Using an Aqueous Two-Phase Polymer-Salt System to Rapidly Concentrate Viruses for Improving the Detection Limit of the Lateral-Flow Immunoassay
Using an Aqueous Two-Phase Polymer-Salt System to Rapidly Concentrate Viruses for Improving the Detection Limit of the Lateral-Flow Immunoassay
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DOI：
10.1002/bit.25316
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发表时间：
2014-12-01
影响因子：
3.8
通讯作者：
Kamei, Daniel T.
中科院分区：
文献类型：
--
作者：
Jue, Erik;Yamanishi, Cameron D.;Kamei, Daniel T.
病毒的即时诊断技术的发展有可能预防大流行病和防范生物战威胁。在这里，我们讨论的方法，使用双水相系统（ATPS）浓缩生物分子之前，侧流免疫测定（LFA），以提高病毒检测。在本文中，我们开发了一种快速PON检测方法，作为我们以前使用胶束ATPS的概念验证研究的扩展。我们提出了我们的调查更快速的聚合物-盐ATPS，可以大大提高测定时间，并表明，含有浓缩的生物分子的相可以提取之前，宏观相分离平衡，而不影响测得的生物分子浓度在该阶段。因此，我们可以显着减少诊断检测的时间与早期提取时间仅为30分钟。使用这种快速ATPS，模型病毒噬菌体M13通过改变两相之间的体积比浓缩约2至10倍。由于提取的富含病毒的相含有高浓度的盐，使LFA中使用的胶体金指示剂不稳定，我们用聚乙二醇（PEG）修饰金纳米探针以提供空间稳定性，并使用这些纳米探针证明LFA检测限提高了10倍。最后，使用MATLAB脚本对有和没有预浓缩步骤的LFA结果进行定量。这种将快速ATPS与LFA相结合的方法对于PON应用具有很大的潜力，特别是通过进一步改变体积比可以实现更大的浓度倍数改进。(C)2014 Wiley Periodicals，Inc.
The development of point-of-need (PON) diagnostics for viruses has the potential to prevent pandemics and protects against biological warfare threats. Here we discuss the approach of using aqueous two-phase systems (ATPSs) to concentrate biomolecules prior to the lateral-flow immunoassay (LFA) for improved viral detection. In this paper, we developed a rapid PON detection assay as an extension to our previous proof-of-concept studies which used a micellar ATPS. We present our investigation of a more rapid polymer-salt ATPS that can drastically improve the assay time, and show that the phase containing the concentrated biomolecule can be extracted prior to macroscopic phase separation equilibrium without affecting the measured biomolecule concentration in that phase. We could therefore significantly decrease the time of the diagnostic assay with an early extraction time of just 30 min. Using this rapid ATPS, the model virus bacteriophage M13 was concentrated between approximately 2 and 10-fold by altering the volume ratio between the two phases. As the extracted virus-rich phase contained a high salt concentration which destabilized the colloidal gold indicator used in LFA, we decorated the gold nanoprobes with polyethylene glycol (PEG) to provide steric stabilization, and used these nanoprobes to demonstrate a 10-fold improvement in the LFA detection limit. Lastly, a MATLAB script was used to quantify the LFA results with and without the pre-concentration step. This approach of combining a rapid ATPS with LFA has great potential for PON applications, especially as greater concentration-fold improvements can be achieved by further varying the volume ratio. (C) 2014 Wiley Periodicals, Inc.