High-throughput, Label-free Promoter Sequence Discovery

描述（由申请人提供）：所提出的工作旨在证明用于检测大分子相互作用的高通量、成本有效且无标记的基于微阵列的方法。特别是，我们提出了一种新的技术，涉及使用微阵列和一种新的无标记的光学检测方法的基础上的谐振腔成像生物传感器（RCIB）。虽然光学谐振器用于传感应用的研究已经进行了多年，但本提案中描述的RCIB的独特功能已经通过波士顿大学最近开发的创新以及光学仪器的进步而实现。主要的促成因素之一是波士顿大学为研究硅上的光电器件而开发的高质量多层介质反射器的发明。这伴随着新技术来感知分层结构上材料的微小变化。共振腔成像生物传感器（RCIB）有望开辟一条基于高通量阵列的蛋白质表达谱分析和基因间调控区发现方法的道路。该提案将重点展示和调整蛋白质寡核苷酸阵列的RCIB检测技术，并评估该技术的灵敏度和特异性。启动子序列和潜在的启动子序列区域将在DNA微阵列上通过电泳合成。将未标记的TF引入微阵列表面，并使其与相应的启动子序列结合。将使用无标记RCIB方法检测和定量TF的存在。本工作将实现以下具体目标：1.演示在SiO2上图案化的0.1 nm高人工结构的RCIB传感和成像; 2.检测结合到生物素化DNA的单层链霉亲和素，所述生物素化DNA以具有64个特征的阵列形式图案化，每个特征的尺寸为50 μ m × 50 μ m; 3.在100个特征的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灵敏度。

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.