Electric Triggering for Enhanced Control of Droplet Generation

Abstract

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its “off-time” is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Membrane protein megahertz crystallography at the European XFEL

“…Only with exceptionally large efforts and large-volume bioreactor culture capability were we able to isolate 1000 mg of PSI from the native sources for this experiment. Ongoing research into pulse injectors 35,36 and future developments in accelerator, detector, and computer technology may potentially alleviate this requirement in the future.…”

Segmented flow generator for serial crystallography at the European X-ray free electron laser

“…With droplets synchronized with the frequency and phase of the X-ray pulses, we expect a further reduction in the volume of sample consumed (potentially down to 1% of continuous injection at the EuXFEL), allowing the exploration of new crystal samples that do not crystallize readily in large volumes and accomplishing structure determination at MHz XFELs in the order of minutes 61 . Efforts to synchronize droplets in a 3D printed droplet generator via electrical triggering are currently being explored 62 . Furthermore, the droplet generation frequency is tunable, presenting a promising future for segmented flow sample delivery of scarce, hard-to-crystallize samples and more efficient data collection for studying macromolecular dynamics at other current and future XFELs.…”

Recent Advances in Microfluidic Technology for Bioanalysis and Diagnostics

“…Gallium electrodes integrated within a microfluidic chip allowed to release droplets on-demand in three different ways. First, through a programmable DC potential, second, under short AC signals, and third, by AC trigger signals to increase the generation frequency . By deforming an aqueous–oil interface inside a microchannel using a pulsed electric field, femtoliter droplets of different viscous solutions were produced on-demand .…”

“…First, through a programmable DC potential, second, under short AC signals, and third, by AC trigger signals to increase the generation frequency. 77 By deforming an aqueous−oil interface inside a microchannel using a pulsed electric field, femtoliter droplets of different viscous solutions were produced ondemand. 78 Another approach employed two Laplace pressure barriers to generate droplets on-demand by first filling a reservoir before pinching off droplets into a main channel.…”