Rapid Single Molecule Detection System for Intracellular Protease Activity

描述(由申请人提供)：该项目将导致基于单分子检测的新细胞筛选技术的发展。基于之前使用荧光半胱氨酸天冬氨酸酶探针的结果，常规工具通常在几个小时后观察到的细胞过程可以在不到一个小时内检测到。我们将扩展这些结果以开发一种微流控细胞扫描系统，该系统可以自动检测荧光探针。利用单分子荧光，可以同时测定多个参数，包括荧光爆发强度(计数分子)、荧光重现时间(扩散校正浓度)和荧光相关时间(荧光相关光谱)。将开发以红色激光激发为特色的新的荧光蛋白酶探针。我们将使用细胞凋亡作为我们的测试模型，并开发caspase特异性探针。蛋白质裂解后，荧光探针将转化为发射波长在660-690 nm附近的荧光分子。光谱的这一区域不受细胞自发荧光的影响，将允许更快的检测。其结果将是一种在活细胞中以高时间分辨率分析生化系统的方法。为了提高我们现有系统的细胞吞吐量，我们将开发一个自动微流控扫描和数据分析系统。细胞将被扫描通过激光光束，使用分段流动和激光捕获。随后将获得单分子数据。软件中的决策模块将比较测量的参数和控制值，并根据蛋白酶活性对细胞进行分类。该项目的最终结果将是一种能够进行多参数荧光检测、单分子灵敏度和自动细胞生产能力的仪器。虽然细胞凋亡和caspase活性被选为测试案例，但这种仪器和方法可以很容易地适用于其他生物医学问题。 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，以高时间分辨率研究细胞内蛋白质-底物相互作用。这种方法将允许以更高的吞吐量和灵敏度来研究生物过程。

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.