The present protocol describes the crystallization of microscopic ice crystals and clathrate hydrates in microfluidic devices, enabling liquid exchange around the formed crystals. This provides unparalleled possibilities to examine the crystallization process and binding mechanisms of the inhibitors.
An accurate mechanistic description of water crystallization is challenging and requires a few key elements: superb temperature control to allow the formation of single microscopic crystals and a suitable microscopy system coupled to the cold stage. The method described herein adds another important feature that includes exchanging solutions around ice and clathrate hydrate crystals. The described system comprises a combination of unique and home-developed instruments, including microfluidics, high-resolution cold stages, and fluorescence microscopy. The cold stage was designed for microfluidic devices and allows for the formation of micron-sized ice/hydrate crystals inside microfluidic channels and the exchange of solutions around them. The temperature resolution and stability of the cold stage is one millikelvin, which is crucial for controlling the growth of these small crystals. This diverse system is used to study the different processes of ice and hydrate crystallization and the mechanism by which the growth of these crystals is inhibited. The protocol describes how to prepare microfluidic devices, how to grow and control microscopic crystals in the microfluidic channels, and how the utilization of the flow of liquids around ice/hydrate crystals affords new insights into the crystallization of water.
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) protect various cold-adapted organisms from frost damages1. AFPs and AFGPs (generalized as AF(G)Ps) inhibit the growth of ice crystals by binding irreversibly to their surfaces and inhibiting further growth due to the Gibbs-Thomson effect2,3,4,5. The resulting gap that forms between the melting temperature, which is largely unchanged, and the newly depressed freezing temperature is called thermal hysteresis (TH) and represents a measurable parameter corresponding to AFP activity6. The use of AFPs to inhibit ice growth has far-reaching and diverse applications, offering potential enhancement in various fields, including cryopreservation, frozen food quality, and protection of cold-exposed crops.
The crystallization of water at low temperatures and high pressures in the presence of small organic molecules results in the formation of clathrate hydrates (or gas hydrates), where the most abundant hydrate is methane hydrate7. The crystallization of methane hydrates in gas/oil flowlines may cause plugs, which might cause explosions due to gas ignition8,9,10. Current efforts to prevent hydrate crystallization in flowlines include using thermodynamic (alcohols and glycols) and kinetic (mainly polymers) inhibitors11,12,13,14. AFPs have also been found to bind to clathrate hydrate crystals and inhibit their growth, which points to the potential use of AFPs to hinder the formation of plugs, thereby providing a greener solution15.
Microfluidics is a prevalent method used to study the properties of fluids at minuscule sample volumes (down to fL) that are flowed through a network of microchannels16. The microchannels follow a pattern created on a silicon wafer (the mold) using lithography17. A commonly used material to fabricate microfluidic devices is polydimethylsiloxane (PDMS), which is inexpensive and relatively simple to work with in research laboratories. The design of the features (channels) is composed with regard to the specific purpose of the device; thus, it can be utilized for a variety of applications, including DNA sensing18, medical diagnosis19 and crystallization processes3,20,21.
The present protocol describes a unique microfluidic method of growing micron-sized ice and hydrate crystals with various inhibitors, including AFPs and AFGPs. For these experiments, Tetrahydrofuran (THF) hydrates were used to mimic the properties of methane gas hydrates22, which require specialized equipment for pressure and temperature control23. Fluorescently labeled AF(G)Ps were used to visualize and analyze the adsorption of the proteins to the crystal surface, and coupled with fluorescent imaging, the microfluidic approach allowed the obtaining of key features of the binding process of these molecules to crystal surfaces.
1. Microfluidic device fabrication
2. Setting up the microfluidic device
3. Formation of single crystals in the microfluidic channels
4. Thermal Hysteresis (TH) activity measurement
NOTE: This step is optional.
5. Solution exchange around single crystals
6. Experiments with clathrate hydrates
Solution exchange with ice crystals
A successful solution exchange around an ice crystal is presented in Figure 3. The time stamp on each snapshot indicates that the solution exchange was relatively fast; however, a slower exchange is possible. The fluorescence intensity coming from the ice-adsorbed AFGP molecules is clearly observed after the exchange is complete (Figure 3, right). A quantitative analysis of the AFP concentration on the ice surface is monitored using a designated region of interest (ROI) tool (Figure 4). In this experiment4, AFP type III (QAE isoform) diluted in 50 mM Tris-HCl (pH 7.8) and 100 mM NaCl was used. The solution is exchanged around a bipyramidal-shaped crystal, and the fluorescence intensity in the solution and on the ice is monitored. The red plot indicating the fluorescence signal in the solution is decreased by a factor of 100 during the solution exchange, while the calculated signal (green plot) on the ice surface stays constant. The calculated signal of the ice-adsorbed molecules was obtained by subtracting the signal coming from the solution (multiplied by a constant that relates to the thickness of the microfluidic channel) from the signal coming from the ice4.
Solution exchange with THF hydrates
Microfluidic experiments with THF hydrates were carried out similarly to the experiments with ice. After the hydrate crystals were allowed to adsorb the inhibitor molecules from the solution, an inhibitor-free solution was injected into the channels. Figure 5 presents THF hydrates after solution exchange with two types of inhibitors: AFGP1-5 labeled with fluorescein isothiocyanate (FITC) (Figure 5A) and safranine O (see Table of Materials), which is a fluorescence dye26 (Figure 5B). This is the first demonstartion of an AFGP binding to the surface of a clathrate hydrate.
Figure 1: A schematic representation of the microfluidic channels used in the present study. Both designs include two inlets and one outlet. Please click here to view a larger version of this figure.
Figure 2: THF hydrates formed in the microfluidic channel after the temperature was cooled to ~-2 °C. The morphology of all crystals presented is a tetrahedron; however, some crystals are oriented differently. Scale bar = 20 µm. Please click here to view a larger version of this figure.
Figure 3: A representative experiment exhibiting solution exchange around a single ice crystal in a microfluidic channel. Initially, the solution contained AFGP1-5 labeled with FITC, and ice-adsorbed AFGPs were not observed. After the solution was exchanged for an AFGP-free solution, the proteins that had been previously adsorbed to the ice surface were clearly detected (image on the right). Scale bar = 25 µm. Please click here to view a larger version of this figure.
Figure 4: A quantitative and qualitative analysis of the AFP concentration on the ice surface. (A) An ice crystal at high AFP solution concentration (before solution exchange). (B) The same crystal after the AFP solution was exchanged with an AFP-free buffer solution. Scale bar = 20 µm. (C) Quantitative analysis of the fluorescence intensity on the ice surface (black) and in the solution (red) during a solution exchange. The green curve represents the calculated intensity on the ice surface. The figure is adapted with permission from reference4. Please click here to view a larger version of this figure.
Figure 5: Single THF hydrate crystals in microfluidic channels after the solution around them (A, AFGP1-5) or (B, Safranine O) was exchanged. The image in (B) is reproduced from reference26. Scale bar = 25 µm. Please click here to view a larger version of this figure.
The present protocol was designed to utilize the combination of microfluidic flow with microscopic crystals in order to reveal new insights into crystal growth and its inhibition. A millikelvin-resolution temperature-controlled cold stage27 enables the control of single microscopic crystals situated inside microfluidic channels, thereby allowing the exchange of solutions around them. While the fabrication of microfluidic devices is standard and similar to common practices17,18, the control over the growth and melting of crystals inside the device is unique and novel. The most critical component in this system is the superb temperature control, which is achieved by using Peltier thermoelectric coolers, feedback from a thermistor that is located close to the sample, and a high-resolution temperature controller that governs the feedback loop.
Another critical step is the solution exchange itself, as the crystals might melt or grow during this process; thus, the temperature must be adjusted during the solution exchange to prevent growth/melting. The formation of crystals in microfluidic channels interferes with the liquid flow and poses the main challenge of this system; thus, the growth of these crystals must be controlled. Here, an IR laser (980 nm) was mounted on the inverted microscope and was used to locally melt unwanted ice/hydrate crystals28. If such a laser cannot be used, the metallic connectors of the microfluidic device can be heated by an additional Peltier thermoelectric cooler, which will melt the ice in the inlet/outlet of the device.
The method described here includes home-developed instruments (cold stage) and requires training, as some of the abovementioned steps are challenging. As the concentration of the solution surrounding the crystals may change even when the flow is not intended, a simple calibration step5 can provide a reliable estimation of the concentration based on the fluorescence signal. Another possible solution to unwanted flow (during TH measurements, for example) is microfluidic valves, which are described in reference4.
This system was also used to explore the growth behavior of D2O ice in H2O liquid, a study that revealed a new phenomenon of microscopic, scalloped ice surfaces27. Thus, microfluidics can be used in the study of various crystalline systems that respond well to temperature changes.
The authors have nothing to disclose.
Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund for support of this research (Grant number 60191-UNI5). The authors would like to thank Prof. Ido Braslavsky for pioneering the use of microfluidic devices to study antifreeze proteins and ice. The authors are thankful to Prof. Arthur DeVries, Prof. Konrad Meister and Prof. Peter Davies for providing antifreeze protein samples.
0.22-micron filters | Fisher Scientific | ||
90-degree bent blunt needles | 18 Gauge | ||
Antifreeze proteins and antifreeze glycoproteins | A gift | See references 5 and 28 | |
Blunt needles | 18 Gauge and 20 Gauge | ||
Bovine Serum Albumin (BSA) | Sigma-Aldrich | ||
Cold stage | Home made | ||
Cover slips | Globe Scientific | 18 X 18 mm, 0.14 mm thickness | |
Glass syringe | |||
Infrared laser 980 nm | Opto Engine LLC | ||
Inverted microscope, Eclipse Ti – S | Nikon | ||
Invisible tape | Staples | ||
lint-free wipe | Kimwipes | ||
Newport 3040 temperature controller | Newport 3040 | ||
NIS-Elements Imaging Software | Nikon | ||
Oil vacuum pump | Harrick Plasma | ||
Plasma cleaner | Harrick Plasma | PDC-32G | |
Polydimethylsiloxane (Dow Corning Sylgard 184 Silicone Elastomer kit) | Dow Corning Syglard | ||
Safranine O | Sigma-Aldrich | S2255-25G | |
Sapphire disc | Ted Pella Inc | 16005-1010 | 25.4 mm diameter, 0.3 mm thickness |
sCMOS Camera, Neo 5.5 | Andor | ||
Tetrahydrofuran (THF) | Sigma-Aldrich | 401757-100ML | |
Tygon Microbore tubing for microfluidic device | Cole-Parmer | 0.020" ID, 0.060"OD, 100 ft/roll. | |
Tygon tubing for water circulation and nitrogen gas | Cole-Parmer | 1/8” ID, 3/16” OD |