MULTI-ANALYTE WAVEGUIDE IMMUNOSENSING

本项目的目标是利用高反射密度 集成光波导（IOWs）开发倏逝波生物传感器 用于高通量遗传筛选。特别是 申请人计划开发核酸杂交测定（也称为 “分子诊断”或“MDx”测定）用于筛选和诊断 遗传性心血管疾病虽然有几种潜在的疾病 目标，他们打算把重点放在长QT综合征（LQTS），因为有一个 这种疾病迫切需要快速和廉价的遗传学方法， 筛查受影响个人的家庭成员。传统诊断 方法（例如，心电图）在大约40%的LQTS病例中是不确定的，因为 受影响的个体表现出正常或临界QT延长 interval.这些人往往去未确诊，导致3000-4000突然 美国每年死亡人数。 LQTS与四个基因（KVLQT 1、HERG、SCN 5A）的遗传多态性有关 和KCNE 1）编码心脏离子通道。目前的方法 评估遗传多态性包括从患有遗传病的人中分离基因， 使用聚合酶链反应（PCR）对个体进行测序，然后 对观察到的突变进行分类然而，这种方法太昂贵， 用于常规患者筛查是耗时的，这导致 所谓的“DNA芯片”的发展，其中包含数百至数千个 在微阵列中，寡核苷酸被固定到单个基底上。患者 用DNA芯片进行筛选包括将感兴趣的基因从细胞中分离出来。 使用PCR检测患者的DNA，并允许PCR产物与 芯片使用落射荧光或共聚焦荧光检测杂交。 显微镜检测过程是耗时的，因为每个阵列元件 连续成像几秒钟或更长时间。此外，仪器 读取芯片所需的费用非常昂贵，花费在10万美元和 20万美元一个典型的设置。因此，测定时间和成本是测定的限制因素。 MDx技术在常规患者筛查和诊断中的应用。 在本申请中对该问题采取的方法是使用集成的 光波导传感器作为DNA芯片的固定化载体。通过做 因此申请人可以显著减少测定时间（至5分钟或更少）， 和仪器成本（我们的原型分析仪的生产成本约为3,000美元， 生产版本的零售价可能约为10，000美元），因为整个 通过电荷耦合器件（CCD）对寡核苷酸阵列进行真实的实时监测 相机此外，真实的时间数据采集使我们能够监控 杂交动力学，这对于鉴定点突变至关重要。

The goal of this project is to exploit the high reflection density of integrated optical waveguides (IOWs) to develop evanescent wave biosensors for applications in high throughput genetic screening. In particular, the applicants plan to develop nucleic acid hybridization assays (also know as "molecular diagnostics" or "MDx" assays) for use in screening and diagnosis of hereditary cardiovascular disease. Although there are several potential disease targets, they intend to focus on long-QT syndrome (LQTS) because there is a compelling need in this disease for rapid and inexpensive methods for genetic screening of family members of affected individuals. Traditional diagnostic methods (e.g., ECG) are equivocal in about 40% of LQTS cases because the affected individual exhibits either a normal or borderline prolonged QT interval. Such individuals often go undiagnosed, resulting in 3000-4000 sudden deaths per year in the United States. LQTS has been linked to genetic polymorphisms in four genes (KVLQT1,HERG, SCN5A & KCNE1) that encode for cardiac ion channels. Present-day methodology for assessing genetic polymorphism involves isolating genes from afflicted individuals using polymerase chain reaction (PCR), sequencing them, and then cataloging the observed mutations. However, this procedure is too expensive and time-consuming for use in routine patient screening, which has lead to development of so-called "DNA chips" that contain hundreds to thousands of oligonucleotides immobilized to a single substrate in a microarray. Patient screening with a DNA chip involves isolating the gene of interest from the patient's DNA using PCR and them allowing the PCR product to hybridize to the chip. Hybridization is detected using either an epifluorescence or confocal microscope. The detection process is time-consuming because each array element is imaged sequentially for a few seconds or more. Moreover, the instrumentation required to read the chips is very expensive, costing between $100,000 and $200,000 for a typical setup. Thus, assay time and cost are limiting factors in the application of MDx technology to routine patient screening and diagnosis. The approach taken in this application to this problem is to use an integrated optical waveguide sensor as the immobilization support for a DNA chip. By doing so the applicants can dramatically reduce assay time (to 5 minutes, or less) and instrumentation cost (our prototype analyzer cost about $3,000 to produce, production versions would probably retail for about $10,000) because the entire oligonucleotide array is monitored in real time by charge-coupled device (CCD) camera. Moreover, data acquisition in real time enables us monitoring hybridization kinetics, which is essential for identifying point mutations.