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，以创建能够进行最佳蛋白酶测定的集成平台。该平台将具有几个显著的优点，包括使用全长底物，多重测定的能力，以及最重要的是，提供蛋白质底物作为蛋白酶反应的饱和浓度的能力，这使得能够确定关键的动力学参数。这些特性代表了蛋白酶测定平台的近乎理想的组合，并且与当今可用的任何其他测定方法都不匹配。 理想的蛋白酶检测平台的开发具有重要的医学意义，因为有数百种蛋白酶直接参与许多重要的人类疾病。例如，蛋白酶切割在疾病进展中重要的蛋白质靶标（底物），如黄病毒科的病毒加工、蛋白质的毒素切割和癌症转移期间细胞外基质的消化。对理想蛋白酶测定的需求进一步受到对蛋白酶抑制剂的需求的驱动（5-10%的药物发现靶向蛋白酶）。具有科学意义的蛋白酶的数量及其巨大的药用价值的组合使得蛋白酶测定成为医学科学中最重要的测定之一。 我们将创建一个接近理想的蛋白酶测定通过实施纳米我们的表面为基础的分析，已成功的多重微球阵列。纳米颗粒蛋白酶测定将使用可切割的荧光融合蛋白底物，通过流式细胞术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，将使用针对多重荧光纳米球阵列分析优化的多参数、高灵敏度流式细胞仪检测颗粒存在、颗粒类型和荧光变化。我们平台的成功开发将通过针对毒素和病毒蛋白酶的体外测定来证明。这些蛋白酶测定将用于推导动力学参数、多重测定和测量抑制剂活性。这些目标将需要多路复用纳米球阵列轴承仿生表面的发展，创建一个高灵敏度的流式细胞仪与阿升分析量，并使用这些集成技术的蛋白酶测定的演示。如果成功，我们的蛋白酶测定形式将易于执行，可复用，提供关键动力学参数（kcat，Km和Ki），并以生物相关的方式显示大蛋白底物，以评估大蛋白酶与蛋白质相互作用（例如外切位点）的贡献。 我们的平台将通过提供用于生物医学研究的最佳蛋白酶测定而具有明确的价值。除此之外，新型纳米颗粒和流式细胞术技术将有助于除本文所述之外的各种分析研究。因此，除了改进的蛋白酶测定，所开发的技术将对科学和生物医学界产生广泛的普遍益处。

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.