纳米功能复合材料的制备及其在生物传感中的应用研究

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英文题名：The Fabrication of Functional Nanocomposite Materials and Their Application in Biosensors 作者：葛磊 论文级别：博士 学科专业名称：材料学 中文关键词：纳米材料 ; 复合材料 ; 光致电化学纸基材 ; 电致化学发光纸基材 ; 电化学纸基材 英文关键词：nanomaterials ; composite material ; photoelectrochemical paper device ; electrochemiluminescence paper device ; electrochemical paper device 学位年度：2014 导师：陶绪堂 ; 程新 学科代码：080502 学位授予单位：山东大学 论文提交日期：2014-05-26

摘要

近几年,随着纳米技术研究的不断深入,纳米复合材料在人类的生活和生产中正显示出不可替代的重要作用。纳米复合材料不仅具有纳米材料本身的大比表面积、高导电性、强机械性能等特点,还具有较高的催化活性、较强的吸附能力、良好的生物相容性等优点,在广泛查阅大量相关文献的基础上,围绕纳米材料在电致化学发光、光致电化学及电化学传感器中的应用,针对生物传感器构建的关键环节即生物传感界面的构建和信号标记放大策略,本研究论文在微流控功能纸基材上引入了一系列不同形貌和结构的纳米复合材料,包括量子点、纳米金、碳纳米管等功能复合材料,实现了微流控功能复合纸基材的高灵敏分析方法的建立。制备了鲁米诺-Au纳米、三联吡啶钌-石墨烯等功能分子组装材料,并将这些纳米复合材料与生物功能大分子进行组装,例如抗体与适配体等,达到提高传感器选择性、延长传感器使用寿命等目的,以满足临床诊断、环境监测等应用的需要。
     1.多孔CdS量子点-碳纳米管复合功能纸基材的制备及光致电化学传感应用研究
     将光致电化学分析方法与微流控功能复合纸基材结合,通过蜡打印技术,构建实现低成本、简单、便携、易处理的光致电化学功能纸基材。对光致电化学功能纸基材进行复合处理,在纤维素纤维表面原位修饰制备CdS量子点-碳纳米管复合材料。该光致电化学功能复合纸基材以鲁米诺-金纳米粒子复合材料为内部光源,以数字万用表为外部终端光电流检测设备。考察了该光致电化学功能复合纸基材的光电流响应。与传统平面电极相比,在内光源与外光源模式下均观察到光致电化学功能复合纸基材对光电流的增强效应。为进一步放大光电流信号,设计制备了固态超级纸电容器,并将其集成到光致电化学功能复合纸基材上,以收集并存储产生的光电流。存储在固态超级纸电容器内的电能可以通过数字万用表短路法瞬间释放出来,并得到放大的光电流(大约放大了.13倍),并可被数字万用表检测。该光致电化学功能复合纸基材摒弃了昂贵复杂的电化学工作站检测机制,并获得比直接光电流检测更高的灵敏度。最后,在该光致电化学功能复合纸基材内构建了夹心式适配体传感界面用于人血清中三磷酸腺苷的检测,线性范围为1.0pmol/L至1.0nmol/L,检测限为0.2pmol/L。最后考察了该光致电化学功能复合纸基材的重现性、特异性与稳定性。
     2.多孔半导体聚合物-纳米金复合功能纸基材的制备及其电化学传感应用
     通过自催化还原生长方法,在纤维素纸纤维表面原位生长制备纳米金导电层,形成微流控功能纸基材上的多孔金纸电极。由于纸的多孔性与大表面积以及金纳米粒子的高导电性,多孔金纸电极极大地提高了纸电极的有效表面积和检测灵敏度。随后,在多孔金纸电极内部的导电纸纤维表面电聚合制备分子印迹聚合物,在微流控功能纸基材上引入分子印迹技术。基于以上制备的分子印迹修饰多孔金纸电极和折纸原理,进一步构建了分子印迹电化学功能复合纸基材用于检测D-谷氨酸。该功能复合纸基材由一个辅助功能卡和四个样品功能片构成。检测线性范围为1.2nmol/L至125.0nmol/L。考察了分子印迹电化学功能复合纸基材的选择性、重现性和稳定性。该分子印迹电化学功能复合纸基材为发展中国家的疾病诊断、公众健康、环境检测等提供了一种灵敏的、特异的、高通量的手段。
     3.电致化学发光复合纸基材的制备及其无线电泳传感应用研究
     将CdS量子点-碳纳米管复合材料通过静电层层修饰技术,组装到纸纤维素纤维表面。利用CdS的电致化学发光特性,首次将微流控功能纸基材与电泳分离技术结合,制备了一种低成本、简单、便携、易处理的微流控电致化学发光电泳纸基材。设计制备柱上铜-金复合双极电极,实现微流控电致化学发光电泳纸基材上的无线电致化学发光检测。该微流控电致化学发光电泳纸基材可在六分钟内将丝氨酸、天冬氨酸与赖氨酸完全分离,分离电压仅为330V。本文还设计制备了一种新型的自制整流器,采用家用电源即可实现上述分离电压。优化实验条件后可得到较高的检测灵敏度。三种氨基酸的检测限分别为：13pmol/L(丝氨酸),34pmol/L(天冬氨酸),0.17nmol/L(赖氨酸)。三种氨基酸电泳谱图的峰高与迁移时间的精密度分别为<5.0%、≤1.5%。该电致化学发光电泳纸基材提供了一种快速、集成、自动化的多组分分离与检测方法。
     4.菱形二氧化钛纳米晶复合材料的制备以及在光致电化学传感器中的应用
     在无水乙醇中,通过溶剂热的方法制备高结晶性的菱形二氧化钛纳米晶。在菱形二氧化钛纳米晶修饰的氧化铟锡导电玻璃上覆盖壳聚糖层后,将抗癌胚抗原抗体通过戊二醛交联共价修饰到电极表面。采用三联吡啶钌配合物作为光致电化学光电流信号分子,抗坏血酸作为自牺牲电子供体,在菱形二氧化钛纳米晶复合材料修饰的氧化铟锡导电玻璃上构建了一种新型的光致电化学免疫传感器。为了进一步增强该传感器在紫外及可见光区内的光电流强度,合成制备了三联吡啶钌配合物-还原石墨烯纳米复合材料(Ru-RGO),其中三联吡啶钌配合物作为电子供体,还原石墨烯作为电子受体,加速光生电子-空穴的分离并抑制其复合。借助免疫反应带来的光电流信号变化,实现了光致电化学免疫传感器测定癌胚抗原。其光电流强度与癌胚抗原浓度的对数成线性关系,线性范围为0.1pg/mL to100ng/mL,检测限为0.059pg/mL。另外该光致电化学免疫传感器还表现出了较高的灵敏度、稳定性、重现性,并为免疫分析开辟了一条新的出路。
     5.量子点-二氧化钛复合薄膜的溶胶凝胶制备与性能研究
     本工作研究了一种新型的CdTe量子点-二氧化钛复合溶胶凝胶膜的制备方法。制备了以巯基乙酸为保护剂的水溶性CdTe量子点,荧光发射颜色分别为绿色、黄色和红色。在最佳制备条件下,水溶性CdTe量子点表现出较高的荧光效率。通过控制钛酸四丁酯在乙醇与聚乙烯基吡咯烷酮溶液中的水解,’制备Ti02溶胶。在Ti02溶胶中加入二乙醇胺以防止CdTe量子点的表面缺陷猝灭。将CdTe量子点嵌入Ti02溶胶膜后,由于CdTe量子点与Ti02之间的相互作用,导致CdTe量子点荧光强度降低。与溶液中的CdTe量子点相比,Ti02溶胶膜内的CdTe量子点荧光发射峰发生轻微蓝移,且蓝移量取决于量子点的性质。红光量子点发生峰蓝移量为1nm,而绿光量子点发生峰蓝移量为7nm,表明红光量子点具有较高的稳定性,且其与Ti02溶胶的相互作用较少。该CdTe量子点-Ti02复合溶胶凝胶膜不仅制备简单,而且具备较高的亮度、多色的光发射以及较高的稳定性等优点,因此CdTe量子点-TiO2复合溶胶凝胶膜将在不同的领域具有较高的应用潜力。
Recently, with the deep development of nanotechnology, nanocomposite has played an important role in our daily life. Nanocomposite not only possesses the advantages of nanomaterials, such as large surface area, good conductivity, and super mechanical property, but also possesses high catalytic activity, robust adsorption capacity, and good biocompatibility. Based on the searching of abundant papers, using the electrochemiluminescent, photoelectrochemical, and electrochemical properties of nanomaterials, nanocomposites with different morphology and structure was constructed in microfluidic paper-based devices, including quantum dot, gold nanoparticles, and carbon nanotubes, to realize high sensitivity on microfluidic paper-based devices. Luminol-gold nanoparticles and Ru(bpy)32+-graphene nanocomposites were prepared to label functional biomacromolecule, such as antibody and aptamer, to enhance the specificity of the biosensors. The use of such high-sensitivity sensors can offer the application of clinical diagonosis and environment monitoring.
     1. Fabrication of CdS Quantum Dot-Carbon Nanotube Functionalized Paper Composite Materials and their Application Research in Photoelectrochemical Biosensors
     In this work, a photoelectrochemical (PEC) method was introduced into a microfluidic paper-based analytical device (μ-PAD), and thus, a truly low-cost, simple, portable, and disposable microfluidic PEC origami device (μ-PECOD) with an internal chemiluminescence light source and external digital multimeter (DMM) was demonstrated. The PEC responses of this μ-PECOD were investigated, and the enhancements of photocurrents in μ-PECOD were observed under both external and internal light sources compared with that on a traditional flat electrode counterpart. As a further amplification of the generated photocurrents, an all-solid-state paper supercapacitor was constructed and integrated into the μ-PECOD to collect and store the generated photocurrents. The stored electrical energy could be released instantaneously through the DMM to obtain an amplified (～13-fold) and DMM detectable current as well as a higher sensitivity than the direct photocurrent measurement, allowing the expensive and sophisticated electrochemical workstation or lock-in amplifier to be abandoned. As a model, sandwich adenosine triphosphate (ATP)-binding aptamers were taken as molecular reorganization elements on this μ-PECOD for the sensitive determination of ATP in human serum samples in the linear range from1.0pM to1.0nM with a detection limit of0.2pM. The specificity, reproducibility, and stability of this μ-PECOD were also investigated.
     2. Fabrication of Porous Semiconductor Polymer-Gold Nanoparticles Functionalized Paper Composite Materials and their Application Research in Electrochemical Biosensors
     Molecular imprinting technique is introduced into microfluidic paper-based analytical devices (μ-PADs) through electropolymerization of molecular imprinted polymer (MIP) in a novel Au nanoparticle (AuNP) modified paper working electrode (Au-PWE). This is fabricated through the growth of a AuNP layer on the surfaces of cellulose fibers in the PWE. Due to the porous morphology of paper as well as the high specific surface area and conductivity of the resulting AuNP layer on the cellulose fibers, the effective surface area and the sensitivity of the Au-PWE is enhanced remarkably. Based on this novel MIP-Au-PWE and the principle of origami, a microfluidic MIP-based electro-analytical origami device (μ-MEOD), comprised of one auxiliary pad surrounded by four sample tabs, is developed for the detection of D-glutamic acid in a linear range from1.2nM to125.0nM with a low detection limit of0.2nM. The selectivity, reproducibility, and stability of this μ-MEOD are investigated. This μ-MEOD would provide a new platform for high-throughput, sensitive, specific, and multiplex assay as well as point-of-care diagnosis in public health, environmental monitoring, and the developing world.
     3. Fabrication of Electrochemiluminescent Paper Nanocompostie and its Application Research in Wireless Electrophoresis Biosensor
     In this work, microfluidic paper-based analytical device (μ-PAD) was further exploited by coupling electrophoretic separation technique for the first time, and a low-cost, simple, portable, and disposable microfluidic paper-based electrophoretic device (μ-PED) with on-column wireless electrogenerated chemiluminescence (ECL) detector (denoted as μ-PED@ECL) was demonstrated. As a proof-of-concept, the performance of this μ-PED@ECL was illustrated by the complete separation and detection of electro-inactive serine, aspartic acid, and lysine within6min, which was realized by household alternating power supply (220V in China) through a novel home-made rectifier, allowing the expensive and sophisticated electrophoretic power supply to be abandoned. Optimizations were performed to achieve both good sensitivity and complete separation with detection limits (3a) of13pM for serine,34pM for aspartic acid, and0.17nM for lysine. The peak height and migration time precisions were<5.0%and<1.5%(n=11) for the three amino acids, respectively. This p.-PED@ECL provided a fast, integrated, and automated potential for multiplex separation and detection on μ-PAD.
     4. Fabrication of Rhombic TiO2Nanocrystal Composites and their Application Research in Photoelectrochemical Biosensor
     A novel photoelectrochemical immunosensor using Ru complex as the photoelectrochemical signal-generating molecule, ascorbic acid (AA) as the sacrificial electron donor based on rhombic TiO2nanocrystals (NCs) modified ITO electrode was developed. In order to enhance the photocatalytic activities under UV-vis or visible light irradiation, Ru complex and reduced graphene oxide (RGO) hybrid (Ru-RGO) was prepared, in which Ru complex acts as an electron donor and RGO serves as an electron acceptor in the hybridized species, which facilitates charge separation and suppresses recombination of photoexcited electron-hole pairs in Ru-RGO. The highly crystalline rhombic TiO2NCs were fabricated through a solvothermal technique in anhydrous ethanol. After the ITO/TiO2electrode was coated with chitosan (CS), carcinoma embryonic antigen (CEA) antibodies were covalently conjugated on the surface of the electrode through glutaraldehyde (GLD). Thus, a photoelectrochemical immunosensor for the detection of CEA was developed by monitoring the changes in the photocurrent signals of the electrode resulting from the immunoreaction. The photocurrents were proportional to the logarithmic CEA concentrations, and the linear range of the developed immunosensor was from0.1pg/mL to100ng/mL and with a detection limit of0.059pg/mL. The proposed method showed high sensitivity, stability, reproducibility, and could become a promising technique for immunoassays.
     5. Composite TiO2Film with Quantum Dots Fabricated Through a Sol-Gel Process
     A novel strategy has been developed to fabricate composite TiO2films with CdTe quantum dots (QDs). Aqueous CdTe QDs with green-, yellow-, and red-emitting were prepared using thioglycolic acid as a capping agent. The QDs revealed high photoluminescence (PL) efficiencies under optimal preparation conditions. TiO2sol was obtained by the controlling hydrolysis of tetrabutyl titanate in ethanol with poly (vinyl pyrrolidone). Diethanolamine was added to prevent the QDs from PL quenching generated by surface defects. After embedding the QDs in composite TiO2film, the PL intensity of the QDs decreased because of the excitation and recombination between the QDs and TiO2. The PL peak wavelength of the QDs in films revealed a slight blue shift compared with their initial ones. The blue shift degree of the PL peaks depended on the properties of the QDs. Red-emitting CdTe QDs revealed a small blue shift of1nm while green-emitting ones revealed a blue shift of7nm. This indicated red-emitting QDs with high stability against incorporation. Facile preparation and excellent properties including high PL brightness, multicolor emission, and high stability make these films important applications in various fields.

引文

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