Multi-parametric Integrated Molecular Detection of SARS-CoV-2 from Biofluids by Adapting Single Extracellular Vesicle Characterization Technologies

摘要 世界卫生组织已确认新型冠状病毒肺炎(新冠肺炎)在全球范围内大流行 因接触严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)。冠状病毒(CoV) 是被膜包裹的正义单链RNA病毒，上面装饰着膜蛋白。这个 SPEKE(S)糖蛋白通过人血管紧张素-1参与病毒对宿主细胞的附着和融合。 转化酶2(HACE2)。新冠肺炎有不同的检测方法，包括核酸、抗原、 以及可用于医院、医疗点和大规模人口检测的血清学检测。核酸 检测是检测SARS-CoV-2的标准方法，包括病毒RNA的扩增 用定量逆转录聚合酶链式反应(qRT-PCR)从鼻咽拭子中提取。 此外，考虑到NPS的侵袭性，唾液被认为是检测的一种替代方法。方法 绕过RNA提取和等温扩增，如环介导的等温扩增 为了提高病毒RNA检测的速度，已经开发了(LAMP)。然而，病毒蛋白的表达 不能用qRT-PCR检测到。另一方面，血清学测试是基于宿主抗体对 病毒(Ig G/Ig M)。虽然这些检测速度很快，但存在严重的假阴性/阳性。此外，他们也不会 检测当前感染。因此，为了缓解目前的医疗危机，新技术能够 同时检测单个病毒水平的病毒RNA/蛋白质和宿主抗体反应 一个集成设备中的体液对于增强新冠肺炎的诊断将具有非常高的价值。 最近，作为细胞外RNA通讯联盟(ERCC2)第二阶段的一部分，我们的小组已经 成功开发了一种微流体技术，能够从生物体液中捕获单个外切体，并 然后同时定量外体表面蛋白和RNA货物。鉴于它们在尺寸上的相似性 和其他特征，我们的技术可以适用于新冠肺炎 诊断。因此，我们建议开发和验证我们的微流体系统的安全使用版本，用于 直接检测SARS-CoV-2。该集成系统能够对增强型多参数进行检测 新冠肺炎诊断。该平台将被设计成同时定量病毒蛋白、病毒RNA、 和宿主抗体(IgG/IgM)在同一样本中，使诊断、疾病状态和预后成为可能 评估。模型系统，包括来自患者血清的宿主IgG/IgM，标准合成囊泡(SVS)，以及 热灭活SARS-CoV-2病毒颗粒(SVV)将被设计并添加到生物液中，以验证和 校准系统。为了证明临床用途，我们的生物芯片技术将被部署和测试，使用 来自两个独立实验室的新冠肺炎患者的不同生物液(系统生物学研究所在 西雅图和俄亥俄州立大学(OSU)哥伦布的韦克斯纳医学中心)。已获得的测量值 从生物芯片中提取的DNA将与标准的qRT-PCR和ELISA法进行比较。将制定一项过渡计划 通过新冠肺炎临床中心申请FDA紧急使用授权(EUA)的生物芯片技术 俄亥俄州立大学韦克斯纳医学中心的检测实验室。还将通过许可方式制定商业化计划 一家生物科技公司。 我们已经组建了一支在纳米生物技术方面拥有丰富知识和经验的多学科团队， 微流体、微纳制造、传染病、临床新冠肺炎患者样本采集和 测试。建议的目标和里程碑如下： 具体目标1：开发一种可同时捕获、固定和表征的集成生物芯片 单一的SARS-CoV-2和Ig G/Ig M蛋白。里程碑。(一)分类、捕获和定量分析 单个病毒中选定的蛋白质和病毒RNA在尖峰实验中具有95%的重复性；(Ii)敏感性 单一病毒检测的重复性为90%，灵敏度比目前的qRT-PCR和ELISA高5倍 方法：研究方法。特定目标2：检测SARS-CoV-2单个病毒及其相关的免疫球蛋白/免疫球蛋白M 新冠肺炎患者。里程碑。(I)临床样本的定量分析，重复性为95%；(Ii)95% 生物芯片技术与实验室QRT-PCR检测SARS-CoV-2的一致性研究 还有伊莉莎。具体目标3：生物芯片技术过渡计划。里程碑。(I)提交文件 向FDA设备和辐射健康中心(CDRH)提供EUA；(Ii)扩大商业化计划 GMP芯片生产。

Abstract The World Health Organization has recognized a global pandemic of novel coronavirus pneumonia (COVID-19) from exposure to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronaviruses (CoVs) are membrane-enveloped positive-sense, single-stranded RNA viruses decorated with membrane proteins. The spike (S) glycoprotein is implicated in the viral attachment and fusion to host cells via the human angiotensin- converting enzyme 2 (hACE2). There are different assays to test for COVID-19, including nucleic acid, antigen, and serological tests that can be used in hospitals, point-of-care, and large-scale population testing. Nucleic acid testing is the standard method for the detection of SARS-CoV-2, which consists of the amplification of viral RNA from nasopharyngeal swabs (NPS) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Furthermore, given the invasive nature of NPS, saliva is being considered an alternative for detection. Methods that bypass RNA extraction, as well as isothermal amplification such as loop-mediated isothermal amplification (LAMP), have been developed to improve the speed of viral RNA detection. However, viral protein expression cannot be detected by qRT-PCR. Serological tests, on the other hand, are based on host antibodies against the virus (IgG/IgM). Although fast, these tests suffer from significant false negative/positive. Besides, they do not detect a current infection. Therefore, to relieve the current healthcare crisis, new technologies capable of simultaneous viral RNA/protein detection at the single virus level and host antibody response detection from a body fluid in an integrated device would be highly valuable for enhanced COVID-19 diagnosis. Recently, our group, as part of Phase 2 of the Extracellular RNA Communication Consortium (ERCC2), has successfully developed a microfluidics technology capable of capturing individual exosomes from biofluids and then simultaneously quantify both exosomal surface proteins and RNA cargo. Given the resemblance in size and other characteristics between exosomes and coronaviruses, our technology can be adapted for COVID-19 diagnosis. Therefore, we propose to develop and validate a safe-to-use version of our microfluidics system for direct detection of SARS-CoV-2. The integrated system is capable of multi-parametric detection for enhanced COVID-19 diagnosis. The platform will be engineered to simultaneously quantify both viral protein, viral RNA, and host antibodies (IgG/IgM) in the same sample, enabling diagnosis, disease status, and prognostic assessment. Model systems, including host IgG/IgM from patient serum, standard synthetic vesicles (SVs), and heat-inactivated SARS-CoV-2 viral particles (SVVs), will be designed and spiked in biofluids to validate and calibrate the system. To demonstrate the clinical utility, our biochip technology will be deployed and tested using different biofluids from COVID-19 patients at two independent laboratories (Institute of Systems Biology in Seattle and The Ohio State University (OSU) Wexner Medical Center in Columbus). Measurements obtained from the biochips will be compared to standard qRT-PCR and ELISA methods. A transition plan will be prepared for FDA Emergency Use Authorization (EUA) application of the biochip technology through a COVID-19 clinical testing laboratory at OSU Wexner Medical Center. A commercialization plan will also be developed via licensing to a biotech company. We have assembled a multi-disciplinary team with extensive knowledge and experience in nanobiotechnology, microfluidics, micro/nano-fabrication, infectious diseases, and clinical COVID-19 patient sample collection and testing. The proposed aims and milestones are given as follows: Specific Aim 1: Development of an integrated biochip to simultaneously capture, fix, and characterize single SARS-CoV-2 and IgG/IgM proteins. Milestones. (i) Sorting, capture, and quantitative analysis of selected proteins and viral RNA in single virus in spike experiments with >95% repeatability; (ii) A sensitivity of single virus detection with >90% repeatability and 5-fold better sensitivity than the current qRT-PCR and ELISA methods. Specific Aim 2: Testing of single SARS-CoV-2 virus and associated IgG/IgM in biofluids from COVID-19 patients. Milestones. (i) Quantitative analysis of clinical samples with >95% repeatability; (ii) 95% of concordance for the detection of SARS-CoV-2 between the biochip technology and the lab-based qRT-PCR and ELISA. Specific Aim 3: Biochip technology transition plan. Milestones. (i) Submission of documentation to the FDA Center for Devices and Radiological Health (CDRH) for EUA; (ii) Scale-up commercialization plan for GMP chip production.