本文選題：電致化學發(fā)光 + 自增強��； 參考：《西南大學》2017年博士論文

【摘要】：電致化學發(fā)光(Electrochemiluminescence,ECL)以電化學為基礎(chǔ),通過電極表面電化學反應(yīng)導致的化學發(fā)光現(xiàn)象進行特定物質(zhì)的定量分析檢測,其同時結(jié)合了電化學的高可控性和化學發(fā)光法的超靈敏性,極大地提高了分析檢測方法的性能,并賦予了其獨特的優(yōu)勢,諸如靈敏度高、線性范圍寬、操作簡單、可控性強、分析快速簡便等。近年來,伴隨著納米技術(shù)及生物技術(shù)的快速發(fā)展,電致化學發(fā)光技術(shù)更是取得了蓬勃的發(fā)展進步,作為一種高效、靈敏的分析檢測技術(shù),在生命分析、環(huán)境監(jiān)測、食品安全等領(lǐng)域備受關(guān)注。目前,電致化學發(fā)光材料正朝著高效、綠色、多功能的方向發(fā)展;而電致化學發(fā)光分析技術(shù)亦正朝著高靈敏、高通量、普適等方向發(fā)展。然而,針對一些特殊研究體系,現(xiàn)有電致化學發(fā)光體系尚存在靈敏度不足、檢測過程復雜繁瑣、適用性不強等方面的缺陷�；诖�,本論文主要從電致化學發(fā)光檢測體系的靈敏度、高通量、適用性等方面出發(fā),通過高效自增強電致化學發(fā)光技術(shù)、原位酶促反應(yīng)增強、核酸擴增放大及高效電致化學發(fā)光材料等方法提高電致化學發(fā)光技術(shù)的分析檢測靈敏度;通過多元分析、競爭反應(yīng)等策略提高電致化學發(fā)光技術(shù)的適用性。在此基礎(chǔ)上,實現(xiàn)了多種疾病標志物及細胞功能檢測研究。本論文的研究工作主要分為以下幾個部分:1.基于自增強復合納米材料的超靈敏細胞傳感器構(gòu)建及其在藥物篩選中的應(yīng)用研究抗癌藥物研究具有極其重要的科學意義及社會價值,尤其是高效、低廉、特異、甚至個體化的抗癌藥物�；诩毎蛲龇治龅乃幬锖Y選評價系統(tǒng)亟需更為靈敏、準確、有效、便捷的分析檢測技術(shù)。靈敏度高、反應(yīng)可控性好、便捷低廉的電致化學發(fā)光技術(shù)具有極大的潛在應(yīng)用價值,然而電致化學發(fā)光材料及共反應(yīng)劑等的生物毒性、分析檢測方法靈敏度等方面尚需進一步改善。本研究提出了一種自增強電致化學發(fā)光納米材料的制備方案。通過羧基化聯(lián)吡啶釕與氨基側(cè)鏈修飾的甲氧基硅烷偶聯(lián),制得功能化的二氧化硅納米材料前體,其中聯(lián)吡啶釕作為電致化學發(fā)光中心,氨基側(cè)鏈作為共反應(yīng)試劑。在此基礎(chǔ)上,通過堿性條件下水解作用,成功制得自增強電致化學發(fā)光納米材料,并成功將其應(yīng)用于基于細胞凋亡分析的抗腫瘤藥效評價系統(tǒng)的構(gòu)建,實現(xiàn)了高效、快速、便捷的抗腫瘤藥效評價。實驗研究同時采用乳腺癌腫瘤細胞(MDA-MB-231)和抗腫瘤藥物(紫杉醇)作為模型研究了所構(gòu)建的抗腫瘤藥效評價系統(tǒng)的適用性,取得了較好的分析效果。該工作提出了自增強電致化學發(fā)光納米材料的設(shè)計構(gòu)建方式,并對其自增強機理進行了相應(yīng)的研究;同時,實現(xiàn)了電致化學發(fā)光分析技術(shù)在亟需的藥物篩選評價系統(tǒng)中的應(yīng)用,進一步拓展了電致化學發(fā)光分析技術(shù)的研究應(yīng)用,為進一步的電致化學發(fā)光技術(shù)及藥物篩選評價等提供參考借鑒。2.基于多元分析的多組份電致化學發(fā)光分析策略研究電致化學發(fā)光分析技術(shù)由于其低背景、高靈敏度、寬線性范圍、低耗用等突出優(yōu)點而倍受關(guān)注。然而,截至到目前為止,絕大多數(shù)的電致化學發(fā)光分析僅僅能在同一界面上實現(xiàn)單一目標物的定量分析。而臨床檢測、環(huán)境分析、食品安全等方面迫切需求一種更為高效、靈敏的多組份電致化學發(fā)光分析檢測技術(shù)。本研究設(shè)計了一種基于多元分析的多組份電致化學發(fā)光分析策略,結(jié)合以雜交連鎖反應(yīng)-滾環(huán)擴增反應(yīng)為基礎(chǔ)的級聯(lián)放大策略,構(gòu)建一種高靈敏的多組份電致化學發(fā)光分析系統(tǒng)。采用心肌損傷類標志物,N末端B型利鈉肽原和肌鈣蛋白I為檢測模型,分別以魯米諾和聯(lián)吡啶釕為信號探針,研究討論了該方法的分析檢測性能。其中,通過多元分析有效地解決了不同電致化學發(fā)光信號探針間相互影響的關(guān)鍵問題;采用HCR-RCA為基礎(chǔ)的級聯(lián)放大策略有效地提高了檢測靈敏度,實現(xiàn)了同一界面多組份的高靈敏分析檢測,為進一步構(gòu)建高通量的電致化學發(fā)光檢測系統(tǒng)奠定了良好的基礎(chǔ)。3.基于競爭法的電致化學發(fā)光免疫傳感技術(shù)用于單一界面多目標物比率分析的研究現(xiàn)今,采用抗腫瘤藥物來消滅腫瘤細胞仍作為抗擊腫瘤的一種重要手段而備受關(guān)注。然而,由于長時間使用藥物等易導致腫瘤細胞耐藥而出現(xiàn)療效差,甚至無療效的情況。若能在腫瘤細胞耐藥早期,通過調(diào)整治療方案,則可以有效地避免或減少細胞耐藥,然而尚缺乏高效、高靈敏、準確的早期細胞耐藥偵檢系統(tǒng)。電致化學發(fā)光分析作為一種高效、靈敏的潛在可用分析技術(shù)而備受關(guān)注。通常細胞耐藥性可以通過P糖蛋白表達情況來確定,而對于P糖蛋白表達分析而言,不僅僅需要對P糖蛋白的高靈敏定量分析,同時需要輔助以細胞計數(shù)或者輔助以另外一種恒定表達蛋白的高靈敏定量分析作為對照,通過計算P糖蛋白與恒定表達蛋白的濃度比值來實現(xiàn)P糖蛋白的表達分析。雖然采用如前所述的基于多元分析的多組份電致化學發(fā)光分析策略可以實現(xiàn)了同一界面上多種目標物的同時檢測。但是,多元分析仍比較耗時,亟需更為便捷有效的檢測方式實現(xiàn)細胞耐藥分析。值得注意的是,通過蛋白質(zhì)表達來評估細胞耐藥程度,僅僅需要耐藥相關(guān)蛋白與恒定表達蛋白的濃度比值即可,而不需要兩者的準確濃度。本研究通過基于免疫識別的目標物轉(zhuǎn)換結(jié)合滾環(huán)擴增信號放大策略和電致化學發(fā)光傳感器表面修飾核酸的競爭反應(yīng)構(gòu)建了一種用于比率分析的高靈敏電致化學發(fā)光分析策略,以P糖蛋白和甘油醛-3-磷酸脫氫酶為檢測模型評估腫瘤細胞耐藥程度。首先,磁珠表面的免疫識別反應(yīng),以夾心法檢測模式,通過固定于磁珠表面生物素化抗體分別特異性識別P糖蛋白和甘油醛-3-磷酸脫氫酶,并進一步結(jié)合核酸標記二抗,實現(xiàn)目標物轉(zhuǎn)化;為了提高分析檢測靈敏度,采用滾環(huán)擴增放大策略,以標記于抗體上的核酸為引發(fā)鏈,延伸得到以滾環(huán)為模板的重復序列,并經(jīng)DSN酶剪切實現(xiàn)1㑳N目標物擴增放大;剪切獲得的重復序列部分一致,能夠與電極表面的捕獲序列形成競爭反應(yīng),并進一步結(jié)合電致化學發(fā)光材料,聯(lián)吡啶釕標記信號探針,通過電致化學發(fā)光信號指示競爭反應(yīng)結(jié)果,進而實現(xiàn)P糖蛋白和甘油醛-3-磷酸脫氫酶的比率檢測,實現(xiàn)對細胞耐藥程度的評估。該比率分析策略亦可推廣于電化學、熒光、化學發(fā)光法等對于多種目標物,諸如蛋白、核酸等的高效、便捷、高靈敏的比率分析測定,同時提供了一種有效策略用于生化分析、臨床診斷,尤其是細胞功能測定等。4.基于時間調(diào)控的檢測范圍可調(diào)式電致化學發(fā)光技術(shù)研究近年來,高靈敏電致化學發(fā)光分析技術(shù)更是得到了長足的發(fā)展進步。然而,截至目前為止,絕大多數(shù)的電致化學發(fā)光分析技術(shù)由于修飾界面單一,僅僅能夠?qū)崿F(xiàn)固定靈敏度及固定檢測范圍內(nèi)的目標物分析。由于同一檢測目標物在不同樣本中的濃度差異懸殊,通常電致化學發(fā)光分析技術(shù)難于直接應(yīng)用于樣本分析檢測,而繁瑣的分離富集/稀釋等操作成為定量分析的隱性必需內(nèi)容之一,這無疑增加了分析檢測工作量,并在一定程度上降低了分析檢測的準確性。以Pb2+離子的分析檢測作為模型,本研究通過在電極表面的核酸特異性雜交,層層組裝核酸網(wǎng)狀結(jié)構(gòu),并于網(wǎng)狀結(jié)構(gòu)內(nèi)部設(shè)計DNA酶,且于網(wǎng)狀結(jié)構(gòu)的雙鏈內(nèi)鑲嵌電致化學發(fā)光材料構(gòu)建針對Pb2+離子的可調(diào)式電致化學發(fā)光傳感器。由Pb2+離子介導的DNA酶循環(huán)剪切所組裝的核酸網(wǎng)狀結(jié)構(gòu),同時釋放電致化學發(fā)光材料,導致電致化學發(fā)光信號降低,以此實現(xiàn)對Pb2+離子的定量分析。而剪切效率不僅與Pb2+離子濃度相關(guān),同時與反應(yīng)時間相關(guān),由此通過控制反應(yīng)時間以調(diào)節(jié)檢測線性范圍、檢測靈敏度等。實驗研究所構(gòu)建的可調(diào)式電致化學發(fā)光策略亦可推廣適用于其他諸如蛋白、核酸等的靈敏可調(diào)式檢測,為環(huán)境安全及臨床檢驗提供一種新型的分析策略。5.基于銥配合物納米材料及原位酶放大的電致化學發(fā)光傳感器研究三苯基吡啶銥配合物作為一種高量子轉(zhuǎn)化效率、高電子轉(zhuǎn)移效率的優(yōu)良電致化學發(fā)光材料,有望以此為基礎(chǔ)構(gòu)建更高檢測靈敏度的電致化學發(fā)光技術(shù)。然而其水溶性差、難于標記等缺陷限制了其在電致化學發(fā)光傳感技術(shù)領(lǐng)域的廣泛應(yīng)用。本研究采用三苯基吡啶銥配合物摻雜二氧化硅納米材料實現(xiàn)了電致化學發(fā)光材料的高效固載,并極大程度地改善了其水溶性。實驗研究以心衰標志物,N末端B型利鈉肽原作為研究模型,探究了基于銥配合物納米材料及原位酶放大的電致化學發(fā)光傳感器的性能。電致化學發(fā)光傳感器采用Nafion分散三苯基吡啶銥配合物摻雜二氧化硅納米材料包覆于電極表面,并進一步偶聯(lián)固定抗N末端B型利鈉肽原抗體來構(gòu)建�；诖�,所制備的電致化學發(fā)光傳感器具有較強的電致化學發(fā)光信號。當檢測樣本中含有N末端B型利鈉肽原時,其能夠通過特異性免疫識別結(jié)合于電極表面,并進一步結(jié)合葡萄糖氧化酶標記的二抗。通過葡萄糖氧化酶催化葡萄糖產(chǎn)生過氧化氫對電致化學發(fā)光信號的猝滅作用實現(xiàn)目標物N末端B型利鈉肽原的定量分析檢測。實驗研究所采用的電致化學發(fā)光分析策略亦可適用于以其他目標物,諸如核酸、細胞等物質(zhì)的高靈敏分析檢測應(yīng)用。
[Abstract]:Electrochemiluminescence (Electrochemiluminescence, ECL) is based on electrochemistry and quantificationally analyses specific substances by electrochemiluminescence caused by electrochemical reaction on the surface of the electrode. It combines the high controllability of electrochemistry and the hyper sensitivity of chemiluminescence, which greatly improves the performance of the analytical method. It has given its unique advantages, such as high sensitivity, wide linear range, simple operation, strong controllability, and rapid and simple analysis. In recent years, with the rapid development of nanotechnology and biotechnology, electrochemiluminescence technology has made vigorous progress, as a highly efficient, sensitive analysis and detection technology, in life analysis, ring At present, electrochemiluminescence materials are developing in the direction of high efficiency, green and multifunction, and electrochemiluminescence analysis technology is developing towards high sensitivity, high throughput and universality. However, the electrochemiluminescence system is still sensitive to some special research systems. This paper is based on the sensitivity, high throughput and applicability of the electrochemiluminescence detection system, through high efficiency autofilt electrochemiluminescence technology, in situ enzymatic reaction enhancement, nucleic acid amplification amplification and high efficiency electrochemiluminescence materials and other aspects. The method improves the sensitivity of electrochemiluminescence technology and improves the applicability of electrochemiluminescence (electrochemiluminescence) by multivariate analysis and competitive reaction. On this basis, the study of various diseases markers and cell function detection is realized. The research work of this paper is divided into the following parts: 1. based on the self enhancement composite nano The construction of super sensitive cell sensor and its application in drug screening are of great scientific significance and social value, especially high efficiency, low, specific, and even individualized anticancer drugs. The drug screening evaluation system based on apoptosis analysis needs more sensitive, accurate, effective and convenient. The electrochemiluminescence technology with high sensitivity, good reaction controllability and convenient and inexpensive electrochemiluminescence has great potential application value. However, the biological toxicity of electrochemiluminescence materials and co reactants and the sensitivity of analytical methods need to be further improved. A kind of self enhanced electrochemiluminescence is proposed in this study. The preparation of nanomaterials is prepared by coupling of carboxyl bipyridine ruthenium with amino side chain modified methoxy silane to prepare functionalized silica nanomaterial precursors, in which Ru is an electrochemiluminescence center and an amino side chain is used as a co reaction reagent. Enhanced electrochemiluminescence nanomaterials have been successfully applied to the construction of an anti-tumor efficacy evaluation system based on apoptosis analysis, which has achieved efficient, rapid and convenient evaluation of antitumor efficacy. The experimental study was built on the use of breast cancer tumor cells (MDA-MB-231) and antitumor drugs (paclitaxel) as models. The application of the anti-tumor efficacy evaluation system has achieved good results. The design and construction of self enhanced electrochemiluminescence nanomaterials were put forward, and the self enhancement mechanism was studied. At the same time, the application of electrochemiluminescence analysis technology in the urgent drug screening evaluation system was realized. One step extended the research and application of electrochemiluminescence analysis technology to provide reference for further electrochemiluminescence technology and drug screening and evaluation..2. based on multicomponent electrochemiluminescence analysis based on multivariate analysis to study electrochemiluminescence analysis technology, because of its low background, high sensitivity, wide linear range and low consumption. So far, most of the electrochemiluminescence analysis can only achieve a single target quantitative analysis at the same interface, and a more efficient and sensitive multi component electrochemiluminescence detection technology is urgently needed in clinical detection, environmental analysis, food safety and so on. In this study, a multicomponent electrochemiluminescence analysis strategy based on multivariate analysis was designed, and a highly sensitive multicomponent electrochemiluminescence analysis system was constructed with a cascade amplification strategy based on the hybridization chain reaction rolling ring amplification reaction. The N terminal B type natriuretic peptide and the troponin I were used. The analysis and detection performance of the method is discussed with Lumino and bipyridine ruthenium as the signal probe, and the key problem of mutual influence between different electrochemiluminescence signal probes is effectively solved by multivariate analysis. The detection sensitivity is effectively improved by using the HCR-RCA based cascade amplification strategy. The high sensitivity analysis and detection of multiple components at the same interface have laid a good foundation for the further construction of high throughput electrochemiluminescence detection system..3. based on competition based electrochemiluminescence immunosensing technology is applied to the analysis of the ratio of multiple targets in a single interface. Nowadays, the use of antitumor drugs to eliminate tumor cells is still done by using antitumor drugs. However, it is not effective and sensitive, however, to avoid or reduce the drug resistance in the early stage of cancer cell resistance. An accurate early cell resistance detection system. Electrochemiluminescence analysis has attracted much attention as a highly efficient, sensitive and potentially useful analytical technique. Generally, cell resistance can be determined by the expression of P glycoprotein. For the analysis of P glycoprotein expression, it is not only required to be highly sensitive and quantitative analysis of P glycoprotein, but also need to be analyzed. The analysis of the expression of P glycoprotein by calculating the ratio of the concentration of P glycoprotein to the constant expressed protein was aided by a cell count or assisted by a high sensitivity quantitative analysis of a constant expressed protein. Although the multi component electrochemiluminescence analysis strategy, as described earlier, could be implemented, the strategy of multicomponent Electrochemiluminescence analysis, as described earlier, could be realized. At the same time, multiple targets are detected at the same interface. However, the multivariate analysis is still more time-consuming and needs more convenient and effective detection methods to realize cell resistance analysis. It is worth noting that the degree of cell resistance is evaluated by protein expression, only the concentration ratio of drug resistance related protein and constant expression protein is needed, but it is not necessary. In this study, a highly sensitive electrochemiluminescence analysis strategy for ratio analysis was constructed by using immune recognition based target conversion combined with rolling ring amplification signal amplification strategy and the competitive reaction of electrochemiluminescence sensor surface modified nucleic acid. The P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase was used as a method. The detection model evaluated the degree of tumor cell resistance. First, the immuno recognition reaction on the surface of the magnetic beads was detected by the sandwich method, and the P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase was identified by the biotinylated antibodies fixed on the surface of the magnetic beads, and the target transformation was further combined with the nucleic acid marker two, in order to improve the analysis and detection. Sensitivity, using the rolling ring amplification strategy, the nucleic acid marked on the antibody as the trigger chain, the repeat sequence of the roll ring as the template, and the amplification and amplification of the 1? N target by the DSN enzyme shear; the repeated sequence parts of the shear are consistent, which can form a competitive reaction with the capture sequence on the electrode surface, and further combine the electromotive force. Chemiluminescent material, bipyridine ruthenium labeled signal probe, indicated the ratio of P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase by electrochemiluminescence signal, and realized the evaluation of the degree of cell resistance. The ratio analysis strategy can also be extended to various targets, such as electrochemistry, fluorescence, chemiluminescence, etc. High efficiency, convenient and highly sensitive ratio analysis, such as protein, nucleic acid, and so on, provides an effective strategy for biochemical analysis, clinical diagnosis, especially cell function determination,.4. based on time regulated detection range of tunable electrochemiluminescence technology, high sensitivity electrochemiluminescence analysis technology is more important. However, so far, most of the electrochemiluminescence analysis techniques have only been able to realize the target analysis in fixed sensitivity and fixed detection range due to the single modification interface. The electrochemiluminescence is usually caused by the difference in the concentration of the same target in different samples. Analysis technology is difficult to be applied directly to sample analysis and detection, and the complicated separation and enrichment / dilution operation becomes one of the recessive content of quantitative analysis, which undoubtedly increases the workload of analysis and detection, and reduces the accuracy of analysis and detection to a certain extent. The analysis of Pb2+ ions is used as a model. This study is carried out through the electrode table. The nucleic acid specific hybridization, the reticular formation of nucleic acid reticulation, and the design of the DNA enzyme in the reticular structure, and the construction of an adjustable electrochemiluminescence sensor for Pb2+ ions in the reticulate double chain of electrochemiluminescence materials. The nucleic acid reticular structure assembled by the Pb2+ ion mediated DNA enzyme cyclic shear was released and released at the same time. The discharge induced chemiluminescence material leads to the reduction of electrochemiluminescence signal to realize the quantitative analysis of Pb2+ ions. The shear efficiency is not only related to the concentration of Pb2+ ions, but also related to the reaction time, thereby regulating the linear range and detecting sensitivity by controlling the reaction time. The chemiluminescence strategy can also be applied to other sensitive and adjustable tests, such as proteins, nucleic acids, etc., and provides a new analytical strategy for environmental safety and clinical testing.5. based on iridium complex nanomaterials and in situ enzyme amplified electrochemiluminescence sensors for the study of the three phenyl pyridine iridium complex as a kind of high quantum conversion efficiency. The excellent electrochemiluminescence materials with high electron transfer efficiency and high electron transfer efficiency are expected to build higher detection sensitivity electrochemiluminescence (electrochemiluminescence) technology on this basis. However, their poor solubility in water, difficult to label and other defects restrict its wide application in the field of electrochemiluminescence sensing technology. This study uses three phenyl pyridine iridium complexes to doping two oxygen. Silicon nanomaterials have realized the high efficiency of electrochemiluminescent materials and greatly improved their water solubility. The performance of electrochemiluminescence sensors based on iridium complex nanomaterials and in situ enzyme amplified electrochemiluminescence sensors was investigated by using the markers of heart failure and the B natriuretic peptide at the end of N. The inductor was coated on the surface of the electrode with Nafion dispersed three phenyl pyridine iridium complex doped silica nanomaterials and further coupled with the immobilized anti N terminal B natriuretic peptide antibody. Based on this, the electrochemiluminescence sensor has a strong electrochemiluminescence signal. When the detection sample contains the N terminal B type natrium At the time of peptide, it can be combined with the surface of the electrode by specific immune recognition, and further combined with the two resistance of glucose oxidase labeling. The quantitative analysis and detection of the B type natriuretic peptide in the N terminal of the target by glucose oxidase catalyzing the quenching of glucose producing hydrogen peroxide to the electrochemiluminescence signal. The electrochemiluminescence analysis strategy can also be applied to high sensitive analytical applications of other targets, such as nucleic acids, cells and other substances.
【學位授予單位】：西南大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：O657.3

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