Generation of two-dimensional concentration-gradient droplet arrays on a two-layer chip for screening of protein crystallization conditions

We developed a two-layer microfluidic chip for the generation of two-dimensional concentration-gradient droplet arrays. All of the channels, including a concentration-gradient formation channel network, an oil phase channel network, and droplet generators, were ingeniously arranged on a single polydimethylsiloxane (PDMS) substrate, which facilitates the fabrication of the chip. Ninety-six lines of subnanoliter droplets with concentration gradients can be formed on a 6-inlet PDMS chip. With the aqueous phase flow rate of 1.0 μL/min and the oil phase flow rate of 35 μL/min, relative standard deviation of the droplet sizes in the same channel was below 5.0 %, while in the 96 channels was 7.0 %. Multiparameter screening of protein crystallization conditions was demonstrated by using the two-dimensional concentration-gradient droplet arrays. Protein, precipitant, and buffer each at 16 concentrations for lysozyme crystallization were tested simultaneously in one 6-inlet chip, and five precipitants each at eight concentrations were screened at the same time in one 10-inlet chip. The concentration-gradient droplet chip proved to be a useful tool for screening crystallization conditions, characterized by low consumption, high throughput, convenience, and automatization.