Droplet-based microfluidics holds great potential for protein crystallization screening by minimizing the crystallization reactions in water-in-oil droplets in the picoliter to nanoliter range.In the previous work,we have developed a sequential operation droplet array (SODA) system for picoliter-scale high throughput screening.The SODA system was built mainly on the basis of a tapered capillary-syringe pump module and a two-dimensional oil-covered droplet array installed on an x-y-z translation stage.Each composite droplet was generated by sequentially assembling sample and reagents in capillary tip and depositing on nanowell chip.The SODA system is a fully-automated microfluidic screening platform that directly interfaces multi-well plate-based (96 or 384 well) sample and reagent storage system with droplet reaction system with picoliter resolution.The ability of the droplet microarray system in drug screening has been well demonstrated in the screening of inhibitors for caspase-1.In this work,we further extended the application of the SODA system in the screening of protein crystallization conditions with protein consumption in nanoliter range for each screening reaction.The SODA system was performed by moving the x-y-z stage to allow the capillary tip to sequentially aspirate protein and precipitant into capillary tip,and then deposit the composite droplet on the chip to form droplet array.The droplet array was incubated at 4 oC for 12 hours.After incubation,the crystals in the droplets were detected with polarized light detection (Figure 1).The SODA system was applied in screening the crystallization conditions of lysozyme using 51 different precipitants from a commercial kit (Figure 1).Each droplet contained 2-nL protein and 2-nL precipitant,and each condition was tested in quintuplicate.The total protein consumption for 255 droplet reactions is 510 nL,which is commonly only enough for conducting one crystallization trial in traditional 96 or 384-well plate-based screening platforms.Taking advantage of flexibly changing the droplet volume and composition for the present system,we investigated the effects of droplet volume on protein crystallization in the range of 0.84 nL to 100 nL (Figure 2).Protein crystals could form in all of these droplets,and the crystal size increased with the droplet volume.The successful crystallization in 0.84-nL droplets demonstrates the feasibility to perform the screening experiment in picoliter scale,which can further reduce the protein consumption.