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基于离子液体和纳米材料的氧化还原蛋白质修饰电极研究
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摘要
离子液体和纳米材料因具有独特的物理化学性质,已在氧化还原蛋白质修饰电极的构置方面引起人们越来越多的关注。本论文基于离子液体和纳米材料,构置了十种氧化还原蛋白质修饰电极,研究了血红蛋白(Hb)、肌红蛋白(Mb)和葡萄糖氧化酶(GOx)的直接电化学和电催化行为。该研究对丰富氧化还原蛋白质的电化学及其生物传感器的研究内容,拓宽离子液体和纳米材料的应用范围具有一定的意义。全文共分四章,作者的主要贡献如下:
     1、采用滴涂法,构置了Hb-HA-BMIMBF4-GCE、Hb-CS-BMIMPF6-SiO2/GCE、Hb-CS-BMIMBF4-clay/GCE和Mb-CS-BMIMBF4-GR/GCE,研究了Hb和Mb的直接电化学和电催化行为。光谱实验表明Hb和Mb在这些复合膜中均保持了其原始的立体构象;电化学实验表明,这四种修饰电极上的Hb和Mb均实现了它们的直接电子转移,且离子液体的存在提高了它们在电极上的电化学响应;并对H2O2均表现出了良好的电催化作用,催化还原电流均与一定浓度范围内的H2O2呈良好的线性关系。研究表明,这些复合膜可为研究氧化还原蛋白质(酶)的电化学行为提供新的研究平台。
     2、采用电化学沉积法,制备了蛋白质的固载膜;采用层层组装法构置了GOx-IL-GNP-IL-SWCNT/GCE、Hb/IL/DNA/PDDA/ITO电极和Mb/DNA/CILE,研究了GOx.Hb和Mb的直接电化学和电催化行为,建立了检测葡萄糖、H2O2和三氯乙酸(TCA)的电化学新方法。实验结果表明,GOx、Hb和Mb在不同固载膜修饰的电极上均出现了峰形良好的、准可逆的氧化还原峰;在GOx-IL-GNP-IL-SWCNT/GCE上GOx对葡萄糖表现出良好的电催化作用,测定葡萄糖的线性范围为2.0×10-6~5.0×10-5mol·L-1,检出限为8.0×10-7mol·L-1(S/N=3),表观米氏常数(KM)为2.21×10-5mol·L-1;在Hb/IL/DNA/PDDA/ITO电极上Hb对H2O2具有良好的电催化活性,催化还原峰电流与1.0×10-6~3.8×10-4mol·L-1范围内的H2O2呈良好的线性关系,检出限为1.0×10-7 mol·L-1(S/N=3);在Mb/DNA/CILE上Mb对H2O2和TCA均具有良好的电催化能力,得到测定H2O2和TCA的线性范围分别为1.0×10-6~1.6×10-4 mol·L-1和5.0×10-4~4.0×10-2mol·L-1,检出限分别为2.0×10-7mol·L-1和8.3×10-5 mol·L-1(S/N=3)。该方法制备的这三种修饰膜可为研究其它氧化还原蛋白质(酶)的直接电化学行为提供良好的研究平台。
     3、以BPPF6和石蜡油为黏合剂,制备了离子液体修饰碳糊电极(CPE-IL);以此电极为基底电极,采用电沉积和层层涂布法,构置了Hb/CoNP/MWCNT/CPE-IL HA/CeO2/Mb/CPE-IL和GG/MgO/Hb/CPE-IL,研究了Hb和Mb的电化学行为,建立了H2O2和TCA的循环伏安测定新方法。实验结果表明,少量石蜡油的加入,可以极大地减小CPE-IL电极的背景电流;BPPF6、CoNP/MWCNT、HA/CeO2和GG/MgO纳米复合膜在促进Hb和Mb与电极之间的直接电子传输中起着非常重要的作用,且这三种修饰电极对H2O2或TCA表现出了良好的电催化作用。对Hb/CoNP/MWCNT/CPE-IL,其电极表面电活性Hb的覆盖度为F=8.92×10-10mol·cm-2,Hb催化H2O2和TCA的KM值分别达到2.61×10-5mol·L-1和2.31×10-4 mol·L-1;以GG/MgO/Hb/CPE-IL为工作电极,得到了宽的H2O2检测范围(2.0×10-7~1.7×10-4 mol·L-1)和很低的检出限(5.0×10-8mol·L-1)。这类修饰电极具有表面易于更新,可批量构置等特点。该方法构置的修饰电极对构建性能优良的第三代电化学生物传感器具有一定意义。
Due to their unique physical and chemical properties, ionic liquids and nanomaterials have been attracted more and more attention in the fabrication of redox proteins modified electrodes. In this dissertation, ten kinds of redox proteins modified electrodes were fabricated based on ionic liquids and nanomaterials and the behaviors of direct electrochemistry and electrocatalysis for hemoglobin (Hb), myoglobin (Mb) and glucose oxidase(GOx) modified on these electrodes were investigated in details. These research are not only significant to enrich research content of the direct electrochemsitry of redox proteins and their interrelated biosensors, but also can broaden the application range of ionic liquids and nanomaterials. The dissertation consists of four chapters. The author's main contributions are summarized and presented as follows:
     1. By casting method, four kinds of protein modified electrodes, including Hb-HA-BMIMBF4-GCE, Hb-CS-BMIMPF6-Si02/GCE, Hb-CS-BMIMBF4-clay/GCE and Mb-CS-BMIMBF4-GR/GCE, were fabricated and their behaviors of direct electrochemistry and electrocatalysis were investigated, respectively. The spectral results revealed that Hb and Mb could retain their native tridimensional structure in these composite films. The electrochemical results indicated that direct electron transfer were all occurred between these two proteins and the underlying electrodes, and their electrchemical response were enhanced obviously, which should be attributed to the presence of ionic liquids. Furthermore, Hb and Mb entrapped in these electrodes exhibited excellent electrocatalytic activities toward the reduction of H2O2 with wide linear ranges. Thus, these composite films could provide novel platform for the study of the electrochemical behavoirs of redox proteins.
     2. Electrodeposition and layer-by-layer assemble methods were employed to prepare modified films. Then, three kinds of protein modified electrodes, including GOx-IL-GNP-IL-SWCNT/GCE, Hb/IL/DNA/PDDA/ITO electrode and Mb/DNA/CILE, were fabricated and their electrochemical behaviors were carefully investigated. The corresponding electrochemical methods were presented for determination of glucose, H2O2 and trichloroacetic acid (TCA), respectively. The experimental results indicated that a pair of of well-defined, quasi-reversible cyclic voltammetric peaks appeared for GOx, Hb and Mb on these modified electrodes, repectively. GOx assembled on GOx-IL-GNP-IL-SWCNT/GCE showed excellent electrocatalytic activity toward the oxidation of glucose. The linear range for the determination of glucose was found to be 2.0×10-6 to 5.0×10-5 mol-L-1 and the detection limit was 8.0×10-7 mol-L-1 (S/N=3). The apparent Michaelis-Menten constant (KM) was further calculated as 2.21×10-5 mol-L-1. Hb modified on Hb/IL/DNA/PDDA/ITO electrode exhibited excellent electrocatalytic activity toward the reduction of H2O2. The reduction peak current had a linear relationship with the concentration of H2O2 in the range from 1.0×10-6 to 3.8×10-4mol-L-1 and the detection limit was 1.O×10-7 mol·L-1 (S/N=3). Mb immobilized on Mb/DNA/CILE showed excellent electrocatalytic activities toward the reduction of H2O2 and TCA with the linear range of 1.0×10-6~1.6×10-4mol·L-1 and 5.0x10-4~4.0×10-2 mol-L-1. The detection limits were 2.0x10-7 mol·L-1 and 8.3x 10-5 mol·L-1 (S/N=3), respectively. Thus, these three kinds of composite films showed potential application for other redox proteins to achieve their direct electron transfer.
     3. Ionic liquid modified carbon paste electrode (CPE-IL) was constructed with the BPPF6 and paraffin as binder. Then CPE-IL was employed as the basal electrode, Hb/CoNP/MWCNT/CPE-IL, HA/CeO2/Mb/CPE-IL and GG/MgO/Hb/CPE-IL were further fabricated by electrodeposition and step-by-step methods. The electrochemical characteristics of Hb and Mb modified on these electrodes were investigated in detail and the corresponding cyclic voltammetry was employed to determine H2O2 and TCA. The results suggested that the addition of a little paraffin could deduce the background current generated mainly by ionic liquid. While, BPPF6, CoNP/MWCNT, HA/CeO2 and GG/MgO composite film acted very important roles in enhancing the direct electron transfer between these two proteins and electrodes, respectively. Furthermore, these two proteins immobilized on the electrodes also showed excellent electrocatalytic activities toward the reduction of H2O2 and TCA. For Hb modified on Hb/CoNP/MWCNT/CPE-IL, the surface concentration of electroactive Hb was calculated to be 8.92×10-10mol-cm-2. The values of KM for H2O2 and TCA were 2.61 x10-5 mol·L-1 and 2.31×10-4mol·L-1, respectively. For Hb immobilized on GG/MgO/Hb/CPE-IL, H2O2 could be detected in a wide linear range of 2.0×10-7~1.7×10-4 mol·L-1 and a very low detection limit of 5.0×10-8 mol-L-1. Therefore, the redox proteins modified electrodes with the CPE-IL as platform render a great potential in fabricating the third-generation biosensor.
引文
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