【作者】 张艳；

【导师】 王帅； 肖菲；

【作者基本信息】 华中科技大学 ， 材料物理与化学， 2018， 博士

【摘要】 癌症对人类的威胁已经成为全球重大的健康问题之一。而癌细胞是产生癌症的病源,具有细胞分化和增殖异常、生长失去控制等生物学特征,这些特征使得癌细胞分泌过氧化氢水平远远超过相应的正常组织细胞。采用电化学方法来检测细胞体系的过氧化氢水平,具有速度快、灵敏度高、仪器简单、易于实现自动连续测量及控制等优点。因此构筑电化学检测平台来快速准确检测细胞内过氧化氢水平有利于癌症早期诊断和筛查,提高治疗效果。碳纳米材料由于生物相容性和导电性好,以及比表面积大等优点,在电催化分析上具有广阔的应用前景。碳基柔性电极基底包括一维、二维和三维碳材料,如一维碳纤维（carbon fiber,CF）,二维石墨烯纸和三维石墨烯泡沫。这些电极具有一些特殊性能,包括化学稳定性高,柔韧性和机械强度好等。因此,碳基功能电极对体内和体外追踪生物信息表现出良好的传感性能。基于上述发展和需求,本论文阐述了四种新型的碳基功能纳米复合材料,并探索其作为柔性自支撑电极在电化学传感器中的应用,建立了癌细胞在正常状态/应激状态下以及接受放疗或化疗后释放过氧化氢水平的电分析技术平台。主要研究内容有以下四个方面:1、基于石墨烯材料的自组装性能和成膜性能、将石墨烯纳米片组装形成三维多孔凝胶、进一步制备成自支撑纸电极,用于对过氧化氢电化学传感系统。我们在还原剂多巴胺辅助下,使得氧化石墨烯的还原和自组装在一个较低温度下进行,并形成了规整的三维多孔离子液体功能化石墨烯凝胶,呈现出典型的分级孔隙结构;同时在石墨烯组装体上原位化学还原氯金酸前驱体形成独特金纳米花,其密度高而且均匀分散在离子功能化石墨烯支架上。复合材料可加工成自支撑的纸质电极。由于材料独特的三维多孔结构和不同组份间的协同作用,对过氧化氢具有良好的电催化活性。将该纸电极应用于检测不同细胞分泌的过氧化氢水平,可以区分不同细胞,并进一步对不同乳腺癌细胞的放疗和化疗效果进行评估。2、为了进一步提高电分析方法的时空分辨率,提高实际样品检测灵敏度,本工作设计了一种新型纤维微电极。通过简单而有效的原位电化学合成,在碳纤维基底上设计和制造了具有多级结构的Au/MnO₂/ERGO。通过结合浸涂法和电化学还原法在碳纤维上包裹一层石墨烯,显著增加裸CF的电导率、比表面积和亲水性;然后通过无模板法电沉积在石墨烯表面覆盖一层致密的MnO₂纳米线,为进一步电沉积活性金纳米粒子提供大表面积和大量成核位点。利用微电极的多级结构特点,以及金纳米粒子的良好分散性,因此合成的分层纳米结构微电极对过氧化氢表现出良好的催化效率,可用于灵敏检测HeLa细胞和HBL-100细胞释放的过氧化氢。3、基于第二章多级结构微电极中MnO₂纳米线层导电性较差,且是无序生长的,导致电极阻抗较大,不利于金纳米粒子的充分利用,而构筑有序三维纳米阵列是增大电极导电性和活性面积的有效方法。因此本章以水热氧化锌纳米阵列为模板,以聚多巴胺为碳源和氮源,在碳纤维上制备了氮掺杂空心碳纳米管阵列,降低了电极阻抗,提高了基底的比表面积和负载位点,减小了金纳米粒子尺寸（5 nm）。将该纳米复合材料用于电化学传感器,由于氮掺杂碳管和金纳米粒子相互协同作用,对过氧化氢具有较高的电催化活性,灵敏度高,线性范围宽（0.5μM⁴.3 mM）。采用该纤维电极实现近细胞检测,通过对比和计算不同种类细胞在应激状态下释放过氧化氢的分子数,建立了间接鉴别不同种类细胞的电化学方法。4、在三维阵列的基础上进一步设计了珊瑚状多级阵列结构,增大电极比表面积和贵金属负载位点,使催化剂与待测溶液充分接触,提高电极检测灵敏度。选用电沉积氧化锌纳米阵列为模板,葡萄糖为前驱体,通过水热自组装在碳纤维上合成了多级阵列球管微纳结构,并通过改变前驱体浓度探究了这种多级阵列结构的形成机理。以该为载体,釆用电活化对电极法,得到了原位负载的铂纳米粒子,提高贵金属的利用率和催化活性。透射电镜和X射线衍射显示了复合材料中纳米粒子的多个晶面,显示纳米粒子分散性较好且粒径分布较为均一（2 nm）。使用该纳米复合物微电极,采用线性扫描伏安法和计时电流-时间等重要的电化学手段,研宄了该催化剂对过氧化氢电化学催化效果,结果表明,纳米催化剂具有较高的催化活性,其柔韧性好,检出限低（50 nM）,线性范围宽（0.1μM¹0.69 mM）,并可用于不同种乳腺细胞在外部物质刺激下释放过氧化氢的检测。更多还原

【Abstract】 With the prolongation of the average life span of human beings,cancer has become increasingly prominent threat and one of the major global health problems.Cancer has some special biological characteristics such as abnormal cell differentiation and proliferation,loss of growth control,which make hydrogen peroxide levels in cancer cells are much higher than the corresponding normal tissue cells.The electrochemical methods have been widely applied in the detection and analysis of small biological molecules due to quick response,high sensitivity,simple instrument,easy to operate and realize further automatic continuous measurement and control.Therefore,constructing an appropriate electrochemical detection platform for quickly and accurately detection of intracellular reactive oxygen species is beneficial to the early diagnosis and treatment of cancer.Carbon nanomaterials have been widely applied in the field of electrocatalytic analysis due to their good electrical conductivity and high specific surface area.The design of flexible electrode substrates has attracted tremendous research efforts.Several flexible one-dimension（1D）,two-dimension（2D）and three-dimension（3D）carbon materials,such as 1D carbon fiber（CF）,2D graphene paper and 3D graphene foam,have been developed in constructing electrochemical biosensors for their special features including high chemical stability,good biocompatibility,and intrinsic flexibility and mechanical strength..Based on the above developments and requirements,four novel carbon-based functional nanocomposites were been prepared and applied in electrochemical biosensors in this paper.We also have established an electroanalytical technology platform for real-time monitoring the released amount of H₂O₂ in cancer cells under normal/stress conditions and after receiving radiotherapy or chemotherapy,providing an important massage for the changes of chemical information in vivo.The main research content has listed as the following:1.In order to achieve the self-assembly of graphene gel at low temperature,the gel can be made as a freestanding paper electrode and its catalytic sensing for hydrogen peroxide,we present the preparation of a new type of 3D porous imidazolium-based ionic liquid（IL）-functionalized graphene framework（GF）by a facile and efficient one-pot self-assembly at low temperature with the assistance of reducing agent dopamine（DA）,exhibiting a typical hierarchical pore structure.Simultaneously,the chloroauric acid precursors were chemically reduced in situ on the IL-GF scaffold,forming highly dense and well dispersed gold nanoflowers,which are dense and uniformly dispersed on the IL-GF scaffold.The composite material was processed into a self-supported paper electrode.Due to the unique three-dimensional porous structure of the material and the synergistic effect between different components,the resultant AuNFs/IL-GF composites have excellent electrocatalytic activity for H₂O₂ detection.Furthermore,the obtain paper electrode can be used to detect the level of H₂O₂ secreted by different cells,which can used for separating different cells,and further evaluated the effect of radiotherapy and chemotherapy of different cancer cells.2.In order to further improve the space-time resolution of the electroanalytical method and the detection sensitivity of the actual sample,a novel microelectrode is designed at this work.A noble Au/MnO₂/ERGO nanocomposites with hierarchical structure on carbon fiber substrate was designed and fabricated by a simple and effective in-situ electrochemical method.Graphene oxide（GO）nanosheets were first coated on bare CF by dip coating.Then the GO was further reduced to graphene under electrochemical production process,which can dramatically increase the electrical conductivity,surface area,and hydrophilicity of CF.Secondly,the surface of graphene wrapped CF has further been covered by twisted and intersectant MnO₂ nanowires through template-free electrodeposition,which provide a large surface area and abundant nucleation sites for the further electrodeposition of electrocatalytically active Au nanoparticles onto it.Utilizing the hierarchical structural characteristics of the microelectrode and the well-dispersed Au nanoparticles,the synthesized nanostructured microelectrode exhibits a high catalytic efficiency for hydrogen peroxide and can be used for the efficient and ultrasensitive electrochemical detection of of H₂O₂ released from HeLa cells and HBL-100 cells.3.Based on the poor conductivity and disordered growth of the MnO₂ nanowire layer in the hierarchical structure microelectrode in Chapter II,resulting in a large electrode impedance,which is not conductive to make full use of Au nanoparticles.And constructing ordered three-dimensional nanoarrays is an effective way to increase the active area of the electrode.Herein,highly ordered nitrogen doped carbon nanotube arrays were wrapped on carbon fiber by using ZnO nanoarrays as templates,polydopamine as a carbon source and nitrogen source,then gold nanoparticles were decorated on the arrays surface by using the reducibility of polydopamine,therefore a new type of flexible nanohybrid microelectrode was synthesized.This method can lower the electrode impedance,increase the specific surface area and load sites,reduce the size of gold nanoparticles to 5 nm.Due to synergistic effect between the nitrogen-doped carbon tube and the loaded gold nanoparticles,the microelectrode not only demonstrates a high electrocatalytic activity towards H₂O₂ reduction,but also high sensitivity,wide liner response range from 0.5μM to 4.3μM.And the fiber electrode can also be placed near the cell to increase accuracy of the current response of different cells under stress conditions.By comparing and calculating the number of H₂O₂ molecules released under different stress states,an electrochemical method for indirect identification of different types of cells was established.4.Based on the three-dimensional nanoarrays,a coral micro-nanostructure arrays of hollow carbon nanotube@carbon nanospheres is further designed on carbon fiber substrate to increase the specific surface area and noble metal loading site of the electrode so that the electrode is in full contact with the tested solution,thereby improving the electrode detection sensitivity.The novel microelectrode is synthesized by hydrothermal self-assembly,which glucose and zinc oxide nanoarrays were used as precursors and sacrificial templates,respectively.The special hierarchical structure dramatically increases the specific surface area of carbon fiber and provides more active sites for supporting the precious metal nanoparticles,which is beneficial to the catalyst in full contact with the test solution.The formation mechanism of this special nanotube-nanosphere structure is discussed by changing the precursor concentration.Using this nanocomposite as a carrier,the surface of the carbon material is electrically activated in H₂SO₄ solution with Pt networks used as counter electrodes,which improved the utilization and catalytic activity of the precious metal.Transmission electron microscopy（TEM）and X-ray diffraction（XRD）showed multiple crystal faces of the Pt nanoparticles in the composite material,indicating that the size of Pt nanoparticles is 2 nm and has uniform distribution.Using this nanocomposite microelectrode,the important electrochemical methods such as linear sweep voltammetry and chronoamperometry were used to study the electrochemical catalytic effect of hydrogen peroxide reduction.The results show that the nanocatalyst has good catalytic activity,good flexibility,low detection limit（50 nM）,wide linear range（range from 0.1μM to 10.69μM）,and can be used to detect the release of hydrogen peroxide from different types of breast cells.更多还原