【作者】 秦霞；

【导师】 陈强；

【作者基本信息】 南开大学 ， 动物学， 2010， 博士

【摘要】 生物传感器作为一种新型的分析手段,具有选择性好、灵敏度高、分析速度快、成本低、能在复杂体系中进行在线连续监测等优点,已被广泛应用于临床医学、环境监测、食品等领域。纳米材料具有优良的物理、化学、电催化性能以及良好的生物相容性,将纳米材料应用于生物传感器的制备可以明显提高传感器的响应性能。本文制备了多种不同形貌的纳米材料,并将这些纳米材料用作电极修饰材料或酶的固定化材料以构建生物传感界面,所制备的传感器对相应的待测物质均表现出较高的灵敏度、较低的检测下限和较快的响应速度。具体结果如下：碳纳米管(CNTs)具有大的比表面积、优良的导电性能和化学稳定性,能很好地促进电活性分子的电子传递,提高响应速率,是一种理想的电极修饰材料。首先用混酸对MWCNTs进行氧化处理,一方面可以截断MWCNTs,降低长径比,使其更容易地固定在电极表面,另一方面可以在其末端和侧壁引入大量羧基等功能团,使其能起到固定其他聚电解质或生物分子的作用。以羧基化MWCNTs为增效材料,以层层自组装技术将胆碱氧化酶(ChOx)和聚二烯丙基二甲基氯化铵(PDDA)交替组装制备(PDDA/ChOx)n复合酶膜,并以(PAA/PVS)3为选择透过性膜构建传感界面。分别研究了MWCNTs和(PAA/PVS)3修饰Pt电极的电化学特征以及复合酶膜的电化学行为。实验结果表明：在所制备的1～10层自组装复合薄膜中,(PDDA/ChOx)8修饰电极性能最优。在工作电位为+0.6 V vs. Ag/AgCl时,实现了对胆碱的无干扰检测,检测的线性范围为0.75μM～0.15mM,灵敏度为10.89μA/mM,检测下限为0.3μM(S/N=3),并表现出良好的稳定性,且MWCNTs的加入提高了传感器的响应灵敏度,降低了检测下限,缩短了响应时间。以共价修饰法将纳米金(GNp)负载于羧基化MWCNTs上,制备MWCNTs-GNp纳米复合材料。利用TEM、EDX、UV-Vis等手段对其微观形貌和元素组成进行了表征,并以之为酶的固定化载体吸附ChOx,将MWCNTs-GNp与ChOx的共混物分散于PDDA中,构建MWCNTs-GNp-ChOx-PDDA复合酶膜修饰Pt电极的传感界面。研究了MWCNTs-GNp修饰电极的电化学特征以及复合酶膜的电化学行为。所制备的胆碱传感器在+0.35 V vs. Ag/AgCl时,对胆碱检测的线性范围为0.001～0.5mM,灵敏度为12.97μA/mM,检测下限为0.3μM(S/N=3)。其响应灵敏度分别是单独使用GNp和MWCNTs的3倍和2倍,GNp和MWCNTs的结合表现出一定的增强效应。在不使用任何模板和催化剂的条件下,以硝酸银为银源,通过控制硝酸银溶液的浓度,利用电化学沉积法在GC电极表面合成了类球形和树枝状的纳米银。利用SEM、TEM、XRD等手段对其微观形貌和物质组成进行了表征,并以树枝状纳米银修饰GC电极为传感界面,研究了纳米银自身的电化学特征及其对过氧化氢的催化行为。结果表明,树枝状纳米银对过氧化氢具有很好的电催化还原活性,在-0.2 V vs. Ag/AgCl电位下,实现了对过氧化氢的灵敏响应,线性范围为0.005～12mM,灵敏度为7.39μA/mM,检测下限为0.5μM(S/N=3),而且有效降低了溶解氧和其他电活性物质的干扰。根据”晶种催化作用机理”,利用L-半胱氨酸诱导合成了直径约200nm的银纳米线。利用SEM、TEM、XRD、UV-Vis等手段对其微观形貌和物质组成进行了表征。将此银纳米线修饰于Pt电极构建传感界面,研究了银纳米线的电化学特征以及银纳米线对过氧化氢的催化行为。所制备的银纳米线可在-0.2 V vs.Ag/AgCl电位下催化过氧化氢的还原,而且还原反应受过氧化氢的扩散控制,反应机理符合电子转移反应和化学反应均不可逆的非均相催化机理。实验还发现,将电极在PBS中循环伏安扫描可以提高传感器的响应灵敏度,这可能是由于扫描后增大了银纳米线的表面积,同时活性位点增多造成的。循环扫描50次后,传感器对过氧化氢检测的线性范围为0.5μM～30 mM,灵敏度为9.45μA/mM,检测下限0.2μM(S/N=3)。在优化银纳米线制备方法的基础上,制备出产量更高的银纳米线,利用SEM、TEM对其微观形貌进行了表征,并将银纳米线修饰在Pt电极表面构建传感界面,应用到对溶液中氯、溴、碘离子的检测。根据卤化银氧化还原电位的不同,银纳米线修饰电极可以对共存的三种卤素离子同时检测。利用循环伏安法研究了修饰电极对三种离子的电化学行为。电极对氯、溴、碘离子检测的线性范围分别为200μM～20 mM、50μM～20 mM和50μM～20 mM,明显要比单独检测时的线性范围窄,表明三种卤素离子同时存在时,互相之间存在竞争干扰。灵敏度分别为0.059μA/mM、0.042μA/mM和0.032μA/mM,优于已报道的同类电极的检测灵敏度。铂的纳米化是提高铂催化剂催化效率的一种重要方法,本论文通过简单的液相化学还原法,以硼氢化钠为还原剂,以聚乙烯醇为保护剂,在水溶液中还原氯铂酸钾,制备了稳定性良好的铂纳米颗粒。利用TEM、XRD等手段对其微观形貌和物质组成进行了表征。将铂纳米颗粒修饰于GC电极构建传感界面,利用循环伏安法研究了其对过氧化氢和甲醇的电催化性能,结果表明铂纳米颗粒修饰电极在甲醇溶液中表现出较好的电催化活性,且在0V电位下能够催化过氧化氢的还原。更多还原

【Abstract】 As a new kind of analytical tool, biosensors which have the characteristics of good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system, have been widely applied in the clinical diagnosis, environmental monitoring, and food control etc. Because of the good physical, chemical, electrocatalytic and biocompatibility properties of nano-materials, applying nano-materials for the fabrication of biosensors will greatly improve the performance of the resulting biosensors. In this thesis, many nano-materials with different morphologies were synthesized using different methods and were applied on the surface of electrodes as modified materials or enzyme immobilizing matrix for the preparation of biosensing interface. The resulting biosensors exhibited high sensitivity, low detection limit and fast response to the corresponding substance, validating the enhancing effect of nano-materials on biosensors’performance. The main results are as follows:Carbon nanotubes (CNTs) represent a new kind of promising carbon material for the modification of electrode owing to the unique electrical properties such as high surface-to-volume ratio, excellent electric ability and high chemical stability. Multi-walled carbon nanotubes (MWCNTs) were firstly treated with a mixture of concentrated sulfuric and nitric acid, which could shorten the chain lengths of CNTs, thereby enabling them to be immobilized easily and stably on an electrode. In addition, carboxylic acid groups could be introduced in the ends and side walls of CNTs during the acid treatment, which can interact with positively charged polyelectrolytes or biomolecules.Amperometric choline biosensors based on MWCNTs and layer-by-layer (LBL) assembled multilayer films composed of poly(diallyldimethylammonium chloride)(PDDA) and choline oxidase (ChOx) were constructed, also a polymer film of (PAA/PVS)3 to be used as a permselective layer. MWCNTs was characterized by transmission electron microscopy (TEM), and properties of the resulting choline biosensors were measured by electrochemical measurements. Among the resulting biosensors, MWCNTs/(PAA/PVS)3/(PDDA/ChOx)8 based choline biosensor was the best. At the applied potential of+0.6 V vs. Ag/AgCl, the biosensor showed a linear range of 0.75μM～0.15 mM, with a sensitivity of 10.89μA/mM, and a detection limit of 0.3μM (S/N=3). Moreover, it exhibited good suppression of interference as well as long-term stability, and MWCNTs played the roles of electron transfer promoting and response speeding, thereby improved the properties of the sensor.MWCNTs-gold nanoparticles (GNp) nanohybride was synthesized and used as an enzyme matrix to immobilize ChOx. PDDA was employed to disperse the mixture of MWCNTs-GNp-ChOx and also used a binder material to electrostatically adsorbe the enzyme. A novel choline biosensor based on the nanocomposite film composed of ChOx, MWCNTs, GNp and PDDA was developed. The microscopic structure and composition of the MWCNTs-GNp hybride were characterized by TEM, energy dispersive X-ray spectroscopy (EDX) and UV-visible asorbance spectroscopy (UV-Vis), and properties of the resulting choline biosensors were monitored by electrochemical measurements. At the applied potential of+0.35 V vs. Ag/AgCl, it showed a linear range of 0.001～0.5 mM, with the sensitivity of 12.97μA/mM, and a detection limit of 0.3μM (S/N=3). Moreover, the biosensor with the nanohybride of MWCNTs-GNp exhibited significant improvement of sensitivity, which was 3 times and 2 times of biosensors with GNp only and MWCNTs only, respectively.Shaped silver nanoparticles (Ag NPs) with like-spheres and well-defined dendrites were fabricated on glassy carbon (GC) electrodes by the direct electrodeposition process from an aqueous solution of AgNO3 in the presence of no template or catalyst, and the shape of Ag NPs was AgNO3 concentration-dependent. Scanning electron microscopy (SEM), powder X-ray diffraction (XRD) and TEM were employed to characterize Ag NPs. The electrochemical behaviors of the silver dendrites and its reduction catalytic activity towards hydrogen peroxide were investigated by electrochemical measurements. At the applied potential of-0.2 V vs. Ag/AgCl, it showed a linear range of 0.005～12 mM, with a sensitivity of 7.39μA/mM, and a detection limit of 0.5μM (S/N=3), meanwhile, eliminating the interference from solved oxygen and other electric active species.Silver nanowires (Ag NWs) with diameter of 200 nm were synthesized by an L-cysteine-assisted poly (vinyl pyrrolidone) (PVP)-mediated polyol route. A novel strategy for fabricating a hydrogen peroxide sensor was developed by direct drop-casting Ag NWs on Pt electrode. UV-Vis, XRD, SEM and TEM were employed to investigate the prepared Ag NWs. The electrochemical properties of Ag NWs and the sensor were characterized by electrochemical measurements. It was found that the sensitivity of the modified electrode would be greatly enhanced after cyclic scan in phosphate buffer solution (PBS), which might be due to the enlarged surface areas and the increased active sites of Ag NWs during the scan. After scanned for 50 cycles, at the applied potential of-0.2 V vs. Ag/AgCl, the resulting sensor showed a linear range of 0.5μM～30 mM, with the sensitivity of 9.45μA/mM, the detection limit of 0.2μM (S/N=3). Furthermore, the sensor exhibited good anti-interference, reproducibility and long-term stability.Based on optimization of Ag NWs synthesis method, by casting the prepared Ag NWs on Pt electrode, a new kind of sensing interface was fabricated for determination of chloride, bromide and iodide in solution. Because of the well-separated redox potential, chloride, bromide and iodide could be simultaneously monitored in a mixture. The electrochemical properties of Ag NWs towards the three kinds of ions were investigated by cyclic voltammetry. By measuring the oxidation peak currents of the respective silver halides, the calibration graph was linear from 200μM～20 mM for chloride,50μM～20 mM for bromide and 50μM～20 mM for iodide, which was narrower than the linear range in single determination. And the sensitivity was 0.059μA/mM,0.042μA/mM and 0.032μA/mM for chloride, bromide and iodide, respectively.It is a significant way to make platinum as nanomaterials to improve its catalytic efficiency. In this thesis, NaHB4 as the reductant, polyvinyl alcohol (PVA) as the stabilizer, the PVA-protected spherical Pt nanoparticles (Pt NPs) were prepared by a reduction method in aqueous solution. The as-prepared Pt NPs were characterized by XRD and TEM, and a new kind of sensing interface was developed by drop-casting the Pt NPs on GC electrode. Cyclic voltammetry was employed to investigate the electrocatalytic activity of Pt NPs towards methanol and hydrogen peroxide. The results demonstrated that the modified electrode exhibited sensitive response for the electro-oxidation of methanol and good catalytic activity to the reduction of hydrogen peroxide, which could be monitored at 0 V vs. Ag/AgCl with good sensitivity.更多还原