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.
Keywords: Dielelctrophoresis; High-throughput; Immunoassay; Microfluidic; Multiplexed.
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