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.

1. Microfluidic device fabrication
<ol>
	<li>Cover the inside surface of a Petri dish with aluminum foil and place the pre-prepared mold into the Petri dish.
	<ol>
		<li>Fabricate the molds using the lithography techniques described in references. The two device designs used in the present work are depicted in Figure 1.</li>
	</ol>
	</li>
	<li>Prepare 30-40 mL of the PDMS mixture by weighing a 1:10 (by weight) mixture of the curing agent and elastomer (see Table of Materials) and mixing continuously for approximately 5 min until the mixture appears white and nearly opaque. 
	NOTE: Place a plastic cup on the weighing scale, pour the elastomer into the cup, and then add the curing agent to achieve a 1:10 weight ratio.</li>
	<li>Pour the PDMS mixture into the Petri dish with the mold and degas in a desiccator until no bubbles remain (approximately 30 min).</li>
	<li>Bake the mold with the liquid PDMS in an oven or on a hot plate at 70 &#176;C until a rubber-like consistency is obtained. This process takes about an hour.</li>
	<li>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. Remove the cut-out PDMS device, placing it upside-down in a new Petri dish. Remove dust and dirt particles from the bottom surface by attaching and removing a piece of adhesive tape (see Table of Materials).</li>
	<li>Use a blunt syringe needle (20 G) to punch out holes in the device based on the imprinted pattern, using a microscope if necessary. Ensure that the hole penetrates through the other side of the device, and use tweezers to remove the punched-out pieces.</li>
	<li>Thoroughly wash a coverslip (18 x 18 mm, 0.14 mm thick) with water and soap, and then with isopropanol. Use air pressure to dry the cleaned coverslip.</li>
	<li>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 HI, and allow some air to enter the plasma cleaner using the fine valve.
	<ol>
		<li>When the viewing window&#39;s color changes from purple to pink, let the plasma cleaner work for 50 s and 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. 
		NOTE: An alternative method to achieve comparable results is heat bonding. For this method, lay the mold onto the coverslip and place it on a hot plate set to 70-80 &#176;C for approximately 60 min.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Secure the needle of a 90&#176; blunt needle (18 G) with a pair of pliers and pull off the plastic syringe connector to remove it. Insert one end of the needle into a Tygon tube (0.020&#34; ID, 0.060&#34; OD, see Table of Materials) and the other end into one of the punched-out holes of the device. Repeat the process for the other holes.</li>
	<li>To remove air bubbles, inject water/buffer into the channels with a glass syringe (a pump is optional, but no pump was used here). Filter all liquids with a 0.22 &#181;m filter before injecting them into the device.</li>
	<li>Use a blocking agent such as a 1% BSA solution to prevent the binding of fluorescence-labeled AFPs onto the PDMS walls. Inject the blocking agent into the inlet channel and allow it to remain in the microchannels for 20 min. Then, inject the buffer solution into the inlet channel to flush out the BSA solution. 
	NOTE: The resulting PDMS device is attached to a coverslip along its bottom surface, connected to tubing in its inlet and outlet holes, and coated with a blocking agent in its channels.</li>
</ol>
2. Setting up the microfluidic device
<ol>
	<li>Apply a small amount of immersion oil to the surface of the copper cold stage (see Table of Materials) and spread it using a lint-free wipe to create a thin layer of oil. Next, place a clean sapphire disc (see Table of Materials) on the created oil layer. 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.</li>
	<li>Hold the device in place and secure the tubing to the outer walls of the aluminium box that houses the cold stage using adhesive tape. Make a note of the placement of each specific tube.</li>
	<li>Use a glass syringe to inject 4-5 &#181;L of AF(G)P solution (see Table of Materials) into the inlet channel. Close the lid of the cold stage. 
	&#8203;NOTE: The concentration of the AFP solutions can be varied and decided based on their fluorescence intensity. In the present protocol, the range of concentrations was 5-40 &#181;L.</li>
</ol>
3. Formation of single crystals in the microfluidic channels
<ol>
	<li>Purge the cold stage with dry air/nitrogen to prevent condensation from forming inside it. Activate a circulating cooling bath to circulate water through the cold stage and serve as a heat sink.</li>
	<li>Initiate the temperature control program and set the temperature to -25 &#176;C. 
	NOTE: Freezing will occur as the sample reaches a temperature of ~-20 &#176;C, which can be observed as a sudden darkening and roughening of the sample. Any objective can be used at this point, preferably the 4x objective, which enables the user to observe the entirety of the microfluidic channels.</li>
	<li>Slowly, increasing the temperature by approximately 1 &#176;C/5 s, approach the sample&#39;s melting point, which can range from -1 to -0.2 &#176;C depending on the buffer used in the AF(G)P solution. As the temperature approaches the melting point, wait until a few single crystals are isolated. 
	NOTE: If needed, unwanted ice can be locally melted using an IR laser (980 nm) (see Table of Materials), which is fixed on the microscope. The laser melts nearby ice crystals as long as it is on.</li>
	<li>At this point, it is recommended to switch to 10x or 20x objectives to better observe single crystals. After obtaining a single crystal in the desired location, grow the crystal by slightly decreasing the temperature (by ~0.01 &#176;C) until the edges of the crystal meet the channel walls.</li>
	<li>Switch to the 50x objective, inject the AF(G)P solution into the channels, and observe the fluorescence intensity increase, indicating that the protein solution was successfully injected into the channels. Perform this step carefully to avoid melting of the ice crystal during the exchange of solutions. Closely monitor and adjust both the flow rate (by applying more/less pressure on the glass syringe) and the temperature during the solution exchange process.</li>
	<li>Allow the proteins enough time to accumulate and bind onto the crystal surface. 
	&#8203;NOTE: This varies based on the accumulation and adsorption rates of the AF(G)P5,25. In a typical experiment, 5-10 min are allowed for AFP accumulation</li>
</ol>
4. Thermal Hysteresis (TH) activity measurement
NOTE: This step is optional.
<ol>
	<li>Determine the melting point of the crystal by adjusting the temperature with small steps of 0.002 &#176;C and observing the highest temperature that a small crystal can be subjected to without it melting.</li>
	<li>On the RAMP function of the temperature controller, set the cooling rate to -0.05 to -0.01 &#176;C/4 s and activate the RAMP. Note the exact temperature at which sudden crystal growth occurs. 
	&#8203;NOTE: This value is called the burst temperature and corresponds to the temperature of freezing. The difference between the melting and freezing temperatures is the TH activity25.</li>
</ol>
5. Solution exchange around single crystals
<ol>
	<li>Ensure that the sample temperature is in the TH gap to prevent the melting or growth of the crystal during the experiment.</li>
	<li>Record the solution exchange process using the NIS Elements imaging program (see Table of Materials) for later fluorescence data analysis. Slowly inject the buffer solution into the second inlet of the microfluidic device, and observe a decrease in the fluorescent signal at a rate that depends on the pressure applied to the syringe.
	<ol>
		<li>Use this visual representation to ensure that the applied pressure is not too high so as not to cause the crystal to melt. See Figure 2 for a sequence of images demonstrating the solution exchange process.</li>
	</ol>
	</li>
	<li>Measure the fluorescence intensity in the microfluidic channel using the imaging program. 
	NOTE: The signal should peak at the region near the surface of the crystal, indicating AFP binding, and should be relatively low in the surrounding solution, which has been flushed with buffer.</li>
	<li>Measure the TH activity after the solution exchange following step 4.</li>
	<li>Repeat the experiment by isolating a new crystal (steps 3.3-3.5). Inject the AFP solution with a glass syringe and repeat the solution exchange (steps 5.1-5.3). Obtain a new single crystal (steps 3.3-3.5) and flow the AFP solution into the channels using the glass syringe.</li>
</ol>
6. Experiments with clathrate hydrates
<ol>
	<li>To obtain THF hydrates, prepare a THF/water solution with a molar ratio of 1:15, which is a volume ratio of 1:3.326. Maintain this ratio in all solutions used in the following experiments, including those containing AF(G)Ps or other inhibitors.</li>
	<li>Follow steps 1 and 2 to prepare the microfluidic device and place it in the cold stage. Set the temperature to -25 &#176;C; the sample will freeze at ~-20 &#176;C as described in step 3.2.</li>
	<li>After the THF solution is frozen, slowly increase the temperature until all the ice has melted. 
	NOTE: The melting point of ice is slightly depressed by the THF.</li>
	<li>To ensure that all the ice crystals melted at the exclusion of the hydrates, hold the temperature at 1 &#176;C for 3 min.</li>
	<li>Set the temperature to -2 &#176;C and observe the abundance of hydrates that appear in the microfluidic channels in the absence of inhibitors. 
	NOTE: THF hydrates are shaped as octahedrons (Figure 2), and in some cases, the crystals are very thin; thus, clear observation might be challenging. In these cases, it is recommended to repeat steps 6.4-6.5 to obtain new crystals.</li>
	<li>Inject the AF(G)P/inhibitor into the microfluidic channel using the glass syringe while adjusting the temperature to ensure that the obtained crystals do not melt/grow. Allow a few minutes for the inhibitor molecules to adsorb to the crystal surface.</li>
	<li>If needed, measure the TH activity following step 4.</li>
	<li>Exchange the solution around the crystals as described in steps 5.2-5.3.</li>
</ol>

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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.

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&#160;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.

<table><tbody><tr><td>0.22-micron filters</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>90-degree bent blunt needles</td><td></td><td></td><td>18 Gauge</td></tr><tr><td>Antifreeze proteins and antifreeze glycoproteins</td><td>A gift</td><td></td><td>See references 5 and 28</td></tr><tr><td>Blunt needles</td><td></td><td></td><td>18 Gauge and 20 Gauge</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Cold stage</td><td>Home made</td><td></td><td></td></tr><tr><td>Cover slips</td><td>Globe Scientific</td><td></td><td>18 X 18 mm, 0.14 mm thickness</td></tr><tr><td>Glass syringe</td><td></td><td></td><td></td></tr><tr><td>Infrared laser 980 nm</td><td>Opto Engine LLC</td><td></td><td></td></tr><tr><td>Inverted microscope, Eclipse Ti - S</td><td>Nikon</td><td></td><td></td></tr><tr><td>Invisible tape</td><td>Staples</td><td></td><td></td></tr><tr><td>lint-free wipe</td><td>Kimwipes</td><td></td><td></td></tr><tr><td>Newport 3040 temperature controller</td><td>Newport</td><td></td><td></td></tr><tr><td>NIS-Elements Imaging Software</td><td>Nikon</td><td></td><td></td></tr><tr><td>Oil vacuum pump</td><td>Harrick Plasma</td><td></td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Polydimethylsiloxane (Dow Corning Sylgard 184 Silicone Elastomer kit)</td><td>Dow Corning Syglard</td><td></td><td></td></tr><tr><td>Safranine O</td><td>Sigma-Aldrich</td><td>S2255-25G</td><td></td></tr><tr><td>Sapphire disc</td><td>Ted Pella Inc</td><td>16005-1010</td><td>&amp;nbsp;25.4 mm diameter, 0.3 mm thickness</td></tr><tr><td>sCMOS Camera, Neo 5.5</td><td>Andor</td><td></td><td></td></tr><tr><td>Tetrahydrofuran (THF)</td><td>Sigma-Aldrich</td><td>401757-100ML</td><td></td></tr><tr><td>Tygon Microbore tubing for microfluidic device</td><td>Cole-Parmer</td><td></td><td>&amp;nbsp;0.020&quot; ID, 0.060&quot;OD, 100 ft/roll.</td></tr><tr><td>Tygon tubing for water circulation and nitrogen gas</td><td>Cole-Parmer</td><td></td><td>1/8&amp;rdquo; ID, 3/16&amp;rdquo; OD</td></tr></tbody></table>

a microfluidic approach for the study of ice and clathrate hydrate crystallization

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64072/64072fig01.jpg" /> 
Figure 1: A schematic representation of the microfluidic channels used in the present study. Both designs include two inlets and one outlet. <a href="https://www.jove.com/files/ftp_upload/64072/64072fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64072/64072fig02.jpg" /> 
Figure 2: THF hydrates formed in the microfluidic channel after the temperature was cooled to ~-2 &#176;C. The morphology of all crystals presented is a tetrahedron; however, some crystals are oriented differently. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64072/64072fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64072/64072fig03.jpg" /> 
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 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64072/64072fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64072/64072fig04v2.jpg" /> 
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 &#181;m. (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. <a href="https://www.jove.com/files/ftp_upload/64072/64072fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64072/64072fig05.jpg" /> 
Figure 5: Single THF hydrate crystals in microfluidic channels after the solution around them (A, AFGP1-5) or (B,&#160;Safranine O) was exchanged. The image in (B) is reproduced from reference26. Scale bar = 25 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64072/64072fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization at JoVE.com

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

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 ...

a-microfluidic-approach-for-study-ice-clathrate-hydrate

Research

JoVE Journal

Chemistry

3.0K Views.  Yeshiva University. Dit protocol is uniek omdat het de gebruiker in staat stelt om de interactie tussen oplosbare moleculen en kristaloppervlakken te bestuderen en te onderzoeken en te meten. Sterk bewijs voor onomkeerbare binding van antivrieseiwitten aan ijs of verkregen met behulp van deze methode. Het belangrijkste voordeel van deze techniek is het vermogen om de groei van micronformaat ijs en hydrateerde kristallen te beheersen en de oplossing eromheen op een gecontroleerde manier uit te wisselen.Pla...

Video: A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Biochemistry

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping, Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Herein, we describe the fabrication and operation of a double-layer microfluidic system made of polydimethylsiloxane (PDMS). We demonstrate the potential of this device for trapping, directing the coordination pathway of a crystalline molecular material and controlling chemical reactions onto on-chip trapped structures.

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. ...

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

Engineering

Methane Hydrate Crystallization on Sessile Water Droplets

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa working pressure, containing a stage for sessile droplets, a sapphire window for visualization, and temperature and pressure transducers. A pressure pump connected to a methane gas cylinder is used to pressurize the cell to 5 MPa. The cooling system is a 10 gallon (37.85 L) tank containing a 50% ethanol solution cooled via ethylene glycol through copper coils. This setup enables the observation of the temperature change associated with hydrate formation and dissociation during cooling and depressurization, respectively, as well as visualization and photography of the morphologic changes of the droplet. With this method, rapid hydrate shell formation was observed at ~-6 &#176;C to -9 &#176;C. During depressurization, a 0.2 &#176;C to 0.5 &#176;C temperature drop was observed at the pressure/temperature (P/T) stability curve due to exothermic hydrate dissociation, confirmed by visual observation of melting at the start of the temperature drop. The "memory effect" was observed after repressurizing to 5 MPa from 2 MPa. This experimental design allows the monitoring of pressure, temperature, and morphology of the droplet over time, making this a suitable method for testing various additives and substrates on hydrate morphology.

We describe a method to form gas hydrate on sessile water droplets to study the effects of various inhibitors, promoters, and substrates on the hydrate crystal morphology.

This paper describes a method to form methane hydrate shells on water droplets. In addition, it provides blueprints for a pressure cell rated to 10 MPa ...

Environment

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Studying Surfactant Effects on Hydrate Crystallization at Oil-Water Interfaces Using a Low-Cost Integrated Modular Peltier Device

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a temperature regulator, visualization techniques, and inner pressure measurements. The temperature control system contains a low-cost, programmable temperature regulator made with solid-state Peltier components. Along with the temperature control system, we incorporated visualization techniques and internal pressure measurements to study hydrate formation and inhibition in the presence of nonionic surfactants. We studied the hydrate-inhibiting ability of nonionic surfactants (sorbitane monolaurate, sorbitane monooleate, PEG-PPG-PEG, and polyoxyethylenesorbitan tristearate) at low (i.e., 0.1 CMC), medium (i.e., CMC), and high (i.e., 10 CMC) concentrations. Two types of crystals were formed: planar and conical. Planar crystals were formed in plain water and low surfactant concentrations. Conical crystals were formed in high surfactant concentrations. The results of the study show that conical crystals are the most effective in terms of hydrate inhibition. Because conical crystals cannot grow past a certain size, the hydrate growth rate as a conical crystal is slower than the hydrate growth rate as planar crystal. Hence, surfactants that force hydrates to form conical crystals are the most efficient. The goal of the protocol is to provide a detailed description of an experimental system that is capable of investigating the cyclopentane hydrate crystallization process on the surface of a water droplet in the presence of surfactant molecules.

We present a protocol to study the formation of hydrates in the presence of nonionic surfactants on the interface of a water droplet submerged in cyclopentane. The protocol consists of building a low-cost, programmable, temperature regulator. The temperature control system is combined with visualization techniques and internal pressure measurements.

We introduce an approach to study the formation and growth of hydrates under the influence of nonionic surfactants. The experimental system includes a ...

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (&#60;1 &#181;L) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies

This paper details the fabrication protocol of microfluidic chips developed for on-chip protein crystallization with the dialysis method and in situ X-ray diffraction experiments. The microfabrication process makes it possible to integrate a semipermeable regenerated cellulose dialysis membrane with any molecular weight cut-off, between two layers of the chip.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins ...

A Microfluidic Approach for the Study of Ice and Clathrate Hydrate Crystallization

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