Liquid crystal based enzyme activity assay

描述（由申请人提供）：本SBIR第1阶段的长期目标是开发优化的组分（纳米结构基质）和方法，用于创建一类新型快速、基于液晶的能够报告酶活性的测定。这项建议的重点是活性的基质金属蛋白酶，因为它们的核心重要性，癌症生物学。需要新的技术，其更具成本效益，劳动强度更低，消耗更少的试剂，可以在更短的时间内进行，可以区分存在的MMP的总量和其活化的级分，并且适合于高通量形式。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。纳米结构的基板与液晶的组合不需要二级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检测提供一个统一的平台。在这个提议中，我们将通过两种方法制造具有纳米级形貌的基底：倾斜沉积金薄膜和通过摩擦共价固定的酶基底，将纳米级顺序引入蛋白质膜的表面。我们将比较评估这些基板的能力：对齐液晶，确定其耐受性的存在下，在细胞培养基中的蛋白质的非特异性吸附，并允许快速定量的基质金属蛋白酶活性。此外，我们将探索策略，允许MMP-9和其活化馏分的特异性鉴定。在这些研究完成后，我们将优化新技术的核心组件，以快速，成本效益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.