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开发。基于先前使用荧光半胱天冬酶探针的结果，通常在几个小时后通过常规工具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，以开发一个微流控细胞扫描系统，可以分析荧光探针在一个自动化的方式。使用单分子荧光，可以同时确定几个参数;包括荧光猝发强度（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）。将开发新的荧光蛋白酶探针，其特征是红色激光激发。我们将使用细胞凋亡作为我们的测试模型，并开发半胱天冬酶特异性探针。在蛋白水解裂解后，荧光探针将转化为具有接近660-690 nm的发射的荧光分子。光谱的该区域不受细胞自发荧光的影响，并且将允许更快的检测。其结果将是一种在活细胞中以高时间分辨率测定生物化学系统的方法。为了提高我们现有系统的细胞通量，将开发自动化微流体扫描和数据分析系统。使用分段流动和激光捕获将细胞扫描通过激光束。随后将获得单分子数据。软件中的决策模块将测量参数与对照值进行比较，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通量的仪器。虽然细胞凋亡和半胱天冬酶活性被选为测试案例，这种仪器和方法可以很容易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，以高时间分辨率研究细胞内蛋白质-底物相互作用。这种方法将使生物过程的研究具有更高的通量和灵敏度。

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.