Generation of Magnetite-Embedded Bacterial Nanocellulose Pellicles

Abstract

Source: Arias, S. L., et al. Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles. J. Vis. Exp. (2016)

This video demonstrates the in situ synthesis of magnetite nanoparticles within a bacterial nanocellulose pellicle produced by Gluconacetobacter xylinus. It explains how magnetic bacterial nanocellulose is generated through iron ion infusion, alkaline precipitation, and purification.

Protocol

1. Synthesis of Polymer-coated Iron Oxide Nanoparticles and Its Deposition in a Bacterial Nanocellulose (BNC) Membrane

1. Bubble 1,000 ml of high-purity water with nitrogen gas in order to remove any dissolved oxygen in the water and replace it with nitrogen.
2. Use a three-neck round-bottom flask to prepare a solution in a 2:1 molar ratio of iron (III) chloride hexahydrate (FeCl₃·6H₂O) and iron (II) chloride tetrahydrate (FeCl₂·4H₂O) diluted with deoxygenated high-purity water. For example, use 5.4 g of FeCl₃·6H₂O and 1.98 g of FeCl₂·4H₂O in 10 ml of deoxygenated high-purity water. If this preparation turns too viscous and difficult to stir, use 0.54 g of FeCl₃·6H₂O and 0.198 g of FeCl₂·4H₂O in 20 ml of deoxygenated high-purity water.
   NOTE: Reduce the exposure time of the FeCl₂·4H₂O to air by weighing this chemical compound as fast as possible. Once introduced in the three-neck round bottom flask, close the three-neck round bottom flask with septum stoppers until it is connected to the nitrogen gas supply and the condenser tube.
3. Use two 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 necks.
4. Place 1 BNC pellicle (15.6 mm of diameter and 2-3 mm of thickness) in the vessel with the reactants. Make sure the sample is completely submerged in the liquid.
5. Connect the remaining neck of the vessel to a condenser tube. Additionally, use a drying tube filled with anhydrous calcium sulfate on top of the condenser tube. Run water through the condenser tube.
6. Seal all of the glass joints with vacuum grease.
7. Heat the solution in a silicone oil bath to 80 °C using a stirring hotplate and hold this temperature until step 1.10. Use a small magnetic stir bar to mix the reactants at 350 rpm for 5 min. Make sure the BNC is appropriately impregnated with the ferrous solution and the reactants are completely dissolved. Keep stirring the mixture until the end of the experiment.
   NOTE: Utilize a thermometer to verify the temperature of the silicone oil. It should be stable to 80 °C.
8. Increase the stirring velocity to 700 rpm and add (by dropping), in a time interval of 5 min, 5 ml of ammonium hydroxide (NH₄OH, 14%) to the 10 ml of ferrous solution using a pipetting needle, which has been also punched in a septum stopper. After the addition of the ammonium hydroxide, the color of the solution changes from yellow/orange to black.
9. Continue stirring the solution at 80 °C for another 5 min. Avoid high-speed stirrings in order to maintain the integrity of the sample. High speeds, i.e., higher than 1,000 rpm, can destroy the sample.
10. Lower the temperature of the solution to 30 °C using the temperature control bottom of the stirring hotplate and keep stirring for another 5 min. Then, turn off the hot plate. At this point, the iron oxide nanoparticles (IONPs) have been incorporated into the BNC mesh.
11. Cool the mixture down to room temperature (RT) and separate the magnetic nanoparticles (MNPs) and BNC using a strong permanent magnet (e.g., 1 Tesla). To do this, transfer the mixture to a vessel flask and then, while keeping the magnet close to the vessel, hold the MNPs and the BNC in place while decanting the supernatant.
    Note: Be careful when handling strong magnets since they can be harmful when used incorrectly. For steps (1.12) - (1.14) and (1.16) use the deoxygenated high-purity water prepared previously in (1.1) to prevent particles from oxidation.
12. Resuspend the MNPs and BNC in 100 ml water. Gently shake the solution to remove all the MNPs that are not strongly incorporated into the BNC. Decant the supernatant again by holding the MNPs and the BNC in place using the magnet.
13. Wash the MNPs and the BNC several times with water until the supernatant reaches neutral pH (pH~7), as measured using a colorimetric strip.
14. Separate the magnetic-functionalized BNC or magnetic bacterial nanocellulose (MBNC) from the MNPs using tweezers and rinse the MBNC several times with water until the water runs clear.
15. Sterilize the MBNC by exposing the MBNC O/N to UV (110-280 nm).
16. Autoclave 500 ml of deoxygenated high-purity water at 120 °C for 20 min and store the MBNC in 20 ml of this water.
17. Aseptically, immerse the sample in 1% of polyethylene glycol (PEG) and stir for 2 hr at RT (37 °C). This procedure improves the biocompatibility and stability of the iron oxide nanoparticles deposited in the BNC, specifically those exposed at the surface. The PEG coating will be distributed over the MBNC 3D network.
    NOTE: Naked IONPs are easily oxidized in air because of their high chemical activity. Even though PEG is considered a non-biodegradable material, its chemical stability depends on the applied biological conditions such as water content, pH, temperature, presence of enzymes, reactive oxygen species, reactive nitrogen species, and others.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glucoacetobacter xylinus	ATCC	700178
Iron(III) Chloride Hexahydrate	Sigma Aldrich	236489-100G
Ammonium Hydroxide	Macron Fine Chemicals	6665-46
Poly(Ethylene Glycol), Average Mn 400	Sigma Aldrich	202398-250G
Iron (II) chloride tetrahydrate	Sigma Aldrich	44939-250G
Anhydrous Calcium Sulfate	W.A. Hammond Drierite	13001
High vacuum grease	Sigma Aldrich	Z273554-1EA
pH test strips	Sigma Aldrich	P4786-100EA
Round-bottom three neck angle type distilling flask	Sigma-Aldrich	CLS4965250
Silicone oil for oil baths	Sigma-Aldrich	85409-250ML
Drying Tube	Chemglass	CG-1295-01
Septum Stopper, Sleeve Type	Chemglass	CG-3022-98
Magnetic stir bar	Chemglass	CG-2001-05
Condenser	Chemglass	CG-1218-01
Temperature Controller	BriskHeat	SDC120JC-A
Stirring Hotplate	Fisher Scientific	11-100-49SH