Here we report a microfluidic device for controlled concentrating of reagents and for multiple-step mixing and reaction.A series of concentrations are often required in chemical analysis,and several microfluidic platforms have been developed to achieve on-chip dilution or concentrating for chemical and biochemical analysis [1,2].However,most of the devices are not appropriate for multistep mixing or reaction.To address this issue,we developed a new multilayer microfluidic device,and we studied cell-free protein expression in the device,mainly concerning the effect of polyethylene glycol (PEG) concentration and amino acid concentration on the expression efficiency.The primary structure of the device includes three vertically aligned parts: a reaction chamber layer,a control channel layer and a thin membrane between the two layers (Figure 1).By exploiting the water permeability of polydimethylsiloxane (PDMS),we can concentrate the reactant solution in the chamber and then load a second reactant into the chamber for reaction.The operation of the device has the following steps.First,the reactant solution is loaded into the chambers by using degassed PDMS [3].Then the solution in the chambers is concentrated by flowing saline solution of high concentration in the control channels which are on top of the chambers [4].After the concentrating process,a second reactant solution is added into the chambers.The concentrating and adding process can be repeated as needed.By adjusting the osmolality of the saline solution in the two control channels,we can generate concentration gradient of the reactant along an array of the chambers (Figure 2).Finally,every chamber is sealed with UV adhesive.Less than 2 μl sample is consumed in total in the experiment.The multilayer PDMS device was applied to the study of cell free protein expression.DNA encoding enhanced Green Fluorescent Protein (eGFP) and in vitro expression agents were loaded into the chamber.In the study,the concentration gradient of PEG and amino acids along the chamber array was generated to study the yield of protein expression.The optimal concentration of PEG for the maximum eGFP yield was identified,which suggested that macromolecular crowding enhanced the efficiency of the protein expression.