- Email: [email protected]
Gum arabic capped copper nanoparticles: Synthesis, characterization, and applications
Journal Pre-proof Gum arabic capped copper characterization, and applications
nanoparticles:
Synthesis,
Prince Chawla, Naveen Kumar, Aarti Bains, Sanju Bala Dhull, Mukul Kumar, Ravinder Kaushik, Sneh Punia PII:
S0141-8130(19)37263-0
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.260
Reference:
BIOMAC 14288
To appear in:
International Journal of Biological Macromolecules
Received date:
8 September 2019
Revised date:
20 November 2019
Accepted date:
30 December 2019
Please cite this article as: P. Chawla, N. Kumar, A. Bains, et al., Gum arabic capped copper nanoparticles: Synthesis, characterization, and applications, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2019.12.260
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© 2020 Published by Elsevier.
Journal Pre-proof Gum arabic capped copper nanoparticles: synthesis, characterization, and applications
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Prince Chawla1, Naveen Kumar2, Aarti Bains4, Sanju Bala Dhull3*, Mukul Kumar5, Ravinder Kaushik1*, Sneh Punia3 1 Shoolini University, Solan, Himachal Pradesh, India 2 Amity University, Jaipur, Rajasthan, India 3 Chaudhary Devi Lal University, Sirsa, Haryana, India 4 Chandigarh Group of Colleges, Mohali, Punjab, India 5 Lovely Professional University, Phagwara, Punjab, India
*Corresponding author Contact e-mail: [email protected]; [email protected] E-mail: [email protected]
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Journal Pre-proof Abstract In present work, we synthesized copper nanoparticles using L-ascorbic acid as a reducing agent and capped them with gum arabic. Based upon spectrometric analysis and particle size distribution(19.60- 35.06 nm) by intensity, a 1% concentrationof gum arabic was selected asthe suitable cappingagentfor copper nanoparticles. Gum arabic capped copper nanoparticles revealed significantly (p<0.05) higher zeta potential valuethan that ofunmodified copper nanoparticles. Energy-dispersive X-ray spectroscopyrevealed the purity of the copper
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nanoparticles, whereas scanning electron microscopy confirmed the polygonal prismatic
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shape of the gum arabic capped copper nanoparticles. As well, TEM analysis confirmed the
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monodispersed nature of thegum arabic cappedcopper nanoparticles.Gum arabic capped
typhimurium
(27mm)than
that
of
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copper nanoparticles showed significantly (p<0.05) higher zone of inhibition for Salmonella other
bacterial
strains.
Approximately95%
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photocatalyticdegradationof methylene blue and crystal violet was observed within 40 and 20
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min. As well, both unmodified and gum arabic cappedcopper nanoparticles were found to be non-toxic to Caco-2 cells during cell viability assay.
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Abbreviations: DLS, Dynamic light scattering, PBS,Phosphate buffer solution, GA,Gum arabic, DMEM, Dulbecco‟s Modified Eagles Medium, DMSO Dimethyl sulfoxide Keywords: Copper nanoparticles; Gum arabic; Dye reduction; Zeta potential; Cytotoxicity; LSPR
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Journal Pre-proof 1. Introduction Copper nanoparticles have gained as the most attractive nanoparticles in the field of multidisciplinary science [1]. Typically, copper nanoparticles are synthesized using several chemical and physical approaches based on the appropriateness of the methods to achieve the desirable applications [2]. Physical methods used for the synthesis of copper nanoparticles implicate expensive instruments and sophisticated techniques (1-2). While, using chemical approaches copper nanoparticles are synthesized by several chemical reductants such as tri-
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sodium citrate, sodium borohydride, hydrazine [3]. Even though chemical approaches have
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been extensively applied for the synthesis of copper nanoparticles but the precarious
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reductants and several organic solvents used for the synthesis are potentially hazardous for
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the environment [2,4].Therefore, the development of affordable solutions is in demand to efficiently reduce the hazardous risks of chemical reductants [4, 5]
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Presently, green synthesis of the nanoparticle is an incipient phenomenonthat
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effectively reduces the risk to the environment by eradicating the hazardous components that are harmful to human health [3-6]. The most common effective approach for obtaining
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copper nanoparticles includes the nucleation of copper ions using L-Ascorbic acid to form nanoparticles [7].The broad-spectrum complications like aggregation and oxidation of copper nanoparticles limit their usage. However, the usage of the appropriate stabilizing agent in the preparation resolves this problem effortlessly [6-10].Though, the presence of a stabilizing component is required to formulate more steady and relatively monodispersed copper nanoparticles [1].Natural components have widely been used either for the reduction of copper ions or for the protection of the formulated copper nanoparticles in the aqueous phase [3-7]. Therefore, among all the biological macromolecules, gum arabic attained decent attention due to reduced toxicity, biocompatibility with the metals, cost-effectiveness and easy
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Journal Pre-proof availability [15]. Surface charge of stabilizer is an important feature to maintain the electrostatic interactions and steric hindrance between monodispersed nanoparticles and water system, therefore, the polyanionic nature of gum arabic is an important feature to maintain the monodispersed nature of nanoparticles [12, 13]. To deal with several biological and inorganic toxic pollutants, metal nanoparticles are being vigorously looked into. Currently, copper nanoparticles are being used as an effective approach to eradicate organic and inorganic pollutants [4-6].
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In addition, copper nanoparticles have attained great interests due to their distinctive
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physicochemical properties that make them effective agents against different classes of
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pollutants [4,5]. Due to differentiated properties, copper nanoparticles exhibits sorbent
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properties that directly used for the elimination of toxic inorganic metal pollutants and organic dyes from industrialized wastewater [6, 14]. The copper nanoparticles are broadly
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used as antibacterial agents because of low toxicity and biodegradable nature [3, 14].These
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copper nanoparticles also exhibit the mechanism of antibacterial properties due to their large surface area relative to the volume that proficientlydecreases the oxygen stream by binding
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with the cell wall that ultimately causes the reduction in respiration. Therefore, the aim of the present study was to improve the oxidative stability of copper nanoparticles and also to improve the bactericidal activity. Keeping this in mind, copper nanoparticles were capped with gum arabic. A major highlight of the current study was the potential application of the capped nanoparticles as anantibacterial against food pathogenic bacteria and photocatalytic dye reduction of methylene blue and crystal violet. 2. Materials and Methods 2.1 Materials Spray-dried gum arabic was procured from Sigma Aldrich, St. Louis, MO, USA. Lascorbic acid, copper sulphate pentahydrate, hydrochloric acid, sulphuric acid, nitric acid, methylene blue trihydrate, crystal violet,
ethanol, hydrogen peroxide,
antibiotic 4
Journal Pre-proof (ciprofloxacin), nutrient agar and nutrient broth were procured from Hi-Media Pvt. Ltd. Mumbai, India. Bacterial cultures (i.e., Staphylococcus aureus (MTCC 3160), Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 3384) and Salmonella typhimurium (MTCC 1357) were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India.Human colon adenocarcinoma (Caco-2 cells) were procured from National Centre for Cell Science, Pune, Maharashtra, India.DMEM (Dulbecco‟s Modified Eagles Medium), DMSO (dimethyl sulfoxide), L-glutamine, and
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streptomycin (sulfate salt) were purchased from Sigma Aldrich, St. Louis, MO, USA; Human
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ferritin ELISA kit (SEA518Hu 96 Tests) from Cloud Clone-Corp. Houston, USA. Plastic
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dishes, plates, and transwell were obtained from Corning (Corning, NY, USA). All the
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chemicals were of „Analytical Reagent‟ grade. Triple distilled water and aqua-regia washed glassware (class “A” certified) was used throughout the experiments.
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2.2 Methods
2.2.1 Synthesis of gum arabic stabilized copper nanoparticles
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Copper nanoparticles were prepared using copper(II)sulphate pentahydrate as a
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precursor and L-Ascorbic acid as a reducing agent [7]. An aqueous solution of L-Ascorbic acid (0.1mM, 50mL) was dropwise added to an aqueous solution of copper(II)sulphate pentahydrate (80℃, 0.1mM, 50mL) under continuous and gentle stirring using magnetic stirrer (M3D, Eltak DIGIMAG, India) for 2h. Later, different concentration of gum arabic (i.e. 0.25, 0.50, 1.0, 1.5,& 2.0%) was added to hot solution of reaction mixture for complete reduction and capping under continuous stirring (1h). The resulting mixture was then transferred to glass vials and stored at room temperature (27℃) for further analysis. 2.2.2 Characterization methods Reduction by L-ascorbic acid and stabilization with gum arabic of copper nanoparticles
was
confirmed
by
the
UV-Visible
analysis
using
UV-Visible
Spectrophotometer (Evolution 201, Thermo Fischer Scientific IndiaPvt.Ltd, Mumbai). The 5
Journal Pre-proof quartz cuvettes of 3.5 ml with 3.5 cm path length were used for the analysis.Zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd. Malvern, WR14 1XZ, UK) was used for the determination of particle size and zeta potential of the copper nanoparticles.The morphology of the selected copper nanoparticles was described by SEM analysis using a JEOLJSM-6510LV (INCA, USA) microscope and the elemental analysis was performed by Energy-dispersive X-ray spectroscopy using a RONTEC‟s EDX system Model QuanTax 200 analyzer.To confirm the homogenous shape of the copper nanoparticles, TEM images were
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acquired using a transmission electron microscope (JEM-1011; JEOL microscope). The
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instrument was used at anaccelerating voltage of 200kV and copper nanoparticles were
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2.3 Application of Copper Nanoparticles
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directly mounted on a carbon-coated copper grid for the analysis.
2.3.1 Antimicrobial efficiencyagainst food pathogenic bacteria
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The agar well dilution method was used to evaluate the antimicrobial activity of
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selected nanoparticle against food pathogenic strains [12]. Mueller Hinton Agar glass plates were prepared and inoculated with the selected microorganisms (~1.5×108 cells/ml) by the
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spread plate method. After 30 min, wells (6mm) in the seeded agar plates were punched using cork borer. For positive control, chloramphenicol was used as a standard antibacterial agent, whereas, DMSO was used as a solvent (negative control). A 10 mL of different concentrations (25, 50, 75, 100 mg/mL) of copper nanoparticles were prepared and constant volume (100μl/well) was poured into the wells, then incubated at 37℃ for 24h. For the reference antibiotic, the concentration of 150mg/ml was used[16]. 2.3.2 Minimum Inhibitory Concentration
(MIC) and
Minimum Bactericidal
Concentrations (MBC) values The MIC values were determined by the microdilution broth method using the microtiterplate method. The plates were prepared by pouring 100μl nutrient broth into each
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Journal Pre-proof well. The 100μl (1mg/mL) aliquot of dispersed copper nanoparticles was added to the first well of the plate. Then, two-fold serial dilution was acquired and 50μl of inoculum was added to each well except for negative control. Chloramphenicol as a positive control and Muller Hinton broth as negative control were used during the assay. The plates were incubated at 37°C for 24h and the lowest sample concentration showing zone of inhibition was considered as MIC value. The lowest concentration of the subculture without visible growth was recorded as a minimum bactericidal concentration (MBC).
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2.3.3 Time-kill study
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The method proposed by [17] was used for the evaluation of the dynamic time-kill
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plots for the selected microorganisms against copper nanoparticles at their MIC values.
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Aliquots of 0.1 ml were taken from each sampleafter 0, 2, 4, 6, 8, 10, 12, 24, 36, 48h interval and were serially dilutedand spread on Muller Hinton agar plates. The plateswere incubated
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at 37°C for 24 h. Colonies were countedmanually and calculated by dilution times.
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2.3.4 Photocatalytic Dye Reduction
The photocatalytic dye reduction ability of the copper nanoparticles was assessed
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using crystal violet (10 mgL-1) and methylene blue (10 mg-1) dye following the method proposed by [18, 19]. Briefly, the experiments of the photocatalytic dye reduction were conducted under the bright sunlight. A 25 ml of dye solution was taken in a conical flask and 0.5 ml of selected copper nanoparticles wereadded under constant stirring. At scheduled time intervals, 2 ml of aliquotsfrom the above solution were taken and centrifuged at 10000 ×g for 10 min. After centrifugation, theabsorption spectra of supernatants were recordedusing a UVVisible spectrophotometer. 2.4 Effect of copper nanoparticles on cell viability by colorimetric (tetrazolium)assay Cytotoxicity of copper nanoparticles was assessed in Caco-2 cells using a tetrazoliumbased colorimetric assay proposed by [20]. Culture (1×105cells) was grownin each well of 96
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Journal Pre-proof well plates and plates were incubated for 24h at 37℃ in a CO2incubator (Company name) for cell attachment. Different concentrations (mg/ml) of copper nanoparticles (solubilizedin media) was added in the wells and incubated for 20h at 37℃ in a CO2 incubator. Media wasremoved after incubation by inverting, flicking and blotting the plate. Washing of cells wascarried out with PBS (100μL/well). To the cells 90μL media and 10μL MTT (5mg/mL ofmedia) wereadded and the plate was gently shaken and incubated for 4 h at 37℃. After incubation,70μL of supernatant was removed and 100μL of dimethyl sulphoxide was added
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in each well tosolubilize the formazan crystals. Optical density was determined with a
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microplate reader usingan absorption spectrum at 570 nm.
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2.5 Statistical Analysis
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Microsoft Excel, 2016 (Microsoft Corp., Redmond, WA) was used for the calculation of standard error mean (SEM). Statistical difference in terms of significant and non-significant
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values was confirmed by one way and two-way analysis of variance and comparison between
3 Results and Discussion
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means was completed by critical difference value [21].
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3.1 Optimization of suitable gum arabic capped copper nanoparticles A rational understanding of the oxidative process turns into a priority in developing strategies for the copper nanoparticle stabilization. In order to prevent the oxidation, capping of copper nanoparticles with different concentrations (0.25-2%) of gum arabic was done. When L-ascorbic acid was added in the hot copper (II) sulfate solution, color change from blue to dark green was visually observed that confirmed the synthesis of copper nanoparticles (Fig 1a).This color change was due to the localized surface plasmon resonance (LSPR) phenomenon in copper nanoparticles as a result of the sp-conduction band to the d-band transition of copper nanoparticles [11]. Similar results werereported in earlier studies, hence confirmed the complete reduction of copper (II) sulfate in the presence of L-ascorbic acid [7,
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Journal Pre-proof 22].Since high surface modifier concentrations are required for the stabilization of the copper nanoparticles and the surface modifier most likely altersthe chemistry of copper nanoparticles, therefore gum arabic capped copper nanoparticles were subjected to spectrophotometric analysis (Fig 1b).Herein, unmodified copper nanoparticles showed surface plasmon resonance at 330 nm. On the other hand, copper nanoparticles stabilized with 0.25 and 0.50% gum arabic did not show a prominent peak at wavelength ranged from 800-290nm, whereas copper nanoparticles stabilized with1, 1.5, and 2% gum arabic showed
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maxima at 330 nm.It can be observed from the spectrometric analysis that gum arabic acted
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both as reducing and capping agents depending upon the concentration.Results are well in
nanoparticles at 330nm.
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accordance with the literature[22] who observed the surface plasmon resonance of copper
3.2 Characterization of gum arabic capped copper nanoparticle
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3.2.1 Particle size and Zeta Potential
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The results of particle size are represented in the (Fig 2a and 2b). The particle size of gum arabic capped copper nanoparticles was ranged from 19.60- 35.06 nm as compared to
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unmodified copper nanoparticles (56.42nm). Copper nanoparticle capped with 0.25 and 0.50% showed non-significant (p<0.05) difference in particle size, whereas copper nanoparticle capped with 1% gum arabic showed least particle size than that of other copper nanoparticles. On the other hand, copper nanoparticles stabilized with 1.5 and 2% gum arabic showed significantly (p<0.05) higher particle size as compared to other concentrations of gum arabic.At low concentration (0.25 and 0.50%), polyanionic droplets of gum arabic served as a nucleation center by binding few copper molecules, therefore, resulted in significantly higher particle size. Whereas,1% of gum arabiccomprehensively transformed the charge dispersal of copper nanoparticle that directly affects thenucleation and monodispersed nature of copper nanoparticles.On the other hand, at higher concentrations of
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Journal Pre-proof gum arabic (1.5 and 2.0 %) bare binding sites of gum arabic increased the particle size of copper nanoparticles. Therefore, based upon UV-Visible spectroscopy, and particle size,copper nanoparticle stabilized with 1% gum arabic was selected for further characterization. Results of zeta potential of copper nanoparticle stabilized with 1% gum arabic are represented in (Fig 2c and 2d). Significant (p<0.05) difference was observed in total charge distribution of gum arabic stabilized copper nanoparticle as compared to unmodified copper nanoparticles. Negative
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charge distribution on gum arabic stabilized copper nanoparticles was attributed due to
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interfacing ofarabino-galacto proteins of gum arabic with polysaccharide moieties that
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extended into the aqueous solution to give rise to a steric and electrostatic stabilization [9].
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3.2.2 SEM, EDS, and TEM of copper nanoparticles
The structural morphologyof copper nanoparticles was evaluated and the results of
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SEM and EDS are represented in (Fig 3a, 3b, 3c, and 3d). Synthesized copper nanoparticles
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were found to be the polygonal prismatic shape [29].As well, the capping of gum arabic on copper nanoparticles did not alter the morphology of the copper nanoparticles and revealed
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the polygonal prismatic shape of copper nanoparticles.In addition, the EDS spectrum illustrated the high intense peak of elemental copper at 1.5keV and 6keV for unmodified copper nanoparticles. In thecase of gum arabic cappedcopper nanoparticles, presence of other elemental carbon (0.5keV) sodium (1.5keV), phosphorus (2.2keV) and chloride (2.5keV) wereobserved that confirmed the capping of gum arabic. Elemental copper was confirmed at 1.5keV and 6keV, respectively. As it was, shown, the EDX spectrum was free of impurities confirming the purity of the gum arabic capped nanoparticles. TEM images (Fig 3e and 3f) also illustrated the closely packed structure of the unmodified copper nanoparticles, whereas copper nanoparticles stabilized with 1% gum arabic showed monodispersed nature of copper
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Journal Pre-proof nanoparticles that could be due to electrostatic and steric interaction of gum arabic with copper nanoparticles. 3.3 MIC and MBC value The antimicrobial efficacy of copper nanoparticles capped with 1% gum arabic was investigated against various pathogenic organisms and the diameter of inhibition zones (mm) around each well is presented in (Fig 4a). Unmodified copper nanoparticles showed significantly (p<0.05) lower zone of inhibition for all the microorganisms in comparison with
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copper nanoparticle capped with 1% gum arabic and positive control, respectively.
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Significantly (p<0.05) higher zone of inhibition exhibited by the gum arabic capped copper
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nanoparticle could be due to the lower particles size [23, 28]. As well, gum arabic capped
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copper nanoparticle showed higher zone of inhibition for Salmonella typhimurium (27mm) followed by Staphylococcus aureus (26mm), Escherichia coli (25mm), and Klebsiella
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pneumonia (25mm). Positive control exhibited, also, significantly (p<0.05) higher zone of
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inhibition (27-30mm) than that of both the copper nanoparticles. As it was, shown, copper nanoparticles capped with 1% gum arabic created oxidative stress by generating reactive
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oxygen species inside the cell membrane and caused the disturbance of the cell membrane by damaging the proteins and DNA [24]. MIC and MBC values of copper nanoparticles are represented in the (Fig 4b and 4c). According to results, copper nanoparticles capped with 1% gum arabic showed significantly (p<0.05) lower MIC and MBC values than that of unmodified copper nanoparticles. In fact, for Staphylococcus aureus and Salmonella typhimurium, copper nanoparticles capped with 1% gum arabic exhibited 3.125mg/ml MIC and 6.25mg/ml MBC values, whereas for Escherichia coli and Klebsiella pneumonia it was 6.25mg/ml and 12.50mg/ml, respectively. In addition, unmodified copper nanoparticles showed 12.50 and 25mg/ml MIC values for
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Journal Pre-proof Staphylococcus aureus, Salmonella typhimurium, Escherichia coli,and Klebsiella pneumonia, whereas 25mg and 50mg/ml MBC values for all the microorganisms.
3.3.1 Time-kill study A time kills dynamic experiment was conducted to compare the antibacterial activity of either unmodified and gum arabic capped copper nanoparticles towards the growth of
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Staphylococcus aureus, Salmonella typhimurium, Escherichia coli,and Klebsiella pneumonia.
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Results of the time-kill study for unmodified and gum arabic capped copper nanoparticles are
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represented in table 1. Copper nanoparticle capped with gum arabic strongly inhibited the
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growth of all microorganisms with increasing time interval than that of unmodified copper nanoparticles. In the case of Staphylococcus aureus, the number of bacterial cells was
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reduced to approximately 101 logCFU/ml during the first 10h, whereas unmodified copper
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nanoparticles reduced approximately 103 logCFU/ml during the first 10h. This inhibitory effect was continuous until 12h and started to increase at 24h. In addition, Salmonella
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typhimurium was more sensitive to copper nanoparticle capped with gum arabic and thenumber of bacterial cells was reduced to 101 logCFU/ml during the first 8h and the inhibitory effect was continued till 24h. As well, in the case of Escherichia coli and Klebsiella pneumonia, copper nanoparticles capped with gum arabic reduced the bacterial cells approximately 101 logCFU/ml during the first 10h. All the microorganisms showed less sensitivity towards unmodified copper nanoparticles and thetime-kill study for copper nanoparticles also supported the results of MIC and MBC. 3.4 Photocatalytic dye reduction Results of photocatalytic dye reduction efficiency of copper nanoparticles are represented in the (Fig 5a, 5b, 5c, and 5d). From the results, copper nanoparticles capped with
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Journal Pre-proof 1% gum arabic reduced the dyes more efficientlyas compared to unmodified copper nanoparticles. Gum arabic capped copper nanoparticle reduced approximately 95% of methylene blue in 30min, whereas unmodified nanoparticles took 60min to reduce the 95% of the dye. Similarly, Gum arabic capped copper nanoparticle reduced approximately 95% of crystal violet in 30min, whereas unmodified nanoparticles reduced 95% of crystal violet in 40 min. Interaction of significant (p<0.05) smaller particle-sized gum arabic capped copper nanoparticles absorbed more photons that created more charge, dynamics, and surface
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trapping. Due to this surface reactivity of the copper nanoparticles surface radicals are formed
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among water, oxygen, and dyes that caused plasmonic decay of small-sized copper
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nanoparticles [25]. Results are in line with the findings of Sinha andAhmaruzzaman, (2017)
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who observed the 95% decay of methylene blue in the presence of green synthesized copper nanoparticles.
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3.5 Effect of copper nanoparticles on Caco-2 cell viability by MTT assay
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Cytotoxicity of copper nanoparticles was assessed and the results of Caco-2 cell viability by MTT assayare represented in the (Fig 5e). From the results, both copper
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nanoparticles were found to be non-toxic to Caco-2 cells. In fact, higher concentration (10mg/ml) of unmodified and gum arabic capped copper nanoparticles showed 95.12 and 97.05% cell viability. Our results correlated well with the literature[26] who studied the cytotoxic effect of albumin coated copper nanoparticles on human breast cancer cells of MDA-MB 231and reported no toxicity of copper nanoparticles even at higher concentration. This could be due to the fact that copper nanoparticles capper with gum arabic did not produce free radicals through the Fenton reaction. As well, gum arabic controlled copper redox potential and inhibit it from contributing in a catalytic cycle to produce toxic components [27].
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Journal Pre-proof 4. Conclusion In conclusion, we synthesizedgum arabic capped copper nanoparticles for enhanced antimicrobial and photocatalytic dye reduction efficacy. The LSPR of gum arabic capped nanoparticles showed no shift in the maxima (330 nm) in comparison with unmodified copper nanoparticles. Based upon particle size, copper nanoparticle capped with 1% gum arabic were found to be the most suitable nanoparticles for enhanced antimicrobial and photocatalytic dye reduction efficiency. Capping with gum arabic did not alter the polygonal
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prismatic shape of the copper nanoparticles. During cytotoxicity study, gum arabic capped
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copper nanoparticles were found non-toxic even at 10 mg/ ml concentration. In addition,
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copper nanoparticles capped with gum arabic were highly oxidative stable and they could be
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directly used as additives for antibacterial formulation. As well, nanoparticles could be used
5. Acknowledgment
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for the purification of waste-water.
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The author recognizes the financial support provided by the Department of Science and Technology, Science for Equity, Empowerment and Development Division, Government
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oxide clusters and nanoparticles. Dalton Transactions, 41(1) (2012) 219-227.
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Journal of Inorg M, 3(7) (2001) 643-646.
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23. O.Yamamoto, Influence of particle size on the antibacterial activity of zinc oxide. Int
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24. C. P. Devatha, K. Jagadeesh, K., M. Patil, Effect of Green synthesized iron nanoparticles by AzardirachtaIndica in different proportions on antibacterial activity.
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Environ. Nanotechnol. Monit. Manage, 9 (2018) 85-94
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25. T. Sinha, M. Ahmaruzzaman, M. (2015). Green synthesis of copper nanoparticles for the efficient removal (degradation) of dye from aqueous phase. Environ SciPollut R,
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22(24) (2015) 20092-20100.
26. M. Azizi, H. Ghourchian, F. Yazdian, F. Dashtestani, F. H. AlizadehZeinabad, (2017). Cytotoxic effect of albumin coated copper nanoparticle on human breast cancer cells of MDA-MB 231. PloS One, 12(11) (2017) e0188639. 27. P.R. Prasad, S. Kanchi, E.B. Naidoo, In-vitro evaluation of copper nanoparticles cytotoxicity on prostate cancer cell lines and their antioxidant, sensing and catalytic activity: One-pot green approach. J. Photochem. Photobiol.161 (2016) 375-382. 28. A. Hanora, M.Ghorab, A.I. El-Batal, F.M.A Mosalam, Synthesis and characterization of gold nanoparticles and their anticancer activity using gamma radiation. JChemical and PharmaceutiRes,8(3) (2016), 405-423.
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Journal Pre-proof 29. G. P. Lee, A.I. Minett, P.C. Innis, G.G. Wallace, (2009). A new twist: controlled shape-shifting of silver nanoparticles from prisms to discs. J of Materials Chem,
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19(44), (2009) 8294-8298.
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Journal Pre-proof Figure 1 (a): Visual confirmation of copper nanoparticles, and Figure1(b): UV-Visible spectrum of unmodified and gum arabic (0.25, 0.50, 1, 1.5, and 2%) capped copper nanoparticles
Figure 2: (a) Average particle size of unmodified copper nanoparticles, (b) average particle size of gum arabic (0.25, 0.50, 1, 1.5, and 2%) capped copper nanoparticles, (c) apparent zeta potential of unmodified copper nanoparticles, and (d) apparent zeta potential of copper nanoparticles capped with 1% gum arabic
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Figure 3: Scanning electron microscopy image of (a) unmodified copper nanoparticles (5000×), (b) copper nanoparticles capped with 1% gum arabic (2500 ×).Energydispersive X-ray spectroscopy image of (c) unmodified copper nanoparticles(d) copper nanoparticles capped with 1% gum arabic. Transmission electron microscopy image of (e) unmodified copper nanoparticles (50nm), and (f) copper nanoparticles capped with 1% gum arabic (50nm).
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Figure 4: (a) Zone of inhibition (b) Minimum Bactericidal Concentrations (MBC) and Minimum Inhibitory Concentration (MIC) values of unmodified and gum arabic (1%) capped copper nanoparticles Data are presented as means±SEM (n=3) a-b
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Means within a row with different lowercase superscript are significantly different (p<0.05) from each other
Figure 5 (a b, c, and d): Photocatalytic methylene blue and crystal violet reduction efficiency of unmodified and 1% gum arabic capped copper nanoparticles. (e) Viability of Caco-2 cells exposed to increasing concentration of unmodified and 1% gum arabic capped copper nanoparticles
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Journal Pre-proof Table 1. Time kill study of unmodified and capped with 1% gum arabic copper nanoparticles against selected microorganisms S. typhimurium (logCFU/ml)
E.coli (logCFU/ml)
K.pneumonia (logCFU/ml)
unmod ified
unmod ified
unmod ified
unmod ified
with 1% gum arabic 105 104 104 103 103 101 101 102 102 103
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105 105 104 104 103 103 103 102 103 104
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105 105 105 104 104 103 103 102 103 104
with 1% gum arabic 105 104 104 103 103 102 101 102 103 103
105 105 104 104 103 103 103 102 103 104
with 1% gum arabic 105 104 104 103 103 101 101 102 103 103
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105 105 104 104 104 103 103 104 104 104
with 1% gum arabic 105 104 104 103 103 101 101 102 104 104
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0 2 4 6 8 10 12 24 36 48
S. aureus (logCFU/ml)
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Time (h)
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Journal Pre-proof CONFLICT OF INTEREST The authors declare no conflict of interest. COMPLIANCE WITH ETHICAL REQUIREMENTS
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This article does not contain any studies with human and animal subjects.
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Journal Pre-proof Highlights
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Eco friendly non-toxic gum arabic capped copper nanoparticles were synthesized SEM micrographs confirmed the cubic shape of copper nanoparticles Gum arabic capped copper nanoparticles were more effective against food pathogenic bacteria. Surface functionalized copper nanoparticles degraded 95% methylene blue and crystal violet
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Figure 1
Figure 2ab
Figure 2cd
Figure 3ad
Figure 3ef
Figure 4
Figure 5ac
Figure 5de
nanoparticles:
Synthesis,
Prince Chawla, Naveen Kumar, Aarti Bains, Sanju Bala Dhull, Mukul Kumar, Ravinder Kaushik, Sneh Punia PII:
S0141-8130(19)37263-0
DOI:
https://doi.org/10.1016/j.ijbiomac.2019.12.260
Reference:
BIOMAC 14288
To appear in:
International Journal of Biological Macromolecules
Received date:
8 September 2019
Revised date:
20 November 2019
Accepted date:
30 December 2019
Please cite this article as: P. Chawla, N. Kumar, A. Bains, et al., Gum arabic capped copper nanoparticles: Synthesis, characterization, and applications, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2019.12.260
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© 2020 Published by Elsevier.
Journal Pre-proof Gum arabic capped copper nanoparticles: synthesis, characterization, and applications
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Prince Chawla1, Naveen Kumar2, Aarti Bains4, Sanju Bala Dhull3*, Mukul Kumar5, Ravinder Kaushik1*, Sneh Punia3 1 Shoolini University, Solan, Himachal Pradesh, India 2 Amity University, Jaipur, Rajasthan, India 3 Chaudhary Devi Lal University, Sirsa, Haryana, India 4 Chandigarh Group of Colleges, Mohali, Punjab, India 5 Lovely Professional University, Phagwara, Punjab, India
*Corresponding author Contact e-mail: [email protected]; [email protected] E-mail: [email protected]
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Journal Pre-proof Abstract In present work, we synthesized copper nanoparticles using L-ascorbic acid as a reducing agent and capped them with gum arabic. Based upon spectrometric analysis and particle size distribution(19.60- 35.06 nm) by intensity, a 1% concentrationof gum arabic was selected asthe suitable cappingagentfor copper nanoparticles. Gum arabic capped copper nanoparticles revealed significantly (p<0.05) higher zeta potential valuethan that ofunmodified copper nanoparticles. Energy-dispersive X-ray spectroscopyrevealed the purity of the copper
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nanoparticles, whereas scanning electron microscopy confirmed the polygonal prismatic
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shape of the gum arabic capped copper nanoparticles. As well, TEM analysis confirmed the
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monodispersed nature of thegum arabic cappedcopper nanoparticles.Gum arabic capped
typhimurium
(27mm)than
that
of
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copper nanoparticles showed significantly (p<0.05) higher zone of inhibition for Salmonella other
bacterial
strains.
Approximately95%
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photocatalyticdegradationof methylene blue and crystal violet was observed within 40 and 20
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min. As well, both unmodified and gum arabic cappedcopper nanoparticles were found to be non-toxic to Caco-2 cells during cell viability assay.
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Abbreviations: DLS, Dynamic light scattering, PBS,Phosphate buffer solution, GA,Gum arabic, DMEM, Dulbecco‟s Modified Eagles Medium, DMSO Dimethyl sulfoxide Keywords: Copper nanoparticles; Gum arabic; Dye reduction; Zeta potential; Cytotoxicity; LSPR
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Journal Pre-proof 1. Introduction Copper nanoparticles have gained as the most attractive nanoparticles in the field of multidisciplinary science [1]. Typically, copper nanoparticles are synthesized using several chemical and physical approaches based on the appropriateness of the methods to achieve the desirable applications [2]. Physical methods used for the synthesis of copper nanoparticles implicate expensive instruments and sophisticated techniques (1-2). While, using chemical approaches copper nanoparticles are synthesized by several chemical reductants such as tri-
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sodium citrate, sodium borohydride, hydrazine [3]. Even though chemical approaches have
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been extensively applied for the synthesis of copper nanoparticles but the precarious
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reductants and several organic solvents used for the synthesis are potentially hazardous for
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the environment [2,4].Therefore, the development of affordable solutions is in demand to efficiently reduce the hazardous risks of chemical reductants [4, 5]
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Presently, green synthesis of the nanoparticle is an incipient phenomenonthat
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effectively reduces the risk to the environment by eradicating the hazardous components that are harmful to human health [3-6]. The most common effective approach for obtaining
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copper nanoparticles includes the nucleation of copper ions using L-Ascorbic acid to form nanoparticles [7].The broad-spectrum complications like aggregation and oxidation of copper nanoparticles limit their usage. However, the usage of the appropriate stabilizing agent in the preparation resolves this problem effortlessly [6-10].Though, the presence of a stabilizing component is required to formulate more steady and relatively monodispersed copper nanoparticles [1].Natural components have widely been used either for the reduction of copper ions or for the protection of the formulated copper nanoparticles in the aqueous phase [3-7]. Therefore, among all the biological macromolecules, gum arabic attained decent attention due to reduced toxicity, biocompatibility with the metals, cost-effectiveness and easy
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Journal Pre-proof availability [15]. Surface charge of stabilizer is an important feature to maintain the electrostatic interactions and steric hindrance between monodispersed nanoparticles and water system, therefore, the polyanionic nature of gum arabic is an important feature to maintain the monodispersed nature of nanoparticles [12, 13]. To deal with several biological and inorganic toxic pollutants, metal nanoparticles are being vigorously looked into. Currently, copper nanoparticles are being used as an effective approach to eradicate organic and inorganic pollutants [4-6].
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In addition, copper nanoparticles have attained great interests due to their distinctive
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physicochemical properties that make them effective agents against different classes of
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pollutants [4,5]. Due to differentiated properties, copper nanoparticles exhibits sorbent
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properties that directly used for the elimination of toxic inorganic metal pollutants and organic dyes from industrialized wastewater [6, 14]. The copper nanoparticles are broadly
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used as antibacterial agents because of low toxicity and biodegradable nature [3, 14].These
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copper nanoparticles also exhibit the mechanism of antibacterial properties due to their large surface area relative to the volume that proficientlydecreases the oxygen stream by binding
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with the cell wall that ultimately causes the reduction in respiration. Therefore, the aim of the present study was to improve the oxidative stability of copper nanoparticles and also to improve the bactericidal activity. Keeping this in mind, copper nanoparticles were capped with gum arabic. A major highlight of the current study was the potential application of the capped nanoparticles as anantibacterial against food pathogenic bacteria and photocatalytic dye reduction of methylene blue and crystal violet. 2. Materials and Methods 2.1 Materials Spray-dried gum arabic was procured from Sigma Aldrich, St. Louis, MO, USA. Lascorbic acid, copper sulphate pentahydrate, hydrochloric acid, sulphuric acid, nitric acid, methylene blue trihydrate, crystal violet,
ethanol, hydrogen peroxide,
antibiotic 4
Journal Pre-proof (ciprofloxacin), nutrient agar and nutrient broth were procured from Hi-Media Pvt. Ltd. Mumbai, India. Bacterial cultures (i.e., Staphylococcus aureus (MTCC 3160), Escherichia coli (MTCC 443), Klebsiella pneumoniae (MTCC 3384) and Salmonella typhimurium (MTCC 1357) were obtained from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India.Human colon adenocarcinoma (Caco-2 cells) were procured from National Centre for Cell Science, Pune, Maharashtra, India.DMEM (Dulbecco‟s Modified Eagles Medium), DMSO (dimethyl sulfoxide), L-glutamine, and
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streptomycin (sulfate salt) were purchased from Sigma Aldrich, St. Louis, MO, USA; Human
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ferritin ELISA kit (SEA518Hu 96 Tests) from Cloud Clone-Corp. Houston, USA. Plastic
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dishes, plates, and transwell were obtained from Corning (Corning, NY, USA). All the
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chemicals were of „Analytical Reagent‟ grade. Triple distilled water and aqua-regia washed glassware (class “A” certified) was used throughout the experiments.
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2.2 Methods
2.2.1 Synthesis of gum arabic stabilized copper nanoparticles
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Copper nanoparticles were prepared using copper(II)sulphate pentahydrate as a
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precursor and L-Ascorbic acid as a reducing agent [7]. An aqueous solution of L-Ascorbic acid (0.1mM, 50mL) was dropwise added to an aqueous solution of copper(II)sulphate pentahydrate (80℃, 0.1mM, 50mL) under continuous and gentle stirring using magnetic stirrer (M3D, Eltak DIGIMAG, India) for 2h. Later, different concentration of gum arabic (i.e. 0.25, 0.50, 1.0, 1.5,& 2.0%) was added to hot solution of reaction mixture for complete reduction and capping under continuous stirring (1h). The resulting mixture was then transferred to glass vials and stored at room temperature (27℃) for further analysis. 2.2.2 Characterization methods Reduction by L-ascorbic acid and stabilization with gum arabic of copper nanoparticles
was
confirmed
by
the
UV-Visible
analysis
using
UV-Visible
Spectrophotometer (Evolution 201, Thermo Fischer Scientific IndiaPvt.Ltd, Mumbai). The 5
Journal Pre-proof quartz cuvettes of 3.5 ml with 3.5 cm path length were used for the analysis.Zeta potential analyzer (Zetasizer Nano ZS, Malvern Instruments Ltd. Malvern, WR14 1XZ, UK) was used for the determination of particle size and zeta potential of the copper nanoparticles.The morphology of the selected copper nanoparticles was described by SEM analysis using a JEOLJSM-6510LV (INCA, USA) microscope and the elemental analysis was performed by Energy-dispersive X-ray spectroscopy using a RONTEC‟s EDX system Model QuanTax 200 analyzer.To confirm the homogenous shape of the copper nanoparticles, TEM images were
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acquired using a transmission electron microscope (JEM-1011; JEOL microscope). The
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instrument was used at anaccelerating voltage of 200kV and copper nanoparticles were
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2.3 Application of Copper Nanoparticles
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directly mounted on a carbon-coated copper grid for the analysis.
2.3.1 Antimicrobial efficiencyagainst food pathogenic bacteria
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The agar well dilution method was used to evaluate the antimicrobial activity of
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selected nanoparticle against food pathogenic strains [12]. Mueller Hinton Agar glass plates were prepared and inoculated with the selected microorganisms (~1.5×108 cells/ml) by the
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spread plate method. After 30 min, wells (6mm) in the seeded agar plates were punched using cork borer. For positive control, chloramphenicol was used as a standard antibacterial agent, whereas, DMSO was used as a solvent (negative control). A 10 mL of different concentrations (25, 50, 75, 100 mg/mL) of copper nanoparticles were prepared and constant volume (100μl/well) was poured into the wells, then incubated at 37℃ for 24h. For the reference antibiotic, the concentration of 150mg/ml was used[16]. 2.3.2 Minimum Inhibitory Concentration
(MIC) and
Minimum Bactericidal
Concentrations (MBC) values The MIC values were determined by the microdilution broth method using the microtiterplate method. The plates were prepared by pouring 100μl nutrient broth into each
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Journal Pre-proof well. The 100μl (1mg/mL) aliquot of dispersed copper nanoparticles was added to the first well of the plate. Then, two-fold serial dilution was acquired and 50μl of inoculum was added to each well except for negative control. Chloramphenicol as a positive control and Muller Hinton broth as negative control were used during the assay. The plates were incubated at 37°C for 24h and the lowest sample concentration showing zone of inhibition was considered as MIC value. The lowest concentration of the subculture without visible growth was recorded as a minimum bactericidal concentration (MBC).
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2.3.3 Time-kill study
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The method proposed by [17] was used for the evaluation of the dynamic time-kill
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plots for the selected microorganisms against copper nanoparticles at their MIC values.
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Aliquots of 0.1 ml were taken from each sampleafter 0, 2, 4, 6, 8, 10, 12, 24, 36, 48h interval and were serially dilutedand spread on Muller Hinton agar plates. The plateswere incubated
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at 37°C for 24 h. Colonies were countedmanually and calculated by dilution times.
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2.3.4 Photocatalytic Dye Reduction
The photocatalytic dye reduction ability of the copper nanoparticles was assessed
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using crystal violet (10 mgL-1) and methylene blue (10 mg-1) dye following the method proposed by [18, 19]. Briefly, the experiments of the photocatalytic dye reduction were conducted under the bright sunlight. A 25 ml of dye solution was taken in a conical flask and 0.5 ml of selected copper nanoparticles wereadded under constant stirring. At scheduled time intervals, 2 ml of aliquotsfrom the above solution were taken and centrifuged at 10000 ×g for 10 min. After centrifugation, theabsorption spectra of supernatants were recordedusing a UVVisible spectrophotometer. 2.4 Effect of copper nanoparticles on cell viability by colorimetric (tetrazolium)assay Cytotoxicity of copper nanoparticles was assessed in Caco-2 cells using a tetrazoliumbased colorimetric assay proposed by [20]. Culture (1×105cells) was grownin each well of 96
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Journal Pre-proof well plates and plates were incubated for 24h at 37℃ in a CO2incubator (Company name) for cell attachment. Different concentrations (mg/ml) of copper nanoparticles (solubilizedin media) was added in the wells and incubated for 20h at 37℃ in a CO2 incubator. Media wasremoved after incubation by inverting, flicking and blotting the plate. Washing of cells wascarried out with PBS (100μL/well). To the cells 90μL media and 10μL MTT (5mg/mL ofmedia) wereadded and the plate was gently shaken and incubated for 4 h at 37℃. After incubation,70μL of supernatant was removed and 100μL of dimethyl sulphoxide was added
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in each well tosolubilize the formazan crystals. Optical density was determined with a
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microplate reader usingan absorption spectrum at 570 nm.
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2.5 Statistical Analysis
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Microsoft Excel, 2016 (Microsoft Corp., Redmond, WA) was used for the calculation of standard error mean (SEM). Statistical difference in terms of significant and non-significant
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values was confirmed by one way and two-way analysis of variance and comparison between
3 Results and Discussion
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means was completed by critical difference value [21].
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3.1 Optimization of suitable gum arabic capped copper nanoparticles A rational understanding of the oxidative process turns into a priority in developing strategies for the copper nanoparticle stabilization. In order to prevent the oxidation, capping of copper nanoparticles with different concentrations (0.25-2%) of gum arabic was done. When L-ascorbic acid was added in the hot copper (II) sulfate solution, color change from blue to dark green was visually observed that confirmed the synthesis of copper nanoparticles (Fig 1a).This color change was due to the localized surface plasmon resonance (LSPR) phenomenon in copper nanoparticles as a result of the sp-conduction band to the d-band transition of copper nanoparticles [11]. Similar results werereported in earlier studies, hence confirmed the complete reduction of copper (II) sulfate in the presence of L-ascorbic acid [7,
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Journal Pre-proof 22].Since high surface modifier concentrations are required for the stabilization of the copper nanoparticles and the surface modifier most likely altersthe chemistry of copper nanoparticles, therefore gum arabic capped copper nanoparticles were subjected to spectrophotometric analysis (Fig 1b).Herein, unmodified copper nanoparticles showed surface plasmon resonance at 330 nm. On the other hand, copper nanoparticles stabilized with 0.25 and 0.50% gum arabic did not show a prominent peak at wavelength ranged from 800-290nm, whereas copper nanoparticles stabilized with1, 1.5, and 2% gum arabic showed
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maxima at 330 nm.It can be observed from the spectrometric analysis that gum arabic acted
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both as reducing and capping agents depending upon the concentration.Results are well in
nanoparticles at 330nm.
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accordance with the literature[22] who observed the surface plasmon resonance of copper
3.2 Characterization of gum arabic capped copper nanoparticle
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3.2.1 Particle size and Zeta Potential
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The results of particle size are represented in the (Fig 2a and 2b). The particle size of gum arabic capped copper nanoparticles was ranged from 19.60- 35.06 nm as compared to
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unmodified copper nanoparticles (56.42nm). Copper nanoparticle capped with 0.25 and 0.50% showed non-significant (p<0.05) difference in particle size, whereas copper nanoparticle capped with 1% gum arabic showed least particle size than that of other copper nanoparticles. On the other hand, copper nanoparticles stabilized with 1.5 and 2% gum arabic showed significantly (p<0.05) higher particle size as compared to other concentrations of gum arabic.At low concentration (0.25 and 0.50%), polyanionic droplets of gum arabic served as a nucleation center by binding few copper molecules, therefore, resulted in significantly higher particle size. Whereas,1% of gum arabiccomprehensively transformed the charge dispersal of copper nanoparticle that directly affects thenucleation and monodispersed nature of copper nanoparticles.On the other hand, at higher concentrations of
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Journal Pre-proof gum arabic (1.5 and 2.0 %) bare binding sites of gum arabic increased the particle size of copper nanoparticles. Therefore, based upon UV-Visible spectroscopy, and particle size,copper nanoparticle stabilized with 1% gum arabic was selected for further characterization. Results of zeta potential of copper nanoparticle stabilized with 1% gum arabic are represented in (Fig 2c and 2d). Significant (p<0.05) difference was observed in total charge distribution of gum arabic stabilized copper nanoparticle as compared to unmodified copper nanoparticles. Negative
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charge distribution on gum arabic stabilized copper nanoparticles was attributed due to
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interfacing ofarabino-galacto proteins of gum arabic with polysaccharide moieties that
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extended into the aqueous solution to give rise to a steric and electrostatic stabilization [9].
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3.2.2 SEM, EDS, and TEM of copper nanoparticles
The structural morphologyof copper nanoparticles was evaluated and the results of
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SEM and EDS are represented in (Fig 3a, 3b, 3c, and 3d). Synthesized copper nanoparticles
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were found to be the polygonal prismatic shape [29].As well, the capping of gum arabic on copper nanoparticles did not alter the morphology of the copper nanoparticles and revealed
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the polygonal prismatic shape of copper nanoparticles.In addition, the EDS spectrum illustrated the high intense peak of elemental copper at 1.5keV and 6keV for unmodified copper nanoparticles. In thecase of gum arabic cappedcopper nanoparticles, presence of other elemental carbon (0.5keV) sodium (1.5keV), phosphorus (2.2keV) and chloride (2.5keV) wereobserved that confirmed the capping of gum arabic. Elemental copper was confirmed at 1.5keV and 6keV, respectively. As it was, shown, the EDX spectrum was free of impurities confirming the purity of the gum arabic capped nanoparticles. TEM images (Fig 3e and 3f) also illustrated the closely packed structure of the unmodified copper nanoparticles, whereas copper nanoparticles stabilized with 1% gum arabic showed monodispersed nature of copper
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Journal Pre-proof nanoparticles that could be due to electrostatic and steric interaction of gum arabic with copper nanoparticles. 3.3 MIC and MBC value The antimicrobial efficacy of copper nanoparticles capped with 1% gum arabic was investigated against various pathogenic organisms and the diameter of inhibition zones (mm) around each well is presented in (Fig 4a). Unmodified copper nanoparticles showed significantly (p<0.05) lower zone of inhibition for all the microorganisms in comparison with
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copper nanoparticle capped with 1% gum arabic and positive control, respectively.
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Significantly (p<0.05) higher zone of inhibition exhibited by the gum arabic capped copper
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nanoparticle could be due to the lower particles size [23, 28]. As well, gum arabic capped
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copper nanoparticle showed higher zone of inhibition for Salmonella typhimurium (27mm) followed by Staphylococcus aureus (26mm), Escherichia coli (25mm), and Klebsiella
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pneumonia (25mm). Positive control exhibited, also, significantly (p<0.05) higher zone of
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inhibition (27-30mm) than that of both the copper nanoparticles. As it was, shown, copper nanoparticles capped with 1% gum arabic created oxidative stress by generating reactive
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oxygen species inside the cell membrane and caused the disturbance of the cell membrane by damaging the proteins and DNA [24]. MIC and MBC values of copper nanoparticles are represented in the (Fig 4b and 4c). According to results, copper nanoparticles capped with 1% gum arabic showed significantly (p<0.05) lower MIC and MBC values than that of unmodified copper nanoparticles. In fact, for Staphylococcus aureus and Salmonella typhimurium, copper nanoparticles capped with 1% gum arabic exhibited 3.125mg/ml MIC and 6.25mg/ml MBC values, whereas for Escherichia coli and Klebsiella pneumonia it was 6.25mg/ml and 12.50mg/ml, respectively. In addition, unmodified copper nanoparticles showed 12.50 and 25mg/ml MIC values for
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3.3.1 Time-kill study A time kills dynamic experiment was conducted to compare the antibacterial activity of either unmodified and gum arabic capped copper nanoparticles towards the growth of
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Staphylococcus aureus, Salmonella typhimurium, Escherichia coli,and Klebsiella pneumonia.
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Results of the time-kill study for unmodified and gum arabic capped copper nanoparticles are
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represented in table 1. Copper nanoparticle capped with gum arabic strongly inhibited the
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growth of all microorganisms with increasing time interval than that of unmodified copper nanoparticles. In the case of Staphylococcus aureus, the number of bacterial cells was
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reduced to approximately 101 logCFU/ml during the first 10h, whereas unmodified copper
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nanoparticles reduced approximately 103 logCFU/ml during the first 10h. This inhibitory effect was continuous until 12h and started to increase at 24h. In addition, Salmonella
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typhimurium was more sensitive to copper nanoparticle capped with gum arabic and thenumber of bacterial cells was reduced to 101 logCFU/ml during the first 8h and the inhibitory effect was continued till 24h. As well, in the case of Escherichia coli and Klebsiella pneumonia, copper nanoparticles capped with gum arabic reduced the bacterial cells approximately 101 logCFU/ml during the first 10h. All the microorganisms showed less sensitivity towards unmodified copper nanoparticles and thetime-kill study for copper nanoparticles also supported the results of MIC and MBC. 3.4 Photocatalytic dye reduction Results of photocatalytic dye reduction efficiency of copper nanoparticles are represented in the (Fig 5a, 5b, 5c, and 5d). From the results, copper nanoparticles capped with
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Journal Pre-proof 1% gum arabic reduced the dyes more efficientlyas compared to unmodified copper nanoparticles. Gum arabic capped copper nanoparticle reduced approximately 95% of methylene blue in 30min, whereas unmodified nanoparticles took 60min to reduce the 95% of the dye. Similarly, Gum arabic capped copper nanoparticle reduced approximately 95% of crystal violet in 30min, whereas unmodified nanoparticles reduced 95% of crystal violet in 40 min. Interaction of significant (p<0.05) smaller particle-sized gum arabic capped copper nanoparticles absorbed more photons that created more charge, dynamics, and surface
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trapping. Due to this surface reactivity of the copper nanoparticles surface radicals are formed
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among water, oxygen, and dyes that caused plasmonic decay of small-sized copper
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nanoparticles [25]. Results are in line with the findings of Sinha andAhmaruzzaman, (2017)
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who observed the 95% decay of methylene blue in the presence of green synthesized copper nanoparticles.
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3.5 Effect of copper nanoparticles on Caco-2 cell viability by MTT assay
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Cytotoxicity of copper nanoparticles was assessed and the results of Caco-2 cell viability by MTT assayare represented in the (Fig 5e). From the results, both copper
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nanoparticles were found to be non-toxic to Caco-2 cells. In fact, higher concentration (10mg/ml) of unmodified and gum arabic capped copper nanoparticles showed 95.12 and 97.05% cell viability. Our results correlated well with the literature[26] who studied the cytotoxic effect of albumin coated copper nanoparticles on human breast cancer cells of MDA-MB 231and reported no toxicity of copper nanoparticles even at higher concentration. This could be due to the fact that copper nanoparticles capper with gum arabic did not produce free radicals through the Fenton reaction. As well, gum arabic controlled copper redox potential and inhibit it from contributing in a catalytic cycle to produce toxic components [27].
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Journal Pre-proof 4. Conclusion In conclusion, we synthesizedgum arabic capped copper nanoparticles for enhanced antimicrobial and photocatalytic dye reduction efficacy. The LSPR of gum arabic capped nanoparticles showed no shift in the maxima (330 nm) in comparison with unmodified copper nanoparticles. Based upon particle size, copper nanoparticle capped with 1% gum arabic were found to be the most suitable nanoparticles for enhanced antimicrobial and photocatalytic dye reduction efficiency. Capping with gum arabic did not alter the polygonal
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prismatic shape of the copper nanoparticles. During cytotoxicity study, gum arabic capped
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copper nanoparticles were found non-toxic even at 10 mg/ ml concentration. In addition,
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copper nanoparticles capped with gum arabic were highly oxidative stable and they could be
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directly used as additives for antibacterial formulation. As well, nanoparticles could be used
5. Acknowledgment
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for the purification of waste-water.
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The author recognizes the financial support provided by the Department of Science and Technology, Science for Equity, Empowerment and Development Division, Government
6. References
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Journal Pre-proof Figure 1 (a): Visual confirmation of copper nanoparticles, and Figure1(b): UV-Visible spectrum of unmodified and gum arabic (0.25, 0.50, 1, 1.5, and 2%) capped copper nanoparticles
Figure 2: (a) Average particle size of unmodified copper nanoparticles, (b) average particle size of gum arabic (0.25, 0.50, 1, 1.5, and 2%) capped copper nanoparticles, (c) apparent zeta potential of unmodified copper nanoparticles, and (d) apparent zeta potential of copper nanoparticles capped with 1% gum arabic
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Figure 3: Scanning electron microscopy image of (a) unmodified copper nanoparticles (5000×), (b) copper nanoparticles capped with 1% gum arabic (2500 ×).Energydispersive X-ray spectroscopy image of (c) unmodified copper nanoparticles(d) copper nanoparticles capped with 1% gum arabic. Transmission electron microscopy image of (e) unmodified copper nanoparticles (50nm), and (f) copper nanoparticles capped with 1% gum arabic (50nm).
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Figure 4: (a) Zone of inhibition (b) Minimum Bactericidal Concentrations (MBC) and Minimum Inhibitory Concentration (MIC) values of unmodified and gum arabic (1%) capped copper nanoparticles Data are presented as means±SEM (n=3) a-b
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Means within a row with different lowercase superscript are significantly different (p<0.05) from each other
Figure 5 (a b, c, and d): Photocatalytic methylene blue and crystal violet reduction efficiency of unmodified and 1% gum arabic capped copper nanoparticles. (e) Viability of Caco-2 cells exposed to increasing concentration of unmodified and 1% gum arabic capped copper nanoparticles
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Journal Pre-proof Table 1. Time kill study of unmodified and capped with 1% gum arabic copper nanoparticles against selected microorganisms S. typhimurium (logCFU/ml)
E.coli (logCFU/ml)
K.pneumonia (logCFU/ml)
unmod ified
unmod ified
unmod ified
unmod ified
with 1% gum arabic 105 104 104 103 103 101 101 102 102 103
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105 105 104 104 103 103 103 102 103 104
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105 105 105 104 104 103 103 102 103 104
with 1% gum arabic 105 104 104 103 103 102 101 102 103 103
105 105 104 104 103 103 103 102 103 104
with 1% gum arabic 105 104 104 103 103 101 101 102 103 103
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105 105 104 104 104 103 103 104 104 104
with 1% gum arabic 105 104 104 103 103 101 101 102 104 104
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0 2 4 6 8 10 12 24 36 48
S. aureus (logCFU/ml)
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Time (h)
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Journal Pre-proof CONFLICT OF INTEREST The authors declare no conflict of interest. COMPLIANCE WITH ETHICAL REQUIREMENTS
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This article does not contain any studies with human and animal subjects.
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Journal Pre-proof Highlights
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Eco friendly non-toxic gum arabic capped copper nanoparticles were synthesized SEM micrographs confirmed the cubic shape of copper nanoparticles Gum arabic capped copper nanoparticles were more effective against food pathogenic bacteria. Surface functionalized copper nanoparticles degraded 95% methylene blue and crystal violet
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Figure 1
Figure 2ab
Figure 2cd
Figure 3ad
Figure 3ef
Figure 4
Figure 5ac
Figure 5de