Department of Mechanical Engineering, National Yang Ming Chiao Tung University, Hsinchu 30010, Taiwan.
Institute of Pharmacology, National Yang Ming Chiao Tung University, Taipei 11221, Taiwan.
Biosensors (Basel). 2022 May 16;12(5):340. doi: 10.3390/bios12050340.
In a biomedical diagnosis with a limited sample volume and low concentration, droplet-based microfluidics, also called digital microfluidics, becomes a very attractive approach. Previously, our group developed a magnetic-beads-based digital microfluidic immunoassay with a bead number of around 100, requiring less than 1 μL of sample volume to achieve a pg/mL level limit of detection (LOD). However, the bead number in each measurement was not the same, causing an unstable coefficient of variation (CV) in the calibration curve. Here, we investigated whether a fixed number of beads in this bead-based digital microfluidic immunoassay could provide more stable results. First, the bead screening chips were developed to extract exactly 100, 49, and 25 magnetic beads with diameters of less than 6 μm. Then, four calibration curves were established. One calibration curve was constructed by using varying bead numbers (50-160) in the process. The other three calibration curves used a fixed number of beads, (100, 49, and 25). The results indicated that the CVs for a fixed number of beads were evidently smaller than the CVs for varying bead numbers, especially in the range of 1 pg/mL to 100 pg/mL, where the CVs for 100 beads were less than 10%. Furthermore, the calculated LOD, based on the composite calibration curves, could be reduced by three orders, from 3.0 pg/mL (for the unfixed bead number) to 0.0287 pg/mL (for 100 beads). However, when the bead numbers were too high (more than 500) or too low (25 or fewer), the bead manipulation for aggregation became more difficult in the magnetic-beads-based digital microfluidic immunoassay chip.
在样本量有限且浓度低的生物医学诊断中，基于液滴的微流控技术（也称为数字微流控技术）成为一种非常有吸引力的方法。之前，我们小组开发了一种基于磁珠的数字微流控免疫分析方法，其磁珠数量约为 100 个，需要的样本量少于 1 μL，可达到 pg/mL 级别的检测限（LOD）。然而，每次测量中的磁珠数量并不相同，导致校准曲线的变异系数（CV）不稳定。在这里，我们研究了在这种基于磁珠的数字微流控免疫分析中，固定数量的磁珠是否可以提供更稳定的结果。首先，开发了磁珠筛选芯片，以精确提取直径小于 6 μm 的 100、49 和 25 个磁珠。然后，建立了四条校准曲线。一条校准曲线是在过程中使用变化的磁珠数量（50-160）构建的。另外三条校准曲线使用固定数量的磁珠（100、49 和 25）。结果表明，固定数量的磁珠的 CV 明显小于变化磁珠数量的 CV，特别是在 1 pg/mL 到 100 pg/mL 的范围内，100 个磁珠的 CV 小于 10%。此外，基于复合校准曲线计算得出的 LOD 可以降低三个数量级，从 3.0 pg/mL（对于未固定的磁珠数量）降低至 0.0287 pg/mL（对于 100 个磁珠）。然而，当磁珠数量过高（超过 500 个）或过低（25 个或更少）时，在基于磁珠的数字微流控免疫分析芯片中，磁珠的聚集操作会变得更加困难。
