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

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

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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和必要的仪器进行分析，以创建一个能够使用最佳蛋白酶测定的集成平台。该平台将具有几个重要的优势，其中包括使用全长底物，多重测定的能力，以及最重要的是，将蛋白质底物作为蛋白酶反应的饱和浓度提供蛋白质底物，从而确定关键动力学参数。这些特性代表了蛋白酶测定平台几乎理想的组合，并且与当今可用的任何其他测定方法不匹配。理想的蛋白酶测定平台的开发具有很大的医学意义，因为有数百种直接参与许多重要人类疾病的蛋白酶。例如，蛋白酶在疾病进展中裂解蛋白质靶标（底物），例如黄素科的病毒加工，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。对蛋白酶抑制剂的需求进一步驱动了对理想蛋白酶测定的需求（5-10％的药物发现靶标蛋白酶）。蛋白酶的蛋白酶数量及其巨大的药物价值的结合使蛋白酶测定在医学中最重要的测定中。我们将通过在纳米级上实施我们的表面测定法，从而创建一个接近理想的蛋白酶测定法，该测定在多重微球阵列上成功。纳米颗粒蛋白酶测定将使用可切合的荧光融合蛋白底物通过流式细胞仪监测从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，将使用对多重荧光纳米球阵列阵列分析进行优化的多参数流式细胞仪检测粒子的存在，粒子类型和荧光变化。通过对毒素和病毒蛋白酶的体外测定，将成功开发我们的平台。这些蛋白酶测定将用于得出动力学参数，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路复用纳米层阵列，创建具有Attoliter分析量的高灵敏度流式细胞仪以及使用这些集成技术的蛋白酶测定的演示。如果成功，我们的蛋白酶测定格式将易于执行，多重，提供关键的动力学参数（KCAT，KM和KI），并以生物学相关的方式显示大蛋白质底物，以促进从大蛋白酶到蛋白质相互作用（例如外ososites）的贡献。我们的平台将通过提供最佳的蛋白酶测定在生物医学研究中具有明显的价值。除此之外，新型的纳米颗粒和流式细胞仪技术除了这里进行的研究外，还将对各种分析研究有用。因此，除了改进的蛋白酶测定法之外，开发的技术还将为科学和生物医学群落带来广泛的一般利益。