The overall goal of this experiment is to observe the effect of relative humidity on structure and tensile properties of regenerated silk fibroin fibers using a microfluidic dry-spinning method. This method can help answer key questions in the biomimetic spinning, such as shear forces and the dry spinning process. The main advantage of this technique is that the microfluidic channel is designed to mimic the spinning duct of a silk worm.

This method can provide insight into the dry spinning process of silk fibroin. This can also be used to the wet spinning process of recombining spider silk. Generally, new individuals new to this method will struggle.

Because, it needs two weeks to prepare the spinning dope and also to insure it has good spinning ability. Visual demonstration of this method is very critical. Because, if you are lack of experience, the spinning dope is very difficult to make, and the spinning time is very essential for you to make.

Start by degumming the Bombyx mori cocoons in an aqueous solution containing 0.5%sodium carbonate. Incubate the submerged cocoons at 100 degrees Celsius for 30 minutes. Then, replace the sodium carbonate solution and repeat the incubation a second time.

When finished, wash the silk with deionized water to remove the sericin. Dry the degummed cocoon silks in air, and then submerge them in 9.0 molar lithium bromide at a weight to weight ratio of 1:10. Incubate the silks at 40 degrees Celsius for two hours until the silks are completely dissolved.

Next, dilute the regenerated silk fibroin solution 1.5 times in 250 milliliter bottles using deionized water. Centrifuge the solution, and then use a vacuum pump to filter it through a double filter paper bed, with a 20 micron mesh size to remove any impurities. Place 250 milliliters of the filtered regenerated silk fibroin solution into cellulose semi-permeable membrane dialysis bags, and dialyze each bag in 10 liters of reverse osmosis deionized water at five degrees Celsius for three days.

Following dialysis, use forced air flow to condense the solution at five degrees Celsius down to 20%of the original weight. Next, add a three molar aqueous solution of calcium chloride to the condensed solution to achieve a final concentration of 1.0 millimoles per gram of calcium ions. Again, concentrate the regenerated silk fibroin solution by forced air flow to 44%of its starting weight.

Begin by boiling a glass slide for 20 minutes in a mixed solution of concentrated sulfuric acid and 30%hydrogen peroxide solution in a 10:1 mixture. When finished, wash the glass slide using deionized water, and then blow dry it using high-purity nitrogen. Next, place the glass slide in a custom-built coating device with a gap of 100 micrometers between the bottom surface of the coating bar and the upper surface of the glass.

Fill the gap with SU-8 photoresist and then spin coat the photoresist at 40.3 times G for 30 seconds to form a uniform film about 85 micrometers thick. Next, place the slide with the photoresist in an oven with a temperature-controlling program. Elevate the temperature from room temperature to 65 degrees Celsius at a rate of two degrees Celsius per minute, and hold the temperature at 65 degrees Celsius for two minutes.

Then, continue to heat the slide from 65 degrees Celsius to 95 degrees Celsius at a rate of two degrees Celsius per minute, and hold the temperature at 95 degrees Celsius for 15 minutes. At this point, turn off the oven and let the slide cool down naturally to room temperature in the oven. Next, place the slide with the photoresist under a UV light and align a photomask containing the proper microchannel above the slide.

Expose the side of the slide to UV light for 12 seconds. Following exposure, place the slide back into the oven, and heat as before. After baking, clean the photoresist ultrasonically in developer solution for 30 seconds.

Then, use isopropanol and developer alternately to wash the glass slide until there is no precipitation on the glass slide. Solidify the photoresist in an oven with a temperature-controlling program. Elevate the temperature from room temperature to 170 degrees Celsius at two degrees Celsius per minute, and hold at 170 degrees Celsius for 30 minutes, and then turn off the oven and cool down naturally to room temperature in the oven.

Next, place the cured SU-8 mold into an aluminum box, and pour 8.8 grams of PDMS pre-polymer over the mold to a height of about five millimeters. Cure the PDMS for 30 minutes at 65 degrees Celsius and 15 minutes at 80 degrees Celsius. Once cured, use a 1.2 millimeter diameter drill bit to drill a hole though the PDMS replica at the beginning of the channel.

Then, place the PDMS channel side up along with a second flat PDMS layer into a plasma coater, and treat with oxygen plasma. Seal the PDMS layer with the channel to a flat PDMS layer immediately after removing them from the plasma chamber. The microchannel is now ready to use.

Inject the regenerated silk fibroin spinning dope into the microchannel at two microliters per minute using a syringe pump. Adjust the relative humidity in the electrospinning area to 40 or 50%relative humidity using a humidifier. Then, turn on a roller so that the surface is moving at three centimeters per second.

Once the humidity has stabilized, touch the regenerated silk fibroin drop and draw a silk fiber into the air. Then, place it onto the roller through the 10 centimeter air gap. The roller will draw the remaining silk fibers.

When finished, store the fibers in a sealed desiccator for 24 hours. The next day, post-treat the fibers by drawing them out four times at 0.9 millimeters per second in 80%ethanol using a custom-built machine. Keep the drawn fiber fixed, and immerse them in 80%ethanol solution for one hour.

Finally, fix the post-treated fibers on a paper frame with a 10 millimeter gauge length. Shown here is an SEM fiber of the final stretched regenerated silk fibroin fiber. These fibers had an average diameter of nine microns.

When measured under tension, the fibers formed under the condition of 40%relative humidity had a slightly higher ultimate tensile strength and maximum strain, but had similar Young's modulus compared with the fibers formed under 50%relative humidity. After watching this video, you should understand how to dry spin a silk fiber from an aqueous silk solution with a bio-inspired microfluidic chip.