A biomimetic nanoparticle protease assay platform

描述(由申请人提供)：我们建议开发仿生纳米球及其分析所需的仪器，以创建一个能够进行最佳蛋白酶分析的集成平台。这个平台将有几个显著的优势，包括使用全长底物，能够进行多重分析，最重要的是，能够提供蛋白质底物作为蛋白酶反应的饱和浓度，从而能够确定关键的动力学参数。这些特性代表了蛋白酶分析平台的近乎理想的组合，目前任何其他可用的分析方法都无法与之匹配。开发一个理想的蛋白水解酶检测平台具有重要的医学意义，因为有数百种蛋白水解酶直接参与人类的许多重要疾病。例如，蛋白水解酶可以分解疾病发展过程中重要的蛋白质靶标(底物)，如黄病毒对病毒的处理、蛋白质的毒素裂解以及癌症转移过程中细胞外基质的消化。对蛋白水解酶抑制剂的需求进一步推动了对理想的蛋白水解酶分析的需求(5%-10%的药物发现是针对蛋白水解酶的)。众多具有科学价值的蛋白酶及其巨大的药用价值相结合，使其成为医学中最重要的检测方法之一。我们将通过在纳米尺度上实现已在多个微球阵列上成功实现的基于表面的分析来创建一种近乎理想的蛋白酶分析。纳米粒子蛋白酶分析将使用可切割的荧光融合蛋白底物，通过流式细胞仪监测微球表面的荧光损失来检测蛋白酶活性。在我们提出的平台中，粒子的存在、粒子类型和荧光变化将使用多参数、高灵敏度的流式细胞仪进行检测，该仪器针对多重荧光纳米球阵列分析进行了优化。我们平台的成功开发将通过对毒素和病毒蛋白酶的体外测试来证明。这些酶分析将被用于推导动力学参数、多重分析和测量抑制剂活性。这些目标将需要开发具有仿生表面的多路复合纳米球阵列，创建具有阿特利特分析体积的高灵敏度流式细胞仪，并使用这些综合技术演示蛋白酶分析。如果成功，我们的蛋白水解酶分析形式将是简单的、多路的，提供关键的动力学参数(kcat、Km和Ki)，并以生物相关的方式展示大的蛋白质底物，从而能够评估大的蛋白酶对蛋白质相互作用(例如，外显子)的贡献。我们的平台将通过提供一种用于生物医学研究的最佳蛋白酶检测而具有明显的价值。除此之外，新的纳米粒子和流式细胞仪技术将用于各种分析研究，除了这里所追求的那些。因此，除了改进的蛋白酶分析外，开发的技术还将对科学界和生物医学界产生广泛的普遍好处。

DESCRIPTION (provided by applicant): We propose to develop biomimetic nanospheres and the necessary instrumentation for their analysis, to create an integrated platform capable of optimal protease assays. This platform will have several significant advantages, which include the use of full-length substrates, the ability to multiplex assays, and, most importantly, the abilty to provide protein substrates as saturating concentrations for protease reactions, which enables the determination of key kinetic parameters. These properties represent a near ideal combination for a protease assay platform and are not matched by any other assay approach available today. The development of an ideal protease assay platform has great medical significance, as there are hundreds of proteases directly involved in many important human diseases. For example, proteases cleave protein targets (substrates) important in the progression of disease, like viral processing by the flaviviridae, toxin cleavage of proteins, and digestion of the extracellular matrix during cancer metastasis. The need for ideal protease assays is further driven by the need for protease inhibitors (5-10% of drug discovery targets proteases). The combination of the number of proteases of scientific interest and their great pharmaceutical value makes the protease assay among the most important assays in medical science. We will create a near ideal protease assay by implementing on the nanoscale our surface based assays that have been successful on multiplex microsphere arrays. Nanoparticle protease assays will use cleavable fluorescent fusion protein substrates to detect protease activity by monitoring the loss of fluorescence from the microsphere surface via flow cytometry. In our proposed platform, particle presence, particle type, and fluorescence changes will be detected using a multiparameter, high-sensitivity flow cytometer optimized for multiplex fluorescent nanosphere array analysis. Successful development of our platform will be demonstrated via in vitro assays against toxin and viral proteases. These protease assays will be used to derive kinetic parameters, multiplex assays, and measure inhibitor activity. These goals will require the development of multiplexed nanosphere arrays bearing biomimetic surfaces, the creation of a high sensitivity flow cytometer with attoliter analytical volumes, and the demonstration of protease assays using these integrated technologies. If successful, our protease assay format will be simple to perform, multiplexable, provide key kinetic parameters (kcat, Km, and Ki), and display large protein substrates in a biologically relevant fashion to enable contributions from large protease to protein interactions (e.g. exosites) to be evaluated. Our platform would have clear value by providing an optimal protease assay for use in biomedical research. Beyond this, the novel nanoparticles and flow cytometry technology will be useful for a variety of analytical studies in addition to those pursued here. Thus, beyond an improved protease assay, the technology developed will have widespread general benefit to the scientific and biomedical communities.