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

用于超灵敏食源性病原体检测的横向流动电化学发光 (LF-ECL) 测试条和便携式读数器

• 批准号: 10483325
• 负责人: John Bruno
• 金额: $ 22.5万
• 依托单位: NANOHMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10483325
• 关键词: Lateral; Flow; Electrochemiluminescent; LF; ECL

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.

摘要摘要 NanoHmics提出将电化学发光(ECL)的极高灵敏度与传统Ru相结合 三联吡啶(Ru(Bpy)3)或甚至更有前景的基于量子点(QDot)的ECL灵敏度具有便携性， 方便和快速(约15分钟)的侧向流动(LF)试纸，以产生一套超灵敏的检测菜单，用于 主要食源性致病菌和病毒的检测。If试纸分析以快速、高度 价格实惠、方便的现场诊断，但通常缺乏敏感性，可能会漏掉食源性病原体的检测 (假阴性结果)，即使在耗时的增菌培养中也是如此。在某些情况下，LF带区开发人员已经 转向荧光以增加敏感度。但是，荧光具有天然的自发荧光背景，来自于激发 样品中的其他生物材料，从而限制了其敏感性。尽管化学发光(CL)可以 由于最初的黑色背景和高信噪比(SNR)，CL非常敏感，因此很难在空间上控制 测试条并需要时间延迟以允许其在测量之前达到稳定平衡，这限制了 再现性。然而，ECL通过控制工作电极电压(仅约1.25V)容易控制，因此 也支持电池供电的手持阅读器。ECL可以随着时间的推移放大或增加信号，每次Ru(Bpy)3 氧化还原“轮”通过电旋转释放出另一个红色光子，从而获得与放射性同位素相当的灵敏度。 方法当ECL信号随时间积分时。更新的基于QDot的ECL技术承诺高达100万- 与Ru(Bpy)3-抗体或适配子标签相比，ECL也增加了一倍。因此，NanoHmics建议在其最近的CDC- 资助的第一阶段和国防部/OSD资助的第二阶段SBIR合同，使其能够开发高度敏感的抗体LF 甲型流感/乙型流感和SARS-CoV-2荧光试纸和使用Qdots的ECL LF分析。这些QDot-LF测试条将是 与基于抗体和DNA适配子-Ru(Bpy)3的LF试纸的ECL产量和检出限的比较 (LOD)第一阶段食源性病原体(最初是李斯特氏菌和沙门氏菌)。获奖的LF条带设计将用于 开发便携式ECL LF试纸读取器，并在第二阶段进行更多食源性细菌和病毒检测，以交付至 公众最敏感和最快速的手持现场食源性致病菌检测，以帮助快速检测和控制 食源性疾病暴发。