Serial femtosecond crystallography (SFX) with an X-ray free-electron laser (XFEL) has enabled the determination of protein structures and protein reaction intermediates in millisecond to microsecond time resolutions. Mix-and-Inject crystallography (MISC) at XFELs enables fast mixing in the magnitude of milliseconds in order to achieve desired reaction time points. For these experiments, numerical simulations of a hydrodynamic flow mixer capable of fast mixing by diffusion has been developed using both COMSOL Multiphysics 5.6 and QuickerSims Computational Fluid Dynamics (CFD) Toolbox for MATLAB. These simulation programs were compared by calculations of mixing times and concentration flow profiles. Mixing times in the range of 1-10 ms were calculated in COMSOL under certain flow rate conditions whereas mixing times in the range of 6-15 ms were calculated with QuickerSims. From these mixing times, reaction intermediates can be varied from sub-millisecond to several hundred millisecond time points for a MISC experiment. Explanations for the discrepancies between the two models were attributed to variations in parameter definitions and meshing. Further analysis on the mixing characteristics were investigated by calculating an analytical solution to the convection-diffusion equation for fluid flow in a two-dimensional rectangular channel. The concentration profile along the width of the channel for the analytical solution was compared with the numerical solution obtained with COMSOL and QuickerSims. Upon comparison, it was determined that the diffusion coefficient may not be a significant factor for the disagreement between the two hydrodynamic flow models.

There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.

An electric field can be applied to a microfluidic device in order to stop particle flow. Electroosmosis, electrophoresis, and dielectrophoresis act on the particles in different directions in the microfluidic channel, and when these forces create zero net force, the particle stops in the channel. The goal of the performed experiments is to investigate whether hydrostatic pressure generated by a syringe pump could help concentrate these particles and separate them from other contents. Introducing precise, adjustable hydrostatic pressure from the syringe pump provides another mechanism for controlling particle behavior. A microfluidic channel was crafted into a device connected to a syringe pump, and videos of 1 µm silica particles in the device were recorded under a microscope in order to show that samples could be infused into the device and concentrated or captured at a specific location in the channel using hydrostatic pressure. Capture of the particles occurred with and without controlled hydrostatic pressure, but these events occurred somewhat consistently at different voltages. In addition, particle movement in the channel with the syringe pump off was originally attributed to the electrokinetic forces. However, when compared to experiments without the syringe pump connected to the device, it became evident that the electrokinetic forces should have moved the particles in the opposite direction and that, in actuality, there is an inherent pressure in the device also affecting particle movement even when the syringe pump is not turned on.