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

采用单细胞外囊泡表征技术对生物体液中的 SARS-CoV-2 进行多参数集成分子检测

基本信息

批准号：
10320988
负责人：
Inyoul Lee
金额：
$ 87.9万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-21 至 2024-06-30
项目状态：
已结题

项目摘要

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.

抽象的世界卫生组织已经认识到新型冠状病毒肺炎的全球大流行（Covid-19）从暴露于严重的急性呼吸综合征冠状病毒2（SARS-COV-2）。冠状病毒（COVS）是膜开发的阳性，单链的RNA病毒，用膜蛋白装饰。这峰值糖蛋白与人血管紧张素 - 转换酶2（HACE2）。有不同的测定法需要测试COVID-19，包括核酸，抗原，以及可用于医院，护理点和大规模人口测试的血清学检查。核酸测试是检测SARS-COV-2的标准方法，该方法由病毒RNA的扩增组成通过定量反向转录聚合酶链反应（QRT-PCR）来自鼻咽拭子（NP）。此外，鉴于NP的侵入性，唾液被认为是检测的替代方法。方法旁路RNA提取以及等温扩增，例如环路介导的等温扩增（灯）已开发以提高病毒RNA检测的速度。但是，病毒蛋白表达 QRT-PCR无法检测到。另一方面，血清学测试基于宿主抗体病毒（IgG/IgM）。尽管很快，但这些测试患有明显的假阴性/阳性。此外，他们没有检测当前感染。因此，为了缓解当前的医疗危机，新技术能够在单个病毒水平上同时进行病毒RNA/蛋白质检测，并从A的宿主抗体反应检测集成装置中的体液对于增强Covid-19诊断而言是非常有价值的。最近，作为细胞外RNA通信联盟（ERCC2）2阶段的一部分，我们的小组具有成功地开发了一种微流体技术，能够从生物流体和然后同时量化外泌体表面蛋白和RNA货物。考虑到大小的相似之处以及外泌体和冠状病毒之间的其他特征，我们的技术可以适应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量化病毒蛋白，病毒RNA，并在同一样品中宿主抗体（IgG/IgM），以实现诊断，疾病状态和预后评估。模型系统，包括患者血清的宿主IgG/IgM，标准合成囊泡（SVS）和热灭活的SARS-COV-2病毒颗粒（SVV）将在生物流体中设计和峰值以验证和校准系统。为了证明临床实用程序，我们的生物芯片技术将被部署和测试来自两个独立实验室的Covid-19患者的不同生物流体（系统生物学研究所西雅图和俄亥俄州立大学（OSU）位于哥伦布的韦克斯纳医学中心）。获得的测量将生物芯片与标准QRT-PCR和ELISA方法进行比较。将准备过渡计划用于FDA紧急使用授权（EUA）通过COVID-19临床应用生物芯片技术的应用在OSU Wexner医疗中心测试实验室。商业化计划也将通过许可制定到一家生物技术公司。我们已经组建了一个具有广泛知识和纳米属技术的知识和经验的多学科团队，微流体，微/纳米制作，感染性疾病和临床covid-19患者样本收集和测试。拟议的目标和里程碑如下：特定目的1：开发综合生物芯片以同时捕获，修复和表征单SARS-COV-2和IgG/IgM蛋白。里程碑。（i）分类，捕获和定量分析在尖峰实验中，单个病毒中选择的蛋白质和病毒RNA，可重复性> 95％；（ii）灵敏度与当前QRT-PCR和ELISA相比，单个病毒检测> 90％的可重复性和5倍更好的灵敏度方法。特定目标2：在生物流体中的单个SARS-COV-2病毒和相关的IgG/IgM的测试 Covid-19患者。里程碑。（i）对重复性> 95％的临床样本的定量分析；（ii）95％在生物芯片技术和基于实验室的QRT-PCR之间检测SARS-COV-2的一致性和Elisa。特定目的3：生物芯片技术过渡计划。里程碑。（i）提交文档到FDA设备和放射健康中心（CDRH）的EUA；（ii）扩大商业化计划 GMP芯片生产。