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

摘要 本提案的目的是开发和验证一套新的低成本、易于使用的即时检测方法 （POCT）可以在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 以实现COVID 19患者的早期诊断、预后和血清学。多路复用的D4-POCT采用 用于诊断和预后芯片中的抗原检测的抗体，以及用于诊断和预后芯片中的抗体检测的抗原。 血清芯片捕获和检测抗体将用于生成S1和N抗原的测定， 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-2 R、IL-6、IL-10和TNF-α 用于预后，S1、N和RBD病毒抗原将用作双- 抗原血清学试验检测人血液和血清中的抗SARS-CoV-2抗体。最佳试剂 将通过利用D4-POCT的多路复用功能来确定用于检测开发的方法 高通量筛选和配对患者来源的抗体和病毒抗原。D4-POCT有三个 件.第一个组件--D4芯片--是通过喷墨打印捕获的微点制造的， 荧光标记的检测试剂（用于抗原检测的抗体或用于抗体检测的抗原） 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，降低背景噪声，产生pg/mL检测限和超过4 log的动态 范围第二个组件是重力驱动的微流体盒，其包裹D4芯片， 通过低成本和高产量的注射成型制造。微流控盒减少了用户暴露 潜在的传染性物质，并最大限度地减少用户交互，因为它只需要添加一滴 将两滴样品滴入样品入口，将两滴洗涤缓冲液滴入洗涤缓冲液入口，从而模拟 用于进行侧流测定的程序，现场使用的免疫测定的金标准。芯片 自动执行定时孵育和冲洗步骤， 从D4芯片的表面去除试剂，留下D4芯片的中心区域准备好用于第三次成像。 D4 Scope是我们开发的一种定制的、低成本的手持式荧光检测器。 D4芯片上的荧光点强度通过芯片上的内置“App”转换为分析物浓度。 D4 Scope、分析物浓度和原始数据（图像）都自动上传到 安全的云服务器D4-POCT的所有组件都易于大批量制造，因为芯片 通过喷墨打印每次测试的皮克试剂来快速制造，微流体盒通过以下步骤制造： D4 Scope采用注塑成型，D4 Scope由现成的部件组装而成。这项提议的影响将是 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，以抗击COVID-19。

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.