Effect of NaOH concentration on antibacterial activities of Cu nanoparticles and the antibacterial mechanism

Journal Pre-proof Effect of NaOH concentration on antibacterial activities of Cu nanoparticles and the antibacterial mechanism

Pengzhao Lv, Lianjie Zhu, Yanmiao Yu, Wenwen Wang, Guokai Liu, Hongguang Lu PII:

S0928-4931(19)33721-X

DOI:

https://doi.org/10.1016/j.msec.2020.110669

Reference:

MSC 110669

To appear in:

Materials Science & Engineering C

Received date:

7 October 2019

Revised date:

17 December 2019

Accepted date:

13 January 2020

Please cite this article as: P. Lv, L. Zhu, Y. Yu, et al., Effect of NaOH concentration on antibacterial activities of Cu nanoparticles and the antibacterial mechanism, Materials Science & Engineering C (2018), https://doi.org/10.1016/j.msec.2020.110669

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof

Effect of NaOH concentration on antibacterial activities of Cu nanoparticles and the antibacterial mechanism Pengzhao Lv, Lianjie Zhu*, Yanmiao Yu, Wenwen Wang, Guokai Liu, Hongguang Lu* School of Chemistry & Chemical Engineering, Tianjin Key Laboratory of Organic

of

Solar Cells and Photochemical Conversion, Tianjin University of Technology, Tianjin

ro

300384, PR China

Abstract

lP

re

(L. Zhu), [email protected] (H. Lu).

-p

* Corresponding authors, Tel. +86-22-60214259, E-mail addresses: [email protected]

na

A series of Cu nanoparticles (NPs) have been prepared by a facile hydrothermal

Jo ur

method at 180 C using different concentrations of NaOH solutions and characterized by XRD, SEM, TEM and FT-IR spectra. Their antibacterial activities were assessed by means of Gram-positive S. aureus and Gram-negative E. coli bacteria, where various dosages (3, 5, 7, 10 mg) of the antibacterial agents were applied, and compared with that of the commercial CuSO4 salt. The antibacterial mechanism was explored in detail based on series of control experiments. The results show that the NaOH concentration affects the crystallinity, crystal size and surface hydroxyl content of the Cu NPs, which significantly influence the antibacterial activities. Compared to the commercial CuSO4 salt, the four Cu samples prepared using no less than 4 mol L-1 1

Journal Pre-proof

of NaOH display excellent antibacterial activities with low concentrations of copper leachates, which is great beneficial to the practical applications. The experimental results support that the highly reactive and soluble copper species in the antibacterial system of the Cu NPs is a Cu (II)-peptide complex, but not the free Cu2+ ions. Keywords: Cu nanoparticles; NaOH effect; Antibacterial activities; Antibacterial

of

mechanism 1 Introduction

ro

A large number of pollutants (e.g. organics, nutriment and microorganisms) are

-p

released into rivers and sea every day, resulting in millions of people around the world

re

suffering from diseases caused by microorganisms (viruses, bacteria and protozoa) in

lP

water every year [1,2]. Thus, many technologies were developed to kill the

na

pathogenic microorganisms [3]. Especially, various antibiotics have been discovered and widely used, which display wonderful effects in fighting diseases for a period.

Jo ur

However, the abuse of antibiotics accelerates the variation of microbial drug resistance [4]. As a result, a growing number of superbugs were reported no more sensitive to most antibiotics in recent years [5], which is serious hazard to human health [6,7]. For instance, Saravanan [8] reported that some Enterobacteriaceae have drug resistance because they can produce a kind of extended spectrum β-lactamases which can hydrolyze a variety of β-lactams including the fourth generation cephalosporins and compromise the efficacy of all β-lactams, except cephamycins and carbapenems. The emergence of nanotechnology offers a new chance to fight against the ever-growing number of antimicrobial-resistant microorganisms specifically by 2

Journal Pre-proof

using metal nanoparticles [9-11]. Nanotechnology has been developed into a promising multidisciplinary field with various significant potentials in pharmaceutical applications, such as anti-cancer, anti-parasite, bactericidal and fungicidal [12-37]. As one kind of important antibacterial materials, inorganic nanomaterials possess pronounced advantages: not

of

easy to produce drug resistance, non-toxic or low-toxic, broad-spectrum antimicrobial, high stability and heat resistance [13-15]. The metal nanomaterials, such as Au [16-20]

ro

and Ag [21-29], have been extensively investigated as efficient antibacterial materials.

-p

Besides, Barabadi systematically collated a series of research about Ag [30-32] and

re

Au [33-35] nanomaterials on anti-cancer and anti-malaria applications. However, the

lP

high cost of Au and Ag limits their applications to certain extent, and Ag is known to

na

accumulate in human body over time, which can result in toxicity, such as argyria [38]. It was reported that the metal NPs may cause genotoxicity due to the increased levels

Jo ur

of reactive oxygen species upon exposure to the metal NPs [39,40]. Nevertheless, highly efficient, cheap and low toxic metal nanomaterials, the Cu NPs, as antibacterial agents still draw great attention.

The Cu NPs have a wide range of applications, such as lubricant additive, electronic products, high-performance catalysts and antibacterial agents etc. The usefulness of Cu NPs as antibacterial agents has been known for a long time and its antibacterial activities have been reported [9,13,14,41-44]. However, the effect of NaOH concentration in the preparation process of the Cu NPs on the antibacterial activities has not been reported, and the antibacterial mechanism of the Cu NPs has 3

Journal Pre-proof

also been rarely concerned [45]. Chatterjee et al. [45] found that the Cu NPs can cause multiple toxic effects such as generation of reactive oxygen species, lipid peroxidation, protein oxidation and DNA degradation in E. coli cells by liberating nascent Cu ions from the Cu NP surface. The release of nascent Cu ions was facilitated by the oxidation of Cu NPs with the simultaneous reduction of the agents such as cells,

of

biomolecules or medium components in the vicinity of the Cu NPs. In the other work [46], however, they found the mechanism of antibacterial action of the CuO NPs was

ro

through the formation of some reactive complex between the CuO NPs and cellular

-p

medium organics. Moreover, Gunawan et al. also reported that the leached

re

copper-peptide complex formed between the CuO NPs and peptide chains induced a

lP

multiple fold increase in intracellular reactive oxygen species generation and reduced

na

the fractions of viable cells, consequently resulting in the overall inhibition of biomass growth [47]. Then, one may wonder if some reactive copper complexes were

Jo ur

also formed between the Cu NPs and medium organics during the antibacterial process, which kills the bacteria instead of the Cu2+ ions. Thus, it is significant to find experimental support to clarify this problem and ascertain the antibacterial mechanism of the Cu NPs. In the present work, a series of Cu NPs were synthesized by simply tuning the concentration of NaOH in the preparation process and their antibacterial activities were studied by absorptiometry using both S. aureus and E. coli as model bacteria. The effect of NaOH concentrations on the antibacterial activities of the Cu NPs was explored and new experimental evidence was provided to explain its antibacterial 4

Journal Pre-proof

mechanism. 2 Experimental 2.1 Preparation of the Cu NPs All chemicals used are analytical grade without further purification. A hydrothermal method has been adopted for preparation of the Cu NPs since it has the

of

characteristics of simple operation, mild reaction conditions and the obtained products have small particle sizes with high crystallinity and minor particle agglomeration. The

ro

preparation process is as follows. After 1 g of copper acetate dissolving in 10 mL

-p

deionized water at room temperature, 6 mL of diluted Poly (acrylic acid) (PAA)

re

solution (1 mL PAA dispersed in 5 mL deionized water) was added to the above

lP

solution under constant stirring and kept for 10 min. When the solution changed to

na

pale bluish green, 0.5 mL of ammonium hydroxide (25%) was added and the solution became blue. Then certain concentration (2, 4, 6, 8, 10 mol L-1) of NaOH solution was

Jo ur

put into the above solution and kept stirring for 10 min. After 5.5 mL of hydrazine hydrate solution (7.3%) was added to the above solution, the mixture was transferred into a Teflon-lined autoclave vessel with 50 mL capacity and heated at 180 ℃ for 1 h. The product was centrifuged and washed for three times by deionized water and ethanol, respectively. Finally, the as-obtained Cu NPs were dried in a vacuum oven at room temperature for 12 h. The Cu NPs prepared using 2, 4, 6, 8 and 10 mol L-1 of NaOH are specified as the sample A, B, C, D and E, respectively. 2.2 Characterizations Powder X-ray diffraction (XRD) was analyzed on a Rigaku ultima IV X-ray 5

Journal Pre-proof diffractometer with Cu Kα radiation, operating at 40 kV and 40 mA. The morphology of the product was observed on a Carl Zeiss MERLINI scanning electron microscopy (SEM) with an operating voltage of 5.0 kV. Transmission electron microscopy (TEM) images were taken on a Tecnai G2 Spirit TWIN transmission electron microscopy operated at 200 kV. The FT-IR spectra were recorded on a Nicolet Avatar 370 Fourier

of

transform infrared spectrometer. The absorbance of the bacterial solution was detected by a MI-100 multi-function water quality analyzer. The laser scanning confocal image

ro

(CLSM) of the bacterial solution was taken on a Nikon A1 laser confocal microscope.

-p

2.3 Antibacterial activity tests

re

To assess the antibacterial activity of the Cu NPs in liquid phase, absorptiometry

lP

was adopted to intuitively trace the Cu-containing ion concentration in the

na

antibacterial process. Referring to our previous works [48,49], the antibacterial activities of the Cu NPs were tested using Gram-negative E. coli and Gram-positive S.

Jo ur

aureus bacteria, where 0.1 mL of 105 CFU·mL-1 bacteria and certain amount of the Cu NPs (310 mg) were put into 1.9 mL of pre-sterilized Luria–Bertani (LB) medium, followed by shake culture at 37℃ for 16 h. The absorbance of the resulting bacteria solutions before and after filtrating by a 0.22 m of microfiltration membrane was measured at the wavelength of 600 nm. For comparison, blank control experiments were performed in the absence of the Cu NPs. The antibacterial rate (AR) of the Cu NPs was calculated by the following formula: AR =[1-(A1-A2)/A0]·100% where A1 and A2 correspond to the absorbances of the bacterial solution before and 6

Journal Pre-proof

after filtration, respectively, in the presence of the Cu NPs, and A0 is the value for the blank control test. 2.4 CLSM measurements Before the CLSM measurement, the bacteria solution (after antibacterial treatment by the Cu NPs) needs to be pretreated by dying the nuclei of bacteria with damaged cytomembrane. Firstly, 1 mL of bacteria solution was taken and put into a pretreated

of

Petri dish at 37 C for 45 min. Then 10 μL of 0.1 g L-1 propidium iodide (PI) dye

ro

solution was added and incubated in dark at 37 C for 15 min. Finally, the bacteria

-p

solution was washed by PBS buffer solution for three times to remove the redundant

re

PI dye. The resulting substance was used to observe fluorescence intensity and

na

3. Results and discussion

lP

morphology of the bacteria under a laser confocal microscope.

3.1 Morphologies and structures of the Cu NPs

Jo ur

The XRD patterns and SEM images (Figure 1) of the series of products prepared by using various concentrations of NaOH solutions demonstrate that the morphologies and crystal phases of the products would not change with increasing concentrations of the NaOH solutions. The Cu NPs were obtained in all the reaction conditions above. But the particle sizes and crystallinity of the Cu NPs are different from each other. According to the XRD patterns (Figure 1f), three diffraction peaks located at 43.5°, 50.6° and 74.3° are identified for the five products, which are indexed to the lattice planes of (111), (200) and (220), respectively, of a cubic phase Cu (JCPDS: 65-9743). But the peak intensities increase gradually with increasing concentrations of the 7

na

lP

re

-p

ro

of

Journal Pre-proof

Jo ur

Figure 1. SEM images of the Cu NPs prepared using various concentrations of NaOH: (A) 2 mol·L-1, (B) 4 mol·L-1, (C) 6 mol·L-1, (D) 8 mol·L-1 and (E) 10 mol·L-1. (F) The corresponding XRD patterns of the samples.

NaOH solutions, indicating gradual enhancement in crystallinity. The average primary crystallite sizes of the Cu NPs are estimated, based on the Cu diffraction peak at 43.5° by using Debye-Scherrer’s formula: D = Kλ/βcosθ [50], which are shown in Table 1 hereinafter. The average primary crystal sizes of the as-prepared Cu NPs gradually increase from 15.7 to 23.3 nm with increasing the concentration of NaOH. The 8

Journal Pre-proof

particle size distributions (Figure 2) of the series of Cu NPs were provided on the basis of the SEM results, which demonstrate that the particle sizes intend increase with increasing the NaOH concentrations. The sizes of the most particles for the samples A-E are in the ranges of 30-50 nm, 20-50 nm, 40-70 nm, 70-110 nm and

na

lP

re

-p

ro

of

80-130 nm, respectively.

Jo ur

Figure 2. Particle size distributions of the series of as-prepared Cu NPs (A-E).

The TEM images (Figure 3) were taken to observe the microstructure of the Cu NPs and further confirm its crystal structure, where the sample E prepared by using 10 mol·L-1 of NaOH solution was chosen as the representative because it has the highest antibacterial activity. Figure 3A displays a solid nature of the Cu NP with many defects on the surface which could be helpful to formation of the soluble copper complex active species in the antibacterial process. The HRTEM image (Figure 3B) by magnifying the chosen area in Figure 3A demonstrates that the inter-planar 9

Journal Pre-proof

spacings for the Cu NP are 0.205, 0.174 and 0.132 nm, corresponding to the (111), (200) and (220) planes, respectively, of a cubic phase Cu, confirming the successful

-p

ro

of

synthesis of the Cu NPs.

re

Figure 3. (A) TEM and (B) HRTEM images of the Cu NPs prepared by using 10

Jo ur

na

lP

mol·L-1 of NaOH solution.

Figure 4. FT-IR spectra of the series of as-prepared Cu NPs (A-E). The inset is the magnification of the chosen black square in the spectra. The FT-IR spectra of the series of as-prepared samples (A-E) were measured to study the surface nature of the Cu NPs obtained under different NaOH concentrations. As shown in Figure 4, weak peaks around 630 cm-1 were observed for the five 10

Journal Pre-proof

samples, which can be attributed to Cu-O stretching vibration [51], originated from slight surface oxidation of the Cu NPs during grinding process in air for preparation of the KBr tablet. The peaks around 3450 and 1635 cm-1 are assigned to stretching vibration of surface -OH groups and bending vibration of H-O-H (adsorbed water) on the Cu NPs, respectively [29,52]. The rather weak peaks at 1398 and 1082 cm-1 could

of

be caused by bending vibration of CH2, wagging of C-H and stretching vibration of C-O [29,52], suggesting the existence of trace of PAA molecules on the Cu NPs.

ro

These results display that the as-obtained Cu NPs (A-E) are rich in surface -OH

-p

groups. Basically, the surface -OH content increases with increasing NaOH

re

concentration in the preparation process of the Cu NPs. The surface hydroxyl content

lP

may influence the antibacterial activity of the Cu NPs.

60

E D C B A

40 20 0

3 mg

5 mg 7 mg Mass of Cu NPs

E.coli

100

Antibacterial ratio/%

Antibacterial ratio/%

80

CuSO4

Jo ur

S.aureus

100

na

3.2 Antibacterial properties of the Cu NPs

80 60 40 20 0

10 mg

CuSO4

E D C B A

3 mg

5 mg 7 mg Mass of Cu NPs

10 mg

Figure 5. Antibacterial ratios of the series of as-prepared Cu NPs (AE) with various dosages and a commercial CuSO4 to both S. aureus and E. coli bacteria.

As an excellent antibacterial material, Cu NPs have attracted much attention in 11

Journal Pre-proof

recent years although they also can be good catalysts and electronic materials [41-44]. In this section, we will focus on the antibacterial activities of the Cu NPs. The antibacterial activities of the five Cu NPs (AE) prepared using various concentrations of NaOH solutions (2, 4, 6, 8 and 10 mol·L-1) were investigated by means of S. aureus and E. coli bacteria, where various dosages (3, 5, 7 and 10 mg) of

of

antibacterial agent, the Cu NPs, were applied to clearly observe evolution of the antibacterial activities. Moreover, the antibacterial activities of a commercial CuSO4

ro

were tested for comparison purpose. As shown in Figure 5, after 16 h exposure to 3

-p

mg of the sample E, the antibacterial ratios to S. aureus and E. coli are 99.8% and

re

41%, respectively, indicating that the antibacterial activity of the sample E to S.

lP

aureus is much higher than that of E. coli. Comparatively, the antibacterial ratio of the

na

sample D to S. aureus is much lower, only 56.3%, suggesting the lower antibacterial activity of the sample D than E. Generally, an antibacterial ratio below 90% is

Jo ur

unsatisfying. Thus, 3 mg of the Cu NPs is not enough to achieve good sterilizing effect simultaneously for both S. aureus and E. coli. Consequently, the loading of the Cu NPs was increased to 5 mg for the antibacterial tests, and this time the sample E displays excellent antibacterial activities to both S. aureus and E. coli with antibacterial ratio of 99.5%. For the sample D, however, the antibacterial ratios to S. aureus and E. coli are 99.5% and 53.6%, respectively, similar to the results in the presence of 3 mg of the sample E. The antibacterial ratios of the sample C to S. aureus and E. coli are even lower, 55.3% and 51.6%, respectively, suggesting the lower antibacterial activities. Further increasing the mass of the Cu NPs to 7 mg, the 12

Journal Pre-proof

antibacterial ratios of the samples D and C to the two kinds of bacteria increase distinctly, more than 98.3%, but they are quite low for the samples B and A, below 35%. When the dosage of the Cu samples was increased to 10 mg, the antibacterial ratios of the sample B become satisfying (>90%) to the two kinds of bacteria, but they are still low for the sample A. These results indicate that the antibacterial activities of

of

the samples A-E follow the order of E > D > C > B > A and they gradually increase with increasing the Cu NPs loading. The dose-dependent toxicity against bacterial

ro

pathogens has also been reported in the literature [25], where the Ag NPs were used as

-p

the antibacterial agents, Vibrio cholera and Shigella flexneri as the model bacteria.

re

Besides, the antibacterial activities of the five samples to the Gram-positive S. aureus

lP

are higher than those of the Gram-negative E. coli, which may be related with the

na

unique structures of the bacteria. Generally, the peptidoglycan layer of Gram-positive bacteria is thicker than that of Gram-negative bacteria, which may make it less

Jo ur

susceptibility to a metal antibacterial agent because the metal NPs are hard to penetrate through the cell bacterial cytoplasm [25]. However, in our liquid phase antibacterial system, exactly opposite experimental results were found. Similar results were also reported in our previous works [48,49] where Cu2-xSe and Cu4O3 antibacterial agents were applied. The higher antibacterial activities of the Cu NPs against the Gram-positive bacteria S. aureus may be because of the numerous pores in the multiple peptidoglycan layers, which are more susceptible of intracellular transduction caused by the soluble Cu (II)-containing ions, leading to cell wall disruption [49]. Compared to the excellent antibacterial activities of the Ag NPs and 13

Journal Pre-proof

Au/Pt composites from other works [12,29], the as-prepared Cu NPs have high antibacterial activities with much less cost and low toxicity, which are beneficial to practical applications. The above results demonstrate that the concentration of NaOH influences the crystallinity, crystal size and surface hydroxyl content of the Cu NPs, which

of

ultimately affect the antibacterial activities. It also implies that the antibacterial activities of the Cu NPs can be tuned by simply adjusting the concentration of NaOH

ro

in the preparation process of the antibacterial agents. For comparison, the antibacterial

-p

ratios of 10 mg of Cu2+ (44 mg CuSO4 powder) to both S. aureus and E. coli were

re

tested, which are slightly lower than 90%. These antibacterial ratios are higher than

lP

those of the sample A, but lower than the other four samples (B, C, D and E),

na

implying that the antibacterial mechanism of the Cu NPs is not simply correlated with

Jo ur

the release of free Cu2+ ions.

Figure 6. Panel of photographs after 24 h incubation, showing the antibacterial effect of the Cu sample E on S. aureus and E. coli: (A) blank control to S. aureus, (B) blank 14

Journal Pre-proof

control to E. coli, (C) S. aureus treated by the Cu NPs and (D) E. coli treated by the Cu NPs.

To further confirm the sterilizing effect of the Cu NPs, 100 L of S. aureus and E. coli bacteria solutions after 16 h incubation at 37 C in the presence of the Cu NPs (5

of

mg of the sample E) were taken and put on two glass panels, respectively, which were further incubated for other 24 h. For comparison, two blank control experiments

ro

containing 100 L of 105 CFU·mL-1 of S. aureus and E. coli bacteria solutions,

-p

respectively, were performed under the same conditions. As shown in Figure 6A and

re

B, without sterilizing treatment by the Cu NPs, both the S. aureus and E. coli bacteria

lP

grow fast, where plenty of bacteria were observed after 24 h incubation. On the

na

contrary, bacteria were hardly observed (Figure 6C and D) after the antibacterial treatment by the Cu NPs, displaying again the excellent antibacterial effect of the Cu

Jo ur

NPs and verifying the accuracy of the antibacterial experiments in Figure 5.

15

lP

re

-p

ro

of

Journal Pre-proof

na

Figure 7. Growth profiles of (A) S. aureus and (B) E. coli bacteria for blank control

Jo ur

and in the presence of the Cu NPs during the incubation time from 6 to 16 h.

We also studied the time-dependent antibacterial effects of the Cu NPs by tracing changes in absorbances of the bacteria solutions in the absence/presence of 5 mg of the sample E during the incubation period from 6 to 16 h. Figure 7 shows the growth profiles of S. aureus and E. coli. Obviously, the absorbances of both S. aureus and E. coli bacteria for the blank control (in the absence of the Cu NPs) increase distinctly over the period from 6 to 16 h, whereas they are hardly changed in the presence of 5 mg of the sample E, displaying a tremendous inhibition of the Cu NPs to the growth of bacteria for the whole incubation period. 16

Journal Pre-proof

ro

of

3.3 Antibacterial mechanism

-p

Figure 8. SEM images of (A,C) S. aureus and (B,D) E. coli bacteria after different

lP

re

treatments: (A,B) without and (C,D) with sterilizing treatment by the Cu NPs.

To explore the antibacterial mechanism of the Cu NPs, we studied the

na

morphological changes of the S. aureus and E. coli bacteria with and without

Jo ur

sterilizing treatment by the Cu NPs, where 3 mg of the sample E was used as the antibacterial agent to ensure part of bacteria viable for observation. As shown in the SEM images in Figures 8A and B, intact spherical and rod-like structures for the blank control of S. aureus and E. coli strains, respectively, were observed. After sterilizing treatment by the Cu NPs for 16 h (Figure 8C and D), however, the bacterial cells of S. aureus and E. coli were destroyed completely or partly, respectively. The S. aureus cells were fragmented into debris without any intact spherical cell structure remained. While, for the E. coli, part of cell structure was preserved, but the surface of the cell had wrinkles, depressions and splitting deformation, similar to the reports 17

Journal Pre-proof

[53,54]. These results indicate that the Cu NPs can sterilize bacteria by damaging the cell structures of the bacteria, and the antibacterial activity to S. aureus is higher than that of the E. coli, consistent with the results in Figure 5. The difference in antibacterial activities to S. aureus and E. coli may be caused by the different structures of the two kinds of bacteria. The cell wall of the E. coli mainly consists of

of

peptidoglycan and outer layers of lipopolysaccharide, lipoprotein, and phospholipids, which are less prone to be attacked by the Cu NPs or Cu (II) ions [49,55,56], resulting

ro

in the lower antibacterial activity for the E. coli with respect to the S. aureus.

-p

To clearly observe the changes in cell structures and survival situation of the

re

bacteria after 16 h antibacterial treatment by the Cu NPs, the CLSM measurements

lP

were performed, where 5 mg of the sample E was used as the antibacterial agent and

na

the concentrations of S. aureus and E. coli were increased to 5109 CFU mL-1 to

Jo ur

ensure partly damaged bacteria available for observation. For comparison purpose,

Figure 9. Bright-field (A1D1) and fluorescence images (A2D2) of S. aureus (A,B) and E. coli (C,D) for blank control (A,C) and after antibacterial treatment by 5 mg of 18

Journal Pre-proof the Cu NPs (B,D) for 16 h. λex=543 nm, λem=560-617 nm.

two blank control experiments (in the absence of the Cu NPs) with 105 CFU mL-1 of S. aureus or E. coli were conducted under the same conditions. As reported in literature [57,58], if the membrane structures of bacteria were inactivated, their nuclei would be

of

stained by PI, which can emit red light in the wavelength range of 560617 nm under excitation of 543 nm light. Thus, PI was used to dye the two kinds of bacteria

ro

collected after 16 incubation in the absence or presence of the Cu NPs, and the images

-p

were taken under bright field and Mito Tracker Red modes, respectively. As shown in

re

Figure 9A and C (for the blank controls), numerous S. aureus and E. coli bacteria

lP

were observed in bright field mode after 16 incubation, but red fluorescence substance

na

(dead cell) was hardly seen in Mito Tracker Red mode, indicating that the membrane structures of the S. aureus and E. coli cells are intact, and the bacteria are viable in the

Jo ur

absence of the Cu NPs. After the antibacterial treatment for 16 h by the Cu NPs (Figure 9B and D), however, the bacteria populations are much smaller than those of the blank controls even if 50000 times higher concentration of bacteria was applied for the test. Moreover, lots of red fluorescence dead cells were observed under Mito Tracker Red mode, displaying that the Cu NPs can inactivate the cytoplasmic membranes of both S. aureus and E. coli bacteria, and further apoptotic bacteria due to changes in membrane permeability. The inactivation of the bacteria could be caused by the Cu-induced reactive oxygen species (ROS), which stimulates DNA strand cleavage, and meanwhile, it doesn’t exclude the cellular uptake of soluble copper [59]. 19

Journal Pre-proof

Figure 9B and D also demonstrate that S. aureus retained fewer intact cells than E. coli under the same conditions, and the cell structure of E. coli changed significantly except the membrane structure was inactivated, which is consistent with the SEM results in Figure 8. To clarify the soluble copper species in the antibacterial system, a series of

of

control experiments were performed. Figure 10A and B clearly show the difference in solution color in the absence and presence of the Cu NPs after 16 h incubation at 37℃,

ro

respectively, indicating the generation of soluble Cu species during the antibacterial

-p

process of the Cu NPs. As for the speciation of the soluble Cu species, there are two

re

main standpoints in literature: Cu2+ ions [45,49] in the case of the Cu and Cu4O3

lP

antibacterial agents and copper-organic complexes [46,47] for the CuO nanomaterials.

na

To clarify this problem and give an insight into the source of cytotoxicity and antibacterial mechanism of the as-prepared Cu NPs, series of control experiments

Jo ur

(Figure 10CH) were conducted. As seen in Figure 10C and D, the color of the LB medium containing 0.01 g of the Cu NPs became glaucous after 16 h oscillation at 37℃, but no any change was observed for the deionized water in the same conditions, suggesting the complexation-mediated high extent of copper leaching in the LB

20

Jo ur

na

lP

re

-p

ro

of

Journal Pre-proof

Figure 10. Photographs of (A) S. aureus-LB solution without antibacterial treatment, (B) S. aureus-LB solution after antibacterial treatment by the Cu NPs, (C) LB medium treated by the Cu NPs, (D) deionized water treated by the Cu NPs, (E) LB medium treated by the CuO NPs, (F) deionized water treated by the CuO NPs and (G,H) solutions obtained after adding NaOH solution into the solution C and E, respectively. (I) UV-Vis spectra of the solutions B, C, E, G, H, standard Cu2+ solution and CuSO4 solution in LB medium. (J) Schematic representation of surface plasmon (electron 21

Journal Pre-proof

cloud) resonance under the effect of an electromagnetic field of the Cu NPs.

medium (not in deionized water), which most likely arises from copper coordination with amino acid side chains from the LB [47]. The corresponding UV-Vis absorption spectra of the series of solutions were measured to affirm the soluble copper species

of

in the antibacterial system. Figure 10I-B and 10I-C demonstrate the same soluble copper species with different concentrations was generated in the presence or absence

ro

of bacteria although the absorption maximum for the solution B slightly blue shifts

-p

from 614 to 600 nm with respect to the solution C due to the existence of bacteria.

re

The lower absorption intensity for the solution B implies that the existence of bacteria

lP

partly depresses formation of the copper-peptide complexes between Cu and LB

na

medium, suggesting that interactions between Cu and bacteria may exist. The experimental results above also confirm that an organic medium is essential for

Jo ur

formation of the reactive copper leachate. Furthermore, the surface plasmon resonance [60] (SPR, arising from the collective oscillations of electrons on the surfaces of the Cu NPs) effect could set up an electromagnetic field around the Cu NPs and boost their interaction with protein molecules [61], resulting in effective resonant energy transfer between the protein molecules and surface plasmons, which could promote the formation of the soluble copper-peptide complexes, as shown in Figure 10J. To verify if the soluble copper is a Cu (II) complex, 0.01 g of CuO sample from our lab was used for the tests in the same conditions (Figure 10E and F). Analogously, 22

Journal Pre-proof

blue copper leachate in the LB medium and colorless liquid in the deionized water were observed, confirming the formation of the blue copper-peptide complexes in the LB medium which possess high redox potential and fast electron transfer, featured by its intense blue color [59,62]. The absorption maximum for the solution E (614 nm) is the same as that of the solution C (Fig. 10I-E and 10I-C), suggesting the same soluble

of

copper species in these two solutions. That is, copper (II)-peptide complexes could be generated in the antibacterial system of the Cu NPs, which are capable of triggering

ro

cellular oxidative stress and causing cytotoxicity [47]. The absorbance of the

-p

copper-peptide complexes is related with S1 pπ → Cu 3𝑑x2 −y2 charge transfer, which

re

occurs when a strong Cu-S bond (for example plastocyanin, a copper-bearing electron

lP

transfer protein) in the trigonal-pyramidal coordination alters the orientation and

na

therefore intersects the lobes of the Cu 3𝑑x2 −y2 orbital [62]. Subsequently, biuret reactions [63] were carried out by adding NaOH to the solutions C and E, resulting in

Jo ur

the solutions G and H (Figure 10G and H), respectively. Immediately, the colors of the solutions C and E changed to pink and purple, respectively, displaying the formation of hydroxyl-peptide-copper multicomponent soluble complex. The absorption peak maximums are located at 543 and 557 nm for the solutions G and H, respectively (Figure 10I-G and 10I-H), corroborating the presence of Cu (II) complexes. The comparison to the absorption peak of a standard Cu2+ solution centered at 809 nm (Figure 10I) further exclude the existence of free Cu2+ ions in the antibacterial system of the Cu NPs.

23

Journal Pre-proof

To further confirm that the Cu NPs react directly with proteins, forming the Cu (II) complex, the absorption spectrum of a CuSO4-LB solution after 16 h incubation was measured. As shown in Figure 10I, the absorption maximum (786 nm) of the CuSO4-LB solution is similar to that of the standard Cu2+ solution (809 nm), but tremendously different from the Cu NPs (614 nm), suggesting that free Cu2+ ions are

of

the main species in the CuSO4-LB solution, but not Cu (II) complex. This experiment affirms indirectly that the Cu NPs react directly with proteins, forming the Cu (II)

ro

complex which is the active species for sterilization. In the CuSO4-LB solution,

-p

however, free Cu2+ ions could be the active species, whose antibacterial effect could

re

be lower than that of the Cu (II) complexes. The slight difference in absorption

Jo ur

na

anions.

lP

maximums of the standard Cu2+ and CuSO4-LB solutions may be caused by the SO42-

Figure 11. Absorbances of the bacteria and Cu (Ⅱ) complex after antibacterial treatment by 7 mg of the Cu NPs (samples A-E).

To further confirm if the copper-peptide complex is the main active species for sterilization, the absorbances of Cu (II) complex and residual bacteria after the 24

Journal Pre-proof

antibacterial treatments were measured, where 7 mg of the series of Cu NPs (samples AE) were used as the antibacterial agents and the two kinds of bacteria were incubated for 16 h at 37 C. As shown in Figure 11, the absorbances of the residual bacteria roughly decrease with increasing concentrations of the copper-peptide complexes, indicating that the main active species for sterilization is Cu (II)

of

complexes. The above results also display that the antibacterial activities of the Cu NPs increase with increasing NaOH concentrations in the preparation process. The

ro

correlation of the NaOH concentration, crystal size of the Cu NPs, absorbance of the

re

-p

Cu (II) complexes and antibacterial ratio is summarized in Table 1. These

lP

Table 1. The relationship of the NaOH concentration, particle size of the Cu NPs,

na

absorbance of the Cu(II) complex and antibacterial ratios of the Cu NPs. Crystal sizes C (NaOH)

Jo ur

of the Cu

Abs. of Cu (II) Antibacterial ratios/% complexes/a.u.

-1

/mol L

samples/nm

S. aureus

E. coli

S. aureus

E. coli

15.7

0.178

0.199

26.3

13.9

4

20.2

0.153

0.151

33.3

22.6

6

20.7

0.360

0.219

98.1

99.1

8

21.5

0.480

0.615

99.7

99.7

10

23.3

0.450

0.706

99.8

99.7

2

experimental results support that the higher the concentration of NaOH is, the higher 25

Journal Pre-proof

crystallinity, the larger crystal size and the more surface -OH groups are the Cu NPs, which cause the more reactive Cu (II) complex leaching and generation of ROS species, and ultimately lead to the higher antibacterial activity. Damm reported that the crystallinity of the silver-polymer composites can influence the rate of Cu2+ ions releasing which decreases with increased crystallinity [64]. Zhang found that the

of

cellular uptake of ligand coated NPs is strongly size-dependent [65], where the optimal radius falls in the range of 25–30 nm. Our experimental results are opposite to

ro

these reports [64,65], suggesting that the antibacterial mechanism of the as-prepared

-p

Cu NPs could be different, where free Cu2+ ions releasing may not play an important

re

role in killing the bacteria and it could not be the Cu NP directly attacking the cells of

lP

the bacteria. We believe that the higher crystallinity of the Cu NPs may be beneficial

na

to formation of the more copper-peptide complexes and ROS due to the higher reactivity and peroxidase/oxidase-like catalytic activity of the Cu NPs. Although the

Jo ur

particle sizes of the five Cu samples are different, their crystal sizes are similar (around 20 nm), which may result in rather minor difference in their antibacterial activities. Besides, the FT-IR results (Figure 4) demonstrate that the surface -OH content of the Cu NPs increases with increasing the NaOH concentration in the preparation process. On the one hand, the more surface -OH groups of the Cu NPs are helpful to formation of the more ROS, for instance OH, due to the enhanced concentration of OH groups on the surface of the Cu NPs. On the other hand, the more surface -OH groups are beneficial to formation of the more copper-peptide complexes owing to the enhanced interactions with protein molecules. Consequently, 26

Journal Pre-proof

the antibacterial activities of the samples A-E increase gradually with increasing concentrations of NaOH. The light color of the copper leachate in Figure 10B and low absorbance in Figure 10I-B also demonstrate that the as-prepared Cu NPs can achieve excellent antibacterial effect in the case of low concentration of copper complex leachate, which is beneficial for the practical applications.

of

Based on the experimental results above, the antibacterial mechanism of the Cu NPs was proposed in the scheme 1. Firstly, the highly reactive and soluble Cu

ro

(II)-peptide complexes were formed due to the reactions between the Cu NPs and LB

-p

medium, which attack the negatively charged cell membranes/walls of the S. aureus

re

and E. coli bacteria. The bacteria were gradually damaged and became wrinkled,

lP

fragmental and debris, accompanying with cleavage of cell substances. The S. aureus

Jo ur

na

contains multiple layers of peptidoglycan with lots of pores which are more

Scheme 1 Antibacterial mechanism of the Cu NPs to S. aureus and E. coli bacteria in LB medium. susceptible of intracellular transduction caused by the soluble Cu (II) complex, 27

Journal Pre-proof

leading to cell wall disruption [30], which explains the higher antibacterial activity of the Cu NPs to S. aureus bacteria than E. coli. The stability of the Cu NPs in the antibacterial system were tested by measuring the SEM image and XRD pattern of the sample E after 16 h antibacterial experiment. As shown in Figure 12, the particle size of the Cu NPs was remained with certain

of

extent of conglomerations. The Cu diffraction peaks are still sharp with high intensities, suggesting the main component of the sample is still Cu although a weak

ro

and broad peak of carbon appears [66]. The remained Cu NPs could still be active for

-p

a longer period of antibacterial performance despite of a slight decrease in

re

antibacterial efficiency due to the conglomeration and deposition of small amount of

lP

carbon from the damaged bacteria. This drawback may be overcome by introducing a

na

polymer into the Cu NPs, forming Cu-polymer composites [51,67,68] where the polymer could be helpful to long term Cu2+ ion release and avoiding particle

Jo ur

conglomeration, thus prolonging the antibacterial activity.

Figure 12. SEM image (A) and XRD pattern (B) of the sample E after 16 h antibacterial experiment. 4. Conclusions 28

Journal Pre-proof

Series of Cu NPs with different crystallinity, crystal sizes and surface -OH contents were prepared successfully by tuning NaOH concentrations in the hydrothermal process, which display obvious difference in antibacterial activities to the two kinds of bacteria, S. aureus and E. coli. The antibacterial activities of the Cu NPs increase distinctly with increasing the concentrations of NaOH, which may be

of

related with the leaching capability of the highly reactive and soluble Cu (II)-peptide complexes and generation of ROS that could be dependent on the crystallinity and

ro

surface -OH content of the Cu NPs. The series of control experiments confirm there

-p

are no free Cu2+ ions in the antibacterial system. Instead, the copper (II)-peptide

re

complexes were formed between the Cu NPs and the peptide chains from LB medium,

lP

which are the main active species for sterilization. The antibacterial mechanism was

na

proposed where the soluble Cu (II)-peptide complexes attack the negatively charged cell membranes /walls of the bacteria, resulting in wrinkled, fragmental and debris of

Jo ur

the bacteria, accompanying with cleavage of cell substances.

Acknowledgements

We thank the supports from the National Natural of Science Foundation of China (No. 21371132).

References [1] M.A. Shannon, P.W. Bohn, M. Elimelech, J.G. Georgiadis, B.J. Marinas, A.M. Mayes, Science and technology for water purification in the coming decades, Nature 29

Journal Pre-proof

452 (2008) 301-310. [2] X. He, D.-P. Yang, X. Zhang, M. Liu, Z. Kang, C. Lin, N. Jia, R. Luque, Waste eggshell membrane-templated CuO-ZnO nanocomposites with enhanced adsorption, catalysis and antibacterial properties for water purification, Chem. Eng. J. 369 (2019) 621-633.

of

[3] W. Wang, G. Huang, J.C. Yu, P.K. Wong, Advances in photocatalytic disinfection of bacteria: Development of photocatalysts and mechanisms, J. Environ. Sci. (China)

ro

34 (2015) 232-247.

-p

[4] E. Weir, A. Lawlor, A. Whelan, F. Regan, The use of nanoparticles in

re

anti-microbial materials and their characterization, Analyst 133 (2008) 835-845.

lP

[5] K. Bush, P. Courvalin, G. Dantas, J. Davies, B. Eisenstein, P. Huovinen, G.A.

na

Jacoby, R. Kishony, B.N. Kreiswirth, E. Kutter, S.A. Lerner, S. Levy, K. Lewis, O. Lomovskaya, J.H. Miller, S. Mobashery, L.J. Piddock, S. Projan, C.M. Thomas, A.

Jo ur

Tomasz, P.M. Tulkens, T.R. Walsh, J.D. Watson, J. Witkowski, W. Witte, G. Wright, P. Yeh, H.I. Zgurskaya, Tackling antibiotic resistance, Nat. Rev. Microbiol. 9 (2011) 894-896.

[6] X. Zhen, C. Xie, K. Pu, Temperature-correlated afterglow of a semiconducting polymer nanococktail for imaging-guided photothermal therapy, Angew. Chem. Int. Ed. Engl. 57 (2018) 3938-3942. [7] V.M. D'Costa, C.E. King, L. Kalan, M. Morar, W.W. Sung, C. Schwarz, D. Froese, G. Zazula, F. Calmels, R. Debruyne, G.B. Golding, H.N. Poinar, G.D. Wright, Antibiotic resistance is ancient, Nature 477 (2011) 457-461. 30

Journal Pre-proof

[8] M. Saravanan, B. Ramachandran, H. Barabadi, The prevalence and drug resistance pattern of extended spectrum beta-lactamases (ESBLs) producing enterobacteriaceae in Africa, Microb. Pathog. 114 (2018) 180-192. [9] A.B. Rezaie, M. Montazer, M.M. Rad, Low toxic antibacterial application with hydrophobic properties on polyester through facile and clean fabrication of nano

of

copper with fatty acid, Mater. Sci. Eng. C 97 (2019) 177-187. [10] M. Alavi, N. Karimi, Biosynthesis of Ag and Cu NPs by secondary metabolites

ro

of usnic acid and thymol with biological macromolecules aggregation and

re

Macromol. 128 (2019) 893-901.

-p

antibacterial activities against multi drug resistant (MDR) bacteria, Int. J. Biol.

lP

[11] G.A. Sotiriou, S.E. Pratsinis, Antibacterial activity of nanosilver ions and

na

particles, Environ. Sci. Technol. 44 (2010) 5649-5654. [12] P. Boomi, G.P. Poorani, S. Palanisamy, S. Selvam, G. Ramanathan, S. Ravikumar,

Jo ur

H. Barabadi, H.G. Prabu, J. Jeyakanthan, M. Saravanan, Evaluation of antibacterial and anticancer potential of polyaniline-bimetal nanocomposites synthesized from chemical reduction method, J. Cluster Sci. 30 (2019) 715-726. [13] N.A. Ismail, K. Shameli, M.M.T. Wong, S.Y. Teow, J. Chew. S.N.A.M. Sukri, Antibacterial and cytotoxic effect of honey mediated copper nanoparticles synthesized using ultrasonic assistance, Mater. Sci. Eng. C 104 (2019) 109899-109908. [14] S.M. Javadhesari, S. Alipour, S. Mohammadnejad, M.R. Akbarpour, Antibacterial activity of ultra-small copper oxide (II) nanoparticles synthesized by mechanochemical processing against S. aureus and E. coli, Mater. Sci. Eng. C 105 31

Journal Pre-proof

(2019) 110011-110020. [15] S. Wu, Z. Weng, X. Liu, K.W.K. Yeung, P.K. Chu, Functionalized TiO2 based nanomaterials for biomedical applications, Adv. Funct. Mater. 24 (2014) 5464-5481. [16] A. Khatua, E. Priyadarshini, P. Rajamani, A. Patel, J. Kumar, A. Naik, M. Saravanan, H. Barabadi, A. Prasad, l. Ghosh, B. Paul, R. Meena, Phytosynthesis,

of

characterization and fungicidal potential of emerging gold nanoparticles using Pongamia pinnata leave extract: A novel approach in nanoparticle synthesis, J. Cluster

ro

Sci. (2019) doi.org/ 10.1007/s10876-019-01624-6.

-p

[17] A. Ahmad, S. Senapati, M.I. Khan, R. Kumar, M. Sastry, Extracellular

re

biosynthesis of monodisperse gold nanoparticles by a novel extremophilic

lP

actinomycete thermomonosporasp, Langmuir 19 (2003) 3550-3553.

na

[18] S. Park, S.H. Cha, I. Cho, S. Park, Y. Park, S. Cho, Y. Park, Antibacterial nanocarriers of resveratrol with gold and silver nanoparticles, Mater. Sci. Eng. C 58

Jo ur

(2016) 1160-1169.

[19] S. Sadhasivam, P. Shanmugam, M. Veerapandian, R. Subbiah, K. Yun, Biogenic synthesis of multidimensional gold nanoparticles assisted by streptomyces hygroscopicus and its electrochemical and antibacterial properties, BioMetals 25 (2012) 351-360. [20] P. Manivasagan, M.S. Alam, K.H. Kang, M. Kwak, S.K. Kim, Extracellular synthesis of gold bionanoparticles by Nocardiopsis sp. and evaluation of its antimicrobial, antioxidant and cytotoxic activities, Bioprocess Biosyst. Eng. 38 (2015) 1167-1177. 32

Journal Pre-proof

[21] S. Sadhasivam, P. Shanmugam, K. Yun, Biosynthesis of silver nanoparticles by Streptomyces hygroscopicus and antimicrobial activity against medically important pathogenic microorganisms, Colloids Surf. B. Biointerfaces 81 (2010) 358-362. [22] S.M. Ghaseminezhad, S. Hamedi, S.A. Shojaosadati, Green synthesis of silver nanoparticles by a novel method: comparative study of their properties, Carbohydr.

of

Polym. 89 (2012) 467-472. [23] L. Karthik, G. Kumar, T. Keswani, A. Bhattacharyya, B.P. Reddy, K.V. Rao,

-p

activity, Nanomedicine 9 (2013) 951-960.

ro

Marine actinobacterial mediated gold nanoparticles synthesis and their antimalarial

re

[24] P. Velmurugan, M. Iydroose, M.H. Mohideen, T.S. Mohan, M. Cho, B.T. Oh,

lP

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract

na

and evaluation of its antibacterial activity, Bioprocess Biosyst. Eng. 37 (2014) 1527-1534.

Jo ur

[25] R. Balachandar, P. Gurumoorthy, N. Karmegam, H. Barabadi, R. Subbaiya, K. Anand, P. Boomi, M. Saravanan, Plant-mediated synthesis, characterization and bactericidal potential of emerging silver nanoparticles using stem extract of Phyllanthus pinnatus: A recent advance in phytonanotechnology, J. Cluster Sci. 30 (2019) 1481-1488. [26] H. Barabadi, B. Tajani, M. Moradi, K.D. Kamali, R. Meena, S. Honary, M.A. Mahjoub, M. Saravanan, Penicillium family as emerging nanofactory for biosynthesis of green nanomaterials: A journey into the world of microorganisms, J. Cluster Sci. 30 (2019) 843-856. 33

Journal Pre-proof

[27] M. Behravan, A.H. Panahi, A. Naghizadeh, M. Ziaee, R. Mahdavi, A. Mirzapour, Facile green synthesis of silver nanoparticles using Berberis vulgaris leaf and root aqueous extract and its antibacterial activity, Int. J. Biol. Macromol. 124 (2019) 148-154. [28] A. Harada, H. Ichimaru, T. Kawagoe, M. Tsushida, Y. Niidome, H. Tsutsuki, T.

activity, Bull. Chem. Soc. Jpn. 92 (2019) 297-301.

of

Sawa, T. Niidome, Gold-treated silver nanoparticles have enhanced antimicrobial

ro

[29] K. Kanagamani, P. Muthukrishnan, K. Shankar, A. Kathiresan, H. Barabadi, M.

-p

Saravanan, Antimicrobial, cytotoxicity and photocatalytic degradation of norfloxacin

re

using Kleinia grandiflora mediated silver nanoparticles, J. Cluster Sci. 30 (2019)

lP

1415-1424.

na

[30] H. Barabadi, O. Hosseini, K.D. Kamali, F.J. Shoushtari, M. Rashedi, H.H. Aminjan, M. Saravanan, Emerging theranostic silver nanomaterials to combat lung

Jo ur

cancer: A systematic review, J. Cluster Sci. (2019) doi.org/ 10.1007/s10876 -019-01639-z.

[31] H. Barabadi, M.A. Mahjoub, B. Tajani, A. Ahmadi, Y. Junejo, M. Saravanan, Emerging theranostic biogenic silver nanomaterials for breast cancer: A systematic review, J. Cluster Sci. 30 (2019) 259-279. [32] H. Barabadi, H. Vahidi, K.D. Kamali, M. Rashedi, O. Hosseini, A.R. Golnaraghi Ghomi, M. Saravanan, Emerging theranostic silver nanomaterials to combat colorectal

cancer:

A systematic

review,

10.1007/s10876-019-01668-8. 34

J.

Cluster

Sci.

(2019)

doi.org/

Journal Pre-proof

[33] H. Barabadi, K.D. Kamali, F.J. Shoushtari, B. Tajani, M.A. Mahjoub, A. Alizadeh, M. Saravanan, Emerging theranostic silver and gold nanomaterials to combat prostate cancer: A systematic review, J. Cluster Sci. 30 (2019) 1375-1382. [34] H. Barabadi, H. Vahidi, K.D. Kamali, O. Hosseini, M.A. Mahjoub, M. Rashedi, F.J. Shoushtari, M. Saravanan, Emerging theranostic gold nanomaterials to combat

of

lung cancer: A systematic review, J. Cluster Sci. (2019) doi.org/ 10.1007/s10876 -019-01650-4.

ro

[35] H. Barabadi, H. Vahidi, K.D. Kamali, M. Rashedi, O. Hosseini, M. Saravanan,

-p

Emerging theranostic gold nanomaterials to combat colorectal cancer: A systematic

re

review, J. Cluster Sci. (2019), doi.org/ 10.1007/s10876-019-01681-x.

lP

[36] H. Vahidi, H. Barabadi, M. Saravanan, Emerging selenium nanoparticles to

-019-01671-z.

na

combat cancer: A systematic review, J. Cluster Sci. (2019) doi.org/ 10.1007/s10876

Jo ur

[37] H. Barabadi, Z. Alizadeh, M.T. Rahimi, A. Barac, A.E. Maraolo, L.J. Robertson, A. Masjedi, F. Shahrivar, E. Ahmadpour, Nanobiotechnology as an emerging approach to combat malaria: A systematic review, Nanomedicine 18 (2019) 221-233. [38] M.C. Fung, D.L. Bowen, Silver products for medical indications: risk-benefit assessment, J. Toxicol. Clin. Toxicol. 34 (2008) 119-126. [39] H. Barabadi, M. Najafi, H. Samadian, A. Azarnezhad, H. Vahidi, M.A. Mahjoub, M. Koohiyan, A. Ahmadi, A systematic review of the genotoxicity and antigenotoxicity of biologically synthesized metallic nanomaterials: Are green nanoparticles safe enough for clinical marketing?, Medicina (Kaunas) 55 (2019) 35

Journal Pre-proof

439-455. [40] K. Mortezaee, M. Najafi, H. Samadian, H. Barabadi, A. Azarnezhad, A. Ahmadi, Redox interactions and genotoxicity of metal-based nanoparticles: A comprehensive review, Chem.-Biol. Interact. 312 (2019) 100814-100827. [41] S. Fuentes, R. Alvina, K. Zegarra, B. Perez, P. Pozo, Antibacterial activities of

of

copper nanoparticles in hybrid microspheres, J. Nanosci. Nanotechnol. 19 (2019) 4512-4519.

ro

[42] A. Perdikaki, A. Galeou, G. Pilatos, I. Karatasios, N.K. Kanellopoulos, A.

-p

Prombona, G.N. Karanikolos, Ag and Cu monometallic and Ag/Cu bimetallic

re

nanoparticle-graphene composites with enhanced antibacterial performance, ACS

lP

Appl. Mater. Interfaces 8 (2016) 27498-27510.

na

[43] S. Mallick, S. Sharma, M. Banerjee, S.S. Ghosh, A. Chattopadhyay, A. Pau, Iodine-stabilized Cu nanoparticle chitosan composite for antibacterial applications,

Jo ur

ACS Appl. Mater. Interfaces 4 (2012) 1313-1323. [44] H. Jing, Z. Yu, L. Li, Antibacterial properties and corrosion resistance of Cu and Ag/Cu porous materials, J. Biomed. Mater. Res. 87A (2008) 33-37. [45] A.K. Chatterjee, R. Chakraborty, T. Basu, Mechanism of antibacterial activity of copper nanoparticles, Nanotechnology 25 (2014) 135101-125112. [46] R. Chakraborty, R.K. Sarkar, A.K. Chatterjee, U. Manju, A.P. Chattopadhyay, T. Basu, A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action, BBA- Gen. Subjects, 1850 (2015) 845-856. 36

Journal Pre-proof

[47] C. Gunawan, W.Y. Teoh, C.P. Marquis, R. Amal, Cytotoxic origin of copper(II) oxide nanoparticles: comparative studies with micron-sized particles, leachate, and metal salts, ACS Nano 5 (2011) 7214-7225. [48] L. Zhu, F. Gao, P. Lv, Y. Zeng, W. Wang, W. Zheng, Facile synthesis of 3D flower-like Cu2-xSe nanostructures via sacrificing template method and the excellent antibacterial activities, CrystEngComm 19 (2017) 7253-7259.

of

[49] W. Wang, L. Zhu P. Lv, G. Liu, Y. Yu, J. Li, Novel Candy-like Cu4O3

ro

microstructure: facile wet chemical synthesis, formation mechanism, and good long-term antibacterial activities, ACS Appl. Mater. Interfaces 10 (2018)

-p

37287-37297.

re

[50] G. Allaedini, P. Aminayi, S.M. Tasirin, Structural properties and optical

na

077161-077168.

lP

characterization of flower-like Mg doped NiO, Aip Advances 5 (2015)

[51] M.N. Rosales, C.A.Á. Orta, M.G.N. Velázquez, H.O. Ortiz, E.H. Hernández,

Jo ur

S.G.S. Rosales, B.L.E. Sánchez, P.G. Morones, R.M.J. Barrera, S.S. Valdes, P.B. Pérez, Effect of plasma modification of copper nanoparticles on their antibacterial properties, Plasma Process. Polym. 11 (2014) 685-693. [52] N. Ba, L. Zhu, H. Li, G. Zhang, J. Li, J. Sun, 3D rod-like copper oxide with nanowire hierarchical structure: Ultrasound assisted synthesis from Cu2(OH)3NO3 precursor, optical properties and formation mechanism, Solid State Sci. 53 (2016) 23-29. [53] D.V. Dharmadhikari, A.S. Phirange, S.G. Sabharwal, A.A. Athawale, Precursordependent structural properties and antibacterial activity of copper oxide, Bull. Mater. 37

Journal Pre-proof

Sci. 41 (2018) 98-119. [54] S.K. Ray, D. Dhakal, J.K. Sohng, S.-Y. Kim, S.W. Lee, Efficient inactivation of Pseudomonas aeruginosa by Cu/Co-α-NiMoO4 in visible light, Chem. Eng. J. 347 (2018) 366-378. [55] N. Ekthammathat, T. Thongtem, S. Thongtem, Antimicrobial activities of CuO

of

films deposited on Cu foils by solution chemistry. Appl. Surf. Sci. 277 (2013) 211-217.

ro

[56] M.M. Momeni, M. Mirhosseini, Z. Nazari, A. Kazempour, M. Hakimiyan,

-p

Antibacterial and photocatalytic activity of CuO nanostructure films with different

re

morphology. J. Mater. Sci.: Mater. Electron. 27 (2016) 8131-8137.

lP

[57] S.W. Tsai, Y.Y. Chen, J.W. Liaw, Compound cellular imaging of laser scanning

2306-2316.

na

confocal microscopy by using gold nanoparticles and dyes, Sensors (Basel) 8 (2008)

Jo ur

[58] Y. Zhang, C. Lin, Q. Lin, Y. Jin, Y. Wang, Z. Zhang, H. Lin, J. Long, X. Wang, CuI-BiOI/Cu film for enhanced photo-induced charge separation and visible-light antibacterial activity, Appl. Catal. B: Environ. 235 (2018) 238-245. [59] L.B. LaCroix, D.W. Randall, A.M. Nersissian, C.W.G. Hoitink, G.W. Canters, J.S. Valentine, E.I. Solomon, Spectroscopic and geometric variations in perturbed blue copper centers: electronic structures of stellacyanin and cucumber basic protein, J. Am. Chem. Soc. 120 (1998) 9621-9631. [60] H. Barabadi, S. Honary, P. Ebrahimi, A. Alizadeh, F. Naghibi, M. Saravanan, Optimization of myco-synthesized silver nanoparticles by response surface 38

Journal Pre-proof

methodology employing Box-Behnken design, Inorg. Nano-Met Chem. 49 (2019) 33-43. [61] M. Dhonde, K. Sahu, V.V.S. Murty, S.S. Nemala, P. Bhargava, Surface plasmon resonance effect of Cu nanoparticles in a dye sensitized solar cell, Electrochim. Acta 249 (2017) 89-95. [62] A.A. Gewirth, E.I. Solomon, Electronic structure of plastocyanin: excited state

of

spectral features, J. Am. Chem. Soc. 110 (1988) 3811-3819.

ro

[63] X. Liu, W. Hu, S. Yang, Z. Li, C. Pei, Y. Zhou, G. Yin, Severe corrosion of

re

Corros. Sci. 94 (2015) 270-274.

-p

copper in a highly alkaline egg white solution due to a biuret corrosion reaction,

[64] C. Damm, H. Munstedt, A. Rosch, The antimicrobial efficacy of polyamide

lP

6/silver nano- and microcomposites, Mater. Chem. Phys. 108 (2008) 61–66.

na

[65] S. Zhang, J. Li, G. Lykotrafitis, G. Bao, S. Suresh, Size-dependent endocytosis

Jo ur

nanoparticles, Adv. Mater. 21 (2009) 419–424. [66] E. Antolini, F. Cardellini, Formation of carbon supported PtRu alloys: an XRD analysis, J. Alloy. Compd. 315 (2001) 118-122. [67] W. Zhang, Y. Zhang, J. Ji, Q. Yan, A. Huang, P. K. Chu, Antimicrobial polyethylene with controlled copper release, J. Biomed. Mater. Res. Part A 83A (2007) 838-844. [68] L. Tamayo, M. Azócar, M. Kogan, A. Riveros, M. Páez, Copper-polymer nanocomposites: An excellent and cost-effective biocide for use on antibacterial surfaces, Mater. Sci. Eng. C 69 (2016) 1391–1409.

39

Journal Pre-proof Author statement

The relevant CRediT roles of the authors are listed as below: authors

relevant CRediT roles

Pengzhao Lv

Synthesis

of

the

Cu

NPs

and

antibacterial experiments, part of data analysis, writing the draft and revise the

of

manuscript partly Lianjie Zhu

The idea for the main work, supervising

ro

the main works, part of data analysis,

-p

revising the draft and manuscript Yanmiao Yu

Characterization of XRD, SEM and

re

TEM before and after antibacterial

Wenwen Wang

lP

experiment, and data analysis Characterization of FT-IR spectra and

na

data analysis

Jo ur

Guokai Liu

Hongguang Lu

Measurement on the absorption spectra in Figure 10 Idea for Figure 9, supervising the experiments, analyzing the data and writing the text of this part

40

Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Lianjie Zhu

朱连杰

Jo ur

na

lP

re

-p

ro

may be considered as potential competing interests:

of

☐The authors declare the following financial interests/personal relationships which

2019.10.10

41

Journal Pre-proof

Jo ur

na

lP

re

-p

ro

of

Graphical abstract

42

Journal Pre-proof Highlights Series of Cu NPs were prepared by tuning the concentrations of NaOH solutions. The Cu NPs exhibit excellent antibacterial activities with lean copper leachate. NaOH concentrations markedly affect the antibacterial activities of the Cu NPs. Antibacterial mechanism of the Cu NPs was explored based on control experiments. 5. Soluble Cu(II)-peptide complex rather than Cu2+ is the active antibacterial species.

Jo ur

na

lP

re

-p

ro

of

1. 2. 3. 4.

43