Summary

便携式纸基免疫测定法与智能手机应用相结合,用于登革热NS1抗原的比色和定量检测

Published: January 26, 2024
doi:

Summary

为了满足登革热的紧急诊断需求,我们在此介绍一种集成了智能手机应用程序的登革热NS1纸质分析设备(DEN-NS1-PAD),用于量化临床血清/血液样本中的登革热NS1抗原浓度。这项创新通过帮助各种医疗机构(甚至是资源有限的医疗机构)的临床决策来加强登革热管理。

Abstract

蚊传播的登革热病毒(DENV)感染是热带和亚热带国家的主要公共卫生问题。每年约有1000万例病例和20,000-25,000例死亡,特别是在儿童中,迫切需要实用的诊断工具。早期感染期间登革热非结构蛋白 1 (NS1) 的存在与细胞因子释放、血管渗漏和内皮功能障碍有关,使其成为重症登革热的潜在标志物。

基于纸张的免疫测定,如侧向层析测定 (LFA) 和微流体纸质分析设备 (PAD),由于其简单、快速、廉价、特异性和易于解释,作为诊断测试越来越受欢迎。然而,用于登革热 NS1 检测的传统纸质免疫测定通常依赖于目视检查,只能产生定性结果。为了解决这一局限性并提高灵敏度,我们提出了一种高度便携的 NS1 登革热检测方法,即纸质分析设备 (PAD),即 DEN-NS1-PAD,它将智能手机应用程序集成为比色和定量阅读器。该开发系统可直接定量临床样本中的NS1浓度。

利用从患者身上获得的血清和血液样本来证明系统原型的性能。结果立即获得,可用于临床评估,无论是在设备齐全的医疗机构还是资源有限的环境中。这种基于纸张的免疫测定与智能手机应用的创新组合为增强登革热NS1抗原的检测和定量提供了一种很有前途的方法。通过提高肉眼能力之外的灵敏度,该系统在改善登革热管理的临床决策方面具有巨大潜力,特别是在偏远或服务不足的地区。

Introduction

登革热病毒 (DENV) 感染是传播速度最快的蚊媒疾病1,全球每年有超过 3.9 亿人感染,有 9600 万例有症状感染,200 万例重症病例,超过 25,000 人死亡 1,2根据世界卫生组织 (WHO) 的数据,估计有 39 亿人面临登革热风险;~70% 居住在亚太国家,主要居住在东南亚3.2019年,向世卫组织报告的登革热病例数为420万例,泰国贡献了至少13.6万例登革热病例和144例登革热感染死亡病例4。泰国的登革热疫情发生在4月至12月的雨季,发生在城市和农村地区,特别是在东北部地区。

登革热病毒感染有不同的临床表现,从亚临床症状、轻度登革热 (DF) 到重度登革出血热 (DHF)。严重 DHF 疾病的主要特征是血管通透性增加,其次是休克和器官功能障碍1。了解可能导致血管渗漏的分子途径对于开发有效的登革热治疗方法非常重要。登革热非结构蛋白 1 (NS1) 是早期病毒感染期间分泌的糖蛋白 5,6作为病毒 RNA 复制的辅助因子7。NS1 可通过与 toll 样受体 4 (TLR4) 和内皮糖萼 8,9 结合来触发细胞因子释放并导致血管渗漏。体外研究表明,NS1与内皮细胞相互作用并诱导细胞凋亡。这种情况会导致内皮功能障碍和血管渗漏10。与血清白细胞介素 (IL)-10 水平相关的 NS1 抗原水平在重症临床疾病患者中显著升高11。登革热 NS1 还通过诱导 IL-10 和抑制 DENV 特异性 T 细胞反应来促进疾病发病机制12,13。此外,登革热NS1蛋白与严重临床疾病有关,发病前3天NS1浓度>600 ng mL-1与DHF14的发生相关。

登革热NS1抗原在DHF患者中的持续存在可作为重症登革热6的标志物。有几种方法可以检测临床样本中的 NS1,例如酶联免疫吸附测定 (ELISA) 和快速检测15。在临床环境中测量 NS1 蛋白浓度的金标准是 ELISA 方法。然而,ELISA方法价格昂贵,需要熟练的人员和实验室设施16。因此,在床旁检测 (POCT) 中检测和定量 NS1 蛋白的技术仍在开发中。在过去十年中,基于纸张的免疫测定,如侧向层析测定(LFAs)和微流体纸质分析设备(μPAD),因其简单、快速、廉价和特异性而成为诊断测试的流行点17,18,19。在基于纸张的免疫测定中,已使用多种标记来生成信号,例如金纳米颗粒 (AuNPs)20、磁性纳米颗粒21,22、量子点23 和荧光材料24,25。AuNPs是纸质免疫测定中最常用的标记物,因为它们的生产成本低廉、易于制造、稳定性高、读数简单。目前,登革热 NS1 的侧向层析测定 (LFA) 在临床环境中的使用很有名26,27。然而,传统的LFA标签检测通常使用肉眼,只能提供定性结果。

在过去十年中,全球已有超过50亿部智能手机被广泛使用,并且有可能开发便携式检测28,29。智能手机具有内置物理传感器、多核处理器、数码相机、USB 端口、音频端口、无线和应用软件等多功能功能,使其适用于各种生物传感器平台30。此外,无线技术允许快速发送数据,并可用于实时和现场监控31。Mudanyali 等人将纸质免疫测定和智能手机相结合,开发了一种便携式、无需设备、快速、低成本且用户友好的 POCT 平台,用于疟疾、结核病和 HIV32。Ling等人报道了一种侧向层析法与智能手机摄像头相结合,可以定量检测牛奶中的碱性磷酸酶活性33。Hou 等人还开发了一种基于智能手机的双模态成像系统,用于在侧向层析测定中对颜色或荧光发出定量信号34。此外,使用智能手机作为比色和定量阅读器可以提高灵敏度,而肉眼无法自信地报告目标35的存在。

DEN-NS1-PAD 36,37,38(以下简称该设备)在登革热诊断方面取得了突破性进展,提供了一种便携且高效的解决方案。该设备使用蜡印微流控纸技术,通过图像处理以高灵敏度和特异性定量 NS1。为了进一步提高其实用性,我们开发了一款用户友好的智能手机应用程序,用于比色和定量阅读。使用来自泰国医院的患者样本进行临床验证强调了其对实时患者评估的直接影响。我们的创新标志着简化的床旁登革热管理取得了关键性进展,有望彻底改变资源有限的医疗保健领域的诊断。

Protocol

泰国曼谷Phramongkutklao医院泰国皇家陆军医疗部机构审查委员会伦理委员会(IRBRTA 1218/2562)批准了这一计划。在进行这项研究时,我们遵守了所有必要的道德规范。 1. 纸基免疫测定法的设备制造 注:纸质免疫测定装置是按照先前建立的方法36,37和泰国专利请求号19010081638制造的。 …

Representative Results

选择制造方法对于确保纸质免疫测定设备中可重复的测定性能至关重要。在我们的研究中,我们在展示基于纸张的免疫测定的背景下探索了各种制造工艺和材料。我们选择的方法利用蜡印系统在纸基微流控设备中产生疏水屏障。这种方法因其简单、快速和一致的结果而脱颖而出。值得注意的是,它具有避免使用光刻胶化学品的优点,因为光刻胶化学品有可能干扰蛋白质吸附并增加纤维素纸的疏水?…

Discussion

基于智能手机的阅读器系统的重要设计参数之一是能够对样品进行可重复的成像处理。在这项研究中,为了简单和方便起见,这些图像是从三个不同的智能手机品牌拍摄的,配备 12-13 MP 摄像头,而无需使用成像盒或配件。图像采集的可变条件(例如相机的分辨率、图像捕获时间、照明条件和环境)可能会影响设备上测试和控制点的颜色强度。通过使用归一化信号强度,将不同图像捕获时间对PAD照…

Disclosures

The authors have nothing to disclose.

Acknowledgements

MHP非常感谢印度尼西亚伊斯兰大学(UII)的奖学金研究基金。作者感谢 Nutchanon Ninyawee 先生在移动应用程序开发过程中提供的宝贵专业知识和帮助,以及他对手稿的贡献。此外,作者感谢泰国科学研究与创新 (TSRI) 基础研究基金:2023 财年(项目编号FRB660073/0164)在吞武里王科技大学的智能医疗保健计划下。

Materials

Materials
0.1 M phosphate-buffered saline (PBS, pH 7.2) 
BBS containing 0.1% Tween 20, 10% sucrose, and 1% casein   the conjugate area treatment and blocking buffer
Borate buffered saline (BBS) (25 mM sodium borate and 150 mM sodium chloride at pH 8.2) supplemented with 1% BSA  the washing buffer during the conjugation process AuNPs with the antibody
Boric acid Merck 10043-35-3
Bovine serum albumin fraction V (BSA)   PAA Lab GmbH (Germany) K41-001 
Casein Merck 9005-46-3
Chromatography paper Grade 2  GE Healthcare 3002-911 
Clear laminate film 3M (Stationery shops)
Disodium hydrogen phosphate Merck 7558-79-4
Double tape side Stationery shops
Goat anti-mouse IgG antibody  MyBiosource (USA) MBS435013
Gold nanoparticles (40 nm)   Serve Science Co., Ltd. (Thailand)
Human IgG polyclonal antibody   Merck AG711-M
Mouse dengue NS1 monoclonal antibody  MyBiosource (USA) MBS834415
Mouse dengue NS1 monoclonal antibody  MyBiosource (USA) MBS834236
NS1 serotype 2 antigens MyBiosource (USA) MBS 568697
PBS 1X containing 0.1% Tween 20 was used as t elution buffer
Plastic backing card 10×30 cm Pacific Biotech Co., Ltd. (Thailand)
Poly-L-lysine (PLL) Sigma Aldrich P4832
Potassium Chloride Merck 104936
Potassium monophosphate Merck 104877
Sodium Chloride Merck 7647-14-5
Sodium tetraborate  Sigma Aldrich 1303-96-4
Sucrose Merck 57-50-1
Tween 20 Sigma Aldrich 9005-64-5
Instruments
CytationTM 5 multimode reader BioTek
Mobile phones Huawei Y7, iPhone 11, Samsung a20
Photo scanner Epson Perfection V30
Oven Memmert
Wax printer  Xerox ColorQube 8880-PS
Software
Could AutoML Vision Object Detection documentation Google Cloud
ImageJ National Institute of Health, Bethesda, MD, USA
Inkscape 0.91 Software

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Cite This Article
Prabowo, M. H., Chalermwatanachai, T., Surareungchai, W., Rijiravanich, P. Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen. J. Vis. Exp. (203), e66130, doi:10.3791/66130 (2024).

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