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）：该第1阶段SBIR的长期目标是开发优化的组件（纳米结构的底物）和用于创建能够报告酶活性的新型快速，液晶基于液晶的测定方法。该提案的重点是基质金属蛋白酶的活性，因为它们对癌症生物学的重要性。需要更具成本效益，劳动力较低的新技术，消耗较少的试剂，可以在更少的时间内进行，可以区分存在的MMP总量及其激活分数，并且可以适应高吞吐量格式。鸭嘴兽TM技术可以成为加速基础研究，药物发现，抗癌药物评估以及预后指标开发的基础。纳米结构底物与液晶的组合不需要次级标签或其他试剂来报告酶活性。它将为许多与癌症生物学相关的多种测定法提供一个统一的平台。在此提案中，我们将通过两种方法来制造具有纳米级地形的底物：斜膜的倾斜沉积，并通过摩擦将纳米级秩序引入蛋白质膜表面的共价固定酶底物摩擦。我们将相对评估这些底物的能力：对齐液晶；确定它们对细胞培养基中蛋白质非特异性吸附的耐受性，并可以快速定量基质金属蛋白酶活性。此外，我们将探讨允许对MMP-9及其激活分数进行特定识别的策略。这些研究完成后，我们将优化一种新技术的核心组成部分，以快速，成本效益和准确的酶活性定量。