Yang Shih-Mo, Lin Qiang, Zhang Hongbo, Yin Ruixue, Zhang Wenjun, Zhang Minchao, Cui Yubao
Biomedical Science and Technology Research Center, School of Mechatronic Engineering and Automation, Shanghai University, Shanghai, China.
Biomedical Science and Technology Research Center, School of Mechatronic Engineering and Automation, Shanghai University, Shanghai, China.
Biosens Bioelectron. 2021 May 15;180:113148. doi: 10.1016/j.bios.2021.113148. Epub 2021 Mar 5.
Digital ELISA is introduced as a novel platform with unique advantages for detecting multiple kinds of single-molecule in the sample. How to improve the sensitivity of detection is the direction of current related research. Here, we report an immunoassay method that applied electrokinetic effects to isolate the individual encoded beads and confine in micro-wells to improve the efficiency of cytokines detection simultaneously. The microfluidic design provided a non-uniform electric field to induce dielectrophoresis (DEP) force and to manipulate the beads. Two wavelengths of excitation light excited the encoded beads for simultaneous detection of reporters. The light was confined to the bottom slide via the principle of total internal reflection. Finally, the concentration of captured cytokines was obtained by picking up each bead from the image and then integrating the intensity of fluorescent light emitted from the reporters. The results demonstrated that the fill percentage of encoded beads was raised from 10-20% to 60-80% via DEP effect. By comparing the fluorescence color of the particle, itself and its surface, the concentration of four target cytokines, IL-2, IL-6, IL-10 and TNF-α, were calculated to the pg/ml level. The spike and recovery experiments verified the efficiency, more than 70% of the target molecules were captured. The reliability of our method was verified by flow cytometry as well. In conclusion, we expect the application of DEP can increase the sensitivity of digital ELISA for multiple rapid detection.
数字酶联免疫吸附测定法作为一种新型平台被引入，在检测样本中的多种单分子方面具有独特优势。如何提高检测灵敏度是当前相关研究的方向。在此，我们报告一种免疫测定方法，该方法应用电动效应来分离单个编码微珠并将其限制在微孔中，以同时提高细胞因子检测效率。微流控设计提供非均匀电场以诱导介电泳（DEP）力并操控微珠。两种激发光波长激发编码微珠以同时检测报告分子。光通过全内反射原理被限制在底部载玻片上。最后，通过从图像中拾取每个微珠并整合报告分子发出的荧光强度来获得捕获的细胞因子浓度。结果表明，通过DEP效应，编码微珠的填充率从10 - 20%提高到了60 - 80%。通过比较颗粒本身及其表面的荧光颜色，计算出四种目标细胞因子IL - 2、IL - 6、IL - 10和TNF - α的浓度达到皮克/毫升水平。加标回收实验验证了该方法的效率，超过70%的目标分子被捕获。流式细胞术也验证了我们方法的可靠性。总之，我们期望DEP的应用能够提高数字酶联免疫吸附测定法对多种物质快速检测的灵敏度。
