【作者】 王学亮；

【导师】 焦奎；

【作者基本信息】 青岛科技大学 ， 应用化学， 2009， 博士

【摘要】 生物传感技术能够捕捉生物体内的各种生物信息,为临床医学诊断、生物医学基础研究等提供人体生理、病理相关信息,因而它的研究已成为迫切需要发展的课题。发展新型功能化的生物医用传感器件将为全面推进人类健康科学的发展提供历史性的契机。纳米技术的出现为该领域的研究发展开辟一个全新的天地,巧夺天工的纳米仿生界面的构建则把生物医用传感的进一步发展推向新的高峰。纳米仿生界面的研究是纳米科技与生命科学的交叉领域,它可以在纳米尺度空间上从分子层次研究生物大分子及其复合体或细胞的结构与功能,解决纳米技术在生物医学领域应用中的基础问题,发展新技术和新方法。各国科研工作者已经在仿生纳米界面上构建了各种生物传感器,用于肝炎,白血病、艾滋病以及SARS等的检测。但是这些研究都还处于起步阶段,寻求方便、快捷的制备和组装纳米材料的方法,构建功能化的仿生纳米界面,发展新的生物传感技术将会成为材料学家、分析化学家以及医学家等共同努力的方向。在本论文中,通过电化学沉积、电聚合、共价键合、吸附和表面滴涂等方法对导电聚合物、多壁碳纳米管（MCNTs）、金纳米粒子（AuNPs）、合金纳米粒子、纳米氧化物等材料进行组合组装,在玻碳电极（GCE）表面构建了用于研究DNA、酶的固定和药物小分子检测的新型纳米仿生界面。利用扫描电子显微镜（SEM）、透射电子显微镜（TEM）、X射线粉体衍射仪（XRD）及紫外可见分光光度法（UV-vis）、循环伏安法（CV）、电化学交流阻抗谱技术（EIS）、微分脉冲伏安法（DPV）、计时电流法、计时库伦分析法等对仿生界面的性质以及DNA、酶和药物小分子在生物界面上的电化学行为进行了探讨,具体内容如下:（1）制备了Fe@Fe₂O₃核壳纳米项链、Fe@Fe₂O₃核壳纳米线及立方纳米Cu2O三种氧化物纳米材料,并通过SEM、TEM及XRD技术对合成的纳米材料进行了表征。利用合成的纳米材料、聚二烯丙基二甲基氯化铵（PDDA）、AuNPs和MCNTs在GCE电极表面构建了PDDA/Fe@Fe₂O₃-AuNPs/PDDA/GCE、PDDA/Fe@Fe₂O₃-MCNTs/PDDA/GCE和PDDA/nanoCu₂O-AuNPs/PDDA/GCE三个用于DNA损伤的纳米仿生界面。用UV-vis法研究了三种界面的性能。以Ru（NH₃）₆³⁺和Co（phen）₃³⁺为电化学探针,通过CV、DPV法研究了DNA在三种界面内的电化学损伤。试验发现DNA的损伤主要发生在阴极处理过程。在阴极处理过程中,界面内发生了Fenton或类Fenton反应,反应生成的活性氧粒子（ROS）进攻并损伤DNA,此反应历程与生物体内重金属损伤DNA的历程十分相像,可用于模拟重金属损伤DNA的活体路线。三个DNA电化学传感器均具有较高的灵敏度和良好的稳定性,有望成为快速检测现存及新化学物质基因毒性的工具。（2）通过电化学聚合反应、共价键合、吸附和滴涂等方式将聚硫堇（PTn）、聚酪氨酸（PTyr）、AuNPs和纳米二氧化锆-聚苯胺复合材料（nanoZrO₂-PAN）修饰于GCE电极表面,分别构建了AuNPs/PTn/GCE和nanoZrO₂-PAN/PTyr/GCE两种用于DNA固定和杂交的仿生界面。利用CV、DPV和EIS法对生物界面的性能及DNA在界面上的固定和杂交进行了研究。通过EIS技术对转基因植物外源基因草丁膦乙酰转移酶基因（PAT基因）片段进行了免试剂检测。PAT基因片段在ss-DNA/AuNPs/PTn/GCE和ss-DNA/nanoZrO₂-PAN/PTyr/GCE电极上的检测范围依次分别为1.0×10^-10 1.0×10^-6 mol/L和1.0×10^-13 1.0×10^-6 mol/L,检测限依次分别为3.2×10^-11 mol/L和2.68×10^-14 mol/L （S/N = 3）。将ss-DNA/AuNPs/PTn/GCE电极用于转基因大豆中提取的外源基因胭脂碱合成酶基因终止子（NOS）的聚合酶链式反应（PCR）扩增产物的检测,结果满意。用nanoZrO₂-PAN制得的传感器与用其它ZrO₂材料制得的传感器相比,具有更宽的线性范围和更低的检测限。两种传感器均具有长期的稳定性、良好的选择性和再生性。（3）利用恒电位电化学沉积技术,在聚苯胺纳米管（nanoPAN）和壳聚糖（CS）复合膜修饰的GCE电极表面电化学合成了金-铂合金纳米粒子（Au-PtNPs）,构建了用于辣根过氧化物酶（HRP）固定的纳米生物界面。在该生物界面上获得了HRP的直接电子转移并据此构建了一种新型的H₂O₂生物传感器。通过CV和EIS法对界面的性能进行了研究。在最佳试验条件下,采用计时电流法,通过往连续搅拌的PBS缓冲溶液中加入等量的H₂O₂研究了传感器对H₂O₂的安培响应。将该传感器用于H₂O₂的测定,响应速度快（< 2 s）,线性范围宽（1.0 2200μmol/L）且检测限低（0.5μmol/L） （S/N = 3）。将该生物传感器应具有较高的灵敏度、良好的重现性和长期稳定性,可用于实际样品中H₂O₂含量的测定。（4）通过CV、线性扫描伏安法、计时库伦分析法,分别研究了林可霉素在Au-PtNPs/nanoPAN/CS/GCE界面上和阿米卡星在nanoPAN/CS/GCE界面上的电化学行为。建立了快速测定两种抗生素的电化学方法。林可霉素在Au-PtNPs/nanoPAN/CS/GCE上和阿米卡星在nanoPAN/CS/GCE上的检测范围依次分别为3.0 100.0 mg/L和10.0 80.0 mg/L,检测限依次分别为1.0 mg/L和8.0 mg/L （S/N = 3）。试验研究计算了林可霉素在电极界面上的电子转移数（n）、动力学参数（nα）及标准速率常数（ks）以及参与电极反应的H⁺数目等。将两种方法分别用于实际样品中抗生素含量的检测均得到了较为理想的结果。更多还原

【Abstract】 Bio-sensing technology can capture a variety of biological information in vivo and provide basic human physiology, pathology-related information for the clinical diagnosis and the basic biomedical research. Therefore, the research related to this subject has become very urgent. The development of the new functional bio-medical sensors will provide a historic opportunity for comprehensively promoting the science progress of the human health. The emergence of nanotechnology opened a new heaven and earth for the development of the research in this field and the construction of the wonderful nano-bionic interface had promoted the development of the biomedical biosensor to a new height. The research of nano-bionic interface is an interdisciplinary field of nanotechnology and life science, which can be used to study the structure and function of biological macromolecules, macromolecule complexes and cell at the molecular level in the nanometer scale space, to resolve the basic problem about nanotechnology applications in the biomedical field and develop new technologies and new methods. A variety of bio-sensors have been constructed on the nano-bionic interface by many countries’scientists, and used for the detection of hepatitis, leukemia, AIDS, SARS and so on. However, these studies are still in the initial stage. Exploring for convenient and fast methods of preparing and assembling nano-materials, building functional bionic nano-interfaces and developing new bio-sensing technology will become a co-endeavor direction of material scientist, analytical chemists, medical scientists and so on.In this paper, the new nano-bionic interfaces were constructed on the surface of glassy carbon electrode （GCE） by assembly or combination of some materials, such as electric polymer, carbon nanotubes, alloy nano-particles and nano-oxides, using the methods of electrodeposition, electro-polymerization, covalent combination, adsorption or casting the sample solution on the surface. The properties of the bio-interface and the electrochemical behaviors of DNA, enzymes and little medicine molecules were characterized by many methods and technologies, such as scanning electronic microscopy （SEM）, transition electronic microscopy （TEM）, X-ray powder diffraction （XRD） and ultraviolet-visible （UV-vis） spectrophotometry, cyclic voltammetry （CV）, electrochemical impedance spectroscopy （EIS）, differential pulse voltammetry （DPV）, chronoamperometry and chronocoulometry. The main content is as follows:（1） The Fe@Fe₂O₃ core-shell nanonecklaces, Fe@Fe₂O₃ core-shell wires and Cu2O nanocubes were synthesized, and their morphologies were characterized by SEM、TEM and XRD patterns. By assembly of the synthesized nanomaterials, poly（dimethyldiallylammonium chloride） （PDDA）, Au nanoparticles （AuNPs） and multi-wall carbon nanotubes （MCNTs） on the surface of GCE electrode, three kinds of nano-bionic interfaces, PDDA/Fe@Fe₂O₃-AuNPs/PDDA/GCE, PDDA/Fe@Fe₂O₃-MCNTs/PDDA /GCE and PDDA/nanoCu₂O-AuNPs/PDDA/GCE, were constructed and the properties of them were studied by UV-vis spectroscopy. The DNA damage on these interfaces during the course of the cathodic treatment were studied by CV and DPV methods using Ru（NH₃）₆³⁺ and Co（phen）₃³⁺ as electrochemical probes. The results showed that the DNA damage mainly happened during the course of the cathodic treatment. When these interfaces were treated by a cathodic process, the Fenton or like-Fenton reaction happened in interfaces and the reactive oxygen species （ROS） were produced. The ROS attacked and caused DNA damage in situ. The DNA damage courses in interface were just like the pathways of the heavy metal induced-DNA damage in vivo. Therefore, these biosensors can be used to mimic heavy metal gene toxicity pathways in vivo and can be used as powerful tools for screening the gene toxicity of chemicals.（2） By electrochemical polymerization, covalent combination, adsorption and casting methods, the polythionine （PTn）, polytyrosine （PTyr）, AuNPs and nanozirconia-polyaniline composite（nanoZrO₂-PAN） were modified on the surface of GCE electrode and two biointerfaces, AuNPs/PTn/GCE and nanoZrO₂-PAN/PTyr/GCE, were constructed. The properties of these two biointerfaces and the immobilization and hybridization of DNA on these surfaces were studied by CV, DPV and EIS. These two biointerfaces can be applied to detect the phosphinothricin acetyltransferase （PAT） gene sequences by a label-free EIS method. On the ss-DNA/AuNPs/PTn/GCE and ss-DNA/nanoZrO₂-PAN/PTyr/GCE, the dynamic detection range of the PAT gene sequences were 1.0×10^-10 1.0×10^-6 mol/L and 1.0×10^-13 1.0×10^-6 mol/L, respectively, and the detection limit were 3.2×10^-11 mol/L and 2.68×10^-14 mol/L （S/N = 3）, respectively. The ss-DNA/AuNPs/PTn/GCE was further used to detect the PCR amplification sample of terminator of nopaline synthase （NOS） from a kind of transgenic modified bean with satisfactory results. Compared with the biosensors constructed by ZrO₂ normal materials, the biosensor composed by nanoZrO2-PAN has wider linear range and lower detection limit.（3） The Au-Pt alloy nanoparticles （Au-PtNPs） were synthesized by electrochemical deposition on the surface of the GCE electrode modified with the composite of polyaniline nanotubes （nanoPAN） and chitosan （CS）. Then horseradish peroxidase （HRP） was immobilized on the surface of Au-PtNPs/nanoPAN/CS and the direct chemistry of HRP was obtained on this surface, based on which a novel H₂O₂ biosensor was constructed. The membrane properties of Au-PtNPs/nanoPAN/CS were studied with CV and EIS. Under the optimal conditions, the amperometric response of H₂O₂ on this biosensor was investigated by adding aliquots of H₂O₂ to a continuous stirring phosphate buffer solution. The biosensor displayed a fast response time （< 2 s） and broad linear response to H₂O₂ in the range from 1.0 to 2200μmol/L with a relatively low detection limit of 0.5μmol/L at 3 times the background noise. Moreover, the biosensor can be applied in practical analysis and exhibited high sensitivity, good reproducibility, and long-term stability.（4） The electrochemical behaviors of lincomycin on Au-PtNPs/nanoPAN/CS/GCE and amikacin on nanoPAN/CS/GCE were studied, respectively, and the electrochemical methods for detection of these two antibiotics were set up. The dynamic detection ranges of lincomycin and amikacin were 3.0 mg/L 100.0 mg/L and 10.0 mg/L 80.0 mg/L, and the detection limits were 8.0 mg/L and 1.0 mg/L （S/N = 3）, respectively. Some electrochemical parameters involved in the redox reaction of lincomycin, such as parameter of kinetic nα, standard rate constant ks and the number of H⁺, were also calculated. Both of the methods can be used in practice.更多还原