Rapid Diagnostic Test for Respiratory Syncytial Virus by Digital Nanobubbles

摘要 呼吸道合胞病毒（RSV）是全球儿科死亡的最常见原因之一。快速RSV 诊断对于抗生素的司法使用，减少疾病在医疗机构中的传播， 由于几种RSV抗病毒疗法即将问世，因此能够迅速治疗。目前的诊断方法 依赖耗时的实验室检测，包括病毒培养和聚合酶链反应（PCR），以及 快速诊断测试（例如，侧流免疫测定（LFA）作为独立诊断不够灵敏。 因此，对于RSV的快速和超灵敏诊断测试存在未满足的需求。等离子体耦合 金纳米颗粒（AuNPs）是一种快速的比色诊断测试，它利用了金纳米颗粒（AuNPs）的光学响应。 在目标识别的过程中，分析其浓度。尽管它操作简单， 等离子体偶联测定是有限的。在这项拟议的工作中，我们的目标是大大提高限制 通过创新的数字纳米气泡检测来检测等离子体偶联测定的LOD。我们特别 提出用识别RSV表面的抗体缀合的AuNP直接检测完整的RSV颗粒 融合（F）蛋白。AuNP结合多种RSV F蛋白并导致等离子体偶联。超短激光脉冲 与单一AuNP相比，由于其增强的吸收，选择性地激活偶联的AuNP。这 更大的光吸收导致纳米级空化气泡，即纳米气泡，其可以容易地测量 气泡引起的瞬态散射单个纳米气泡生成导致敏感的数字 通过“开”和“关”信号进行检测。我们的初步结果表明~3个数量级的改善 检测RSV的LOD。在这项拟议的工作中，我们将首先通过优化AuNP来创新AuNP探针， 制剂（大小，浓度和共轭），研究非球形纳米颗粒，以更有效地 病毒结合，并探索用于受控结合的不对称抗体包被的Janus纳米颗粒。接下来， 我们将设计和建造一个原型装置，用于自动样品加载，阅读和数据处理。 数字纳米气泡测定。最后，我们将使用临床RSV分离株（来自不同的遗传毒株）测试该检测试剂盒 和临床标本。通过与现有的PCR和快速诊断试验的比较，我们将建立临床上的 数字纳米气泡测试的灵敏度和特异性。该检测快速（< 30分钟），低成本（AuNP试剂 成本类似于侧流免疫测定），并且对RSV高度敏感和特异。此外，直接 病毒颗粒的检测消除了大量样品制备如核酸提取的需要。 我们的项目的成功将满足快速和敏感的RSV诊断的迫切需求，并解决一个主要的 RSV感染儿科患者的医疗保健需求。

ABSTRACT Respiratory syncytial virus (RSV) is one of the most common causes to pediatric death globally. Rapid RSV diagnostics is important for judicial use of antibiotics, to reduce disease spread in healthcare facilities, and to enable prompt treatment since several RSV antiviral therapies are on the horizon. Current diagnostic methods rely on time-consuming laboratory-based tests including virus culture and polymerase chain reaction (PCR), and rapid diagnostic tests (e.g., lateral flow immunoassay, LFA) are not sufficiently sensitive as standalone diagnosis. Therefore, there is an unmet need for rapid and ultrasensitive diagnostic tests for RSV. The plasmonic coupling assay is a rapid colorimetric diagnostic test that makes use of the optical response of gold nanoparticles (AuNPs) during the process of target recognition to analyze its concentration. Despite its easy operation, the sensitivity of the plasmonic coupling assay is limited. In this proposed work, we aim to substantially improve the limit of detection (LOD) of the plasmonic coupling assay by innovative digital nanobubble detection. Specifically, we propose to directly detect intact RSV particles with antibody-conjugated AuNPs that recognize the RSV surface fusion (F) protein. AuNPs bind to multiple RSV F proteins and lead to plasmonic coupling. Ultrashort laser pulse selectively activates coupled AuNPs due to their enhanced absorption compared with a single AuNP. This greater optical absorption leads to nanoscale cavitation bubbles, i.e. nanobubbles, which can be measured easily from the bubble-induced and transient scattering. Single nanobubble generation leads to a sensitive digital detection with “on” and “off” signals. Our preliminary results suggest ~3 orders of magnitude improvement of LOD in detecting RSV. In this proposed work, we will firstly innovate the AuNP probe by optimizing the AuNP formulation (size, concentration and conjugation), investigating the non-spherical nanoparticle for more efficient virus binding, and exploring the asymmetric antibody-coated Janus nanoparticles for controlled binding. Next, we will design and build a prototype device for automated sample loading, reading, and data processing for the digital nanobubble assay. Lastly, we will test this assay with clinical RSV isolates (from diverse genetic strains) and clinical specimens. By comparing with current PCR and rapid diagnostic tests, we will establish the clinical sensitivity and specificity for the digital nanobubble test. This test is fast (< 30 minutes), low-cost (AuNP reagent cost is similar to lateral flow immunoassay), and highly sensitive and specific for RSV. Furthermore, the direct detection of virus particles eliminates the need of extensive sample preparation such as nucleic acid extraction. Success of our project will meet urgent demands of rapid and sensitive RSV diagnostics and address a major healthcare need for pediatric patients that are affected by RSV.