Generation of Magnetite-Embedded Bacterial Nanocellulose Pellicles

Take a bacterial nanocellulose pellicle, a gelatinous sheet of cellulose fibers.

Place it into a three-neck flask containing a solution of iron salts.

Attach a condenser and run water through it to prevent volume loss during heating. Then, purge the flask with nitrogen gas through an inlet to prevent iron oxidation.

Heat and stir the flask in a silicone oil bath to promote diffusion of iron salts into the pellicle.

Add an alkali slowly to raise the pH, causing the iron salts to form magnetite nanoparticles within the pellicle matrix.

Lower the temperature and continue stirring to ensure uniform nanoparticle distribution.

Cool the mixture, then transfer it to a clean vessel.

Using a magnet, retain the pellicle and decant unincorporated materials.

Wash the pellicle repeatedly with deoxygenated water until the pH becomes neutral and the supernatant appears clear.

Transfer the pellicle and sterilize using UV exposure, then immerse it in sterile deoxygenated water for further studies.

Bubble 1000 milliliters of high-purity water with nitrogen gas to remove any dissolved oxygen in the water and replace it with nitrogen.

Use a three-neck round-bottom flask to prepare a solution in a 2 to 1 molar ratio of Iron (III) chloride hexahydrate and Iron (II) chloride tetrahydrate, diluted with the deoxygenated high-purity water. Use 2 necks of the vessel to provide a constant entrance and output of nitrogen gas by connecting the nitrogen gas supply to a needle punched in a septum stopper and fixed to the vessel's neck. Place one BNC pellicle in the vessel with the reactants.

Make sure the sample is completely submerged in the liquid. Then, fill all of the glass joints with vacuum grease. Connect the remaining neck of the vessel to a condenser tube topped with a drying tube filled with anhydrous calcium sulphate.

Run water through the condenser tube. Next, heat the solution in a silicone oil bath to 80 degrees Celsius, using a stirring hot plate, and hold this temperature. Use a small magnetic stir bar to mix the reactants at 350 RPM for 5 minutes.

Make sure the BNC is appropriately impregnated with the ferrous solution and the reactants are completely dissolved. Increase the stirring velocity to 700 RPM. In a time interval of five minutes, add five milliliters of ammonium hydroxide to the 10 milliliters of ferrous solution using a pipetting needle that has been punched in a septum stopper. After addition of the ammonium hydroxide, the color of the solution changes from yellow-orange to black.

Continue stirring the solution at 80 degrees Celsius for another five minutes. Avoid high-speed stirrings to maintain the integrity of the sample. Lower the temperature of the solution to 30 degrees Celsius using the temperature control bottom of the stirring hot plate, and keep stirring for another five minutes.

Then, turn off the hot plate. At this point, the iron oxide nanoparticles have been incorporated into the BNC mesh. Next, cool the mixture down to room temperature and transfer it to a vessel flask to separate the magnetic nanoparticles, or MNPs, and BNC with a strong permanent magnet.

To do this, keep the magnet close to the vessel to hold the MNPs and BNC in place while decanting the supernatant. Resuspend the MNPs and BNC in 100 milliliters of water. Gently shake the solution to remove all the MNPs that are not strongly incorporated into the BNC.

Then, decant the supernatant again by holding the MNPs and the BNC in place using the magnet. Wash the MNPs and the BNC several times with water until the supernatant reaches neutral pH, as measured using a colorimetric strip. Separate the magnetic functionalized BNC, or MBNC from the MNPs, using tweezers, and rinse the MBNCs several times with water until the water runs clear.

Sterilize the MBNC by exposing it overnight to UV. Store the MBNC in 20 milliliters of deoxygenated high-purity water that has been autoclaved at 120 degrees Celsius for 20 minutes. Aseptically, immerse the sample in one percent of polyethylene glycol, or PEG and stir for two hours at room temperature. Coating with PEG improves the biocompatibility and stability of the iron oxide nanoparticles deposited in the BNC, as it is distributed over the MBNC 3D network.