EAGER: T1-iSDA - Bringing NAAT-like sensitivity to (nearly) instrument-free measurement of protein concentration

EAGER：T1-iSDA - 为（几乎）无需仪器的蛋白质浓度测量带来类似 NAAT 的灵敏度

• 批准号: 1842440
• 负责人: Paul Yager
• 金额: $ 9.97万
• 依托单位: University of Washington
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2019-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1842440&HistoricalAwards=false
• 关键词: EAGER; T1; iSDA; Bringing; NAAT

Sensitivity matters in testing for the presence and concentration of many small molecules and proteins; so does the length of time between capturing a sample and getting the results of the measurement. As a consequence, there is a big push to develop sensitive test for such molecules that can be made anywhere by anyone and in a few minutes. This is true if you are trying to differentiate a heart attack from indigestion, and if you are trying to measure pollutants in the water supply, for two examples. Many of these molecules to be detected, or "analytes", are not nucleic acids (DNA and RNA) and are therefore not detectable by highly-sensitive nucleic acid amplification tests that can detect a few copies of a target. New methods for sensitive protein detection use large instruments, and must be in a well-funded laboratory. The overall goal of this project is to enable measurement of low-concentration protein analytes rapidly and at low cost anywhere, particularly in homes or in low-resource settings. This project will combine in a single device low-cost low-sensitivity paper-based lateral flow tests with signal enhancement using isothermal nucleic acid amplification. This one-year project targets the development of this ultrasensitive protein detection in a prototype low-cost easy-to-use platform that will be suited to point-of use operation in low-resource labs and homes. In the long run, the aim is to make the platform commercially available for a wide range of protein detection problems not previously tractable because of the need for a centralized laboratory and highly-trained personnel. The aim is to combine three known technologies into a common platform that will allow rapid detection of fewer than a million copies of a protein in a format the combines: 1) lateral-flow immunoassay tests on nitrocellulose strips to capture specific protein targets, 2) detection of the presence of the assembled protein stack sandwiching the protein target by incorporating a nucleic acid template attached to the detection antibody, 3) amplifying said template in situ using an isothermal nucleic amplification method (iSDA) also embodied in a paper system and supported by a simple heater on a printed circuit board, and 4) detecting the amplification process by fluorescence in real time within the paper using a cell phone and a black box containing two optical filters to cover the camera?s flash and camera. Preliminary data has shown that the combination of a common lateral flow immunoassay detecting an influenza nucleoprotein protein with the iSDA can produce 10,000-fold enhancement of sensitivity of the test. However, the 4 processes have not been combined in a cartridge format that runs quickly and avoids poor rinsing of the labeled detection antibody (which produces a false positive signal). The goal of the project is to integrate the processes into a single rapid convenient sample-to-result prototype instrument that will be simple to use for anyone familiar with modern smart phones. It will take up no more desk space that a smart phone, should need no power other than the battery of the phone to run multiple tests, allowing the device to run anywhere, and to communicate data immediately to centralized facilities over the internet.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在检测许多小分子和蛋白质的存在和浓度时，灵敏度很重要;捕获样品和获得测量结果之间的时间长度也很重要。 因此，有一个很大的推动力来开发这种分子的敏感测试，可以在任何地方由任何人在几分钟内制造。 如果你试图区分心脏病发作和消化不良，如果你试图测量供水中的污染物，这是正确的，举两个例子。 这些待检测的分子或“分析物”中的许多不是核酸（DNA和RNA），并且因此不能通过可检测靶的几个拷贝的高灵敏度核酸扩增测试来检测。 用于敏感蛋白质检测的新方法使用大型仪器，并且必须在资金充足的实验室中进行。 该项目的总体目标是能够在任何地方快速、低成本地测量低浓度蛋白质分析物，特别是在家庭或低资源环境中。 该项目将联合收割机结合在一个单一的设备，低成本，低灵敏度的纸基侧流测试与信号增强使用等温核酸扩增。 这个为期一年的项目的目标是在一个原型低成本易于使用的平台上开发这种超灵敏的蛋白质检测，该平台将适用于低资源实验室和家庭的使用点操作。 从长远来看，目标是使该平台商业化，用于解决以前由于需要集中实验室和训练有素的人员而无法处理的各种蛋白质检测问题。 其目的是将联合收割机三种已知技术结合到一个通用平台中，该平台将允许快速检测不到一百万个拷贝的蛋白质，其格式如下：1）在硝酸纤维素条上进行侧流免疫测定试验以捕获特异性蛋白质靶，2）通过掺入附着于检测抗体的核酸模板来检测包裹蛋白质靶的组装蛋白质堆叠的存在，3）使用等温核酸扩增方法（iSDA）原位扩增所述模板，所述方法也包含在纸系统中并由印刷电路板上的简单加热器支撑，以及4）使用手机和包含两个滤光器以覆盖照相机的黑盒，通过荧光在纸内真实的实时检测扩增过程。的闪光灯和相机。 初步数据显示，检测流感核蛋白的常见侧流免疫测定与iSDA的组合可以使测试的灵敏度提高10，000倍。 然而，这4个过程尚未以快速运行并避免标记检测抗体冲洗不良（产生假阳性信号）的检测盒形式组合。 该项目的目标是将这些过程集成到一个快速方便的样品到结果的原型仪器中，该仪器对于任何熟悉现代智能手机的人来说都很简单。 它将不再占用智能手机的桌面空间，除了手机电池外，不需要其他电源即可运行多个测试，允许设备在任何地方运行，并通过互联网立即将数据传输到集中设施。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。