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陷 基于基因和抗原/抗体的诊断的可用性有限、敏感性和/或特异性不足。 基于测试导致假阴性/阳性测试结果的比率相对较高，这进一步导致了失败 患者隔离以及卫生当局和公众之间的混乱是当前的根本局限性。 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 当前的测试需要劳动密集型的、基于实验室的样品制备方案。 用于病毒裂解、遗传物质提取、分离材料纯化、酶促热循环 我们寻求专业人士对病毒核酸序列的扩增以及对复杂结果的解释。 快速和直接病原体检测、识别和定量的新范例，其中完整的病毒粒子 通过其独特的表面表位特征直接识别，产生的荧光信号是 立即由基于智能手机的便携式荧光计捕获，以实现对 SARS 的特异性识别。 针对 CoV-2 病毒粒子，我们定制了一种基于 DNA 纳米结构 (DDN) 的捕获探针，该探针包含 大分子“网”，其顶点与 SARS-CoV-2 三聚体的空间内和空间模式精确匹配 刺突糖蛋白簇，并整合了 SARS-CoV-2 刺突特异性靶向适体的网状阵列 设计用于在与多价和模式中的尖峰结合时获得最大亲和力和特异性 当暴露于测试样本时，例如溶液中的唾液或鼻咽拭子材料， DNA菱形“病毒网”快速、选择性地结合完整的病毒体，从而触发释放 我们成功开发了一种基于智能手机的仪器，可以检测和量化荧光。 即时检测 (POC) 设置中的荧光信号 因此，荧光信号从病毒网中释放出来。 我们基于智能手机的荧光计可以在 POC 设置中轻松检测到与 SARS-CoV-2 的结合。 首次提议将 DDN 捕获探针和智能手机荧光计结合起来，开发和 展示一种快速、室温、单步、病毒特异性和超灵敏的诊断方法 COVID-19 可以在护理点采集样本后立即进行，并提供结果 我们的目标包括在 POC 环境中开发 COVID-19，并进行技术上稳健的检测。 我们的研究将以其敏感性、特异性、重现性和成本效益的表征结束。 使用临床标本对系统进行初步验证并与黄金标准进行直接比较 实验室PCR测试。

项目成果

Self-Assembly of DNA Nanostructures in Different Cations.

DNA 纳米结构在不同阳离子中的自组装。

• DOI: 10.1002/smll.202300040
• 发表时间: 2023-06-01
• 期刊: Small
• 影响因子: 13.3
• 作者: Arlin Rodriguez;Dhanush G;avadi;avadi;Johnsi Mathivanan;Tingjie Song;B. Madhanagopal;Hannah Talbot;Jia Sheng;Xing Wang;A. Ch;rasekaran;rasekaran
• 通讯作者: rasekaran

Designer DNA NanoGripper.

设计师DNA NanoGripper。

• 发表时间: 2023-04-27
• 作者: Zhou, Lifeng;Xiong, Yanyu;Cooper, Laura;Shepherd, Skye;Song, Tingjie;Dwivedy, Abhisek;Rong, Lijun;Wang, Tong;Cunningham, Brian T;Wang, Xing
• 通讯作者: Wang, Xing

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

SARS-CoV-2 刺突蛋白的突变和进化。

• 发表时间: 2022-03-19
• 作者: Magazine, Nicholas;Zhang, Tianyi;Wu, Yingying;McGee, Michael C;Veggiani, Gianluca;Huang, Weishan
• 通讯作者: Huang, Weishan

Review of HIV Self Testing Technologies and Promising Approaches for the Next Generation.

艾滋病毒自我检测技术和下一代有前途的方法的回顾。

• 发表时间: 2023-02-20
• 作者: Bacon, Amanda;Wang, Weijing;Lee, Hankeun;Umrao, Saurabh;Sinawang, Prima Dewi;Akin, Demir;Khemtonglang, Kodchakorn;Tan, Anqi;Hirshfield, Sabina;Demirci, Utkan;Wang, Xing;Cunningham, Brian T
• 通讯作者: Cunningham, Brian T