苯丙氨酸二肽—石墨烯复合界面的构建及其在电化学传感中的应用

发布时间：2018-05-11 21:30

本文选题：石墨烯 + 苯丙氨酸二肽　；参考：《郑州大学》2017年博士论文

【摘要】：“工欲善其事,必先利其器”。修饰界面的构建是电化学传感的核心,也是提高电化学传感灵敏度和选择性的有效途径之一。寻找合适的能用于电化学传感的新功能结构材料,设计新的构建途径一直都是电化学传感研究的热点。本论文以苯丙氨酸二肽(FF)-石墨烯(rGO)复合物为对象,采用自组装、脉冲电位和Langmuir-Blodgett(LB)膜三种方法,制备了不同微观形貌的生物复合界面;以此为基础,基于牛血红蛋白(Hb)、葡萄糖氧化酶(GOD)、细胞色素c(Cyt.c)和醇溶蛋白抗体(Anti-gliadin),构建了对H_2O_2、葡萄糖、CH_3NO_2和醇溶蛋白等目标分子具有高选择性和高灵敏度的电化学生物传感器。论文具体工作如下:1.苯丙氨酸二肽及其衍生物的合成、表征采用Fmoc多肽固相合成法,通过氨基酸分子间氨基与羧基脱水缩合,合成了FF和衍生肽(FAF,FAAAF,FFA,FFAAA和二茂铁甲酸-FF(Fc-FF))。利用反相高效液相色谱、电喷雾质谱对合成的肽进行分析、分离和表征,结果显示所合成的肽均为目标肽,且纯度均在95%以上。2.苯丙氨酸二肽-石墨烯的自组装特性及其电化学生物传感应用通过在FF的自组装过程中加入rGO,在温和的室温条件下,成功制备了具有高密度特性的自组装FF纳米线-rGO(FFNW-rGO)复合膜。优化FFNW-rGO复合膜的制备条件,筛选得到了具有良好导电性的FFNW-rGO复合膜。以FFNW-rGO复合膜作为生物传感材料,将Hb固载到修饰电极表面,构建了H_2O_2的电化学生物传感器。分别通过扫描电镜实验、紫外-可见光谱实验、电化学实验揭示了,Hb已经成功固载到电极表面;在Nafion/FFNW-rGO薄膜中,Hb保持了原有结构构象,没有发生变性;同时验证了Nafion/FFNW-rGO复合薄膜为Hb的直接电子转移构筑了良好的微环境,能有效地促进电极表面与Hb之间的直接电子转移。在优化条件下,该电化学生物传感器对H_2O_2的还原表现出良好的催化活性,其催化电流与H_2O_2的浓度,在5.0×10-7 mol L-1~5.0×10-4 mol L-1范围内呈线性关系;将该法应用到实际样品的测定,结果令人满意。3.基于脉冲电位法构建的苯丙氨酸二肽-石墨烯复合膜电化学传感研究首次提出以氧化石墨烯(GO)和苯丙氨酸二肽-石墨烯(FF-GO)分散液为前驱,采用脉冲电位法,一步实现GO的还原和电极修饰,构建了电化学还原石墨烯(ErGO)和苯丙氨酸二肽-电化学还原石墨烯(FF-ErGO)修饰界面。在ErGO修饰电极上,研究了左旋多巴(L-dopa)在石墨烯修饰电极上的电化学行为,采用各种电化学方法计算了相关电化学参数。在优化条件下,建立了在尿酸(UA)和抗坏血酸(AA)存在情况下,L-dopa的简便、快速和灵敏的分析方法。此外,以FF-ErGO修饰电极为平台,将Hb作为模型分子,研究了Cs-Hb/FF-ErGO修饰电极的直接电化学行为及对CH_3NO_2的电催化特性。进一步设计并提出一步、简便、快速的方法直接将葡萄糖氧化酶(GOD)嵌入FF-ErGO中,构建了新型葡萄糖电化学生物传感器。通过循环伏安法研究了GOD在复合薄膜中的直接电化学行为及对葡萄糖的检测特性。4.基于多肽(FFAAA)-石墨烯复合LB膜构建的电化学生物传感器提出并筛选出多肽-石墨烯(FFAAA-rGO)为LB膜成膜材料分子,讨论了石墨烯和FFAAA-rGO的成膜性能;通过原子力显微镜(AFM)和电化学方法对石墨烯和FFAAA-rGO复合LB膜的结构形貌和电化学特性进行了表征。基于LB膜的特性,成功提出简单、方便的方法将细胞色素c(Cyt.c)直接嵌入FFAAA-rGO复合LB膜中,构建了基于Cyt.c的H_2O_2电化学生物传感器。紫外-可见光谱证明了Cyt.c在FFAAA-rGO复合LB膜中,能保持原有结构构象;电化学实验展示了FFAAA-rGO复合LB膜能有效地促进Cyt.c与电极表面之间的直接电子转移。另外,该修饰电极具有较小的表观米氏常数(app MK),说明FFAAA-rGO复合LB膜能为Cyt.c的直接电子转移构筑良好的微环境,因而能对H_2O_2具有高催化活性以及更高的亲和力。此外,以FFAAA-rGO复合LB膜界面为平台,通过缩合反应,将醇溶蛋白抗体(Anti-gliadin)固定于FFAAA-rGO复合LB膜界面,构建了醇溶蛋白的免疫传感器。由于FFAAA-rGO复合LB膜具有比表面积大和导电性能好的特点,能显著增大固定抗体的固载量,从而提高传感器的灵敏度。在最优条件下,该电化学免疫传感器对醇溶蛋白检测线性范围为:5.0~80.0 ng mL-1,检出限为1.0 ng mL-1。该方法用于实际样品中醇溶蛋白的检测,结果令人满意。
[Abstract]:The construction of the modified interface is the core of the electrochemical sensing. It is also one of the effective ways to improve the sensitivity and selectivity of electrochemical sensing. To find a new functional structure material for electrochemical sensing and to design a new way of construction is the hot spot of the electrochemical sensing research. With phenylalanine two peptide (FF) - graphene (rGO) complex as the object, using three methods of self-assembly, pulse potential and Langmuir-Blodgett (LB) membrane, the biological complex interface of different micromorphology was prepared. Based on this, it was based on bovine hemoglobin (Hb), glucose oxidase (GOD), cytochrome c (Cyt.c) and gliadin antibody (Anti-gliadin). A high selectivity and high sensitivity electrochemical biosensor for target molecules such as H_2O_2, glucose, CH_3NO_2 and gliadin was constructed. The specific work of this paper is as follows: synthesis of 1. phenylalanine two peptide and its derivatives, characterization by solid-phase synthesis of Fmoc peptides, synthesis of FF by condensation of amino acid amino acid and carboxyl group of amidic acid And derived peptides (FAF, FAAAF, FFA, FFAAA and two metallocalonylic acid -FF (Fc-FF)). The synthesized peptides were analyzed, separated and characterized by reversed phase high performance liquid chromatography (RP HPLC) and electrospray ionization mass spectrometry (ESI MS). The results showed that the synthesized peptides were all target peptides, and the self-assembly characteristics and electrochemical biosensor of the.2. phenylpropane two peptide - graphene were more than 95% of the purity. By adding rGO in the self-assembly process of FF, the self assembled FF nanowire -rGO (FFNW-rGO) composite membrane with high density properties was successfully prepared under mild room temperature conditions. The preparation conditions of the FFNW-rGO composite membrane were optimized. The good conductivity of the FFNW-rGO complex membrane was screened. The composite membrane of FFNW-rGO was used as a biosensor. Hb was immobilized to the surface of the modified electrode, and the electrochemical biosensor of H_2O_2 was constructed. By scanning electron microscope experiment, ultraviolet visible spectrum experiment, the electrochemical experiment revealed that Hb had been successfully immobilized on the surface of the electrode. In the Nafion/FFNW-rGO film, Hb kept the conformation of the original structure without denaturation; at the same time, the Nafion/FFNW-r was verified. The GO composite film provides a good micro environment for the direct electron transfer of Hb, which can effectively promote the direct electron transfer between the electrode surface and the Hb. Under the optimum conditions, the electrochemical biosensor exhibits good catalytic activity for the reduction of H_2O_2, and its catalytic current and H_2O_2 concentration at 5 * 10-7 mol L-1~5.0 x 10-4 mol L-1 A linear relationship was found in the range, and the method was applied to the determination of real samples. The results were satisfactory. The electrochemical sensing of the phenylalanine two peptide and graphene composite membrane based on the pulsed potential method for the first time proposed that the precursor of graphene oxide (GO) and phenylalanine two peptide - graphene (FF-GO) dispersions were used as the precursors for the first time, and the pulse potential method was used in one step to realize.3.. The electrochemical reduction of graphene (ErGO) and phenylalanine two peptide - electrochemical reduction Shi Moxi (FF-ErGO) modification interface was constructed. The electrochemical behavior of Zo do Ba (L-dopa) on the graphene modified electrode was studied on the ErGO modified electrode. The electrochemical parameters were calculated by various electrochemical methods. Under the condition of the presence of uric acid (UA) and ascorbic acid (AA), the simple, rapid and sensitive analysis method of L-dopa was established. In addition, the direct electrochemical behavior of Cs-Hb/FF-ErGO modified electrode and the electrocatalytic properties of the Cs-Hb/FF-ErGO modified electrode were studied on the platform of FF-ErGO modified electrode and Hb as a model molecule. A step was further designed and proposed. A simple and rapid method of embedding glucose oxidase (GOD) directly into FF-ErGO and constructing a new glucose electrochemical biosensor was constructed. The electrochemical behavior of GOD in the composite film and the detection characteristic of glucose in the composite film were studied by cyclic voltammetry. The electrochemical biosensing based on the peptide (FFAAA) - graphene composite LB membrane was constructed. The peptide graphene (FFAAA-rGO) was selected as a membrane forming material for LB membrane, and the film forming properties of graphene and FFAAA-rGO were discussed. The morphology and electrochemical properties of graphene and FFAAA-rGO composite LB films were characterized by atomic force microscopy (AFM) and electrochemical methods. Based on the properties of LB films, it was simple and convenient. The method of embedding cytochrome c (Cyt.c) directly into the FFAAA-rGO composite LB membrane and constructing a H_2O_2 electrochemical biosensor based on Cyt.c. The UV visible spectrum shows that Cyt.c can maintain the original structure conformation in the FFAAA-rGO compound LB membrane, and the electrochemical experiment shows that the FFAAA-rGO compound LB membrane can effectively promote the between the surface of the Cyt.c and the surface of the electrode. In addition, the modified electrode has a small apparent Michaelis constant (APP MK), which indicates that the FFAAA-rGO composite LB film can create a good microenvironment for direct electron transfer of Cyt.c, thus can have high catalytic activity and higher affinity to H_2O_2. In addition, the FFAAA-rGO composite LB membrane interface is used as a platform, through condensation reaction, The antibody of gliadin (Anti-gliadin) was immobilized on the interface of FFAAA-rGO composite LB membrane, and the immune sensor of gliadin was constructed. Because the FFAAA-rGO composite LB film has the characteristics of large specific surface area and good electrical conductivity, it can significantly increase the fixed amount of the immobilized antibody and improve the sensitivity of the sensor. Under the optimal condition, the electrochemical immunization is used. The linear range of detection of gliadin by sensor is 5.0~80.0 ng mL-1, and the detection limit is 1 ng mL-1.. The method is used for the detection of gliadin in actual samples. The results are satisfactory.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.1

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