A Rapid and Sensitive Technology for Direct Sensing of Intact SARS-CoV-2 Virions Using Designer DNA Nanostructure Probes and a Smartphone Fluorimeter

使用设计 DNA 纳米结构探针和智能手机荧光计直接感测完整 SARS-CoV-2 病毒粒子的快速灵敏技术

• 批准号: 10196257
• 负责人: Brian T. Cunningham
• 金额: $ 42.22万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-08 至 2024-09-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196257
• 关键词: 2019-nCoV; Address; Affinity; Agar Gel Electrophoresis; Antigens; Architecture; Avidity; Base Sequence; Binding; Biological Assay; Biosensor; COVID-19; COVID-19 assay; COVID-19 diagnosis; COVID-19 diagnostic; COVID-19 pandemic; Cell surface; Cellular Phone; Cessation of life; Clinical; Communicable Diseases; Complex; Confusion; Country; Custom; Cytolysis; DNA; DNA Viruses; Dengue Virus; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Direct Costs; Engineering; Epitopes; Exposure to; Failure; Fluorescence; Genes; Genetic Materials; Glycoproteins; Goals; Gold; Health; Industrialization; Laboratories; Ligands; Materials Testing; Measures; Membrane Glycoproteins; Modeling; Monitor; Nanostructures; Nucleic Acid Amplification Tests; Nucleic Acids; Pathogen detection; Patients; Pattern; Performance; Positive Test Result; Preparation; Property; Proteins; Protocols documentation; Quarantine; Reagent; Reporter; Reproducibility; Reverse Transcriptase Polymerase Chain Reaction; Saliva; Sampling; Sensitivity and Specificity; Severe Acute Respiratory Syndrome; Shapes; Signal Transduction; Specificity; Specimen; Structure; Surface; Surface Plasmon Resonance; System; Technology; Temperature; Test Result; Testing; Time; Transmission Electron Microscopy; Validation; Viral; Viral Genome; Virion; Virus; amplification detection; antibody test; aptamer; authority; base; clinical application; coronavirus disease; cost; cost effective; cost effectiveness; design; diagnostic assay; env Gene Products; fluorophore; improved; instrument; laboratory experience; microscopic imaging; nanoscale; nasopharyngeal swab; novel; pandemic disease; particle; point of care; point-of-care diagnosis; portability; sample collection; sensor; stem

Abstract The rapid development of the COVID-19 pandemic reveals the shortcomings of current technologies for diagnosis. The limited availability, insufficient sensitivity and/or specificity of gene-based and antigen/antibody- based tests resulted in relatively high rates of false negative/positive test results, which further led to failure of patient quarantine and confusion among health authorities and the public. The fundamental limitations of current gene-based assays stem from their reliance upon amplification and detection of specific nucleic acid sequences within the viral genome. The current test requires labor-intensive, laboratory-based sample preparation protocols for virus lysis, extraction of genetic materials, purification of the isolated materials, thermal cycling for enzymatic amplification of viral nucleic acid sequences, and interpretation of complex results by professionals. We seek a new paradigm for rapid and direct pathogen detection, identification, and quantification in which the intact virions are directly recognized through their distinct surface epitope features, and the resultant fluorescent signal is immediately captured by a portable, smartphone-based fluorimeter. To achieve specific recognition of SARS- CoV-2 virions, we customized a designer DNA nanostructure (DDN)-based capture probe that harbors a macromolecular “net” whose vertices precisely match the intra- and inter-spatial pattern of SARS-CoV-2 trimeric spike glycoprotein clusters, and integrates a net-shaped array of SARS-CoV-2 spike specific-targeting aptamers that are designed for maximum affinity and specificity when binding with spikes in a polyvalent and pattern- matching fashion. When exposed to a test sample, such as saliva or nasopharyngeal swab material in solution, the DNA rhombus-shaped “virus nets” rapidly and selectively bind intact virions to trigger the release of fluorescence. We have successfully developed a smartphone-based instrument that can detect and quantify fluorescent signals in point-of-care (POC) settings. Thus, the fluorescent signal released from the virus net upon binding to SARS-CoV-2 can be readily detected by our smartphone-based fluorimeter in POC settings. We propose to combine DDN capture probes and a smartphone fluorimeter for the first time, to develop and demonstrate a rapid, room temperature, single-step, virus-specific, and ultrasensitive diagnostic assay for COVID-19 that can be performed immediately after sample collection at the point of care, and provide a result in < 5 minutes. Our aims include development of a COVID-19 assay in POC settings, and statistically robust characterization of its sensitivity, specificity, reproducibility and cost-effectiveness. Our study will conclude with a preliminary validation of the system using clinical specimens and direct comparison against a gold-standard laboratory PCR test.

抽象的 COVID-19大流行的快速发展揭示了当前技术的缺点 诊断。基于基因和抗原/抗体的有限的可用性，敏感性不足和/或特异性 基于测试导致相对高的假阴性/阳性测试结果，这进一步导致失败 卫生当局和公众之间的患者隔离和混乱。当前的基本限制 基于基因的测定源于其对特定核酸序列的扩增和检测的依赖 在病毒基因组中。当前的测试需要实验室密集型，基于实验室的样品准备协议 对于病毒裂解，遗传材料的提取，纯化材料的纯化，用于酶促的热循环 病毒核酸序列的扩增以及专业人士对复杂结果的解释。我们寻求一个 用于快速和直接病原体检测，鉴定和定量的新范式，其中完整的病毒 通过其不同的表面表位特征直接识别，所得的荧光信号为 立即被便携式，基于智能手机的荧光计捕获。为了获得对SARS的特定认识 COV-2病毒，我们定制了一个设计师DNA纳米结构（DDN）基于捕获探针 大分子“网”的顶点与SARS-COV-2三聚体的空间间和空间间模式完全匹配 尖峰糖蛋白簇，并集成了一系列的SARS-COV-2 SPIKE特定靶向适体 当与多价和图案中的尖峰结合时，其设计为最大亲和力和特异性 匹配的时尚。当暴露于测试样品中，例如溶液中的唾液或鼻咽拭子材料时， DNA菱形形状的“病毒网”迅速，有选择地结合完整的病毒，以触发释放 荧光。我们已经成功地开发了一种基于智能手机的乐器，该工具可以检测和量化 荧光信号（POC）设置。那，从病毒网释放的荧光信号在 我们的基于智能手机的荧光计在POC设置中很容易检测到与SARS-COV-2的结合。我们 首次结合DDN捕获问题和智能手机荧光计的建议，开发和 展示快速，室温，单步，病毒特异性和超敏感性诊断测定法 Covid-19可以在护理点收集样品后立即执行，并提供结果 在<5分钟内。我们的目的包括在POC环境中开发COVID-19分析，以及统计上强大的 表征其灵敏度，特异性，可重复性和成本效益。我们的研究将包括 使用临床标本对系统进行初步验证，并直接比较金标准 实验室PCR测试。

项目成果

Self-Assembly of DNA Nanostructures in Different Cations.

• DOI: 10.1002/smll.202300040
• 发表时间: 2023-06
• 期刊: Small
• 影响因子: 13.3
• 作者: Arlin Rodriguez;Dhanush Gandavadi;Johnsi Mathivanan;Tingjie Song;B. Madhanagopal;Hannah Talbot;Jia Sheng;Xing Wang;A. Chandrasekaran

Mutations and Evolution of the SARS-CoV-2 Spike Protein.

• DOI: 10.3390/v14030640
• 发表时间: 2022-03-19
• 期刊: Viruses
• 作者: Magazine N;Zhang T;Wu Y;McGee MC;Veggiani G;Huang W
• 通讯作者: Huang W