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

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/10320988
• 关键词: 2019-nCoV; ACE2; Antibodies; Antibody Response; Award; Biological Assay; Biological Models; Biometry; Biotechnology; Body Fluids; Bypass; COVID-19; COVID-19 detection; COVID-19 diagnosis; COVID-19 patient; COVID-19 test; Cells; Characteristics; Clinical; Clinical Microbiology; Column Chromatography; Communicable Diseases; Communication; Coronavirus; Detection; Development; Devices; Diagnosis; Disease; Doctor of Philosophy; Documentation; Emergency Situation; Engineering; Enzyme-Linked Immunosorbent Assay; FDA Emergency Use Authorization; Fluorescence; Glycoproteins; Healthcare; Hospitals; Human; Immunoglobulin G; Immunoglobulin M; In Situ; Individual; Infection; Institutes; Knowledge; Label; Laboratories; Licensing; Location; Measurement; Mediating; Medical center; Membrane; Membrane Proteins; Methods; Microfluidics; Molecular; Molecular Sieve Chromatography; Nanotechnology; Nature; Nucleic Acid Amplification Tests; Nucleic Acids; Ohio; Particulate; Patients; Phase; Physicians; Plasma; Pneumonia; Polymerase Chain Reaction; Population; Production; Proteins; RNA; RNA Viruses; Radiologic Health; Reader; Research Personnel; Retroviridae; Reverse Transcription; Risk; SARS-CoV-2 exposure; SARS-CoV-2 positive; Saliva; Sampling; Sensitivity and Specificity; Serology test; Serum; Services; Signal Transduction; Sorting - Cell Movement; Speed; Surface; System; Systems Biology; Technology; Testing; United States National Institutes of Health; Universities; Validation; Vesicle; Viral; Viral Antibodies; Viral Load result; Viral Proteins; Virus; World Health Organization; antigen test; base; biochip; commercialization; comparative; design; detection method; emerging pathogen; exosome; experience; experimental study; extracellular; extracellular vesicles; fluorescence microscope; improved; interest; isothermal amplification; microfluidic technology; multidisciplinary; nanobiotechnology; nanofabrication; nasopharyngeal swab; new technology; novel coronavirus; pandemic disease; particle; point of care; prognostic; protein expression; research clinical testing; saliva sample; sample collection; scale up; standard of care; viral RNA; viral detection

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 世界卫生组织已确认新型冠状病毒肺炎（COVID-19）已在全球大流行 因暴露于严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2)。冠状病毒 (CoV) 是有膜包膜的正义单链 RNA 病毒，上面装饰有膜蛋白。这 刺突 (S) 糖蛋白参与病毒通过人血管紧张素与宿主细胞的附着和融合 转换酶 2 (hACE2)。有不同的检测方法可用于检测 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 (NPS) 中提取。 此外，鉴于 NPS 的侵入性，唾液被认为是检测的替代方法。方法 绕过 RNA 提取以及等温扩增，例如环介导的等温扩增 (LAMP) 的开发是为了提高病毒 RNA 检测的速度。然而，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）。虽然速度很快，但这些测试存在明显的假阴性/阳性现象。此外，他们不 检测当前感染。因此，为了缓解当前的医疗危机，新技术能够 在单一病毒水平上同时检测病毒 RNA/蛋白质，并检测宿主抗体反应 集成设备中的体液对于增强 COVID-19 诊断非常有价值。 最近，我们的团队作为细胞外 RNA 通讯联盟 (ERCC2) 第二阶段的一部分， 成功开发了一种微流体技术，能够从生物流体中捕获单个外泌体，并 然后同时定量外泌体表面蛋白和 RNA 货物。鉴于尺寸相似 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，我们建议开发并验证我们的微流体系统的安全使用版本 直接检测 SARS-CoV-2。该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、 和宿主抗体 (IgG/IgM) 在同一样本中，从而实现诊断、疾病状态和预后 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 (SV) 和 热灭活的 SARS-CoV-2 病毒颗粒 (SVV) 将被设计并掺入生物液中以验证和 校准系统。为了展示临床实用性，我们的生物芯片技术将使用 两个独立实验室（美国系统生物学研究所）提供了来自 COVID-19 患者的不同生物液 西雅图和俄亥俄州立大学 (OSU) 哥伦布韦克斯纳医学中心）。获得的测量结果 来自生物芯片的结果将与标准 qRT-PCR 和 ELISA 方法进行比较。将制定过渡计划 FDA 紧急使用授权 (EUA) 通过 COVID-19 临床应用生物芯片技术 俄勒冈州立大学韦克斯纳医学中心的测试实验室。还将通过许可制定商业化计划 到一家生物技术公司。 我们组建了一支多学科团队，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 患者样本采集和 测试。拟议的目标和里程碑如下： 具体目标 1：开发可同时捕获、固定和表征的集成生物芯片 单一 SARS-CoV-2 和 IgG/IgM 蛋白。里程碑。 (i) 分类、捕获和定量分析 在刺突实验中选择单一病毒中的蛋白质和病毒RNA，重复性>95%； (ii) 灵敏度 单病毒检测重复性 >90%，灵敏度比当前 qRT-PCR 和 ELISA 高 5 倍 方法。具体目标 2：在生物体液中检测单一 SARS-CoV-2 病毒和相关 IgG/IgM COVID-19 患者。里程碑。 (i) 临床样本的定量分析重复性 >95%； (二) 95% 生物芯片技术与基于实验室的 qRT-PCR 检测 SARS-CoV-2 的一致性 和酶联免疫吸附试验。具体目标 3：生物芯片技术转型计划。里程碑。 (i) 提交文件 向 FDA 设备和放射健康中心 (CDRH) 提交 EUA； (ii) 扩大商业化计划 GMP芯片生产。

项目成果

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

设计可调谐微图案阵列分析来分选单个细胞外囊泡和颗粒，从而原位检测 RNA 和蛋白质。

• 发表时间: 2023-11
• 作者: Zhang, Jingjing;Rima, Xilal Y;Wang, Xinyu;Nguyen, Luong T H;Huntoon, Kristin;Ma, Yifan;Palacio, Paola Loreto;Nguyen, Kim Truc;Albert, Karunya;Duong;Walters, Nicole;Kwak, Kwang Joo;Yoon, Min Jin;Li, Hong;Doon;Hisey
• 通讯作者: Hisey

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.

从固定化微凝胶中微流体收获乳腺癌肿瘤球体衍生的细胞外囊泡，用于单囊泡分析。

• 发表时间: 2022-06-28
• 影响因子: 6.1
• 作者: Rima, Xilal Y;Zhang, Jingjing;Nguyen, Luong T H;Rajasuriyar, Aaron;Yoon, Min Jin;Chiang, Chi;Walters, Nicole;Kwak, Kwang Joo;Lee, L James;Reátegui, Eduardo
• 通讯作者: Reátegui, Eduardo