- Email: [email protected]
Microfluidics in structural biology: smaller, faster… better
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Microfluidics in structural biology: smaller, faster. . . better Carl Hansen and Stephen R Quake Microfluidic technologies promise unprecedented savings in cost and time through the integration of complex chemical and biological assays on a microfabricated chip. Recent advances are making elements of this vision a reality, facilitating the first large-scale integration of microfluidic plumbing with biological assays. The power of miniaturization lies not only in achieving an economy of scale, but also in exploiting the unusual physics of fluid flow and mass transport on small length scales to realize precise and efficient assays that are not accessible with macroscopic tools. Diverse applications ranging from time-resolved studies of protein folding to highly efficient protein crystal growth suggest that microfluidics may become an indispensable tool in biology. Addresses Department of Applied Physics, California Institute of Technology, MS 128-95, Pasadena, CA 91125, USA e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:538–544 This review comes from a themed issue on Biophysical methods Edited by Brian T Chait and Keith Moffat 0959-440X/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2003.09.010
Abbreviations FID free interface diffusion MSL multilayer soft lithography
Introduction In the same way that integrated circuits have revolutionized computation, microfluidics seeks to integrate high levels of wet laboratory functionality on a microfabricated fluidic chip. By processing samples on the nanoliter scale, microfluidic technologies have the potential to realize increased speed and sensitivity, while at the same time achieving an unprecedented economy of scale, dramatically increasing throughput and reducing cost. Over the past decade, there has been a tremendous effort to devise methods of fabricating miniaturized plumbing that exploit this analogy with the semiconductor industry on many levels [1]. In this perspective, we will provide a brief introduction to microfluidics, describe some of the basic physics of fluids in small dimensions and review recent applications of this technology to structural biology.
Fabrication of microfluidic devices Although there is a thriving MEMS (micro-electromechanical systems) community making silicon-based Current Opinion in Structural Biology 2003, 13:538–544
valves and microfluidic devices, alternative fabrication techniques using nontraditional materials, including hydrogels [2], plastics and elastomers [3,4], are gaining popularity and have been developed for the quick and inexpensive fabrication of passive and active microfluidic devices. In particular, the technique of soft lithography [3,4], in which polymer replicas are molded from a micromachined master, has been widely adopted. Soft lithography allows the rapid reproduction of features as small as 80 nm, creating a soft and inexpensive negative of the original master. This technique has found diverse applications, including patterned surface deposition [5,6], photonics [7,8] and microfluidic device fabrication [9]. An extension of soft lithography, multilayer soft lithography (MSL) enables facile and inexpensive large-scale integration of valves on chip [10]. MSL uses consecutive soft lithography molding and bonding steps to generate complex multilayer fluidic devices. In this way, planar channel structures, separated by only a thin flexible elastomeric membrane, may be integrated into a monolithic polymer chip. The orthogonal crossing of channel structures in two adjacent layers creates a deflectable membrane valve. Because of the compliance of the membrane, a hermetic seal may be easily achieved at moderate actuation pressures, even in the presence of particulates or imperfections. The low Young’s modulus of the elastomer (1 MPa versus 100 GPa for crystalline silicon) allows the closing of valves with areas as small as 100 mm2, so that many thousands of active valves may be integrated into an area of only a few square centimeters [11] (Figure 1). Just as a transistor serves as the fundamental component in a modern integrated circuit, a true mechanical valve provides a fluidic switch from which higher order components such as pumps [10], mixers [12] and fluidic logic may be constructed. These building blocks may be further assembled for the large-scale integration of fluidic networks [11]. By applying these large-scale networks in massively parallel and combinatorial structures, a single microliter of reagent may be spread over many independent assays to achieve a true economy of scale.
Fluid physics at the microscale Beyond simply realizing economies of scale and integration, the unique physics of fluid flow and mass transport that are manifested on the micrometer length scale enable novel biological assays that are otherwise impractical or even impossible [13,14]. For example, www.current-opinion.com
Microfluidics in structural biology Hansen and Quake 539
Figure 1
Optical micrograph of an LSI (large-scale integration) microfluidic comparator chip. The chip has 2056 active valves and features an addressable storage array that enables 256 distinct subnanoliter reactions. Binary arrays of valves (pink) create fluidic logic for complex fluidic control by limited control lines. Valve and flow structures have been filled with food dye for visualization purposes [11].
the large surface-to-volume ratio achieved in microfabricated flow structures may be exploited to enhance detector sensitivity by greatly increasing the interaction of a sensing element with a large sample [15,16]. Perhaps the most striking feature of microfluidic devices is the dominant role of viscosity in mass transport phenomena. The Reynolds number (Re) describes the relative importance of inertial forces to viscous forces in a fluid: Re ¼
rUL m
(1)
where r is the fluid density (g/cm3), U is the characteristic velocity (cm/s), L is the characteristic channel dimension (cm) and m is the viscosity of the fluid (g/cms). For most flows in small channels, the Reynolds number is small, which means that the flow is laminar and the familiar turbulent mixing of macroscopic devices vanishes. In practice, a liquid such as water has Re < 1 in channels less than 100 micrometers in diameter for velocities up to 1 cm/s. www.current-opinion.com
In this regime of low Re, two adjacent fluids flowing down a single channel maintain a well-defined interface and mix only by diffusion. By creating networks of these diffusive contacts, this controlled mixing may be used to generate complex and temporally stable gradients [17]; these gradients have proved useful in the study of chemotaxis [18,19]. Diffusion, which is extremely slow on macroscopic length scales, becomes an efficient mechanism of mixing when the characteristic length scales become small. Strategies for accelerating mixing in microfluidic devices rely on reducing the characteristic length of the laminar streams by repetitive folding of the fluid onto itself [12], by induced chaotic advection [20] or by hydrodynamic focusing [21]. By making the transverse length scale of the laminar streams very small, extremely rapid diffusive mixing may be accomplished, thereby enabling time-resolved studies of chemical reactions to be conducted with submillisecond resolution [21]. In one such study [22], the rapid diffusion of a chemical denaturant (urea) from a Current Opinion in Structural Biology 2003, 13:538–544
540 Biophysical methods
tightly focused stream containing a model protein (blactoglobulin) was used to change the chemical environment of the protein on the millisecond timescale. This rapid mixing strategy was used to make time-resolved small-angle X-ray scattering measurements of protein folding intermediates [22]. Viscosity plays another important role in microfluidic reactors by suppressing the onset of buoyancy-driven convection. This property is unique to reactors with micrometer-scale dimensions and is discussed in detail below. It facilitates a robust, efficient and scaleable implementation of crystallization by free interface diffusion (FID). Protein crystal growth has thus emerged as a new application that is ideally suited to the microfluidic environment, benefiting from both the economy of scale and the properties of mass transport on the microscale.
Microfluidics in crystallization Obtaining high-quality crystals of macromolecule targets is an outstanding problem in structural biology. Whereas significant technological advances in synchrotron radiation sources, phasing techniques [23,24] and model building [25] have accelerated structure determination efforts, crystallization remains a challenging bottleneck. As there is currently no way to predict crystallization conditions a priori, the identification of initial conditions often requires an empirical search of thousands of chemical conditions. The large number of experiments required to uncover successful crystallization conditions therefore represents a formidable obstacle to determining the structure of many important biological macromolecules. This problem is exacerbated by the fact that natural crystallography targets such as large complexes and integral membrane proteins are generally available in only milligram quantities. The ability to conduct crystallization trials in nanoliter reaction volumes can facilitate experiments with scarce samples that would otherwise not be feasible. Even in cases in which reasonable amounts of target molecule are available, conducting experiments with ultra-small volumes makes more exhaustive and aggressive crystallization trials possible. Automated crystallization
Automation has been used extensively to scale down the two most commonly used crystallization techniques: microbatch and vapor diffusion [26–28]. State-of-the-art liquid-handling robots can dispense volumes as small as 10 nL and can significantly improve both reproducibility and throughput. However, liquid-handling robots are complex systems requiring substantial dedicated space and are expensive to both purchase and maintain, making them unavailable to most crystallization laboratories. Furthermore, because dispensing robots are sensitive to Current Opinion in Structural Biology 2003, 13:538–544
solution viscosity and surface tension, they must be properly calibrated for each working fluid and have difficulty dispensing small volumes of the highly viscous solutions (such as MPD and PEG) used in crystallography. Having undergone a transition from a labor-intensive ‘art’ to an automated industrial process, protein crystallization is in a state reminiscent of that of computation before the integrated circuit. Although large robotic workstations can, by brute force, increase the chance of crystallization success through the sheer number of screening experiments, they have not necessarily changed or improved the efficiency of the crystallization method itself [29]. In the same way that the integrated circuit has changed computation, alternative and complementary technologies made possible by miniaturization will bring about major advances in crystallography. Free interface diffusion
Crystallization in microfluidic devices offers the opportunity to obtain savings in reagent consumption while exploiting the physics of micrometer-scale mass transport to implement crystallization trials by FID. This type of diffusion has long been recognized as a highly efficient means of detecting crystallization conditions and has distinct advantages over microbatch or vapor diffusion techniques [30]. Despite the favorable kinetics of FID, however, this method has not been widely adopted in the protein crystallography community for several reasons. In conventional FID setups, a liquid–liquid interface is created by carefully introducing microliter volumes of the two solutions into opposite ends of a capillary tube. Establishing a good interface is a delicate and laborintensive process that is not amenable to high-throughput screening trials. Typically, the introduction of the second solution causes transient convection, resulting in a poorly defined interface. The drawbacks of large sample volume, delicate dispensing and poorly defined interface have thus inhibited the use of FID as either a routine or a large-scale automated screening technique. Even when a well-defined fluidic interface can be created, buoyancy-driven convection due to density differences in the solutions causes complex mixing at the interface. To avoid this unwanted mixing, capillaries must be stored with the long axis parallel to gravity and the more dense solution on the bottom. This configuration creates a stable fluidic interface, but often causes nucleated crystals to fall away from the interface and out of the optimal growth conditions. It was therefore proposed that FID would only realize its practical advantages in microgravity environments where gravityinduced convection is eliminated [31,32]. However, the unusual properties of fluid flow in microfluidic devices make it both possible and practical to implement nearly ideal FID conditions in terrestrial devices. The relevant non-dimensional number that governs the onset of convection in a closed fluidic system is the Grashof www.current-opinion.com
Microfluidics in structural biology Hansen and Quake 541
number, Gr, which measures the ratio of buoyant to viscous forces: aDcgL3 (2) v2 where a is the solutal expansivity [defined as the specific change in density (r) as a function of solute concentration (c). a ¼ ðdr=dcÞ/r, where both r and c are mass density with units mg/cm3 so that units of a are cm3/mg], Dc is the difference in solute concentration (mg/cm3), g is the acceleration of gravity (cm/s2), L is the characteristic dimension of the container (cm) and n is the kinematic viscosity (cm2/s). Gr ¼
At low Grashof numbers (i.e. Gr < 1), the interface between two distinct solutions brought into intimate fluidic contact is stable. Inspection of Equation 2 suggests that this condition may be achieved at high solution viscosity [33], in microgravity environments [34] or at small characteristic length scales. The strong dependence of the Grashof number on length implies that, at dimensions that are relevant for microfluidic devices, convection is suppressed and mass transport is dominated by diffusion. Microfluidic crystallization reactors
A highly reproducible and scaleable implementation of microfluidic FID has been demonstrated in elastomeric devices by exploiting the gas permeability of this
material. Figure 2a shows a simple unit cell designed to conduct three FID experiments at different fluid mixing ratios. Three pairs of microfluidic chambers, with welldefined volumes, are in fluid contact via a fluidic interface that is controlled by a microfabricated valve. With this central interface valve closed, the two chambers are first dead-end filled with different solutions (Figure 2b). As fluid enters each chamber, it compresses the gas ahead of it, causing the gas to diffuse quickly into the permeable bulk material. Once both chambers have been completely filled, containment valves are actuated, isolating each reaction from the rest of the chip. Deactivation of the interface valve establishes a well-defined fluidic interface between the chambers, allowing them to mix by diffusion (Figure 2c). Because the amount of each reagent participating in the reaction is precisely defined by the volumes of the microwells, this technique allows control over the end point of each reaction. The parallel implementation of 48 unit cells in a single crystallization chip (Figure 2d) facilitates 144 simultaneous FID protein crystallization reactions while consuming only 3.0 mL of protein solution — only 20 nL per assay. The counterdiffusion of two solution reservoirs through a constricted channel is distinct from conventional FID in capillaries. FID in capillaries of constant cross-section results in a complicated spatiotemporal evolution of the concentration profiles. The small cross-section of the
Figure 2
(a)
(b)
1 mm (c)
1 mm (d)
1 mm Current Opinion in Structural Biology
Microfluidic crystallization chips. (a) Microfluidic unit cell designed to implement three FID assays at three mixing ratios. Microwells with volumes of 5 nL, 12.5 nL and 20 nL are coupled to create final mixing ratios of 1:4, 1:1 and 4:1 with a fixed total volume of 25 nL. (b) With the central interface valve closed, solutions are dead-end loaded into the chambers, forcing the ambient air to diffuse into the bulk elastomer. (c) Containment valves (top and bottom) isolate the unit cell and the interface valve is opened to allow diffusive mixing between the coupled chambers. (d) A microfluidic chip with 48 of the unit cell structures shown in (a). The chip has 432 active valves, controlled by only two control lines, and performs 144 simultaneous FID crystallization experiments using only 3 mL of protein sample. www.current-opinion.com
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connecting channel (10 mm 100 mm), compared with that of the chambers (300 mm 100 mm), acts as a highimpedance connection that slows the equilibration process and localizes the concentration gradient to the length of the channel [35]. The lack of an appreciable gradient within the chambers implies that, for a given diffusion constant and mixing ratio, the rate of equilibration depends only on two characteristic lengths: the length of the connecting channel and the ratio of the well volume to the channel cross-sectional area [36]. Modifying the channel geometry therefore enables intuitive and accurate control over the kinetics of equilibration without changing the chemical evolution of the reaction. The decoupling of the kinetics and thermodynamics of diffusive equilibration has important implications for crystal
optimization, where it is often desirable to approach crystallization conditions slowly while conserving the successful thermodynamic variables. Large-scale crystallization studies
The crystallization chip may be used to conduct a large number of nanoliter-scale assays without compromising success rates. In a recent study comparing the effectiveness of chip-based FID experiments with conventional microbatch and vapor diffusion techniques over a broad range of standard and novel crystallization targets [37], chip-based crystallization experiments were found to produce more hits. This technique has proved to be effective in crystallizing large complexes (Figure 3a), membrane proteins (Figure 3b) and macromolecule
Figure 3
Optical micrograph of macromolecule crystals grown by chip-based FID. (a) Crystal of a bacterial 70S ribosome (C Hansen, A Vila-Sanjurjo, J Cate, unpublished data). (b) Crystals of the integral membrane protein aquaporin (C Hansen, R Stroud, K Self, unpublished data). (c) Crystal of the catalytic core domain of bacterial primase. Crystals were detected in 11 conditions of the commercial sparse matrix screens Crystal Screen 1 (Hampton Research http://www.hamptonresearch.com) and Emerald Biostructures Wizard I (deCODE http://www.decode.com/emeraldbiostructures); vapor diffusion and microbatch techniques produced only precipitation using the same screens. (d) Crystals of the type II topoisomerase ATPase domain bound to ADP grown in less than 12 hours using chip-based FID; crystallization by microbatch required incubation for more than a week. (e) Cluster of crystals of the hydroxylase domain of the cytochrome P450 BM-3 mutant 139-3 [38] grown in 10 nL microwells by FID. (f) Large single crystal of the hydroxylase domain of the cytochrome P450 BM-3 mutant 139-3 grown in 300 nL microwells by FID. This crystal was grown under conditions identical to those in (e), but with slower equilibration due to the larger well volume. Scale bars, 100 mm. Current Opinion in Structural Biology 2003, 13:538–544
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targets that have been unsuccessfully screened using conventional methods (Figure 3c). Furthermore, crystal growth in chips was generally found to be faster than that achieved by traditional means, with hits occurring between 3 hours and 7 days of incubation. Figure 3d shows large single crystals of a type II topoisomerase ATPase domain grown in less than 12 hours on a chip; in microbatch experiments, crystals appeared after 1 week of incubation. Ultimately, the success of any crystallization method depends on the ability to get high-quality crystals into the X-ray beam. It has been shown that large single crystals can be grown and harvested directly from chip reactions and used for high-resolution diffraction studies [37]. In cases where large crystals cannot be obtained from small-volume experiments, it is necessary to export successful conditions to larger volume formats. Because the kinetics of chip-based FID is very different from that of vapor diffusion and microbatch, it is not generally the case that hits may be directly exported to these macroscopic methods. Once chemical conditions conducive to crystallization have been identified, however, chip-based hits may be exported to traditional (microbatch and vapor diffusion) formats by subsequent refinement around the successful chip conditions. Alternatively, the thermodynamics of FID may be preserved in a growth chip with larger volume wells to provide a high-correspondence method of transporting initial hits from the chip (Figure 3e) to larger volumes (Figure 3f).
Conclusions Breakthroughs in microfluidic technology are rapidly bringing the long-awaited potential of microfluidics to fruition. The recent acceleration of progress in this field has allowed levels of complexity and functionality to be routinely achieved on chip that only a short time ago seemed unattainable. Judicious application of these tools to important biological problems that can benefit both from the economy of scale and from the physical properties that are manifested on the micrometer length scale will ensure that the full impact of these advances is felt in the near future. As discussed above, one problem — protein crystallization — has been identified as an ideal application of this technology. A simple microfluidic device has proved to be highly effective at detecting crystallization conditions by exploiting the properties of mass transport that are characteristic of small length scales. More sophisticated control and flow architectures will facilitate the development of tools with increased functionality and applicability to studies both in and beyond protein crystallography. It is possible to envisage that, in the not too distant future, microfluidic devices will enable many new innovative tools in structural biology for the optimization of growth conditions, on-chip protein purification and solubility www.current-opinion.com
analysis, and the integration of in vitro protein synthesis with the crystal growth process.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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10. Unger M, Chou H, Thorsen T, Scherer A, Quake S: Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 2000, 288:113-116. 11. Thorsen T, Maerkl SJ, Quake SR: Microfluidic large-scale integration. Science 2002, 298:580-584. Using MSL, the authors demonstrate the first large-scale integration of fluidic networks on a chip. Extending an analogy with microelectronic circuits, addressable fluidic networks with thousands of microfabricated valves are used for parallel cell screening assays and the implementation of a microfluidic memory array. 12. Chu H, Unger MA, Quake SR: A microfabricated rotary pump. Biomedical Microdevices 2001, 3:323-330. 13. Manz A, Eijkel JCT: Miniaturization and chip technology. What can we expect? Pure Appl Chem 2001, 73:1555-1561. 14. Beebe D, Mensing G, Walker G: Physics and applications of microfluidics in biology. Annu Rev Biomed Eng 2002, 4:261-286. This review provides a good introduction to the essential properties of flow and mass transport that are manifested in microfluidic devices. 15. Lavigne JJ, Savoy S, Clevenger MB, Ritchie JE, McDoniel B, Yoo SJ, Anslyn EV, McDevitt JT, Shear JB, Neikirk D: Solutionbased analysis of multiple analytes by a sensor array: toward the development of an ‘‘electronic tongue’’. J Am Chem Soc 1998, 120:6429-6430. 16. Hofmann O, Voirin G, Niedermann P, Manz A: Three-dimensional microfluidic confinement for efficient sample delivery to biosensor surfaces. Application to immunoassays on planar optical waveguides. Anal Chem 2002, 74:5243-5250. 17. Dertinger SKW, Chiu DT, Jeon NL, Whitesides GM: Generation of gradients having complex shapes using microfluidic networks. Anal Chem 2001, 73:1240-1246. Current Opinion in Structural Biology 2003, 13:538–544
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18. Jeon NL, Baskaran H, Dertinger SKW, Whitesides GM, Van de Water L, Toner M: Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device. Nat Biotechnol 2002, 20:826-830. Laminar flow and controlled diffusion are used to produce controlled and temporally stable chemical gradients in a microfluidic channel. These gradients are used to study the chemotaxis of neutrophils in the presence of linear and nonlinear chemical gradients. 19. Mao HB, Cremer PS, Manson MD: A sensitive, versatile microfluidic assay for bacterial chemotaxis. Proc Natl Acad Sci USA 2003, 100:5449-5454.
29. Chayen EN: Tackling the bottleneck of protein crystallization in the post- genomic era. Trends Biotechnol 2002, 20:98-98. 30. Salemme FR: A free interface diffusion technique for the crystallization of proteins for X-ray crystallography. Arch Biochem Biophys 1972, 15:533-539. 31. McPherson A: Crystallization of Biological Macromolecules, edn 1. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1999.
20. Stroock A, Dertinger S, Ajdari A, Mezic I, Stone H, Whitesides G: Chaotic mixer for microchannels. Science 2002, 295:647-651.
32. Wang YP, Han Y, Pan JS, Wang KY, Bi RC: Protein crystal growth in microgravity using a liquid/liquid diffusion method. Microgravity Sci Tec 1996, 9:281-283.
21. Knight JB, Vishwanath A, Brody JP, Austin RH: Hydrodynamic focusing on a silicon chip: mixing nanoliters in microseconds. Phys Rev Lett 1998, 80:3863-3866.
33. Garcia-Ruiz JM, Novella ML, Moreno R, Gavira JA: Agarose as crystallization media for proteins. I: Transport processes. J Cryst Growth 2001, 232:165-172.
22. Pollack L, Tate MW, Finnefrock AC, Kalidas C, Trotter S, Darnton NC, Lurio L, Austin RH, Batt CA, Gruner SM et al.: Time resolved collapse of a folding protein observed with small angle x-ray scattering. Phys Rev Lett 2001, 86:4962-4965. Hydrodynamic focusing by laminar flow allows ultrafast mixing by reducing the required diffusion length to the micrometer scale. The authors combine this technique with small-angle X-ray scattering to make timeresolved measurements of protein folding intermediates.
34. Delucas L, Smith CD, Smith H, Vijaykumar S, Senadhi S, Ealick S, Carter D, Snyder R, Weber P, Salemme F et al.: Protein crystal growth in microgravity. Science 1989, 246:651-654.
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35. Hansen C, Sommer M, Self K, Berger J, Quake S: Micro-crystallization. In Protein Crystallography in Drug Discovery. Edited by Babine R, Abdel-Meguid: Wiley-VCH; 2003. 36. Andrew SPS: A simple method of measuring gaseous diffusion coefficients. Chem Eng Sci 1955, 4:269-272.
27. Stevens RC, Wilson IA: Industrializing structural biology. Science 2001, 293:519-520.
37. Hansen CL, Skordalakes E, Berger JM, Quake SR: A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc Natl Acad Sci USA 2002, 99:16531-16536. A simple and robust method for the parallel metering of solutions in microfluidic devices is described. This technique enables the simultaneous implementation of hundreds of FID crystallization assays using only 20 nL of sample per assay. The small characteristic length scale of these reactions allows the suppression of gravity-induced convection, so that a stable interface is easily maintained. The unique kinetics of purely diffusive mixing on the micrometer scale results in the detection of more crystallization conditions and faster crystal growth.
28. Goodwill KE, Tennant MG, Stevens RC: High-throughput x-ray crystallography for structure-based drug design. Drug Discov Today 2001, 6:S113-S118.
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Current Opinion in Structural Biology 2003, 13:538–544
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Microfluidics in structural biology: smaller, faster. . . better Carl Hansen and Stephen R Quake Microfluidic technologies promise unprecedented savings in cost and time through the integration of complex chemical and biological assays on a microfabricated chip. Recent advances are making elements of this vision a reality, facilitating the first large-scale integration of microfluidic plumbing with biological assays. The power of miniaturization lies not only in achieving an economy of scale, but also in exploiting the unusual physics of fluid flow and mass transport on small length scales to realize precise and efficient assays that are not accessible with macroscopic tools. Diverse applications ranging from time-resolved studies of protein folding to highly efficient protein crystal growth suggest that microfluidics may become an indispensable tool in biology. Addresses Department of Applied Physics, California Institute of Technology, MS 128-95, Pasadena, CA 91125, USA e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:538–544 This review comes from a themed issue on Biophysical methods Edited by Brian T Chait and Keith Moffat 0959-440X/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2003.09.010
Abbreviations FID free interface diffusion MSL multilayer soft lithography
Introduction In the same way that integrated circuits have revolutionized computation, microfluidics seeks to integrate high levels of wet laboratory functionality on a microfabricated fluidic chip. By processing samples on the nanoliter scale, microfluidic technologies have the potential to realize increased speed and sensitivity, while at the same time achieving an unprecedented economy of scale, dramatically increasing throughput and reducing cost. Over the past decade, there has been a tremendous effort to devise methods of fabricating miniaturized plumbing that exploit this analogy with the semiconductor industry on many levels [1]. In this perspective, we will provide a brief introduction to microfluidics, describe some of the basic physics of fluids in small dimensions and review recent applications of this technology to structural biology.
Fabrication of microfluidic devices Although there is a thriving MEMS (micro-electromechanical systems) community making silicon-based Current Opinion in Structural Biology 2003, 13:538–544
valves and microfluidic devices, alternative fabrication techniques using nontraditional materials, including hydrogels [2], plastics and elastomers [3,4], are gaining popularity and have been developed for the quick and inexpensive fabrication of passive and active microfluidic devices. In particular, the technique of soft lithography [3,4], in which polymer replicas are molded from a micromachined master, has been widely adopted. Soft lithography allows the rapid reproduction of features as small as 80 nm, creating a soft and inexpensive negative of the original master. This technique has found diverse applications, including patterned surface deposition [5,6], photonics [7,8] and microfluidic device fabrication [9]. An extension of soft lithography, multilayer soft lithography (MSL) enables facile and inexpensive large-scale integration of valves on chip [10]. MSL uses consecutive soft lithography molding and bonding steps to generate complex multilayer fluidic devices. In this way, planar channel structures, separated by only a thin flexible elastomeric membrane, may be integrated into a monolithic polymer chip. The orthogonal crossing of channel structures in two adjacent layers creates a deflectable membrane valve. Because of the compliance of the membrane, a hermetic seal may be easily achieved at moderate actuation pressures, even in the presence of particulates or imperfections. The low Young’s modulus of the elastomer (1 MPa versus 100 GPa for crystalline silicon) allows the closing of valves with areas as small as 100 mm2, so that many thousands of active valves may be integrated into an area of only a few square centimeters [11] (Figure 1). Just as a transistor serves as the fundamental component in a modern integrated circuit, a true mechanical valve provides a fluidic switch from which higher order components such as pumps [10], mixers [12] and fluidic logic may be constructed. These building blocks may be further assembled for the large-scale integration of fluidic networks [11]. By applying these large-scale networks in massively parallel and combinatorial structures, a single microliter of reagent may be spread over many independent assays to achieve a true economy of scale.
Fluid physics at the microscale Beyond simply realizing economies of scale and integration, the unique physics of fluid flow and mass transport that are manifested on the micrometer length scale enable novel biological assays that are otherwise impractical or even impossible [13,14]. For example, www.current-opinion.com
Microfluidics in structural biology Hansen and Quake 539
Figure 1
Optical micrograph of an LSI (large-scale integration) microfluidic comparator chip. The chip has 2056 active valves and features an addressable storage array that enables 256 distinct subnanoliter reactions. Binary arrays of valves (pink) create fluidic logic for complex fluidic control by limited control lines. Valve and flow structures have been filled with food dye for visualization purposes [11].
the large surface-to-volume ratio achieved in microfabricated flow structures may be exploited to enhance detector sensitivity by greatly increasing the interaction of a sensing element with a large sample [15,16]. Perhaps the most striking feature of microfluidic devices is the dominant role of viscosity in mass transport phenomena. The Reynolds number (Re) describes the relative importance of inertial forces to viscous forces in a fluid: Re ¼
rUL m
(1)
where r is the fluid density (g/cm3), U is the characteristic velocity (cm/s), L is the characteristic channel dimension (cm) and m is the viscosity of the fluid (g/cms). For most flows in small channels, the Reynolds number is small, which means that the flow is laminar and the familiar turbulent mixing of macroscopic devices vanishes. In practice, a liquid such as water has Re < 1 in channels less than 100 micrometers in diameter for velocities up to 1 cm/s. www.current-opinion.com
In this regime of low Re, two adjacent fluids flowing down a single channel maintain a well-defined interface and mix only by diffusion. By creating networks of these diffusive contacts, this controlled mixing may be used to generate complex and temporally stable gradients [17]; these gradients have proved useful in the study of chemotaxis [18,19]. Diffusion, which is extremely slow on macroscopic length scales, becomes an efficient mechanism of mixing when the characteristic length scales become small. Strategies for accelerating mixing in microfluidic devices rely on reducing the characteristic length of the laminar streams by repetitive folding of the fluid onto itself [12], by induced chaotic advection [20] or by hydrodynamic focusing [21]. By making the transverse length scale of the laminar streams very small, extremely rapid diffusive mixing may be accomplished, thereby enabling time-resolved studies of chemical reactions to be conducted with submillisecond resolution [21]. In one such study [22], the rapid diffusion of a chemical denaturant (urea) from a Current Opinion in Structural Biology 2003, 13:538–544
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tightly focused stream containing a model protein (blactoglobulin) was used to change the chemical environment of the protein on the millisecond timescale. This rapid mixing strategy was used to make time-resolved small-angle X-ray scattering measurements of protein folding intermediates [22]. Viscosity plays another important role in microfluidic reactors by suppressing the onset of buoyancy-driven convection. This property is unique to reactors with micrometer-scale dimensions and is discussed in detail below. It facilitates a robust, efficient and scaleable implementation of crystallization by free interface diffusion (FID). Protein crystal growth has thus emerged as a new application that is ideally suited to the microfluidic environment, benefiting from both the economy of scale and the properties of mass transport on the microscale.
Microfluidics in crystallization Obtaining high-quality crystals of macromolecule targets is an outstanding problem in structural biology. Whereas significant technological advances in synchrotron radiation sources, phasing techniques [23,24] and model building [25] have accelerated structure determination efforts, crystallization remains a challenging bottleneck. As there is currently no way to predict crystallization conditions a priori, the identification of initial conditions often requires an empirical search of thousands of chemical conditions. The large number of experiments required to uncover successful crystallization conditions therefore represents a formidable obstacle to determining the structure of many important biological macromolecules. This problem is exacerbated by the fact that natural crystallography targets such as large complexes and integral membrane proteins are generally available in only milligram quantities. The ability to conduct crystallization trials in nanoliter reaction volumes can facilitate experiments with scarce samples that would otherwise not be feasible. Even in cases in which reasonable amounts of target molecule are available, conducting experiments with ultra-small volumes makes more exhaustive and aggressive crystallization trials possible. Automated crystallization
Automation has been used extensively to scale down the two most commonly used crystallization techniques: microbatch and vapor diffusion [26–28]. State-of-the-art liquid-handling robots can dispense volumes as small as 10 nL and can significantly improve both reproducibility and throughput. However, liquid-handling robots are complex systems requiring substantial dedicated space and are expensive to both purchase and maintain, making them unavailable to most crystallization laboratories. Furthermore, because dispensing robots are sensitive to Current Opinion in Structural Biology 2003, 13:538–544
solution viscosity and surface tension, they must be properly calibrated for each working fluid and have difficulty dispensing small volumes of the highly viscous solutions (such as MPD and PEG) used in crystallography. Having undergone a transition from a labor-intensive ‘art’ to an automated industrial process, protein crystallization is in a state reminiscent of that of computation before the integrated circuit. Although large robotic workstations can, by brute force, increase the chance of crystallization success through the sheer number of screening experiments, they have not necessarily changed or improved the efficiency of the crystallization method itself [29]. In the same way that the integrated circuit has changed computation, alternative and complementary technologies made possible by miniaturization will bring about major advances in crystallography. Free interface diffusion
Crystallization in microfluidic devices offers the opportunity to obtain savings in reagent consumption while exploiting the physics of micrometer-scale mass transport to implement crystallization trials by FID. This type of diffusion has long been recognized as a highly efficient means of detecting crystallization conditions and has distinct advantages over microbatch or vapor diffusion techniques [30]. Despite the favorable kinetics of FID, however, this method has not been widely adopted in the protein crystallography community for several reasons. In conventional FID setups, a liquid–liquid interface is created by carefully introducing microliter volumes of the two solutions into opposite ends of a capillary tube. Establishing a good interface is a delicate and laborintensive process that is not amenable to high-throughput screening trials. Typically, the introduction of the second solution causes transient convection, resulting in a poorly defined interface. The drawbacks of large sample volume, delicate dispensing and poorly defined interface have thus inhibited the use of FID as either a routine or a large-scale automated screening technique. Even when a well-defined fluidic interface can be created, buoyancy-driven convection due to density differences in the solutions causes complex mixing at the interface. To avoid this unwanted mixing, capillaries must be stored with the long axis parallel to gravity and the more dense solution on the bottom. This configuration creates a stable fluidic interface, but often causes nucleated crystals to fall away from the interface and out of the optimal growth conditions. It was therefore proposed that FID would only realize its practical advantages in microgravity environments where gravityinduced convection is eliminated [31,32]. However, the unusual properties of fluid flow in microfluidic devices make it both possible and practical to implement nearly ideal FID conditions in terrestrial devices. The relevant non-dimensional number that governs the onset of convection in a closed fluidic system is the Grashof www.current-opinion.com
Microfluidics in structural biology Hansen and Quake 541
number, Gr, which measures the ratio of buoyant to viscous forces: aDcgL3 (2) v2 where a is the solutal expansivity [defined as the specific change in density (r) as a function of solute concentration (c). a ¼ ðdr=dcÞ/r, where both r and c are mass density with units mg/cm3 so that units of a are cm3/mg], Dc is the difference in solute concentration (mg/cm3), g is the acceleration of gravity (cm/s2), L is the characteristic dimension of the container (cm) and n is the kinematic viscosity (cm2/s). Gr ¼
At low Grashof numbers (i.e. Gr < 1), the interface between two distinct solutions brought into intimate fluidic contact is stable. Inspection of Equation 2 suggests that this condition may be achieved at high solution viscosity [33], in microgravity environments [34] or at small characteristic length scales. The strong dependence of the Grashof number on length implies that, at dimensions that are relevant for microfluidic devices, convection is suppressed and mass transport is dominated by diffusion. Microfluidic crystallization reactors
A highly reproducible and scaleable implementation of microfluidic FID has been demonstrated in elastomeric devices by exploiting the gas permeability of this
material. Figure 2a shows a simple unit cell designed to conduct three FID experiments at different fluid mixing ratios. Three pairs of microfluidic chambers, with welldefined volumes, are in fluid contact via a fluidic interface that is controlled by a microfabricated valve. With this central interface valve closed, the two chambers are first dead-end filled with different solutions (Figure 2b). As fluid enters each chamber, it compresses the gas ahead of it, causing the gas to diffuse quickly into the permeable bulk material. Once both chambers have been completely filled, containment valves are actuated, isolating each reaction from the rest of the chip. Deactivation of the interface valve establishes a well-defined fluidic interface between the chambers, allowing them to mix by diffusion (Figure 2c). Because the amount of each reagent participating in the reaction is precisely defined by the volumes of the microwells, this technique allows control over the end point of each reaction. The parallel implementation of 48 unit cells in a single crystallization chip (Figure 2d) facilitates 144 simultaneous FID protein crystallization reactions while consuming only 3.0 mL of protein solution — only 20 nL per assay. The counterdiffusion of two solution reservoirs through a constricted channel is distinct from conventional FID in capillaries. FID in capillaries of constant cross-section results in a complicated spatiotemporal evolution of the concentration profiles. The small cross-section of the
Figure 2
(a)
(b)
1 mm (c)
1 mm (d)
1 mm Current Opinion in Structural Biology
Microfluidic crystallization chips. (a) Microfluidic unit cell designed to implement three FID assays at three mixing ratios. Microwells with volumes of 5 nL, 12.5 nL and 20 nL are coupled to create final mixing ratios of 1:4, 1:1 and 4:1 with a fixed total volume of 25 nL. (b) With the central interface valve closed, solutions are dead-end loaded into the chambers, forcing the ambient air to diffuse into the bulk elastomer. (c) Containment valves (top and bottom) isolate the unit cell and the interface valve is opened to allow diffusive mixing between the coupled chambers. (d) A microfluidic chip with 48 of the unit cell structures shown in (a). The chip has 432 active valves, controlled by only two control lines, and performs 144 simultaneous FID crystallization experiments using only 3 mL of protein sample. www.current-opinion.com
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connecting channel (10 mm 100 mm), compared with that of the chambers (300 mm 100 mm), acts as a highimpedance connection that slows the equilibration process and localizes the concentration gradient to the length of the channel [35]. The lack of an appreciable gradient within the chambers implies that, for a given diffusion constant and mixing ratio, the rate of equilibration depends only on two characteristic lengths: the length of the connecting channel and the ratio of the well volume to the channel cross-sectional area [36]. Modifying the channel geometry therefore enables intuitive and accurate control over the kinetics of equilibration without changing the chemical evolution of the reaction. The decoupling of the kinetics and thermodynamics of diffusive equilibration has important implications for crystal
optimization, where it is often desirable to approach crystallization conditions slowly while conserving the successful thermodynamic variables. Large-scale crystallization studies
The crystallization chip may be used to conduct a large number of nanoliter-scale assays without compromising success rates. In a recent study comparing the effectiveness of chip-based FID experiments with conventional microbatch and vapor diffusion techniques over a broad range of standard and novel crystallization targets [37], chip-based crystallization experiments were found to produce more hits. This technique has proved to be effective in crystallizing large complexes (Figure 3a), membrane proteins (Figure 3b) and macromolecule
Figure 3
Optical micrograph of macromolecule crystals grown by chip-based FID. (a) Crystal of a bacterial 70S ribosome (C Hansen, A Vila-Sanjurjo, J Cate, unpublished data). (b) Crystals of the integral membrane protein aquaporin (C Hansen, R Stroud, K Self, unpublished data). (c) Crystal of the catalytic core domain of bacterial primase. Crystals were detected in 11 conditions of the commercial sparse matrix screens Crystal Screen 1 (Hampton Research http://www.hamptonresearch.com) and Emerald Biostructures Wizard I (deCODE http://www.decode.com/emeraldbiostructures); vapor diffusion and microbatch techniques produced only precipitation using the same screens. (d) Crystals of the type II topoisomerase ATPase domain bound to ADP grown in less than 12 hours using chip-based FID; crystallization by microbatch required incubation for more than a week. (e) Cluster of crystals of the hydroxylase domain of the cytochrome P450 BM-3 mutant 139-3 [38] grown in 10 nL microwells by FID. (f) Large single crystal of the hydroxylase domain of the cytochrome P450 BM-3 mutant 139-3 grown in 300 nL microwells by FID. This crystal was grown under conditions identical to those in (e), but with slower equilibration due to the larger well volume. Scale bars, 100 mm. Current Opinion in Structural Biology 2003, 13:538–544
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targets that have been unsuccessfully screened using conventional methods (Figure 3c). Furthermore, crystal growth in chips was generally found to be faster than that achieved by traditional means, with hits occurring between 3 hours and 7 days of incubation. Figure 3d shows large single crystals of a type II topoisomerase ATPase domain grown in less than 12 hours on a chip; in microbatch experiments, crystals appeared after 1 week of incubation. Ultimately, the success of any crystallization method depends on the ability to get high-quality crystals into the X-ray beam. It has been shown that large single crystals can be grown and harvested directly from chip reactions and used for high-resolution diffraction studies [37]. In cases where large crystals cannot be obtained from small-volume experiments, it is necessary to export successful conditions to larger volume formats. Because the kinetics of chip-based FID is very different from that of vapor diffusion and microbatch, it is not generally the case that hits may be directly exported to these macroscopic methods. Once chemical conditions conducive to crystallization have been identified, however, chip-based hits may be exported to traditional (microbatch and vapor diffusion) formats by subsequent refinement around the successful chip conditions. Alternatively, the thermodynamics of FID may be preserved in a growth chip with larger volume wells to provide a high-correspondence method of transporting initial hits from the chip (Figure 3e) to larger volumes (Figure 3f).
Conclusions Breakthroughs in microfluidic technology are rapidly bringing the long-awaited potential of microfluidics to fruition. The recent acceleration of progress in this field has allowed levels of complexity and functionality to be routinely achieved on chip that only a short time ago seemed unattainable. Judicious application of these tools to important biological problems that can benefit both from the economy of scale and from the physical properties that are manifested on the micrometer length scale will ensure that the full impact of these advances is felt in the near future. As discussed above, one problem — protein crystallization — has been identified as an ideal application of this technology. A simple microfluidic device has proved to be highly effective at detecting crystallization conditions by exploiting the properties of mass transport that are characteristic of small length scales. More sophisticated control and flow architectures will facilitate the development of tools with increased functionality and applicability to studies both in and beyond protein crystallography. It is possible to envisage that, in the not too distant future, microfluidic devices will enable many new innovative tools in structural biology for the optimization of growth conditions, on-chip protein purification and solubility www.current-opinion.com
analysis, and the integration of in vitro protein synthesis with the crystal growth process.
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