Macromolecular crystallization with microfluidic free-interface diffusion

Fluidigm Corp. released the Topaz 1.96 and 4.96 crystallization chips in the fall of 2004. Topaz 1.96 and 4.96 are the latest evolution of Fluidigm's microfluidics crystallization technologies that enable ultra-low-volume rapid screening for macromolecular crystallization. Topaz 1.96 and 4.96 are similar to each other but represent a major redesign of the Topaz system and have substantially improved ease of automation and ease of use, improved efficiency and even further reduced the amount of material needed. With the release of the new Topaz system, Fluidigm continues to set the standard in low-volume crystallization screening, which is having an increasing impact in the field of structural genomics and more generally in structural biology. It is likely that further optimization and increased utility of the Topaz crystallization system will emerge. It is also probable that further innovation and the emergence of competing technologies will be seen.     

“Connecting worlds – a view on microfluidics for a wider application”

From its birth, microfluidics has been referenced as a revolutionary technology and the solution to long standing technological and sociological issues, such as detection of dilute compounds and personalized healthcare. Microfluidics has for example been envisioned as: (1) being capable of miniaturizing industrial production plants, thereby increasing their automation and operational safety at low cost; (2) being able to identify rare diseases by running bioanalytics directly on the patient’s skin; (3) allowing health diagnostics in point-of-care sites through cheap lab-on-a-chip devices. However, the current state of microfluidics, although technologically advanced, has so far failed to reach the originally promised widespread use.In this paper, some of the aspects are identified and discussed that have prevented microfluidics from reaching its full potential, especially in the chemical engineering and biotechnology fields, focusing mainly on the specialization on a single target of most microfluidic devices and offering a perspective on the alternate, multi-use, “plug and play” approach. Increasing the flexibility of microfluidic platforms, by increasing their compatibility with different substrates, reactions and operation conditions, and other microfluidic systems is indeed of surmount importance and current academic and industrial approaches to modular microfluidics are presented. Furthermore, two views on the commercialization of plug-and-play microfluidics systems, leading towards improved acceptance and more widespread use, are introduced. A brief review of the main materials and fabrication strategies used in these fields, is also presented. Finally, a step-wise guide towards the development of microfluidic systems is introduced with special focus on the integration of sensors in microfluidics. The proposed guidelines are then applied for the development of two different example platforms, and to three examples taken from literature.With this work, we aim to provide an interesting perspective on the field of microfluidics when applied to chemical engineering and biotechnology studies, as well as to contribute with potential solutions to some of its current challenges.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

Synthetic symmetrization in the crystallization and structure determination of CelA from Thermotoga maritima

Protein crystallization continues to be a major bottleneck in X‐ray crystallography. Previous studies suggest that symmetric proteins, such as homodimers, might crystallize more readily than monomeric proteins or asymmetric complexes. Proteins that are naturally monomeric can be made homodimeric artificially. Our approach is to create homodimeric proteins by introducing single cysteines into the protein of interest, which are then oxidized to form a disulfide bond between the two monomers. By introducing the single cysteine at different sequence positions, one can produce a variety of synthetically dimerized versions of a protein, with each construct expected to exhibit its own crystallization behavior. In earlier work, we demonstrated the potential utility of the approach using T4 lysozyme as a model system. Here we report the successful application of the method to Thermotoga maritima CelA, a thermophilic endoglucanase enzyme with low sequence identity to proteins with structures previously reported in the Protein Data Bank. This protein had resisted crystallization in its natural monomeric form, despite a broad survey of crystallization conditions. The synthetic dimerization of the CelA mutant D188C yielded well‐diffracting crystals with molecules in a packing arrangement that would not have occurred with native, monomeric CelA. A 2.4 Å crystal structure was determined by single anomalous dispersion using a seleno‐methionine derivatized protein. The results support the notion that synthetic symmetrization can be a useful approach for enlarging the search space for crystallizing monomeric proteins or asymmetric complexes.     

A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.     

Microfabricated devices in biotechnology and biochemical processing

In the past few years, interdisciplinary science and technologies have converged to create exciting challenges and opportunities, which involve a new generation of integrated microfabricated devices. These new devices are referred to as 'lab-on-a-chip' or Micro Total Analysis Systems. Their development involves both established and evolving technologies, which include microlithography, micromachining, Micro Electro Mechanical Systems technology, microfluidics and nanotechnology. This review summarizes the key device subject areas and the basic interdisciplinary technologies, and gives a better understanding of how these technologies can be used to provide appropriate technical solutions to fundamental problems. Important applications for this novel 'synergized' technology in chemical and biotechnological processing, in addition to the application of simulation methods in the development of microfabricated devices, will also be discussed.     

Monitoring the Differentiation and Migration Patterns of Neural Cells Derived from Human Embryonic Stem Cells Using a Microfluidic Culture System

Microfluidics can provide unique experimental tools to visualize the development of neural structures within a microscale device, which is followed by guidance of neurite growth in the axonal isolation compartment. We utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stem cells (hESCs). We co-cultured hESCs with PA6 stromal cells, and isolated neural rosette-like structures, which subsequently formed neurospheres in suspension culture. Tuj1-positive neural cells, but not nestin-positive neural precursor cells (NPCs), were able to enter the microfluidics grooves (microchannels), suggesting that neural cell-migratory capacity was dependent upon neuronal differentiation stage. We also showed that bundles of axons formed and extended into the microchannels. Taken together, these results demonstrated that microfluidics technology can provide useful tools to study neurite outgrowth and axon guidance of neural cells, which are derived from human embryonic stem cells.     

Crystallization for the Downstream Processing of Proteins

Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low‐molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale‐up.     

Carboxylic acids in crystallization of macromolecules: learning from successful crystallization experiments

The production of macromolecular crystals suitable for structural analysis is one of the most important and limiting steps in the structure determination process. Often, preliminary crystallization trials are performed using hundreds of empirically selected conditions. Carboxylic acids and/or their salts are one of the most popular components of these empirically derived crystallization conditions. Our findings indicate that almost 40 % of entries deposited to the Protein Data Bank (PDB) reporting crystallization conditions contain at least one carboxylic acid. In order to analyze the role of carboxylic acids in macromolecular crystallization, a large-scale analysis of the successful crystallization experiments reported to the PDB was performed. The PDB is currently the largest source of crystallization data, however it is not easily searchable. These complications are due to a combination of a free text format, which is used to capture information on the crystallization experiments, and the inconsistent naming of chemicals used in crystallization experiments. Despite these difficulties, our approach allows for the extraction of over 47,000 crystallization conditions from the PDB. Initially, the selected conditions were investigated to determine which carboxylic acids or their salts are most often present in crystallization solutions. From this group, selected sets of crystallization conditions were analyzed in detail, assessing parameters such as concentration, pH, and precipitant used. Our findings will lead to the design of new crystallization screens focused around carboxylic acids.     

Crystallization of membrane proteins from media composed of connected-bilayer gels

The use of hydrated-lipid gels in which the bilayer is an infinitely periodic (or at least continuous), three-dimensional structure offers a relatively new approach for the crystallization of membrane proteins. While excellent crystals of the Halobacterial rhodopsins have been obtained with such media, success remains poor in extending their use to other membrane proteins. Experience with crystallization of bacteriorhodopsin has led us to recognize a number of improvements that can be made in the use of such hydrated-gel media, which may now prove to be of general value for the crystallization of other membrane proteins.     

Air bubble-initiated biofabrication of freestanding,semi-permeable biopolymer membranes in PDMS microfluidics

Membrane functionality in microfluidics is critical for sample separation, concentration, compartmentalization, filtration, pumping, gradient generation, gas–liquid exchange, and other processes. Integration of functional membranes in microfluidics, however, is nontrivial. Here, we report a simple approach for biofabricating freestanding, semi-permeable biopolymer membranes in microfluidics, initiated with intentionally trapped air bubbles caught within specifically designed polydimethylsiloxane (PDMS) apertures. Pressure-driven dissipation of air bubbles through the gas permeable PDMS facilitates local and quiescent contact of two oppositely charged polyelectrolyte polysaccharides forming a layered or sandwiched membrane. This polyelectrolyte complex membrane (PECM) is permeable to ions including hydroxyl ions, which further facilitates layer-by-layer assembly of membrane stratum. Assembled membranes that bridge the 40-μm apertures are sufficiently strong to withstand >1 atmosphere hydrostatic pressure. Further, the semi-permeable membranes allow for programmed generation of small molecule gradients while preventing protein efflux. We envision the simplicity of fabrication, which requires no reagents or complicated valving, when coupled with the functional properties of the membrane polysaccharides, will find utility in cell and tissue studies including preclinical drug screening and toxicity analyses.     

Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases

Crystals of transmembrane proteins may be grown from detergent solutions or in a matrix of membranous lipid bilayers existing in a liquid crystalline state and forming a cubic phase (in cubo). While crystallization in micellar solutions appears analogous to that for soluble proteins, crystallization in lipidic matrices is poorly understood. As this method was shown to be applicable to several membrane proteins, understanding its mechanism will facilitate a rational design of crystallization, minimizing the laborious screening of a large number of parameters. Using polarization microscopy and low-angle X-ray diffraction, experimental evidence is provided to support a mechanistic model for the in cubo crystallization of bacteriorhodopsin in a lipid matrix. Membrane proteins are thought to reside in curved lipid bilayers, to diffuse into patches of lower curvature and to incorporate into lattices which associate to form highly ordered three-dimensional crystals. Critical testing of this model is necessary to generalize it to other membrane proteins.     

Quantitative Detection of Pharmaceuticals Using a Combination of Paper Microfluidics and Wavelength Modulated Raman Spectroscopy

Raman spectroscopy has proven to be an indispensable technique for the identification of various types of analytes due to the fingerprint vibration spectrum obtained. Paper microfluidics has also emerged as a low cost, easy to fabricate and portable approach for point of care testing. However, due to inherent background fluorescence, combining Raman spectroscopy with paper microfluidics is to date an unmet challenge in the absence of using surface enhanced mechanisms. We describe the first use of wavelength modulated Raman spectroscopy (WMRS) for analysis on a paper microfluidics platform. This study demonstrates the ability to suppress the background fluorescence of the paper using WMRS and the subsequent implementation of this technique for pharmaceutical analysis. The results of this study demonstrate that it is possible to discriminate between both paracetamol and ibuprofen, whilst, also being able to detect the presence of each analyte quantitatively at nanomolar concentrations.     

Ion channel electrophysiology via integrated planar patch-clamp chip with on-demand drug exchange

Planar patch clamp has revolutionized characterization of ion channel behavior in drug discovery primarily via advancement in high throughput. Lab use of planar technology, however, addresses different requirements and suffers from inflexibility to enable wide range of interrogation via a single cell. This work presents integration of planar patch clamp with microfluidics, achieving multiple solution exchanges for tailor-specific measurement and allowing rapid replacement of the cell-contacting aperture. Studies via endogenously expressed ion channels in HEK 293T cells were commenced to characterize the device. Results reveal the microfluidic concentration generator produces distinct solution/drug combination/concentrations on-demand. Volume-regulated chloride channel and voltage-gated potassium channels in HEK 293T cells immersed in generated solutions under various osmolarities or drug concentrations show unique channel signature under specific condition. Excitation and blockage of ion channels in a single cell was demonstrated via serial solution exchange. Robustness of the reversible bonding and ease of glass substrate replacement were proven via repeated usage of the integrated device. The present approach reveals the capability and flexibility of integrated microfluidic planar patch-clamp system for ion channel assays.     

Anaerobic crystallization of proteins

Crystallization has been a bottleneck in the X-ray crystallography of proteins. Although many techniques have been developed to overcome this obstacle, the impurities caused by chemical reactions during crystallization have not been sufficiently considered. Oxidation of proteins, which can lead to poor reproducibility of the crystallization, is a prominent example. Protein oxidization in the crystallization droplet causes inter-molecular disulfide bridge formation, formation of oxidation film, and precipitation of proteins. These changes by oxidation are typically irreversible. The best approach for preventing protein oxidization during crystallization is anaerobic crystallization. Here we review the anaerobic crystallization of proteins, which was originally developed to trap a reaction intermediate of the enzyme in the crystal. We also summarize representative anaerobic crystallizations from our laboratory and the general setup of anaerobic crystallization.     

Large‐scale identification of membrane proteins with properties favorable for crystallization

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large‐scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high‐value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.     

A new approach to the crystallization of proteins

A new method for the crystallization of proteins was developed. Saturated solutions of a protein for crystallization are prepared by chromatography of adsorbed proteins from an insoluble polysaccharide matrix using gradients with decreasing ionic strength, decreasing concentration of organic solvents or an appropriate shift in pH. Insulin, a-chymotrypsin and lysozyme for which the X-ray crystal structures are known, were crystallized by this method. X-ray diffraction photographs were taken to demonstrate the quality of the crystals. In addition to this analytical application, the method may be useful for the crystallization and the simultaneous purification of larger amounts of proteins.     

Building bio-assays with magnetic particles on a digital microfluidic platform

Digital microfluidics (DMF) has emerged as a promising liquid handling technology for a variety of applications, demonstrating great potential both in terms of miniaturization and automation. DMF is based on the manipulation of discrete, independently controllable liquid droplets, which makes it highly reconfigurable and reprogrammable. One of its most exclusive advantages, compared to microchannel-based microfluidics, is its ability to precisely handle solid nano- and microsized objects, such as magnetic particles. Magnetic particles have become very popular in the last decade, since their high surface-to-volume ratio and the possibility to magnetically separate them from the matrix make them perfect suitable as a solid support for bio-assay development. The potential of magnetic particles in DMF-based bio-assays has been demonstrated for various applications. In this review we discuss the latest developments of magnetic particle-based DMF bio-assays with the aim to present, identify and analyze the trends in the field. We also discuss the state-of-the art of device integration, current status of commercialization and issues that still need to be addressed. With this paper we intend to stimulate researchers to exploit and unveil the potential of these exciting tools, which will shape the future of modern biochemistry, microbiology and biomedical diagnostics.     

A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

Wetting Kinetics: an Alternative Approach Towards Understanding the Enhanced Dissolution Rate for Amorphous Solid Dispersion of a Poorly Soluble Drug

Developing amorphous solid dispersions of water-insoluble molecules using polymeric materials is a well-defined approach to improve the dissolution rate and bioavailability. While the selected polymer plays a vital role in stabilizing the amorphous solid dispersion physically, it is equally important to improve the dissolution profile by inhibiting crystallization from the supersaturated solution generated by dissolution of the amorphous material. Furthermore, understanding the mechanism of dissolution rate enhancement is of vital importance. In this work, wetting kinetics was taken up as an alternative approach for understanding the enhanced dissolution rate for amorphous solid dispersion of a poorly soluble drug. While cilostazol (CIL) was selected as the model drug, povidone (PVP), copovidone, and hypromellose (HPMC) were the polymers of choice. The concentrations against time profiles were evaluated for the supersaturated solutions of CIL in the presence and absence of the selected polymers. The degree of supersaturation increased significantly with increase in polymer content within the solid dispersion. While povidone was found to maintain the highest level of supersaturation for the greatest length of time both in dissolution and solution crystallization experiments, copovidone and hypromellose were found to be the less effective as crystallization inhibitor. The ability of polymers to generate and maintain supersaturated drug solutions was assessed by dissolution studies. The wetting kinetics was compared against the solid dispersion composition to establish a correlation with enhanced dissolution rate.KEY WORDS: Cilostazol, Crystallization inhibition, Solid dispersions, Supersaturated solutions, Wetting kinetics