Multiplexed efficient on-chip sample preparation and sensitive amplification-free detection of Ebola virus.
Multiplexed efficient on-chip sample preparation and sensitive amplification-free detection of Ebola virus.
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DOI：
10.1016/j.bios.2016.12.071
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发表时间：
2017-05-15
影响因子：
12.6
通讯作者：
Mathies RA
中科院分区：
文献类型：
--
作者：
Du K;Cai H;Park M;Wall TA;Stott MA;Alfson KJ;Griffiths A;Carrion R;Patterson JL;Hawkins AR;Schmidt H;Mathies RA
已经开发了一种用于埃博拉病毒检测的自动化微流体样品制备多路复用器（SPM）并对其进行了评估。由微型阀控制的计量气泡用于改善珠-溶液混合，从而增强靶埃博拉病毒RNA与结合到珠的捕获探针的杂交。该方法使用热稳定的4-甲酰基苯甲酰胺官能化（4FB）磁珠，而不是具有高密度捕获探针的链霉亲和素包被的珠，以提高靶捕获效率。利用SPM中的芯片上浓度协议和抗共振反射光波导（ARROW）生物传感器芯片的单分子检测能力，在没有靶扩增的情况下，临床样品的检测限为0.021 pfu/mL。该RNA靶标捕获效率比使用链霉亲和素珠的先前结果高两个数量级，并且检测限（LOD）提高了10倍。该技术的宽动态范围涵盖了整个临床适用的浓度范围。此外，目前的样品制备时间约为1小时，比以前的工作快8倍。这种多路复用的小型化样品制备微器件建立了旨在开发下一代床旁（POC）检测系统的关键技术。
An automated microfluidic sample preparation multiplexer (SPM) has been developed and evaluated for Ebola virus detection. Metered air bubbles controlled by microvalves are used to improve bead-solution mixing thereby enhancing the hybridization of the target Ebola virus RNA with capture probes bound to the beads. The method uses thermally stable 4-formyl benzamide functionalized (4FB) magnetic beads rather than streptavidin coated beads with a high density of capture probes to improve the target capture efficiency. Exploiting an on-chip concentration protocol in the SPM and the single molecule detection capability of the antiresonant reflecting optical waveguide (ARROW) biosensor chip, a detection limit of 0.021 pfu/mL for clinical samples is achieved without target amplification. This RNA target capture efficiency is two orders of magnitude higher than previous results using streptavidin beads and the limit of detection (LOD) improves 10X. The wide dynamic range of this technique covers the whole clinically applicable concentration range. In addition, the current sample preparation time is ~1 hour which is eight times faster than previous work. This multiplexed, miniaturized sample preparation microdevice establishes a key technology that intended to develop next generation point-of-care (POC) detection system.