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A nanoparticle-based sensor for visual detection of multiple mutations

机译：基于纳米粒子的传感器，用于视觉检测多个突变

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Disposable dipstick-type DNA biosensors in the form of lateral flow strips are particularly useful for genotyping in a small laboratory or for field testing due to their simplicity, low cost and portability. Their unique advantage is that they enable visual detection inminutes without the use of instruments. In addition, the dry-reagent format minimizes the pipetting, incubation and washing steps. In this work, we significantly enhance the multiplexing capabilities of lateral flow strip biosensors without compromising their simplicity. Multiplex genotyping is carried out by polymerase chain reaction (PCR) followed by a single primer extension reaction for all target alleles, in which a primer is extended and biotin is incorporated only if it is perfectly complementary to the target. Multiallele detection is achieved by multiple test spots on the membrane of the sensor, each comprising a suspension of polystyrene microspheres functionalized with capture probes. The products of the primer extension reaction hybridize, through specific sequence tags, to the capture probes and are visualized by using antibiotin-conjugated gold nanoparticles. This design enables accommodation of multiple spots in a small area because the microspheres are trapped in the fibres of the membrane and remain fixed in site without any diffusion. Furthermore, the detectability is improved because the hybrids are exposed on the surface of the trapped microspheres rather than inside the pores of the membrane. We demonstrate the specificity and performance of the biosensor for multiallele genotyping.
机译：侧向流动条形式的一次性量油尺型DNA生物传感器由于其简单，低成本和便携性，特别适用于小型实验室的基因分型或现场测试。它们的独特优势是无需使用仪器即可在几分钟内进行视觉检测。另外，干试剂形式最大程度地减少了移液，孵育和洗涤步骤。在这项工作中，我们在不影响其简单性的情况下，显着增强了横向流条生物传感器的复用能力。通过聚合酶链反应（PCR）进行多重基因分型，然后对所有靶标等位基因进行单引物延伸反应，其中引物被延伸并且仅在与靶标完全互补时才掺入生物素。多等位基因检测是通过传感器膜上的多个测试点实现的，每个测试点都包含一个用捕获探针功能化的聚苯乙烯微球的悬浮液。引物延伸反应的产物通过特定的序列标签与捕获探针杂交，并通过使用抗生物素偶联的金纳米颗粒进行可视化。这种设计可以在一个小区域内容纳多个斑点，因为微球被捕获在膜的纤维中，并保持固定在位而没有任何扩散。此外，由于杂化物暴露在被捕获的微球的表面而不是膜孔的内部，因此提高了可检测性。我们证明了生物传感器的多等位基因分型的特异性和性能。

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