PROTEIN CRYSTALLIZATION USING MICROFLUIDIC TECHNOLOGIES Submitted by

Sandeep Kannan PID A53010454 MAE 207

PROTEIN CRYSTALLIZATION USING MICROFLUIDIC TECHNOLOGIES

1. Introduction: Proteins are essential part of biological environment, which takes part in most of the cellular activities. Protein crystallization techniques are used for separation and purification of various biological products, drug design etc. It is very important to study the structure of proteins for designing drugs, identifying malfunctioning of proteins, diseases etc. X-ray diffraction technique is the most common method used to study the structure of proteins, which requires pure protein crystals. Protein crystals are very fragile and so its crystallization is a challenging process. Most proteins can be crystallized when the solutions in which they are dissolved gets saturated in appropriate conditions. The goal of protein crystallization is to obtain high quality protein crystals without impurities, large enough to diffract X-ray beam in studies like X-ray diffraction crystallography. Traditionally proteins are crystallized in macroscale, which had lot of disadvantages, like poor quality of crystal composition, concentration may not be uniform etc. Conventionally there are mainly four methods for protein crystallization; microbatch process, vapor diffusion, free interface diffusion (FID) and dialysis. All of these methods can be implemented with microfluidic approaches, with the use of specially designed microfluidic chips. There are basically three microfluidic approaches for protein crystallization, namely valve based system, droplet based system and system based on slip-chip approach. The advantages of using microfluidic technology are the miniaturization of the experimental set up, which are already present on a larger scale. Secondly, It is also possible to perform experiments on rare and expensive proteins on small or trial volumes. Since it is on micro scale, precise control of molecular diffusion and nucleation of protein crystals are possible. 2. Microfluidic approaches: The conventional protein crystallization methods can be used with ultra-low volume and corresponding modifications in devices for microfluidic protein crystallization. The three types of microfluidic approaches [1], are valve-based system, droplet-based system and system based on slip chip. All these systems use nanoliter volumes for crystallization process. 2.1 Valve-based system: In this system the protein sample, precipitants and buffer are added to micro-chambers and allowed to mix by flowing into mixing chamber by the operation of some pneumatic valves. Here loading precipitants and proteins, and mixing them are all initiated by separately operating respective pneumatic valves. Inside the mixing chamber crystallization is initiated as a result of diffusion.

2.2 Slipchip based system: Slipchip based system uses relatively movable plates, which contains nanoliter volume wells for accommodating proteins and precipitants. The solutions are loaded into the nanowells. When the plate is moved relative to the other, wells filled with protein gets isolated from the loading channel and come in contact with the wells filled with reagents, and hence crystallization occurs. The volume of precipitant and protein depends on the volume of the wells, hence there is no need of a metering system. 2.3 Droplet based system: In this type of approach, crystallization takes place inside a droplet. The droplet is introduced into an immiscible carrier fluid under motion. Carrier fluid is usually an inert oil. The main advantage is that, hundreds of fluid droplets of varying composition can be introduced by changing the flow rates of the streams of aqueous reagent, protein, and buffer, by flowing these fluids into a fluorinated carrier fluid in single experimental set up. 3. Valve based free interface diffusion (FID): At the beginning, there were only valve-less electro-kinetic and pressure-driven systems. These systems werent vigorous and scalable and were highly dependent on fluid properties. Mechanical valves served better, but it was extremely difficult to fabricate in such a small scale. To overcome these problems, valve-nanbased system is introduced, in which pneumatic valves are used. Valve based FID [2] is basically a straight forward method in which a protein and a precipitant are placed into contact along a microfluidic free interface and allowed to mix by diffusion without convective flow across the interface. Since the protein and the precipitants are in direct contact, true interface diffusion occurs and the adverse effects of convection and turbulence are avoided. Free interface diffusion is also called as liquidliquid diffusion. A typical schematic of a valve based FID is shown in the figure 1.

Figure 1 [6]: Schematic of a valve based FID chip, macromolecule (protein) will be loaded at one end and precipitant at the other end.

Microfluidic devices in this system are generally made of silicone elastomer using soft lithography process and then plumbing components are integrated into it. In this system, after

proper priming of the micro-chambers, aqueous reagents are loaded into different chambers. At the start of the process all the valves of the fluidic circuit are open and protein and the crystallizing agents are introduced into the chamber. The microchannels are then closed, and those connecting top and bottom chambers are opened with the help of pneumatic valves integrated in the chip. The crystallizing agent diffuses in the biomolecule chamber and triggers crystallization. Each module is divided into three pairs of chambers with volumes of 520 nl. This helps in obtaining different concentration of protein crystals. Hansen et al developed a vigorous and scalable valve based FID crystallization method called Barrier interface method (BIM). BIM is independent of fluid properties and has a great accuracy up to picoliter scale. The sample wastes are negligible in this method. BIM process consists of mixing of various salts, precipitants, buffers and proteins, allowing it for pure diffusion for crystallization. 3.1 Device fabrication: The device for this crystallization is made of multilayer silicon elastomer, sealed on a glass substrate, forming a glass-elastomer hybrid. The controls and channels for the flow is fabricated using multilayer soft-lithography. High-resolution transparency masks are used for better monitoring of the crystallization process. The mask for the control structure is made 1.5% larger than the required size, in order to accommodate the size shrink occurring during curing. These devices are cured usually at 80C. the liquid silicone elastomer is poured in the mold of the control and flow structures and after curing for one hour, these structures are aligned according to the design and micro-wells are introduced by punching with luer stub. It is hard baked at 125C for acquiring good mechanical strength. The glass substrate is reusable and elastomer chips are disposed after single use. 3.2 Loading and crystallization: As a first step, the crystallization chambers are primed by pressurized outgas. Here a liquid is injected into the channel with pressure, which forces the gas inside the chamber to expel out. The chambers are isolated from the rest of the area by three micro-fabricated pneumatic valves. The barrier valve placed between the two sets of chambers is closed and is primed with two different fluids. The two containment valves which isolates the chambers from rest of the chip is opened and two fluids, the protein to be crystallized and the precipitant and buffer solution is filled into the corresponding chambers (refer figure 1). The two containment valves are opened to isolate the chambers from the rest of the chip. The barrier valve is opened, the two fluids mixes by pure diffusion. For successful protein crystallization, the concentrated solution of protein molecules must be supersaturated, where crystal phase is energetically favorable. So when this state is maintained, nucleation and growth occurs. Supersaturation can be induced through solution pH, salt concentration,

Figure 2. [2] a) Part of a chip showing three pairs of compound reaction chambers. Containment valves are at the top and bottom and barrier valve at the middle. A 15m elastomer membrane is used to control the flow at the valves. b) Reagents are loaded using pressurized outgas priming method. Lower wells are loaded with water and upper wells are loaded with 13 mm bromophenol blue sodium salt (Orange G). c) A gradient in dye concentration can be noticed during diffusion. Containment valves at the top and bottom isolates the wells from rest of the chip during incubation. Barrier valve at the middle will be left opened during incubation. The image shows complete mixing after two hours.

mixing ratios. The micro-wells are of volume 5, 12.5 and 20 nl approximately. A labile region of a two-dimensional phase diagram supports rapid nucleation giving large number of low quality small nucleus. In the metastable region, these nucleuses grow forming high quality large crystals. The final protein concentration is determined by the mixing ratios. Proteins usually crystallized with this technique are lysozyme, glucose isomerase, bovine trypsin, beef-liver catalase, ATPase, mycobacterial RNase etc. 3.3 The main advantages of microfluidic free interface diffusion are: a) Ultra low volume, microfluidic FID uses very low volume of protein to crystalize, generally of the order of micro-liter to nanoliter, best suited for trial experiments. b) Rapid result, crystals form in few days once the experiment is set up, compared to other methods which takes several days or even weeks to obtain results. c) The elastomer used to make the chip is generally translucent, which makes it common to obtain the images of crystallization process. d) Since FID is a pure diffusive process, the mixing will be gradual and there wont by any steep concentration gradients as in convective mixing. 4. Slipchip: Slipchip [3] method basically consists of two glass plates, which can be moved relative to each other. The bottom plates have an array of wells, which will be loaded with the reagents of crystallization and the top plate with the protein. There are no valves or pumps used in this system. The top plate will act as a lid for the wells of the bottom plate. Also the top plate is designed to have a complementary pattern of the bottom plate. Wells of the bottom plates have channels connected for loading the reagents. 4.1 Fabrication: Material used for fabrication is soda-lime glass plate having chromium and photo-resist coating. Conventional photolithography technique is used to cut channels and wells on the

glass substrate. Wells are generally of the order of 300m*300m, and has a depth approximately 60m. After cleaning the etched glass plates are exposed for oxygen plasma treatment and it is made hydrophobic by silanizing in a vacuum desiccator. Ethanol and nitrogen gas are used to clean and dry the chip. 4.2 Loading and crystallization: Firstly, the reagents for crystallization are introduced and this process is called pre-loading. The reagent plugs are deposited into the wells of bottom plate, according to the desired configuration. Narrow Teflon tubes, inclined at 45 to the wells are used to load reagents. Plugs are dropped into the wells with the help of a micromanipulator. FC-40 fluid is used to prevent any evaporation due to its low vapor pressure. FC-40 also helps in filling up the defects on the chip.

Figure 3. [3] Schematically shows the step-by-step process in slipchip crystallization. a) Shows the reagents loaded in the wells of bottom plate. b) Both plates are aligned together with preloaded bottom wells. c) and d) Shows the protein being loaded into the wells of top plate through the duct etched on the bottom plate. e) The top plate is slipped in a position for wells of both plates to align together for reaction. f) The diffusion reaction is initiated and crystals started growing.

After preloading the plates are arranged in such a way that, the channels of bottom plates are in contact with the wells of top plates, this helps to load the protein into the wells of top plate. Protein is loaded manually by means of a pipettor. Protein is loaded into the wells of top plate through the channels etched on the bottom plate after moving the plate by a small distance, in a position such that wells of top plate and channels are connected. In order to bring the protein in contact with the reagents, the top plate is slipped relative to the bottom. All the loaded reagents and proteins come in contact according to the desired configuration. Diffusion begins, leading to the formation of small nucleus. In the metastable region the nucleus starts growing, forming fine protein crystals. If the wells are made very thin or shallow, rapid mixing is possible, even for larger proteins. For deeper wells, mixing

can be accelerated. When the plates are moved relatively, shear is generated at the wells, leading to chaotic advection. In addition, the reagents and proteins are mostly of different densities, and hence due to surface tension at the interface, Marangoni convection is also possible. Examples of proteins crystallized with slipchip method are dihydrofolate reductase, thymidylate synthase, ferritin etc. 4.3 Few advantages of slipchip crystallization method are, a) There is no valves or pumps involved in the process; hence no external equipment is needed for the operation. b) Very reliable at nanoliter scale with a wide range of solutions and proteins for crystallization. c) Bonding is absent in slipchip fabrication. d) Multiple experiments are possible simultaneously on the same chip, due to more number of wells. e) Low labor cost and high efficiency. f) Miniaturized volume etc. 5. Droplet based system: In this system, crystallization is taking place in small aqueous droplets of size nearly 7 nL of which volume of protein stands up to 4 nL. This method is highly employed in genome sequencing projects for obtaining the tertiary structures of protein. Crystallization occurs in the labile region on phase diagram, which is a narrow region where nucleation occurs but not precipitation. This microfluidic system uses only minimum amount of reagents, which makes it well suited for crystallizing rare and expensive proteins. In droplet based systems [4], manual mixing of reagents are not advisable, as it requires at least 100 nL of protein per trial and hence robotic systems have to be adopted. A disadvantage of robotic system is it is very expensive, so it is not available in small laboratories.

Figure 4 [6]: Schematic diagram of droplet based system. Protein, crystallizing agent and buffer are loaded from the top with the help of syringes and carrier fluid is loaded to displace the droplets along the micro-channel, perpendicular to the direction of protein loading. Crystallization takes place inside the channel.

In droplet based system, crystallization takes place inside a micro-channel inside a PDMS or hydrophobic glass, usually a cross sectional area of 150 * 100 m2. The aqueous reagents and protein are loaded in syringes, which are connected to corresponding channels for introducing into the main channel where crystallization occurs. This is schematically shown in figure 4. Another syringe containing fluorinated oil is connected to the main micro-channel perpendicular to the channels of aqueous reagents. It is called as carrier fluid. The carrier fluid is immiscible and inert with the aqueous solution. Syringe pumps are used to activate the syringes. Reagents and protein mixes in the channel when syringes are activated, which is in turn introduced into the flowing stream oil, where they form droplets spontaneously. The volume of aqueous droplets introduced into the oil stream depends on the volumetric flow rate at the time of formation of droplets, which can be controlled by the syringe pump. Carrier fluid is usually pumped at 10 to 12 nL s-1 depending on the size of the micro-channel and droplet size required. There are no valves employed in this system. Therefore by varying the flow rate different concentration and composition are possible on variety of droplet sizes. This makes it possible for hundreds of varying concentration protein can be crystallized at a time in the same device. Once the required numbers of droplets are formed inside the crystallizing micro-channel, pumping is stopped, the syringes are disconnected and the inlets are sealed. Thus the flow is stopped and it is allowed o incubate at constant temperature to form fine protein crystals.

Figure 5 [4] a), b) and c) shows the reagents for screening experiment are introduced into the moving stream of carrier fluid. The volume of NaCl is reduced gradually and buffer is increased, hence the concentration of NaCl also decreases in the droplet, which is represented in the color of the droplets. d) Graphical representation of relative concentration of fluids introduced vs total volume dispensed. e) Polarized microphotograph of droplets containing protein crystals. Crystals are formed at locations inside droplets, favorable for crystallization.

Zheng et al conducted the screening experiment using precipitant polyethylene glycol (PEG, 30% w/v aqueous PEG 6000, viscosity 16 cP), with stock solution of NaCl (1.0 M, viscosity 1 cP) and concentrated solution of lysozyme (100 mg/mL) protein. In the experiment the flow rates were varied with three concentrations of NaCl and four concentrations of PEG and two concentrations of lysozyme. Carrier fluid used was 10:1 mixture of C14F12 and C6F13(CH2)2OH. The result of the experiment is shown in figure 5. Due to the different reaction conditions used, lysozyme crystals were formed only at regions with conditions favorable for crystallization. This is shown in figure 5e, where crystals are formed only in some regions inside the droplet. 5.1 Advantages of droplet based systems: a) It uses only up to 4 nL of protein per droplet and the re is a potential to scale it further lower, as aqueous droplets of volumes ranging from 100 nL to 10 pL can reliably formed. b) This process is rapid and hence reduces manual labor. Large number of droplets can be formed by syringes in a matter of minutes. c) Materials like glass, plastic can be used to make the chip, if they are made hydrophobic. d) It can handle 5 to 10 number of solutions on the chip during the trial work. Thus multidimensional phase diagrams can be provided easily. e) It has a potential application of crystallizing other macromolecules too. 6. Membrane protein crystallization: A membrane protein is a protein molecule attached with cell membranes. Membrane protein plays vital role in daily biological processes. Malfunctioning of any such protein can cause several diseases. So it is important to know the structure of membrane proteins by crystallization for treatments, drug design etc. Additional consideration has to be taken into account while crystallizing membrane proteins. It can be crystallized using lipidic mesophase (a state between solid and liquid) like lipidic cubic phase (LCP). Perry et al [5] developed a

Figure 6 [5]: Optical micrograph of membrane protein crystallizing chip (Left). Magnified view, with two protein chambers at the bottom and lipid chamber between them. Precipitant chamber and crystallization chambers are at the top (Right).

microfluidic system to crystallize membrane proteins, similar to valve based approach, using pneumatic valves with some additional complexity. The crystallizing chip is made of PDMS material, schematically shown in figure 6. Functionally there are two sets of pneumatic valves, isolation valves to control the flow direction and injection valves to control the flow rates. Since the membrane proteins are relatively viscous, the chip is designed to have short channel lengths for the ease of movement. Protein will be pumped into two bottom chambers placed on either side with volume 4.9 nL and lipid chamber of volume 9.6 nL placed between them.

Figure 7 [5]: Optical micrograph of a part of membrane protein crystallization chip. a) Protein and lipid are pumped to respective chambers. b) Isolating valves between the two chambers are opened and the solutions are mixed. d) and e) The solution is circulated between the chambers to get a homogeneous mixture. f) Solution ready to move into crystallizing chamber.

Above these chambers, there are crystallization chamber where crystallization is carried out and precipitant chamber where precipitants are stored. All these chambers are connected with isolating valves to control the flow rate. A round shape is employed for the chambers to avoid dead volume minimizing concentration graduate. By actuating the valves, protein is moved from protein chamber to lipid chamber as shown in figure 7 and allowed to mix with lipids. The proteinlipid mix is moved back to protein chambers by actuating respective valves. A tendril flow is obtained while flowing from one chamber to other and a whorl flow is occurs inside the chamber.Both tendril and whorl flows results in chaotic advection. This cycle can be repeated to get a homogeneous mixture. The mesophase is then transferred into crystallization chamber. Precipitant solution is injected into the crystallization chamber. Next, the entire chip is sealed and placed in a dark room for incubation. Within few days, membrane protein crystals can be obtained. Perry et al used bacteriarhodopsine membrane protein in their trial experiment. Precipitant was 2.5 M Sorenson phosphate buffer at 5.5 pH. The main advantages of this technique are, it is highly useful if the solutions mixing have large

differences in viscosity. Also if the protein is highly viscous even in microfluidic scale this method can be employed. The preparations necessary for obtaining the mesophase in membrane protein crystallization has been reduced by 1000 times by this technique. Additionally, large number of trial volumes helps in studying the interactions between the fluids in detail. 7. Conclusion: The three microfluidic approaches for protein crystallization was described, namely valvebased system, slipchip based system and droplet-based system. Conventional protein crystallization methods can be implemented with these three microfluidic approaches, which overcome most of the disadvantages associated with macro-scale approach. High quality protein crystals can be obtained when worked on micro-scale, best suited for X-ray crystallography. In addition microfluidic method for crystallizing membrane protein is also described, a modified form of valve-based crystallization, which is relatively a complex process, using LCP. With numerous advantages, microfluidic protein crystallization techniques will be very much useful for the biomedical industry.

References:
1. Liang Li and Rustem F. Ismagilow 2010. Protein crystallization using microfluidic technologies based on valves, droplets, and slipchip. 2. Hansen CL, Skordalakes E, Berger JM, Quake SR. 2002. A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion. Proc. Natl. Acad. Sci. USA 99:1653136 3. Du WB, Li L, Nichols KP, Ismagilov RF. 2009. SlipChip. Lab Chip 9:228692 4. Zheng B, Roach LS, Ismagilov RF. 2003. Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets. J. Am. Chem. Soc. 125:1117071 5. Perry SL, Roberts GW, Tice JD, Gennis RB, Kenis PJA. 2009. Microfluidic generation of lipidic mesophases for membrane protein crystallization. Cryst. Growth Des. 9:256669 6. Sauter C, Dhouib K and Lorber B 2007, From macrofluidics to microfluidics for the crystallization of biological macromolecules.