This research was supported by the Chung-Ang University research grant in 2022 (during Professor Jonghoon Choi&#39;s sabbatical leave).&#160;This research was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (No. RS-2023-00275006), and the Korea Environment Industry &#38; Technology Institute (KEITI) grant funded by the Korea Ministry of Environment (MOE) (No. 2022002980003). Clinical MRSA and P. aeruginosa were obtained from Chung-Ang University Hospital.

1. Preparation of the GO/Cu nanocomposites
NOTE: The size and morphology of Cu nanoparticles that grow on the GO nanosheets are determined by the degree of GO oxidation, the concentration of the Cu precursor, and the concentration of the reducing agent26.
<ol>
	<li>Prepare 10 mL of 1 mg/mL GO suspension in a glass bottle. Sonicate the GO suspension for 1 h until GO is well dispersed in distilled (DI) water.</li>
	<li>Prepare a 20 mM CuCl2 s...

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

<ol>
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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibiotic+resistance.">Molecular mechanisms of antibiotic resistance.</a> Nat Rev Microbiol. 13 (1), 42-51 (2015).</li><li>S&#225;nchez-L&#243;pez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-based+nanoparticles+as+antimicrobial+agents:+An+overview.">Metal-based nanoparticles as antimicrobial agents: An overview.</a> Nanomaterials (Basel). 10 (2), 292 (2020).</li><li>Makabenta, J. M. 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Herein, we report a low-cost and simple method for preparing GO nanosheets deposited with Cu nanoparticles, which would be a potentially efficient method for eradicating antibiotic-resistant bacteria. The critical step in synthesizing GO/Cu nanocomposites is thoroughly dispersing GO and CuCl2 in the solution and maintaining an elevated temperature while mixing them. Further, the redox step must be conducted quickly because the reducing agent causes a reduction reaction as soon as it comes in contact with water...

Bacterial infections have a significant impact on public health. Pathogenic bacteria, in particular, can evade the body&#39;s protective mechanisms and cause diseases1. Antibiotics are widely used to treat bacterial infections. However, the inappropriate use of antibiotics has precipitated the emergence of antibiotic-resistant bacteria. Currently, nosocomial infections attributable to antibiotic-resistant bacteria have caused notable complications in healthcare facilities2. Unfortunately, bacteria have resistance mechanisms for all current antibiotics3. Therefore, developing new antibiotics is essential,&#160;though&#160;there is also a high probability that resistance mechanisms will emerge.
Metal nanoparticles have emerged as promising agents for combating antibiotic-resistant bacteria because of their effective antibacterial properties4,5,6. It is difficult for bacteria to develop resistance mechanisms against metal nanoparticles because they do not bind to any specific bacterial receptors7. In particular, silver, copper, and zinc oxide nanoparticles are the focus of extensive research because of their remarkable antibacterial efficacy8,9,10,11,12,13. The toxicity of metal nanoparticles toward bacteria is attributed to three primary mechanisms: 1) the release of metal ions, 2) the generation of reactive oxygen species (ROS), such as &#8226;OH and &#8226;O2&#8722;, and 3) physical interaction and attachment4,14.
In this study, graphene oxide/copper (GO/Cu) nanocomposites were developed as an antimicrobial agent. Copper (Cu) efficiently kills pathogens that encounter its surface and interferes with bacterial gene replication. However, it is an essential microelement in the human body, and it is less toxic to mammalian cells because it has homeostatic mechanisms that regulate the concentrations of Cu inside the cells4. When Cu nanoparticles are oxidized, they generate Cu ions, which have a relatively high affinity for negatively charged bacteria15,16. Cu ions bind to the cellular structures (e.g., proteins, membranes, and DNA), disrupting cell functions17. By synthesizing Cu nanoparticles on the surface of graphene oxide (GO), the release rate of metal ions can be controlled18,19,20. GO also shows an antibacterial effect by preventing bacterial adhesion with a rough surface through oxidative stress or by forming ROS21,22. Moreover, GO/Cu nanocomposites release more ROS than GO through the chemical action of Cu2+, which damages bacterial proteins and DNA, leading to the death of bacteria23,24.
This paper outlines the protocol for synthesizing GO/Cu nanocomposites and introduces an antimicrobial testing approach against clinical bacteria MRSA and P. aeruginosa, the most common multidrug-resistant bacteria strains that cause nosocomial infections25. This protocol aims to introduce a facile chemical reduction method for synthesizing GO/Cu nanocomposites that can impede the growth of antibiotic-resistant bacteria. We use copper(II) chloride (CuCl2) and sodium borohydride (NaBH4) as a precursor and reducing agent, respectively. The protocol also describes the specifics of applying these nanocomposites to bacteria using the microdilution broth method. To exclude errors due to the absorption interference of metal nanoparticles and make accurate evaluations, the antibacterial ability is evaluated using the colony counting method.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Clinical MDR bacterial strains&#160;</td>
			<td>Chung-Ang University Hospital (Seoul, South Korea)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) chloride dihydrate</td>
			<td>Duksan</td>
			<td>10125-13-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Field Emission Scanning Electron Microscope (FE-SEM)</td>
			<td>Carl Zeiss</td>
			<td>SIGMA</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphene oxide</td>
			<td>Sigma</td>
			<td>796034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Borohydride</td>
			<td>Sigma</td>
			<td>71320</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>JEOL</td>
			<td>JEM-2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>BD difco</td>
			<td>236950</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic Soy Broth</td>
			<td>BD difco</td>
			<td>211825</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of antibacterial graphene oxide/copper nanocomposites

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to antibiotics. Developing&#160;antimicrobial agents that can replace existing antibiotics is essential because antibiotic-resistant bacteria have resistance mechanisms for all current antibiotics and can promote nosocomial infections. To address this challenge, in this study, we propose graphene oxide/copper (GO/Cu) nanocomposites as antibacterial materials that can replace the existing antibiotics. GO/Cu nanocomposites are characterized by transmission electron microscopy and scanning electron microscopy. They show that copper (Cu) nanoparticles are well-grown on the graphene oxide sheets. Additionally, a microdilution broth method is used to confirm the efficacy of the antimicrobial substance against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa), which are frequently implicated in nosocomial infections. Specifically, 99.8% of MRSA and 84.7% of P. aeruginosa are eliminated by 500 &#181;g/mL of GO/Cu nanocomposites. Metal nanocomposites can eradicate antibiotic-resistant bacteria by releasing ions, forming reactive oxygen species, and physically damaging the bacteria. This study demonstrates the potential of antibacterial GO/Cu nanocomposites in eradicating antibiotic-resistant bacteria.

Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

Antibiotics are widely used to treat bacterial infections, but overusing them has led to bacteria becoming resistant. To address this problem we've been working on developing anti bacteria metallic nanocomposites which make a tougher bacteria to develop resistance mechanisms. The advantage of this protocol is that it is a low cost and easy method for making geo 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 disinfectants or capping agents making it an option for biomedical applications. Our group is focused on the developing bio comparable metallic nanocomposites for infection treatment.Therefore we are working on coding these nanocomposites onto surfaces or using them to deliver drugs aiming to effectively eradicate antibody resistant bacteria and their biofilms in real world infection scenarios.

Transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) analyses were performed to confirm the formation of GO/Cu nanocomposites. Figure 1A,B show that heterogeneous Cu nanoparticles were grown on the GO sheets. As shown in Figure 1C, the EDS mapping confirmed that the particles on the GO sheet were Cu nanoparticles.
<p class="jove_content biglegend" fo:keep-together.within-p...

Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Antibiotics are currently the most used antibacterial treatment for killing bacteria. However, bacteria develop resistance when continually overexposed to ...

fabrication-of-antibacterial-graphene-oxidecopper-nanocomposites

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Bioengineering

Development of Antibacterial Graphene Oxide/Copper Nanocomposites to Combat Antibiotic-Resistant Pathogens

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 G for 10 minutes at room temperature. Discard the supernatant after centrifugation. To wash the GO copper nano composite 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 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 minus 60 degrees Celsius under a vacuum overnight until the nano composites are completely dry.Store the nano composite powder at minus 20 degrees Celsius until use. Transmission in 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.

Antibacterial Potential of Graphene Oxide/Copper Nanocomposites Against Antibiotic-Resistant Nosocomial Infection-Causing Bacteria 

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 one times 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 micropipette 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.

364 Views.  Chung-Ang University.  Herein, we introduce graphene oxide/copper (GO/Cu) nanocomposites as an antibacterial nanomaterial. The antibacterial effectiveness of the GO/Cu nanocomposites was evaluated against both antibiotic-resistant gram-positive and gram-negative bacteria.

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

Summary