Multiplex point-of-care test for diagnosis, prognosis and serology of COVID19

用于 COVID19 诊断、预后和血清学的多重即时检测

基本信息

批准号：
10297706
负责人：
Ashutosh Chilkoti
金额：
$ 50.27万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-03 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10297706
关键词：
2019-nCoV Address Adsorption Antibodies Antigens Archives Area Biological Assay Biological Markers Blood Blood group antigen S Blood specimen Buffers CCL7 gene COVID-19 COVID-19 detection COVID-19 diagnosis COVID-19 patient CXCL10 gene Cells Clinical Collection Contact Tracing Custom Data Detection Deterioration Development Devices Diagnosis Drops Early Diagnosis Ensure Exposure to Fibrin fragment D Fluorescence Force of Gravity Geometry Glass Glycols Gold Hand Health Home Human Image Immunoassay Individual Injections Intelligence Interleukin-10 Interleukin-6 Intervention Label Length Liquid substance Methacrylates Microfluidics Molds Monitor Noise Nose Nucleocapsid Nucleocapsid Proteins Patient Care Patient Monitoring Patients Performance Pharyngeal structure Plasma Polymethyl Methacrylate Population Prevalence Printing Procedures Prognosis Proteins Quarantine Reagent Recombinants Reproducibility Resistance Risk Running SARS-CoV-2 antibody SARS-CoV-2 antigen SARS-CoV-2 infection Sampling Scanning Secure Sensitivity and Specificity Serology Serology test Serum Severe Acute Respiratory Syndrome Severities Signal Transduction Spottings Surface Swab Symptoms TNF gene Testing Text Thick Time Validation Viral Viral Antigens Whole Blood antibody detection antigen detection assay development cloud platform cost detection limit detector epidemiology study fight against fluorescence imaging high risk high throughput screening imaging platform lateral flow assay manufacturing process multiplex detection nasal swab oropharyngeal swab performance tests point of care testing point-of-care diagnostics prognostic rapid diagnosis receptor binding sample archive screening tool

项目摘要

ABSTRACT The objective of this proposal is to develop and validate a new set of low-cost, easy-to-use, point-of-care tests (POCTs) that can simultaneously detect multiple biomarkers within 30 minutes with high sensitivity and specificity to enable early diagnosis, prognosis, and serology of COVID19 patients. The multiplexed D4-POCT employs antibodies for antigen detection in the diagnosis and prognosis chips, and antigens for antibody detection in the serology chip. Capture and detection antibodies will be used to generate assays for S1 and N antigens from nasal/throat swab collection media for diagnosis, D-dimer, IL-2R, IL-6, IL-10 and TNF-α in human blood or serum for prognosis, and S1, N, and RBD viral antigens will be used as capture and detection reagents in a double- antigen serological assay to detect anti-SARS-CoV-2 antibodies in human blood and serum. Optimal reagents for assay development will be identified by leveraging the multiplexing capabilities of the D4-POCT to perform high-throughput screening and pairing of patient derived antibodies and viral antigens. The D4-POCT has three components. The first component—the D4 chip—is fabricated by inkjet printing microspots of capture and fluorescently labeled detection reagents (antibodies for antigen detection or antigens for antibody detection) directly on a protein- and cell-resistant poly(oligoethylene glycol methacrylate) brush that is grafted from the surface of glass, which reduces background noise, yielding pg/mL limit-of-detection and over 4 logs of dynamic range. The second component is a gravity-driven microfluidics cassette that encases the D4 chip and is fabricated by low-cost and high-throughput injection molding. The microfluidics cassette reduces user exposure to potentially infectious material and minimizes user interaction, as it only requires the addition of a drop of sample into the sample inlet and two drops of wash buffer into the wash buffer inlet —thereby mimicking the procedure used to carry out lateral flow assays, the gold standard of immunoassay for field use. The chip automatically performs a timed incubation and rinse step that removes the sample and any unbound detection reagent from the surface of the D4 chip, leaving the central area of the D4 chip ready for imaging by the third component—the D4Scope—a customized, low-cost, hand-held fluorescence detector that we have developed. The fluorescent spot intensities on the D4 chip are converted to analyte concentration by a built-in “App” on the D4Scope, and analyte concentrations and the raw data —the images— are both automatically uploaded to a secure cloud server. All components of the D4-POCT are easy to fabricate at high volumes as the chips are rapidly manufactured by inkjet printing picograms of reagents per test, the microfluidics cassette is fabricated by injection molding, and the D4Scope is assembled from off-the shelf parts. The impact of this proposal will be the development and clinical validation of POCTs for diagnosis, prognosis and serology—all built on the same platform— that can be globally deployed in the fight against COVID-19.

抽象的该提案的目标是开发和验证一套新的低成本、易于使用的即时检测 (POCT) 可在 30 分钟内同时检测多种生物标志物，具有高灵敏度和特异性采用多重 D4-POCT 来实现 COVID19 患者的早期诊断、预后和血清学检测。诊断和预后芯片中用于抗原检测的抗体，以及诊断和预后芯片中用于抗体检测的抗原捕获和检测抗体将用于生成 S1 和 N 抗原测定。用于诊断的鼻/咽拭子采集介质，人血液或血清中的 D-二聚体、IL-2R、IL-6、IL-10 和 TNF-α 用于预后，S1、N 和 RBD 病毒抗原将用作双特异性抗体中的捕获和检测试剂。抗原血清学测定检测人血液和血清中的抗 SARS-CoV-2 抗体。将通过利用 D4-POCT 的多重功能来执行检测开发 D4-POCT 具有三种功能，即对患者来源的抗体和病毒抗原进行高通量筛选和配对。第一个组件——D4 芯片——是通过喷墨打印捕获和微点来制造的。荧光标记检测试剂（抗原检测用抗体或抗体检测用抗原）直接在抗蛋白质和细胞的聚（低聚乙二醇甲基丙烯酸酯）刷上，该刷是从玻璃表面，可降低背景噪音，产生 pg/mL 检测限和超过 4 个对数的动态第二个组件是重力驱动的微流体盒，它封装了 D4 芯片。微流体盒通过低成本和高通量注塑成型制造，减少了用户的暴露。潜在的传染性材料，并最大限度地减少用户交互，因为它只需要添加一滴将样品放入样品入口，并将两滴洗涤缓冲液放入洗涤缓冲液入口 - 从而模仿用于进行侧流测定的程序，这是现场使用的免疫测定的黄金标准。自动执行定时孵育和冲洗步骤，去除样品和任何未结合的检测从 D4 芯片表面去除试剂，使 D4 芯片的中心区域准备好由第三个成像组件 - D4Scope - 我们开发的定制、低成本、手持式荧光检测器。 D4 芯片上的荧光点强度通过 D4 芯片上的内置“App”转换为分析物浓度 D4Scope、分析物浓度和原始数据（图像）都会自动上传到 D4-POCT 的所有组件都易于大批量制造，因为芯片是安全的云服务器。微流体盒通过喷墨打印每次测试的试剂皮克来快速制造，微流体盒的制造方法是注塑成型，D4Scope 由现成零件组装而成。该提案的影响将是。用于诊断、预后和血清学的 POCT 的开发和临床验证——所有这些都建立在相同的基础上平台——可以在全球范围内部署来对抗 COVID-19。