An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Serial femtosecond crystallography has opened new opportunities in structural biology. Light-triggered systems have enabled time-resolved experiments, but most enzymes are not light-dependent, and many reactions rely on ligand diffusion into active sites. This work presents a drop-on-drop sample delivery system for enzyme-catalyzed reactions in microcrystal slurries. Ligand solutions are delivered as bursts of picoliter drops onto larger crystal-containing drops, inducing turbulent mixing and transporting the mixture to the X-ray interaction region with temporal control. Mixing is demonstrated using fluorescent dyes, numerical simulations, and time-resolved serial femtosecond crystallography, showing rapid ligand diffusion through microdroplets. The drop-on-drop method is widely applicable to serial crystallography, particularly for enzyme reactions with small molecule substrates.