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>

<ol>
	<li>Bar Dolev, M., Braslavsky, I., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-Binding+Proteins+and+Their+Function.">Ice-Binding Proteins and Their Function.</a> Annual Review of Biochemistry. 85 (1), 515-542 (2016).</li><li>Raymond, J. A., DeVries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+inhibition+as+a+mechanism+of+freezing+resistance+in+polar+fishes.">Adsorption inhibition as a mechanism of freezing resistance in polar fishes.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (6), 2589-2593 (1977).</li><li>Celik, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (4), 1309-1314 (2013).</li><li>Drori, R., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+are+antifreeze+proteins+in+solution+essential+for+ice+growth+inhibition.">When are antifreeze proteins in solution essential for ice growth inhibition.</a> Langmuir. 31 (21), 5805-5811 (2015).</li><li>Meister, K., DeVries, A. L., Bakker, H. J., Drori, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+glycoproteins+bind+irreversibly+to+ice.">Antifreeze glycoproteins bind irreversibly to ice.</a> Journal of the American Chemical Society. 140 (30), 9365-9368 (2018).</li><li>Braslavsky, I., Drori, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LabVIEW-operated+Novel+Nanoliter+Osmometer+for+Ice+Binding+Protein+Investigations.">LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations.</a> Journal of Visualized Experiments. (72), e4189 (2013).</li><li>Ruppel, C. D., Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+of+climate+change+and+methane+hydrates.">The interaction of climate change and methane hydrates.</a> Reviews of Geophysics. 55 (1), 126-168 (2017).</li><li>Mozaffar, H., Anderson, R., Tohidi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+alcohols+and+diols+on+PVCap-induced+hydrate+crystal+growth+patterns+in+methane+systems.">Effect of alcohols and diols on PVCap-induced hydrate crystal growth patterns in methane systems.</a> Fluid Phase Equilibria. 425, 1-8 (2016).</li><li>Sa, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+methane+and+natural+gas+hydrate+formation+by+altering+the+structure+of+water+with+amino+acids.">Inhibition of methane and natural gas hydrate formation by altering the structure of water with amino acids.</a> Scientific Reports. 6, 31582 (2016).</li><li>Lederhos, J. P., Long, J. P., Sum, A., Christiansen, R. L., Sloan, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+kinetic+inhibitors+for+natural+gas+hydrates.">Effective kinetic inhibitors for natural gas hydrates.</a> Chemical Engineering Science. 51 (8), 1221-1229 (1996).</li><li>Sun, T., Davies, P. L., Walker, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+for+the+Inhibition+of+Gas+Hydrates+by+&#945;-Helical+Antifreeze+Proteins.">Structural Basis for the Inhibition of Gas Hydrates by &#945;-Helical Antifreeze Proteins.</a> Biophysical Journal. 109 (8), 1698-1705 (2015).</li><li>Zeng, H., Wilson, L. D., Walker, V. K., Ripmeester, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antifreeze+proteins+on+the+nucleation,+growth,+and+the+memory+effect+during+tetrahydrofuran+clathrate+hydrate+formation.">Effect of antifreeze proteins on the nucleation, growth, and the memory effect during tetrahydrofuran clathrate hydrate formation.</a> Journal of the American Chemical Society. 128 (9), 2844-2850 (2006).</li><li>Zeng, H., Wilson, L. D., Walker, V. K., Ripmeester, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibition+of+tetrahydrofuran+clathrate-hydrate+formation+with+antifreeze+protein.">The inhibition of tetrahydrofuran clathrate-hydrate formation with antifreeze protein.</a> Canadian Journal of Physics. 81 (1-2), 17-24 (2003).</li><li>Walker, V. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifreeze+proteins+as+gas+hydrate+inhibitors.">Antifreeze proteins as gas hydrate inhibitors.</a> Canadian Journal of Chemistry. 93 (8), 839-849 (2015).</li><li>Gordienko, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+green+hydrate+inhibitor:+imaging+antifreeze+proteins+on+clathrates.">Towards a green hydrate inhibitor: imaging antifreeze proteins on clathrates.</a> PloS One. 5 (2), 8953 (2010).</li><li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+the+future+of+microfluidics.">The origins and the future of microfluidics.</a> Nature. 442 (7101), 368-373 (2006).</li><li>Cole, R. H., Tran, T. M., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+emulsion+generation+using+a+polydimethylsiloxane+(PDMS)+co-axial+flow+focus+device.">Double emulsion generation using a polydimethylsiloxane (PDMS) co-axial flow focus device.</a> Journal of Visualized Experiments. (106), e53516 (2015).</li><li>Dutse, S. W., Yusof, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+lab-on-chip+systems+in+DNA-based+biosensing:+An+overview.">Microfluidics-based lab-on-chip systems in DNA-based biosensing: An overview.</a> Sensors. 11 (6), 5754-5768 (2011).</li><li>Burklund, A., Tadimety, A., Nie, Y., Hao, N., Zhang, J. X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+diagnostic+microfluidics.">Advances in diagnostic microfluidics.</a> Advances in Clinical Chemistry. 95, 1-72 (2020).</li><li>Sui, S., Perry, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+From+crystallization+to+serial+time-resolved+crystallography.">Microfluidics: From crystallization to serial time-resolved crystallography.</a> Structural Dynamics. 4 (3), 032202 (2017).</li><li>Haleva, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+cold-finger+device+for+the+investigation+of+ice-binding+proteins.">Microfluidic cold-finger device for the investigation of ice-binding proteins.</a> Biophys Journal. 111 (6), 1143-1150 (2016).</li><li>Vlasic, T. M., Servio, P. D., Rey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THF+hydrates+as+model+systems+for+natural+gas+hydrates:+Comparing+their+mechanical+and+vibrational+properties.">THF hydrates as model systems for natural gas hydrates: Comparing their mechanical and vibrational properties.</a> Industrial and Engineering Chemistry Research. 58 (36), 16588-16596 (2019).</li><li>Chen, W., Pinho, B., Hartman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flash+crystallization+kinetics+of+methane+(sI)+hydrate+in+a+thermoelectrically-cooled+microreactor.">Flash crystallization kinetics of methane (sI) hydrate in a thermoelectrically-cooled microreactor.</a> Lab on a Chip. 17 (18), 3051-3060 (2017).</li><li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscale+patterning.">Soft lithography for micro- and nanoscale patterning.</a> Nature Protocols. 5 (3), 491-502 (2010).</li><li>Drori, R., Celik, Y., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-binding+proteins+that+accumulate+on+different+ice+crystal+planes+produce+distinct+thermal+hysteresis+dynamics.">Ice-binding proteins that accumulate on different ice crystal planes produce distinct thermal hysteresis dynamics.</a> Journal of the Royal Society Interface. 11 (98), 20140526 (2014).</li><li>Soussana, T. N., Weissman, H., Rybtchinski, B., Drori, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption-inhibition+of+clathrate+hydrates+by+self-assembled+nanostructures.">Adsorption-inhibition of clathrate hydrates by self-assembled nanostructures.</a> ChemPhysChem. 22 (21), 2182-2189 (2021).</li><li>Drori, R., Holmes-Cerfon, M., Kahr, B., Kohn, R. v., Ward, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+and+unsteady+morphologies+at+ice+interfaces+driven+by+D2O-H2O+exchange.">Dynamics and unsteady morphologies at ice interfaces driven by D2O-H2O exchange.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (44), 11627-11632 (2017).</li><li>Deng, J., Apfelbaum, E., Drori, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice+growth+acceleration+by+antifreeze+proteins+leads+to+higher+thermal+hysteresis+activity.">Ice growth acceleration by antifreeze proteins leads to higher thermal hysteresis activity.</a> Journal of Physical Chemistry B. 124 (49), 11081-11088 (2020).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 3040</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>&#160;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>&#160;0.020&#34; ID, 0.060&#34;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&#8221; ID, 3/16&#8221; 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

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JoVE Journal

Chemistry

2.9K Views.  Yeshiva University. 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.

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 ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in understanding contaminant mass transport and the ability to decontaminate materials. If a material is contaminated, over time, the transport of highly toxic chemicals (such as chemical warfare agent species) out of the material can result in vapor exposure or transfer to the skin, which can result in percutaneous exposure to personnel who interact with the material. Due to the high toxicity of chemical warfare agents, the release of trace chemical quantities is of significant concern. Mapping subsurface concentration distribution and transport characteristics of absorbed agents enables exposure hazards to be assessed in untested conditions. Furthermore, these tools can be used to characterize subsurface reaction dynamics to ultimately design improved decontaminants or decontamination procedures. To achieve this goal, an inverse analysis mass transport modeling approach was developed that utilizes time-resolved mass spectroscopy measurements of vapor emission from contaminated paint coatings as the input parameter for calculation of subsurface concentration profiles. Details are provided on sample preparation, including contaminant and material handling, the application of mass spectrometry for the measurement of emitted contaminant vapor, and the implementation of inverse analysis using a physics-based diffusion model to determine transport properties of live chemical warfare agents including distilled mustard (HD) and the nerve agent VX.

In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.

The ability to directly characterize chemical transport and interactions that occur within a material (i.e., subsurface dynamics) is a vital component in ...

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

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. ...

Sulfate Separation by Selective Crystallization with a Bis-iminoguanidinium Ligand

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, 1,4-benzene-bis(iminoguanidinium) (BBIG), is demonstrated. The ligand is synthesized as the chloride salt (BBIG-Cl) by in situ imine condensation of terephthalaldehyde with aminoguanidinium chloride in water, followed by crystallization as the sulfate salt (BBIG-SO4). Alternatively, BBIG-Cl is synthesized ex situ in larger scale from ethanol. The sulfate separation ability of the BBIG ligand is demonstrated by selective and quantitative crystallization of sulfate from seawater. The ligand can be recycled by neutralization of BBIG-SO4 with aqueous NaOH and crystallization of the neutral bis-iminoguanidine, which can be converted back into BBIG-Cl with aqueous HCl and reused in another separation cycle. Finally, 35S-labeled sulfate and &#946; liquid scintillation counting are employed for monitoring the sulfate concentration in solution. Overall, this protocol will instruct the user in the necessary skills to synthesize a ligand, employ it in the selective crystallization of sulfate from aqueous solutions, and quantify the separation efficiency.

A protocol for in situ aqueous synthesis of a bis(iminoguanidinium) ligand and its utilization in selective separation of sulfate is presented.

A simple and effective method for selective sulfate separation from aqueous solutions by crystallization with a bis-guanidinium ligand, ...

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

A protocol for the microfluidic spinning and microstructure characterization of regenerated silk fibroin monofilament is presented.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

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 ...

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the methodology for non-substituted pyridines in 1957 in a 12 h process, but no X-ray suitable crystals were obtained. The substituted ring used in the methodology presented here clearly influenced the addition of water molecules into the asymmetric unit, which confers a different nucleophilic strength in 1. The X-ray suitable crystal compound 1 was possible due to the stabilization of the negative charge in the oxygen by the presence of two water molecules where the hydrogen atoms donate positive charge into the ring; such water molecules serve well to construct a supramolecular interaction. The hydrated molecules may be possible for the alkaline system that is reached by adjusting the pH to 10. Importantly, the double methyl substituted ring and a reaction time of 5 h, makes it a more versatile method and with wider chemical applications for future ring insertions.

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Herein, we report the synthesis and crystallization of 3,5-lutidine N-oxide dehydrate by a simple protocol that differs from the classical synthesis of pyridine N-oxide. This protocol utilizes different starting material and involves less reaction time to yield a new solvated supramolecular structure, which crystallizes under slow evaporation.

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the ...