Rapid Single Molecule Detection System for Intracellular Protease Activity

细胞内蛋白酶活性快速单分子检测系统

• 批准号: 7930154
• 负责人: Dimitri Pappas
• 金额: $ 17.38万
• 依托单位: TEXAS TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7930154
• 关键词: Apoptosis; Apoptotic; Binding; Biochemical; Biological; Biological Assay; Biological Process; Caspase; Cell physiology; Cells; Cleaved cell; Computer software; Data; Data Analyses; Decision Making; Detection; Development; Diffusion; Dyes; Event; Flow Cytometry; Fluorescence; Fluorescent Probes; Goals; Hour; Induction of Apoptosis; Inhibition of Apoptosis; Lasers; Left; Life; Measurement; Measures; Methodology; Methods; Microfluidics; Microscopy; Modeling; Molecular; Molecular Probes; Monitor; Pathway interactions; Peptide Hydrolases; Pharmaceutical Preparations; Process; Proteins; Recurrence; Reporting; Research; Research Personnel; Resolution; S Phase; Scanning; Screening procedure; Spectrum Analysis; Speed; Substrate Interaction; System; Techniques; Technology; Testing; Time; analytical method; base; caspase-8; improved; innovation; instrument; instrumentation; novel; public health relevance; response; single molecule; tool

DESCRIPTION (provided by applicant): This project will result in the development of new cell-screening technologies based on single molecule detection. Based on previous results using fluorogenic caspase probes, cellular processes that are normally observed after several hours by conventional tools can be detected in less than one hour. We will extend these results to develop a microfluidic cell-scanning system that can assay fluorogenic probes in an automated fashion. Using single molecule fluorescence, several parameters can be determined simultaneously; including fluorescence burst intensity (counting molecules), fluorescence recurrence time (diffusion-corrected concentration), and fluorescence correlation time (fluorescence correlation spectroscopy). New fluorogenic protease probes will be developed that feature red-laser excitation. We will use apoptosis as our test model, and develop caspase-specific probes. Upon proteolytic cleavage, the fluorogenic probe will be converted to a fluorescent molecule with an emission near 660-690 nm. This region of the spectrum is free from cell autofluorescence and will allow for even faster detection. The result will be a method for assaying a biochemical system, in living cells, with high temporal resolution. In an effort to improve the cell throughput of our existing system, an automated microfluidic scanning and data analysis system will be developed. Cells will be scanned past the laser beam using segmented flow and laser trapping. Single molecule data will be subsequently obtained. A decision-making module in the software will compare measured parameters to control values and classify cells according to protease activity. The end result of this project will be an instrument capable of multiparameter fluorescence detection, single molecule sensitivity, and automated cell throughput. While apoptosis and caspase activity are chosen as a test case, this instrumentation and methodology can be adapted easily to other biomedical problems. PUBLIC HEALTH RELEVANCE (provided by the applicant): We will develop new molecular probes, instrumentation, and methods to study intracellular protein-substrate interactions with high temporal resolution. This methodology will allow biological processes to be studied with higher throughput and sensitivity.

描述（由申请人提供）：该项目将基于单分子检测来开发新的细胞筛查技术。基于使用荧光caspase探针的先前结果，可以在不到一小时的时间内检测到几个小时后通常在几个小时后观察到的细胞过程。我们将扩展这些结果，以开发一个微流体扫描系统，该系统可以自动化的方式测定荧光探针。使用单分子荧光，可以同时确定几个参数。包括荧光爆发强度（计数分子），荧光复发时间（扩散校正浓度）和荧光相关时间（荧光相关光谱法）。将开发新的荧光蛋白酶探针，具有红色激动激发。我们将使用凋亡作为我们的测试模型，并开发caspase特异性探针。蛋白水解裂解后，荧光探针将转换为荧光分子，其发射接近660-690 nm。该频谱的该区域不含细胞自动荧光，将允许更快的检测。结果将是一种在活细胞中分析生化系统，具有高时间分辨率的方法。为了改善现有系统的细胞吞吐量，将开发自动的微流体扫描和数据分析系统。通过分段流和激光捕获，将将细胞扫描过激光束。随后将获得单分子数据。软件中的决策模块将根据蛋白酶活性比较测量的参数以控制值和分类。该项目的最终结果将是一种能够使用多参数荧光检测，单分子灵敏度和自动细胞吞吐量的仪器。虽然选择了凋亡和胱天蛋白酶活性作为测试案例，但该仪器和方法可以轻松地适应其他生物医学问题。 公共卫生相关性（由申请人提供）：我们将开发新的分子探针，仪器和方法来研究具有高时间分辨率的细胞内蛋白质 - 基底相互作用。该方法将允许以更高的吞吐量和灵敏度研究生物学过程。