Force-Clamp Rheometry for Characterizing Protein-based Hydrogels

Summary

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 'clamping' of the force experienced to the desired protocol.

Abstract

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 enables the tethering of extremely low-volume hydrogel samples (< 5 µL) 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.

Introduction

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 'pull', 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 level^1,2,3 and at the macroscopic level^4,5,6,7. Similarly, osmolytes increase the stability of single proteins^8,9, leading to a decrease in the viscoelastic response of hydrogels, for the same force conditions^7,10.

Several approaches have been implemented to synthesize protein-based hydrogels, by either using physical interactions^11,12 or covalent cross-linking^4,13. Covalent reactions allow for fixed cross-linking locations and these hydrogels 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 hydrogels^13,18. While DMA can apply constant force protocols to biomaterials, it requires Young's moduli over 10 kPa, and large sample volumes of more than 200 µL¹⁹. 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 best^4,15. Furthermore, most biological tissues are softer than 10 kPa. Several approaches were developed for biological samples, especially in the study of muscle elasticity^20,21. 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 extension^4,22. 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'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 diameters < 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 'clamping' 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'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.

Protocol

1. Reagents Solution Preparation

1. 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 > 1 mM.
2. Prepare stocks of ammonium persulfate (APS) (1 M) and tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)₃]²⁺) (6.67 mM) solutions by dissolving APS and [Ru(bpy)₃]²⁺ powders in the Tris buffer.

2. Protein-based Hydrogel Synthesis

1. Fix a 23 G needle on a 1 mL syringe with a pressed plunger.
2. Cut a 10 cm polytetrafluoroethylene (PTFE) tube (with an inner diameter of 0.022 in and an outer diameter of 0.044 in) using a razor blade. Attach the needle and syringe to one end of the PTFE tube.
3. Insert the second end of the tube into a silane solution and fill the tube by retracting the syringe plunger. Leave the tube for ~30 min.
4. Remove the silane solution and dry the tube with compressed air.
   NOTE: Make sure that all of the silane solution is dried and that no residue is left in the tube.
5. Mix the protein solution with APS and [Ru(bpy)3]²⁺ in a 1.5 mL tube using a constant volume ratio [e.g., 15:1:1 or 15:0.5:0.5 (v:v:v)].
6. Vortex the photoactive solution until it is mixed completely.
7. Centrifuge the mix at maximum speed (e.g., 14,000 x g) to remove any bubbles from the solution.
8. 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.
9. Place the loaded tube ~10 cm away from a 100 W mercury lamp to prevent heating it and keep it there for up to 30 min at room temperature (Figure 1B).
   NOTE: In some cases, the exposure time to light can be as low as 30 s. Shorter gelation times are used here for fluorescent gels, to limit photobleaching.
10. Remove the tube from the needle and cut the edges of the tube near the hydrogel ends with a razor blade.
11. Use a blunted 24 G needle to extrude the hydrogel into the Tris solution (Figure 1C).
    NOTE: Blunted needles are used to avoid any notches or damage to the hydrogel sample.
12. Visually inspect the gels for any defects that might form during the extrusion or due to bubbles and discard the gels with defects.

3. Protein-based Hydrogel Attachment and Force-Clamp Rheometer Set-up

1. Start the instrument control program. Turn on the voice-coil motor. Set the coil position to a value toward the end of the range (e.g., 7.5 mm).
   NOTE: The voice-coil position is recommended to be toward the end of the maximum movement range, to maximize the possible extension of the hydrogel.
2. Displace the hooks in the z-direction and align them at the bend in the x-direction (which is the pulling coordinate; see Figure 2B). Record the values of the micrometer screws for the x-direction.
3. Cut 2 sterile sutures into strands of equal length (2 - 3 cm; see Figure 3A and B).
4. Tie a loose double overhand knot into each of the strands and place the 2 loops on the hook connected to the force sensor (Figure 3C and 3D).
5. Fill the experimental chamber with Tris buffer and transfer the hydrogel sample into the filled chamber using medical tweezers.
6. Place the voice coil and force sensor hooks close to the solution surface and align the hooks in all directions using the x/y/z-positioning manipulators.
7. Using medical tweezers, hang both sides of the protein hydrogel sample on the hooks connected to the voice coil and force sensor (Figure 3C).
8. Tighten 1 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 (Figure 3D).
9. Repeat step 3.8 for the loop connected to the force sensor (Figure 3D).
   NOTE: Avoid an extreme tightening of the sutures to prevent any structural damage and transversal cutting of the hydrogel sample.
10. 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 in step 3.2. Cut the excess lengths of the sutures using medical scissors (Figure 3D).
11. Move the attached hydrogel using z-manipulators along the z-axis toward the experimental chamber to immerse the hydrogel in the experimental solution.
12. Align the hydrogel sample in y-z using the manipulators such that the gel is not under any stress.
13. 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.
14. Record the position of both manipulators for the voice coil motor and the sensor and use the difference between these values and the ones measured in step 3.2 to calculate the exact separation between the tethering hooks at the start of the experiment.
15. Set the range for the slack curve to ~1.5 - 2 mm and measure the gel slack (Figure 4A).
    NOTE: For each slack measurement, try to keep the start of the slack regime near the initial voice coil position, allowing for an optimal number of data points to fit the 2 regimes (Figure 4A). The gel length can be determined with a micron resolution using the separation between the hooks and the intersection between the 2 regimes in the slack curve (see also step 5.1). As the force sensor might drift with time due to variations in the experimental conditions, the part of the slack curve where the gel is not under force reports on this possible drift. The program controlling the instrument compensates automatically for this difference when sending the set-point command to the PID loop (Figure 4A inset).

4. Protein-based Hydrogel Characterization using Controlled Force-Ramp and Constant-Force Measurements

1. Force-ramp experiments
   1. To perform a force-ramp cycle by increasing the force at the desired loading rate (e.g., 0.01 mN/s), input the starting and final forces and the duration of the protocol as a flipped "V". Then, hold the gel at 0 mN (or low force) for > 200 s to allow the protein domains to refold and the gel elasticity to recover.
   2. Save the trace.
2. Constant-force experiments
   1. Perform a constant-force protocol by applying a low force (e.g., 0.1 mN) for 30 s and then increase the force to a constant force (e.g., 1 mN) for a defined amount of time (e.g., 120 s), followed by quenching the force back to the same low value (e.g., 0.1 mN) for > 300 s to allow the protein domains to refold and the gel elasticity to recover.
   2. Following the first pulse, adjust the PID settings to maximize the response time of the feedback loop (see Figure 2D).
      NOTE: For stiff gels and for small changes in force, the response time of the loop is limited by the electronics of the force sensor and the response time of the coil, and can be as low as 5 ms⁷. For softer gels and large changes in force, the response time is dictated by the elasticity of the hydrogels (Figure 2D).
   3. Save the trace.

5. Data Analysis

1. Utilizing the measured separation between the hooks and the calculated coil position, when the gel starts to experience force (Δx in the Figure 4A insert), compute the gel length L using the equation:
   L = L₀ + ∆x
   Here, L₀ is the separation between the hooks, measured from the position of the micrometer screws before the experiment (step 3.14).
   NOTE: For gels with low protein concentrations that do not result in a complete cross-linking, the measured length will change from trace-to-trace. Also, over long periods of time, proteins inside hydrogels might experience aging effects²⁵, which result in an overall lengthening of the gel.
2. Normalize the measured extension to the gel length to obtain the strain.
3. Normalize the measured force to the transversal surface area by using the inner diameter of the tube used for the polymerization.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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