ABSTRACT
High-speed liquid micro-jets are used to rapidly and repeatedly deliver protein microcrystals to focused and pulsed X-ray beams in the method of serial femtosecond crystallography. However, the current continuous flow of crystals is mismatched to the arrival of X-ray pulses, wasting vast amounts of an often rare and precious sample. Here, we introduce a method to address this problem by periodically trapping and releasing crystals in the liquid flow, creating locally concentrated crystal bunches, using an optical trap integrated in the microfluidic supply line. We experimentally demonstrate a 30-fold increase of particle concentration into 10 Hz bunches of 6.4 µm diameter polystyrene particles. Furthermore, using particle trajectory simulations, a comprehensive description of the optical bunching process and parameter space is presented. Adding this compact optofluidics device to existing injection systems would thereby dramatically reduce sample consumption and extend the application of serial crystallography to a greater range of protein crystal systems that cannot be produced in high abundance. Our approach is suitable for other microfluidic systems that require synchronous measurements of flowing objects.
ABSTRACT
Serial femtosecond crystallography is a new method for protein structure determination utilizing intense and destructive X-ray pulses generated by free-electron lasers. The approach requires the means to deliver hydrated protein crystals to a focused X-ray beam and replenish them at the repetition rate of the pulses. A liquid-jet sample delivery system where a gas dynamic virtual nozzle is printed directly on a silicon-glass microfluidic chip using a 2-photon-polymerization 3D printing process is implemented. This allows for rapid prototyping and high-precision production of nozzles to suit the characteristics of a particular sample and opens up the possibility for high-throughput and versatile sample delivery systems that can integrate microfluidic components for sample detection, characterisation, or control. With the hybrid system described here, stable liquid jets with diameters between 1.5 µm at liquid flow rate of 1.5 µl/min and more than 20 µm at liquid flow rate of 100 µl/min under atmospheric and vacuum conditions are generated. The combination of 2D lithography with direct 3D printing may streamline the integration of free-form-features and also facilitate scale-up production of such integrated microfluidic devices that may be useful in many other applications such as flow cytometry and optofluidics.
ABSTRACT
A concept for the determination of concentrations in microchannels using FT-IR spectroscopy in transmission is presented. The fundamental idea of spatially resolved measurements along several measuring points was implemented in a single-channel microreactor. Compared to existing microreactor setups for the analysis of fast chemical reactions or mixing processes, the presented concept enables longer residence times at appropriate resolution. Once steady-state conditions were reached in the reactor, mid-infrared spectra were collected at different locations. Information throughout the considered conversion range is available, which is of great importance to analyze inhibitory effects, next to the kinetic constants (vmax and KM). Therefore, this technology enables a rapid screening of (bio-)catalysts, substrate specificity and process conditions. In particular, the analysis of real substrates instead of model substrates and the possibility to follow side reactions and follow-up reactions during enzymatic catalysis open a broad field of application.
