Liquid crystal based enzyme activity assay

基于液晶的酶活性测定

• 批准号: 6787091
• 负责人: Barbara A. Israel
• 金额: $ 20万
• 依托单位: PLATYPUS TECHNOLOGIES, LLC
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787091
• 关键词: antibody; bioassay; bioengineering /biomedical engineering; collagen; enzyme activity; extracellular matrix; gold; liquid crystal; metalloendopeptidases; neoplasm /cancer diagnosis; optical polarization; proteolysis; surface coating; surface property; technology /technique development

DESCRIPTION (provided by applicant): The long-term goal of this Phase 1 SBIR is to develop optimized components (nanostructured substrates) and methods for use in the creation of a novel class of rapid, liquid crystal based assays capable of reporting enzyme activity. This proposal focuses on the activity of matrix metalloproteinases because of their central importance to cancer biology. There is a need for new technologies that are more cost effective, less labor intensive, consume less reagents, can be performed in less time, can distinguish between the total amount of MMP present and its activated fraction, and are amenable to high throughput formats. Platypus TM technology can be the foundation for such assays to accelerate basic research, drug discovery, evaluation of anti-cancer drugs and development of prognostic indicators. The combination of nanostructured substrates with liquid crystals requires no secondary labels or additional reagents to report enzyme activity. It will provide a unified platform for many diverse assays relevant to cancer biology. In this proposal, we will fabricate substrates with nanoscale topography by two methods: oblique deposition of thin films of gold and by rubbing of covalently immobilized enzyme substrates that introduce nanoscale order into the surface of the protein film. We will comparatively evaluate the ability of these substrates to: align liquid crystals; determine their tolerance to the presence of non-specific adsorption of proteins in cell media and to allow rapid quantification of matrix metalloproteinase activity. Additionally, we will explore strategies that allow specific identification of MMP-9 and its activated fraction. At the completion of these studies, we will have optimized the core component of a new technology for rapid, cost effective and accurate quantification of enzyme activity.

描述（由申请人提供）：第一阶段 SBIR 的长期目标是开发优化的组件（纳米结构底物）和方法，用于创建能够报告酶活性的新型快速液晶检测方法。该提案重点关注基质金属蛋白酶的活性，因为它们对癌症生物学至关重要。需要更具成本效益、劳动力密集度较低、试剂消耗更少、可以在更短的时间内完成、能够区分存在的 MMP 总量及其活化部分、并且适合高通量形式的新技术。 Platypus TM 技术可以成为此类检测的基础，以加速基础研究、药物发现、抗癌药物评估和预后指标的开发。纳米结构底物与液晶的组合不需要二次标记或额外的试剂来报告酶活性。它将为与癌症生物学相关的许多不同检测提供一个统一的平台。在本提案中，我们将通过两种方法制造具有纳米级形貌的基材：金薄膜的倾斜沉积和通过共价固定酶基材的摩擦，将纳米级有序引入蛋白质膜的表面。我们将比较评估这些基板的以下能力：排列液晶；确定它们对细胞介质中蛋白质非特异性吸附存在的耐受性，并允许快速定量基质金属蛋白酶活性。此外，我们将探索能够特异性识别 MMP-9 及其激活部分的策略。这些研究完成后，我们将优化新技术的核心组件，以实现快速、经济有效且准确的酶活性定量。