Vapor diffusion is the most common method to grow protein crystals suitable for X-ray diffraction analysis.In the method,small droplets containing protein and precipitant are allowed to equilibrate by vapor diffusion with a solution of a lower initial chemical potential of the solvent.For the vapor diffusion method,water equilibration rate plays a key role in screening successful crystallization conditions [1].Unfortunately,it is impossible to predict an optimal water equilibration rate between the protein solution and the precipitant solution.Thus,a laborious screening of a wide range of different conditions is typically required to obtain X-ray quality crystals of proteins for structure-function studies.To ease the laborious and time consuming task of screening optimal crystallization conditions,we present a microfluidic device with a linear vapor diffusion gradient for rapid optimization of protein crystallization.As shown in figure 1,the device contains a microchannel network with a repeated sequence of loops and each loop contains a trapping chamber to immobilize a protein droplet.Meanwhile,a coverslip with a hanging droplet containing precipitant solution is invertedly placed onto the inlet port.This configuration creates a gradient of distance from the growth droplets to the precipitant solution(Figure 1b),thus resulting in vapor diffusion rate gradient for rapid screening of different rates of supersaturation [2].To demonstrate the feasibility of this device,we first tested its capabilities of generating vapor diffusion rate gradient.As shown in Figure 2,the volume of water loss of each droplet in the device is varied from drop-to-drop,and a gradient of the rate of water loss can be observed in the droplet array.This suggests that a gradient of vapor diffusion rate was built in the device,which allows exploring more crystallization space in an individual screening experiment.Thus this device can be used to rapidly narrow down the search space for suitable crystallization conditions.To further evaluate the functionality of the proposed chip,we applied it to the crystallization of lysozyme as a model protein(Figure 3).The results clearly show that crystal growth is dependent on the vapor diffusion rate.Especially,a slower vapor diffusion rate of protein growth solution generally resulted in smaller populations of larger protein crystals.