器的研究
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摘要
长期以来，由于电化学免疫传感器具有设计制造简单、高灵敏度、价格低廉而被广泛研究并已在生物检测中逐步得到了应用。然而，如何将生物活性组分有效地固定在电极表面上的固定化方法、降低甚至消除蛋白质在传感器上的非特异性吸附以及免疫传感器的重现性和重复使用性能等方面存在的问题阻碍了电化学免疫传感器的发展和应用。其中有效的生物活性组分的固定方法是构建性能优良的生物传感器的关键步骤，据此本研究论文发展了五种新型的生物活性组分在电极上的固定化技术，并以此来构建电化学免疫传感器。主要内容如下：
(1)在第二章和第三章发展了基于邻氨基硫酚在金电极上进行自组装和电催化聚合形成低聚合物膜技术的电容型电化学免疫传感器。采用邻氨基硫酚低聚合物膜固定生物活性组分，构建电容型免疫传感器的优势在于一方面其表面存在自由的氨基可以用戊二醛活化或结合纳米金来固定抗体构建电容免疫传感器；另一方面，邻氨基硫酚低聚合物膜可完全封闭金电极表面，而且所形成的低聚合膜为一种有机半导体膜，可赋予传感器具有较高的初始电容值，以提高其检测范围及检测灵敏度。采用循环伏安法研究这类传感器的基本性能如法拉第电流效应、离子的不可通透性等，均表明该低聚合膜能很好的满足构建电容免疫传感器的要求。采用恒电位脉冲计时安培法可确定传感器的电容值，实验结果表明，用戊二醛活化邻氨基硫酚低聚合物膜固定转铁蛋白抗体而构建的传感器，对溶液中转铁蛋白检测的浓度的线性范围为1.25-80.0ng／ml，检测下限为0.12ng／ml。而采用结合纳米金来进一步固定转铁蛋白抗体所构建的电容免疫传感器，对溶液中转铁蛋白的测定的浓度线性范围为0.1-1000ng／ml，检测下限达0.08ng／ml。
(2)κ型角叉皂素(κ-LC)为带磺酸基的天然聚电解质，是一种具有强电负性的物质，将κ-LC与蛋白质偶联，可以增强蛋白质的负电荷性而使通过静电自组装技术固定的蛋白质的稳定性得到提高。本论文的第四章采用此技术构建了亲和柱用于流动免疫分析方法检测了溶液中NH IgG，其检测的浓度的线性范围为0.1-1000μg／ml，该亲和柱用于免疫分析具有重复性较好、非特异性吸附影响低等特点。
褐藻酸钠与壳聚糖之间可通过静电自组装而形成牢固的小分子可透过性的天然高分子膜。可利用褐藻酸钠这一特性可构建电化学免疫传感器，即将褐藻酸钠通过EDC活化与目标生物活性组相联，将壳聚糖通过包埋技术固定在碳糊电极里；这样，负载有生物活性组分的褐藻酸钠与固定在电极表面的壳聚糖分子之间发生静电自组装的同时也将
摘要
生物组分固定到电极表面而完成了电化学免疫传感器的构建。第五章描述了采用该技术
固定日本血吸虫抗体构建了一种新的日本血吸虫电化学免疫传感器。实验结果表明该传
感器对日本血吸虫抗原检测的浓度线性范围为0.64一40雌/m1，检测下限为0.40阳
/m1，且该固定化方法具有重复性好、蛋白质的非特异性吸附影响较低的特点。
(3) TriS改性的褐藻酸钠或通过肤胺而固定在金电极表面上的TriS改性的褐藻酸
钠在CaZ十存在下引发固定抗体构建了电化学免疫传感系统和免疫传感器。通过对转铁蛋
白免疫反应体系来评价这两种技术固定抗体技术的可行性。实验证明此免疫传感器具有
重现性好、重复使用性能良好及蛋白质的非特异性吸附影响低等特点。用该传感器检测
溶液中转铁蛋白的浓度的线性范围为0.2一30阳/m1，其检测下限可达0.05雌/m1;
而采用免疫传感系统分析转铁蛋白的浓度线性范围为5.0一35协g/m1，其检测下限为
2.3雌/ml，也具有蛋白质的非特异性吸附影响低等特点，但实验操作较传感器稍繁
琐。
(4)纳米金(胶体金)可用于吸附固定抗体，己在免疫分析中得到广泛的应用。近
年来的研究发现，纳米金能降低一些物质在电极上发生反应的氧化还原电位，并催化这
些物质在电极上的电化学氧化还原反应。利用纳米金的上述特征，第八章设计了非标记
的电化学免疫传感器用于直接测定溶液中的对氧磷含量。研究发现在纳米金存在下，对
氧磷分子上的硝基能在一165mV的电位下被还原，在一O，smV电位下可被氧化;而无纳米
金存在下，其电化学还原电位大于一800mV.应用该传感器检测溶液中对氧磷浓度的上限
为1920林g/L，其检测下限可达12哪/L。
(5)该电极用于吸附固定转铁蛋白抗体构建了转铁蛋白电化学免疫传感器。实验表
明，采用该电极研制的传感器在检测溶液中电活性组分时，较石蜡碳糊电极具有更高的
灵敏度，且该电极能用简单的抛光技术处理而重复使用;采用竞争免疫分析方法及计时
安培测量技术，该传感器能在浓度范围为0.5一70.。协g/ml实现对转铁蛋白的检测，其
检测下限达0.35林g/m1，且具有良好的重现性、重复使用性和较低的蛋白质非特性吸
附性能。
Electrochemical immunosensor are widely used for the assay of biological analytes. The advantages of this approach including their simple-design, high-sensitivity and low-cost attract substantial research efforts directed to the developments of some new electrochemical immunosensors in the last two decades. However, the method of immobilization, the prevention or elimination of nonspecific interactions, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors.
Focused on these said topics, several new methods for immobilizing immuno-species to construct immunosensor have been developed in the presented paper and described as follows:
(1) Development of capacitance immunosensors based on the oligomer of o-aminobenzenethiol polymerized electrochemically was described in both the chapter 2 and 3. The main advantage of this strategy can be described as follows: the oligomer of o-aminobenzenethiol has been chem-adsorbed firmly on the surface of crystalline gold electrode and the organic semiconductor with free amino-groups formed can be employed as a platform to react with glutareldehyde or combined with nano-Au for immobilization of transferrin antiserum. Its above-described performances can meet the requirements for fabricating capacitance immunosensor such as impermeability to electroactive species in solution and as thin as possible to guarantee the sensitivity of immunosensor. The cyclic voltammetry set up was applied to evaluate the performances of the immunosensor (such as impermeability). The potentiostatic pulse technique was applied to measure the capacitance value of the immunosensor. The transferrin was determined in the range of
1. 25 - 80.0 ng/ral with detection limit of 0. 12 ng / ml for immunosensor employed glutareldehyde for immobilization of antibodies as well as in the range of 0. 1 ?lOOng/ml with detection limit of 80 pg /ml
Abstract
for immunosensor employed nano-Au for immobilization of antibodies.
(2.1) K - Lambda-carrageenan ( K -LC ) or alginate is the negatively charged natural polyelectrolyte with numerous negatively charged sulphonate groups on their backbone. Cross-linked with K -LC or alginate , proteins can be further negatively charged and immobilized on the substrate further firmly by the self-assembly technique. The proposed method was realized by flow immunoassay strategy in an immunoaffinity column based on electrostatic self-Assembly (see chapter 4). The flow immunoassay system was used to detect the concentration of NH IgG in the range of 0. 1-1000 g/ml.
(2.2) A self-assembly event between chitosan and alginate is a known phenomena. The strategy of self-assembly event was exploited to fabricate the electrochemical immunosensor in chapter 5. The interesting proteins were modified with alginate by cross-linking technique employing EDC as the promoter. Chitosan was encapsulated in carbon paste electrode. The self-assembly events between Chitosan and alginate loaded with proteins will complete the immobilization of interesting proteins on the transducer. The chronoamperometric set up was applied to measure the reduced current values of 1, 4-benzo quinone at peak potential of - 0. 150mV when horseradish peroxidase as a label as well as both hydroquinone and hydrogen peroxide (H202) as the substrates were employed in electrochemical immunoassay.
It is in chapter 5 that the schistosoma japonium (Sj) system was employed to test the feasibility of the above-mentioned strategy. The sensor exhibited a linear response to SjAg in the concentration range 0.64 to 40 gmL-1 with the detection limit of 0.64 gmL-1.
(3) Both the tris-derived alginate and tris-derived alginate fixed on the surface of gold electrode by coupling with cysteine self-assembled layer on the gold electrode were employed as the platforms for immobilizing antibody to develop the electrochemical immunoassay system or electrochemical immunosensor in the presence of Ca2+. The reproducibility of the immunosensor was realized by being washed in 0. 1 0
(1)在第二章和第三章发展了基于邻氨基硫酚在金电极上进行自组装和电催化聚合形成低聚合物膜技术的电容型电化学免疫传感器。采用邻氨基硫酚低聚合物膜固定生物活性组分，构建电容型免疫传感器的优势在于一方面其表面存在自由的氨基可以用戊二醛活化或结合纳米金来固定抗体构建电容免疫传感器；另一方面，邻氨基硫酚低聚合物膜可完全封闭金电极表面，而且所形成的低聚合膜为一种有机半导体膜，可赋予传感器具有较高的初始电容值，以提高其检测范围及检测灵敏度。采用循环伏安法研究这类传感器的基本性能如法拉第电流效应、离子的不可通透性等，均表明该低聚合膜能很好的满足构建电容免疫传感器的要求。采用恒电位脉冲计时安培法可确定传感器的电容值，实验结果表明，用戊二醛活化邻氨基硫酚低聚合物膜固定转铁蛋白抗体而构建的传感器，对溶液中转铁蛋白检测的浓度的线性范围为1.25-80.0ng／ml，检测下限为0.12ng／ml。而采用结合纳米金来进一步固定转铁蛋白抗体所构建的电容免疫传感器，对溶液中转铁蛋白的测定的浓度线性范围为0.1-1000ng／ml，检测下限达0.08ng／ml。
(2)κ型角叉皂素(κ-LC)为带磺酸基的天然聚电解质，是一种具有强电负性的物质，将κ-LC与蛋白质偶联，可以增强蛋白质的负电荷性而使通过静电自组装技术固定的蛋白质的稳定性得到提高。本论文的第四章采用此技术构建了亲和柱用于流动免疫分析方法检测了溶液中NH IgG，其检测的浓度的线性范围为0.1-1000μg／ml，该亲和柱用于免疫分析具有重复性较好、非特异性吸附影响低等特点。
褐藻酸钠与壳聚糖之间可通过静电自组装而形成牢固的小分子可透过性的天然高分子膜。可利用褐藻酸钠这一特性可构建电化学免疫传感器，即将褐藻酸钠通过EDC活化与目标生物活性组相联，将壳聚糖通过包埋技术固定在碳糊电极里；这样，负载有生物活性组分的褐藻酸钠与固定在电极表面的壳聚糖分子之间发生静电自组装的同时也将
摘要
生物组分固定到电极表面而完成了电化学免疫传感器的构建。第五章描述了采用该技术
固定日本血吸虫抗体构建了一种新的日本血吸虫电化学免疫传感器。实验结果表明该传
感器对日本血吸虫抗原检测的浓度线性范围为0.64一40雌/m1，检测下限为0.40阳
/m1，且该固定化方法具有重复性好、蛋白质的非特异性吸附影响较低的特点。
(3) TriS改性的褐藻酸钠或通过肤胺而固定在金电极表面上的TriS改性的褐藻酸
钠在CaZ十存在下引发固定抗体构建了电化学免疫传感系统和免疫传感器。通过对转铁蛋
白免疫反应体系来评价这两种技术固定抗体技术的可行性。实验证明此免疫传感器具有
重现性好、重复使用性能良好及蛋白质的非特异性吸附影响低等特点。用该传感器检测
溶液中转铁蛋白的浓度的线性范围为0.2一30阳/m1，其检测下限可达0.05雌/m1;
而采用免疫传感系统分析转铁蛋白的浓度线性范围为5.0一35协g/m1，其检测下限为
2.3雌/ml，也具有蛋白质的非特异性吸附影响低等特点，但实验操作较传感器稍繁
琐。
(4)纳米金(胶体金)可用于吸附固定抗体，己在免疫分析中得到广泛的应用。近
年来的研究发现，纳米金能降低一些物质在电极上发生反应的氧化还原电位，并催化这
些物质在电极上的电化学氧化还原反应。利用纳米金的上述特征，第八章设计了非标记
的电化学免疫传感器用于直接测定溶液中的对氧磷含量。研究发现在纳米金存在下，对
氧磷分子上的硝基能在一165mV的电位下被还原，在一O，smV电位下可被氧化;而无纳米
金存在下，其电化学还原电位大于一800mV.应用该传感器检测溶液中对氧磷浓度的上限
为1920林g/L，其检测下限可达12哪/L。
(5)该电极用于吸附固定转铁蛋白抗体构建了转铁蛋白电化学免疫传感器。实验表
明，采用该电极研制的传感器在检测溶液中电活性组分时，较石蜡碳糊电极具有更高的
灵敏度，且该电极能用简单的抛光技术处理而重复使用;采用竞争免疫分析方法及计时
安培测量技术，该传感器能在浓度范围为0.5一70.。协g/ml实现对转铁蛋白的检测，其
检测下限达0.35林g/m1，且具有良好的重现性、重复使用性和较低的蛋白质非特性吸
附性能。
Electrochemical immunosensor are widely used for the assay of biological analytes. The advantages of this approach including their simple-design, high-sensitivity and low-cost attract substantial research efforts directed to the developments of some new electrochemical immunosensors in the last two decades. However, the method of immobilization, the prevention or elimination of nonspecific interactions, the reproducibility and the reusability still remain to be solved in the design and applications of these sensors.
Focused on these said topics, several new methods for immobilizing immuno-species to construct immunosensor have been developed in the presented paper and described as follows:
(1) Development of capacitance immunosensors based on the oligomer of o-aminobenzenethiol polymerized electrochemically was described in both the chapter 2 and 3. The main advantage of this strategy can be described as follows: the oligomer of o-aminobenzenethiol has been chem-adsorbed firmly on the surface of crystalline gold electrode and the organic semiconductor with free amino-groups formed can be employed as a platform to react with glutareldehyde or combined with nano-Au for immobilization of transferrin antiserum. Its above-described performances can meet the requirements for fabricating capacitance immunosensor such as impermeability to electroactive species in solution and as thin as possible to guarantee the sensitivity of immunosensor. The cyclic voltammetry set up was applied to evaluate the performances of the immunosensor (such as impermeability). The potentiostatic pulse technique was applied to measure the capacitance value of the immunosensor. The transferrin was determined in the range of
1. 25 - 80.0 ng/ral with detection limit of 0. 12 ng / ml for immunosensor employed glutareldehyde for immobilization of antibodies as well as in the range of 0. 1 ?lOOng/ml with detection limit of 80 pg /ml
Abstract
for immunosensor employed nano-Au for immobilization of antibodies.
(2.1) K - Lambda-carrageenan ( K -LC ) or alginate is the negatively charged natural polyelectrolyte with numerous negatively charged sulphonate groups on their backbone. Cross-linked with K -LC or alginate , proteins can be further negatively charged and immobilized on the substrate further firmly by the self-assembly technique. The proposed method was realized by flow immunoassay strategy in an immunoaffinity column based on electrostatic self-Assembly (see chapter 4). The flow immunoassay system was used to detect the concentration of NH IgG in the range of 0. 1-1000 g/ml.
(2.2) A self-assembly event between chitosan and alginate is a known phenomena. The strategy of self-assembly event was exploited to fabricate the electrochemical immunosensor in chapter 5. The interesting proteins were modified with alginate by cross-linking technique employing EDC as the promoter. Chitosan was encapsulated in carbon paste electrode. The self-assembly events between Chitosan and alginate loaded with proteins will complete the immobilization of interesting proteins on the transducer. The chronoamperometric set up was applied to measure the reduced current values of 1, 4-benzo quinone at peak potential of - 0. 150mV when horseradish peroxidase as a label as well as both hydroquinone and hydrogen peroxide (H202) as the substrates were employed in electrochemical immunoassay.
It is in chapter 5 that the schistosoma japonium (Sj) system was employed to test the feasibility of the above-mentioned strategy. The sensor exhibited a linear response to SjAg in the concentration range 0.64 to 40 gmL-1 with the detection limit of 0.64 gmL-1.
(3) Both the tris-derived alginate and tris-derived alginate fixed on the surface of gold electrode by coupling with cysteine self-assembled layer on the gold electrode were employed as the platforms for immobilizing antibody to develop the electrochemical immunoassay system or electrochemical immunosensor in the presence of Ca2+. The reproducibility of the immunosensor was realized by being washed in 0. 1 0
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