Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Building 345B, DK-2800 Kongens Lyngby, Denmark.
Analyst. 2017 Sep 8;142(18):3441-3450. doi: 10.1039/c7an01023k.
We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.
我们报告了一种光磁技术，该技术使用两种不同的检测方法，针对登革热病毒在理想和非理想实验室条件下的实时分子检测进行了优化。第一种方法基于检测链霉亲和素包被的磁性纳米颗粒与生物素化的 LAMP 扩增子连接后的流体力学体积。我们在理想的无污染实验室环境中，在 20 分钟内证明了对亚飞摩尔浓度的登革热 DNA 靶标浓度的检测。第二种检测方法基于功能化磁性纳米颗粒与 LAMP 扩增子环的序列特异性结合。熔解研究表明，真正的阳性和虚假的扩增子具有不同的熔点，这允许我们对它们进行区分。这与随后对 LAMP 扩增子的实时序列特异性鉴别研究结果一致。特异性结合通过与 LAMP 延伸阶段出现的多个位点（环）结合导致磁性纳米颗粒的聚集。通过由于自由纳米颗粒的耗尽导致光磁信号的损耗来监测纳米团簇的形成。经过序列特异性验证后，我们声称在 20 分钟的 LAMP 后，可以检测到低至 100 fM 的登革热靶标，背景污染为 100 fM。
