Liquid crystal based enzyme activity assay

描述(由申请人提供)：该第一阶段SBIR的长期目标是开发优化的组件(纳米结构底物)和方法，用于创建一种能够报告酶活性的新型快速、基于液晶的分析方法。这项建议侧重于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、更少的劳动力密集、消耗更少的试剂、能够在更短的时间内完成、能够区分存在的总量和其活化部分、并且能够适应高通量格式的新技术。鸭嘴兽TM技术可以作为此类检测的基础，以加快基础研究、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结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的分析提供一个统一的平台。在这个方案中，我们将通过两种方法来制备具有纳米级形貌的底物：倾斜沉积金薄膜和摩擦共价固定化的酶底物，将纳米级的有序引入到蛋白质膜的表面。我们将比较评估这些底物的能力：排列液晶；确定它们对细胞培养液中存在的蛋白质的非特异性吸附的耐受性，并允许快速定量基质金属蛋白酶活性。此外，我们还将探索允许特定鉴定基质金属蛋白酶-9及其活性部分的策略。在这些研究完成后，我们将优化一项新技术的核心组成部分，以实现快速、经济有效和准确的酶活性定量。

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.