本文選題：生物分子探針 + 作用機制��； 參考：《東南大學(xué)》2016年博士論文

【摘要】：腫瘤的早期檢測對挽救生命至關(guān)重要,能夠明顯提高患者的生存率。發(fā)展簡便易行、特異性高、敏感性強的腫瘤檢測與診斷技術(shù),已成為一個亟待解決的難題。在本論文的研究工作中,我們研究和拓展了新型二茂鐵碳硼烷衍生物(FcCB)的生物學(xué)效用,并進(jìn)一步將其用作生物分子探針,實現(xiàn)了腫瘤細(xì)胞的高靈敏識別與檢測。與此同時,我們采用液滴電化學(xué)系統(tǒng)和光譜學(xué)手段研究了FcCB的電化學(xué)及其光譜學(xué)性質(zhì),推理了其氧化還原機制。進(jìn)一步地,采用多種電化學(xué)和光譜學(xué)方法研究了FcCB與生物大分子的相互作用過程,計算其結(jié)合位點數(shù)和結(jié)合常數(shù)。在此基礎(chǔ)上,將該生物分子探針用于白血病細(xì)胞的識別與檢測,并進(jìn)行了臨床樣本的分析。同時,論文進(jìn)行了基于電化學(xué)-表面等離子體激元共振技術(shù)(SPR)的細(xì)胞傳感研究,制備了氨基苯硼酸原位修飾的SPR芯片,用于腫瘤細(xì)胞的傳感與分析；使用原位電化學(xué)-SPR技術(shù)動態(tài)監(jiān)測了生物分子探針與腫瘤細(xì)胞相互作用過程,并用于細(xì)胞活性的評價。具體內(nèi)容如下：1)碳硼烷衍生物的光電性質(zhì)及其與生物大分子相互作用研究首先構(gòu)建了液滴電化學(xué)系統(tǒng),以減少樣本的用量,提高檢測效率。液滴電化學(xué)系統(tǒng)由電化學(xué)單元、XYZ三軸滑動平臺及成像系統(tǒng)組成。使用液滴電化學(xué)系統(tǒng)及光譜方法研究了FcCB的氧化還原性質(zhì)。FcCB具有1對可逆的氧化還原峰和1個不可逆的氧化還原峰,分別歸屬于二茂鐵基團和環(huán)戊烯結(jié)構(gòu)；FcCB的電化學(xué)性質(zhì)與電解質(zhì)的pH值密切相關(guān),電解質(zhì)溶液的pH值為3.0～8.0時,峰電位與pH值呈線性關(guān)系,其斜率值接近59 mV/pH單位,說明反應(yīng)過程中質(zhì)子轉(zhuǎn)移數(shù)目和電子轉(zhuǎn)移數(shù)目相等；電化學(xué)及光譜結(jié)果表明FcCB的氧化還原過程涉及兩步單電子-單質(zhì)子轉(zhuǎn)移的氧化還原反應(yīng)。電化學(xué)、紫外光譜及熒光光譜結(jié)果表明FcCB與血紅蛋白具有較強的相互作用,結(jié)合常數(shù)在104M-1數(shù)量級；FcCB能夠猝滅血紅蛋白的熒光,為動態(tài)和靜態(tài)混合猝滅機制,且靜態(tài)猝滅常數(shù)和動態(tài)猝滅常數(shù)接近；當(dāng)FcCB和血紅蛋白濃度比小于2時,結(jié)合位點數(shù)為1,結(jié)合常數(shù)為5.67×103 M-1,而FcCB和血紅蛋白濃度比大于2時,結(jié)合位點數(shù)為2,結(jié)合常數(shù)為1.06×108 M-1；FcCB能夠結(jié)合到血紅蛋白的疏水區(qū)域,進(jìn)而改變血紅蛋白的構(gòu)象,使其三級結(jié)構(gòu)變得疏松甚至解聚。2) FcCB生物分子探針在腫瘤細(xì)胞識別中的應(yīng)用研究拓展了碳硼烷衍生物FcCB的生物醫(yī)學(xué)應(yīng)用,將其用作生物分子探針,用于腫瘤細(xì)胞的識別與檢測及臨床樣本分析。與不可逆峰相比,FcCB的可逆峰具有更寬的線性范圍,更低的檢出限,使其在生物傳感檢測中的應(yīng)用成為可能。研究表明,FcCB與正常及病變的白細(xì)胞作用后,其電化學(xué)響應(yīng)發(fā)生特異性改變。正常組的峰電位相比FcCB本身向負(fù)電位方向移動,而白血病組的峰電位則向正電位方向移動。因此其峰電位可以用作細(xì)胞識別與檢測的特征信號。多種統(tǒng)計學(xué)分析結(jié)果顯示正常組和白血病組的峰電位位移具有顯著性差異,因此FcCB可以用于臨床樣本的特異性識別與檢測。本方法為腫瘤的早期診斷和臨床療效監(jiān)控等方面提供了新的方法與技術(shù)手段,具有潛在而重要的應(yīng)用前景。3)基于氨基苯硼酸原位修飾的SPR生物傳感研究制備了氨基苯硼酸原位修飾的SPR芯片,接觸角和紅外光譜結(jié)果進(jìn)一步表明氨基苯硼酸修飾到了芯片表面。氨基苯硼酸修飾的SPR芯片對葡萄糖分子具有良好的檢測靈敏度,檢出限為0.512mM,同時能夠進(jìn)行再生進(jìn)而重復(fù)使用。進(jìn)一步地,氨基苯硼酸修飾的SPR芯片可以用于腫瘤細(xì)胞的傳感與檢測。隨著時間的增加,HepG2細(xì)胞吸附到芯片表面,SPR響應(yīng)逐漸增大,最后趨于穩(wěn)定,達(dá)到平臺。隨著細(xì)胞濃度的增加,SPR響應(yīng)達(dá)到平臺所用的時間逐漸降低,且參比通道所用時間大與樣品通道。同時,HepG2細(xì)胞在樣品通道上的SPR響應(yīng)明顯高于參比通道,樣品通道的檢測靈敏度遠(yuǎn)高于參比通道。在5×103～1×106細(xì)胞/mL濃度范圍內(nèi)與細(xì)胞的數(shù)量的對數(shù)值呈正相關(guān),樣品通道對HepG2細(xì)胞的檢測限低于1000細(xì)胞/mL。這種非標(biāo)記、實時、快速的檢測方法對腫瘤細(xì)胞的傳感與檢測提供了新的分析手段。4)基于電化學(xué)-SPR技術(shù)的生物活性分子和瘤細(xì)胞相互作用的研究采用了電化學(xué)-SPR聯(lián)用方法評估了柔紅霉素處理后HepG2細(xì)胞的活性。首先優(yōu)化了細(xì)胞芯片的制備條件,進(jìn)而在實時記錄SPR響應(yīng)的同時,采集細(xì)胞外柔紅霉素的電化學(xué)信號。SPR響應(yīng)來源于SPR芯片表面吸附細(xì)胞形態(tài)和質(zhì)量以及液體環(huán)境折射率等因素的變化,電化學(xué)信號則歸屬于細(xì)胞外殘留、未被細(xì)胞吸收的柔紅霉素分子。實驗結(jié)果表明,細(xì)胞的SPR響應(yīng)及柔紅霉素的電化學(xué)信號均是濃度和時間依賴的。隨著柔紅霉素濃度和孵育時間的增加,細(xì)胞凋亡率增加,有更多的細(xì)胞從SPR芯片上脫落,導(dǎo)致芯片覆蓋率及芯片表面的折射率降低,因此SPR響應(yīng)逐漸降低。同時,隨著孵育時間的增加,細(xì)胞吸收培養(yǎng)基中的柔紅霉素分子,使得細(xì)胞外殘留的柔紅霉素含量減小,因此其電化學(xué)信號發(fā)生降低；另一方面,細(xì)胞凋亡后釋放部分柔紅霉素分子到培養(yǎng)基中,因此隨后的電化學(xué)信號略有增加。與此同時,MTT和顯微學(xué)結(jié)果驗證了電化學(xué)-SPR的結(jié)果。進(jìn)一步地,SPR響應(yīng)和細(xì)胞存活率呈良好的線性關(guān)系,可以用于評語細(xì)胞活性。因此,該方法具有非標(biāo)記、實時動態(tài)及原位分析等特點,在臨床療效評估、藥物分析等方面具有巨大的應(yīng)用潛力。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R730.2
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本文編號：1979559