Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are widely used to treat bacteria infections, but overusing them has lead to bacteria becoming resistant. To address this problem we've been working on developing antibacterial metallic nanocomposites, which make it tough for bacteria to develop resistance mechanisms. The advantage of this protocol is that it is a low-cost and easy method for making GO/copper nanocomposites compared to other protocols.

This makes it practical for large scale production, which is great for commercial use. Also, our method doesn't need extra surfactants or capping agents, making it an option for biomedical applications. Our group is focused on developing biocompatible metal nanocomposites for a wound infection treatment.

Therefore, we are working on coating these nanocomposites onto surfaces or using them to deliver drugs, aiming to effectively eradicate antibiotic-resistant bacteria and their biofilms in real-world infection scenarios. To begin, prepare 10 milliliters of one milligram per milliliter graphene oxide, or GO, suspension in a glass bottle. Sonicate the GO suspension for one hour until the GO is well dispersed in distilled water.

Prepare a 20 millimolar cupric chloride solution in a glass vial and sonicate the solution until cupric chloride is well dispersed in distilled water. Next, add 10 milliliters of the 20 millimolar cupric chloride solution to the GO suspension and sonicate the mixture at 70 degrees Celsius for one hour. Prepare a 20 millimolar sodium borohydride solution in a chemical fume hood immediately before use.

Add 20 milliliters of the solution to the GO and cupric chloride mixture while stirring with a magnetic bar at 200 rpm. Then transfer the mixture to a centrifuge tube and spin it at 23, 000 x g for 10 minutes at room temperature. Discard the supernatant after centrifugation to wash the GO/copper nanocomposite mixture.

Resuspend it with 10 milliliters of distilled water and sonicate the solution to disperse the precipitate evenly. Next, centrifuge the solution again at 23, 000 x g for 10 minutes at room temperature and remove the supernatant. Then add one milliliter of distilled water to the nano composites and sonicate the mixture to disperse the sediment evenly throughout the liquid phase.

After transferring the sonicated mixture to another tube, freeze dry the solution at 60 degrees Celsius under a vacuum overnight until the nanocomposites are completely dry. Store the nanocomposite powder at 20 degrees Celsius until use. Transmission and scanning electron microscopy images confirmed the growth of heterogeneous copper nanoparticles on the GO sheets, and energy dispersive X-ray spectroscopy mapping verified that the particles were copper nanoparticles.

To begin, prepare the graphene oxide copper nanocomposites. On day one, inoculate one colony of the bacteria from the agar plate into 10 milliliters of broth using a loop. Incubate the broth at 37 degrees Celsius using a shaking incubator set at 200 rpm for 24 hours.

On day two, dilute the nanocomposite mixture using DPBS to obtain three different concentrations and prepare the control solutions for the experiment. Then dispense 100 microliters of the nanocomposite suspension and control solutions into 96-well plates. Based on the CFUs, dilute the bacterial suspension to 1 x 10 to the power of six CFU per milliliter in tryptic soy broth.

Inoculate 100 microliters of the diluted bacterial suspension into the sample wells in the 96 well plate and incubate the plate at 37 degrees Celsius in a shaking incubator Set at 200 rpm for 24 hours. On day three, vigorously mix the sample and bacterial suspensions with a 200 microliter micropipet before serially diluting the sample bacteria mixture tenfold with sterile distilled water. Then spread 100 microliters of the diluted bacterial suspension on an agar plate with a spreader and incubate the plate at 37 degrees Celsius for 24 hours.

On day four, count the bacterial colonies and determine the CFU values to confirm the antibacterial activity of the nanocomposites using the equation. The nanocomposites demonstrated significant antibacterial efficacy against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, eradicating 99.8%and 84.7%of those bacteria, respectively, at 500 micrograms per milliliter concentration.