【作者】 谭月明；

【导师】 谢青季；

【作者基本信息】 湖南师范大学 ， 分析化学， 2010， 博士

【摘要】 生物／化学催化剂修饰电极已广泛用于传感检测、能源和电合成等领域。与化学催化剂相比,生物催化剂通常选择性更好、催化效率更高、反应条件更温和。生物电催化是生物传感和生物燃料电池的重要学科基础,创新和优化酶等生物催化材料在电极上的高效固定方法对实现高效生物电催化至关重要。此外,电化学技术集合成、分离与分析功能于一体,若以电极作为工作平台,建立生物／化学催化体系的表征新方法,对于研究催化反应过程与机理颇具意义。本学位论文中,我们综述了压电传感技术、酶生物传感器、酶生物燃料电池和酶催化聚合的近期进展,采用压电电化学等方法对几个生物／化学催化体系进行了较详细的研究,主要内容如下：1.首次采用电化学噪声(ECN)装置测试了无隔膜葡萄糖／空气生物燃料电池(BFC)和单极葡萄糖BFC的性能。无隔膜葡萄糖／空气BFC的阳极采用明胶-多壁碳纳米管(MWCNTs)固定葡萄糖氧化酶(GOx)和二茂铁,阴极采用聚吡咯-MWCNTs固定漆酶和2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)二胺盐(ABTS)。在含有40 mM葡萄糖的醋酸缓冲溶液(pH 5.0)中,磁力搅拌下,无隔膜葡萄糖／空气BFC的短路电流为85μA cm-2,开路电压为0.29 V,最大输出功率密度为8μW cm-2。该BFC在100 kΩ外阻负载下,在上述溶液中连续放电15小时,电池输出电流降至初始值的78.9%。经双通道压电石英晶体微天平(QCM)监测,发现电池性能下降主要是因为阴极所固定的电子媒介体ABTS的泄漏所致。单极葡萄糖BFC中,阴极液为酸性KMnO4溶液,阳极液为含40 mM葡萄糖的磷酸缓冲溶液(pH 7.0),阴极室和阳极室间以Nafion 117质子交换膜隔开。以ECN装置测得此电池短路电路为202μAcm-2,开路电压为1.24 V,最大输出功率密度为115μW cm-2,与无隔膜葡萄糖／空气BFC相比,电池输出功率有显著提高。此外,还比较了阳极有无MWCNTs修饰时单极葡萄糖BFC的性能,发现修饰MWCNTs后输出功率提高到未修饰时的1.8倍。ECN装置有望成为研究BFC的一种实时、灵敏而简便的手段。2.通过简便的材料改性,使生物高分子壳聚糖(CS)用于酸性介质中BFC和生物传感研究成为可能。先通过CS和戊二醛(GA)反应制得GA功能化的CS (GAfCS),再与漆酶(Lac)反应形成Lac-GAfCS复合膜。QCM研究表明,该膜在弱酸性溶液中有较好的稳定性。ABTS存在下,Lac-GAfCS-MWCNTs/玻碳电极(GCE)能很好地催化氧气的还原,并研究了催化活性对溶液pH的依赖性。以Lac-GAfCS-MWCNTs/GCE为阴极、GOx-GAfCS-MWCNTs/GCE为阳极、Nafion膜为隔膜,构建了葡萄糖／空气BFC。采用ECN装置测得该BFC最大输出功率为9.6μW cm-2,开路电压为0.19 V,短路电流为114μA cm-2。此外,基于Lac-GAfCS-MWCNTs/GCE在pH 3.0的B-R缓冲溶液中检测了邻苯二酚,线性范围为0.1～50μM,检测限为20 nM。与直接采用GA一锅法交联固定Lac所制(?)Lac-GA-CS和Lac-GA相比,采用大分子交联剂GAfCS(即两步法)对酶活性的影响更小,因而更适合固定酶用于研制BFC和生物传感器。3.在少量交联剂存在下,使酶先键合到壳聚糖,再进行一锅法电沉积,可明显提高酶负载量和所制生物传感器的检测灵敏度(与无预交联的常规CS电沉积固定酶技术相比)。基本的实验流程如下(以模型酶GOx为例)：以低浓度GA(0.08 wt%)将GOx键合到CS链上,再通过电还原过氧化氢以增加电极表面的pH,可电沉积得到CS-GA-GOx复合膜。基于酸碱滴定模型对CS-GA-GOx的电沉积行为进行了理论探讨,采用电化学压电石英晶体微天平(EQCM)技术对电沉积过程进行了实验监测。在0.7 V vs SCE检测电位下,所制第一代酶电极(CS-GA-GOx/Ptnano/Au)的灵敏度高达102μA mM-1 cm-2,是传统电沉积方法(未将GOx连接到CS上)所制CS-GOx/Ptnano/Au电极的13倍。以紫外可见分光光度法测定了有／无GA时、加碱沉淀CS复合物后的上清液中GOx的含量,结果表明GA处理可明显增加沉积复合物中的酶负载量。以电化学方法研究了GA处理对GOx活性的影响,发现该低浓度GA处理GOx几乎不影响酶活性。此外,通过一系列实验,可靠地证明了所提出的预交联方法具有较高的普适性,包括改变预交联方式(1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺(EDC/N-羟基琥珀酰亚胺(NHS)激活)、电沉积方式(水还原)、传感模式(第二代)、电极面积(5μm半径Pt微电极)及固定酶的种类(碱性磷酸酶)。因电沉积法已广泛用于固定生物大分子,而提高生物大分子的负载量和生物活性一直是重要的科学问题,这里提出的将生物大分子预先连接到电沉积前躯物上并实现高负载量、高活性固定生物大分子的方法有望在生物技术研发方面得到广泛应用。4.通过酶(漆酶Lac)催化聚合途径,合成了新型聚合物-酶-MWCNTs复合物膜并将之用于生物传感和BFC研究。采用紫外光谱、循环伏安法(CV)、QCM和扫描电镜等手段,考察了Lac对多巴胺(DA)的催化氧化和聚合。将DA、Lac和MWCNTs混合溶液滴加在GCE上制备了聚多巴胺(PDA)-Lac-MWCNTs/GCE,该电极检测氢醌的灵敏度达643μA mM-1cm-2,检测限为20nM(S/N=3)。与以苯胺、邻苯二胺和邻氨基酚为聚合底物相比,以DA为聚合底物所制的电极性能更好。将DA、GOx、Lac和MWCNTs混合溶液滴加在Pt电极上制备了PDA-GOx-Lac-MWCNTs/Pt电极,该电极检测葡萄糖的灵敏度达68.6μA mM-1 cm-2。此外,主要通过MWCNTs的吸附效应制备了PDA-Lac-MWCNTs-ABTS/GCE,该电极能有效催化氧气的还原,用作无隔膜葡萄糖／氧气BFC的阴极得满意结果。这种基于酶催化聚合的“绿色”生物固定平台有望用于制备多种多功能纳米聚合物膜,在生物技术和应用领域发挥作用。5.在含有GOx的水溶液中,以Lac催化氧化和聚合去甲肾上腺素(NA)制得复合物,再通过金电极上的电聚合制备了酶膜和葡萄糖生物传感器。采用紫外可见分光光度法和电化学方法研究了Lac对NA的催化氧化行为。0.7V vs SCET下,检测葡萄糖的灵敏度为38μAmM-1 cm-2,检测限为0.4μM。该葡萄糖传感器的性能明显优于传统电聚合法(无预氧化步骤)所制葡萄糖传感器。采用EQCM和紫外光谱法测定了固定化GOx的质量比活性,发现预氧化-电聚合所固定的GOx保持着很高的活性。6.采用双通道EQCM研究了水溶液中普鲁士蓝(PB)薄膜修饰的两金电极上的两电极循环伏安电化学行为,归属了普鲁士白、PB、普鲁士黄三者间的转变过程,以及金基底和PB膜内所夹带的Fe(CN)63-/Fe(CN)64-杂质的氧化还原峰,为UV-Vis光谱电化学实验所支持。考察了两电极体系中PB对过氧化氢的催化还原。此外,还研究了PB粉末的两电极固态电化学。夹在两喷金的铟锡氧化物(ITO)电极间的PB粉末的两电极固态循环伏安图和两PB修饰金电极在水溶液中的两电极循环伏安图相似,说明发生了类似的电极反应。双通道EQCM有望成为研究其他物质或材料的两电极系统电化学行为的高效技术。7.为研究8-羟基喹啉型类锰(Ⅲ)配合物催化剂(Q3MnⅢ)催化烯烃环氧化的机理,采用液相CV和QCM技术研究了Q3MnⅢ、Q3FeⅢ、5-NO2-8-QMnⅢCl和salen-MnⅢCl催化剂。CV和QCM研究表明,六齿配位的Q3MnⅢ催化剂中轴向Mn-O键可被打开而形成羟基,转变为五齿配位结构,Q3MnⅢ催化烯烃环氧化的高催化效率应与Q3MnⅢ催化剂在反应介质中的配位模式转变有关。CV和QCM技术可为液相化学催化过程和机理研究提供大量有用信息,有望在催化科学中获得进一步应用。更多还原

【Abstract】 Bio/chemocatalyst modified electrodes have been widely used in bio/chemosensing, energy science, and electrosynthesis. Reactions using biocatalysts can proceed with higher efficiency and better selectivity under milder conditions than those using chemical catalysts. Bioelectocatalysis is virtually the discipline base of biosensors and biofuel cells (BFCs), and it is thus important to efficiently immobilize enzymes and other biocatalysts on electrodes for bioelectrocatalysis. In addition, the electrochemical techniques feature the three-in-one integration of synthesis, separation and analysis functions, it is thus of great significance to develop new characterization methods with the electrode as a research platform for bio/chemocatalysis systems. In this dissertation, recent advances in quartz crystal microbalance (QCM), enzymatic biosensors, enzymatic BFCs, and enzyme-catalyzed polymerization are briefly reviewed, and detailed studies on several bio/chemocatalysis systems are carried out using many instrumental analysis techniques including electrochemical quartz crystal microbalance (EQCM). The main contents are as follows.1. An electrochemical noise (ECN) device is utilized for the first time to study and characterize a glucose/air membraneless BFC and a monopolar glucose BFC. In the glucose/air membraneless BFC, ferrocene (Fc) and glucose oxidase (GOx) were immobilized on a multiwalled carbon nanotubes (MWCNTs)/Au electrode with a gelatin film at the anode; and laccase (Lac) and an electron mediator,2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), were immobilized on a MWCNTs/Au electrode with polypyrrole at the cathode. This BFC was performed in a stirred acetate buffer solution (pH 5.0) containing 40 mM glucose in air, with a maximum power density of 8μW cm-2, an open-circuit cell voltage of 0.29 V, and a short-circuit current density of 85μA cm-2, respectively. The cell current at the load of 100 kQ retained 78.9% of the initial value after continuous discharging for 15 h in a stirred acetate buffer solution (pH 5.0) containing 40 mM glucose in air. The performance decrease of the BFC resulted mainly from the leakage of the ABTS mediator immobilized at the cathode, as revealed by the two-channel quartz crystal microbalance technique. In addition, a monopolar glucose BFC was performed with the same anode as that in the glucose/air membraneless BFC in a stirred phosphate buffer solution (pH 7.0) containing 40 mM glucose, and a carbon cathode in Nafion-membrane-isolated acidic KMnO4, with a maximum power density of 115μW cm-2 an open-circuit cell voltage of 1.24 V, and a short-circuit current density of 202μA cm-2, respectively, which are superior to those of the glucose/air membraneless BFC. A modification of the anode with MWCNTs for the monopolar glucose BFC increased the maximum power density by a factor of 1.8. The ECN device is highly recommended as a convenient, real-time and sensitive technique for BFC studies.2. A simple chemical modification on the popular biocompatible biopolymer chitosan (CS) makes it feasible as an acid-resistant film matrix for biosensing and BFC applications in acidic media. To covalently immobilize Lac from Trametes versicolor that shows its maximum enzymatic activity in acidic solutions, CS was chemically modified with glutaraldehyde (GA) to form GA functionalized CS (GAfCS), which was then allowed to react with Lac to form a Lac-GAfCS composite that is robust in acidic solutions (two-step protocol), as confirmed by QCM and durability tests. The Lac-GAfCS-multiwalled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE), Lac-GAfCS-MWCNTs/GCE, exhibited good catalytic activity towards O2 reduction in the presence of 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), and the pH-dependent enzymatic activity of the immobilized Lac towards O2 reduction was examined. A glucose/air biofuel cell was fabricated, with the Lac-GAfCS-MWCNTs/GCE and a glucose oxidase (GOx)-GAfCS-MWCNTs/GCE as the biocathode and the bioanode in Nafion-membrane-separated acetate buffer solutions (pH 5.0), respectively. The biofuel cell output a maximum power density of 9.6μW cm-2, an open-circuit cell voltage of 0.19 V, and a short-circuit current density of 114μA cm-2, respectively, as measured with an ECN device. Furthermore, the Lac-GAfCS-MWCNTs/GCE was applied to determine catechol in Britton-Robinson buffer solution (pH 3.0), with a linear range of 0.1-50μM and a limit of detection of 20 nM. In comparison with the direct use of GA for one-pot Lac-GA-CS or Lac-GA crosslinking to immobilize enzyme, the use of macromolecular GAfCS in the proposed two-step protocol was proven to be less harmful to enzymatic activity and thus more suitable for immobilizing Lac to construct the biofuel cell and biosensor.3. Pre-crosslinking enzyme molecules to CS with low-concentration crosslinker and then one-pot electrodeposition of the resultant complex can increase the enzyme load and sensitivities of thus-prepared biosensors (vs. conventional CS electrodeposition). GOx is chosen to examine the proposed protocol in detail. GOx was crosslinked to CS with low-concentration GA (0.08 wt%), and the electroreduction of added H2O2 increased the electrode-surface pH and triggered the electrodeposition of a GOx-GA-CS composite film. The GOx-GA-CS electrodeposition was monitored by an electrochemical quartz crystal microbalance and is theoretically discussed based on an electrogenerated base-to-acid titration model. The prepared first-generation enzyme electrode (CS-GA-GOx/Ptnano/Au) exhibits a current sensitivity as high as 102μA mM-1 cm-2 at 0.70 V vs SCE, being 13 times that of the CS-GOx/Ptnano/Au prepared similarly but without precrosslinking GOx to CS. UV-Vis spectrophotometric determination of the GOx remains in the supernatant liquids after pH-induced CS precipitation suggested a high enzyme load in the GOx-GA-CS film, and amperometric measurements suggested a negligible decrease in the enzymatic activity of GOx after its reaction with the low-concentration GA. Also, the proposed protocol works well for the precrosslinking manner of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysulfosuccinimide activation, the water-electroreduction-triggered CS electrodeposition, the second-generation biosensing mode, a 5.0-μm-radius Pt microelectrode, and immobilization of alkaline phosphatase for phenyl phosphate biosensing. Because electrodeposition has been so widely used to immobilize biomacromolecules, and it is always an important topic to increase the load and activity of the immobilized biomacromolecule, the proposed protocol of pretethering the target biomacromolecule to the electrodeposition precusor for immobilization of the biomacromolecule at high load/activity is recommended for wide applications.4. The facile preparation of polymer-enzyme-MWCNTs cast films accompanying in-situ Lac-catalyzed polymerization is described for electrochemical biosensing and biofuel cell applications. Lac-catalyzed polymerization of dopamine (DA) as a new substrate was examined in detail by UV-Vis spectroscopy, cyclic voltammetry (CV), QCM, and scanning electron microscopy. Casting the aqueous mixture of DA, Lac and MWCNTs on a GCE yielded a robust polydopamine (PDA)-Lac-MWCNTs/GCE that can sense hydroquinone (HQ) with 643-μA mM-1 cm-2 sensitivity and 20-nM detection limit (S/N=3). The DA substrate yielded the best biosensing performance, as compared with aniline, o-phenylenediamine or o-aminophenol as the substrate for similar Lac-catalyzed polymerization. Casting the aqueous mixture of DA, GOx, Lac and MWCNTs on a Pt electrode yielded a robust PDA-GOx-Lac-MWCNTs/Pt electrode that exhibits glucose-detection sensitivity of 68.6μA mM-1 cm-2. In addition, ABTS was also co-immobilized to yield a PDA-Lac-MWCNTs-ABTS/GCE that can effectively catalyze the reduction of O2, and it was successfully used as the biocathode of a membraneless glucose/02 BFC in pH 5.0 Britton-Robinson buffer. The proposed biomacromolecule-immobilization platform based on enzyme-catalyzed polymerization may be useful for preparing many other multifunctional polymeric bionanocomposites for wide applications.5. The electropolymerization of L-noradrenaline (NA) after Lac-catalyzed preoxidation to efficiently immobilize GOx at an Au electrode is described for sensitive amperometric biosensing of glucose. The Lac-catalyzed preoxidation of NA was studied by UV-vis spectrophotometry and electrochemical technique. The prepared glucose biosensor displayed a sensitivity of 38μA mM-1 cm-2 and a limit of detection of 0.4μM at 0.7 V vs. SCE, which are obviously better than those prepared via preoxidation-free conventional electropolymerization. The immobilized GOx retained high enzymatic specific activity, as quantified by EQCM and UV-vis spectrophotometry. The proposed Lac-catalyzed preoxidation strategy may have application potential in many fields, such as biosensing, biocatalysis, and BFCs.6. A two-channel EQCM is used to investigate the cyclic voltammetric behavior of two Prussian blue (PB) film-modified Au electrodes in a two-electrode configuration in aqueous solution. The redox peaks observed in the two-electrode cyclic voltammogram are assigned to the intrinsic redox transitions among the Everitt’s salt, PB, and Prussian yellow for the film itself, the redox process of the Au substrate and the redox process of small-quantity ferri-/ferrocyanide impurities entrapped in the PB film, as also supported by UV-Vis spectroelectrochemical data. The electrocatalytic reduction of H2O2 was also examined in two-electrode system. The profile of the two-electrode solid-state cyclic voltammogram for the PB powder sandwiched between two gold-coated indium-tin oxide electrodes is similar to that for two PB-modified Au electrodes in aqueous solution, implying similar origins for the corresponding redox peaks. The two-channel EQCM method is expected to become a highly effective technique for the studies of the two-electrode electrochemical behaviors of many other species/materials.7. To investigate the catalytic mechanism of 8-quinolinolato manganese(III) complexes (Q3MnⅢ) epoxidation system, CV and QCM were employed to study catalytic Q3MnⅢ, Q3FeⅢ,5-nitryl-8-quinolinolato MnⅢCl and salen-MnⅢCl complexes. The results showed that the high catalytic efficiency for the epoxidation of olefins with Q3MnⅢcatalysts should be due to their special hexadentate binding structures that could be easily converted to the corresponding pentadentate with pendant hydroxyl groups via opening an axial Mn-O bond in the reaction media. The CV and QCM tests have given us much useful information on the liquid-phase catalysis mechanism, which are highly expected to find wider applications in catalysis science and technology.更多还原