Nanobody-Based Electrochemical Biosensor for Real-Time Detection of Aerosolized SARS-CoV2

摘要/项目总结 截至9月初，2019年冠状病毒（COVID-19）已感染620万美国人，造成19万人死亡 2020年（世卫组织网站）;死亡率约为3%。在检测不足和不明症状之间 如果将感染者的人数估计为1000人，实际感染人数可能比报告的高6至24倍。SARS-CoV-2 （CoV-2）是该疾病的潜在病毒，会导致一系列症状;在某些病例中，会出现严重的呼吸道感染 可能导致住院和死亡的呼吸障碍疾病。CoV-2是人与人之间传播的， 人经鼻和喉粘膜吸入病毒后， 接触受污染的表面或吸入雾化的病毒。不幸的是，COVID-19很可能是 到2021年及以后都很普遍。 我们必须提高检测CoV-2的能力。首先，需要测试来诊断个体， 有症状或无症状，以减少社区传播。第二，监控聚集区， 空气传播的病毒，可以告知关闭空间或实施消毒和缓解的决定， 一个地区。我们提出在两种检测装置中使用电化学生物传感器，1）诊断装置， 用于即时检测CoV-2的呼吸测醉器和2）用于实时，连续监测 很大的空间。 我们开发了一种新型的超灵敏的，基于抗体的电化学生物传感器来检测CoV-2 重复结合域（RBD）刺突蛋白。该技术基于微免疫电极（MIE） 生物传感器由Cirrito实验室开创，用于研究神经变性环境中的蛋白质动力学（2，3）。 生物传感器使用伏安法来测量酪氨酸氨基酸的氧化;氧化是酪氨酸氨基酸的释放。 电子，生物传感器测量为电流的变化。抗体共价连接到 电极表面以提供选择性。我们的原型CoV-2生物传感器对2毫微微克/毫升敏感， 与目前对低皮克/毫升范围敏感的几种CoV-2抗原检测相比， 该提案将首先（目标1）优化我们的CoV-2生物传感器，以检测CoV-2病毒颗粒，以及测试 几个参数来增加灵敏度和寿命。目标2将建立一个测试呼气测醉器，将利用 喷雾器，以产生含有气溶胶液滴的载有病毒的空气，所述气溶胶液滴类似于含有限定的 浓度的CoV-2病毒颗粒。目标3将在真实环境中测试机载生物传感器。Co-I Chakrabarty的实验室具有模仿现实世界环境条件的独特能力，特别是 在大气气溶胶的情况下，测试和优化生物传感器的性能所必需的， 外地部署。大气条件包括相对湿度（RH）和温度，以及常见的 室内空气中的污染物 寻找新的方法来检测CoV-2，并创建一个平台来检测其他和未来 病原体，将使我们能够限制病毒在整个社区的传播，在当前和未来 流行病

ABSTRACT/PROJECT SUMMARY Coronavirus 2019 (COVID-19) has afflicted 6.2 million Americans and killed 190,000 as of early September 2020 (WHO website); a roughly 3% mortality. Between a shortage in testing and unidentified asymptomatic individuals, the actual number of those infected could be 6 to 24-fold higher than that reported. SARS-CoV-2 (CoV-2), the virus underlying the disease, results in a range of symptoms; in select cases a severe respiratory illness that impedes breathing that could lead to hospitalization and death. CoV-2 is transmitted person-to- person via inhalation of the virus through mucosal membranes of the nose and throat from transfer after touching a contaminated surface or by inhaling aerosolized virus. Unfortunately, COVID-19 is likely to be prevalent well into 2021 and beyond. We must increase our ability to test for CoV-2. First, testing is needed to diagnose individuals that are symptomatic or asymptomatic to reduce community spread. And second, monitoring gathering areas for airborne virus that could inform the decision to shutdown a space or implement disinfection and mitigation of an area. We propose to use an electrochemical biosensor in two detection devices, 1) a diagnostic breathalyzer for instant detection of CoV-2 and 2) an airborne detector for real-time, continuous surveillance of a large space. We have developed a novel ultra-sensitive, antibody-based electrochemical biosensor to detect CoV-2 repeat binding domain (RBD) spike protein. The technology is based on a micro-immunoelectrode (MIE) biosensor pioneered by the Cirrito laboratory to study protein dynamics in the setting of neurodegeneration (2,3). The biosensor uses voltammetry to measure the oxidation of tyrosine amino acids; oxidation is the release of electrons that the biosensor measures as a change in current. Antibodies are covalently attached to the electrode surface to provide selectivity. Our prototype CoV-2 biosensor is sensitive to 2 femtogram/ml, compared to several current CoV-2 antigen tests that are sensitive to the low picogram/ml range. The proposal will first (Aim 1) optimize our CoV-2 biosensor to detect CoV-2 viral particles, as well as test several parameters to increase sensitivity and longevity. Aim 2 will build a test breathalyzer that will utilize a nebulizer to generate virus laden air containing aerosol droplets similar to a breath that contain defined concentrations of CoV-2 viral particles. Aim 3 will test the airborne biosensor in a realistic environment. Co-I Chakrabarty’s laboratory has unique capabilities of mimicking real-world environmental conditions, especially in the context of atmospheric aerosols, necessary for testing and optimizing the biosensor’s performance for field deployment. Atmospheric conditions include relative humidity (RH) and temperature, as well as common airborne pollutants found indoors. Finding novel means to detect the CoV-2, as well as create a platform to detect other and future pathogens, would enable us to limit the viral spread throughout the community in the current and future pandemics.