本文選題：傳感器陣列 + 卟啉��； 參考：《重慶大學》2011年博士論文

【摘要】：細胞代謝產(chǎn)物與細胞生命活動過程密切相關(guān),可以準確的反應細胞生理病理狀態(tài)。與基因或者蛋白質(zhì)相比,細胞間信號轉(zhuǎn)導、能量傳遞等細胞內(nèi)生命過程多發(fā)生在代謝層面,通過細胞代謝產(chǎn)物進行調(diào)控。對細胞代謝產(chǎn)物中的生物分子定性定量檢測,并尋找細胞微量代謝物與生理病理變化的對應關(guān)系,對于醫(yī)學診斷具有相當重要的作用。目前,在生物分子及生物體系狀態(tài)的檢測中常用的方法是大型儀器檢測,大型儀器雖然保證了檢測的準確率,卻帶來了高額的檢測費用和低下的檢測效率;電子鼻/舌等新興技術(shù)雖然在構(gòu)建原理和加工制備等方面已較為完善,但是由于敏感材料的選擇性和穩(wěn)定性問題也無法廣泛應用于實際檢測中。本研究立足于開發(fā)一種新型可視陣列傳感器系統(tǒng),以實現(xiàn)對生物分子及生物體系狀態(tài)的快速檢測。 可視化傳感陣列是通過模擬哺乳動物味覺/嗅覺識別過程,將多種具有一定特異性和敏感性的廣譜識別材料作為敏感單元,以目標混合物作為獨立樣本進行分析,獲取其特征指紋,并利用模式識別算法實現(xiàn)對目標物質(zhì)的特異性識別與鑒定,在混合物檢測方面具有獨特的優(yōu)勢。其中,卟啉及其衍生物因其良好的生物相容性和光敏性能成為彩色可視傳感器研究熱點之一,以其作為傳感元件具有靈敏度高,穩(wěn)定性好,易于修飾改性等特點。本研究以卟啉及其衍生物作為敏感材料構(gòu)建彩色可視化傳感陣列,并應用這種陣列傳感器、對氨基酸、蛋白質(zhì)及細胞代謝產(chǎn)物的檢測,可以實現(xiàn)對正常細胞和病理細胞代謝周期的特異性檢測,有望為藥物的體外篩選提供有效平臺。所完成的主要工作和結(jié)論如下: 1.完成了卟啉可視陣列傳感器系統(tǒng)材料的合成與篩選 以氨基酸為檢測對象,采用Adler法設(shè)計并合成了水溶性磺酸基取代卟啉衍生物(PS1和PS4)以及卟啉金屬衍生物(P1-P5)。通過分析卟啉、卟啉衍生物及指示劑染料與十種氨基酸反應后紫外可見光譜變化,說明卟啉及其衍生物和指示劑染料可以與氨基酸結(jié)合程度不同,對氨基酸具有一定的親和力和選擇性,產(chǎn)生可用于分析的模式信號。并用類似方法篩選得到一個含有36個敏感單元的彩色可視檢測陣列,通過各個傳感受體的交叉響應,用于生物分子的檢測。 2.完成了卟啉可視陣列傳感器系統(tǒng)的構(gòu)建工作 詳細闡述了卟啉可視陣列傳感器系統(tǒng)構(gòu)建的設(shè)計方案及檢測方法,對卟啉可視陣列傳感器系統(tǒng)進行了硬件設(shè)計、軟件設(shè)計及傳感芯片設(shè)計。通過硬件設(shè)計實現(xiàn)了傳感芯片反應前后的圖像采集,通過軟件設(shè)計實現(xiàn)了反應前后芯片圖像的處理、可視化指紋響應差譜圖的輸出和數(shù)據(jù)的保存。并利用溶膠凝膠法,按照TEOS:水:鹽酸:乙醇:染料(卟啉、卟啉衍生物及pH指示劑)體積比為25:4:3:15:10的比例制備溶膠凝膠溶液,通過毛細管點樣構(gòu)建了一種6×6的傳感陣列,得到了一種對生物分子和生物體系具有識別能力和廣譜識別效果的傳感芯片,為人工舌檢測系統(tǒng)的構(gòu)建奠定基礎(chǔ)。 3.應用構(gòu)建的卟啉可視陣列傳感器系統(tǒng)開展了對生命活動中重要生物分子氨基酸、糖類化合物和蛋白質(zhì)的檢測研究 氨基酸、糖類化合物和蛋白質(zhì)是構(gòu)成生命體的基本物質(zhì),是細胞代謝的基礎(chǔ)物質(zhì)。利用所構(gòu)建的可視化陣列傳感系統(tǒng),對十種常見的氨基酸、九種糖類化合物及其混合物以及七種蛋白質(zhì)及混合物和三種熱變性蛋白進行了檢測,獲得了其特異性指紋圖譜。結(jié)合紫外可見光譜和密度泛函理論,從理論分析和化學計算角度分析了卟啉PS1與氨基酸間相互作用機理,表明氨基酸的殘基結(jié)構(gòu)是影響其與卟啉分子間相互作用的關(guān)鍵。采用聚類分析(HCA)、主成分分析(PCA)以及判別分析(LDA)等模式識別方法對彩色陣列的RGB分析結(jié)果顯示。分析結(jié)果顯示,本文構(gòu)建的可視陣列傳感器系統(tǒng)能夠顯著的區(qū)別不同的氨基酸,并且反映出不同氨基酸之間的性質(zhì)和結(jié)構(gòu)差異;通過對糖類化合物及其混合物以及蛋白質(zhì)及其混合體系以及變性蛋白的檢測,結(jié)果表明,可視傳感陣列可以顯示出出幾類糖類化學我和蛋白之間在性質(zhì)和結(jié)構(gòu)上的真實差異。 4.應用傳感陣列檢測系統(tǒng)實現(xiàn)了對內(nèi)皮細胞生長周期的鑒別 利用可視化卟啉傳感陣列對人臍靜脈內(nèi)皮細胞(HUVEC)培養(yǎng)1-10d的細胞代謝液進行了檢測,檢測結(jié)果數(shù)據(jù)采用聚類分析和主成分分析(PCA)進行了計算和分析,并結(jié)合MTT比色法和紫外可見光譜法對陣列與細胞代謝的響應進行了驗證。實驗結(jié)果顯示,通過陣列響應的可視差圖可以對HUVEC培養(yǎng)1-10d的細胞代謝液進行顯著區(qū)別。聚類分析、主成分分析和判別分析結(jié)果表明,彩色可視傳感陣列能較好的區(qū)分人臍靜脈內(nèi)皮細胞不同培養(yǎng)時間下的生長代謝差異,并具有較好的重復性和可重現(xiàn)性。另外,將陣列指紋圖譜的歐氏距離與紫外可見光譜的峰值點進行綜合分析,表明所構(gòu)建的可視陣列傳感系統(tǒng)在檢測過程中能夠有效捕捉細胞代謝過程中代謝物的特征,在細胞識別、探究細胞生命活動規(guī)律、細胞研究和藥物篩選等領(lǐng)域具有潛在應用價值。 5.基于微流控技術(shù)實現(xiàn)對正�；ず筒±砘ぜ毎淖R別與檢測 介紹了微流控芯片的制備加工過程,考察了該體系對細胞分離和培養(yǎng)的效果,并利用可視化傳感器陣列檢測了正�；ぜ毎�(NHS)和關(guān)節(jié)炎滑膜細胞(RA)培養(yǎng)1-9天的代謝液的變化。HCA和PCA分析的結(jié)果表明彩色可視傳感陣列可以有效辨別不同培養(yǎng)時間下的細胞生長代謝差異。對細胞培養(yǎng)1-9d生長代謝液檢測的共57個樣本總體結(jié)果LDA分析準確率也高達94%,說明充分表明本文所用彩色可視傳感陣列對細胞代謝液具有很好的識別效果,可以準確的識別細胞代謝液中微量成分的變化。上述內(nèi)容說明可視傳感陣列系統(tǒng)不僅能為類風濕性關(guān)節(jié)炎致病機理的研究提供一個良好的研究平臺,同時在相關(guān)抗關(guān)節(jié)炎藥物、抗血管生成藥物的篩選方面也有潛在的應用價值。
[Abstract]:Cell metabolism products are closely related to the process of cell life activity, which can accurately reflect the physiological and pathological state of cells. Compared with genes or proteins, the intercellular life processes such as signal transduction and energy transfer occur at the metabolic level, and are regulated by the metabolites of cells. Quantitative detection and finding the correspondence between cell trace metabolites and physiological and pathological changes is very important for medical diagnosis. At present, large instrument detection is commonly used in the detection of biological molecules and biological system states. Although large instruments have guaranteed the accuracy of detection, they bring high detection fees. The new technologies, such as electronic nose / tongue, have been improved in construction principle and processing, but the problem of selectivity and stability of sensitive materials can not be widely used in actual detection. This study is based on developing a new type of optic array sensor system to realize biological molecules. And the rapid detection of the state of the biological system.
By simulating the taste / smell recognition process of mammal, a variety of wide spectrum recognition materials with certain specificity and sensitivity are used as sensitive units. The target mixture is analyzed as an independent sample, and its characteristic fingerprint is obtained. The specific identification and identification of the target material are realized by the pattern recognition algorithm. In addition, porphyrin and its derivatives have become one of the hotspots in the study of color visual sensors because of their good biocompatibility and photosensitivity. In this study, porphyrin and its derivatives are sensitive to their sensitivity, stability and modification. The application of this array sensor to the detection of amino acid, protein and cell metabolites can be used to detect the specific metabolic cycle of normal and pathological cells. It is expected to provide an effective platform for the screening of drugs in vitro. The main work and conclusions are as follows:
1. we completed the synthesis and screening of porphyrin visible array sensor materials.
Water soluble sulfonated porphyrin derivatives (PS1 and PS4) and porphyrin metal derivatives (P1-P5) were designed and synthesized by Adler method. The UV visible spectrum changes after reaction of porphyrin, porphyrin derivative and indicator dye and ten kinds of amino acids were analyzed. The results showed that porphyrin and its derivatives and indicator dyes were available. With the degree of binding to amino acids, the amino acids have a certain affinity and selectivity, producing a pattern signal that can be used for analysis. A color visual detection array containing 36 sensitive units is screened by similar methods, and is used for the detection of biomolecules through the cross response of each sensor receptor.
2. completed the construction of porphyrin visible array sensor system.
The design scheme and detection method of the porphyrin visual array sensor system are elaborated in detail. The hardware design, software design and sensor chip design of the porphyrin visual array sensor system are carried out. The image collection before and after the reaction of the sensor chip is realized through the hardware design, and the chip image before and after the reaction is realized through the software design. With the sol-gel method, sol-gel solution was prepared by sol-gel method in the proportion of TEOS: water: hydrochloric acid: ethanol: dye (porphyrin, porphyrin derivative and pH indicator) in proportion to 25:4:3:15:10, and a 6 * 6 sensor array was constructed by capillary point sample, and a kind of generation was obtained. The sensor and chip have the ability of identifying and broad-spectrum recognition, which lays the foundation for the construction of artificial tongue detection system.
3. the porphyrin visible array sensor system has been applied to the detection of important biomolecules amino acids, carbohydrates and proteins in life activities.
Amino acids, carbohydrates and proteins are the basic substances that constitute the life body and the basic substance of cell metabolism. Using the visualized array sensing system constructed, ten kinds of common amino acids, nine kinds of carbohydrates and their mixtures, seven kinds of proteins and mixtures and three kinds of thermal denaturation proteins have been detected. Specific fingerprints. The interaction mechanism between porphyrin PS1 and amino acids is analyzed from theoretical analysis and chemical calculation with the combination of UV visible spectrum and density functional theory. It is shown that the structure of the amino acid residues is the key to influence the interaction between porphyrin and porphyrin molecules. Cluster analysis (HCA), principal component analysis (PCA) and discriminant are used. Analysis (LDA) and other pattern recognition methods show the results of RGB analysis of color array. The analysis results show that the visual array sensor system constructed in this paper can distinguish different amino acids significantly, and reflect the properties and structural differences between different amino acids, and the combination of sugar compounds and their mixtures and proteins and their mixing. The system and the detection of denatured proteins show that the visual sensing array can show the true differences in the nature and structure of several kinds of sugar chemistry between me and the protein.
4. the application of sensor array detection system has realized the identification of endothelial cell growth cycle.
A visual porphyrin sensor array was used to detect the metabolic fluid of 1-10d in human umbilical vein endothelial cells (HUVEC). The results were calculated and analyzed by cluster analysis and principal component analysis (PCA), and the response of the array and cell metabolism was verified by MTT colorimetry and UV visible spectroscopy. The results show that the visual difference diagram of the array response can be significantly different from the cell metabolism fluid in the HUVEC culture of 1-10d. Cluster analysis, principal component analysis and discriminant analysis show that the color visual sensing array can distinguish the difference in the growth and metabolism of human umbilical vein endothelial cells at different incubation times and have good reproducibility. In addition, the Euclidean distance of the array fingerprint and the peak value of UV visible spectrum are synthetically analyzed. It shows that the constructed visual array sensing system can effectively capture the characteristics of metabolites during cell metabolism, explore cell life activities, cell research and drugs in cell recognition. The fields of screening are of potential application value.
5. identification and detection of normal synovium and pathological synovial cells based on microfluidics technology
The process of microfluidic chip preparation was introduced. The effect of the system on cell separation and culture was investigated. The changes of metabolic fluid in normal synovial cells (NHS) and arthritis synovial cells (RA) were detected by visual sensor array for 1-9 days. The results of.HCA and PCA analysis showed that the color visual sensing array could be effectively identified. The difference of cell growth and metabolism at different culture times. The total results of 57 samples for the detection of 1-9d growth and metabolic fluid in cell culture were also 94%, which indicated that the color visual sensing array used in this paper had a good recognition effect on the cell metabolism fluid and could accurately identify the microamount in the cell metabolism fluid. The above contents indicate that the visual sensing array system can not only provide a good research platform for the research on the pathogenesis of rheumatoid arthritis, but also have potential application value in the screening of anti arthritis drugs and antiangiogenic drugs.
【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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