新型纳米催化剂的设计及用于特定细胞与细胞中重要小分子的检测

【作者】 吴萍；

【导师】 蔡称心；

【作者基本信息】 南京师范大学 ， 生物物理化学， 2013， 博士

【摘要】 细胞是生物体形态结构和生命活动的基本单元。在细胞中存在着一些具有特殊生理功能的活性小分子物质,如H202、02等；它们在细胞生物过程中的作用与其在细胞内的含量密切相关。如当细胞中H202浓度较低时,它参与细胞信号转导、多种因子细胞生物学效应的启动,并且控制细胞的增殖、分化与凋亡等；而当其高浓度较高时,则会引起细胞膜脂质过氧化、细胞内蛋白质和酶的变性、DNA的损伤等,进而造成细胞的死亡、机体的炎症、甚至心血管疾病、神经元退变及肿瘤疾病的发生。因此,对细胞内一些重要活性小分子进行实时、动态的检测不仅能帮助我们从分子水平理解它们介导生理过程的机制,而且能够有助于揭示这些活性小分子与人类相关疾病之间的内在规律。电化学方法具有响应快、专一性强和操作简单等优点,成为细胞中一些活性小分子检测的常用技术手段,该方法不仅能够准确测量细胞中活性小分子的含量,更能实时监测活性小分子从细胞中向胞外释放的动力学过程。而掌握纳米材料的电催化特性,尤其是理解检测对象在纳米电催化剂表面氧化还原反应的机制,从而选择合适的纳米材料构建具有高电催化活性的表界面,能够极大程度提高电化学检测方法的灵敏度与专一性。本论文制备了一系列纳米电催化剂,研究了细胞中H202含量的电化学检测方法、细胞中H202的释放过程动力学以及细胞中H202的胞吐过程等；本论文还用密度泛函理论(DFT)阐明了H2O2和O2分子在纳米电催化剂(如氮掺杂石墨烯及石墨炔等)表面的电催化还原反应的机制。本论文的主要内容及结果如下：1.制备了辣根过氧化物酶-凹土(HRP-Atta)纳米复合催化剂并将其应用于巨噬细胞RAW264.7中H2O2含量的电化学检测。利用物理吸附法将HRP固定到凹土表面制备得到HRP-Atta纳米复合催化剂,用透射电镜(TEM)、原子力显微镜(AFM)、红外光谱(FTIR)、紫外-可见光谱(UV-vis)、圆二色光谱(CD)等对其进行了表征,研究结果表明,HRP分子能够通过静电作用成功吸附在凹土表面并能很好地维持其天然的空间结构。电化学实验结果表明,HRP分子在凹土表面能够进行有效的直接电子转移反应,其伏安曲线上呈现出一对良好的氧化还原峰,氧化峰电位(Epa)和还原峰电位(Epc)分别为-295和-360mV (vs. SCE,100mV/s);进一步的研究结果表明,HRP-Atta纳米复合催化剂对H2O2的电化学还原反应具有良好的催化性能,对H2O2的定量测定具有响应速率快(2s)、线性范围宽(0.2-150umol/L).灵敏度高(55.8±2.9μA L/mmol cm2)及检测限低(0.05±0.01μmol/L)等优点。将HRP-Atta复合催化剂应用于RAW264.7细胞中H202含量的电化学检测,结果表明该复合物可以准确地测定细胞中H202的含量为(0.29±0.05) mmol/L,并且不受细胞内其他活性氧物质(ROS)和电活性物质如OCl-和抗坏血酸等的干扰。2.建立了一种制备氮掺杂石墨烯(N-graphene)的简单方法。该方法以水合肼为还原剂,在水热条件下一步实现氧化石墨烯的还原及氮原子的掺杂从而制备得到N-graphene o利用TEM、AFM、FTIR、拉曼光谱(Raman)和X-射线光电子能谱(XPS)等对制备的N-graphene进行了表征。结果表明,所得到的N-graphene具有单层结构,N元素含量为4.5%,其中吡啶型N、毗咯型N、石墨型N及氧化吡啶型N分别为28%、49%、19%及4%。循环伏安实验结果表明N-graphene对H202电化学还原具有高的催化活性,还原起峰电位分别比不掺杂的石墨烯及玻碳正移了230和280mV,还原电流密度也分别比不掺杂的石墨烯及玻碳提高到2倍和25倍。将N-graphene应用于活细胞如嗜中性粒细胞、RAW264.7细胞及人乳腺癌细胞MCF-7中H202的电化学监测,结果表明N-graphene具有良好的生物相容性及高的稳定性,能用于细胞中H202释放动力学及不同外界刺激条件下细胞胞吐过程的研究。3.利用DFT方法研究了N-graphene中N掺杂微观构型对H202电还原反应催化活性的影响。分别研究了H202在石墨烯、吡啶型N-aphene、吡咯型N-graphene及石墨型N-graphene表面的吸附以及电催化还原过程的机制。结果表明：H202在石墨烯及N-graphene表面的吸附过程属于弱的物理吸附,而当反应体系中引入质子和电子后,一系列化学反应随之发生,包括H202分子中的O-O键的断裂、-OH的生成、-OH以C-O键的形式在石墨烯及N-graphene表面的化学吸附及两个H20分子的生成。对H202还原的反应能及还原反应每个步骤的可逆电势进行了计算；结果表明,H202在石墨烯及N-graphene表面的电还原反应为自发的反应过程,并且石墨烯纳米催化剂对H202还原反应的电催化活性顺序为吡啶型N-graphene>吡咯型N-graphene>石墨型N-graphene>石墨烯。我们还从静电势及最高占有轨道(HOMO)与最低空未占轨道(LUMO)之间能级差的角度就N掺杂构型对H202电还原反应催化活性的影响规律进行了解释。4.采用DFT方法研究了新型二维碳材料石墨炔表面的氧气电催化还原反应(ORR)的机制。我们首先对石墨炔的构型进行了优化,并对其表面的电荷分布进行了分析,发现石墨炔结构中的碳原子分别以Sp-及Sp2-杂化状态的C≡C及C=C键模式存在,其表面电荷分布明显不均匀,存在大量的带有正电荷的碳原子,能够为O2及OOH+分子的吸附及催化反应提供多个位点。我们还对石墨炔表面的ORR反应机制进行了研究；结果表明,O2及OOH+分子在石墨炔表面通过C-O化学键形成了有效的化学吸附,吸附能分别为-1.48eV和-4.28eV,并且在石墨炔表面发生了O-O键的断裂从而实现了4电子的还原反应；我们通过反应能及可逆电势的计算证明了石墨炔表面的ORR反应属于自发的放热反应,说明石墨炔对ORR反应具有高的电催化活性,并从HOMO-LUMO之间能级差的角度对石墨炔具备的高催化活性进行了解释。5.制备了适配体-Ag-Au纳米复合物(aptamer-Ag-Au),并将其应用于人乳腺癌细胞的拉曼(Raman)检测及近红外光疗。用光还原法以核酸适配体S2.2为模板合成了aptamer-Ag-Au纳米复合物,并用UV-vis及TEM对其进行了表征。相差(DIC)、荧光、Raman光谱及TEM结果表明,制备的纳米复合物可以通过S2.2和MCF-7细胞膜表面的粘蛋白MUC1之间的特异性作用结合在MCF-7细胞表面,从而特异性地识别MCF-7细胞,而该复合物与其他类型的肿瘤细胞(如人肝癌细胞HepG2)或正常乳腺上皮细胞MCF-10A等不能发生特异性结合。制备的aptamer-Ag-Au复合物还具有高的表面增强拉曼(SRER)活性,其Raman增强因子能够达到106,因此可以应用于MCF-7细胞的高灵敏及高特异性Raman检测。进一步将所制备的aptamer-Ag-Au应用于MCF-7细胞的光热治疗,研究结果表明,aptamer-Ag-Au复合物能吸收近红外光并将光能转化为热能传递给靶细胞MCF-7,在功率0.25W/cm2的近红外光光照1h能有效地将MCF-7细胞的活性降低到3%,并能避免对周围正常细胞及组织的损害。更多还原

【Abstract】 Numerous bioactive small moleculer species are ubiquitous in living cells, such as H2O2, O2et al., which show various physicological functions and play the key roles in life course. However, the mechanism of these bioactive species on the physiological events is closely related to their cellular level. At a low concentration, they act as a messenger in cellular signal transduction and signal amplification, play an important role as the regulator for a number of oxidative stress-related states, control the proliferation, differentiation and apoptosis of cells. Whereas at a high concentration, they can damage major cellular constituents and harm to living organisms, causing a number of pathological events ranging from aging, diabetes, neurodegeneration, even to cancer. Therefore, the quantitative determination of cellular bioactive species concentration could lead to a better understanding of the clinical consequences of the enhancement in their concentration as well assisting in studies designed to elucidate the biological effect of them in cells. Electrochemical detection method takes advantage of fast response, high specifity, and ease operation. More important, electrochemical detection not only can realize real-time determination of concentration of bioactive species with a great precision, but also can provide the dynamic information on the release process of these bioactive species from cells. Elucidating the detailed electron transfer (ET) mechanism of the redox molecules at the surface of novel electrocatalysts not only helps to understand the major factors that control the ET processes, but also make better design of the new synthesized electrocatalysts, which will significantly improve the detection sensitivity and specificity.In this paper, we synthesized a series of nanocatalysts, and applied them into electrochemical measurement of the concentration of cellular H2O2(the most important representative of small and bioactive moleculer species in cells) and monitor its dynamic release process from the cells. We also chose the nitrogen-doped grapheme and graphyne as study model, studied the mechanism of these bioactive small molecule (H2O2, O2) on their electrocatalytic reduction reations at the surface of nanocatalysts. The main results are as follows:1. Developed a new method for reliable determination of cellular ROS (reactive oxygen species) concentration could lead to a better understanding of the clinical consequences of the enhancement in ROS concentration, elucidating the relationship between ROS and environmental stresses and lipid peroxidation, and assisting in studies of the biological effect of ROS in cells. This work developed and validated an electrochemical approach for measuring the concentration of H2O2in RAW264.7macrophage cells. This approach is based on the electrocatalytic reduction of the releasing H2O2at the biosensor of HRP-Attapulgite/GC, which was fabricated by depositing the horseradish peroxidase-attapulgite nanohybrids on the glassy carbon (GC) electrode. The biosensor exhibited a rapid response (less than2s), a wide linear range (0.2to150μmol/L), a high sensitivity (55.8±2.9μA L/mmol cm2), a low detection limit (0.05±0.01μmol/L), as well as good stability and repeatability due to using the natural mineral (attapulgite) as the enzyme immobilization substrate. In addition, some common coexisting ROS and compounds in biological system such as hypochlorite (OC1-), nitric oxide (NO·), peroxynitrite (ONOO-), and ascorbic acid (AA) etc., did not cause any interference due to the use of a low operating potential (-400mV, versus SCE). Moreover, the developed approach can also be used for studying the effects of the stimulator loading on the generation of H2O2in cells. Therefore, this work has demonstrated a simple and effective sensing platform for detection of concentration of cellular H2O2in cells such as RAW264.7cells, which has potential utility to cellular biology and pathophysiology.2. Modulating the electronic characteristics of graphene is of great technological importance for improving and expanding its applications. Chemical doping with other elements is a promising way to achieve this goal. This work reports a facile synthesis of the nitrogen-doped graphene (N-graphene) at low temperature. This method, which involves the steps of graphite oxidation, exfoliation, and chemical reduction with the use of hydrazine as a reducing agent, can simultaneously realize the reduction of graphene oxide and doping graphene with nitrogen atoms. The spectroscopic results demonstrate that the N-graphene with N/C atomic ratio up to～4.5%can be prepared, and the doping N atoms consist of the pyridinic, pyrrolic, graphitic, and oxidized nitrogen structures with the surface atomic compositions of～28%,49%,19%, and4%, respectively. The prepared N-graphene exhibits the superior electrocatalytic activity toward H2O2reduction, and the contribution of the doped N atoms to the enhanced electrocatalytic activity is explained in details based on density functional theory (DFT) calculation. Moreover, the N-graphene is further used for studying the dynamic process of H2O2(a common representative of reactive oxygen species, ROS, in the living cells) releasing from living cells such as neutrophil, RAW264.7macrophage, and MCF-7cells. The results presented here has opened a new way for synthesizing the N-graphene, and also developed a new platform for a reliable collection of kinetic information on cellular ROS release. The approach established in this work could be potentially useful in study of downstream biological effects of various stimuli in physiology and pathology.3. Elucidating the detailed electron transfer (ET) mechanism of the redox molecules at the surface of novel materials with different microstructural features will helps to understand the mechanism on electrocatalytic reaction. We report here a density functional theory (DFT) study on the microscopically understanding the detailed effects of the bonding configuration of nitrogen-doped graphene (N-graphene) within the carbon lattice such as including pyridinic, pyrrolic, and graphitic N on the activity and mechanistic processes of the H2O2reduction. We simulated the adsorption process of H2O2, analyzed the mechanistic processes and calculated the reversible potential of each reaction step of H2O2reduction reaction at N-graphene. The simulation results indicate that the adsorption of H2O2on the pristine and N-graphene surface occurs via a physisorption without the formation of chemical bond. When H+is introduced into the system, a series of reactions can occur including the breakage of the O-O bond, formation of an O-C chemical bond between oxygen and graphene, and the creation of water molecules. The results also indicate a decrease in the energy of the system and a positive value of the reversible potential for each reaction step. The calculated results on the relative energy of the each reaction step and the value of the onset potential for H2O2reduction suggested the reactivity of pristine and N-graphene has an order of pyridinic N-graphene> pyrrolic N-graphene> graphitic N-graphene> pristine graphene. We also proposed an explanation for this dependence of reactivity order on the doping bond configuration in N-graphene based on the electrostatic potential calculation. The results presented here should help to understand microscopically the dependence of the reactivity N-graphene on its microstructure, inspire the study of heteroatom-doped graphenes to improve their catalytic efficiency, and provide a theoretical framework to analyze their catalytic activities.4. Graphyne, a new two-dimensional periodic carbon allotrope with a one-atom-thick sheet of carbon built from triple-and double-bonded units of two sp-and sp2-hybridized carbon atoms, has been shown in recent studies to have the potential for high-density hydrogen and lithium storage. We report here a density functional theory (DFT) study of an oxygen reduction reaction (ORR) involving graphyne and demonstrate that graphyne is a good, metal-free electrocatalyst for ORRs in acidic fuel cells. We optimized the geometrical structure, calculated the charge densities on each carbon atom in the graphyne, and simulated the each step of the ORR reaction involving graphyne. The simulation results indicate that the distribution of the charge density at each carbon atom on the graphyne plane is not uniform and that a large number of positively charged carbon atoms, which are beneficial to the adsorption of O2and OOH+molecules, can behave as catalytic sites to facilitate ORRs. When H+is introduced into the system, a series of reactions can occur including the formation of an O-C chemical bond between oxygen and graphyne, breakage of O-O bond, and the creation of water molecules. The results also indicate a decrease in the energy of the system and a positive value of the reversible potential for each reaction step on the graphyne surface. In addition, a spontaneous electron transformation process occurs during the ORR along a four-electron pathway. The results presented here should lead to an improvement in the catalytic efficiency of carbon nanomaterials and provide a theoretical framework for the analysis of their catalytic activity. This paper highlights the urgent need for new experimental syntheses for graphyne.5. The aptamer (S2.2)-guided Ag-Au nanostructures (aptamer-Ag-Au) have been synthesized by photoreduction and validated by UV-vis spectra and transmission electron microscopy (TEM) images. Differential interference contrast (DIC), fluorescence, and TEM images indicated that the aptamer-Ag-Au nanostructures can be targeted to the surface of human breast cancer cells (MCF-7) with high affinity and specificity. This targeting is completed via the specific interaction between S2.2aptamer (a25-base oligonucleotide) and MUC1mucin (a large transmembrane glycoprotein, whose expression increased at least10-fold at MCF-7cells in primary and metastatic breast cancers). However, the nanostructure cannot be targeted with HepG2(human liver cancer cells) or MCF-10A cells (human normal breast epithelial cells) due to these cells are MUCl-negative expressed. Moreover, the synthesized nanostructures exhibited a high SERS activity. Based on these results, a new assay has been proposed for high specifically detecting MCF-7cells, even down to a single cell. This assay can also discriminate MCF-7cells from MCF-10A cells and different cancer cell lines such as HepG2cells. In addition, the aptamer-Ag-Au nanostructures have high capability of adsorption NIR irradiation and are able to perform photothermal therapy of MCF-7cells at a very low irradiation power density (0.25W/cm2) without causing the destroy of the healthy cells and the surrounding normal tissues. Therefore, the proposed assay is significant for diagnosis of the tumors in its nascent stage. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have a great potential useful in photothermal therapy of the cancers.更多还原