Multiplexed digital ELISA in picoliter droplets based on enzyme signal amplification block and precisely decoding strategy: A universal and practical biodetection platform

Ultrasensitive multiplexed digital ELISA technologies are important in clinical applications, as one of the most sensitive methods for sub-femtomolar protein detection. Compared to microwell arrays, droplet microfluidics with high-throughput droplet generation and unlimited number of partitions are the more ideal candidate for multiplexed digital biodetection. Here, a universal and practical multiplexed droplet-based digital ELISA (ddELISA) strategy was innovatively developed, and it combined fluorescent barcodes as carriers to identify multiplexed targets with the imaging based decoded method of barcodes using short and long exposure times to improve decoding accuracy. Imaging decoding of 25-plexed 3 µm dual-color barcodes in droplets with diameters of 30 µm was achieved, demonstrating that constructed bead droplet microfluidics has a high multiplexed capability. To overcome the challenge of slow mono-enzyme catalytic rate in picoliter droplets, a horseradish peroxidase polymer (polyHRP) was introduced into 14 pL microdroplets, which shortened the detection time to 3 min. As a proof of concept, 5-plexed barcodes were chosen as microcarriers to detect five cytokines in one reaction by the established ddELISA, and LODs in the sub-fM range were achieved, resulting in an up to 3–40,000-fold improvement over multiplexed suspension chip. Thus, ddELISA we developed paves a promising way for ultrasensitive multiplexed biodetection.