Metal Enhancement for Near-IR Fluorescence Based Membrane Assays

描述（由申请人提供）：定位在银纳米颗粒附近的荧光分子会产生增加的荧光强度，同时增加光稳定性和减少寿命。人们对这些银结构进行了非常详细的研究，因为它们在沉积到固体表面（如玻璃或石英）时能够增加荧光强度。然而，关于银纳米结构增强沉积在多孔膜表面的荧光团的能力的信息很少。本提案的总体目标是利用金属增强荧光来增加膜底物上的近红外荧光发射。基于膜的检测，如western blots，通常用于临床环境，以确认其他筛选试验建议的严重诊断，并在研究环境中表征和定量感兴趣的蛋白质。与可见光谱中产生的背景相比，基于荧光的近红外分析具有显著降低背景的额外优势，有助于降低检测极限。从长远来看，这项工作应该适用于大多数利用荧光的系统，以一种将荧光染料捕获在膜底物上的方式。近红外金属增强荧光膜表面应该提供一个可靠和有效的程序，以增加荧光发射和降低检测的限制，提供一个工具，是兼容当前的蛋白质组学和基因组学应用。未来的努力可以使用金属增强荧光来提高膜基DNA、蛋白质和膜上抗体阵列的可检测性。该项目专注于开发一种用于Western和dot blot分析的增强试剂，该试剂集成了低本底近红外LI-COR染料与金属增强荧光（MEF）的使用，以产生更敏感的基于膜的分析，能够将检测极限降低一个数量级。这些Western和dot blot检测灵敏度的提高将有助于分析分子、细胞和细胞间过程，以及这些过程相互之间的关系。

DESCRIPTION (provided by applicant): Fluorescent molecules that are positioned in close proximity to silver nanoparticles produce increased fluorescence intensity along with increased photostability and decreased lifetimes. These silver structures have been studied in great detail for their ability to increase fluorescent intensities when deposited onto solid surfaces, such as glass or quartz. Little information exists, however, on the ability of silver nanostructures to enhance fluorophores deposited onto porous membrane surfaces. The overall objective of this proposal is to use metal enhanced fluorescence to increase near- infrared fluorescence emissions on membrane substrates. Membrane based assays, such as western blots are routinely used in clinical settings to confirm serious diagnoses suggested by other screening tests, and in research settings to characterize and quantitate proteins of interest. Fluorescence based assays in the near-infrared offer the additional advantage of significantly lowering the background compared to that generated in the visible spectrum, helping to lower the limit of detection. In the long term, this work should be applicable to most systems that utilize fluorescence in a manner that traps the fluorescent dyes on a membrane substrate. Near-infrared metal enhanced fluorescence on membrane surfaces should provide a reliable and efficient procedure to increase fluorescent emissions and decrease the limit of detection, providing a tool that is compatible with current proteomic and genomic applications. Future endeavors could use metal enhanced fluorescence to increase the detectability of membrane based DNA, protein, and antibody arrays on membranes. This project focuses on developing an enhancement reagent for Western and dot blot analyses, which integrates the use of low background near-infrared LI-COR dyes with Metal Enhanced Fluorescence (MEF), to produce more sensitive membrane based assays capable of reducing the limit of detection by an order of magnitude. Advances in sensitivity for these Western and dot blot assays will aid in the analysis of molecular, cellular, and inter-cellular processes, and the relationship of these processes with respect to one another.