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是如此。因此，人们大力推动对此类分子进行灵敏测试，这种测试可以在任何地方、任何人在几分钟内完成。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。许多待检测的分子或“分析物”不是核酸（DNA和RNA），因此无法通过高灵敏度的核酸扩增测试检测到，这种测试可以检测到目标的几个拷贝。灵敏蛋白检测的新方法使用大型仪器，而且必须在资金充足的实验室进行。该项目的总体目标是在任何地方，特别是在家庭或资源匮乏的环境中，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、低灵敏度的基于纸张的横向流动测试和等温核酸扩增的信号增强。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。其目的是将三种已知技术结合到一个共同的平台上，该平台将允许快速检测少于100万份的蛋白质拷贝。1)在硝化纤维素条上进行横向流动免疫分析，以捕获特定的蛋白质靶标；2)通过将核酸模板与检测抗体结合，检测夹在蛋白质靶标上的组装蛋白堆栈的存在；3)使用等温核酸扩增法（iSDA）原位扩增所述模板，该方法也体现在纸系统中，并由印刷电路板上的简单加热器支持。4)在纸上实时检测荧光放大过程，使用手机和一个包含两个光学滤光片的黑盒子来覆盖相机。S闪光灯和相机。初步数据表明，将检测流感核蛋白蛋白的普通侧流免疫分析法与iSDA相结合，可使该试验的灵敏度提高10,000倍。然而，这4个过程还没有结合在一个快速运行的盒式格式中，并避免对标记的检测抗体进行不良冲洗（这会产生假阳性信号）。该项目的目标是将这些过程集成到一个单一的快速方便的样品到结果的原型仪器中，对于熟悉现代智能手机的人来说，这将是简单易用的。它不会像智能手机那样占用更多的桌面空间，除了手机电池外，不需要任何电源就可以运行多个测试，允许设备在任何地方运行，并通过互联网将数据立即传输到集中设施。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.