UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.
i3N|CENIMAT, Departamento de Ciência dos Materiais, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal.
Sensors (Basel). 2020 Mar 14;20(6):1624. doi: 10.3390/s20061624.
Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 µL volume, attaining a limit of detection of five copies/µL under 60 min. This technology was applied to quantify a cancer biomarker, , but it can be further extended to any other disease biomarker.
微流控（MF）技术的进步已经被应用于最先进的分子诊断平台的开发，其中等温扩增方案允许进一步简化 DNA 检测和定量方案。MF 与环介导等温扩增（LAMP）的结合是当今基于芯片的新一代分子检测设备的焦点，旨在实现快速和自动化的核酸分析。在这里，我们将 MF 与液滴数字 LAMP（ddLAMP）结合在一个集成的设备中，该设备允许生成液滴、目标扩增和绝对定量。这个多层 3D 芯片是在不到 30 分钟的时间内开发的，使用了一种低成本且极其灵活的生产工艺，该工艺利用了“ shrinky-dinks ”聚苯乙烯片上的直接激光写入技术。ddLAMP 和目标定量直接在芯片上进行，显示出目标浓度与阳性液滴分数之间的高度相关性。我们通过对 5 到 500,000 个拷贝/反应的目标进行扩增验证了这个集成芯片。此外，在 10 μL 体积的芯片上进行了扩增，在 60 分钟内达到了 5 拷贝/μL 的检测限。这项技术被应用于定量分析一种癌症生物标志物 ，但它可以进一步扩展到任何其他疾病生物标志物。
