Designing Silk-silk Protein Alloy Materials for Biomedical Applications

The overall goal of this procedure is to demonstrate how to fabricate various protein alloy materials using silk silk composite films with electronic circuits. As an example. This is accomplished by first regenerating individual protein solutions from two different species of silkworm cocoons.

The second step is to mix two protein solutions with different ratios and generate a series of blending solutions. Next, a thin film fabrication technique is used to fabricate electronic circuits on glass substrates. The final step is to coat the blending solutions onto the glass substrates and transfer the electronic circuits to the dried protein.

Composite films Compare with the traditional synthetic polymer materials. Protein materials can provide better biocompatibility, biodegradability, and tenability in human body. Blending proteins with different ratio is a simple technological approach to generate alloy materials with tuneable, physical, chemical, or biological properties.

Extending this technique to implanted biomedical systems can provide electronic monitoring, enhancing diagnosis and therapy in medical treatments. In addition, the technique can be used to develop devices that can be absorbed into the body after they're no longer needed. In this video, we'll use the protein blends of the wild TOSA silk, as well as domestic mulberry silk, as an example to provide useful protocols regarding this topic, including how to produce protein alley solutions as well as fabricate alley materials using electronic circuits.

The major advantage of our circuits coating technique compared to other methods like evaporation, is that the protein materials aren't subject to high heat directly or through radiation during our coating process. To begin this procedure, cut raw, wild tssa silk cocoons at a weight of three grams. Fill a two liter glass speaker with distilled water, then place the glass speaker on a hot stage.

Cover it with aluminum foil and heat to boiling. Remove the aluminum foil cover and add three grams of sodium bicarbonate slowly into the boiling water, allowing it to completely dissolve. Once the sodium bicarbonate has completely dissolved, add the wild silk cocoons into the boiling water and allow the mixture to boil for two to three hours.

After boiling, carefully remove the protein fibers from the solution with a spatula and squeeze them to remove the excess water. Next, immerse the fibers in a two liter beaker with cold distilled water and wash the fibers twice for 30 minutes each to completely remove the impure residues from the fiber surface. After washing, dry the fibers in a fume hood for at least 12 hours.

Melt 48.784 grams of calcium nitrate in a glass speaker to form a liquid at 65 degrees Celsius for dissolving the wild silk protein fibers. Then combine the fibers and solvent at a ratio of one gram of fiber to 10 milliliters of solvent. After allowing the fibers to dissolve at 95 degrees Celsius for five to 12 hours, pour the wild silk solution into a 12 milliliter dialysis tubing and dialyze against two liters of distilled water.

Change the distilled water frequently to remove the calcium nitrate solvents in the solution. After three days, collect the protein solutions from the dialysis cassettes and place them into 13, 000 RPM rated tubes. Following this centrifuge, the solutions for one hour at 3, 500 RPM at four degrees Celsius.

Three times to remove deposits when finished. Store the final solutions in a four degrees Celsius refrigerator after collecting another natural protein such as domesticated mulberry silk. Repeat the above process with an appropriate soap and dissolving solvent until the final protein water solution with measured concentration is obtained.

Next, slowly mix a one weight percent wild silk solution with a one weight percent domesticated silk solution using a pipette store. The final solution at four degrees Celsius To avoid protein aggregation, to fabricate an electrical circuit pattern on a glass substrate first, clean a glass slide using some degreasing solvent such as hinox and an ultrasonic cleaner for five minutes. Then clean the glass slide for five minutes in acetone and five minutes in methanol.

Blow the glass slide dry using dry nitrogen gas, which is generated by the boil off from a 180 liter liquid nitrogen doer. At this point, introduce the substrate materials into the deposition chamber and evacuate the load lock to a pressure of 30 militar. Then open the gate valve between the load lock and the main deposition chamber and introduce the substrate into the chamber.

Turn on the Argonne gas and the pressure regulator and control the pressure to the desired deposition pressure in order to remove oxide layers and contaminants from the target pre sputter for several minutes. After opening the shutter, sputter the metal onto the substrate using a deposition rate to achieve the desired thickness. Once the sputtering process is complete, remove the coated glass slide from the chamber.

Using a spinner spin a positive photo, resist coating onto the surface of the film. After the resist is spun onto the film, soft Bake at 90 degrees Celsius for five minutes to dry the resist. When finished, place a contact mask with an image of the device firmly against the resist.

Expose the photo resist to a 1000 watt UV light source for 10 seconds. Following this, place the film in the photo resist developer until the projected image appears. Immediately after the image appears, dip the film in deionized water to stop the developer from working on the unexposed photo.Resist.

Then blow the film dry with dry nitrogen gas. Place the film into an oven at 120 degrees Celsius for 15 minutes to hard bake the photo resist After the film has cooled, place it in an etching solution until the metal not protected by the photo. Resist lifts off.

Dip the film in water to stop the etching. Next, rinse the film with acetone to remove the hardened photo. Resist then rinse the film with methanol and blow dry them with dry nitrogen.

Once the coated glass is ready, drop different protein alloy solutions onto the glass surface. Using a pipette, dry it for at least 12 hours in the fume hood to obtain protein alloy films on the glass. Following this, peel off the protein alloy films with the thin metal patterns from the glass substrate using forceps To obtain insoluble protein alloy materials, place the dry film into a 60 degrees Celsius vacuum oven at 25 kilo pascals with a water dish on the bottom of the chamber.

Pump out the air in the oven, allowing the water vapors to al the samples for at least two hours. Once the vacuum has been released and the samples have been removed from the oven, peel off the water insoluble film from the substrate using forceps. Finally, test the electrical resistance of the metal pattern on the protein alloy films and compare it to the original pattern on the glass.

Typical protein protein interactions could contain electrostatic attractions, hydrogen bonding, hydrophobic hydrophilic interactions, as well as dipole solvent counter ion, and in tropic effects between the protein specific domains. The effects of these interactions can be predicted by computational simulations during the stretching simulation. One protein can form an elastic network like springs, providing super elasticity for the materials, while another can serve as particles with strong physical cross linkers for the materials.

Dynamic structural transitions can be induced in different domains for storing and releasing energy or providing additional mechanical support. The protein alloy solutions can be formed into various biomaterials with tunable properties, including material matrixes for thermal, mechanical, optical, electrical, chemical, or biomedical application. In this demonstration, the room temperature electrical resistivity of the electric circuits on the protein films was measured using two terminal and four terminal techniques.

In the two terminal measurement, the films had a resistivity of 3.6 times 10 to the negative seventh oh meters, approximately 20 times larger than the bulk. A higher resistivity measured in films in comparison to the bulk is typical due to the already constrained current path and inability of charge carriers to avoid defects. By changing the ratio of protein components, we can control biophysical properties of the final materials such as their elasticity, strength, surface roughness, or surface charge.

This properties would ultimately impact the functions and behaviors of the tissues attached to the material. Therefore, protein alloy system offers us a new pathway to produce implantable medical devices or materials with tuneable functions in the future. After watching this video, you should have a good understanding of how to fabricate circuits on these materials, and we hope the protocols we provided in the article will benefit both research scientists and clinical doctors in multiple biomedical fields in the future.