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 新冠肺炎疫情的快速发展暴露了当前技术的缺陷 诊断。基于基因和抗原/抗体的可获得性有限、敏感性和/或特异性不足- 基于测试的测试导致较高的假阴性/阳性测试结果，这进一步导致 患者隔离以及卫生当局和公众之间的混乱。电流的根本限制 基于基因的分析源于它们依赖于特定核酸序列的扩增和检测 在病毒基因组中。目前的检测需要以实验室为基础的劳动密集型样品制备方案 用于病毒裂解、遗传物质的提取、分离材料的纯化、酶的热循环 病毒核酸序列的扩增，以及专业人员对复杂结果的解释。我们寻求一种 用于快速和直接病原体检测、鉴定和量化的新范例，其中完整的病毒粒子 通过其不同的表面表位特征直接识别，所产生的荧光信号是 立即被便携式智能手机荧光仪捕捉到。为实现对SARS的具体认识-- CoV-2病毒粒子，我们定制了一种基于设计者DNA纳米结构(DDN)的捕获探针，该探针包含 其顶点与SARS-CoV-2三聚体的空间内和空间间模式精确匹配的大分子“网” 刺激性糖蛋白簇，并整合SARS-CoV-2刺激性靶向适配子的网状阵列 当与多价和图案中的尖峰结合时，它们被设计为最大的亲和力和特异性- 相配的时尚。当接触到测试样本时，例如唾液或鼻咽拭子材料在溶液中， 脱氧核糖核酸菱形“病毒网”快速和选择性地结合完整的病毒粒子，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仪器，可以检测和量化 护理点(POC)设置中的荧光信号。因此，从病毒网络释放的荧光信号 在POC设置中，我们的智能手机荧光仪可以很容易地检测到与SARS-CoV-2的结合。我们 首次提出将DDN捕获探头与智能手机荧光仪相结合，开发和 展示一种快速、室温、一步、病毒特异性和超灵敏的诊断方法 新冠肺炎，可在护理点采集样本后立即执行，并提供结果 &lt；5分钟。我们的目标包括在PoC环境下开发新冠肺炎检测，并具有统计稳健性 对其敏感性、特异性、重复性和成本效益进行表征。我们的研究将以 使用临床标本对该系统进行初步验证，并与金标准进行直接比较 实验室的聚合酶链式反应检测。

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.