Plasmon-enhanced Lateral Flow Assay for Multiplexed Detection of SARS-CoV-2 RNA and Antigens in Point-of-Care Settings

横向流动测定法（LFAs）是最简单、最快、最便宜的医疗点（POC）和家庭生物诊断平台之一，为个性化和人群水平的传染病筛查提供了广阔的潜力。传统的lfa依靠金纳米颗粒作为标记，在目标生物标志物存在的情况下产生可检测的颜色。然而，依赖金纳米颗粒作为标记的LFAs的灵敏度往往不足以检测低浓度的生物标志物，从而在疾病进展的早期阶段导致假阴性。该项目旨在探索使用超亮荧光标签将LFA的灵敏度提高100倍以上。该项目还旨在设计和制造一个个人手持荧光计，该荧光计将与连接的移动设备进行无线通信，以测量来自LFA测试条的荧光信号。利用超亮纳米标签和个人手持式荧光计，设想一个同时检测SARS-CoV-2（引起COVID-19的病毒）RNA和抗原的新型诊断平台。同时检测病毒RNA和抗原可以潜在地提高诊断的准确性和准确地指示疾病的阶段。该项目还包括开发一项新的纳米生物传感器暑期学校活动，旨在向中学生介绍纳米技术和生物传感器，目的是激发对科学/工程的兴趣，同时在代表性不足的群体中培养追求这些主题的信心。迫切需要新一代生物传感器技术，该技术应具有仪器成本低、分析方法简单、灵敏度高、定量准确和严格有效性的综合特点。该项目的最终目标是设计并实现一种在资源有限的环境下检测和定量严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）抗原和RNA的多路等离子体增强荧光侧流试验（p-LFA）。具体目标包括：(i)设计、实现并验证用于同时检测和定量SARS-CoV-2 RNA和抗原的超灵敏多路p-LFA；（二）设计高效的样品处理方法，同时检测2019冠状病毒病（COVID-19）患者鼻咽拭子和唾液中的RNA和抗原；（iii）构建和测试智能手机接口的便携式p-LFA读取器，用于在护理点（POC）和资源有限的环境中部署拟议的生物传感技术。该项目是一个变革性的进步，因为它寻求建立一种新的生物诊断平台，其灵敏度超过标准的实验室检测，一种简单的分析工作流程，与POC和患者家中的自我检测兼容，快速从样品到答案，以及一种廉价/便携式仪器。如果成功，这种无扩增的检测方法可以在同一条LFA试纸上对SARS-CoV-2 RNA和抗原进行空间多路检测和定量，从而缩短了从样本到答案的时间，并有可能将处于感染阶段的个体与已通过感染阶段的个体区分开来。除COVID外，同时检测RNA和抗原广泛适用于各种传染病的高度特异性和敏感性诊断，包括埃博拉、流感和人类免疫缺陷病毒。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Lateral flow assays (LFAs) are amongst the simplest, fastest, and cheapest point-of-care (POC) and at-home biodiagnostic platforms and offer a broad potential for individualized and population-level screening for infectious diseases. Conventional LFAs rely on gold nanoparticles as labels to produce visually detectable color in the presence of the target biomarker. However, the sensitivity of LFAs relying on gold nanoparticles as labels is often insufficient to detect low concentrations of biomarkers, resulting in false negatives at the early stage of disease progression. This project seeks to explore the use of an ultrabright fluorescent label for improving the sensitivity of the LFA by more than 100-fold. The project also aims to design and build a personal handheld fluorimeter that will communicate wirelessly with a linked mobile device to measure the fluorescence signal from an LFA test strip. Harnessing the ultrabright nanolabel and the personal handheld fluorimeter, a novel diagnostic platform that simultaneously detects SARS-CoV-2 (the virus causing COVID-19) RNA and antigen is envisioned. Simultaneous detection of viral RNA and antigen can potentially improve diagnostic accuracy and accurately indicate the stage of illness. The project also involves the development of a new Nano-Biosensors Summer School activity intended to introduce middle school students to nanotechnology and biosensors, with the goal of stimulating interest in science/engineering while instilling confidence in pursuing these topics among under-represented groups.There is a pressing need for a new generation of biosensor technologies that encompass the combined characteristics of low-cost instrumentation, simple assay methods, high sensitivity, accurate quantitation, and rigorous validity. The ultimate goal of the project is to design and realize a multiplexed plasmon-enhanced fluorescence-based lateral flow assay (p-LFA) for the detection and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and RNA in resource-limited settings. Specific objectives include: (i) Design, realize and validate an ultrasensitive multiplexed p-LFA for simultaneous detection and quantification of SARS-CoV-2 RNA and antigen; (ii) Devise efficient sample processing methods for simultaneous detection of RNA and antigens in nasopharyngeal swabs and saliva from coronavirus disease 2019 (COVID-19) patients; and (iii) Build and test a smart phone-interfaced portable p-LFA reader for deploying the proposed biosensing technology in point-of-care (POC) and resource-limited settings. The project represents a transformative advance in that it seeks to establish a novel biodiagnostic platform with sensitivity exceeding that of the standard laboratory tests, a simple assay workflow compatible with self-testing at the POC and patient home, rapid sample-to-answer, and an inexpensive/portable instrument. If successful, the proposed amplification-free assay enables spatially-multiplexed detection and quantification of SARS-CoV-2 RNA and antigens on the same LFA strip, which shortens the sample-to-answer time and potentially differentiates individuals in the infectious stage of the illness from those who have passed it. Beyond COVID, simultaneous detection of RNA and antigen is broadly applicable for highly specific and sensitive diagnosis of various infectious diseases, including Ebola, Influenza, and Human Immunodeficiency Virus.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.