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

摘要 这项建议的目标是开发和验证一套新的低成本、易于使用的医疗点测试 (POCT)，可在30分钟内同时检测多个生物标志物，具有高灵敏度和特异性 使柯萨奇病毒19患者的早期诊断、预后和血清学成为可能。多路复用的D4-POCT采用 诊断和预后芯片中用于抗原检测的抗体，以及用于抗体检测的抗原 血清学芯片。捕获和检测抗体将用于产生S1和N抗原的分析 人血或血清中D-二聚体、IL-2R、IL-6、IL-10和肿瘤坏死因子-α的检测 对于预后，将使用S1、N和RBD病毒抗原作为捕获和检测试剂，在双重 抗原血清学检测人血和血清中抗SARS-CoV-2抗体。最佳试剂 将通过利用D4-POCT的多路传输能力来确定化验开发 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 组件。第一个组件-D4芯片-是通过喷墨打印捕获的微点和 荧光标记检测试剂(用于检测抗原的抗体或用于检测抗体的抗原) 直接涂在耐蛋白质和细胞的聚甲基丙烯酸寡乙二醇酯刷子上，这种刷子是从 玻璃表面，可降低背景噪音，检测极限为pg/m L，动态超过4对数 射程。第二个组件是包裹D4芯片的重力驱动微流体盒，它是 采用低成本、高产量的注塑成型。微流体盒减少了使用者的接触 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 样品进入样品入口，两滴洗涤缓冲液进入洗涤缓冲器入口-从而模拟 用于进行横向流动分析的程序，这是现场使用的免疫分析的黄金标准。芯片 自动执行定时孵化和漂洗步骤，移除样品和任何未结合的检测 D4芯片表面的试剂，使D4芯片的中心区域准备好第三次成像 组件-D4Scope-我们开发的定制、低成本手持荧光检测器。 D4芯片上的荧光点强度通过内置的应用程序转换为分析物浓度 D4Scope，分析物浓度和原始数据-图像-都自动上载到 安全的云服务器。D4-POCT的所有组件都很容易像芯片一样大批量制造 通过喷墨打印每个测试试剂的皮克快速制造，微流体盒是由 注塑成型，D4Scope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--都建立在相同的基础上 平台-可以在全球部署以对抗新冠肺炎。

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.