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>

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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 th...

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 ...

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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7.9K 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 ...

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 ...

Challenges in Rheological Characterization of Highly Concentrated Suspensions &#8212; A Case Study for Screen-printing Silver Pastes

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the manifold requirements during processing and application of such complex fluids. In this investigation, measuring protocols for a conclusive assessment of different process relevant rheological parameters have been evaluated. This includes the determination of yield stress, viscosity, wall slip velocity, structural recovery after large deformation and elongation at break as well as tensile force during filament stretching.
The importance of concomitant video recordings during parallel-plate rotational rheometry for a significant determination of rheological quantities is demonstrated. The deformation profile and flow field at the sample edge can be determined using appropriate markers. Thus, measurement parameter settings and plate roughness values can be identified for which yield stress and viscosity measurements are possible. Slip velocity can be measured directly and measuring conditions at which plug flow, shear banding or sample spillover occur can be identified clearly. 
Video recordings further confirm that the change in shear moduli observed during three stage oscillatory shear tests with small deformation amplitude in stage I and III but large oscillation amplitude in stage II can be directly attributed to structural break down and recovery. For the pastes investigated here, the degree of irreversible, shear-induced structural change increases with increasing deformation amplitude in stage II until a saturation is reached at deformations corresponding to the crossover of G' and G'', but the irreversible damage is independent of the duration of large amplitude shear. 
A capillary breakup elongational rheometer and a tensile tester have been used to characterize deformation and breakup behavior of highly filled pastes in uniaxial elongation. Significant differences were observed in all experiments described above for two commercial screen-printing silver pastes used for front side metallization of Si-solar cells.

A protocol for a robust and application relevant rheological characterization of highly concentrated suspensions is presented. Silver pastes used for screen-printing application in solar cell production are employed as model systems.

A comprehensive rheological characterization of highly concentrated suspensions or pastes is mandatory for a targeted product development meeting the ...

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 ...

Chemistry

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 ...

Bioengineering

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 ...

Studying Large Amplitude Oscillatory Shear Response of Soft Materials

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl sulfoxide (DMSO) and xanthan gum in water &#8212; two concentrated polymer solutions used as viscosifiers in foods, enhanced oil recovery, and soil remediation. Understanding the nonlinear rheological behavior of soft materials is important in the design and controlled manufacturing of many consumer products. It is shown how the response to LAOS of these polymer solutions can be interpreted in terms of a clear transition from linear viscoelasticity to viscoplastic deformation and back again during a period. The LAOS results are analyzed via the fully quantitative Sequence of Physical Processes (SPP) technique, using free MATLAB-based software. A detailed protocol of performing a LAOS measurement with a commercial rheometer, analyzing nonlinear stress responses with the freeware, and interpreting physical processes under LAOS is presented. It is further shown that, within the SPP framework, a LAOS response contains information regarding the linear viscoelasticity, the transient flow curves, and the critical strain responsible for the onset of nonlinearity.

We present a detailed protocol outlining how to perform nonlinear oscillatory shear rheology on soft materials, and how to run the SPP-LAOS analysis to understand the responses as a sequence of physical processes.

We investigate the sequence of physical processes exhibited during large amplitude oscillatory shearing (LAOS) of polyethylene oxide (PEO) in dimethyl ...

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 ...

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi River basin due to its highly efficient filter feeding mechanism. The characteristic organs called gill rakers (GRs), found in many such filter feeders, facilitate the efficient filtration of food particles such as phytoplankton that are of a few microns in size.
The motivation to investigate the rheology of the GR mucus stems from our desire to understand its role in aiding the filter feeding process in the silver carp. The mucus-rich fluid, in a &#8216;thick and sticky&#8217; state may facilitate the adhesion of food particulates. The permeation and transport through the GR membrane are&#160;facilitated by the action of external shear forces that induce varying shear strain rates. Therefore, mucus rheology can provide a vital clue to the tremendous outcompeting nature of the silver carp within the pool of filter feeding fish. Based on this it was posited that GR mucus may provide an adhesive function to food particles and act as a transport vehicle to assist in the filter feeding process.
The main objective of the protocol is to determine the yield stress of the mucus, attributed to the minimum shear stress required to initiate flow at which irreversible plastic deformation is first observed across a structured viscoelastic material. Accordingly, rheological properties of the GR mucus, i.e., viscosity, storage, and loss moduli, were investigated for its non-Newtonian, shear-thinning nature using a rotational rheometer. &#160;
A protocol presented here is employed to analyze the rheological properties of mucus extracted from the gill rakers of a silver carp, fished at Hart Creek location of the Missouri River. The protocol aims to develop an effective strategy for rheological testing and material characterization of mucus assumed to be a structured viscoelastic material.

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

This protocol presents a method to perform rheology characterization of mucus that resides on gill rakers (GRs) of the silver carp. Viscoelastic characteristics of GR-mucus, obtained by measuring viscosity, storage and loss moduli, are evaluated for the apparent yield stress to understand the filter feeding mechanism in GRs.

The silver carp, Hypophthalmichthys molitrix, is an invasive planktivorous filter feeder fish that infested the natural waterways of the upper Mississippi ...

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

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

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Przeglądaj więcej filmów

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

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

Challenges in Rheological Characterization of Highly Concentrated Suspensions — A Case Study for Screen-printing Silver Pastes

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

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

Studying Large Amplitude Oscillatory Shear Response of Soft Materials

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

Macro-Rheology Characterization of Gill Raker Mucus in the Silver Carp, Hypophthalmichthys molitrix

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids