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 solution in a glass vial. Sonicate the CuCl2 solution until CuCl2 is well dispersed in DI water.</li>
	<li>Add 10 mL of the 20 mM CuCl2 solution to the GO solution and sonicate the mixture at 70 &#176;C for 1 h.</li>
	<li>Prepare a 20 mM NaBH4 solution. 
	CAUTION: This procedure must be conducted in a chemical fume hood. A redox reaction occurs as soon as the reducing agent NaBH4 is introduced into water. Prepare the NaBH4 solution immediately before the experiment.</li>
	<li>Preparing the mixture of NaBH4, GO, and CuCl2
	<ol>
		<li>Add 20 mL of the 20 mM NaBH4 solution to the GO and CuCl2 mixture while stirring with a magnetic bar at 200 rpm. After adding the NaBH4 solution, stir continuously for 30 min. 
		CAUTION: This procedure must be conducted in a chemical fume hood.</li>
	</ol>
	</li>
	<li>Centrifugation of the mixture
	<ol>
		<li>Transfer the mixture from step 1.5.1 to a centrifuge tube. Centrifuge the solution at 23,000 &#215; g for 10 min at room temperature. Discard the supernatant. 
		CAUTION: When operating the centrifuge, always maintain balance by placing tubes of equal weight.</li>
	</ol>
	</li>
	<li>Resuspend the GO/Cu nanocomposite mixture with 10 mL of DI water and sonicate them to enable the precipitate to disperse evenly throughout the solution.</li>
	<li>Centrifuge the solution at 23,000 &#215; g for 10 min at room temperature; then remove the supernatant.</li>
	<li>Repeat steps 1.7-1.8 once more to remove the unreacted chemicals.</li>
	<li>Add 1 mL of distilled water to the GO/Cu nanocomposites and sonicate the mixture to disperse the sediment evenly throughout the liquid phase. 
	NOTE: Collect all GO/Cu nanocomposite solutions in one conical tube.</li>
	<li>Freeze-dry the solution at &#8722;60 &#176;C under a vacuum overnight until the GO/Cu nanocomposites are completely dried and obtain GO/Cu nanocomposite powder.</li>
	<li>Store the GO/Cu nanocomposite powder at &#8722;20 &#176;C until use.</li>
</ol>
2. Preparation of bacteria for the antibacterial test
CAUTION: This procedure must be conducted in a biological safety cabinet with an alcohol lamp. Bacterial waste must be autoclaved before disposal. When handling antibiotic-resistant bacteria, gloves, gowns, and masks must be worn, and hands must be washed using soap or hand sanitizer after the experiment. Always thoroughly disinfect; if contamination occurs in the experimental area, immediately disinfect it with 70% ethanol.
<ol>
	<li>Bacterial culture media preparation
	<ol>
		<li>Mix 20 g of tryptic soy agar and 500 mL of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
		<li>Prepare the agar plate by pouring 15 mL of the agar solution into a Petri dish before the agar hardens. Store the agar plate at 4 &#176;C until use. 
		NOTE: This must be done quickly before the agar solution hardens. To prevent contamination, sterilize the agar plate with UV for 15 min during hardening.</li>
		<li>Mix 15 g of tryptic soy broth (TSB) and 500 mL of distilled water in a flask. Sterilize the mixture in an autoclave at 121 &#176;C for 15 min.</li>
		<li>Aliquot 40-50 mL of the broth solution into a 50 mL conical tube. Store the broth at 4 &#176;C until use. 
		NOTE: To prevent contamination, sterilize the aliquoted broth with UV for 15 min.</li>
	</ol>
	</li>
	<li>Bacterial culture
	<ol>
		<li>Inoculate the MSRA or P. aeruginosa stock solution on the agar plate using a loop. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h.</li>
		<li>Inoculate one colony of the bacteria into 10 mL of the broth using a loop. Incubate the broth at 37 &#176;C using a shaking incubator at 200 rpm for 24 h.</li>
		<li>Repeat steps 2.2.1-2.2.2 once more to obtain bacteria with the same characteristics.</li>
		<li>After 24 h, serially dilute the bacterial solution tenfold using sterile distilled water. Inoculate 100 &#181;L of the diluted bacterial suspension onto the agar plate and spread it with a spreader. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h. 
		NOTE: The dilution level is dependent on the bacteria. Aim to prepare less than 100 colonies in an agar plate.</li>
		<li>Count the bacterial colonies after the 24 h incubation to determine the colony-forming unit (CFU) using equation (1). 
		<img alt="Equation 1" src="/files/ftp_upload/66762/66762eq01.jpg" style="margin: auto;" /> (1)</li>
		<li>Repeat steps 2.2.4-2.2.5 at least 3x to confirm whether the CFU of the bacteria is relatively constant.</li>
	</ol>
	</li>
</ol>
3. Antibacterial test using the microdilution broth method
<ol>
	<li>Day 1) Inoculate one colony of the bacteria from the agar plate into 10 mL of the broth using a loop. Incubate the broth at 37 &#176;C using a shaking incubator at 200 rpm for 24 h.</li>
	<li>Day 2) Prepare the GO/Cu nanocomposite mixture in at least three concentrations using Dulbecco&#39;s phosphate-buffered saline (DPBS). Sonicate the GO/Cu nanocomposite suspension until the GO/Cu nanocomposites are well dispersed in DPBS. 
	NOTE: In this protocol, 500, 250, 125, and 62.5 &#181;g/mL of the GO/Cu nanocomposite mixture were tested.</li>
	<li>Prepare the control solutions. The negative control is DPBS, and the positive control is 1% of a penicillin/streptomycin solution in DPBS that will kill the bacteria.</li>
	<li>Add 100 &#181;L of the GO/Cu nanocomposite suspension and the control solutions into 96-well plates. Add all samples in triplicate. 
	NOTE: Sterilize samples with UV for 15 min before applying them to bacteria.</li>
	<li>Based on the CFU after 24 h of incubation, dilute bacterial suspension using TSB to 1 &#215; 106 CFU/mL. 
	NOTE: The initial MRSA and P. aeruginosa culture suspension concentrations in our study, according to step 2.2.5, are 4.5 &#215; 109 CFU/mL and 3 &#215; 109 CFU/mL, respectively. Bacterial concentrations of 1 &#215; 106 CFU/mL are obtained by diluting the cultures 4,500x and 3,000x, respectively.</li>
	<li>Inoculate 100 &#181;L of the 1 &#215; 106 CFU/mL diluted bacterial suspension into the sample wells in the 96-well plates. Incubate the 96-well plates at 37 &#176;C using a shaking incubator at 200 rpm for 24 h. 
	NOTE: The final concentration of bacteria is 5 &#215; 105 CFU/mL after mixing with 100 &#181;L of sample.</li>
	<li>Day 3) Mix the sample and bacterial suspensions vigorously with a 200 &#181;L micropipette tip. Serially dilute the sample-bacteria mixture tenfold with sterile distilled water. 
	NOTE: The dilution level is dependent on the bacteria. Aim to prepare less than 100 colonies in an agar plate.</li>
	<li>Inoculate 100 &#181;L of the diluted bacterial suspension onto the agar plate and spread it with a spreader. Incubate the agar plate at 37 &#176;C using a shaking incubator for 24 h.</li>
	<li>Day 4) Count the bacterial colonies and determine the CFU values to confirm the antibacterial activity of the GO/Cu nanocomposites using equation (2). 
	<img alt="Equation 2" src="/files/ftp_upload/66762/66762eq02.jpg" style="margin: auto;" /> (2)</li>
</ol>

Antibacterial Graphene Oxide/Copper Nanocomposites for Resistant Bacteria

<ol>
	<li>Doron, S., Gorbach, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+infections:+Overview.">Bacterial infections: Overview.</a> International Encyclopedia of Public Health. , 273-282 (2008).</li><li>Van Duin, D., Paterson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-resistant+bacteria+in+the+community:+An+update.">Multidrug-resistant bacteria in the community: An update.</a> Infect Dis Clin North Am. 34 (4), 709-722 (2020).</li><li>Blair, J. M. A., Webber, M. A., Baylay, A. J., Ogbolu, D. O., Piddock, L. J. 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. V., 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=Nanomaterial-based+therapeutics+for+antibiotic-resistant+bacterial+infections.">Nanomaterial-based therapeutics for antibiotic-resistant bacterial infections.</a> Nat Rev Microbiol. 19 (1), 23-36 (2021).</li><li>Liang, X., Govindaraju, S., Yun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+applicability+of+polyaniline+coated+gold+nanorods:+A+study+of+antibacterial+and+redox+activity.">Dual applicability of polyaniline coated gold nanorods: A study of antibacterial and redox activity.</a> BioChip J. 12 (2), 137-145 (2018).</li><li>Ni&#241;o-Mart&#237;nez, N., Salas Orozco, M. F., Mart&#237;nez-Casta&#241;&#243;n, G. A., Torres M&#233;ndez, F., Ruiz, F. <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+bacterial+resistance+to+metal+and+metal+oxide+nanoparticles.">Molecular mechanisms of bacterial resistance to metal and metal oxide nanoparticles.</a> Int J Mol Sci. 20 (11), 2808 (2019).</li><li>Kim, J., Choi, Y., Park, J., Choi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin-gallic+acid+microcomplexes+release+go/cu+nanomaterials+to+eradicate+antibiotic-resistant+microbes+and+their+biofilm.">Gelatin-gallic acid microcomplexes release go/cu nanomaterials to eradicate antibiotic-resistant microbes and their biofilm.</a> ACS Infect Dis. 9 (2), 296-307 (2023).</li><li>Jang, J., 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=Development+of+antibiofilm+nanocomposites:+Ag/cu+bimetallic+nanoparticles+synthesized+on+the+surface+of+graphene+oxide+nanosheets.">Development of antibiofilm nanocomposites: Ag/cu bimetallic nanoparticles synthesized on the surface of graphene oxide nanosheets.</a> ACS Appl Mater Interfaces. 12 (32), 35826-35834 (2020).</li><li>Jang, J., Choi, Y., Tanaka, M., Choi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+silver/graphene+oxide+nanocomposites+for+antibacterial+and+antibiofilm+applications.">Development of silver/graphene oxide nanocomposites for antibacterial and antibiofilm applications.</a> J Ind Eng Chem. 83, 46-52 (2020).</li><li>Bankier, C., 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=Synergistic+antibacterial+effects+of+metallic+nanoparticle+combinations.">Synergistic antibacterial effects of metallic nanoparticle combinations.</a> Sci Rep. 9 (1), 16074 (2019).</li><li>Mendes, C. R., 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=Antibacterial+action+and+target+mechanisms+of+zinc+oxide+nanoparticles+against+bacterial+pathogens.">Antibacterial action and target mechanisms of zinc oxide nanoparticles against bacterial pathogens.</a> Sci Rep. 12 (1), 2658 (2022).</li><li>Yoon, S., 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=Biologically+benign+multi-functional+mesoporous+silica+encapsulated+gold/silver+nanorods+for+anti-bacterial+applications+by+on-demand+release+of+silver+ions.">Biologically benign multi-functional mesoporous silica encapsulated gold/silver nanorods for anti-bacterial applications by on-demand release of silver ions.</a> BioChip J. 13 (4), 362-369 (2019).</li><li>Slavin, Y. N., Asnis, J., H&#228;feli, U. O., Bach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+nanoparticles:+Understanding+the+mechanisms+behind+antibacterial+activity.">Metal nanoparticles: Understanding the mechanisms behind antibacterial activity.</a> J Nanobiotechnology. 15 (1), 65 (2017).</li><li>Ismail, N. A., 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=Sonochemical+synthesis+of+a+copper+reduced+graphene+oxide+nanocomposite+using+honey+and+evaluation+of+its+antibacterial+and+cytotoxic+activities.">Sonochemical synthesis of a copper reduced graphene oxide nanocomposite using honey and evaluation of its antibacterial and cytotoxic activities.</a> Front Mol Biosci. 9, 995853 (2022).</li><li>Tu, Y., 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=Remarkable+antibacterial+activity+of+reduced+graphene+oxide+functionalized+by+copper+ions.">Remarkable antibacterial activity of reduced graphene oxide functionalized by copper ions.</a> Adv Funct Mater. 31 (13), 2008018 (2021).</li><li>Li, M., 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=Toward+a+molecular+understanding+of+the+antibacterial+mechanism+of+copper-bearing+titanium+alloys+against+staphylococcus+aureus.">Toward a molecular understanding of the antibacterial mechanism of copper-bearing titanium alloys against staphylococcus aureus.</a> Adv Healthc Mater. 5 (5), 557-566 (2016).</li><li>Yang, Z., 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=Long-term+antibacterial+stable+reduced+graphene+oxide+nanocomposites+loaded+with+cuprous+oxide+nanoparticles.">Long-term antibacterial stable reduced graphene oxide nanocomposites loaded with cuprous oxide nanoparticles.</a> J Colloid Interface Sci. 533, 13-23 (2019).</li><li>Yang, Y., 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=Graphene+oxide/copper+nanoderivatives-modified+chitosan/hyaluronic+acid+dressings+for+facilitating+wound+healing+in+infected+full-thickness+skin+defects.">Graphene oxide/copper nanoderivatives-modified chitosan/hyaluronic acid dressings for facilitating wound healing in infected full-thickness skin defects.</a> Int J Nanomedicine. 15, 8231-8247 (2020).</li><li>Mao, M., 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=Graphene+oxide-copper+nanocomposites+suppress+cariogenic+streptococcus+mutans+biofilm+formation.">Graphene oxide-copper nanocomposites suppress cariogenic streptococcus mutans biofilm formation.</a> Int J Nanomedicine. 16, 7727-7739 (2021).</li><li>Ravikumar, V., Mijakovic, I., Pandit, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+graphene+oxide+contributes+to+alteration+of+key+stress-related+and+membrane+bound+proteins.">Antimicrobial activity of graphene oxide contributes to alteration of key stress-related and membrane bound proteins.</a> Int J Nanomedicine. 17, 6707-6721 (2022).</li><li>Afreen, G., Shoeb, M., Upadhyayula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+reactive+oxygen+species+generated+from+rgo/cds+qd+heterostructure+for+photodegradation+and+disinfection+of+pollutants+in+waste+water.">Effectiveness of reactive oxygen species generated from rgo/cds qd heterostructure for photodegradation and disinfection of pollutants in waste water.</a> Mater Sci Eng C. 108, 110372 (2020).</li><li>Yang, F., 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=Fabrication+of+graphene+oxide/copper+synergistic+antibacterial+coating+for+medical+titanium+substrate.">Fabrication of graphene oxide/copper synergistic antibacterial coating for medical titanium substrate.</a> J Colloid Interface Sci. 638, 1-13 (2023).</li><li>Rajapaksha, P., 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=Antibacterial+properties+of+graphene+oxide-copper+oxide+nanoparticle+nanocomposites.">Antibacterial properties of graphene oxide-copper oxide nanoparticle nanocomposites.</a> ACS Appl Bio Mater. 2 (12), 5687-5696 (2019).</li><li>Sachdeva, C., Satyamoorthy, K., Murali, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+interplay+in+skin+and+chronic+wounds.">Microbial interplay in skin and chronic wounds.</a> Curr Clin Microbiol Rep. 9 (3), 21-31 (2022).</li><li>Ramos, M. K., Zarbin, A. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene/copper+oxide+nanoparticles+thin+films+as+precursor+for+graphene/copper+hexacyanoferrate+nanocomposites.">Graphene/copper oxide nanoparticles thin films as precursor for graphene/copper hexacyanoferrate nanocomposites.</a> Appl Surf Sci. 515, 146000 (2020).</li><li>Fivenson, E. M., 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=A+role+for+the+gram-negative+outer+membrane+in+bacterial+shape+determination.">A role for the gram-negative outer membrane in bacterial shape determination.</a> PNAS. 120 (35), e2301987120 (2023).</li><li>Wu, C., 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=Copper-containing+mesoporous+bioactive+glass+scaffolds+with+multifunctional+properties+of+angiogenesis+capacity,+osteostimulation+and+antibacterial+activity.">Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity.</a> Biomater. 34 (2), 422-433 (2013).</li><li>Zhang, W., 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=Graphene+oxide-copper+nanocomposite-coated+porous+cap+scaffold+for+vascularized+bone+regeneration+via+activation+of+hif-1&#945;.">Graphene oxide-copper nanocomposite-coated porous cap scaffold for vascularized bone regeneration via activation of hif-1&#945;.</a> Adv Healthc Mater. 5 (11), 1299-1309 (2016).</li><li>Labauve, A. E., Wargo, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+laboratory+maintenance+of+pseudomonas+aeruginosa.">Growth and laboratory maintenance of pseudomonas aeruginosa.</a> Curr Protoc Microbiol. 25 (1), 6E.1.1-6E.1.8 (2012).</li><li>Lin, J., 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=The+icmf3+locus+is+involved+in+multiple+adaptation-+and+virulence-related+characteristics+in+pseudomonas+aeruginosa+pao1.">The icmf3 locus is involved in multiple adaptation- and virulence-related characteristics in pseudomonas aeruginosa pao1.</a> Front Cell Infect Microbiol. 5, 70 (2015).</li></ol>

Dr. Jonghoon Choi is the CEO/Founder, and Dr. Yonghyun Choi is the CTO of the Feynman Institute of Technology at the Nanomedicine Corporation.

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><tbody><tr><td>Antibiotic-Antimycotic</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Clinical MDR bacterial strains&amp;nbsp;</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.

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.
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Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

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.

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 ...

fabrication-of-antibacterial-graphene-oxidecopper-nanocomposites

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Research

JoVE Journal

Bioengineering

713 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.

Video: Author Spotlight: Metallic Nanocomposites to Eliminate Antibiotic-Resistant Bacteria

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees of freedom to control electronic devices. In particular, the behavior of graphene&#8217;s charge carriers in response to potentials from charged Coulomb impurities is predicted to differ significantly from that of most materials. Scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS) can provide detailed information on both the spatial and energy dependence of graphene&#39;s electronic structure in the presence of a charged impurity.&#160;The design of a hybrid impurity-graphene device, fabricated using controlled deposition of impurities onto a back-gated graphene surface, has enabled several novel methods for controllably tuning graphene&#8217;s electronic properties.1-8 Electrostatic gating enables control of the charge carrier density in graphene and the ability to reversibly tune the charge2 and/or molecular5 states of an impurity. This paper outlines the process of fabricating a gate-tunable graphene device decorated with individual Coulomb impurities for combined STM/STS studies.2-5 These studies provide valuable insights into the underlying physics, as well as signposts for designing hybrid graphene devices.

This paper details the fabrication process of a gate-tunable graphene device, decorated with Coulomb impurities for scanning tunneling microscopy studies. Mapping the spatially dependent electronic structure of graphene in the presence of charged impurities unveils the unique behavior of its relativistic charge carriers in response to a local Coulomb potential.

Owing to its relativistic low-energy charge carriers, the interaction between graphene and various impurities leads to a wealth of new physics and degrees ...

Engineering

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using visible light irradiation with plasmonic nanoparticles. The plasmonic nanoparticle is used to improve the reduction efficiency of GO. It only takes 30 min&#160;at RT by illuminating the solutions with Xe-lamp, the r-GO solutions can be obtained by completely removing gold nanoparticles through simple centrifugation step. The spherical gold nanoparticles (AuNPs) as compared to the other nanostructures is the most suitable plasmonic nanostructure for r-GO preparation. The reduced graphene oxide prepared using visible light and AuNPs was equally qualitative as chemically reduced graphene oxide, which was supported by various analytical techniques such as UV-Vis spectroscopy, Raman spectroscopy, powder XRD and XPS. The reduced graphene oxide prepared with visible light shows excellent quenching properties over the fluorescent molecules modified on ssDNA and excellent fluorescence recovery for target DNA detection. The r-GO prepared by recycled AuNPs is found to be of same quality with that of chemically reduced r-GO. The use of visible light with plasmonic nanoparticle demonstrates the good alternative method for r-GO synthesis.

A simple protocol for the preparation of reduced graphene oxide using visible light and plasmonic nanoparticle is described.

Present work demonstrates the simple, chemical free, fast, and energy efficient method to produce reduced graphene oxide (r-GO) solution at RT using ...

Preparation of ZnO Nanorod/Graphene/ZnO Nanorod Epitaxial Double Heterostructure for Piezoelectrical Nanogenerator by Using Preheating Hydrothermal

Well-aligned ZnO nanostructures have been intensively studied over the last decade for remarkable physical properties and enormous applications. Here, we describe a one-step fabrication technique to synthesis freestanding ZnO nanorod/graphene/ZnO nanorod double heterostructure. The preparation of the double heterostructure is performed by using thermal chemical vapor deposition (CVD) and preheating hydrothermal technique. In addition, the morphological properties were characterized by using the scanning electron microscopy (SEM). The utility of freestanding double heterostructure is demonstrated by fabricating the piezoelectric nanogenerator. The electrical output is improved up to 200% compared to that of a single heterostructure owing to the coupling effect of the piezoelectricity between the arrays of ZnO nanorods on the top and bottom of graphene. This unique double heterostructure have a tremendous potential for applications of electrical and optoelectrical devices where the high number density and specific surface area of nanorod are needed, such as pressure sensor, immuno-biosensor and dye-sensitized solar cells.

One-step fabrication method for obtaining freestanding epitaxial double heterostructure is presented. This approach could achieve ZnO coverage with a higher number density than that of the epitaxial single heterostructure, leading to a piezoelectric nanogenerator with an increased output electrical performance.

Well-aligned ZnO nanostructures have been intensively studied over the last decade for remarkable physical properties and enormous applications. Here, we ...

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding

The assembly of two-dimensional (2D) graphene into three-dimensional (3D) polyhedral structures while preserving the graphene's excellent inherent properties has been of great interest for the development of novel device applications. Here, fabrication of 3D, microscale, hollow polyhedrons (cubes) consisting of a few layers of 2D graphene or graphene oxide sheets via an origami-like self-folding process is described. This method involves the use of polymer frames and hinges, and aluminum oxide/chromium protection layers that reduce tensile, spatial, and surface tension stresses on the graphene-based membranes when the 2D nets are transformed into 3D cubes. The process offers control of the size and shape of the structures as well as parallel production. In addition, this approach allows the creation of surface modifications by metal patterning on each face of the 3D cubes. Raman spectroscopy studies show the method allows the preservation of the intrinsic properties of the graphene-based membranes, demonstrating the robustness of our method.

Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding

Here, we present a protocol for fabrication of 3D graphene-based polyhedrons via origami-like self-folding.

The assembly of two-dimensional (2D) graphene into three-dimensional (3D) polyhedral structures while preserving the graphene's excellent inherent ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

 Zinc Oxide Nanoparticles Synthesis and Analysis of its Antibacterial Effects

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant bacterial strains in hospitals has been attributed to the extensive and varied use of antibiotics, further exacerbating the problem of antibiotic resistance. Metal nanomaterials have been widely studied as an alternative solution for eradicating antibiotic-resistant bacterial cells. Metallic nanoparticles attack bacterial cells through various mechanisms, such as the release of antibacterial ions, generation of reactive oxygen species, or physical disruption, against which bacteria cannot develop resistance. Among the actively researched antimicrobial metal nanoparticles, zinc oxide nanoparticles, which are FDA-approved, are known for their biocompatibility and antibacterial properties. In this study, we focused on successfully developing a precipitation method for synthesizing zinc oxide nanoparticles, analyzing the properties of these nanoparticles, and conducting antimicrobial tests. Zinc oxide nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), ultraviolet/visible spectroscopy, and X-ray diffraction (XRD). Antibacterial tests were conducted using the broth microdilution test with the multidrug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. This study demonstrated the potential of zinc oxide nanoparticles in inhibiting the proliferation of antibiotic-resistant bacteria.

Author Spotlight: Exploring the Antibacterial Effects of Zinc Oxide Nanoparticles in Overcoming Antibiotic Resistance

In this study, zinc oxide nanoparticles were synthesized using a precipitation method. The antibacterial effect of the synthesized particles was tested against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa bacterial strains.

Nosocomial bacterial infections have become increasingly challenging due to their inherent resistance to antibiotics. The emergence of multidrug-resistant ...

Fabrication of Antibacterial Graphene Oxide/Copper Nanocomposites

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Chapters in this video

Fabrication of Gate-tunable Graphene Devices for Scanning Tunneling Microscopy Studies with Coulomb Impurities

Visible-light Induced Reduction of Graphene Oxide Using Plasmonic Nanoparticle

Preparation of ZnO Nanorod/Graphene/ZnO Nanorod Epitaxial Double Heterostructure for Piezoelectrical Nanogenerator by Using Preheating Hydrothermal

Graphene Coatings for Biomedical Implants

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

Fabrication of Three-Dimensional Graphene-Based Polyhedrons via Origami-Like Self-Folding

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Preparation of Zinc Oxide Nanoparticles and the Evaluation of their Antibacterial Effects