Funding for this work was provided by the Procter &amp; Gamble Co. and the American Chemical Society Petroleum Research Fund (54462-DNI7). Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund for partial support of this research. The authors would like to acknowledge Dr. Marco Caggioni for helpful discussions.

1. Fabrication of the Microfluidic Device
<ol>
	<li>Microfluidic stamp fabrication. 
	Note: This step requires the use of volatile materials and should be done in a chemical fume hood.
	<ol>
		<li>Use a negative printed design with the same dimensions as the glass slide (75&#215;50 mm), the channels colored white, and the background colored black (See Figure 1). Print this design on a clear acetate sheet (transparency) with a resolution of 1200 dpi.</li>
		<li>If the dark section of the transparency still allows light through, layer several negatives and adhere using double sided tape.</li>
		<li>Turn on the high intensity ultraviolet (UV) light source and allow it to warm up to a constant output (approximately 30 min). 
		Note: UV protective eyewear should be worn when using the high intensity UV light source.</li>
		<li>Fill three 150 mL Petri dishes with acetone, ethanol and distilled water.</li>
		<li>Place a blank transparency on a flat surface close to the high output UV source, but not directly under the UV light. This will provide a base to fabricate the microfluidic stamp.</li>
		<li>Outline the corners of a 75 &#215; 50 mm glass slide in the center of the blank transparency using a permanent marker and place four glass spacers (approximately 30 &#215; 30 &#215; 1 mm) in the corners of the outlined rectangle.</li>
		<li>Pour approximately 5 mL of UV curable thiol:ene resin in the center of the spacers, then carefully place a 75 x 50 x 1 mm glass slide onto the glass spacers, so that the glue is completely covered by the glass slide with no air bubbles.</li>
		<li>Place the transparency with printed negative on top of the glass slide and move all the above components (from bottom to top: blank transparency, glass spacers and UV glue, glass slide, and negative printed transparency) by dragging the blank transparency carefully under the UV light source.</li>
		<li>Allow the UV light to shine through the negative onto the resin and cure. The cure time can be adjusted to change the height of the channel. A 45 s cure time results in a channel height of 1 mm, using our light source.</li>
		<li>Move the components away from the UV source and remove the negative printed transparency and glass slide. The glass slide with the channels fabricated in UV glue will now be referred to as the microfluidic stamp.</li>
		<li>Discard the transparency and glass spacers.</li>
		<li>Dip the microfluidic stamp in the acetone bath, followed by the ethanol bath. Repeat this step twice. Acetone will degrade the UV glue, so do not leave acetone on the stamp for greater than 10 s.</li>
		<li>While holding the stamp, submerge the stamp in the water and use a cotton swab to remove the rest of the unreacted resin. Do not directly wipe the cured portions, as it will make the channels rough.</li>
		<li>Place the stamp on a paper towel and return to the UV source for at least 30 minutes to ensure complete curing.</li>
	</ol>
	</li>
	<li>Polydimethylsiloxane (PDMS) molding
	<ol>
		<li>Pour 70 g of PDMS base into a clear cup and add the cross-linking agent to the base in a weight ratio of 1:10 crosslinker to base. These are the manufacturer recommended ratios. Do not use a glass container.</li>
		<li>Mix the cross-linker and base thoroughly with a metal stirrer; the mixture should be turbid due to entrapped air bubbles once properly mixed.</li>
		<li>Put the PDMS into a vacuum oven and pull vacuum on the mixed PDMS. Turn off vacuum if the solution begins to overflow from the cup, then resume vacuum after the overflow subsides. The total pressure in the vacuum oven does not matter, as the vacuum only speeds up the degassing process. Leave in the chamber until all bubbles have been evacuated from the mixture (approximately 60 min).</li>
		<li>Place the microfluidic stamp in an empty 150 mm diameter plastic Petri dish, then slowly pour the PDMS into the Petri dish, completely covering the microfluidic stamp. Pour close to the surface of the Petri dish to minimize bubbles reforming in the PDMS.</li>
		<li>Cover the Petri dish and place in a 55 &#176;C oven overnight to cure. Once completely cured, cut the patterned PDMS using a knife and remove from the stamp. Retain excess PDMS for constructing the solvent basin (Protocol step 1.6.3) and PDMS stoppers (Protocol step 2.2.1.1).</li>
		<li>Using a 0.5 mm biopsy punch, cut holes into the following locations: one at each corner, one in the suction chamber near the edge of the channel, six in the sample chamber symmetrically placed 60&#176; apart, and one in the center of the sample chamber. To ensure symmetrically placed holes print a pattern on a sheet of paper and place under the sample chamber. Since the PDMS is clear, you can easily see where the holes need to be placed.</li>
	</ol>
	</li>
	<li>Preparation of sol-gel solution to create glass walls in microfluidic device
	<ol>
		<li>In a 100 mL jar, add 25 mL of 90% ethanol, 25 mL of pH 4 (0.0001 M) hydrochloric acid solution, 25 mL of 98% tetraethoxysilane, and 25 mL of 98% methyltriethoxysilane. Place the 100 mL solution, now referred to as the preconverted fluid, in the microwave uncovered for 10 seconds, then place in an 80 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Device assembly
	<ol>
		<li>Place both the patterned PDMS and a 75 &#215; 50 &#215; 0.10 mm glass slide into the plasma cleaner and set the 3-way valve to the off position.</li>
		<li>Turn on the vacuum pump and allow one minute to evacuate the chamber.</li>
		<li>Put the 3-way valve to flow controller position and let the chamber equilibrate for 5 seconds. The flow controller position allows a small flow rate of air to enter the plasma cleaner, low enough to keep the chamber at low pressures. Turn on the radio frequency (RF) switch to medium for 40 seconds, then turn off the RF switch and vacuum pump.</li>
		<li>Place the 3-way valve to the open position to return the chamber to atmospheric conditions. Remove the patterned PDMS and glass slide.</li>
		<li>Carefully adhere the patterned PDMS to the glass slide by putting the two surfaces in contact with each other.</li>
		<li>Apply UV curable resin to the seams around the patterned PDMS and cure for 5 min under low intensity UV light.</li>
	</ol>
	</li>
	<li>Fabrication of glass walls in the microfluidic channels. 
	Note: This step must be completed within 30 minutes of plasma treatment, as it relies on PDMS surface changes that occur during plasma treatment. The thickness of the layer will be approximately 5-10 &#181;m. This step requires the use of volatile materials and should be done in a chemical fume hood.
	<ol>
		<li>Set a hotplate to 100 &#176;C and prepare four syringes (three 30 mL and one 3 mL) with 18 gauge needles and approximately 30 cm length of clear thermoplastic tubing.</li>
		<li>Fill three 30 mL syringes with ethanol, chloroform and air, respectively. Fill one 3 mL syringe with preconverted fluid (from Protocol step 1.3.1.)</li>
		<li>Insert clear thermoplastic tubing using stainless steel connectors into each of the holes in the patterned PDMS, with the exception of one corner hole. This hole will be used as an inlet, while all others will be outlets.</li>
		<li>Fill the microfluidic device with preconverted fluid from the syringe, then place the microfluidic device on the 100 &#176;C hot plate so the bottom glass is touching the surface of the hot plate.</li>
		<li>Flow 3 mL of preconverted fluid through the microfluidic device over 10 seconds. The thickness of the glass walls is adjusted by changing the flow rate of the preconverted fluid.</li>
		<li>Remove the microfluidic device from the hotplate. Replace the preconverted solution syringe with the air syringe and push out any excess preconverted fluid.</li>
		<li>Replace the air syringe with the chloroform syringe and slowly flow 15 mL of chloroform through the microfluidic device. Replace the chloroform syringe with the ethanol syringe and slowly flow 30 mL of ethanol through the microfluidic device. These steps should take approximately 1 min each.</li>
		<li>Replace the ethanol syringe with the air syringe and flow air through the microfluidic device until dry.</li>
	</ol>
	</li>
	<li>Application of glass supports and solvent basin
	<ol>
		<li>Cut 75 &#215; 10 &#215; 1 mm strips of glass from 75 &#215; 25 &#215; 1 mm glass slides.</li>
		<li>Apply UV curable resin to the glass strips. Place the microfluidic device onto the strips, with the PDMS side facing up. Move under low intensity UV light source for 5 minutes.</li>
		<li>Cut a 30 &#215; 30 mm square of PDMS. Using a biopsy punch, cut a hole large enough to cover the 10 mm sample chamber.</li>
		<li>Place the PDMS square and microfluidic device into the plasma cleaner, then put the 3-way valve to the off position and turn on the vacuum pump.</li>
		<li>Allow one minute to evacuate chamber. Put the 3-way valve to the flow controller position and let the chamber equilibrate for 5 seconds.</li>
		<li>Turn on RF switch to medium for 40 seconds, then turn off RF switch and vacuum pump.</li>
		<li>Place the 3-way valve to the open position to return the chamber to atmospheric conditions.</li>
		<li>Remove the solvent basin and microfluidic device from the plasma chamber and adhere by contacting the surfaces. Be sure to place the solvent basin over the sample chamber.</li>
	</ol>
	</li>
</ol>
2. &#181;2rheology Procedure
<ol>
	<li>Preparation of soft matter samples
	<ol>
		<li>Washing probe particles
		<ol>
			<li>Pipette water and 0.5 &#181;m probes into a microcentrifuge tube at a ratio of water to probes of 10:1. Mix thoroughly using pipette mixing, then place the microcentrifuge tube into the microcentrifuge and spin at 4600 x g for 10 minutes. 
			NOTE: The probe solution used in this work is received suspended in water with an initial concentration of 2.6% solids/volume, and the final concentration of probes used in the sample is 0.1% solids/volume.</li>
			<li>Remove the microcentrifuge tube and pipette out the supernatant. Replace the supernatant with DI water. Repeat centrifugation for a total of three times.</li>
			<li>Place microcentrifuge tube in the sonicator and sonicate on low for 15 minutes to remove any aggregates.</li>
		</ol>
		</li>
		<li>Combining probes and soft matter. 
		NOTE: For this procedure, a colloidal gel was used as the soft matter sample.
		<ol>
			<li>On a 75 &#215; 50 mm glass slide, measure 1 mL of sample material. Pipette 40 &#181;L of probe solution into the center of the sample.</li>
			<li>Gently fold the sample with a metal spatula until completely combined. Scoop the sample into a microcentrifuge tube and centrifuge at 2340 x g for 15 s to remove entrapped air.</li>
			<li>Fill a 1 mL syringe fitted with an 18 gauge needle and clear thermoplastic tubing with the probe/HCO mixture.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Consecutive phase transitions induced by fluid exchange
	<ol>
		<li>Filling the microfluidic device
		<ol>
			<li>To create PDMS stoppers, using excess PDMS from Protocol step 1.2.5, cut a 5 mm square section of PDMS. Using a 0.5 mm diameter biopsy punch, cut a hole halfway into the PDMS stopper. Insert a stainless steel connector into the 0.5 mm diameter hole.</li>
			<li>Connect thermoplastic tubing to three of the corner inlet channels and the suction chamber outlet channel.&#160;Completely fill the device with water using a syringe connected to the device. Ensure there are no bubbles in the sample chamber or in the microfluidic channels. Block the remaining corner hole with PDMS stoppers.</li>
			<li>The solvent basin should be partially filled from step 2.2.1.2; if it is not filled, fill the solvent basin with water.</li>
			<li>Using the syringe from Protocol step 2.1.2.3, inject 10 &#181;L of sample/probe mixture through the center channel into the sample chamber at approximately 2 &#181;L/s, then use a PDMS stopper to block the center channel in the sample chamber.</li>
		</ol>
		</li>
		<li>Collecting microrheological data
		<ol>
			<li>Turn the camera settings to those optimized to minimize static and dynamic particle tracking errors, then switch to the 63&#215; water immersion objective and pipette a drop of water onto the lens.</li>
			<li>Place the microfluidic device on the microscope stage and raise the objective until it is focused on the sample.</li>
			<li>Take videos of the Brownian motion of the probes over time intervals appropriate for the total length of a phase change. For gelation, continue taking videos until probe movement has been completely stopped. 
			&#8203;NOTE: We collect data every 10 min. If we start with a degradation experiment, data are collected until the probes are completely diffusing.</li>
		</ol>
		</li>
		<li>Exchanging fluids in the sample chamber (gravity flow, exchange of lower density fluids with higher density fluids)
		<ol>
			<li>Remove the microfluidic device from the microscope stage.</li>
			<li>Suction the water out of the solvent basin using a transfer pipette, then pipette 4 mL of higher density fluid into the solvent basin.</li>
			<li>Return the microfluidic device to the microscope stage and repeat to Step 2.2.2.3</li>
		</ol>
		</li>
		<li>Exchanging fluids in the sample chamber (suction flow, exchange of higher density fluids with lower density fluids)
		<ol>
			<li>Remove the microfluidic device from the microscope stage and remove the PDMS stopper from the suction chamber.</li>
			<li>Insert a syringe equipped with an 18 gauge needle and clear thermoplastic tubing to the suction chamber channel, and hook the syringe up to a syringe pump. Set the syringe pump to pull at 1 mL/min.</li>
			<li>Remove excess gelling agent in the solvent basin and rinse three times with water by filling the solvent basin and then suctioning out the rinse fluid.</li>
			<li>Begin suction with the syringe pump while adding water to the solvent basin for one minute. Do not let the solvent basin empty completely, as this will pull air into the sample chamber.</li>
			<li>Remove the syringe from the microfluidic device and replace the PDMS stopper on the suction chamber.</li>
			<li>Return the microfluidic device to the microscope stage and continue taking samples</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Continue taking samples at regular intervals during degradation/gelation cycles until the desired number of cycles is completed or there are insufficient probes for measurement.</li>
</ol>

<ol>
	<li>Mitchell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-downsizing+large-scale+biology.">Microfluidics-downsizing large-scale biology.</a> Nat. Biotech. 19, 717-721 (2001).</li><li>Haber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+commercial+applications;+an+industry+perspective.">Microfluidics in commercial applications; an industry perspective.</a> Lab Chip. 6, 1118-1121 (2006).</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, 368-373 (2006).</li><li>Huang, X., Raghavan, S. R., Terech, P., Weiss, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+kinetic+pathways+generate+organogel+networks+with+contrasting+fractality+and+thixotropic+properties.">Distinct kinetic pathways generate organogel networks with contrasting fractality and thixotropic properties.</a> J. Am. Chem. Soc. 128, 15341-15352 (2006).</li><li>Larsen, T. H., Schultz, K. M., Furst, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microrheology+near+the+liquid-solid+transition.">Hydrogel microrheology near the liquid-solid transition.</a> Korea-Aust. Rheol. J. 20, 165-173 (2008).</li><li>Larsen, T. H., Furst, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microrheology+of+the+liquid-solid+transition+during+gelation.">Microrheology of the liquid-solid transition during gelation.</a> Phys. Rev. Lett. 100, 146001 (2008).</li><li>Schultz, K. M., Baldwin, A. D., Kiick, K. L., Furst, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+rheological+screening+to+identify+conditions+of+biomaterial+hydrogelation.">Rapid rheological screening to identify conditions of biomaterial hydrogelation.</a> Soft Matter. 5, 740-742 (2009).</li><li>Switzer, L. H., Klingenberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocculation+in+simulations+of+sheared+fiber+suspensions.">Flocculation in simulations of sheared fiber suspensions.</a> Int. J. Multiph. Flow. 30, 67-87 (2004).</li><li>Wehrman, M. D., Lindberg, S., Schultz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+dynamic+transition+of+hydrogenated+castor+oil+gels+measured+via+multiple+particle+tracking+microrheology.">Quantifying the dynamic transition of hydrogenated castor oil gels measured via multiple particle tracking microrheology.</a> Soft Matter. 12, 6463-6472 (2016).</li><li>Wehrman, M. D., Milstrey, M. J., Lindberg, S., Schultz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+&#181;2rheology+to+quantify+rheological+properties+during+repeated+reversible+phase+transitions+of+soft+matter.">Using &#181;2rheology to quantify rheological properties during repeated reversible phase transitions of soft matter.</a> Lab Chip. 17, 2085-2094 (2017).</li><li>Wehrman, M. D., Lindberg, S. E., Schultz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+shear+on+the+structure+and+rheological+properties+of+a+hydrogenated+castor+oil+colloidal+gel+during+dynamic+phase+transitions.">Impact of shear on the structure and rheological properties of a hydrogenated castor oil colloidal gel during dynamic phase transitions.</a> J. Rheol. , (2018).</li><li>Loh, 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=Dual-responsive+"reversible+micelles".">Dual-responsive "reversible micelles".</a> J. Appl. Polym. Sci. 127, 992-1000 (2013).</li><li>Kern, F., Zana, R., Candau, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+properties+of+semidilute+and+concentrated+aqueous+solutions+of+cetyltrimethylammonium+chloride+in+the+presence+of+sodium+salicylate+and+sodium+chloride.">Rheological properties of semidilute and concentrated aqueous solutions of cetyltrimethylammonium chloride in the presence of sodium salicylate and sodium chloride.</a> Langmuir. 7, 1344-1351 (1991).</li><li>Trappe, V., Prasad, V., Cipelletti, L., Segre, P. N., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jamming+phase+diagram+for+attractive+particles.">Jamming phase diagram for attractive particles.</a> Nature. 411, 772-775 (2001).</li><li>Philipse, A. P., Wierenga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+density+and+structure+formation+in+gels+and+clusters+of+colloidal+rods+and+fibers.">On the density and structure formation in gels and clusters of colloidal rods and fibers.</a> Langmuir. 14, 49-54 (1998).</li><li>Schultz, K. M., Bayles, A. V., Baldwin, A. D., Kiick, K. L., Furst, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+high+resolution+screening+of+biomaterial+hydrogelators+by+mu2rheology.">Rapid, high resolution screening of biomaterial hydrogelators by mu2rheology.</a> Biomacromolecules. 12, 4178-4182 (2011).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+digital+video+microscopy+for+colloidal+studies.">Methods of digital video microscopy for colloidal studies.</a> J. Colloid Interface Sci. 179, 298-310 (1996).</li><li>Mason, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+viscoelastic+moduli+of+complex+fluids+using+the+generalized+Stokes--Einstein+equation.">Estimating the viscoelastic moduli of complex fluids using the generalized Stokes--Einstein equation.</a> Rheol. Actac. 39, 371-378 (2000).</li><li>Mason, T. G., Ganesan, K., van Zanten, J. H., Wirtz, D., Kuo, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+tracking+microrheology+of+complex+fluids.">Particle tracking microrheology of complex fluids.</a> Phys. Rev. Lett. 79, 3282-3285 (1997).</li><li>Mason, T. G., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+frequency-dependent+linear+viscoelastic+moduli+of+complex+fluids.">Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids.</a> Phys. Rev. Lett. 74, 1250-1253 (1995).</li><li>Squires, T. M., Mason, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+mechanics+of+microrheology.">Fluid mechanics of microrheology.</a> Annu. Rev. Fluid Mech. 42, 413-438 (2010).</li><li>Gittes, F., Schnurr, B., Olmsted, P. D., MacKintosh, F. C., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+viscoelasticity:+shear+moduli+of+soft+materials+determined+from+thermal+fluctuations.">Microscopic viscoelasticity: shear moduli of soft materials determined from thermal fluctuations.</a> Phys. Rev. Lett. 79, 3286-3289 (1997).</li><li>Mai, D. J., Brockman, C., Schroeder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+systems+for+single+DNA+dynamics.">Microfluidic systems for single DNA dynamics.</a> Soft Matter. 8 (41), 10560-10572 (2012).</li><li>Tanyeri, M., Ranka, M., Sittipolkul, N., Schroeder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic-based+hydrodynamic+trap:+design+and+implementation.">A microfluidic-based hydrodynamic trap: design and implementation.</a> Lab Chip. 11, 1786-1794 (2011).</li><li>Lee, J. S., Dylla-Spears, R., Teclemariam, N. P., Muller, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+four-roll+mill+for+all+flow+types.">Microfluidic four-roll mill for all flow types.</a> Appl. Phys. Lett. 90, 074103 (2007).</li><li>Crocker, J. C., Grier, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+digital+video+microscopy+for+colloidal+studies.">Methods of digital video microscopy for colloidal studies.</a> J. Colloid Interface Sci. 179 (1), 298-310 (1996).</li><li>Mason, T. G., Weitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+frequency-dependent+linear+viscoelastic+moduli+of+complex+fluids.">Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids.</a> Phys. Rev. Lett. 74 (7), 1250 (1995).</li><li>Schultz, K. M., Furst, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+rheology+in+a+microfluidic+device.">High-throughput rheology in a microfluidic device.</a> Lab on a chip. 11, 3802-3809 (2011).</li><li>Abate, A. R., Lee, D., Do, T., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+coating+for+PDMS+microfluidic+channels+by+sol-gel+methods.">Glass coating for PDMS microfluidic channels by sol-gel methods.</a> Lab Chip. 8, 516-518 (2008).</li><li>Happel, J., Brenner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Low Reynolds Number Hydrodynamics: with special applications to particulate media. , (1965).</li></ol>

There are no disclosures for this work.

The two-layer microfluidic device (Figure 1) can be easily made by following well-documented microfluidic fabrication techniques29. Glass supports are added to the bottom of the device to decrease vibrational effects on probe movement. The glass slide is very thin (0.10 mm) in order to accommodate the working distance of the microscope objective. This makes the device susceptible to small vibrations in the building and sample environment that are then measured with the high-speed camera. The glass supports successfully negate these external stimuli. The short working distance objective is chosen to collect precise MPT data which requires: (1) at least 4 pixels per particle and (2) a limited working distance to avoid imaging particles that are not in the 2D plane.
The design of this device, Figure 1a-c, is based on previous investigations that combine microrheology with microfluidics. Schultz and Furst developed &#181;2rheology, using the same microfluidic fabrication technique used for our work. Their device was designed to create 50 - 100 samples of hydrogel materials, separated by a continuous phase, with gradients in both the polymer backbone and cross-linker concentration16,29. Additionally, techniques using converging microfluidic streams have been shown to be useful for the trapping of single particles or macromolecules25,26. We have combined the fabrication technique by Schultz and Furst and their idea of taking microrheological measurements in a microfluidic device in our new device and have scaled-up microfluidic trapping designs. In our design, we create 1 mm square channels that are rounded when a layer of glass is made on the inside of the channels using sol-gel chemistry30. The channel dimensions are chosen to avoid interactions between the probe particles and the walls of the microfluidic device31. For our probe size (0.5 &#181;m) and channel height (1 mm) the hydrodynamic force from the glass on the probe is on the order of F &#8776; 10-18 N, indicating this force from the glass wall will not have a noticeable effect on the movement of the probe particles31. Additionally, probe particles would have to be within 10 &#181;m of the wall to impact the motion of the particles due to particle interactions with the wall. All data is collected more than 10 &#181;m from the wall. Glass is fabricated in the microfluidic channels to prevent solvent uptake by PDMS during our experimental time scale, which is on the order of hours. By using a fabrication technique that allows for 10 &#181;L sample sizes (corresponding to a channel height of 1 mm) and converging microfluidic streams, we can scale up the trapping method from single molecules to 10 &#181;L.
The 2-layer design is incorporated to prevent flow instabilities that occur in a single layer device. During early iterations of this work the entire device was one layer. Two input streams (each with the same fluid, either water or gelling agent) started from two corners of the device and flowed only through the long channels running the length of the device, tangential to the sample chamber. However, any flow instability between the channels resulted in flow through the sample chamber and a loss of sample, ending the experiment. The second layer, along with the addition of the large suction chamber, removes these instabilities by only using one suction source and creating equal pressure around the sample, trapping the sample during experiments. The second layer has a very simple design and is meant to hold the new surrounding fluid above the sample chamber.
&#181;2rheology data are successfully collected using the above protocol. The first stage of the setup, filling the device, is critical to a successful experiment. Improper filling can result in bubbles in the channels or in the sample chamber, which will negatively impact both the microrheology and the function of the microfluidic device (Figure 2). The easiest way to avoid bubbles in the microfluidic device is by completely filling the syringe (making sure that there are no bubbles) prior to filling the device. Alternatively, bubbles can be removed by either introducing a large flow rate of solvent (by pressing the plunger of the syringe harder) or by tapping the device gently until the bubbles move to an exit channel. Bubbles within the sample chamber can cause directed motion of probes at the air-liquid interface. MPT measurements require probes to undergo purely Brownian motion to measure material properties. Bubbles in the microfluidic channels also affect flow during fluid exchange, which can then cause changes in pressure and move the gel sample out of the chamber. Both of these issues can be avoided by ensuring that the device and solvent chamber are completely filled with fluid prior to sample injection. The sample input step will impart some shear on the sample, however it does not negatively impact the rheological properties and structure of the material. This was verified by bulk rheological measurements of samples loaded without shear and samples loaded onto the rheometer using the same input techniques as the microfluidic device. After the device is properly filled, the procedure outline above ensures proper function during experiments.
There is minimal material loss during fluid exchange. This is evident during experiments by: (1) the ability of the colloidal material to have enough fibers to continue to form a gel network and (2) the concentration of probe particles remaining high enough to collect statistically significant MPT measurements. Additionally, a single sample is capable of up to nine phase transitions, further indicating minimal loss during fluid exchange. The total number of observable transitions depends on the amount of probes lost due to diffusion out of the sample when it is in the sol phase. Probes are diffusive when the material is a sol, and a small amount of them will diffuse out of the sample after each gel-sol transition. The amount of probes will limit the amount of transitions possible for the device.
We have demonstrated that this device can be used to measure material properties and determine microstructure of soft matter during repeated phase transitions. The symmetry of the inlet ports entering the sample chamber traps a sample in place, allowing repeated phase transitions on a single sample without loss of the sample. The device design can be easily adapted to different systems, including polymeric hydrogels that change structure due to changes in pH or biological and surfactant materials that are responsive to changes in salt concentration, allowing reproducible results to determine the rheological properties and structure of a gel system during consecutive phase changes.

Phase transitions in soft matter can change the scaffold structure, which has implications in the processing and final stability of the material1,2,3. The characterization of soft materials during dynamic phase transitions provides essential information about the relationship between structural evolution and equilibrium structure and rheological properties. For example, many home care products require a phase change during consumer use. Also, during manufacturing, processing steps, including dilution and mixing, can impart shear affecting the rheological properties and final microstructure of the product. Understanding the rheological properties throughout a phase change ensures that the product performs as designed. Additionally, if forces alter the starting rheology of the material during manufacturing, phase transitions can yield unexpected and undesired results, changing the intended function and effectiveness.At the critical gelation point, defined as the point where the material transitions from a solution of associated colloids or polymers to a sample-spanning gel network, material properties change drastically with slight changes to association. Any modification to the structure at the critical gel point can impact the end product4. During these dynamic transitions, soft materials have weak mechanical properties and measurements that use classical experimental techniques can be within the measurement noise limit5,6,7. To account for this, techniques such as microrheology, which is sensitive in the low moduli range (10-3 - 4 Pa), are used to characterize the weak incipient gel during dynamic evolution. Some materials are susceptible to changes in microstructure due to external forces, which presents a challenge during characterization, as any transfer of material or fluid can affect the structure and, ultimately, the final material properties. To avoid altering the material microstructure, we have developed a microfluidic device that can exchange the environmental fluid around a sample while minimizing shear. By exchanging the fluid environment, changes in rheological properties and microstructure are measured during phase transitions with minimal contributions from shear. The device is combined with multiple particle tracking microrheology (MPT) in a technique called &#181;2rheology. This technique is used to quantify material properties during consecutive phase changes of a gel in response to an external driving force. The technique will be illustrated using a fibrous colloidal gel, hydrogenated castor oil (HCO)9,10,11.
Gel scaffolds can undergo changes in association and dissociation due to their sample environment12,13,14,15. The driving force for gelation and degradation are material specific and must be tailored for each material of interest. &#181;2rheology can be used to characterize gel systems that respond to external stimuli, including colloidal and polymeric networks. Altering pH, osmotic pressure or salt concentration are examples of driving forces that can induce changes in the material microstructure. For example, HCO undergoes controlled phase transitions by creating an osmotic pressure gradient. When a concentrated HCO gel sample (4 wt% HCO) is submerged in water, the attractive forces between colloidal particles weaken, causing degradation. Alternatively, when a dilute solution of HCO (0.125 wt% HCO) is contacted with a hydrophilic material (referred to as the gelling agent and composed of mostly glycerin and surfactant), the attractive forces return, causing gelation. This gel system will be used to show the operation of the device as a tool for measuring consecutive phase transitions on a single sample9,10. To characterize these gel scaffolds during dynamic transitions and the delicate incipient gel structure at the critical phase transition, we use MPT to characterize these materials with high spatio-temporal resolution.
Microrheology is used to determine gel properties and structure, especially at the critical transition, of an array of soft materials, including colloidal and polymeric gels5,6,9,16. MPT is a passive microrheological technique that uses video microscopy to record the Brownian motion of fluorescent probe particles embedded within a sample. The particle positions throughout the videos are precisely determined to within 1/10th of a pixel using classical tracking algorithms17,18. The ensemble averaged mean-squared displacement (MSD, (&#916;r2(t))) is calculated from these particle trajectories. The MSD is related to material properties, such as the creep compliance, using the Generalized Stokes-Einstein Relation17,19,20,21,22,23. The state of the material is determined by calculating the logarithmic slope of the MSD curve as a function of lag time, &#945;,
<img alt="Equation 1" src="/files/ftp_upload/57429/57429eq1.jpg" />
where t is the lag time, and comparing it to the critical relaxation exponent, n. n is determined using time-cure superposition, a well-documented technique that was modified to analyze MPT data by Larsen and Furst6. By comparison of n to &#945; the state of the material is quantitatively determined. When &#945; &#62; n the material is a sol, and when &#945; &#60; n the material is a gel. Previous work has characterized the HCO system using microrheology to determine the critical relaxation exponent9. Using this information, we precisely determine when the material transitions from a gel to a sol during an experiment. Additionally, the non-Gaussian parameter, &#945;NG, can be calculated to determine the extent of structural heterogeneity of a system,
<img alt="Equation 2" src="/files/ftp_upload/57429/57429eq2.jpg" />
where &#916;x(t) is the one-dimensional particle movement in the x direction. Using MPT, we can characterize a single phase transition, but by characterizing materials with MPT in a microfluidic device, we are able to manipulate the surrounding fluid environment and collect data of several phase transitions on a single gel sample.
This microfluidic device is designed to investigate the critical transitions of a single gel sample that undergoes phase changes in response to changes in the surrounding fluid environment. The device exchanges fluid surrounding the sample when it is either in the gel or sol state by locking the sample in place to induce a phase transition while minimizing shear. A solvent basin is located directly above the sample chamber, which are connected by six symmetrically spaced inlet channels. This symmetry allows for the exchange of fluid from the solvent basin to the sample chamber while creating equal pressure around the sample, locking it in place. There have been several studies that use this technique for single particle and DNA trapping, but this work scales up the volume from single molecules to samples that are approximately 10 &#181;L24,25,26. This unique design also enables real-time microrheological characterization during phase transitions.
&#181;2rheology is a robust technique that is applicable to many soft matter systems. The technique described in this paper was designed for colloidal gels, but it can be easily adapted to other materials such as polymer or micellar solutions. With this technique, we determine not just how phase transitions affect the equilibrium material properties, but also how different processing steps can have lasting effects on the rheological evolution of the material and the final scaffold structure and properties.

<table><tbody><tr><td>150 x 15 mm Petri Dish</td><td>Corning, Inc.</td><td>351058</td><td></td></tr><tr><td>75 x 50 x 0.15 mm glass slide</td><td>Fisher Scientific</td><td>Custom</td><td></td></tr><tr><td>75 x 50 x 1.0 mm glass slide</td><td>Fisher Scientific</td><td>12-550-C</td><td></td></tr><tr><td>75 x 25 x 1.0 mm glass Slide</td><td>Fisher Scientific</td><td>12-550-A3</td><td></td></tr><tr><td>22 x 22 Glass cover slips</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Acetone, 99.5%</td><td>VWR Analytical</td><td>67-64-1</td><td></td></tr><tr><td>Low intensity UV source</td><td>UVP</td><td>UVL-56</td><td></td></tr><tr><td>Chloroform, 99.9%</td><td>Fisher Chemical</td><td>C298-500</td><td></td></tr><tr><td>Cotton Swabs</td><td>Q-tips</td><td>83289205</td><td></td></tr><tr><td>Ethanol, 90%</td><td>Fisher Chemical</td><td>A962-4</td><td></td></tr><tr><td>Fluoresbrite&amp;reg; YG Carboxylate Microspheres 0.50&amp;micro;m</td><td>Polysciences, Inc.&amp;nbsp;</td><td>15700-10</td><td></td></tr><tr><td>High-Intensity UV Lamp</td><td>Spectroline Corp.</td><td>SB-100P</td><td></td></tr><tr><td>Hot plate</td><td>Corning, Inc.</td><td>PC-420</td><td></td></tr><tr><td>Hydrochloric Acid, 6N</td><td>Ricca Chemical Company</td><td>3750-32</td><td></td></tr><tr><td>Methyltriethyoxysilane, 98%</td><td>Acros Organics</td><td>174622500</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma, Inc.</td><td>PDC-32G</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Robert McKwown Company</td><td>2065622</td><td></td></tr><tr><td>Sonicator</td><td>Branson, Emerson Electric</td><td>1800</td><td></td></tr><tr><td>Steel connectors, ID 0.023 inch</td><td>New England Small Tube Corp.</td><td>Custom</td><td></td></tr><tr><td>Tetraethoxysilane, 98%</td><td>Alfa Aesar</td><td>A14965</td><td></td></tr><tr><td>Thiol-ene Resin (UV curable)</td><td>Norland Products, Inc.&amp;nbsp;</td><td>NOA81</td><td></td></tr><tr><td>Transparency</td><td>Staples Inc.&amp;nbsp;</td><td>21828</td><td></td></tr><tr><td>Tygon tubing, ID 1/32 inch</td><td>McMaster-Carr</td><td>E-3603</td><td></td></tr><tr><td>Vacuum oven</td><td>Fisher Scientific</td><td>282A</td><td></td></tr><tr><td>Biopsy punch 8 mm</td><td>World Precision Instruments</td><td>504535</td><td></td></tr><tr><td>Bioposy punch 0.5 mm</td><td>World Precision Instruments</td><td>504528</td><td></td></tr><tr><td>Syringe, 30 mL</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Syringe, 3 mL</td><td>BD</td><td>309651</td><td></td></tr><tr><td>Needle, 18 gauge</td><td>BD</td><td>305195</td><td></td></tr><tr><td>Microcentrifuge tube, 1.5 mL</td><td>Eppendorf</td><td>22-36-320-4</td><td></td></tr><tr><td>High-speed Camera</td><td>Vision Research</td><td>Miro M120&amp;nbsp;</td><td></td></tr><tr><td>Microscope</td><td>Carl Zeiss AG</td><td>Zeiss Observer, Z1</td><td></td></tr><tr><td>Syringe pump</td><td>New Era Pump Systems</td><td>NE-300</td><td></td></tr><tr><td>Hydrogenated castor oil</td><td>Procter &amp;amp; Gamble</td><td>N/A</td><td></td></tr><tr><td>Af&amp;iacute;cio MP 6002 Printer</td><td>Ricoh Company, Ltd.</td><td>415877</td><td></td></tr></tbody></table>

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.

A two-layered microfluidic device is constructed with PDMS (Figure 1a,b), which is patterned on a microfluidic stamp. The design of the stamp is shown in Figure 1c. Improper experimental setup can result both in errors in passive microrheology and microfluidic flows during surrounding fluid exchange (Figure 2). Examples of improper experimental setup are detailed in the Discussion section. During device operation, the surrounding fluid around a gel sample is exchanged. This fluid exchange occurs when the material is in both the gel and the sol phase. This microfluidic device design has the ability to exchange fluid without substantial loss of gelling material and embedded probe particles. Two types of fluid exchanges are used, gravity flow (when going from lower to higher density fluid) or suction flow (when going from higher to lower density fluid). In our HCO gel system, suction flow is used to induce degradation while gravity flow is used to induce gelation. Both flows impart minimal shear on the sample, with shear on the order of 0.01 Pa and 1 Pa for suction and gravity flow, respectively. These shears are under the yield stress of the gel, which is on the order of 10 Pa9,10.
The state, structure and rheological properties of the gel sample are quantitatively characterized using multiple particle tracking microrheology (MPT). In MPT, probe particles are tracked using classic tracking algorithms27,28. The ensemble averaged mean-squared displacement (MSD) is calculated from particle trajectories (Figure 3) during successive phase transitions. Microrheology data (Figure 3) show that successive phase transitions are achieved in the HCO gel system. The MSD curves alternate between &#945; &#8594; 0 (gel) and &#945; &#8594; 1 (sol). The magnitude of the MSD curves is above the lower measurable MSD limit of this experimental setup (0.001 &#181;m2). This limit is determined experimentally by adhering probes to glass in a sample chamber, which is done by allowing particles to settle under gravity overnight. The ensemble averaged MSD of the arrested probe particles is measured to obtain the lower limit of the MSD, which is dependent on the specific experimental apparatus.
The logarithmic slope of the MSD curve (&#945;) and the non-Gaussian parameter (&#945;NG) are calculated and the state of the material is quantitatively determined by comparison to the critical relaxation exponent (n) (Figure 4). For this experiment, using HCO as a model gel, a total of nine transitions are measured. The material starts as a gel (&#945; &#8594; 0) and five degradations (&#945; &#8594;&#160;1) and four gelations (&#945; &#8594; 0) are measured over 1,500 minutes. The timing of each phase transition is material dependent. Phase changes are induced by the exchange of fluid. When there is no fluid exchange, as shown by the extended period in the sol phase between 800 - 1400 min, probe particles remain within the sample and there are no changes to the rheological properties. Once a fluid exchange takes place, the material again gels.
The data we obtain from this device provides information on the rheological properties and structure of the material in several ways. We do not measure changes in equilibrium rheological properties from repeated phase changes. This is apparent by &#945; in each phase returning to the same value. When the material is in the sol phase, it reaches &#945;, = 0.90, and when in the gel phase, &#945; = 0.20. The value of &#945; in the sol phase indicates that the material retains some structure even after degradation. A colloidal system that has completely degraded into a solution of colloidal particles will have &#945; = 1.0, while the equilibrium value in the sol phase for HCO is &#945; = 0.90, indicating some structure is retained. Additionally, colloidal rearrangement can occur immediately after fluid exchange, which is indicated by an increase in &#945;. Finally, the non-Gaussian parameter, &#945;NG, which quantifies heterogeneity of the material, shows that the gel undergoes an increase in structural heterogeneity during degradation (gel to sol) transition. This is apparent by the peak in &#945;NG.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57429/57429fig1.jpg" /> 
Figure 1:&#160;Microfluidic device. (a) Image of the two-layer microfluidic device that traps a sample in place while the surrounding fluid is exchanged. The first layer has two chambers, one for the sample and another for suction. Holes are located in each of the corners, as well as one in the suction chamber and seven in the sample chamber. Above the sample chamber a second layer of PDMS is adhered to the device to act as a solvent basin. (b) The sample is injected into the sample chamber through the middle channel. The sample is locked in place during fluid transfer by symmetric inlet streams into the sample chamber creating equal pressure around the sample. (c) The design used to create the microfluidic stamp for the 1st layer of the device. The chambers each have a diameter of 10 mm, while the channels have a width of 1 mm. The resulting height for both the channels and chambers is 1 mm after 45 s of UV exposure. Reproduced from Wehrman et al. 201710 with permission from The Royal Society of Chemistry. <a href="/files/ftp_upload/57429/57429fig1large.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/57429/57429fig2.jpg" /> 
Figure 2: Image of an improperly filled microfluidic device. Air bubbles injected into the device can have negative effects on both microrheological data (due to directed motion of particles at the gas-liquid interface) and microfluidic flows during solution exchange. <a href="/files/ftp_upload/57429/57429fig2large.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/57429/57429fig3.jpg" /> 
Figure 3:&#160;Mean-squared displacement curves of a hydrogenated castor oil gel during degradation (a,c) and gelation (b,d). The 2nd (a,b) and 3rd (c,d) phase transitions are shown out of 9 total transitions. The critical transition point (gel - sol or sol - gel) is denoted by a dashed line at n = &#945; = 0.77 and represents each phase change with MSD curves above the line are a sol and below a gel. Reproduced from Wehrman et al. 201710 with permission from The Royal Society of Chemistry. <a href="/files/ftp_upload/57429/57429fig3large.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/57429/57429fig4.jpg" /> 
Figure 4:&#160;Logarithmic slope (, closed) and non-Gaussian parameter (&#945;NG, open) for a single sample of 4 wt% hydrogenated castor oil gel during repeated phase transitions. The critical transition is denoted by a horizontal dashed line (n = 0.77) and was determined from previous microrheological experiments9,10. Vertical lines indicate solvent exchange, with white backgrounds indicating that there is water in the solvent basin and shaded grey areas that there is gelling agent in the solvent basin. A change in the color between white and grey indicates a reverse of the osmotic gradient. If the color does not change at a vertical line, this indicates that the sample chamber is being re-flushed with the same solvent. This occurs when there is insufficient fluid removed, and often happens when the higher density fluid is in the sample chamber due to its large viscosity. Reproduced from Wehrman et al. 201710 with permission from The Royal Society of Chemistry. <a href="/files/ftp_upload/57429/57429fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions at JoVE.com

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

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 ...

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7.8K Views.  Lehigh University. 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.

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

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is presented. The SANS shear cell consists of a concentric cylinder Couette geometry that is sealed and rotating about a horizontal axis so that the vorticity direction of the flow field is aligned with the neutron beam enabling scattering from the 1-2 plane of shear (velocity-velocity gradient, respectively). This approach is an advance over previous shear cell sample environments as there is a strong coupling between the bulk rheology and microstructural features in the 1-2 plane of shear. Flow-instabilities, such as shear banding, can also be studied by spatially resolved measurements. This is accomplished in this sample environment by using a narrow aperture for the neutron beam and scanning along the velocity gradient direction. Time resolved experiments, such as flow start-ups and large amplitude oscillatory shear flow are also possible by synchronization of the shear motion and time-resolved detection of scattered neutrons. Representative results using the methods outlined here demonstrate the useful nature of spatial resolution for measuring the microstructure of a wormlike micelle solution that exhibits shear banding, a phenomenon that can only be investigated by resolving the structure along the velocity gradient direction. Finally, potential improvements to the current design are discussed along with suggestions for supplementary experiments as motivation for future experiments on a broad range of complex fluids in a variety of shear motions.

A shear cell is developed for small-angle neutron scattering measurements in the velocity-velocity gradient plane of shear and is used to characterize complex fluids. Spatially resolved measurements in the velocity gradient direction are possible for studying shear-banding materials. Applications include investigations of colloidal dispersions, polymer solutions, and self-assembled structures.

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Chemistry

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Bioengineering

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for the purpose of investigating phase change of those liquid segments. The magnetic beads were externally controlled using a magnet, allowing for the beads to bridge the air valve between the adjacent liquid segments. A hydrophobic coating was applied to the inner surface of the tube to enhance the separation between two liquid segments. The applied magnetic field formed an aggregate cluster of magnetic beads, capturing a certain liquid amount within the cluster that is referred to as carry-over volume. A fluorescent dye was added to one liquid segment, followed by a series of liquid transfers, which then changed the fluorescence intensity in the neighboring liquid segment. Based on the numerical analysis of the measured fluorescence intensity change, the carry-over volume per mass of magnetic beads has been found to be ~2 to 3 &#181;l/mg. This small amount of liquid allowed for the use of comparatively small liquid segments of a couple hundred microliters, enhancing the feasibility of the device for a lab-in-tube approach. This technique of applying small compositional variation in a liquid volume was applied to analyzing the binary phase diagram between water and the surfactant C12E5 (pentaethylene glycol monododecyl ether), leading to quicker analysis with smaller sample volumes than conventional methods.

Here, we present a protocol to investigate multi-component phase diagrams using externally controlled magnetic beads as liquid carriers in a lab-in-tube approach. This approach can aid in applications that seek to gather further information on phase change in complex liquid systems.

Magnetic beads with ~1.9 &#181;m average diameter were used to transport microliter volumes of liquids between contiguous liquid segments with a tube for ...

Dielectric RheoSANS &#8212; Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids

A procedure for the operation of a new dielectric RheoSANS instrument capable of simultaneous interrogation of the electrical, mechanical and microstructural properties of complex fluids is presented. The instrument consists of a Couette geometry contained within a modified forced convection oven mounted on a commercial rheometer. This instrument is available for use on the small angle neutron scattering (SANS) beamlines at the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR). The Couette geometry is machined to be transparent to neutrons and provides for measurement of the electrical properties and microstructural properties of a sample confined between titanium cylinders while the sample undergoes arbitrary deformation. Synchronization of these measurements is enabled through the use of a customizable program that monitors and controls the execution of predetermined experimental protocols. Described here is a protocol to perform a flow sweep experiment where the shear rate is logarithmically stepped from a maximum value to a minimum value holding at each step for a specified period of time while frequency dependent dielectric measurements are made. Representative results are shown from a sample consisting of a gel composed of carbon black aggregates dispersed in propylene carbonate. As the gel undergoes steady shear, the carbon black network is mechanically deformed, which causes an initial decrease in conductivity associated with the breaking of bonds comprising the carbon black network. However, at higher shear rates, the conductivity recovers associated with the onset of shear thickening. Overall, these results demonstrate the utility of the simultaneous measurement of the rheo-electro-microstructural properties of these suspensions using the dielectric RheoSANS geometry.

Here, we present a procedure for the measurement of simultaneous impedance, rheology and neutron scattering from soft matter materials under shear flow.

A procedure for the operation of a new dielectric RheoSANS instrument capable of simultaneous interrogation of the electrical, mechanical and ...

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

High-Contrast and Fast Photorheological Switching of a Twist-Bend Nematic Liquid Crystal

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such as on-demand switchable adhesion technologies, actuators, molecular clutches, and nano-/microscopic mass transporters. Recently it was found that through a special solid-liquid transition, rheological properties can exhibit significant changes, thus providing suitable smart viscoelastic materials. However, designing materials with such a property is complex, and forward and backward switching times are usually long. Therefore, it is important to explore new working mechanisms to realize solid-liquid transitions, shorten the switching time, and enhance the contrast of rheological properties during switching. Here, a light-induced crystal-liquid phase transition is observed, which is characterized by means of polarizing light microscopy (POM), photorheometry, photo-differential scanning calorimetry (photo-DSC), and X-ray diffraction (XRD). The light-induced crystal-liquid phase transition presents key features such as (1) fast switching of crystal-liquid phases for both forward and backward reactions and (2) a high contrast ratio of viscoelasticity. In the characterization, POM is advantageous in offering information on the spatial distribution of LC molecule orientations, determining the type of liquid crystalline phases appearing in the material, and studying the orientation of LCs. Photorheometry allows measurement of a material&#8217;s rheological properties under light stimuli and can reveal the photorheological switching properties of materials. Photo-DSC is a technique to investigate thermodynamic information of materials in darkness and under light irradiation. Lastly, XRD allows studying of microscopic structures of materials. The goal of this article is to clearly present how to prepare and measure the discussed properties of a photorheological material.

This protocol demonstrates the preparation of a photorheological material that exhibits a solid phase, various liquid crystalline phases, and an isotropic liquid phase by increasing temperature. Presented here are methods for measuring the structure-viscoelasticity relationship of the material.

Smart viscoelastic materials that respond to specific stimuli are one of the most attractive classes of materials important to future technologies, such ...

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

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Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Microfluidic Mixers for Studying Protein Folding

Phase Diagram Characterization Using Magnetic Beads as Liquid Carriers

Dielectric RheoSANS — Simultaneous Interrogation of Impedance, Rheology and Small Angle Neutron Scattering of Complex Fluids

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

High-Contrast and Fast Photorheological Switching of a Twist-Bend Nematic Liquid Crystal