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

脂质过氧化物及其产物是细胞信号转导途径中的第二信使，与血小板活化因子类似，可诱导细胞保护酶，刺激蛋白激酶，介导程序性细胞死亡，刺激中性粒细胞粘附和平滑肌细胞增殖，对脂质过氧化产物的鉴定和定量将有助于加深对脂质过氧化的酶学、生物化学、and biodisposition生物disposal处置of these biologicalmediators生物介质.目前检测细胞或亚细胞组分中这些产物的方法包括分光光度测定法和液相色谱法，但都不能实时产生数据。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。 该仪器将包含一个细胞孵育装置，该装置具有适合与高速气相色谱（HSGC）一起使用的低温聚焦入口，并将与各种平台兼容。细胞将在37 ℃烘箱中在配有专用盖的标准组织培养瓶中培养。瓶盖用来隔离烧瓶内的产品，同时提供空气和CO2。直接监测将通过在低温聚焦阱上浓缩气态产物以注入HSGC系统来实现。该项目的长期目标是开发一种集成的模块化培养箱/入口，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，尽管这项工作将侧重于脂质过氧化产物的表征。对于入口设计测试和概念验证工作，脂质过氧化将在大鼠肝脏分离的微粒体组分中被刺激，从而产生相对大量的产物。将使用飞行时间（TOF）质谱法完成检测。TOF仪器的高扫描速率与高速GC兼容，并且数据的第二维有利于过氧化物产物的阳性鉴定。这对于复杂和动态的生物系统来说是一个决定性的优势。第二阶段将建造一个专门的培养箱，用于细胞培养。将对最终阀门配置进行测试和优化。为了研制出一种适合于专利申请和市场销售的仪器，将对仪器的耐用性进行详细的研究。

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.