摘要
本论文分三部分,第一、第二部分分别利用聚电解质/碳酸钙微球的静电作用和电化学预处理法,实现了铁氰化钾和硫堇两种电子媒介体的有效固定。分别构筑了抗坏血酸传感器和无酶葡萄糖生物传感器;第三部分基于溶胶-凝胶法共价固定辣根过氧化物酶(HRP),制备了一种新型过氧化氢传感器。
1、通过将聚丙烯基胺盐酸盐(PAH)静电组装到聚对苯乙烯磺酸钠(PSS)掺杂的多孔碳酸钙微球表面上,Fe(CN)63?离子通过静电作用吸附到PAH层表面,成功制备一种新型电活性材料。将得到的电子媒介体材料嵌入到壳聚糖(CS)溶胶-凝胶基质中,构筑了抗坏血酸(AA)电化学传感器。并考察了该传感器在测定AA方面的应用。Fe(CN)63?对AA的催化氧化电位为0.27 V,与AA在裸玻碳电极上直接催化氧化电位(0.4 V)相比明显发生负移。该电化学传感器响应快速,具有良好的重现性和稳定性。测定AA的线性范围是1.0×10-6 ~ 2.143×10-3 M,其检测限是7.0×10-7 M。
2、利用电化学预处理法共价固定硫堇(Th)分子,通过聚多巴胺(PDA)对得到的Th修饰电极的负载作用,实现了特异性亲和受体伴刀豆球蛋白(Con A)分子的固定化,建立了一种方便、有效的检测葡萄糖的新方法,构筑了新型无酶葡萄糖生物传感器。PDA作为功能性“双面胶”,利用自身的粘附性和Schiff反应,得到功能性界面Th/PDA/Con A。通过Con A与葡萄糖特异性结合导致Th电化学信号的改变,从而实现了葡萄糖的定量检测。该传感器表现出良好的重现性、稳定性和抗干扰能力,对葡萄糖响应的线性范围为1.0×10?6 ~ 1×10?4 M,检测限为7.5×10?7 M。
3、基于一步多聚糖溶胶-凝胶反应原位共价固定HRP制备了新型H2O2生物传感器。硅氧烷是一种含有环氧基和三甲氧基官能团的无机材料,利用生物高聚物?壳聚糖(CS)的-NH2与γ-环氧丙氧丙基三甲氧基硅烷(GPTMS)的环氧基团之间的反应形成的网络结构将HRP实现固定。固定在这种具有高生物相容性杂化材料中的酶,能够保持高的酶催化活性,对H2O2具有快速的响应。生物传感器对H2O2响应的线性范围是2.0×10-7 ~ 4.6×10-5 M,其检测限是8.1×10-8 M。
There are three parts in this thesis. Two new and simple strategies were established for fabricating of electrochemical biosensor based on immobilization of electron mediators (Fe(CN)63? and Thionine) by electrostatic interaction of polyelectrolyte-calcium carbonate microsphere (the first chapter) and electrochemically pretreatment (the second chapter). A novel H2O2 biosensor was also fabricated based on in-situ covalent immobilization of horseradish peroxidase (HRP) through sol–gel process in the third part.
1. A novel electro-active material was successfully prepared with the Fe(CN)63? ions loaded by electrostatic interaction onto the layer of poly(allylamine) hydrochloride (PAH), which was firstly assembled on the prepared poly(Sodium 4-styrenesulfonate) (PSS)-doped porous calcium carbonate microspheres (CaCO3). Further, an electrochemical sensor toward ascorbic acid (AA) detection was constructed with the use of above electro-active materials embedded into a chitosan (CS) sol-gel matrix as electron mediators. The electrocatalytic oxidation of AA by ferricyanide was observed at the potential of 0.27 V, which was evidently negative-shifted compared with that by direct electrochemical oxidation of AA on the glassy carbon electrode at the potential of 0.4 V. The experimental parameters including pH value of testing solution and applied potential for detection of AA were optimized. The current electrochemical sensor not only exhibited a good reproducibility and storage stability but also showed a fast amperometric response to AA in a linear range (1.0×10?6 to 2.143×10?3 M), a low detection limit (7.0×10?7 M).
2. A convenient and effective strategy based on biospecific binding affinity of Concanavalin A (Con A) through polydopamine (PDA) mussel-inspired coatings on Thionine (Th) modified glassy carbon electrode was used to develop a non-enzyme-based glucose biosensor for the detection of glucose. In the proposed electrodeposition strategy, PDA was applied as functional“Double-sided adhesive-type”for inspired incorporating of Th and Con A due to its adhesive mechanisms and Schiff base reaction. With the peak current response of the Th moiety changed by biospecific binding Con A-glucose complex, quantitative measurement of glucose could be achieved. The current electrochemical sensor not only exhibited a excellent reproducibility, storage stability and high anti-interferent ability, but also showed broader linear response from 1.0×10?6 to 1×10?4 M with a low detection limit of 7.5×10?7 M to glucose. As a result, the proposed economical, efficient methodology was potentially attractive for evaluating clinical health risk.
3. A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H2O2) biosensor based on in-situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide incorporated sol–gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H2O2 and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H2O2 ranged from 2.0×10-7 to 4.6×10-5 M with a detection limit of 8.1×10-8 M. The apparent Michaelis-Menten constant was determined to be 45.18μM. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.
引文
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