We acknowledge financial support from Research Growth Initiative (Award No. 101X340), National Science Foundation, Major Research Instrumentation Program (Grant No. PHY-1626450), Greater Milwaukee Foundation (Shaw Award) and University of Wisconsin System (Applied Research Grant).

1. Reagents Solution Preparation
<ol>
	<li>Prepare a starting protein solution by dissolving/diluting the protein of interest to the desired concentration, using a Tris buffer [20 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl, pH 7.4]. 
	NOTE: The smallest protein concentration for which cross-linking leads to hydrogels depends on the protein used and is typically &#62; 1 mM.</li>
	<li>Prepare stocks of ammonium persulfate (APS) (1 M) and tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]2+</s...

<ol>
	<li>Cao, Y., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+chemical+denaturants+affect+the+mechanical+folding+and+unfolding+of+proteins?.">How do chemical denaturants affect the mechanical folding and unfolding of proteins?.</a> Journal of Molecular Biology. 375 (1), 316-324 (2008).</li><li>Carrion-Vazquez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+unfolding+of+a+single+protein:+a+comparison.">Mechanical and chemical unfolding of a single protein: a comparison.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3694-3699 (1999).</li><li>Wiita, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+chemistry+of+thioredoxin+catalysis+with+force.">Probing the chemistry of thioredoxin catalysis with force.</a> Nature. 450 (7166), 124-127 (2007).</li><li>Lv, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designed+biomaterials+to+mimic+the+mechanical+properties+of+muscles.">Designed biomaterials to mimic the mechanical properties of muscles.</a> Nature. 465 (7294), 69-73 (2010).</li><li>Plumere, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+redox+hydrogel+protects+hydrogenase+from+high-potential+deactivation+and+oxygen+damage.">A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage.</a> Nature Chemistry. 6 (9), 822-827 (2014).</li><li>Kong, N., Peng, Q., Li, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+Designed+Dynamic+Protein+Hydrogels+with+Reversibly+Tunable+Mechanical+Properties.">Rationally Designed Dynamic Protein Hydrogels with Reversibly Tunable Mechanical Properties.</a> Advanced Functional Materials. 24 (46), 7310-7317 (2014).</li><li>Khoury, L. R., Nowitzke, J., Shmilovich, K., Popa, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+Biomechanical+Properties+of+Protein-Based+Hydrogels+Using+Force-Clamp+Rheometry.">Study of Biomechanical Properties of Protein-Based Hydrogels Using Force-Clamp Rheometry.</a> Macromolecules. 51 (4), 1441-1452 (2018).</li><li>Auton, M., Rosgen, J., Sinev, M., Holthauzen, L. M. F., Bolen, D. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-Based+Rubber-Like+Hydrogels.">Elastin-Based Rubber-Like Hydrogels.</a> Biomacromolecules. 17 (7), 2409-2416 (2016).</li><li>Fusi, L., Brunello, E., Yan, Z., Irving, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thick+filament+mechano-sensing+is+a+calcium-independent+regulatory+mechanism+in+skeletal+muscle.">Thick filament mechano-sensing is a calcium-independent regulatory mechanism in skeletal muscle.</a> Nature Communications. 7, (2016).</li><li>McDonald, K. 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The authors have nothing to disclose.

Herein, we describe a force-clamp rheometry technique to investigate the biomechanical response of low-volume protein-based hydrogels. Additionally, a protocol is provided to synthesize a uniform cylindrical low-volume protein hydrogel sample. A protocol is also presented which describes how to tie different types of protein-based hydrogels with various elasticities without causing any mechanical deformation or damage to the protein-based hydrogel samples or slippage of the gel on the hooks. The analog PID system, togeth...

Apart from having unique physical properties, protein-based hydrogels hold the promise of revolutionizing force spectroscopy by enabling the measurement of several billion molecules in one &#39;pull&#39;, thus enabling the study of proteins in crowded environments, similar to those encountered in skin and other tissues. Protein domains remain folded inside hydrogels, allowing the study of their biomechanical response to force, binding partners, and chemical conditions. Additionally, the biomechanical response of protein domains inside hydrogels resembles the response seen with single-molecule force spectroscopy techniques. For example, chemical denaturants and oxidizing agents decrease the stability of the folded state, both at the single protein domain level1,2,3 and at the macroscopic level4,5,6,7. Similarly, osmolytes increase the stability of single proteins8,9, leading to a decrease in the viscoelastic response&#160;of hydrogels, for the same force conditions7,10.
Several approaches have been implemented to synthesize protein-based hydrogels, by either using physical interactions11,12 or covalent cross-linking4,13. Covalent reactions allow for fixed cross-linking locations and these hydrogels&#160;can recover the initial state upon a removal of the mechanical or chemical perturbations. A successful approach for covalent cross-linking relies on forming covalent carbon-carbon bonds between exposed tyrosine amino acids using ammonium persulfate (APS) as an oxidant and a ruthenium (II) salt as an initiator (Figure 1)14. Upon exposure to white light, a solution of concentrated proteins can be turned into a hydrogel. By controlling when the reaction starts, the protein-APS mix can be injected into any casting form, such as polytetrafluoroethylene (PFTE) tubes (Figure 1B and 1C), allowing the use of an extremely small solution volume15. Furthermore, the use of white light to trigger the cross-linking reaction results in a limited bleaching of fluorescent proteins and allows the formulation of composite hydrogels with fluorescent markers (Figure 1). Other protein-based hydrogel formation methods use cross-linking based on the SpyTag-SpyCatcher covalent interaction16, amine cross-linking via glutaraldehyde13, or biotin-streptavidin interactions17.
Dynamic mechanical analysis (DMA) is currently a technique extensively used to study polymer-based hydrogels13,18. While DMA can apply constant force protocols to biomaterials, it requires Young&#39;s moduli over 10 kPa, and large sample volumes of more than 200 &#181;L19. Due to these limitations, protein hydrogels are generally too soft to be investigated by this technique. As engineered polyproteins are harder to synthesize than polymers, since they require a living system to produce, such high volumes are inefficient, at best4,15. Furthermore, most biological tissues are softer than 10 kPa. Several approaches were developed for biological samples, especially in the study of muscle elasticity20,21. These techniques can also operate under feedback to apply constant force but are optimized for samples with small diameters (in the micron range) exposed to force for very short times (typically less than 1 s).
Protein-based hydrogels were successfully studied with modified rheometry techniques. For example, casting the hydrogel in a ring shape allows the use of extensional rheometry to measure the change in the experienced force as a function of extension4,22. Other approaches for studying the rheological properties of protein-based hydrogels use controlled shear-stress rheometry. These techniques can also achieve low sample volume and tolerate soft materials. However, these methods lack the ability to mimic the pulling forces that cause protein unfolding in vivo, and Young&#39;s modulus is calculated based on complex theories that require various assumptions and corrections23.
We have recently reported a new approach that utilizes a small volume of proteins, polymerized inside tubes with&#160;diameters &#60; 1 mm. Our first implementation of this technique was operating in length-clamp mode, where the gel was extended following the desired protocol15. In this method, the proteins experience a continuous change in both extension and force while the domains unfold, making the data interpretation cumbersome. Recently, we have reported a new force-clamp rheometry technique, where a feedback loop can expose low-volume protein hydrogels to a predefined force protocol7 (Figure 2). An analog PID system compares the force measured by the force sensor with the set point sent from the computer and adjusts the gel extension by moving the voice coil to minimize the difference between the two inputs. This &#39;clamping&#39; of the force now allows for new types of experiments to measure the biomechanics of protein hydrogels.
In the force-ramp mode, a tethered protein hydrogel experiences a constant increase and decrease of force with time. The PID compensates for any viscoelastic deformation by changing the extension in a non-linear way, depending on the type of protein and hydrogel formulation. The main advantage of force ramp is that it allows the quantification of standard parameters, such as Young&#39;s modulus and energy dissipation, due to an unfolding and refolding of protein domains.
In constant-force mode, the applied force changes in a step-like fashion. In this mode, the gel extends and contracts elastically when the force is increased or decreased, respectively, followed by a time-dependent deformation. This viscoelastic deformation, taking place while the gel experiences a constant force, is directly related to domain unfolding/refolding. In a simplified way, this extension can be seen as the equivalent of several billion single molecule traces averaged together and measured all at once. Constant-force protocols can be used to study the creep and relaxation of protein hydrogels as a function of force and time. As a function of force, for BSA-based protein hydrogels, we have recently shown that there is a linear dependency between the elastic and viscoelastic extension and recoil with the applied strain7.
Here we detail the operation of a force-clamp rheometer using composite gels made from a mixture of protein L (8 domains24, depicted as L8) and a protein L-eGFP construct (L-eGFP), which makes the overall hydrogel fluorescent and easy to demonstrate.

<table>
	<tbody>
		<tr>
			<td>SI-KG4A force transducer</td>
			<td>World Precision Instruments (WPI)</td>
			<td>SI-KG4A</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear Voice Coil Motor</td>
			<td>Equipement Solutions</td>
			<td>LFA2010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Rocky Mountain Biologicals (RMBIO)</td>
			<td>BSA-AAF-1XG / 100 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma</td>
			<td>Sigma-Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>248614-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(bipyridine)ruthenium(II) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>544981-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>EXPRESS MEDICAL SUPPLIES&#160;6-0 NYLON SUTURE 12/PK</td>
			<td>Fisher Scientific</td>
			<td>NC0395626</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL Syringe Only, Luer-Lok Tip</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Silane, Sigmacote</td>
			<td>Sigma-Aldrich</td>
			<td>SL2-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbore PTFE Tubing, 0.022&#34;ID x 0.042&#34;OD, 100 ft/roll</td>
			<td>Cole-Parmer</td>
			<td>EW-06417-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 23 Gauge</td>
			<td>Healthcare Supply Pros</td>
			<td>305194</td>
			<td></td>
		</tr>
		<tr>
			<td>Jensen Global JG24-1.5X Red IT Dispensing Tips - 24 gauge</td>
			<td>KIMCO</td>
			<td>JG24-1.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>USH-103D USHIO 100W Short Arc Mercury Lamp</td>
			<td>ALB</td>
			<td>USH-103D USHIO</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Medical scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The computer code and CAD design of the custom parts can be made available on request to the corresponding author.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

force-clamp rheometry for characterizing protein-based hydrogels

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog proportional-integral-derivative (PID) system to apply controlled-force protocols on cylindrical protein-based hydrogel samples, which are tethered between a linear voice-coil motor and a force transducer. During operation, the PID system adjusts the extension of the hydrogel sample to follow a predefined force protocol by minimizing the difference between the measured and set-point forces. This unique approach to protein-based hydrogels&#160;enables the tethering of extremely low-volume hydrogel samples (&#60; 5 &#181;L) with different protein concentrations. Under force-ramp protocols, where the applied stress increases and decreases linearly with time, the system enables the study of the elasticity and hysteresis behaviors associated with the (un)folding of proteins and the measurement of standard elastic and viscoelastic parameters. Under constant-force, where the force pulse has a step-like shape, the elastic response, due to the change in force, is decoupled from the viscoelastic response, which comes from protein domain unfolding and refolding. Due to its low-volume sample and versatility in applying various mechanical perturbations, force-clamp rheometry is optimized to investigate the mechanical response of proteins under force using a bulk approach.

Figure 1A shows the scheme of the photoactive reaction used to synthesize the L-EGP/L8 hydrogel. Figure 1B shows the hydrogel mixture in the PTFE tube before and after the photoactivation. Figure 1C presents the extruded L-eGFP-L8 hydrogel inside a Tris solution. The hydrogel sample has no structural defects such as notches. Hydrogels with clearly visible damage should be discar...

Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

A new force-clamp rheometry technique is used to investigate the mechanical properties of low-volume protein-based hydrogel samples tethered between a voice-coil motor and a force sensor. An analog proportional-integral-derivative (PID) system allows for the &#39;clamping&#39; of the force experienced to the desired protocol.

Here, we describe a force-clamp rheometry method to characterize the biomechanical properties of protein-based hydrogels. This method uses an analog ...

The main advantages of this technique are that it utilizes a PID, which is a proportional integral derivative system to apply controlled force protocols to protein hydrogel samples, and it utilizes small sample volume. The protocols for the forces clamp allow for straight-forward interpretation of the data while low volumes are critical when working with hard to produce proteins that are available only in small amounts. The implications of this technique extend toward developing and characterizing new biometric materials with durable elasticity, using down-folding, refolding transition characteristic for proteins.This method can answer questions in tissue and biomaterial mechanics through enabling the measurements of billions of protein molecules in one pull and simulating crowded environments specific to biological tissues. Begin this procedure with reagent solutions preparation as described in the text protocol. To synthesize the protein-based hydrogel, first fix a 23-gauge needle on a one milliliter syringe with a pressed plunger.Then, cut a 10 centimeter polytetrafluoroethylene, or PTFE, tube using a razor blade. Attach the needle and syringe to one end of the PTFE tube. Insert the second end of the tube into a silane solution and fill the tube by retracting the syringe plunger.Leave the tube for approximately 30 minutes. Then, remove the silane solution and dry the tube with compressed air. Now, mix the protein solution with APS and Tris(bipyridine)ruthenium(II)chloride in a 1.5 milliliter tube using a constant volume ratio.Vortex the photoactive solution until it is mixed completely. Then, centrifuge the mix at maximum speed to remove any bubbles from the solution. Bubbles can form while loading the hydrogel photoactive mix into the Teflon tube, leading to sample damage.To prevent bubble formation, keep the Teflon tube end in the solution mix during the loading process and retract the syringe plunger slowly. Insert the open end of the treated PTFE tube into the photoactive mixture and draw the solution into the tube by retracting the syringe plunger. Now, place the loaded tube approximately 10 centimeters away from a 100 watt mercury lamp to prevent heating it and keep it there for up to 30 minutes at room temperature.Remove the tube from the needle and cut the edges of the tube near the hydrogel ends with a razor blade. Then, use a blunted 24-gauge needle to extrude the hydrogel into the Tris solution. Visually inspect the gels for any defects that might form during the extrusion or due to bubbles and discard any defective gels.Start the instrument control program and turn on the voice coil motor. Then, set the coil position to a value toward the end of the range. Displace the hooks in the Z direction and align them at the bend in the X direction.Then, record the values of the micrometer screws for the X direction. Now, tie a loose double-overhand knot at the end of the suture strand, such that the diameter of the loop is about four millimeters. Then, cut the loop off the strand.Repeat to form a second loop. Then, place the two loops on the hook connected to the force sensor. Fill the experimental chamber with Tris buffer and transfer the hydrogel sample into the filled chamber using medical tweezers.Place the voice coil and force sensor hooks close to the solution surface and align the hooks in all directions with the XYZ positioning manipulators. Using medical tweezers, hang both sides of the protein hydrogel sample on the hooks connected to the voice coil and force sensor. A typical error is an over-tightening of the suture loops around the hydrogel samples during the attachment process.This may lead to a notch formation and cutting of the hydrogel sample. Carefully tighten one suture loop around the hydrogel sample on the voice coil hook by holding both ends of the suture loop with medical tweezers and pulling them simultaneously. Repeat this step for the loop connected to the force sensor.Tighten the suture loops on the bends of each hook to prevent any slippage. Use these bends as reference points to find the zero separation between the hooks. Cut the excess lengths of the sutures using medical scissors.Move the attached hydrogel using Z manipulators along the Z axis towards the experimental chamber to immerse the hydrogel in the experimental solution. Align the hydrogel sample in YZ using the manipulators such that the gel is not under any stress. Zero the force sensor and separate the two hooks using the X micrometer stages until the gel starts to experience force.Once this happens, slightly turn back the micrometer screw in the X direction. Record the position of both manipulators for the voice coil motor and the sensor. Then, use the difference between these values and the ones previously measured to calculate the exact separation between the tethering hooks at the start of the experiment.To perform a force-ramp cycle by increasing the force at the desired loading rate, input the starting and final force values, as well as the duration of the protocol, which appears as a flipped V, followed by a constant low force for about 200 seconds to allow the protein domains to refold before the next cycle. Perform a constant-force protocol by applying a low force for 30 seconds. Then, increase the force to a constant force for a defined amount of time, followed by quenching the force back to the same low value for greater than 300 seconds to allow the protein domains to refold and the gel elasticity to recover.Finally, perform data analysis as described in the text protocol. Each measurement starts with a slack curve. By fitting two lines, the zero force on the sensor and the true gel length is determined.The true gel length is calculated from the intersection of the fits and the position of the micrometer screws. The force-clamp rheometry system can apply two different types of protocols. In the force ramp mode, the hydrogel sample experiences a force changing protocol with time that resembles an inverted V.In constant force mode, the applied stress changes in a step-like pattern.During measurements, the PID system adjusts the hydrogel extension by changing the coil position to follow the pre-defined set point from the force protocol. The strain is then calculated by dividing the measured extension to the true gel length. The stress is determined by dividing the applied force to the cross-sectional area of the hydrogel sample.Force Ramp traces are best represented as stress versus strain. The Young's modulus can be calculated from the slope measure during the loading phase. The hysteresis gives the energy dissipation coming from unfolding and refolding the protein.Constant force traces are best represented as a function of time. The change in strain can be used to measure the unfolding and refolding rates by fitting a double exponential. While attempting this procedure, it's important to remember to dry any silane from the tube before adding the protein, to centrifuge the protein mix to remove bubbles, to check the bubbles after injecting the protein mix inside the tube and to discard any mechanically damaged hydrogels after extrusion from the tube.Generally, individuals new to this method will struggle at first with attaching the gels to the hooks using the surgical sutures without causing damage to the hydrogel sample. Extruding the gel from the tube can damage the hydrogel as well. This method enables the applications of constant force protocols on low volume hydrogel samples.These experiments allow the decoupling of the elastic and the viscoelastic behaviors and the study of protein unfolding and folding mechanics in a bulk approach. After its development, this technique allows researchers in the field of material sciences to explore new soft biomaterials such as engineered polyproteins-based hydrogels that have an excellent potential for serving as tissue engineering scaffolds, drug delivery systems and biological ink for 3-D printing. Not only does this method provide insight into the mechanics of protein-based hydrogels, it can also be applied to other systems such as measuring the isometric response of muscle fibers or the elasticity of tissues such as skin.

force-clamp-rheometry-for-characterizing-protein-based-hydrogels

Research

JoVE Journal

Engineering

6.8K Görüntüleme.  University of Wisconsin-Milwaukee. The main advantages of this technique are that it utilizes a PID, which is a proportional integral derivative system to apply controlled force protocols to protein hydrogel samples, and it utilizes small sample volume. The protocols for the forces clamp allow for straight-forward interpretation of the data while low volumes are critical when working with hard to produce proteins that are available only in small amounts. The implications of this technique extend toward developing and character...