Lateral Flow-Electrochemiluminescent (LF-ECL) Test Strips and Portable Reader for Ultrasensitive Foodborne Pathogen Detection

Summary Abstract Nanohmics proposes to couple the extreme sensitivity of electrochemiluminescence (ECL) from traditional ruthenium trisbipyridine (Ru(bpy)3) or even more promising quantum dot (Qdot)-based ECL sensitivity with the portability, convenience and speed (~ 15 minutes) of lateral flow (LF) test strips to produce a menu of ultrasensitive assays for detection of the major foodborne pathogenic bacteria and viruses. LF strip assays are known for being rapid, highly affordable, and facile onsite diagnostics, but generally lack sensitivity and may miss detection of foodborne pathogens (false negative results) at low levels even in time-consuming enrichment cultures. In some cases, LF strip developers have turned to fluorescence for added sensitivity. But, fluorescence has innate autofluorescent background from the excitation of other biological materials in samples, thus limiting its sensitivity. And although chemiluminescence (CL) can be ultrasensitive due to its initial black background and high signal to noise ratios (SNRs), CL is difficult to control spatially on a test strip and requires temporal delays to allow it to reach stable equilibrium prior to measurement which limits reproducibility. However, ECL is simple to control by controlling the working electrode voltage (only about 1.25V), thus enabling battery-operated handheld readers too. ECL can amplify or accrue the signal over time each time the Ru(bpy)3 redox "wheel" is turned electrically to release another red photon, thus giving sensitivity comparable to radioisotopic methods when the ECL signal is integrated over time. Newer Qdot-based ECL techniques promise as much as a million- fold increase in ECL over Ru(bpy)3-antibody or aptamer tags as well. Thus, Nanohmics proposes to build on its recent CDC- funded Phase I and DoD/OSD-funded Phase II SBIR contracts that are enabling it to develop highly sensitive antibody LF test strips for Influenza A/B and SARS-CoV-2 fluorescent and ECL LF assays using Qdots. These Qdot-LF test strips will be compared with antibody- and DNA aptamer-Ru(bpy)3-based LF test strips in terms of ECL output and limits of detection (LOD) for foodborne pathogens (initially Listeria and Salmonella) in Phase I. The winning LF strip design will be used for development of a portable ECL LF test strip reader and more foodborne bacterial and virus assays in Phase II to deliver to the public the most sensitive and rapid handheld onsite foodborne pathogen tests to aid in rapid detection and control of foodborne disease outbreaks.