Plasmonic Detection of Free-Bilirubin in Neonates

描述(申请人提供)：在过去的几年里，我们的实验室开发了一种新的基于荧光的现象，这一现象现在对临床生物技术具有深远的影响。这项新技术导致最近发表了50多篇同行评议的文章，一本经过编辑的精装本，并推出了一本新的同行评议的斯普林格杂志，题为《血浆》。这一新现象依赖于荧光团与金属纳米颗粒的相互作用，可以增加荧光物种的量子产率，提供空间局域激发，甚至可以显著提高荧光团的光稳定性。我们的新技术清楚地表明，这些新颖的效应可以导致每个荧光团产生高达100万倍的光子，当应用于分析传感平台时，可以提供快速、简单的高灵敏度分析。在这方面，在最近完成的NIH授予PI的奖项下，我们能够开发出一种增强的RNA靶标检测分析平台，它具有PCR和ELISA的灵敏度，但最深刻的是，不需要任何扩增步骤。在这份R21提案中，我们组建了一个由光谱学家、生物统计学家和临床医生组成的多学科团队，以开发一种高灵敏度的分析方法来检测新生儿血清中的游离胆红素，这是一个具有密集研究和高度临床重要性的领域，因为它能够跨越血脑屏障并参与各种神经疾病。目前，游离胆红素的检测费时费力，至今还没有标准化的方法。这里介绍的金属增强荧光(MEF)无胆红素检测方法具有检测灵敏度高、操作简单、一次性使用等优点，甚至有可能在世界各地的实验室实现标准化。MEF测定的功能如下：人血清白蛋白(HSA)分别结合和运输血清游离胆红素和结合胆红素，固定在亲水性共聚物中，自身固定在玻片上。当含有以血清白蛋白为主的结合胆红素(&gt；99%)的血清被洗过分析表面时，游离胆红素扩散到聚合物中，与嵌入的人血清白蛋白结合，并由于其对表面银纳米结构的定位而变得固有的高荧光。我们打算使用实验室和临床样本优化和确定检测的灵敏度，最重要的是，将我们的结果与“UB分析仪”进行统计比较，“UB分析仪”是美国仅有的三种用于测量游离胆红素的仪器之一。这项研究旨在开发一种高度新颖的新生儿全血游离胆红素检测传感平台。如今，美国还没有商业工具可用。鉴于游离胆红素已经与晚期神经疾病联系在一起，那么这项提议所支持的技术将在保护新生儿和成人健康方面发挥重要作用。

DESCRIPTION (provided by applicant): Over the last couple of years our laboratories have developed a new fluorescence based phenomenon, which is now having profound implications in clinical biotechnology. This new technology has led to the recent publication of over 50 peer reviewed articles, one edited hard bound volume and the launch of a new peer reviewed Springer Journal, entitled Plasmonics. This new phenomenon relies on the interactions of fluorophores with metallic nanoparticles and can increase the quantum yield of fluorescing species, provide spatially localized excitation and can even substantially improve fluorophore photostability. Our new technology clearly demonstrates that these novel effects can result in up to a million-fold more photons per fluorophore, which when applied to assay sensing platforms, provides for quick and simple high sensitivity assays. In this regard, under a recently completed NIH award to the PI, we were able to develop an enhanced assay platform for RNA target detection which has the sensitivity of PCR and ELISA, but most profoundly, without any amplification steps. In this R21 proposal we have assembled a multidisciplinary team of Spectroscopists, Biostatisticians and Clinicians alike, to develop a high sensitivity assay for the detection of free unbound Bilirubin in neonatal serum, an area of intense research and high clinical importance due to its ability to cross the blood brain barrier and participate in various neurological disorders. At present, tests for free unbound bilirubin are arduous, time consuming, and no approach has to date been standardized. The Metal-Enhanced Fluorescence (MEF) free bilirubin assay described here, offers high sensitivity detection, simple operation, can be made disposable and has the potential to even be standardized in laboratories around the world. The MEF assay functions as follows: Human Serum Albumin (HSA), which binds and transports serum free and bound bilirubin respectively, is immobilized in a hydrophilic copolymer, itself immobilized onto a glass slide. When serum, containing serum albumin predominantly bound bilirubin (> 99 %) is washed across the assay surface, free bilirubin diffuses into the polymer, binds to the embedded HSA and becomes intrinsically highly fluorescent due to its locality to surface silver nanostructures. We intend to optimize and determine the sensitivity of the assay using both laboratory and clinical samples and most importantly, statistically compare our results with the "UB analyzer", one of only 3 instruments in the USA designed to measure free bilirubin. This proposed research aims to develop a highly novel sensing platform for whole blood free Bilirubin detection in neonates. No commercial instrument is available in the US today. Given that free bilirubin has been linked to later stage neurological disorders, then the technology underpinned by this proposal goes some significant ways to safeguard both neonatal and in-turn adult health.