Name: force-clamp rheometry for characterizing protein-based hydrogels
Uploaded: 2018-08-21T15:00:00
Description: Watch this Scientific Journal Video about Force-Clamp Rheometry for Characterizing Protein-based Hydrogels at JoVE.com

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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmolyte+effects+on+protein+stability+and+solubility:+A+balancing+act+between+backbone+and+side-chains.">Osmolyte effects on protein stability and solubility: A balancing act between backbone and side-chains.</a> Biophysical Chemistry. 159 (1), 90-99 (2011).</li><li>Popa, I., Kosuri, P., Alegre-Cebollada, J., Garcia-Manyes, S., Fernandez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+dependency+of+biochemical+reactions+measured+by+single-molecule+force-clamp+spectroscopy.">Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy.</a> Nature Protocols. 8 (7), 1261-1276 (2013).</li><li>Aioanei, D., Brucale, M., Tessari, I., Bubacco, L., Samori, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worm-Like+Ising+Model+for+Protein+Mechanical+Unfolding+under+the+Effect+of+Osmolytes.">Worm-Like Ising Model for Protein Mechanical Unfolding under the Effect of Osmolytes.</a> Biophysical Journal. 102 (2), 342-350 (2012).</li><li>Wheeldon, I. R., Gallaway, J. W., Barton, S. C., Banta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectrocatalytic+hydrogels+from+electron-conducting+metallopolypeptides+coassembled+with+bifunctional+enzymatic+building+blocks.">Bioelectrocatalytic hydrogels from electron-conducting metallopolypeptides coassembled with bifunctional enzymatic building blocks.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (40), 15275-15280 (2008).</li><li>Sathaye, 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=Rheology+of+peptide-+and+protein-based+physical+hydrogels:+are+everyday+measurements+just+scratching+the+surface?.">Rheology of peptide- and protein-based physical hydrogels: are everyday measurements just scratching the surface?.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 7 (1), 34-68 (2015).</li><li>Ma, X., 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+Biocompatible+and+Biodegradable+Protein+Hydrogel+with+Green+and+Red+Autofluorescence:+Preparation,+Characterization+and+In+Vivo+Biodegradation+Tracking+and+Modeling.">A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and In Vivo Biodegradation Tracking and Modeling.</a> Scientific Reports. 6, 19370 (2016).</li><li>Fancy, D. A., Kodadek, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+for+the+analysis+of+protein-protein+interactions:+rapid+and+efficient+cross-linking+triggered+by+long+wavelength+light.">Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (11), 6020-6024 (1999).</li><li>Saqlain, F., Popa, I., Fernandez, J. M., Alegre-Cebollada, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Strategy+for+Utilizing+Voice+Coil+Servoactuators+in+Tensile+Tests+of+Low+Volume+Protein+Hydrogels.">A Novel Strategy for Utilizing Voice Coil Servoactuators in Tensile Tests of Low Volume Protein Hydrogels.</a> Macromolecular Materials and Engineering. 300 (3), 369-376 (2015).</li><li>Sun, F., Zhang, W. B., Mahdavi, A., Arnold, F. H., Tirrell, 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=Synthesis+of+bioactive+protein+hydrogels+by+genetically+encoded+SpyTag-SpyCatcher+chemistry.">Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (31), 11269-11274 (2014).</li><li>Thompson, M. 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=Self-assembling+hydrogels+crosslinked+solely+by+receptor-ligand+interactions:+tunability,+rationalization+of+physical+properties,+and+3D+cell+culture.">Self-assembling hydrogels crosslinked solely by receptor-ligand interactions: tunability, rationalization of physical properties, and 3D cell culture.</a> Chemistry. 21 (8), 3178-3182 (2015).</li><li>Kocen, R., Gasik, M., Gantar, A., Novak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+behaviour+of+hydrogel-based+composites+for+tissue+engineering+under+mechanical+load.">Viscoelastic behaviour of hydrogel-based composites for tissue engineering under mechanical load.</a> Biomedical Materials. 12 (2), (2017).</li><li>Desai, M. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++dependence+of+loaded+shortening+in+rat+skinned+cardiac+myocytes+and+skeletal+muscle+fibres.">Ca2+ dependence of loaded shortening in rat skinned cardiac myocytes and skeletal muscle fibres.</a> Journal of Physiology-London. 525 (1), 169-181 (2000).</li><li>Wu, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+designed+synthetic+protein+hydrogels+with+predictable+mechanical+properties.">Rationally designed synthetic protein hydrogels with predictable mechanical properties.</a> Nature Communications. 9, (2018).</li><li>Bharadwaj, N. A., Ewoldt, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-point+parallel+disk+correction+for+asymptotically+nonlinear+oscillatory+shear.">Single-point parallel disk correction for asymptotically nonlinear oscillatory shear.</a> Rheologica Acta. 54 (3), 223-233 (2015).</li><li>Valle-Orero, J., Rivas-Pardo, J. A., 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=Multidomain+proteins+under+force.">Multidomain proteins under force.</a> Nanotechnology. 28 (17), 174003 (2017).</li><li>Valle-Orero, J., 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+Deformation+Accelerates+Protein+Ageing.">Mechanical Deformation Accelerates Protein Ageing.</a> Angewandte Chemie International Edition. 56 (33), 9741-9746 (2017).</li><li>Fang, J., 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=Forced+protein+unfolding+leads+to+highly+elastic+and+tough+protein+hydrogels.">Forced protein unfolding leads to highly elastic and tough protein hydrogels.</a> Nature Communications. 4, (2013).</li><li>Gungor-Ozkerim, P. S., Inci, I., Zhang, Y. S., Khademhosseini, A., Dokmeci, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinks+for+3D+bioprinting:+an+overview.">Bioinks for 3D bioprinting: an overview.</a> Biomaterial Science. , (2018).</li></ol>

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 ...

Research

JoVE Journal

Engineering

Bringing the Visible Universe into Focus with Robo-AO

The angular  resolution of ground-based optical telescopes is limited by the degrading  effects of the turbulent atmosphere. In the absence of an ...

Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM

Organic photovoltaic  (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale  inhomogeneity of OPV materials affects performance ...

Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition

Nanosecond Pulsed  Laser Deposition (PLD) in the presence of a background gas allows the deposition  of metal oxides with tunable morphology, structure, ...

The Preparation of Electrohydrodynamic Bridges from Polar Dielectric Liquids

Horizontal and vertical liquid bridges are simple and powerful tools for exploring the interaction of high intensity electric fields (8-20 kV/cm) and ...

Selective Area Modification of Silicon Surface Wettability by Pulsed UV Laser Irradiation in Liquid Environment

The wettability of silicon (Si) is one of the important parameters in the technology of surface functionalization of this material and fabrication of ...

Detection and Recovery of Palladium, Gold and Cobalt Metals from the Urban Mine Using Novel Sensors/Adsorbents Designated with Nanoscale Wagon-wheel-shaped Pores

Developing low-cost, efficient processes for recovering and recycling palladium, gold and cobalt metals from urban mine remains a significant challenge in ...

Writing Bragg Gratings in Multicore Fibers

Fiber Bragg gratings in multicore fibers can be used as compact and robust filters in astronomical and other research and commercial applications. Strong ...

Characterizing Far-infrared Laser Emissions and the Measurement of Their Frequencies

The generation and subsequent measurement of far-infrared radiation has found numerous applications in high-resolution spectroscopy, radio astronomy, and ...

Experimental Methods for Investigation of Shape Memory Based Elastocaloric Cooling Processes and Model Validation

Shape Memory Alloys (SMA) using elastocaloric cooling processes have the potential to be an environmentally friendly alternative to the conventional vapor ...

Orientational Transition in a Liquid Crystal Triggered by the Thermodynamic Growth of Interfacial Wetting Sheets

In liquid crystal (LC) physical chemistry, molecules near the surface play a great role in controlling bulk orientation. Thus far, mainly to achieve ...