This protocol is unique cause it allows the user to study and examine and measure the interaction between soluble molecules and crystal surfaces. Strong evidence for irreversible binding of antifreeze proteins to ice or obtained using this method. The main advantage of this technique is the ability to control the growth of micron size ice and hydrate crystals and exchange the solution around them in a controlled manner.
To begin, place the pre-prepared mold in a glass Petri dish covered with aluminum foil. Then prepare 30 to 40 milliliters of the PDMS mixture by weighing a 1 to 10 mixture of the curing agent and elastomer and mixing continuously for approximately five minutes until the mixture appears white and nearly opaque. Next, pour the PDMS mixture into the Petri dish with the mold and degas in a desiccator until no bubbles remain.
Bake the mold with the liquid PDMS in an oven or on a hot plate at 70 degree Celsius until a rubber-like consistency is obtained. Then cut the device out by tracing around the features with a scalpel, taking care to push forward with the scalpel instead of down, since the mold is fragile. After removing the cutout PDMS device, place it upside down in a new Petri dish.
Using a blunt syringe, 20 gauge needle, punch out holes in the device based on the imprinted pattern. Then insert the cleaned PDMS and coverslip into the plasma cleaner. Close the valves and turn on the power, vacuum and pump.
Allow the plasma cleaner to run for about a minute. Set the RF to high and allow some air to enter the plasma cleaner using the fine valve. When the viewing windows color changes from purple to pink, let the plasma cleaner work for 50 seconds to turn the RF off.
Keep the pump on for a minute, and after turning it off, gradually open the main valve to allow air into the plasma cleaner. Then press the PDMS surface onto the cleaned coverslip and confirm that they are bonded by observing no detachment when pulling up slightly on the coverslip. After securing the needle of a 90 degrees blunt needle with a pair of pliers, insert one end of the needle into a Tygon tube and the other end into one of the punched out holes of the device while repeating the process for the other holes.
Apply a small amount of immersion oil to the surface of the copper cold stage and spread it using a lint free wipe to create a thin layer of oil. Next, place clean sapphire disc on the created oil layer. Then apply a droplet of immersion oil onto the center of the sapphire disc and position the PDMS device onto the drop such that the features of the device are aligned over the viewing hole of the cold stage.
After holding the device in place, secure the tubing to the outer walls of the aluminum box that houses the adhesive tape. Using a glass syringe, inject four to five microliters of antifreeze protein solution into the inlet channel and close the lid of the cold stage. Initiate the temperature control program and set the temperature to minus 25 degree Celsius.
Then slowly increase the temperature by approximately one degree Celsius per five seconds. Approach the samples melting point, which can range from minus 1 to minus 0.2 degree Celsius, depending on the buffer used in the antifreeze protein solution. To better observe single crystals, switch to 10x or 20x objectives.
And after obtaining a single crystal in the desired location, grow the crystal by slightly decreasing the temperature until the ends of the crystal meet the channel walls. After switching to the 50x objective, inject the antifreeze protein solution into the channels and observe the fluorescence intensity increase, indicating that the protein solution was successfully injected into the channels. To record the solution exchange process, use the NIS-Elements Imaging Program, ensuring that the applied pressure is not too high and slowly inject the buffer solution into the second inlet of the microfluidic device.
Observe a decrease in the fluorescent signal at a rate that depends on the pressure applied to the syringe. To obtain THF hydrates after preparing a THF water solution with a molar ratio of 1 to 15, inject the solution into the microfluidic device. After the THF water solution is frozen, slowly increase the temperature until all the ice has melted at the exclusion of the hydrates and hold the temperature at one degree Celsius for three minutes.
Set the temperature to minus two degree Celsius and observe the abundance of hydrates that appear in the microfluidic channels in the absence of inhibitors. Then inject the antifreeze protein or inhibitor into the microfluidic channel using the glass syringe while adjusting the temperature to ensure that the obtained crystals do not melt or grow and allow a few minutes for the inhibitor molecules to adsorb to the crystal surface. Perform the solution exchange by injecting the inhibitor free solution into the channel.
Take images of the crystal before and after the solution exchange and analyze the fluorescence intensity on the crystal and in the solution using the imaging program. A successful solution exchange around an ice crystal was performed indicating that the solution exchange was relatively fast. However, a slower exchange is possible.
The fluorescence intensity coming from the ice adsorbed antifreeze glycoproteins molecules was clearly observed after the exchange was complete. A quantitative analysis of the antifreeze proteins concentration was monitored and the fluorescence intensity was determined in the solution and on the ice, indicating the fluorescence signal in the solution is decreased by a factor of 100 during the solution exchange, while the calculated signal on the ice surface stays constant. Microfluidic experiments with THF hydrates were carried out where an inhibitor free solution was injected into the channels after the hydrate crystals were allowed to adsorb the inhibitor molecules.
The THF hydrates were observed after solution exchange with two types of inhibitors, including antifreeze glycoproteins labeled with fluorescein isothiocyanate and Safranin O, a fluorescent dye. The critical steps of this procedure are the formation and isolation of a single crystal in the microfluidic channels and the exchange of solution around it. This method can be used with other crystal materials that are highly temperature sensitive, in an effort to understand the mechanism by which inhibitors interact with these crystals.
The ability to exchange solutions around crystals paved the way for researchers to identify key insights into the binding mechanism of antifreeze proteins and to discover a new phenomenon of isotope effect on ice growth.
