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

摘要 Nanohydrate提出将传统钌的电化学发光（ECL）的极端灵敏度 三联吡啶（Ru（bpy）3）或更有前途的基于量子点（Qdot）的ECL灵敏度， 方便和快速（约15分钟）的侧向流（LF）试纸，以产生超灵敏度测定菜单， 主要食源性致病菌和病毒的检测。已知LF条测定快速、高度 负担得起的，方便的现场诊断，但通常缺乏灵敏度，可能会错过食源性病原体的检测 即使在耗时的富集培养物中，也能以低水平检测到假阴性结果。在某些情况下，LF条开发人员 转向荧光以增加灵敏度。但是，荧光具有来自激发的固有自发荧光背景 样品中的其他生物材料，从而限制了其灵敏度。虽然化学发光（CL）可以 由于其初始黑色背景和高信噪比（SNR），CL是超灵敏的， 并且需要时间延迟以允许其在测量之前达到稳定的平衡， 再现性然而，通过控制工作电极电压（仅约1.25V）来控制ECL是简单的，因此 也可以使用电池供电的手持阅读器。ECL可以随着时间的推移放大或累积信号，每次Ru（bpy）3 氧化还原“轮子”被电转动，释放出另一个红色光子，从而产生与放射性同位素相当的灵敏度。 当ECL信号随时间积分时的方法。较新的基于Qdot的ECL技术有望达到100万- ECL也比Ru（bpy）3-抗体或适体标签增加了一倍。因此，纳米技术公司建议在其最近的疾病预防控制中心的基础上， 由美国国防部/战略事务部资助的第一阶段和第二阶段SBIR合同，使其能够开发高灵敏度抗体LF 使用Qdots的甲型/B流感和SARS-CoV-2荧光和ECL LF检测试纸。这些Qdot-LF试纸将 在ECL输出和检测限方面，与基于抗体和DNA适体-Ru（bpy）3的LF试纸条进行比较 (LOD)食源性病原体（最初是李斯特菌和沙门氏菌）的第一阶段。获奖的LF带材设计将用于 在第二阶段开发便携式ECL LF试纸条阅读器和更多食源性细菌和病毒检测， 向公众提供最灵敏和快速的手持式现场食源性病原体检测，以帮助快速检测和控制 食源性疾病暴发。

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.