Novel Instrumentation For Direct On-Line Monitoring of Biological Samples in Real-Time With High-Speed Gas Chromatography

过氧化脂质或其产物最近被认为是信号转导通路中的生物第二信使，诱导细胞保护酶，刺激蛋白激酶，介导细胞程序性死亡，并以类似于血小板激活因子的方式刺激中性粒细胞黏附和平滑肌细胞增殖。鉴定和定量脂质过氧化产物将加深对这些生物介体的酶学、生物化学和生物处置的理解。目前细胞或亚细胞组分中这些产物的检测方法包括分光光度分析和高效液相色谱法，但不能实时产生数据。本项目将开发实时监测组织培养中生物过程的仪器和方法。两名全日制研究生将协助研究和实施过程。该仪器将包括一个带有冷冻聚焦入口的细胞孵化装置，适合与高速气相色谱(HSGC)一起使用，并将与各种平台兼容。细胞将在37摄氏度的烤箱中培养，在标准的组织培养瓶中配备专门的盖子。瓶盖用于隔离烧瓶内的产品，同时提供空气和二氧化碳。直接监测将通过将气体产品集中在低温聚焦捕集器上以注入HSGC系统来完成。该项目的长期目标是开发一个集成的、模块化的孵化器/进水口，为实时监测生物系统提供新的能力。该仪器可用于许多系统的研究，尽管这项工作将重点放在脂质过氧化产物的表征上。为了进气设计测试和概念验证工作，将在从大鼠肝脏分离的微粒体部分中刺激脂质过氧化，从而产生相对大量的产物。检测将用飞行时间(TOF)质谱仪完成。TOF仪器的高扫描速率与高速GC兼容，第二维数据有助于对过氧化产物的阳性识别。对于复杂和动态的生物系统来说，这是一个决定性的优势。在该项目的第二阶段将建造一个专门的孵化器，用于细胞培养。最终的阀门配置将进行测试和优化。为了开发出一种适合专利申请和市场销售的仪器，将对仪器的坚固性进行详细的测试。

Lipid peroxides or their products have recently been cited as biological secondmessengers in signal transduction pathways that induce cytoprotective enzymes, stimulateprotein kinases, mediate programmed cell death, and stimulate neutrophil adhesion andsmooth muscle cell proliferation in a manner similar to platelet activating factor.Identification and quantification of lipid peroxidation products will enhanceunderstanding of the enzymology, biochemistry, and biodisposition of these biologicalmediators. The current detection methods for these products in cells or subcellularfractions include spectrophotometric assays and liquid chromatography, though none arecapable of generating data in real-time.Instrumentation and methods to monitor biological processes in tissue cultures in real-timewill be developed in this project. Two full-time graduate students will assist in the research and implementation process. The instrument will incorporate a cell incubation device with a cryofocusing inlet suitable for use with high-speed gas chromatography (HSGC), and willbe compatible with various platforms. Cells will be cultured in a 37 degrees C oven in standard tissue culture flasks fitted with specialized caps. The caps serve to isolate theproducts within a flask while providing air and CO2. Direct monitoring will be accomplished by concentrating gaseous products on a cryofocusing trap for injection into a HSGC system. The long-term goal of this project is to develop an integrated, modular incubator/inlet that will provide new capabilities for monitoring biological systems in real-time. The instrument could be used to study many systems, although this work will focus on the characterization of lipid peroxidation products.For inlet design testing and proof of concept work, lipid peroxidation will be stimulatedin the isolated microsomal fraction from rat liver, which generates relatively largeamounts of products. Detection will be accomplished with time of flight (TOF) massspectrometry. The high scanning rates of TOF instruments are compatible with high speedGC, and the second dimension of data facilitates positive identification of peroxidationproducts. This is a decided advantage for complex and dynamic biological systems. Aspecialized incubator will be constructed in the second phase of the project for use withcell cultures. Final valving configurations will be tested and optimized. The ruggednessof the instrument will be examined in detail, in order to develop an instrument suitablefor patent application and marketing.