Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

Transcript

We are able to produce versatile, biocompatible, and biodegradable silk micro-motors for use in micro-mixing applications such as enzyme assay enhancement, lava wood chip diagnostics, and environmental monitoring and remediation. Utilizing reactive inkjet printing to create enzyme and Marangoni-powered silk micro-stirrers allows us to alter shapes, sizes, catalyst distributions, and blend other moieties into the structures rapidly and easily, thereby controlling trajectory, behavior and functions. Reactive inkjet printing can also be applied to produce micro-motor devices with other aqueous reactive inks apart from silk, which demonstrates the great versatility of this method.

The person demonstrating the procedure will be PhD student, Ana Jimenez-Franco from my laboratory. First, cut five grams of clean silk cocoons into one-centimeter squared small pieces using scissors. In an extraction hood, boil two liters of deionized water in a two-liter beaker on a magnetic hot plate.

Add a magnetic stir bar to the beaker of deionized water. Then slowly add 4.24 grams of sodium carbonate to the water to avoid boiling over, and allow it to dissolve. Once the solution starts to boil again, add the cut pieces of the silk cocoons to the solution.

Ensure that all the silk is submerged, and heat the solution, with constant stirring for 90 minutes. Cover the beaker with aluminum foil. After 90 minutes, remove the extracted fibroin fibers from the sodium carbonate solution with a glass rod.

Wash the fibroin fibers three times with one liter of pre-heated deionized water, gradually decreasing the temperature for each washing. Spread out the fibroin fibers on a 750-milliliter borosilicate glass crystallizing dish and place them in a drying oven overnight at 60 degrees Celsius under atmospheric pressure. Once dry, store the fibroin fibers in a closed container at room temperature.

Prepare a ternary solution containing 4.8 grams of deionized water, 3.7 grams of ethanol and 3.1 grams of calcium chloride. First dissolve the calcium chloride in deionized water and then add the ethanol. Place a 100-milliliter two-necked round-bottom flask into a water bath on top of a magnetic hot plate.

Then add the ternary solution to the flask. Next, place a thermometer in one of the necks of the flask to accurately monitor the solution temperature. Cover the other neck with aluminum foil to prevent drying out the solution due to evaporation.

Then, heat the solution to 80 degrees Celsius. When the temperature of the solution is stable at 80 degrees Celsius, remove the aluminum foil and add one gram of dried fibroin to the solution. Add a small magnetic stir bar to ensure that the solution is mixed well throughout the dissolution process.

Cover the second neck of the flask with aluminum foil to minimize evaporation and leave to dissolve for 90 minutes. After 90 minutes of dissolution, allow the fibroin solution to cool to room temperature for 10 minutes. Tie a knot on one of the ends of a 15-centimeter long dialysis tube.

Wash the tube for a few minutes with running deionized water from the tap. Open the other end of the dialysis tube and add the fibroin solution. Using a metal clamp, close the other end of the dialysis tube ensuring that it is closed as tightly as possible.

Attach one of the ends of the dialysis tube via a screw cap to an empty 30-milliliter plastic vial to allow the dialysis tube to float in water. Next, place the dialysis tube in a two-liter beaker containing two liters of deionized water. Change the water at regular intervals and check the conductivity of the water every time it is changed to follow the dialysis process.

After dialysis is complete, cut one end of the dialysis tube with scissors and pour the solution into a series of 1.5 milliliter tubes. Centrifuge for five minutes at 16000 x G to remove any particles inside the fibroin solution. Then, collect the supernatant in a 30-milliliter plastic vial and store it at four degrees Celsius.

For printing the main body of the artificial, self-propelled micro-stirrers, or spms's, mix the fibroin solution, polyethylene glycol 400 and deionized water to make 1.5 milliliters of Ink A.For printing the catalytic engine of the spms's, mix the fibroin solution, polyethylene glycol 400, catalase, and deionized water to make 1.5 milliliters of Ink B.Prepare 1.5 milliliters of Ink C by dissolving 0.05 mg per milliliter of Coomassie Brilliant Blue and methanol. Use jetting devices with 80-micrometer nozzle diameter for printing the inks on the silicon substrate, placed on the stage at a working distance between the nozzle and the silicon wafer substrate of around five millimeters. Load inks A, B, and C into three reservoirs and then adjust the back pressure using the back pressure valve for each individual channel to ensure that the ink is not dripping from the jetting devices.

Next, adjust the jetting parameters for each channel to ensure that each ink gives a good stable droplet formation. Print the silk fibroin ink layer by layer alternating with methanol on a clean polished silicon wafer substrate. Print two batches of fibroin spms's with 200-layer and 100-layer thickness respectively.

To remove the samples off the silicon wafers, immerse them in deionized water and gently agitate until detachment occurs. Clean a nine-centimeter glass Petri dish with deionized water and ensure that the surface is dust-free. Add 10 milliliters of pre-filtered five percent hydrogen peroxide to the clean and dry Petri dish and leave to settle.

Light up the bottom of the Petri dish with a cool white LED light source. Then, use a high-speed camera with macro-zoom lens to capture the motion from above. Now, wash the printed silk stirrers for 10 minutes by submerging them in deionized water to remove any unbound polyethylene glycol 400.

Carefully take one washed stirrer with the tip of a sterile syringe needle and place it in the center of the Petri dish. When the washed stirrer touches the hydrogen peroxide fuel, bubbles start forming around the engine and circular motion of the stirrer is observed. When the system appears stable, press record in the recording software to start capturing the video.

Perform tracking of the micro-stirrers on a frame by frame basis, tracking each end of the stirrers. Stable droplets formed from the jetting devices will enable the higher definition of the printed samples as shown here. Depending on the ink jet printers used in the droplet size, the distance between each printed droplet needs to be adjusted in such a way that they overlap to generate connected lines.

When the silk micro-stirrers are placed in the hydrogen peroxide fuel solution, the surface morphology of the stirrers is altered due to the bubbles being released from the inner structures, generating small pores. Still video frames of two representative 100-layer and 200-layer micro-stirrers and five percent hydrogen peroxide fuel are shown here. The red and green lines indicate the trajectories tracked.

The rotational velocity can be determined by the rate change of orientation as shown here. Comparison of 100-layer and 200-layer catalase doped micro-stirrers shows a distinctive increase in rotational velocity of approximately 0.6 fold, from 60 plus or minus six rpm to 100 plus or minus 10 rpm. To produce printable inks for this procedure, it is important that the fibers are completely dissolved during the dissolution process.

This technique has allowed us to produce micro-stirrers powered by various mechanisms, suitable for stirring and contamination-detection studies as recently published by us in the journal, Small.

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Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

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