The overall goal of this procedure is to produce antibacterial nanocomplexes consisting of carbon nanotubes with silver nanoparticles synthesized on the nanotube's surfaces. This method can be used in the development of novel bacterial nanomaterials that have low toxicity to humans while having strong septic properties to diverse kinds of bacteria and biofilms. The protocol describes the evaluation of nanocomplexes in eradicating bacteria while maintaining their bio-comparability with humans.
To begin the procedure, place 30 to 50 milligrams of MWCNTs in a 50 milliliter vial. Using a micropipette fitted with one milliliter pipette tips, slowly add eight milliliters of a three-to-one by volume solution of sulfuric acid and nitric acid to the MWCNTs. Wait 30 minutes for the exothermic reaction to finish, then sonicate the solution at 60 to 80 degrees celsius and 160 watts until the MWCNTs settle at the bottom of the vial, which usually takes about one hour.
Transfer the mixture to a centrifuge tube. Use a micropipette to carefully add 30 milliliters of distilled water drop-wise to the carboxylic acid functionalized MWCNTs, pausing to allow the mixture to cool as needed. Then, centrifuge the mixture at 12, 000 G for 15 minutes.
Remove the supernatant with a pipette equipped with a one milliliter pipette tip. Add five milliliters of ethanol to the solid and rinse the walls of the vial with additional ethanol. Centrifuge the mixture again with the same settings and check the pH of the supernatant with pH paper.
Continue washing the solids by centrifugation in ethanol until the supernatant pH is neutral, then remove the supernatant and add one milliliter of distilled water to the precipitate. Disperse the carboxylic acid functionalized MWCNTs in the water and freeze dry the dispersion at minus 60 degrees celsius under vacuum. Place 10 milligrams of freeze-dried carboxylic acid functionalized MWCNTs in a 50 milliliter conical centrifuge tube.
Add 30 milliliters of distilled water to the tube. Combine six milliliters of 0.1 normal silver nitrate and 18 milliliters of distilled water in another 50 milliliter conical tube. Sonicate both mixtures at 60 degrees celsius and 160 watts for one hour.
Then, while still sonicating the MWCNT dispersion at 60 degrees celsius and 160 watts, add the silver nitrate solution drop-wise to the MWCNT dispersion using a pipette equipped with a one milliliter pipette tip. Once addition is complete, continue sonicating the mixture for one hour, then centrifuge the mixture for 15 minutes at 12, 000 G at room temperature and remove the supernatant. Re-disperse the silver MWCNTs in 30 milliliters of distilled water by sonication at 24 degrees celsius for 30 minutes.
Repeat the centrifugation in 30 milliliters of distilled water and removal of the supernatant twice. Next, disperse the washed silver MWCNTs in one milliliter of distilled water by sonication for five minutes. Add five milliliters of ethanol to the dispersion and stir the mixture at room temperature for one hour.
Then centrifuge the mixture for 15 minutes at 12, 000 G at room temperature and check the pH of the supernatant. Remove the supernatant and re-disperse the mixture in five milliliters of ethanol by vortexing. Continue washing the precipitate in ethanol by centrifugation until the supernatant pH is neutral.
Finally, disperse the washed silver MWCNTs in one milliliter of distilled water. Freeze dry the dispersion at minus 70 degrees celsius under vacuum for 24 hours and store the dispersion in a sealed container. To begin preparing the test, mix and autoclave 18.12 grams of R2A agar and one liter of distilled water.
Allow the gel to cool at room temperature. When the gel is ready to pour, place 10 milliliters of gel in 90 millimeter Petri dishes and allow the agar to harden. In a fume hood, use a loop and needle sterilized in alcohol flame to streak 100 microliters of a methylobacterium species mixture on the surface of the culture medium.
Incubate the dishes at 30 degrees celsius for 48 hours, then mix 3.12 grams of R2A broth with one liter of distilled water and autoclave the medium at 121 degrees celsius for 15 minutes. Use a sterilized loop and needle to transfer the colonies to the R2A growth medium. Incubate the sample in a shaking incubator at 30 degrees celsius and 180 RPM.
Measure the optical density at 600 nanometers hourly until the optical density is greater than 0.7. Next, prepare another Petri dish containing 10 milliliters of R2A agar. Then mix 3.12 grams of R2A broth and eight grams of agar with one liter of distilled water.
Autoclave the mixture at 121 degrees celsius for 15 minutes. Mix 100 microliters of the methylobacterium culture into the agar mixture. Pour 10 milliliters of the methylobacterium-containing gel over the bottom layer of R2A agar in the Petri dish, then prepare a dispersion of the desired concentration of freeze-dried silver MWCNTs in distilled water.
Use a micropipette to load 50 microliters of this dispersion onto a six-by-one millimeter sterile paper disk. Apply distilled water to another sterile paper disk as a control. Dry and sterilize both disks in a vacuum chamber under UV light for 30 minutes.
Then place the silver MWCNT sample and the control on the agar and incubate the agar plate at 30 degrees celsius for 48 hours. Measure the zone of inhibition around the silver MWCNT sample when finished. Prior to the test, autoclave triple-distilled water at 121 degrees celsius for 15 minutes.
Prepare a 1000 microgram per milliliter dispersion of carboxylic acid functionalized MWCNTs and 50 microgram per milliliter, 40 microgram per milliliter, and 30 microgram per milliliter dispersions of silver MWCNTs in this sterile water. To begin the antibacterial testing procedure, prepare a methylobacterium culture as previously described and monitor its optical density at 600 nanometers. When the optical density is at maximum, innoculate 100 microliters of the methylobacterium culture into three milliliters of fresh R2A culture medium.
Place 500 microliters of this solution in each of the five 15 milliliter conical tubes. Add 100 microliters of methanol to one of the tubes to prepare the methanol reference sample. Prepare a sample for each of the four dispersions in the same way.
Incubate the samples and the remaining methylobacterium control culture at 180 RPM and 30 degrees celsius until the optical density of the control indicates maximal activity. Use a sterilized spreader to spread each sample on solid R2A agar gel in Petri dishes. Incubate the dishes at 30 degrees celsius for 48 hours.
Count the resultant bacterial colonies and determine the minimum bacteriocidal concentration. Silver MWCNTs were successfully synthesized using the described procedure. The average deposited silver particle size was approximately 3.83 nanometers.
Methylobacterium growth was not observed when cultured with 50 microgram per milliliter or 40 microgram per milliliter dispersions of silver MWCNTs. The bacterial population was reduced 100, 000 times relative to the control sample when cultured with a 30 microgram per milliliter dispersion of silver MWCNTs. A zone of inhibition was observed around a sample prepared from a 10 microgram per milliliter dispersion of silver MWCNTs.
No zone of inhibition was observed for a sample prepared from a one microgram per milliliter dispersion. Some cytotoxicity was observed when a 40 microgram per milliliter dispersion of silver MWCNTs was added. No significant cytotoxicity was observed when a 30 microgram per milliliter dispersion was added.
A trypan blue assay confirmed that there was minimal cytotoxicity when a 30 microgram per milliliter dispersion of silver MWCNTs was added. This protocol would help researchers in preparing nanocomplexes that have metal nanoparticles that act directly on carbon nanomaterials. Following this protocol, researchers would also be able to evaluate the antibacterial properties of the prepared nanocomplexes.
