Protease chain reactions for molecular analysis of cancer markers

描述(申请人提供)：我们的目标是开发一种蛋白链式反应技术(PROR)，能够对癌症标记物进行超灵敏和高度定量的检测。酶链式反应的基本成分是酶-抑制物复合体。当蛋白水解酶与抑制物结合时是不活跃的，但一旦释放，就能够裂解抑制物并释放更多的游离蛋白水解酶。微量的游离蛋白酶启动链式反应，最终导致所有的蛋白酶从抑制复合体中释放出来。蛋白水解酶完全释放前的滞后时间由游离水解酶的初始浓度决定。第一阶段项目开发了第一代自放大复合体，形成了procR检测技术的核心。所有第一阶段的里程碑都大大超过了。在第二阶段，我们将开发基于自放大络合物的功能强大的分子传感器。传感器由三个元件组成：1)响应分析物存在的传感元件；2)放大和量化来自检测元件的信号的处理/计算元件；以及3)产生光信号的换能器元件。通过这种结合，将建造极其复杂的酶传感器来检测癌症标记物。因此，三个实验目标是：1)设计传感元件；2)设计增强的处理/计算元件；以及3)设计信号组件。从根本上说，蛋白质链式反应是一台功能强大的模拟计算机，具有两个关键特征，极大地促进了目标分子的检测。1)它可以将特定目标分子的浓度转换为时间信号。2)它可以产生巨大的信号放大，类似于通过聚合酶链式反应(PCR)扩增DNA。因此，检测是可能的，因为目标分子产生的最终可观察信号可能非常大，并且信号开始之前的时间延迟与目标分子的浓度精确相关。其长期目标是开发酶抑制复合体作为酶纳米处理器，它可以组合在一起检测多个信号，并用多个逻辑门控制输出。

DESCRIPTION (provided by applicant): Our objective is to develop a Protease Chain Reaction technology (ProCR) that will enable ultra- sensitive and highly-quantitative detection of cancer markers. The basic element of a protease chain reaction is a protease-inhibitor complex. The protease is inactive when bound to the inhibitor but, once freed, is capable of cleaving the inhibitor and releasing additional free protease. A trace of free protease initiates the chain reaction and eventually causes the release of all protease from the inhibitory complex. The lag time preceding the full release of protease is determined by the initial concentration of free protease. The Phase I project developed a first-generation of self-amplifying complexes that form the core of the ProCR detection technology. All Phase I milestones were significantly exceeded. In Phase II we will develop powerful molecular sensors based on self- amplifying complexes. A sensor consists of three elements: 1) a sensing element that responds to the presence of an analyte; 2) the processing/computational element that amplifies and quantifies the signal from the detection element; and 3) the transducer element that produces an optical signal. Through this combination, extremely sophisticated enzymatic sensors will be built to detect cancer markers. Accordingly the three experimental Aims are to: 1) Engineer sensing elements; 2) Engineer enhanced processing/computational elements; and 3) Engineer signaling components. Fundamentally, a protein chain reaction is a powerful analogue computer with two key characteristics that greatly facilitate the detection of target molecules. 1) It can convert th concentration of a specific target molecule into a time signature. 2) It can create enormous signal amplification, analogous to the amplification of DNA by a polymerase chain reaction (PCR). Thus detection is enabled because the final observable signal produced by a target molecule can be very large and the time lag until onset of the signal is precisely correlated with the concentration of target molecule. The long term goal is to develop protease-inhibitor complexes as enzymatic nano-processors which can be combined to detect multiple signals and to control output with multiple logic gates.