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（COVID-19）全球大流行 严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）。冠状病毒（CoV） 是膜包膜正义单链RNA病毒，修饰有膜蛋白。的 刺突（S）糖蛋白参与病毒通过人血管紧张素- 转化酶2（hACE 2）。有不同的检测方法来检测COVID-19，包括核酸，抗原， 以及可用于医院、护理点和大规模人群检测的血清学检测。核酸 检测是检测SARS-CoV-2的标准方法，包括病毒RNA的扩增 通过定量逆转录聚合酶链反应（qRT-PCR）从鼻咽拭子（鼻咽拭子）中获得。 此外，鉴于唾液的侵入性，唾液被认为是检测的替代品。方法 其绕过RNA提取，以及等温扩增如环介导等温扩增 （LAMP），已经被开发用于提高病毒RNA检测的速度。然而，病毒蛋白表达 不能通过qRT-PCR检测。另一方面，血清学测试是基于针对 病毒（IgG/IgM）。虽然快速，但这些测试遭受显著的假阴性/阳性。此外，他们不 检测当前感染。因此，为了缓解当前的医疗危机，能够 在单一病毒水平上同时检测病毒RNA/蛋白质，并从 集成设备中的体液对于增强COVID-19诊断非常有价值。 最近，我们的小组，作为细胞外RNA通讯联盟（ERCC 2）第2阶段的一部分， 成功开发了能够从生物流体中捕获单个外泌体的微流体技术， 然后同时定量外泌体表面蛋白和RNA货物。鉴于大小相似 以及外泌体和冠状病毒之间的其他特征，我们的技术可以适用于COVID-19 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， 直接检测SARS-CoV-2集成系统能够进行多参数检测，以增强 COVID-19诊断该平台将被设计为同时定量病毒蛋白，病毒RNA， 和宿主抗体（IgG/IgM）在同一样品中，使诊断，疾病状态，和预后 考核模型系统，包括来自患者血清的宿主IgG/IgM、标准合成囊泡（SV）和 热灭活的SARS-CoV-2病毒颗粒（SVV）将被设计并加入生物液体中，以验证和 校准系统。为了证明临床实用性，我们的生物芯片技术将使用 在两个独立的实验室中，来自COVID-19患者的不同生物流体（ 西雅图和哥伦布的俄亥俄州州立大学（OSU）Wexner医学中心）。获得的测量 将来自生物芯片的检测结果与标准qRT-PCR和ELISA方法进行比较。将制定过渡计划 通过COVID-19临床试验申请FDA紧急使用授权（EUA）生物芯片技术 OSU Wexner医疗中心的检测实验室。还将通过许可制定商业化计划 一家生物科技公司 我们已经组建了一个多学科的团队，在纳米生物技术方面拥有丰富的知识和经验， 微流体、微/纳米制造、传染病和临床COVID-19患者样本采集， 试验.拟议的目标和里程碑如下： 具体目标1：开发集成生物芯片，同时捕获，固定和表征 单一SARS-CoV-2和IgG/IgM蛋白。Mildly. (i)分类、捕获和定量分析 在加标实验中，单一病毒中的选定蛋白质和病毒RNA具有>95%的可重复性;（ii） 单一病毒检测，重复性>90%，灵敏度比当前的qRT-PCR和ELISA高5倍 方法.具体目标2：检测来自以下国家的生物液体中的单一SARS-CoV-2病毒和相关IgG/IgM 新冠肺炎患者。Mildly. (i)临床样品的定量分析，重复性>95%;（ii）95% 生物芯片技术与实验室qRT-PCR检测SARS-CoV-2的一致性 和ELISA。具体目标3：生物芯片技术转型计划。Mildly. (i)提交文件 向FDA器械和放射卫生中心（CDRH）提交EUA;（ii） 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.