Paper-based Vertical Flow Immunoassay (VFI) for detection of bio-threat pathogens
Paper-based Vertical Flow Immunoassay (VFI) for detection of bio-threat pathogens
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DOI：
10.1016/j.talanta.2018.08.043
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发表时间：
2019-01-01
期刊：
影响因子：
6.1
通讯作者：
Zenhausern, Frederic
中科院分区：
文献类型：
--
作者：
Chen, Peng;Gates-Hollingsworth, Marcellene;Zenhausern, Frederic
目前，鉴定病原体、病毒、毒素等潜在威胁国家安全和公共卫生的生物制剂的标准方法主要依靠微生物培养。这种方法耗时，需要精密的设备和训练有素的人员，而这些在偏远地区或有需要的地方往往是不具备的。因此，迫切需要一种快速、简便、灵敏的检测生物威胁剂的方法。我们报告了一种基于纸张的垂直流动免疫测定（VFI）装置，可以克服这些限制。该VFI装置利用纳米多孔硝化纤维素膜封装在不锈钢过滤器支架中。当样品被推过被捕获抗体预功能化的膜时，形成一个夹心实验，并产生比色信号以反映目标抗原的存在。通过对多孔膜内抗原抗体结合过程的理论分析，我们确定了两个关键因素-膜孔径和样品流量可以优化，以提高检测灵敏度。然后，通过实验研究，以伪伯克霍尔德菌（类鼻疽病的病原体）为模型病原体，证明了其效果。伪芽孢杆菌VFI是基于针对伪芽孢杆菌表面荚膜多糖（CPS）的免疫测定。实验结果与理论一致，增大流速（最大1.06 mm/s）和减小膜孔径（最小0.1 μ m）可使灵敏度提高至少5倍。在缓冲溶液中添加CPS的VFI检测限为0.02 ng/mL。开发的VFI显示出在各种临床和资源受限条件下作为检测生物威胁剂的护理点工具的巨大潜力。
Currently, the standard method for identifying biological agents of potential threats to national security and public health, such as pathogens, virus, and toxins, mainly rely on microbiological cultivation. This method is time-consuming and it requires sophisticated equipment and well-trained personnel, which are often unavailable in remote areas or at point-of-need. Therefore, an alternative rapid, simple, and sensitive method for detecting bio-threat agents is in crucial need. We report a paper-based Vertical Flow Immunoassay (VFI) device that can overcome these limitations. The VFI device utilizes a nanoporous nitrocellulose membrane encapsulated in a stainless steel filter holder. As the sample is pushed through the membrane, which is pre-functionalized with capture antibody, a sandwich assay is formed and colorimetric signal is generated to reflect the presence of target antigens. Through theoretical analyses of antigen-antibody binding process inside a porous membrane, we identified two critical factors-membrane pore size and sample flow rate that can tie optimized to improve the assay sensitivity. Then, the effects were demonstrated through experimental studies using Burkholderia pseudomallei (the causative agent of melioidosis) as a model pathogen. The B. pseudomallei VFI was based on an immunoassay targeting the B. pseudomallei surface capsular polysaccharide (CPS). The experimental results agreed well with the theory showing that increasing the flow speed (up to 1.06 mm/s) and reducing the membrane pore size (down to 0.1 mu m) could improve the sensitivity by at least 5 times. The VFI's limit-of-detection for CPS spiked in buffer solution was determined to be 0.02 ng/mL. The developed VFI shows great potential as a point of-care tool for detection of bio-threat agents in a variety of clinical and resource-restricted conditions.