基于可视阵列传感对细胞代谢过程的研究

发布时间：2018-05-21 18:16

本文选题：传感器阵列 + 卟啉　；参考：《重庆大学》2011年博士论文

【摘要】：细胞代谢产物与细胞生命活动过程密切相关,可以准确的反应细胞生理病理状态。与基因或者蛋白质相比,细胞间信号转导、能量传递等细胞内生命过程多发生在代谢层面,通过细胞代谢产物进行调控。对细胞代谢产物中的生物分子定性定量检测,并寻找细胞微量代谢物与生理病理变化的对应关系,对于医学诊断具有相当重要的作用。目前,在生物分子及生物体系状态的检测中常用的方法是大型仪器检测,大型仪器虽然保证了检测的准确率,却带来了高额的检测费用和低下的检测效率;电子鼻/舌等新兴技术虽然在构建原理和加工制备等方面已较为完善,但是由于敏感材料的选择性和稳定性问题也无法广泛应用于实际检测中。本研究立足于开发一种新型可视阵列传感器系统,以实现对生物分子及生物体系状态的快速检测。可视化传感阵列是通过模拟哺乳动物味觉/嗅觉识别过程,将多种具有一定特异性和敏感性的广谱识别材料作为敏感单元,以目标混合物作为独立样本进行分析,获取其特征指纹,并利用模式识别算法实现对目标物质的特异性识别与鉴定,在混合物检测方面具有独特的优势。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为彩色可视传感器研究热点之一,以其作为传感元件具有灵敏度高,稳定性好,易于修饰改性等特点。本研究以卟啉及其衍生物作为敏感材料构建彩色可视化传感阵列,并应用这种阵列传感器、对氨基酸、蛋白质及细胞代谢产物的检测,可以实现对正常细胞和病理细胞代谢周期的特异性检测,有望为药物的体外筛选提供有效平台。所完成的主要工作和结论如下: 1.完成了卟啉可视阵列传感器系统材料的合成与筛选以氨基酸为检测对象,采用Adler法设计并合成了水溶性磺酸基取代卟啉衍生物(PS1和PS4)以及卟啉金属衍生物(P1-P5)。通过分析卟啉、卟啉衍生物及指示剂染料与十种氨基酸反应后紫外可见光谱变化,说明卟啉及其衍生物和指示剂染料可以与氨基酸结合程度不同,对氨基酸具有一定的亲和力和选择性,产生可用于分析的模式信号。并用类似方法筛选得到一个含有36个敏感单元的彩色可视检测阵列,通过各个传感受体的交叉响应,用于生物分子的检测。 2.完成了卟啉可视阵列传感器系统的构建工作详细阐述了卟啉可视阵列传感器系统构建的设计方案及检测方法,对卟啉可视阵列传感器系统进行了硬件设计、软件设计及传感芯片设计。通过硬件设计实现了传感芯片反应前后的图像采集,通过软件设计实现了反应前后芯片图像的处理、可视化指纹响应差谱图的输出和数据的保存。并利用溶胶凝胶法,按照TEOS:水:盐酸:乙醇:染料(卟啉、卟啉衍生物及pH指示剂)体积比为25:4:3:15:10的比例制备溶胶凝胶溶液,通过毛细管点样构建了一种6×6的传感阵列,得到了一种对生物分子和生物体系具有识别能力和广谱识别效果的传感芯片,为人工舌检测系统的构建奠定基础。 3.应用构建的卟啉可视阵列传感器系统开展了对生命活动中重要生物分子氨基酸、糖类化合物和蛋白质的检测研究氨基酸、糖类化合物和蛋白质是构成生命体的基本物质,是细胞代谢的基础物质。利用所构建的可视化阵列传感系统,对十种常见的氨基酸、九种糖类化合物及其混合物以及七种蛋白质及混合物和三种热变性蛋白进行了检测,获得了其特异性指纹图谱。结合紫外可见光谱和密度泛函理论,从理论分析和化学计算角度分析了卟啉PS1与氨基酸间相互作用机理,表明氨基酸的残基结构是影响其与卟啉分子间相互作用的关键。采用聚类分析(HCA)、主成分分析(PCA)以及判别分析(LDA)等模式识别方法对彩色阵列的RGB分析结果显示。分析结果显示,本文构建的可视阵列传感器系统能够显著的区别不同的氨基酸,并且反映出不同氨基酸之间的性质和结构差异;通过对糖类化合物及其混合物以及蛋白质及其混合体系以及变性蛋白的检测,结果表明,可视传感阵列可以显示出出几类糖类化学我和蛋白之间在性质和结构上的真实差异。 4.应用传感阵列检测系统实现了对内皮细胞生长周期的鉴别利用可视化卟啉传感阵列对人脐静脉内皮细胞(HUVEC)培养1-10d的细胞代谢液进行了检测,检测结果数据采用聚类分析和主成分分析(PCA)进行了计算和分析,并结合MTT比色法和紫外可见光谱法对阵列与细胞代谢的响应进行了验证。实验结果显示,通过阵列响应的可视差图可以对HUVEC培养1-10d的细胞代谢液进行显著区别。聚类分析、主成分分析和判别分析结果表明,彩色可视传感阵列能较好的区分人脐静脉内皮细胞不同培养时间下的生长代谢差异,并具有较好的重复性和可重现性。另外,将阵列指纹图谱的欧氏距离与紫外可见光谱的峰值点进行综合分析,表明所构建的可视阵列传感系统在检测过程中能够有效捕捉细胞代谢过程中代谢物的特征,在细胞识别、探究细胞生命活动规律、细胞研究和药物筛选等领域具有潜在应用价值。 5.基于微流控技术实现对正常滑膜和病理滑膜细胞的识别与检测介绍了微流控芯片的制备加工过程,考察了该体系对细胞分离和培养的效果,并利用可视化传感器阵列检测了正常滑膜细胞(NHS)和关节炎滑膜细胞(RA)培养1-9天的代谢液的变化。HCA和PCA分析的结果表明彩色可视传感阵列可以有效辨别不同培养时间下的细胞生长代谢差异。对细胞培养1-9d生长代谢液检测的共57个样本总体结果LDA分析准确率也高达94%,说明充分表明本文所用彩色可视传感阵列对细胞代谢液具有很好的识别效果,可以准确的识别细胞代谢液中微量成分的变化。上述内容说明可视传感阵列系统不仅能为类风湿性关节炎致病机理的研究提供一个良好的研究平台,同时在相关抗关节炎药物、抗血管生成药物的筛选方面也有潜在的应用价值。
[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.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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