生物分子探针与肿瘤细胞相互作用机制的光谱—电化学研究

发布时间：2018-06-05 00:29

  本文选题：生物分子探针 + 作用机制　； 参考：《东南大学》2016年博士论文

【摘要】：肿瘤的早期检测对挽救生命至关重要,能够明显提高患者的生存率。发展简便易行、特异性高、敏感性强的肿瘤检测与诊断技术,已成为一个亟待解决的难题。在本论文的研究工作中,我们研究和拓展了新型二茂铁碳硼烷衍生物(FcCB)的生物学效用,并进一步将其用作生物分子探针,实现了肿瘤细胞的高灵敏识别与检测。与此同时,我们采用液滴电化学系统和光谱学手段研究了FcCB的电化学及其光谱学性质,推理了其氧化还原机制。进一步地,采用多种电化学和光谱学方法研究了FcCB与生物大分子的相互作用过程,计算其结合位点数和结合常数。在此基础上,将该生物分子探针用于白血病细胞的识别与检测,并进行了临床样本的分析。同时,论文进行了基于电化学-表面等离子体激元共振技术(SPR)的细胞传感研究,制备了氨基苯硼酸原位修饰的SPR芯片,用于肿瘤细胞的传感与分析；使用原位电化学-SPR技术动态监测了生物分子探针与肿瘤细胞相互作用过程,并用于细胞活性的评价。具体内容如下：1)碳硼烷衍生物的光电性质及其与生物大分子相互作用研究首先构建了液滴电化学系统,以减少样本的用量,提高检测效率。液滴电化学系统由电化学单元、XYZ三轴滑动平台及成像系统组成。使用液滴电化学系统及光谱方法研究了FcCB的氧化还原性质。FcCB具有1对可逆的氧化还原峰和1个不可逆的氧化还原峰,分别归属于二茂铁基团和环戊烯结构；FcCB的电化学性质与电解质的pH值密切相关,电解质溶液的pH值为3.0～8.0时,峰电位与pH值呈线性关系,其斜率值接近59 mV/pH单位,说明反应过程中质子转移数目和电子转移数目相等；电化学及光谱结果表明FcCB的氧化还原过程涉及两步单电子-单质子转移的氧化还原反应。电化学、紫外光谱及荧光光谱结果表明FcCB与血红蛋白具有较强的相互作用,结合常数在104M-1数量级；FcCB能够猝灭血红蛋白的荧光,为动态和静态混合猝灭机制,且静态猝灭常数和动态猝灭常数接近；当FcCB和血红蛋白浓度比小于2时,结合位点数为1,结合常数为5.67×103 M-1,而FcCB和血红蛋白浓度比大于2时,结合位点数为2,结合常数为1.06×108 M-1；FcCB能够结合到血红蛋白的疏水区域,进而改变血红蛋白的构象,使其三级结构变得疏松甚至解聚。2) FcCB生物分子探针在肿瘤细胞识别中的应用研究拓展了碳硼烷衍生物FcCB的生物医学应用,将其用作生物分子探针,用于肿瘤细胞的识别与检测及临床样本分析。与不可逆峰相比,FcCB的可逆峰具有更宽的线性范围,更低的检出限,使其在生物传感检测中的应用成为可能。研究表明,FcCB与正常及病变的白细胞作用后,其电化学响应发生特异性改变。正常组的峰电位相比FcCB本身向负电位方向移动,而白血病组的峰电位则向正电位方向移动。因此其峰电位可以用作细胞识别与检测的特征信号。多种统计学分析结果显示正常组和白血病组的峰电位位移具有显著性差异,因此FcCB可以用于临床样本的特异性识别与检测。本方法为肿瘤的早期诊断和临床疗效监控等方面提供了新的方法与技术手段,具有潜在而重要的应用前景。3)基于氨基苯硼酸原位修饰的SPR生物传感研究制备了氨基苯硼酸原位修饰的SPR芯片,接触角和红外光谱结果进一步表明氨基苯硼酸修饰到了芯片表面。氨基苯硼酸修饰的SPR芯片对葡萄糖分子具有良好的检测灵敏度,检出限为0.512mM,同时能够进行再生进而重复使用。进一步地,氨基苯硼酸修饰的SPR芯片可以用于肿瘤细胞的传感与检测。随着时间的增加,HepG2细胞吸附到芯片表面,SPR响应逐渐增大,最后趋于稳定,达到平台。随着细胞浓度的增加,SPR响应达到平台所用的时间逐渐降低,且参比通道所用时间大与样品通道。同时,HepG2细胞在样品通道上的SPR响应明显高于参比通道,样品通道的检测灵敏度远高于参比通道。在5×103～1×106细胞/mL浓度范围内与细胞的数量的对数值呈正相关,样品通道对HepG2细胞的检测限低于1000细胞/mL。这种非标记、实时、快速的检测方法对肿瘤细胞的传感与检测提供了新的分析手段。4)基于电化学-SPR技术的生物活性分子和瘤细胞相互作用的研究采用了电化学-SPR联用方法评估了柔红霉素处理后HepG2细胞的活性。首先优化了细胞芯片的制备条件,进而在实时记录SPR响应的同时,采集细胞外柔红霉素的电化学信号。SPR响应来源于SPR芯片表面吸附细胞形态和质量以及液体环境折射率等因素的变化,电化学信号则归属于细胞外残留、未被细胞吸收的柔红霉素分子。实验结果表明,细胞的SPR响应及柔红霉素的电化学信号均是浓度和时间依赖的。随着柔红霉素浓度和孵育时间的增加,细胞凋亡率增加,有更多的细胞从SPR芯片上脱落,导致芯片覆盖率及芯片表面的折射率降低,因此SPR响应逐渐降低。同时,随着孵育时间的增加,细胞吸收培养基中的柔红霉素分子,使得细胞外残留的柔红霉素含量减小,因此其电化学信号发生降低；另一方面,细胞凋亡后释放部分柔红霉素分子到培养基中,因此随后的电化学信号略有增加。与此同时,MTT和显微学结果验证了电化学-SPR的结果。进一步地,SPR响应和细胞存活率呈良好的线性关系,可以用于评语细胞活性。因此,该方法具有非标记、实时动态及原位分析等特点,在临床疗效评估、药物分析等方面具有巨大的应用潜力。
[Abstract]:Early detection of tumor is very important for saving life, and it can obviously improve the survival rate of the patients. It has become a difficult problem to develop a simple and easy to develop tumor detection and diagnosis technology with high specificity and sensitivity. In this paper, we have studied and expanded the new type of two ferrocene carbon borane derivative (FcCB). At the same time, we use liquid drop electrochemical system and spectroscopy to study the electrochemical and spectroscopic properties of FcCB, and deduce its oxidation-reduction mechanism. Further, we use a variety of electrochemical and spectroscopic methods. The interaction process of FcCB with biomolecules was studied, and the number of binding sites and binding constants were calculated. On this basis, the biomolecular probe was used to identify and detect leukemia cells and to analyze the clinical samples. At the same time, the paper carried out the cell transmission based on the electrochemical surface plasmon resonance technique (SPR). An in situ modified SPR chip was prepared for the sensing and analysis of tumor cells. In situ electrochemical -SPR technique was used to dynamically monitor the interaction between biomolecular probes and tumor cells, and used for evaluation of cell activity. The specific contents are as follows: 1) the photoelectric properties of the derivatives of carbon borane and their biological properties. The study of macromolecule interaction first constructed a droplet electrochemical system to reduce the amount of samples and improve the detection efficiency. The droplet electrochemical system is composed of electrochemical units, XYZ three axis sliding platform and imaging system. The redox properties of FcCB have been studied by the liquid drop electrochemical system and spectral method, and the redox property of.FcCB has 1 pairs of reversible oxidation. The reduction peak and 1 irreversible redox peaks belong to the ferrocene group and the structure of cyclopentene, respectively. The electrochemical properties of FcCB are closely related to the pH value of the electrolyte. When the pH value of the electrolyte solution is 3 to 8, the peak potential and the pH value are linear. The slope value is close to 59 mV/ pH units, indicating the number of proton transfer in the reaction process and the number of proton transfer. The number of electron transfer is equal; the electrochemical and spectral results show that the redox process of FcCB involves two step single electron mono proton transfer redox reaction. The results of electrochemical, UV and fluorescence spectra show that FcCB has strong interaction with hemoglobin, and the binding constant is at the 104M-1 order of magnitude; FcCB can quench the hemoglobin. The fluorescence is the dynamic and static quenching mechanism, and the static quenching constant and the dynamic quenching constant are close. When the FcCB and hemoglobin concentration ratio is less than 2, the number of binding sites is 1, the binding constant is 5.67 * 103 M-1, while the number of binding sites is 2 and the binding constant is 1.06 * 108 M-1 when the concentration ratio of FcCB and hemoglobin is greater than 2; FcCB can be combined. To the hydrophobic area of hemoglobin, then change the conformation of hemoglobin, make the three-stage structure loose and even disassemble.2), the application of FcCB biomolecular probe in tumor cell recognition extends the biomedical application of the carbon borane derivative FcCB, and uses it as a biologic probe for the identification and detection of tumor cells and its presence. Analysis of bed samples. Compared with the irreversible peak, the reversible peak of FcCB has a wider linear range and a lower detection limit, making it possible for its application in biosensor detection. The study shows that the electrochemical response of FcCB to the normal and diseased white cells changes specifically. The peak potential of the normal group is compared to the negative potential of the FcCB itself. The peak potential of the leukemia group moves toward the positive potential. So the peak potential can be used as the characteristic signal of cell recognition and detection. The results of statistical analysis show that the peak potential displacement of the normal group and the leukemia group is significant difference, so FcCB can be used for the specific identification and detection of clinical samples. Methods a new method and technique was provided for the early diagnosis of tumor and the monitoring of clinical effect. It has potential and important application prospect.3) based on the SPR biosensor of amino benzboric acid in situ modification, the in situ modified SPR chip of amino benzboric acid was prepared. The results of contact angle and infrared spectrum further indicated that amino benzonate was used. The acid modified to the surface of the chip. The amino benzboric acid modified SPR chip has a good detection sensitivity to the glucose molecule. The detection limit is 0.512mM, and it can be regenerated and reused. Further, the SPR chip modified by amino benzyl boric acid can be used for the transmission and detection of tumor cells. As time increases, HepG2 cells suck. On the surface of the chip, the response of SPR gradually increased and finally stabilized to reach the platform. As the cell concentration increased, the time used by the SPR response to the platform gradually decreased, and the time of the reference channel was larger than the sample channel. At the same time, the SPR of the HepG2 cells in the sample channel should be significantly higher than the reference channel and the detection sensitivity of the sample channel. It is far higher than the reference channel. The number of cells is positively correlated with the number of cells in the /mL concentration range of 5 x 103~1 x 106 cells. The detection limit of sample channel to HepG2 cells is lower than that of /mL. in 1000 cells. In real time, the rapid detection method provides a new analytical method for the detection and detection of tumor cells..4 based on electrochemical -SPR technology. The study of the interaction between bioactive molecules and tumor cells used the electrochemical -SPR method to evaluate the activity of HepG2 cells after daunorubicin treatment. First, the preparation conditions of the cell chip were optimized, and the response of the SPR was recorded in real time. The response of the electrochemical signal.SPR of the extracellular daunorubicin was collected from the SPR chip table. The changes in the morphology and mass of the surface adsorbed cells and the refractive index of the liquid environment, the electrochemical signals belong to the unabsorbed daunorubicin molecules. The results show that the SPR response and the electrochemical signals of the daunorubicin are both concentration and time dependent. With the concentration of daunorubicin and the incubation time In addition, the rate of cell apoptosis increased and more cells fell off the SPR chip, causing the chip coverage and the decrease of the refractive index on the surface of the chip, so the SPR response gradually decreased. At the same time, the cells absorbed the daunorubicin molecules in the medium with the increase of incubation time, which reduced the content of the residual daunorubicin in the extracellular matrix, so the electricity was reduced. The chemical signals were reduced; on the other hand, after the apoptosis was released, some daunorubicin molecules were released into the medium, and the subsequent electrochemical signals increased slightly. At the same time, the results of the electrochemical -SPR were verified by MTT and microscopy. Further, the response of the SPR to the cell survival rate was in good linear relationship with the cell survival rate, which could be used in the evaluation cells. Therefore, this method has the characteristics of non label, real-time dynamic and in situ analysis, and has great potential in clinical efficacy evaluation and drug analysis.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2
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本文编号：1979559