Rapid Single Molecule Detection System for Intracellular Protease Activity

项目描述（由申请人提供）：本项目将发展基于单分子检测的新型细胞筛选技术。根据以前使用荧光caspase探针的结果，通常用传统工具需要几个小时才能观察到的细胞过程可以在不到一个小时的时间内检测到。我们将扩展这些结果，以开发一种微流体细胞扫描系统，该系统可以自动检测荧光探针。采用单分子荧光法，可同时测定多个参数；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）。新型荧光蛋白酶探针将以红色激光激发为特征。我们将以细胞凋亡为实验模型，开发caspase特异性探针。在蛋白水解裂解后，荧光探针将转化为荧光分子，其发射波长在660- 690nm附近。这个光谱区域不受细胞自身荧光的影响，可以更快地进行检测。结果将是一种分析生物化学系统的方法，在活细胞中，具有高时间分辨率。为了提高现有系统的细胞通量，我们将开发一种自动微流控扫描和数据分析系统。细胞将被扫描通过激光束使用分段流和激光捕获。随后将获得单分子数据。软件中的决策模块将比较测量参数以控制值，并根据蛋白酶活性对细胞进行分类。这个项目的最终结果将是一个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酶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。

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.