X-ray crystallography is the leading technique for determining the atomic structure of biological molecules. The developers of this technique have maintained this position by continuously pushing the hardware and software boundaries such that data-collection from micron sized crystals are now possible. However, to break through the micron barrier will require a paradigm shift. Currently, the majority of samples are mounted on cryo-cooled pins. Mounting and aligning a micron and nano metre sized crystal to an X-ray incident beam is beyond the limits of electromechanical goniometers whose inherent limitations such as movement backlash impair practicable manipulation and alignment on this scale. Currently proposed alternatives have had varying degrees of success, but have yet to unlock the coveted submicron sample-loading domain. We propose a novel sample loading methodology, which combines elements from microfluidics and optical tweezing. The device is an optofluidic chip and nano-tweezers system that traps samples via evanescent fields emitted from silicon nitride waveguides transmitting a 1064 nm laser beam. Crystals can be aligned in solution, negating the need for pins and cryo-cooling systems. This paper presents a further development of the system's application. The highly beneficial implementation of microfluidics was further exploited by upgrading the system to allow for the control of one or more microfluidic ports and flow streams. This not only improved the tweezing process but it also created the ability to introduce multiple samples and reagents. Combining these during both the sample feeding and tweezing stages has allowed for the possibility of using the x-ray crystallographic method to capture ongoing reactions as they occur. These 'time resolved' nano crystal experiments are currently beyond the capabilities of the crystallographic method but remain the ultimate quest of many structural biology projects as they could reveal the complex structural changes of enzymes as they react in real time with their substrates. The optofluidic chip is currently being used to explore the mechanism of the beta-lactamases; several families of proteins which catalyse the hydrolysis of beta-lactam antibiotics. These proteins are ideal for this task, not only because the biological question is exceptionally relevant in a post-antibiotic world [1], but also several substrates undergo a colorimetric change during catalysis so the X-ray diffraction data can be obtained whilst monitoring the activity of the enzyme. These optofluidic chips have potential application for both synchrotrons light sources and X-FEL beamlines
