Department of Bioscience and Biotechnology , National Taiwan Ocean University , Keelung 20224 , Taiwan.
Biomedical Engineering Research Center, Department of Electronic Engineering , Chang Gung University , Taoyuan 33302 , Taiwan.
ACS Sens. 2019 Jun 28;4(6):1543-1551. doi: 10.1021/acssensors.9b00054. Epub 2019 May 16.
Many serious public health emergencies around the globe are caused by viral epidemics. Thus, developing a reliable method for viral screening is in high demand. Multiplex assays for simultaneous detection and fast screening of high-risk pathogens are especially needed. This study employs metal nanoparticles to generate specific mass spectral signals for different RNA viruses, which enables simultaneous detection of whole viruses by laser desorption/ionization mass spectrometry (LDI-MS). We developed a nanoparticle-based sandwich immunosorbent assay as a sensing platform for the detection of viruses and viral nonstructural protein by LDI-MS. Cellulose acetate membrane (CAM) serves as the substrate for the fabrication of the sandwich immunosorbent assay with the advantages of clean mass spectra and high enrichment of analytes. Antibody-modified metal nanoparticles (Ab-MNPs; M = Au or Ag) act as metallic biocodes for the LDI-MS detection. The signal amplification readout for the virus is through the pulsed laser-induced formation of metal cluster ions ([M ]; n = 1-3) from the Ab-MNPs which specifically bind on the CAM. Our sensing system is effective for the detection of intact viruses [Enterovirus 71 (EV71) and Japanese encephalitis virus (JEV)], nonstructural protein 1 (NS1) of Zika virus (ZIKV), EV71-spiked human serum samples, and the simultaneous detection of EV71 and ZIKV. Our probe efficiently detects EV71 in real clinical serum samples with >95% agreement with RT-qPCR results. This high-throughput LDI-MS viral detection system is simple, reliable, and high-throughput. We believe this platform has the potential to be employed for the routine screening of patients with viral infections.
全球许多严重的公共卫生突发事件都是由病毒流行引起的。因此，开发一种可靠的病毒筛选方法是非常有需求的。特别需要能够同时检测和快速筛选高危病原体的多重分析方法。本研究利用金属纳米粒子为不同的 RNA 病毒产生特定的质谱信号，从而通过激光解吸/电离质谱（LDI-MS）实现对整个病毒的同时检测。我们开发了一种基于纳米粒子的三明治免疫吸附测定法作为通过 LDI-MS 检测病毒和病毒非结构蛋白的传感平台。醋酸纤维素膜（CAM）作为制备三明治免疫吸附测定法的基底，具有清洁质谱和高分析物富集的优点。抗体修饰的金属纳米粒子（Ab-MNP；M = Au 或 Ag）作为 LDI-MS 检测的金属生物码。通过脉冲激光诱导 Ab-MNP 形成金属簇离子（[M]；n = 1-3）来进行病毒信号放大读数，这些金属簇离子特异性地结合在 CAM 上。我们的传感系统有效地用于检测完整的病毒[肠道病毒 71（EV71）和日本脑炎病毒（JEV）]、寨卡病毒（ZIKV）的非结构蛋白 1（NS1）、EV71 加标人血清样本，以及同时检测 EV71 和 ZIKV。我们的探针能够高效地检测真实临床血清样本中的 EV71，与 RT-qPCR 结果的一致性超过 95%。这种高通量 LDI-MS 病毒检测系统简单、可靠且高通量。我们相信这个平台有潜力用于常规筛选病毒感染患者。
