Protease chain reactions for molecular analysis of cancer markers

描述（由申请人提供）：我们的目标是开发一种蛋白酶链式反应技术（ProCR），该技术将能够实现癌症标志物的超灵敏和高度定量检测。蛋白酶链式反应的基本要素是蛋白酶-抑制剂复合物。当与抑制剂结合时，蛋白酶是无活性的，但是一旦释放，能够切割抑制剂并释放另外的游离蛋白酶。微量的游离蛋白酶启动链式反应，并最终导致所有蛋白酶从抑制复合物中释放出来。蛋白酶完全释放之前的滞后时间由游离蛋白酶的初始浓度决定。第一阶段项目开发了第一代自放大复合物，构成了ProCR检测技术的核心。第一阶段的所有里程碑都已大大超过。在第二阶段，我们将开发基于自放大复合物的强大分子传感器.传感器由三个元件组成：1）响应分析物的存在的感测元件; 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）产生光学信号的换能器元件。通过这种组合，将建立非常复杂的酶传感器来检测癌症标志物。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）设计信令组件。从根本上说，蛋白质链式反应是一种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成时间信号。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，能够进行检测，因为由靶分子产生的最终可观察信号可能非常大，并且直到信号开始的时间滞后与靶分子的浓度精确相关。长期目标是开发蛋白酶抑制剂复合物作为酶纳米处理器，其可以组合以检测多个信号并通过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.