Spatial barcoding-enabled highly multiplexed immunoassay with digital microfluidics

Digital microfluidics (DMF), facilitating independent manipulation of microliter samples, provides an ideal platform for immunoassay detection; however, suffering limited multiplexity. To address the need, herein we described a digital microfluidics (DMF) platform that realizes spatial barcoding on the Teflon-coated indium tin oxide (ITO) glass side to fulfill highly multiplexed immunoassay (10+) with low-volume samples (∼4 μL) in parallel, representing the highest multiplexing recorded to date for DMF-actuated immunoassay. Planar-based spatial immobilization of multiple capture antibodies was realized on a Teflon-coated ITO glass side, which was then used as the top plate of the DMF device. Droplets containing analytes, secondary antibodies, and fluorescent signaling reporters with low volume, which were electrically manipulated by our DMF control system, were shuttled sequentially along the working electrodes to complete the immuno-reaction. Evaluation of platform performance with recombinant proteins showed excellent sensitivity and reproducibility. To test the feasibility of our platform in analyzing multiplex biomarkers of the immune response, we used lipopolysaccharide-stimulated macrophages as a model system for protein secretion dynamics studies. As a result, temporal profiling of pro-inflammatory cytokine secretion dynamics was obtained. The spatial barcoding strategy presented here is easy-to-operate to enable a more comprehensive evaluation of protein abundance from biological samples, paving the way for new opportunities to realize multiplexity-associated applications with the DMF platform.