Institut Curie, Centre de Recherche, Paris, France.
Lab Chip. 2013 Jun 21;13(12):2344-9. doi: 10.1039/c3lc50353d. Epub 2013 May 3.
Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.
虽然被动免疫凝集分析仅包含一个步骤且操作简单，但通常不适应高通量分析，并且定量步骤需要昂贵且庞大的设备。在这里，我们展示了一种低成本、多模式和高通量的免疫凝集分析，该分析依赖于磁珠（MBs）、液滴微流控和磁镊的结合。抗体包被的 MBs 被用作均相中的捕获支撑物。在免疫相互作用之后，生成并在特氟隆管中输送含有 MBs 和分析物的油包水液滴。当在磁镊之间通过时，包含在液滴中的 MBs 被磁场限制以增强聚集速率和动力学。当释放磁场时，液滴中的内部再循环流产生有利于 MBs 再分散的剪切力。在存在分析物的情况下，该系统保留特异性相互作用，并且 MBs 保持聚集状态，而在非特异性分析物的情况下，颗粒发生再分散。分析物定量程序依赖于 MBs 在液滴中的再分散速率。已经研究和优化了不同参数（例如磁场强度、流速和 MBs 浓度）对聚集性能的影响。尽管本文描述的免疫凝集分析在灵敏度方面可能无法与酶联免疫吸附测定（ELISA）竞争，但它在试剂消耗（分析在亚微升液滴中进行）和平台成本方面具有主要优势，从而导致非常便宜的分析。此外，全自动分析程序提供具有高于现有技术的通量的可重复分析。我们以 100 nM 的检测限检测了 100nL 样品体积中的生物素化碱性磷酸酶，分析速度为 300 次/小时。
