A biomimetic nanoparticle protease assay platform

仿生纳米颗粒蛋白酶检测平台

基本信息

批准号：
8582398
负责人：
STEVEN W GRAVES
金额：
$ 18.51万
依托单位：
UNIVERSITY OF NEW MEXICO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8582398
关键词：
Acetylcholine Active Sites Affinity Apoptosis Bacillus anthracis Binding Biological Assay Biology Biomedical Research Biomimetics Blood coagulation Bontoxilysin Botulinum Toxin Type A Cessation of life Chimeric Proteins Cleaved cell Communities Complex Custom Dengue Detection Development Digestion Disease Disease Progression Distant Ensure Excision Extracellular Matrix Flaviviridae Flow Cytometry Fluorescence Fluorescence Resonance Energy Transfer Future Goals HIV Health Hepatitis C Human Immune response Kinetics Label Lead Length Light Lipid Bilayers Measurement Measures Mechanics Medical Medicine Membrane Microbe Microspheres Modeling Modification Monitor Nanosphere Nature Neoplasm Metastasis Neurons Optics Organelles Paralysed Pathogenesis Peptide Hydrolases Peptides Pharmacologic Substance Polyproteins Polystyrenes Process Property Proteins Proteolysis Reaction Research Research Personnel Resolution Retroviridae Science Serotyping Signal Transduction Silicon Dioxide Solutions Substrate Interaction Surface Technology Therapeutic Time Toxic effect Toxin Viral West Nile virus Work Wound Healing anthrax lethal factor base botulinum botulinum toxin type G cellular targeting drug discovery enzyme mechanism fluorophore high throughput screening human disease improved in vitro Assay inhibitor/antagonist instrument instrumentation interest maspin multiplex detection nanoparticle nanoscale neglect novel particle prevent protease So public health relevance receptor success syntaxin tool

项目摘要

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.

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着的优势，其中包括使用全长底物、多重检测的能力，以及最重要的是，能够为蛋白酶反应提供饱和浓度的蛋白质底物，从而能够确定关键动力学参数。这些特性代表了蛋白酶检测平台近乎理想的组合，并且是当今可用的任何其他检测方法所无法比拟的。开发理想的蛋白酶检测平台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），例如黄病毒科的病毒加工、蛋白质的毒素切割以及癌症转移期间细胞外基质的消化。对蛋白酶抑制剂的需求进一步推动了对理想蛋白酶检测的需求（5-10% 的药物发现以蛋白酶为目标）。大量具有科学意义的蛋白酶及其巨大的药用价值使蛋白酶测定成为医学科学中最重要的测定之一。我们将通过在纳米尺度上实施我们在多重微球阵列上取得成功的基于表面的测定来创建近乎理想的蛋白酶测定。纳米颗粒蛋白酶测定将使用可裂解的荧光融合蛋白底物，通过流式细胞术监测微球表面荧光的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进行优化的多参数、高灵敏度流式细胞仪来检测颗粒存在、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导出动力学参数、多重测定和测量抑制剂活性。这些目标将需要开发带有仿生表面的多重纳米球阵列，创建具有阿托升分析体积的高灵敏度流式细胞仪，以及使用这些集成技术演示蛋白酶测定。如果成功，我们的蛋白酶检测格式将易于执行、可多重使用、提供关键动力学参数（kcat、Km 和 Ki），并以生物学相关的方式显示大蛋白底物，以实现大蛋白酶对蛋白质相互作用的贡献（例如外部位点））进行评估。我们的平台通过提供用于生物医学研究的最佳蛋白酶测定而具有明显的价值。除此之外，新型纳米粒子和流式细胞术技术将可用于除此处所追求的研究之外的各种分析研究。因此，除了改进蛋白酶测定之外，所开发的技术还将给科学界和生物医学界带来广泛的普遍利益。