呼吸道病毒引起的流行病,比如SARS-CoV-1/2流感病毒,和呼吸道合胞病毒,给人类造成了严重后果和大量死亡。在感染的早期阶段检测这种呼吸道病毒可以通过防止病毒传播来帮助控制疾病。然而,呼吸道病毒种类和亚型的多样性,它们的快速基因突变,并且在感染的早期阶段有限的病毒释放给它们的检测带来了挑战。这项工作报告了一种多路复用的微流控免疫测定芯片,用于同时检测八种具有明显感染人群的呼吸道病毒,即,甲型流感病毒,乙型流感病毒,呼吸道合胞病毒,SARS-CoV-2,人类博卡病毒,人类偏肺病毒,腺病毒,和人类副流感病毒。对纳米酶的纳米材料(Au@Pt纳米颗粒)进行了优化,以提高标记效率并显著提高检测灵敏度。使用Nanozyme结合抗体在40分钟内用肉眼和酶标仪以0.1pg/mL的检测极限检测病毒蛋白。此外,在免疫测定中针对每种病毒的保守蛋白筛选特异性抗体,临床样本检测显示出很高的特异性,八种病原体之间没有交叉反应性。此外,微流控芯片免疫分析显示出很高的准确性,与临床样品检测的RT-PCR方法相比,阳性/阴性符合率为97.2%/94.3%。因此,这种提出的方法提供了一种方便的,快速,和同时检测八种呼吸道病毒的灵敏方法,这对病毒感染的早期诊断具有重要意义。重要的是,它可以被广泛用于检测病原体和生物标志物,只取代抗原特异性抗体。
Pandemics caused by respiratory viruses, such as the SARS-CoV-1/2, influenza virus, and respiratory syncytial virus, have resulted in serious consequences to humans and a large number of deaths. The detection of such respiratory viruses in the early stages of infection can help control diseases by preventing the spread of viruses. However, the diversity of respiratory virus species and subtypes, their rapid antigenic mutations, and the limited viral release during the early stages of infection pose challenges to their detection. This work reports a multiplexed microfluidic immunoassay chip for simultaneous detection of eight respiratory viruses with noticeable infection population, namely, influenza A virus, influenza B virus, respiratory syncytial virus, SARS-CoV-2, human bocavirus, human metapneumovirus, adenovirus, and human parainfluenza viruses. The nanomaterial of the nanozyme (Au@Pt nanoparticles) was optimized to improve labeling efficiency and enhance the detection sensitivity significantly. Nanozyme-binding antibodies were used to detect viral proteins with a limit of detection of 0.1 pg/mL with the naked eye and a microplate reader within 40 min. Furthermore, specific antibodies were screened against the conserved proteins of each virus in the immunoassay, and the clinical sample detection showed high specificity without cross reactivity among the eight pathogens. In addition, the microfluidic chip immunoassay showed high accuracy, as compared with the RT-PCR assay for clinical sample detection, with 97.2%/94.3% positive/negative coincidence rates. This proposed approach thus provides a convenient, rapid, and sensitive method for simultaneous detection of eight respiratory viruses, which is meaningful for the early diagnosis of viral infections. Significantly, it can be widely used to detect pathogens and biomarkers by replacing only the antigen-specific antibodies.