Droplets isolated array: A universal platform of delaying molecule cross-contamination between microdroplets for digital enzyme-based immunoassay

摘要

Digital biodetection in picoliter-volume microdroplets has great potential for ultra-sensitivity detection due to the wide development of microfluidic technology and the high throughput of droplets generation. However, the diffusion of enzyme catalytic fluorescent products between droplets makes signal reading confused and inaccurate. Here, a spherical Poly (acrylic acid) Brushes immobilized Alkaline phosphatase (SP-AKP) as signal amplification block was synthesized and introduced into 14 pL droplet, and the SP-AKP digital detection was realized within only 6 min, which significantly improved the catalytic efficiency and shortened the detection time. Furthermore, aimed to decrease the diffusion rate of fluorophore between droplets, we proposed a Droplets Isolated Array (DIA) where droplets were trapped in chambers to keep them isolated. The DIA could trap -34,000 droplets once and reach 82.6 % of droplet coverage with high throughput and efficiency. Compared to conventional non-isolated droplets array, the DIA delayed the diffusion of fluorophore, and the reading time of droplet signal could be extended from 1 min to 30 min, which greatly improved the operability of the detection system and ensured the accuracy of the results. Finally, an accurate signal identification makes the limit of detection of SP-AKP achieve 309 aM.