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.
