MULTI-ANALYTE WAVEGUIDE IMMUNOSENSING

本项目的目标是开发高反射密度。 集成光波导(IOW)开发逝去波生物传感器 用于高通量基因筛查。尤其是， 申请者计划开发核酸杂交分析(也称为 “分子诊断”或“MDX”分析)用于筛查和诊断 遗传性心血管疾病。尽管有几种潜在的疾病 他们打算关注长QT综合征(LQTS)，因为有一种 这种疾病迫切需要快速和廉价的基因检测方法 对受影响个人的家庭成员进行筛查。传统诊断 在大约40%的LQTS病例中，方法(例如，心电图)是模棱两可的，因为 受累个体的QT间期正常或接近延长 时间间隔。这样的人经常得不到诊断，导致3000-4000人突然 美国每年的死亡人数。 LQTS与四个基因(KVLQT1、HERG、SCN5A)的遗传多态有关 &KCNE1)，编码心脏离子通道。当今的方法论 评估遗传多态涉及从患者中分离基因 使用聚合酶链式反应(PCR)的个体，对它们进行测序，然后 对观察到的突变进行分类。然而，这个程序太昂贵了，而且 在常规患者筛查中使用耗时，这导致 开发包含成百上千个DNA芯片的所谓“DNA芯片” 微阵列中固定在单一底物上的寡核苷酸。有耐心的 使用DNA芯片进行筛选包括将感兴趣的基因从 患者的DNA使用聚合酶链式反应，并允许聚合酶链式反应产物与 奇普。使用荧光或共聚焦检测杂交 显微镜。检测过程很耗时，因为每个阵列元素 被连续成像几秒钟或更长时间。此外，仪器设备 读取芯片的费用非常昂贵，价格在10万美元到10万美元之间 典型设置为200,000美元。因此，化验时间和成本是限制 MDX技术在常规患者筛查和诊断中的应用。 此应用程序中解决此问题的方法是使用集成的 光波导传感器作为DNA芯片的固定支架。通过做 因此，申请者可以极大地减少化验时间(至5分钟或更短) 和仪器成本(我们的原型分析仪的生产成本约为3,000美元， 生产版本可能零售价约为10,000美元)，因为整个 利用电荷耦合器件(CCD)实时监测寡核苷酸阵列 摄影机。此外，实时数据采集使我们能够监控 杂交动力学，这是识别点突变的关键。

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.