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

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；捕获样本和获得测量结果之间的时间长度也很重要。因此，人们大力推动开发对这种分子的敏感测试，任何人都可以在任何地方在几分钟内进行这种测试。这是正确的，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，举两个例子。许多待检测的分子或“分析物”不是核酸(DNA和RNA)，因此不能被可以检测到几个拷贝的目标的高灵敏度核酸扩增测试检测到。检测敏感蛋白质的新方法使用大型仪器，而且必须在资金充足的实验室里进行。该项目的总体目标是能够在任何地方，特别是在家庭或低资源环境中，以低成本快速测量低浓度蛋白质分析物。该项目将在单一设备中结合低成本、低灵敏度的纸基横向流动测试与使用等温核酸放大的信号增强。这个为期一年的项目的目标是在一个低成本、易于使用的原型平台上开发这种超灵敏的蛋白质检测，该平台将适合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，以解决以前由于需要一个中央实验室和训练有素的人员而无法解决的各种蛋白质检测问题。其目的是将三种已知技术结合到一个公共平台中，该平台将允许以组合的格式快速检测少于一百万个拷贝的蛋白质：1)在硝酸纤维素条上的横向流动免疫分析测试以捕获特定的蛋白质靶标，2)通过将核酸模板结合到检测抗体上来检测夹在蛋白质靶标之间的组装的蛋白质堆栈的存在，3)使用也包含在纸系统中并由印刷电路板上的简单加热器支撑的等温核酸扩增方法(ISDA)原位扩增所述模板，4)用一部手机和一个装有两个滤光片的黑匣子盖住摄像机--S闪光灯和摄像机，在纸内实时检测荧光放大过程。初步数据表明，将检测流感核蛋白的普通横向流动免疫分析与ISDA相结合，可以将检测的灵敏度提高1万倍。然而，这4种方法并没有组合成一种快速运行并避免标记的检测抗体(产生假阳性信号)冲洗不良的卡盒形式。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，任何熟悉现代智能手机的人都可以简单地使用它。它不会占用比智能手机更多的桌面空间，不需要手机电池以外的电力来运行多个测试，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中央设施。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.