Liquid crystal based enzyme activity assay

描述（由申请人提供）：该1期SBIR的长期目标是开发优化的组分（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。由于基质金属蛋白酶在癌症生物学中的核心重要性，本研究主要关注基质金属蛋白酶的活性。需要一种成本效益更高、劳动强度更低、消耗更少的试剂、可以在更短的时间内完成、可以区分存在的MMP总量及其活化部分、并且适合高通量格式的新技术。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结合不需要二次标记或额外的试剂来报告酶的活性。它将为许多与癌症生物学相关的不同分析提供一个统一的平台。在这个提议中，我们将通过两种方法来制造具有纳米级形貌的底物：倾斜沉积金薄膜和通过摩擦共价固定酶底物，将纳米级秩序引入蛋白质膜的表面。我们将比较评估这些衬底的能力：排列液晶；测定它们对细胞培养基中蛋白质非特异性吸附的耐受性，以便快速定量测定基质金属蛋白酶的活性。此外，我们将探索允许特异性鉴定MMP-9及其活化部分的策略。在这些研究完成后，我们将优化一种新技术的核心组件，用于快速，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.