This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Currently, microfluidics technology is being applied to develop micromixers for time resolved cryo-electron microscopy (TRCEM) application. TRCEM requires fast and homogeneous premixing of tiny amounts of macromolecules in the time scale of milliseconds or less. Work is also just beginning in which this technology will be applied to the problems of specimen deposition onto EM grids and rapid freezing. Our goal for the remainder of this grant is to design and test a microfluidics-based device that allows TRCEM experiments to be conducted routinely with millisecond time resolution and requires only the small amounts of biomolecules that are often available to researchers. The nano-device design and fabrication is conducted in collaboration with Dr. Toh Ming and Dr. Zonghuan Lu at Rensselaer Polytechnic Institute via subcontract from the P41 grant. Very few publications (Tittor et al., 2002;Walker et al., 1999) have appeared over the past decade employing TRCEM on sub-second time scales. Aside from the ongoing efforts in our laboratory, the other existing technologies involve depositing the macromolecular complex on the specimen grid by the standard blotting technique and then initiating the reaction as the grid is being plunged into cryogen by spraying it with a small-molecule reactant (Berriman and Unwin, 1994;White et al., 2003;White et al., 1998) or exposing the grid to a brief pulse of UV irradiation to activate a photo-sensitive reactant, either a chemically synthesized """"""""caged"""""""" molecule or a naturally photosensitive component (Menetret et al., 1991;Subramaniam et al., 1993). Probably the most notable achievement to date of sub-second TRCEM was the trapping and structural determination of the acetylcholine receptor in its transiently open state by Unwin over 15 years ago (Unwin, 1995). In large part, the objective of this TRD in the last few years has focused on developing a device that combines rapid mixing and spraying to deposit a thin layer of aqueous biological samples on cryo-EM grids, i.e., to achieve sample mixing in the submillisecond time scale, to incubate the mixture for a particular time scale of milliseconds or more, and then to spray the sample onto TEM grids. A major advantage of the method we have developed is that it allows the reaction to occur in bulk solution and not on the EM grid where reactants can undergo interactions (e.g. irreversible adsorption) to the carbon support film. Fig. 1. Micromixer development. (a) Configuration of the micromixer, with detailed dimensions labeled. SEM images on the bottom show the mixing channel as fabricated using DRIE etching technology (left), and the details of half of the butterfly mixing unit (right). (b) Fluorescence microscopy images of mixing fluorescein and Fluoro Sphere Red in the fabricated mixer at various flowrates. (C) Experimental and simulated mixing indices showing essentially complete mixing at flow rates above 4 ?L/sec. (D) Activity of malate dehydrogenase is preserved at various flowrates. To achieve this objective, we developed and evaluated numerous microfluidic mixer designs for rapid sample mixing with economical sample volumes. These designs included T- or Tesla-shaped mixers, hemicircular- and butterfly-shaped in-channel mixing elements, and their combinations. Initial evaluations of the designs were done using three-dimensional Computational Fluid Dynamics (CFD) simulations. Guided by the CFD results, physical devices were fabricated using micro-machining technologies (including DRIE, deep reactive ion etching) and were tested to assess their efficiency for rapid mixing. A novel micromixer that combined two T-shaped premixers and four in-channel butterfly mixing elements (Fig. 1a) displayed mixing performance characteristics that should satisfy most requirements of TRCEM. The mixer utilizes a chaotic advection mixing strategy to achieve submillisecond mixing, while sample consumption is small at several Fig. 2. Monolithic microfluidic devices for mixing and spraying (a) Detailed schematic of monolithic device (micromixer/sprayer) design showing (1) device design layout, (2) micromixer configuration, (3) SEM image of the mixer, (4) external atomization nozzle design, (5) SEM image of the nozzle, (6) device top view, and (7) device bottom view. (b) The droplet size distributions for two of the devices with the external atomization nozzles, showing the droplet size mainly in the range of 10-30 ?m. Fig. 3. Experimental setup with microspray generation shown: (a) Diagram of the experimental system, including syringe pump for liquid transport, monolithic device for mixing and spraying, and cryo-EM grid for droplet collection. (b) Photo of the entire experimental system setup. (c) Close-up of the device with fittings, which is mounted on a plastic holder. A tweezers (connected to the plunger) with an EM grid placed at the exit of the spray nozzle. (d) Fine microspray (blue arrows) generated by the device with the liquid flow rate at 6.0 ?L/s, and with nitrogen gas pressure at 50 psi (set by nitrogen tank regulator). microliters per second. The simulation results showed that the mixing structures generated chaotic advection in the mixer by forming secondary vortex flows in the microchannels. For example, at a flow rate of 6.0 ?L/s the calculated mixing index is 99%, and the mean residence time is less than 0.5 msec for 95% of the molecules. Experimental mixing tests of the actual devices indicated that the mixer performance is consistent with the simulation results, as shown in Figure 1 (b,c). To determine whether the devices have deleterious effects on protein function we also conducted enzymatic activity tests of malate dehydrogenase, which was passed through the micromixer at various flow rates up to 10 ?L/s. Full activity, assessed by comparison to enzyme that had not gone through the mixer, was retained at all flow rates tested (Fig. 1d). These results are a good indicator that the device will be useful for biophysical characterization of the dynamics of functioning or assembling macromolecular complexes. More recent studies using ribosomal protein translation assays and assembly of ribosomal subunits have thus far given no indications of any damage caused by the microdevices. The next challenge was to deposit reaction mixtures that had passed through the micromixers on to EM grids such that they could be frozen with minimal delay. Several years ago our lab developed a mechanically machined air-assisted sprayer consisting of a 100-?m diameter plastic-tubing liquid nozzle surrounded by a ~300 ?m conical plastic air nozzle (Barnard and Wagenknecht, 2005). We attempted to mate this sprayer to various mixers (a simple T mixer and one of the micromixers described above), but results were unsatisfactory. For example, this system was impractical for reaction times on the order of milliseconds. However, our experience with this sprayer was valuable. It has led us to the design and fabrication of an integrated monolithic device that combines the micromixers described above with a miniaturized air-assisted sprayer that can achieve processing (mixing + reaction) times as short as a few milliseconds. Silicon and Pyrex glass wafers were used as substrates but other materials can also be used, such as PDMS, PMMA, or other suitable biocompatible polymers. The device's performance for generating micron size droplets was tested. Ribosome-ferritin mixing experiments were carried out to verify its mixing efficiency. Several methods were used to evaluate the activities of biological macromolecules after passing them through the device, including 3D cryo-EM reconstruction, UV-spectroscopy, sucrose-gradient ultracentrifugation, and assays of ribosomal activity. Three types of devices were tested based on their nozzle configurations, including external or internal atomization nozzles. All the devices successfully generated microsprays at flow rates of 4-6 ?L/s. A solution of ferritin was used as tracer when sprayed on TEM grids to detect the droplet size distribution. The two devices with the external atomization spray nozzle configuration, shown in Figure 2a, more stably generated microdroplet sprays than the other types of sprayers, and yielded adequate coverage of the grid by droplets. The droplet sizes are in the range of tens of micrometers (as shown in Figure 2b), which generally fulfilled the droplet size requirement, although the large number of droplets greater than 25 ?m in diameter is not optimal for good spreading for cryo-EM. Figure 3 shows the experimental setup for TRCEM, including a photograph of the device in operation while generating a microdroplet-containing spray. Two devices are currently being tested, one with a total processing time of 10 msec and the other 42 msec (about 5 msec of this time is for spraying onto the grid and plunging into cryogen). We are currently utilizing this experimental setup for ribosome subunit association experiments with the goal of discovering intermediate states in the reassociation of 30S and 50S ribosomal subunits to form 70S monosomes. We have successfully imaged and determined 3D reconstructions of ribosomes that were passed through the devices (Figure 4). The resolution attained was the same as that obtained for a similar number of particles imaged by conventional (non-time-resolved) cryo-EM. Further, biophysical and biochemical characterization of ribosomes and ribosomal subunits that have passed through the device also indicated no loss of structural or functional activity. Experiments to characterize the reassociation of the E. coli ribosome from 30S and 50S subunits in the monolithic devices have commenced, and the preliminary results show that reassociation is occurring in the monolithic devices. Figure 5 (top panel) shows some selected examples of images that have been identified as re-assembled ribosomes. Already, some atypical images have been observed, but it is premature to identify these as true intermediates in reassociation. Several issues should be addressed to improve the performance of our current experimental system. First, the average droplet size is larger than is optimal for data collection;larger droplets tend not to spread sufficiently thin for optimal contrast. Related to this issue, enhancing the hydrophilicity of the grid surface would promote better surface wetting and droplet spreading. The most important factors that need to be extensively investigated are, first, time resolution control, which will allow a broader range of biological applications and, second, reduced sample consumption, especially for valuable biological samples whose amounts are usually very limited. Other issues related to system improvement include temperature and humidity control to obtain adjustable and reproducible reaction rates, and to facilitate the integration of monolithic device into the environments commonly found in laboratories that conduct cryo-EM experiments. Fig. 4. Cryo-EM of ribosomes and ferritin that were mixed and sprayed by the monlithic device #2. Upper left: low magnification micrograph showing the edge of a droplet that has spread sufficiently thin for image. Right: High magnification view of a hole containing thin ice and showing presence of both ferritin (dark structures) and ribosomes (lighter structures). Lower left: Surface representation of a three-dimensional reconstruction of the ribosome determined from images such as that shown in B. Resolution is 18.9?. Adapted from Lu et al. J. Struct. Biol.. References: Barnard D and T Wagenknecht. 2005. Pneumatic Micro-Sprayer for Millisecond Time Resolution in Cryo-Electron Microscopy. Microscopy &Microanalysis 11:290-291. Berriman J and N Unwin. 1994. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56:241-252. Subramaniam, S., M.Gerstein, D.Oesterhelt, and R.Henderson. 1993. Electron Diffraction Analysis of Structural Changes in the Photocycle of Bacteriorhodopsin. EMBO J. 12:1-8. Tittor, J., S.Paula, S.Subramaniam, J.Heberle, R.Henderson, and D.Oesterhelt. 2002. Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes. J. Mol. Biol. 319:555-565. Unwin, N. 1995. Acetylcholine receptor channel imaged in the open state. Nature 373:37-43. Walker, M., X.-Z.Zhang, W.Jiang, J.Trinick, and H.D.White. 1999. Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution cryomicroscopy: evidence that the start of the crossbridge power stroke in muscle has variable geometry. Proc. Natl. Acad. Sci. USA 96:465-470. White, H.D., K.Thirumurugan, M.L.Walker, and J.Trinick. 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144:246-252. White, H.D., M.L.Walker, and J.Trinick. 1998. A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy. J. Struct. Biol. 121:306-313. In the previous reporting period, the following abstracts were presented: + Lu, Z., McMahon, J., Mohamed, H., Barnard, D., Shaikh, T.R., Wagenknecht, T. and Lu, T.M. 2008. Microfluidic Mixing System for Time Resolved Cryo-Electron Microscopy. Microscopy and Microanalysis 14:1598-1599. + Barnard, D., Lu, Z., Shaikh, T.R., Mohamed, H., Buttle, K., McMahon, J., Meng, X., Lu, T.M. and Wagenknecht, T. 2008. 1576-Pos Development of a Reaction Mixer/Micro-Nebulizer for Time-Resolved Cryo-Electron Microscopy of Macromolecular Systems. Biophys. J. 94:1576.
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