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

University Of Washington, Seattle WA

Investigators

Abstract

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.