Inertial mixing of acoustically levitated droplets for time-lapse protein crystallography

Varying the chemical consistency of acoustically levitated droplets opens up an in situ study of chemical and biochemical reactions in small volumes.However,the opti-mization of the mixing time and the minimization of the positional instability induced by solution dispensing are necessary for practical applications such as the study of the transient state of macromolecules crystallography during the ligand binding pro-cesses.For this purpose,we study the inertial mixing in a configuration compatible with the room-temperature crystallography using the acoustic levitation diffractometer,therein solution drops ejected at high velocity collide and coalesce with droplets dis-pensed on acoustically levitated and rotating polymer thin-film sample holders.With the proposed method,we are able to achieve the mixing time of∼0.1 s for sub-micro and a few microliter droplets.The observed short mixing time is ascribed to the rapid penetration of the solution into the droplets and confirmed by a computational fluid dynamic simulation.The demonstrated accelerated solution mixing is tested in a pilot time-lapse protein crystallography experiment using the acoustic levitation diffrac-tometer.The results indicate the detection of transient ligand binding state within 2 s after the solution dispensing,suggesting the feasibility of the proposed method for studying slow biochemical processes.