High-throughput, Label-free Promoter Sequence Discovery

描述(由申请人提供)：拟议的工作旨在展示一种基于高通量、成本效益和无标记的微阵列检测大分子相互作用的方法。特别是，我们提出了一种新的技术，包括使用微阵列和一种新的基于共振腔成像生物传感器(RCIB)的无标记光学检测方法。虽然使用光学谐振器进行传感应用的研究已有多年，但本提案中描述的RCIB的独特功能是由波士顿大学最近开发的创新以及光学仪器的进步实现的。主要的促成因素之一是波士顿大学为研究硅上的光电子器件而开发的高质量多层介质反射器的发明。伴随而来的是感知分层结构上材料微小变化的新技术。共振腔成像生物传感器(RCIB)有望为基于高通量阵列的蛋白质表达谱和发现基因间调控区的方法开辟道路。该提案将侧重于展示和采用蛋白质寡核苷酸阵列的RCIB检测技术，并评估该技术的敏感性和特异性。启动子序列和潜在的启动子序列区域将在DNA微阵列上进行光刻合成。未标记的转录因子将被引入微阵列表面，并允许与其相应的启动子序列结合。将使用无标记的RCIB方法来检测和定量TF的存在。这项工作将实现以下具体目标：1.展示基于二氧化硅的0.1 nm高人工结构的RCIB传感和成像；2.以阵列的形式检测与生物素化的DNA结合的链霉亲和素单层，每个大小为50微米×50微米；3.在100个特征微阵列上展示不同转录因子(TF)与其相应启动子序列结合的特异性和敏感性。

DESCRIPTION (provided by applicant): The proposed work is aimed at demonstrating a high throughput, cost effective, and label-free microarray- based method for detecting macromolecular interactions. In particular, we propose a novel technology that involves the use of microarrays and a new label-free optical detection method based on the Resonant Cavity Imaging Biosensor (RCIB). While the use of optical resonators for sensing applications has been studied for many years, the unique capabilities of RCIB described in this proposal have been enabled by recent innovations developed at Boston University as well as advancements in optical instrumentation. One of the primary enabling factors is the invention of high-quality multi-layer dielectric reflectors developed at Boston University for the study of optoelectronic devices on silicon. This is accompanied by new techniques to sense small changes in material on layered structures. Resonant Cavity Imaging Biosensor (RCIB) promises to open the way to high throughput array based methods for profiling protein expression and for discovering intergenic regulatory regions. This proposal will focus on demonstrating and adapting RCIB detection techniques for protein oligonucleotides arrays, and assessing the sensitivity and specificity of the technology. Promoter sequences and potential promoter sequence regions will be photolithographically synthesized on a DNA microarray. Unlabeled TFs will be introduced to the microarray surface and allowed to bind to their corresponding promoter sequences. The presence of TFs will be detected and quantified using the label-free RCIB method. This work will achieve the following specific aims: 1. Demonstrate RCIB sensing and imaging of 0.1 nm high artificial structures patterned on SiO2; 2. Detect monolayer of streptavidin bound to biotinylated DNA patterned in an array format with 64 features each 50 mu m x 50 mu m in size; 3. Demonstrate specificity and sensitivity of different transcription factors (TFs) binding to their corresponding promoter sequences on a 100-feature microarray.