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

摘要 COVID-19大流行的快速发展揭示了当前技术的缺陷， 诊断.基于基因和抗原/抗体的检测方法的可用性有限，灵敏度和/或特异性不足， 基于的测试导致相对较高的假阴性/阳性测试结果率，这进一步导致 病人隔离以及卫生当局和公众之间的混乱。电流的基本局限性 基于基因的测定源于它们对特定核酸序列的扩增和检测的依赖 在病毒基因组中。目前的测试需要劳动密集型，基于实验室的样品制备方案 用于病毒裂解、遗传物质的提取、分离物质的纯化、用于酶促的热循环 病毒核酸序列的扩增以及由专业人员对复杂结果的解释。我们寻求一种 一种新的快速和直接的病原体检测、鉴定和定量的范例，其中完整的病毒体 通过其独特的表面表位特征直接识别，并且所得的荧光信号是 立即被便携式智能手机荧光计捕获。为了达到对SARS的具体认识- CoV-2病毒体，我们定制了一种基于设计师DNA纳米结构（DDN）的捕获探针， 其顶点精确匹配SARS-CoV-2三聚体的内部和内部空间模式的大分子“网” 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 其被设计为在与多价和模式的刺突结合时具有最大的亲和力和特异性- 搭配时尚当暴露于测试样品时，例如溶液中的唾液或鼻咽拭子材料， DNA菱形“病毒网”迅速并选择性地结合完整的病毒体， 荧光。我们已经成功开发了一种基于智能手机的仪器， 荧光信号在床旁护理（POC）设置。因此，当病毒感染时从病毒网释放的荧光信号 结合SARS-CoV-2可以很容易地检测到我们的智能手机为基础的荧光仪在POC设置。我们 首次提出将联合收割机DDN捕获探针和智能手机荧光计相结合，开发和 证明了一种快速、室温、单步、病毒特异性和超灵敏的诊断检测方法， COVID-19可以在护理点采集样本后立即进行，并提供结果 在< 5分钟。我们的目标包括在POC环境中开发COVID-19检测方法， 它的灵敏度，特异性，重现性和成本效益的表征。我们的研究将以 使用临床样本对系统进行初步验证，并与金标准进行直接比较 实验室PCR检测

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.