Materials Science and Engineering C 35 (2014) 205–211
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of water-soluble Cu/PAA composite flowers and their antibacterial activities Binjie Li a,b, Yuanyuan Li a,b, Yonghui Wu b, Yanbao Zhao b,⁎ a b
Institute of Immunology, Henan University, Kaifeng 475004, PR China Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 22 October 2013 Accepted 2 November 2013 Available online 14 November 2013 Keywords: Water-soluble Cu/PAA flower Synthesis Antibacterial activity
a b s t r a c t Water-soluble copper/polyacrylic acid (Cu/PAA) composites were synthesized by a facile solution-phase reduction route. The Cu/PAA composites presented flower-like architecture, consisting of several intercrossing sheets, and reaction conditions had an important effect on their morphologies. Their antibacterial activity towards the bacterial strains such as Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were evaluated by the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), cup diffusion method and optical density (OD600). Results indicated that Cu/PAA flower is selective in its antibacterial action. It displays more effective antibacterial activity against B. subtilis than other three stains, and better bactericidal activity against S. aureus, E. coli and P. aeruginosa than B. subtilis. There is no bactericidal ability against B. subtilis in the tested concentration range, which indicates that B. subtilis may be a copper-tolerant bacterium. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Recently, microbial threats on human health and safety have become a serious public concern, and various antimicrobial agents have been developed for curing and preventing diseases in public health hygiene and antifouling in biomedical industry [1–4]. Especially, inorganic antibacterial agents such as metal ions, metal oxides and metal or inorganic nanoparticles (NPs), have attracted increasing attention in medical applications due to their unique properties and amenability to biological functionalization [5–8]. The main advantages of using inorganic antibacterial agents when compared with organic antimicrobial agents are their stability, robustness, and long-term effect [9,10]. Among them, silver-based materials are of special interest owing to their broad spectrum inhibitory and strong bactericidal effect [11,12]. The use of colloidal silver as antibacterial agent has become very important in recent years, following the well-known use of silver ions as antibacterial agent [13,14]. For example, Taglietti et al. reported the antibacterial activity of glutathione–silver NPs [15]. Lok et al. demonstrated that the antibacterial activity of silver NPs is dependent on chemisorbed Ag+ ions, which is readily formed owing to extreme sensitivity to oxygen [16]. However, the use of silver-based materials has to face the restrictive applications attributed to expensive cost, colorchanging and accumulating toxicity [17]. In this regard, copper-based
⁎ Corresponding author. Tel.: +86 378 2820579; fax: +86 379 3881358. E-mail address: [email protected] (Y. Zhao). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.006
materials are attractive alternative because in addition to their lowcost and easy release out of human body they are known to have significant antibacterial properties [18,19]. Copper ions are also broad-spectrum biocide and can effectively inhibit the growth of bacteria, fungi and algae [20]. With the development of nanotechnology, many studies have been focused on Cu-based nanosized antibacterial agent [21,22]. For example, gelatin stabilized copper NPs displayed highly antibacterial activity against Escherichia coli at low concentration [23]. However, like other metal NPs, use of Cu NPs still poses instability, easy reunion and poor water soluble issues, causing deterioration of antibacterial property [24]. To address the above issues, Cu NPs are generally deposited on the surface of substrates, embedded in a suitable matrix or in the form of composite to improve their antibacterial property [25,26]. For example, Cu NPs/cellulose films show efficient antibacterial activity [27]. At present, there is still a need for the preparation of water soluble and stable Cu based antibacterial agent with long-term effects. Here, we report an aqueous-phase synthetic route to Cu/PAA composite with tunable size and shape. The synthesis involves the reduction of copper (II) salts with hydrazine in presence of poly (acrylic acid) (PAA) capping agent. The PAA molecules have significant content of carboxyl groups, which can bind on the surface of Cu NPs and make them water-soluble and stable [28,29]. By adding different amounts of PAA and hydrazine, Cu/PAA composites with tunable morphologies were obtained. The effect of reaction conditions on the morphology of resulting products was also studied. Furthermore, the antibacterial tests against four strains were performed by MIC, MBC and cup diffusion method.
206
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
2. Experimental
2.3. Characterization of Cu/PAA architectures
2.1. Materials
The crystalline phase of the sample was examined by X-ray diffractometer (X'Pert Philips, Holland) with Cu–Kα radiation (λ = 0.15406 nm) and the operating voltage and current were maintained at 40 kV and 40 mA, respectively. Their morphology and composition were characterized by scanning electron microscope (JEOL JSM5600LV, Japan) and Fourier transform infrared (Nicolet, AVATAR360, America), respectively. UV–Vis absorption spectra were recorded using an ultraviolet–visible absorption spectroscopy (Perkin Elmer, Lambda35, America). The optical density (OD600) was collected by UV–Vis spectrophotometer (Oppler, 752N, China).
Polyacrylic acid (PAA, Mw ≈ 800–1000), absolute ethanol and hydrazine hydrate (N2H4⋅ H2O, 80%) were supplied from Tianjin Kermel Chemical Reagent Company (Tianjin, China). Copper hydroxide was bought from Sinopharm Chemical Reagent Company (Shanghai, China). All the chemical reagents used were of analytical grade without further purification. Nutrient broth (NB, BR) and nutrient agar (NA, BR) were received from Beijing Aoboxing biotech Company (Beijing, China). Gram-positive Staphylococcus aureus (S. aureus, ATCC 35696), Bacillus Subtilis (B. subtilis, ATCC 21332), Gram-negative Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) and E. coli (E. coli, ATCC 23282) were selected as bacterial strains (China center of industrial culture collection, China).
2.4. Antibacterial tests To investigate the antibacterial ability of Cu/PAA flower, grampositive S. aureus, B. subtilis, Gram-negative P. aeruginosa and E. coli were selected as indicators. All disks and materials were sterilized in an autoclave before experiment. For MIC test, the samples with different concentrations were dispersed in sterilized tubes with 2 mL broth media by twofold serial dilution method. Then 20 μL bacteria suspensions (108 CFU/mL) were added into serial tubes, respectively. Finally, the tubes were incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration of samples that inhibited visible bacteria growth by turbidimetric method after incubation. For MBC test, the melt nutrient agar was spread onto plates, and then the bacterial suspensions with different concentration of sample were coated on the agar plates. Finally, the agar plates were incubated at 37 °C for 24 h.
2.2. Synthesis of Cu/PAA flower In a typical experiment, a mixture of Cu(OH)2 (0.2 mmol) and various amount of PAA were added into 40 mL distilled water under vigorous stirring at 60 °C for 30 min, giving a blue emulsion. The PAA amount varied from 0.2 to 0.5 g. After the solution was stirred for 30 min at 60 °C, a solution of N2H4⋅H2O (0.3–1.2 mL) was added to the solution with constant stirring and kept reaction for 2 h. The color of the solution gradually changed from blue to wine red, which indicated the formation of Cu/PAA composite sample. At last, the solution was centrifuged, washed and dried to get Cu/PAA sample.
c
(111)
Intensity(a.u.)
a
(200) (220) (311)
JCPDS No. 85-1326 10
20
30
40
50
60
70
80
90
2θ/degree
b
d
Transmittance(%)
1.0
0.9
0.8
0.7
634 cm -1 3425 cm
-1
2925 c m
-1
1703 cm
-1
0.6 1560 cm
4000
3500
3000
2500
2000
-1
1500
1402 cm
1000
-1
500
Wavenumber/cm-1 Fig. 1. SEM (a), XRD (b) and FT-IR (c) characterization of Cu/PAA flower TEM (b) image of Cu nanoparticles prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and 0.2 g PAA.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
The MBC may be defined as the minimal concentration of samples in which the number of survival bacteria was less than 5 on the agar plates. The antibacterial activities were also conducted by cup diffusion method. First, the melted LB nutrient agar was poured into the Petri dishe, and then 50 μL bacterial suspensions were uniformly coated onto the agar plates after solidification. Second, the sterilized oxford cups were gently placed on the lawn bacteria in LB agar plates, and the sample solutions with different concentrations were added into the cups. Meanwhile, 0.9% of sterilized saline was selected as contrast sample dropped into the central cups. Finally, these agar plates were incubated at 37 °C for 24 h. The antibacterial effect was evaluated by the diameter of visible transparent inhibitory zone. The antibacterial activity was also studied by the bacterial growth kinetics in broth media. The Cu/PAA flower was first dissolved into beef broth to yield serial broth solutions with concentration of 0.15 μg/mL, 0.29 μg/mL, 0.59 μg/mL, 1.17 μg/mL, 2.34 μg/mL and 4.69 μg/mL, respectively. Then, the serial broth solutions were mixed with 4 mL of bacterial broth in 10-mL culture tube. All the cultures were allowed to grow at 37 °C in a gyratory shaker at 125 rpm. At set time intervals, these tubes were withdrawn to monitor bacterial growth by measuring the optical density (OD) of the aliquots at 600 nm. Finally the OD values were plotted against the time to obtain the growth curve.
207
a
b
3. Results and discussion 3.1. XRD, FI-IR and morphological characterization Fig. 1 gives the SEM, XRD and FT-IR characterizations of the sample prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and 0.2 g PAA. It is clearly seen that the obtained sample shows flowerlike structures, consisting of several smaller sheets, and each flower is three-dimensional in appearance with a diameter 3 μm (Fig. 1a). After full desorption of surface PAA, Cu NPs are obtained (Fig. 1b). The Cu NPs presented spherical shape and their diameter was in the range of 30 nm to 70 nm. The XRD pattern of the synthesized Cu/PAA flower is shown in Fig. 1c. The characteristic peaks were observed at scattering angles (2θ) of 43.3°, 50.4°, 74.2° and 89.9°, corresponding to (111), (200), (220) and (311) crystal planes, respectively, of facecentered-cubic (fcc) of copper (JCPDS no. 85-1326). Furthermore, no other peaks of impurities such as cuprous oxide or copper oxide are observed, indicating the formation of metal copper under current synthetic condition. In FTIR spectrum (Fig. 1d), the absorption peaks at 3425 cm− 1 and 2925 cm− 1 correspond to the stretching vibration of \OH and C\H groups, respectively. The peak at 1703 cm− 1 is attributed to the stretching vibration of C_O in carboxyl. The typical absorption peaks at 1560 cm− 1 and 1402 cm− 1 can be assigned to the asymmetry and symmetry stretching vibration of \COO• group. These results indicate that the obtained flowerlike sample is Cu/PAA composite.
3.2. Effects of polyacrylic acid Fig. 2 depicts the SEM images of the samples prepared with 1.2 mL N2H4⋅H2O, 0.2 mmol Cu(OH)2 and different concentrations of PAA. In the presence of 0.1 g PAA, the sample mainly consists of irregular sheets and the surface of sheet is smooth (Fig. 2a). Interestingly, with a moderate amount of PAA (0.2 g), wonderfully flower-like sample is obtained (Fig. 1a). Increasing the amount of PAA to 1.0 g, the main sample is a simple-flower shape, which is composed of three intercrossing rhomboic sheets (Fig. 2b). Further increasing the amount of PAA to 2 g, the obtained sample is also flower-like, consisting of several sheets, and some sheets transit from rhombus to hexagon (Fig. 2c). Hence, varying the PAA amount could induce a change in the shape of Cu/PAA composite.
c
Fig. 2. SEM images of Cu/PAA samples prepared with at 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and different amount of PAA. (a) 0.1 g; (b) 1 g; (c) 2 g.
3.3. Effects of N2H4⋅H2O Fig. 3 gives the SEM images of samples obtained with 0. 2 mmol Cu(OH)2 and 0.5 g PAA with different amount of N2H4⋅H2O. When the amount of N2H4⋅ H2O is 0.3 mL, the sample is spherical in shape and has an average diameter of 350 nm (Fig. 3a). Increasing the amount of N2H4⋅ H2O to 0.6 mL, the sample consists of intercrossing sheets, with small particles on the surface of sheet (Fig. 3b). With increasing the amount of N2 H4 ⋅ H2 O to 1.2 mL, uniformly thin sheet is obtained (Fig. 3c). It is clear that high amount of N2H4⋅ H2O is propitious to Cu/PAA sheet. 3.4. The optical properties of Cu/PAA samples Fig. 4 displays UV–Vis absorption spectra and photos (insert) of Cu/PAA solution obtained with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O, 0.2 g PAA and different reaction time. When the reaction time is 5 min, there appears a typical absorption peak at 575 nm, and the
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
absorption peak can be attributed to the excitation of plasmon resonance or interband transition, which is a characteristic property of the metallic nature of nanosized copper [30]. With extending the reaction time, the absorption peak exhibits slightly blue-shift, which indicates the stability of nanosized Cu in Cu/PAA composite. The appearance of this absorption peak further indicates the reduction of copper ions into nanosized copper under our synthetic conditions.
a
0.6 0.5
3.5. Formation mechanism According to the above analysis, the possible formation mechanism of Cu/PAA composite might be deduced. Cu(OH)2 would provide low concentration of Cu2+ ions in aqueous environments [Ksp (Cu(OH)2), 2.2 × 10− 20]. PAA molecule is soluble long-chain compound with plenty of carboxyl (\COOH) groups, which can coordinate Cu2+ ions in solution. When Cu2 + ions binding in PAA chains are reduced to form Cu NPs, the PAA molecules can coat in situ on the surface of Cu NPs and hinder the growth of Cu NPs. Meanwhile, the binding Cu NPs would link PAA chains together and affect the movement of PAA chains. Adjusting the amount of N2H4⋅ H2O or PAA would affect the size of Cu
0.7
Absorbance
208
0.4
5 min 10 min 30 min 1h 2h
0.3 0.2 0.1 0.0
400
500
600
700
800
Wavelength/nm
b
a
Fig. 4. (a) UV–Vis absorption spectra and (b) photograph of Cu/PAA samples prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O, 0.2 g PAA and different reaction time.
NPs and PAA flexibility. PAA chains with moderate flexibility would form regular aggregates in solution and proper crosslinking is conducive to the formation of flower-like structure. Therefore, the reaction conditions had an important effect on the morphology of Cu/PAA composite.
b
c
3.6. Antibacterial activities Table 1 gives the MIC and MBC values of Cu/PAA flower prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅ H2O and 0.2 g PAA. According to the reactant amounts, the mass ratio of Cu:Cu/PAA is 0.4: 1. Here, the concentration is the equivalent copper content in Cu/PAA flower. It is clear that Cu/PAA flower exhibits excellent antibacterial activity against the tested bacteria, and MIC value is different for each strain. The MIC values for S. aureus, B. subtilis, E. coli and P. aeruginosa are 2.34 μg/mL, 0.29 μg/mL, 0.59 μg/mL and 0.59 μg/mL, respectively, which indicate that Cu/PAA flower can effectively release active Cu2+ or Cu particles. Interestingly, Cu/PAA sample presents more effective antibacterial activity against B. subtilis than S. aureus. For example, the MIC value against B. subtilis is 8 times less than that against S. aureus. The MBC values indicate that Cu/PAA flower could more effectively prevent growth of P. aeruginosa. For both E. coli and S. aureus, the sample shows identical bactericidal abilities. However, there is no bactericidal ability against B. subtilis within the test concentration, indicating that B. subtilis may be a copper-tolerant bacterium.
Table 1 The values of MIC and MBC of Cu/PAA flower against four strains.
Fig. 3. SEM images of Cu/PAA samples prepared with at 0.2 mmol Cu(OH)2, 0.5 g PAA and different amount of N2H4⋅H2O. (a) 0.3 mL; (b) 0.6 mL; (c) 1.2 mL.
Strains
MIC (μg/mL)
MBC (μg/mL)
S. aureus B. subtilis E. coli P. aeruginosa
2.34 0.29 0.59 0.59
9.38 – 9.38 4.69
represents no bactericidal activity.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
209
Fig. 5. Antibacterial tests of Cu/PAA flower against four strains measured by cup diffusion method.
The antibacterial activity of Cu/PAA flower against tested strains was also measured by cup diffusion method. Here, the concentrations of sample are 0.15 μg/mL 0.29 μg/mL, 0.59 μg/mL, 1.17 μg/mL, 2.34 μg/mL, 4.69 μg/mL, 9.38 μg/mL, 18.75 μg/mL, 37.5 μg/mL, 75 μg/mL, 150 μg/mL and 300 μg/mL, corresponding to the number from one to twelve, respectively. For S. aureus, the antibacterial activity is not observed when the concentration is less than 18.75 μg/mL (Fig. 5 a2). When the concentration is increased to 37.5 μg/mL, there appears a transparent growth inhibition ring with the diameter of 17.8 mm. It is clear that the concentration is higher and the inhibition ring is bigger (Fig. 5 a3). For B. subtilis, E. coli and P. aeruginosa, the inhibition rings could be observed at a relatively lower concentration of 4.69 μg/mL. Similarly, with increasing the concentration of sample, the inhibition ring becomes bigger. These results indicate that Cu/PAA flower has excellent antibacterial effect against tested strains. The antibacterial test was also performed by the bacterial growth curve. The time-dependent changes in the quantity of bacterial growth were monitored by measuring OD600. Fig. 6 gives the growth curve of tested bacteria with different concentration of Cu/PAA flower. It is clear that Cu/PAA at all tested concentrations has strong suppression of proliferation of tested strains. For S. aureus, when the concentration is 2.34 μg/mL or 4.69 μg/mL, the sample could inhibit completely the
growth of S. aureus during the whole 48 h (Fig. 6a). However, when the concentration is below MIC, for example, 1.17 μg/mL, it couldn't completely inhibit the growth of S. aureus within 48 h, causing a growth delay of S. aureus. For B. subtilis, the solution of 0.29 μg/mL or 0.59 μg/mL could completely inhibit the growth of bacteria. Similarly, the growth curve of B. subtilis at the concentration of 0.15 μg/mL also shows a lag phase (Fig. 6b). Similar phenomena are also observed in the case of Gram-negative E. coli and P. aeruginosa (Fig. 6c and d). In order to investigate the interaction between bacteria and Cu/PAA, SEM microscopy was used to evaluate the change in morphology of treated bacteria. Fig. 7 gives the SEM images of both native and treated bacteria. It is clear that the morphology of native S. aureus is spherical and has smooth surface, in contrast, the treated S. aureus has rough surface (Fig. 7a). Similarly, native E. coli, B. subtilis and P. aeruginosa are rodlike shape and have smooth surface (Fig. 7 b1, c1 and d1), whereas the treated bacterial cells are significantly changed and show major damage, which are characterized by coarse surfaces, pits or broken cells (Fig. 7b2, c2 and d2). These results can be attributed to the death of bacteria owing to the inhibition of bacteria division by being treated with the samples. In addition, we also have performed the antibacterial test of CuSO4 and CuCl2 solution (shown in SI). Free Cu2+ ions display identical MIC
210
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
0.7
0.40
a
0.6
0.30
0.5 control 1.17 g/mL 2.34 g/mL 4.69 g/mL
0.4 0.3
OD600
OD600
b
0.35
0.25 0.20 control 0.15 g/mL 0.29 g/mL 0.59 g/mL
0.15
0.2
0.10
0.1
0.05
0.0
0.00 0
6
12
18
24
30
36
42
48
0
6
12
18
Time/h 0.7
0.40
c
0.6
30
36
42
48
d
0.35 0.30
0.5 0.4
control 0.29 g/mL 0.59 g/mL 1.17 g/mL
0.3
OD600
OD600
24
Time/h
0.25 0.20 control 0.29 g/mL 0.59 g/mL 1.17 g/mL
0.15
0.2
0.10
0.1
0.05
0.0
0.00 0
6
12
18
24
30
36
42
48
Time/h
0
6
12
18
24
30
36
42
48
Time/h
Fig. 6. Bacterial growth curve in liquid media. Different concentrations of Cu/PAA flower were added to S. aureus (a), B. subtilis (b), E. coli (c) and P. aeruginosa (d) culture. The growth of the bacteria was monitored measuring the OD600. Each data point represents a minimum of three independent experiments shown with standard error of the mean.
values for S. aureus, B. subtilis and P. aeruginosa strains and different MBC values for four tested strains, which are different from that of Cu/PAA flower. The antibacterial mechanism of Cu/PAA flower might be similar to that of Cu ions. First, Cu NPs embedded in Cu/PAA flower could diffuse to the surface of composite. Then the exposed Cu NPs would release slowly Cu2+ ions by oxidization to environment medium and the release effect of Cu ions is significant. The concentration of Cu2+ ions is depended on the release and oxidation of Cu NPs and the morphology of composite would affect the release of Cu NPs. Therefore, the antibacterial activity of Cu/PAA flower is different from that of free Cu2+ ions. 4. Conclusion In this paper, we reported a simple route to prepare water-soluble Cu/PAA composite, which presented flower-like architecture, consisting of several intercrossing sheets. Their morphologies could be tuned by adjusting reaction condition, and increasing amount of PAA and reductant is advantageous to obtain regular sample. The Cu/PAA composite is selective in its antibacterial action, and displays effective antibacterial and bactericidal activity against S. aureus, E. coli and P. aeruginosa. Acknowledgment Financial support of this work from the National Science Foundation (no. 21271062) and the Construction Fund of Henan province and Department of Education (no. SBGJ090515) is gratefully acknowledged. Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.11.006. References [1] B. Krajewska, P. Wydro, A. Jańczyk, Biomacromolecules 12 (2011) 4144–4152.
[2] H.-S. Lee, D.M. Eckmann, D. Lee, N.J. Hickok, R.J. Composto, Langmuir 27 (2011) 12458–12465. [3] W.-L. Du, S.-S. Niu, Y.-L. Xu, Z.-R. Xu, C.-L. Fan, Carbohydr. Polym. 75 (2009) 385–389. [4] Y. Li, L. Yang, Y. Zhao, B. Li, L. Sun, H. Luo, Mater. Sci. Eng. C 33 (2013) 1808–1812. [5] S. Ghosh, A. Saraswathi, S. Indi, S. Hoti, H. Vasan, Langmuir 28 (2012) 8550–8561. [6] M. Li, L. Zhu, D. Lin, Environ. Sci. Technol. 45 (2011) 1977–1983. [7] G.A. Sotiriou, S.E. Pratsinis, Environ. Sci. Technol. 44 (2010) 5649–5654. [8] M. Li, M.E. Noriega-Trevino, N. Nino-Martinez, C. Marambio-Jones, J. Wang, R. Damoiseaux, F. Ruiz, E.M.V. Hoek, Environ. Sci. Technol. 45 (2011) 8989–8995. [9] M. Lv, S. Su, Y. He, Q. Huang, W. Hu, D. Li, C. Fan, S.-T. Lee, Adv. Mater. 22 (2010) 5463–5467. [10] K.R. Raghupathi, R.T. Koodali, A.C. Manna, Langmuir 27 (2011) 4020–4028. [11] B. Yu, K.M. Leung, Q. Guo, W.M. Lau, J. Yang, Nanotechnology 22 (2011) 115603–115611. [12] Z. Xiu, Q. Zhang, H.L. Puppala, V.L. Colvin, P.J. Alvarez, Nano Lett. 12 (2012) 4271–4275. [13] M. Gao, L. Sun, Z. Wang, Y. Zhao, Mater. Sci. Eng. C 33 (2013) 397–404. [14] M. Liong, B. France, K.A. Bradley, J.I. Zink, Adv. Mater. 21 (2009) 1–6. [15] A. Taglietti, Y.A.D. Fernandez, E. Amato, L. Cucca, G. Dacarro, P. Grisoli, V. Necchi, P. Pallavicini, L. Pasotti, M. Patrini, Langmuir 28 (2012) 8140–8148. [16] C.-N. Lok, C.-M. Ho, R. Chen, Q.-Y. He, W.-Y. Yu, H. Sun, P.K.-H. Tam, J.-F. Chiu, C.-M. Che, J. Biol. Inorg. Chem. 12 (2007) 527–534. [17] M.C. Fung, D.L. Bowen, Clin. Toxicol. 34 (1996) 119–126. [18] N. Cioffi, L. Torsi, N. Ditaranto, L. Sabbatini, P.G. Zambonin, G. Tantillo, Appl. Phys. Lett. 85 (2004) 2417–2419. [19] M. Raffi, S. Mehrwan, T.M. Bhatti, J.I. Akhter, A. Hameed, W. Yawar, M.M.ul. Hasan, Ann. Microbiol. 60 (2010) 75–80. [20] W.-L. Du, Y.-L. Xu, Z.-R. Xu, C.-L. Fan, Nanotechnology 19 (2008) 085707–085711. [21] Y. Wei, S. Chen, B. Kowalczyk, S. Huda, T.P. Gray, B.A. Grzybowski, J. Phys. Chem. C 114 (2010) 15612–15616. [22] Feng Gao, Huan Pang, Shuoping Xu, Qingyi Lu, Chem. Commun. (2009) 3571–3573. [23] A.K. Chatterjee, R.K. Sarkar, A.P. Chattopadhyay, P. Aich, R. Chakraborty, T. Basu, Nanotechnology 23 (2012) 085103–085113. [24] S. Mallick, S. Sharma, M. Banerjee, S.S. Ghosh, A. Chattopadhyay, A. Paul, ACS Appl. Mater. Interfaces 4 (2012) 1313–1323. [25] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255–5262. [26] Y.H. Kim, D.K. Lee, H.G. Cha, C.W. Kim, Y.C. Kang, Y.S. Kang, J. Phys. Chem. B 110 (2006) 24923–24928. [27] B. Jia, Y. Mei, L. Cheng, J. Zhou, L. Zhang, ACS Appl. Mater. Interfaces 4 (2012) 2897–2902. [28] Y. Wang, A.V. Biradar, G. Wang, K.K. Sharma, C.T. Duncan, S. Rangan, T. Asefa, Chem. Eur. J. 16 (2010) 10735–10743. [29] Y. Wang, T. Asefa, Langmuir 26 (2010) 7469–7474. [30] A. Yanase, H. Komiyama, Surf. Sci. 248 (1991) 11–19.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
211
Fig. 7. SEM images of S. aureus, B. subtilis, E. coli and P. aeruginosa before (left) and after (right) treated with Cu/PAA flower solution. (a) S. aureus; (b) B. subtilis; (c) E. coli; (d) P. aeruginosa.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of water-soluble Cu/PAA composite flowers and their antibacterial activities Binjie Li a,b, Yuanyuan Li a,b, Yonghui Wu b, Yanbao Zhao b,⁎ a b
Institute of Immunology, Henan University, Kaifeng 475004, PR China Key Laboratory for Special Functional Materials, Henan University, Kaifeng 475004, PR China
a r t i c l e
i n f o
Article history: Received 19 June 2013 Received in revised form 22 October 2013 Accepted 2 November 2013 Available online 14 November 2013 Keywords: Water-soluble Cu/PAA flower Synthesis Antibacterial activity
a b s t r a c t Water-soluble copper/polyacrylic acid (Cu/PAA) composites were synthesized by a facile solution-phase reduction route. The Cu/PAA composites presented flower-like architecture, consisting of several intercrossing sheets, and reaction conditions had an important effect on their morphologies. Their antibacterial activity towards the bacterial strains such as Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) were evaluated by the minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC), cup diffusion method and optical density (OD600). Results indicated that Cu/PAA flower is selective in its antibacterial action. It displays more effective antibacterial activity against B. subtilis than other three stains, and better bactericidal activity against S. aureus, E. coli and P. aeruginosa than B. subtilis. There is no bactericidal ability against B. subtilis in the tested concentration range, which indicates that B. subtilis may be a copper-tolerant bacterium. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Recently, microbial threats on human health and safety have become a serious public concern, and various antimicrobial agents have been developed for curing and preventing diseases in public health hygiene and antifouling in biomedical industry [1–4]. Especially, inorganic antibacterial agents such as metal ions, metal oxides and metal or inorganic nanoparticles (NPs), have attracted increasing attention in medical applications due to their unique properties and amenability to biological functionalization [5–8]. The main advantages of using inorganic antibacterial agents when compared with organic antimicrobial agents are their stability, robustness, and long-term effect [9,10]. Among them, silver-based materials are of special interest owing to their broad spectrum inhibitory and strong bactericidal effect [11,12]. The use of colloidal silver as antibacterial agent has become very important in recent years, following the well-known use of silver ions as antibacterial agent [13,14]. For example, Taglietti et al. reported the antibacterial activity of glutathione–silver NPs [15]. Lok et al. demonstrated that the antibacterial activity of silver NPs is dependent on chemisorbed Ag+ ions, which is readily formed owing to extreme sensitivity to oxygen [16]. However, the use of silver-based materials has to face the restrictive applications attributed to expensive cost, colorchanging and accumulating toxicity [17]. In this regard, copper-based
⁎ Corresponding author. Tel.: +86 378 2820579; fax: +86 379 3881358. E-mail address: [email protected] (Y. Zhao). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.006
materials are attractive alternative because in addition to their lowcost and easy release out of human body they are known to have significant antibacterial properties [18,19]. Copper ions are also broad-spectrum biocide and can effectively inhibit the growth of bacteria, fungi and algae [20]. With the development of nanotechnology, many studies have been focused on Cu-based nanosized antibacterial agent [21,22]. For example, gelatin stabilized copper NPs displayed highly antibacterial activity against Escherichia coli at low concentration [23]. However, like other metal NPs, use of Cu NPs still poses instability, easy reunion and poor water soluble issues, causing deterioration of antibacterial property [24]. To address the above issues, Cu NPs are generally deposited on the surface of substrates, embedded in a suitable matrix or in the form of composite to improve their antibacterial property [25,26]. For example, Cu NPs/cellulose films show efficient antibacterial activity [27]. At present, there is still a need for the preparation of water soluble and stable Cu based antibacterial agent with long-term effects. Here, we report an aqueous-phase synthetic route to Cu/PAA composite with tunable size and shape. The synthesis involves the reduction of copper (II) salts with hydrazine in presence of poly (acrylic acid) (PAA) capping agent. The PAA molecules have significant content of carboxyl groups, which can bind on the surface of Cu NPs and make them water-soluble and stable [28,29]. By adding different amounts of PAA and hydrazine, Cu/PAA composites with tunable morphologies were obtained. The effect of reaction conditions on the morphology of resulting products was also studied. Furthermore, the antibacterial tests against four strains were performed by MIC, MBC and cup diffusion method.
206
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
2. Experimental
2.3. Characterization of Cu/PAA architectures
2.1. Materials
The crystalline phase of the sample was examined by X-ray diffractometer (X'Pert Philips, Holland) with Cu–Kα radiation (λ = 0.15406 nm) and the operating voltage and current were maintained at 40 kV and 40 mA, respectively. Their morphology and composition were characterized by scanning electron microscope (JEOL JSM5600LV, Japan) and Fourier transform infrared (Nicolet, AVATAR360, America), respectively. UV–Vis absorption spectra were recorded using an ultraviolet–visible absorption spectroscopy (Perkin Elmer, Lambda35, America). The optical density (OD600) was collected by UV–Vis spectrophotometer (Oppler, 752N, China).
Polyacrylic acid (PAA, Mw ≈ 800–1000), absolute ethanol and hydrazine hydrate (N2H4⋅ H2O, 80%) were supplied from Tianjin Kermel Chemical Reagent Company (Tianjin, China). Copper hydroxide was bought from Sinopharm Chemical Reagent Company (Shanghai, China). All the chemical reagents used were of analytical grade without further purification. Nutrient broth (NB, BR) and nutrient agar (NA, BR) were received from Beijing Aoboxing biotech Company (Beijing, China). Gram-positive Staphylococcus aureus (S. aureus, ATCC 35696), Bacillus Subtilis (B. subtilis, ATCC 21332), Gram-negative Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) and E. coli (E. coli, ATCC 23282) were selected as bacterial strains (China center of industrial culture collection, China).
2.4. Antibacterial tests To investigate the antibacterial ability of Cu/PAA flower, grampositive S. aureus, B. subtilis, Gram-negative P. aeruginosa and E. coli were selected as indicators. All disks and materials were sterilized in an autoclave before experiment. For MIC test, the samples with different concentrations were dispersed in sterilized tubes with 2 mL broth media by twofold serial dilution method. Then 20 μL bacteria suspensions (108 CFU/mL) were added into serial tubes, respectively. Finally, the tubes were incubated at 37 °C for 24 h. The MIC was defined as the lowest concentration of samples that inhibited visible bacteria growth by turbidimetric method after incubation. For MBC test, the melt nutrient agar was spread onto plates, and then the bacterial suspensions with different concentration of sample were coated on the agar plates. Finally, the agar plates were incubated at 37 °C for 24 h.
2.2. Synthesis of Cu/PAA flower In a typical experiment, a mixture of Cu(OH)2 (0.2 mmol) and various amount of PAA were added into 40 mL distilled water under vigorous stirring at 60 °C for 30 min, giving a blue emulsion. The PAA amount varied from 0.2 to 0.5 g. After the solution was stirred for 30 min at 60 °C, a solution of N2H4⋅H2O (0.3–1.2 mL) was added to the solution with constant stirring and kept reaction for 2 h. The color of the solution gradually changed from blue to wine red, which indicated the formation of Cu/PAA composite sample. At last, the solution was centrifuged, washed and dried to get Cu/PAA sample.
c
(111)
Intensity(a.u.)
a
(200) (220) (311)
JCPDS No. 85-1326 10
20
30
40
50
60
70
80
90
2θ/degree
b
d
Transmittance(%)
1.0
0.9
0.8
0.7
634 cm -1 3425 cm
-1
2925 c m
-1
1703 cm
-1
0.6 1560 cm
4000
3500
3000
2500
2000
-1
1500
1402 cm
1000
-1
500
Wavenumber/cm-1 Fig. 1. SEM (a), XRD (b) and FT-IR (c) characterization of Cu/PAA flower TEM (b) image of Cu nanoparticles prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and 0.2 g PAA.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
The MBC may be defined as the minimal concentration of samples in which the number of survival bacteria was less than 5 on the agar plates. The antibacterial activities were also conducted by cup diffusion method. First, the melted LB nutrient agar was poured into the Petri dishe, and then 50 μL bacterial suspensions were uniformly coated onto the agar plates after solidification. Second, the sterilized oxford cups were gently placed on the lawn bacteria in LB agar plates, and the sample solutions with different concentrations were added into the cups. Meanwhile, 0.9% of sterilized saline was selected as contrast sample dropped into the central cups. Finally, these agar plates were incubated at 37 °C for 24 h. The antibacterial effect was evaluated by the diameter of visible transparent inhibitory zone. The antibacterial activity was also studied by the bacterial growth kinetics in broth media. The Cu/PAA flower was first dissolved into beef broth to yield serial broth solutions with concentration of 0.15 μg/mL, 0.29 μg/mL, 0.59 μg/mL, 1.17 μg/mL, 2.34 μg/mL and 4.69 μg/mL, respectively. Then, the serial broth solutions were mixed with 4 mL of bacterial broth in 10-mL culture tube. All the cultures were allowed to grow at 37 °C in a gyratory shaker at 125 rpm. At set time intervals, these tubes were withdrawn to monitor bacterial growth by measuring the optical density (OD) of the aliquots at 600 nm. Finally the OD values were plotted against the time to obtain the growth curve.
207
a
b
3. Results and discussion 3.1. XRD, FI-IR and morphological characterization Fig. 1 gives the SEM, XRD and FT-IR characterizations of the sample prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and 0.2 g PAA. It is clearly seen that the obtained sample shows flowerlike structures, consisting of several smaller sheets, and each flower is three-dimensional in appearance with a diameter 3 μm (Fig. 1a). After full desorption of surface PAA, Cu NPs are obtained (Fig. 1b). The Cu NPs presented spherical shape and their diameter was in the range of 30 nm to 70 nm. The XRD pattern of the synthesized Cu/PAA flower is shown in Fig. 1c. The characteristic peaks were observed at scattering angles (2θ) of 43.3°, 50.4°, 74.2° and 89.9°, corresponding to (111), (200), (220) and (311) crystal planes, respectively, of facecentered-cubic (fcc) of copper (JCPDS no. 85-1326). Furthermore, no other peaks of impurities such as cuprous oxide or copper oxide are observed, indicating the formation of metal copper under current synthetic condition. In FTIR spectrum (Fig. 1d), the absorption peaks at 3425 cm− 1 and 2925 cm− 1 correspond to the stretching vibration of \OH and C\H groups, respectively. The peak at 1703 cm− 1 is attributed to the stretching vibration of C_O in carboxyl. The typical absorption peaks at 1560 cm− 1 and 1402 cm− 1 can be assigned to the asymmetry and symmetry stretching vibration of \COO• group. These results indicate that the obtained flowerlike sample is Cu/PAA composite.
3.2. Effects of polyacrylic acid Fig. 2 depicts the SEM images of the samples prepared with 1.2 mL N2H4⋅H2O, 0.2 mmol Cu(OH)2 and different concentrations of PAA. In the presence of 0.1 g PAA, the sample mainly consists of irregular sheets and the surface of sheet is smooth (Fig. 2a). Interestingly, with a moderate amount of PAA (0.2 g), wonderfully flower-like sample is obtained (Fig. 1a). Increasing the amount of PAA to 1.0 g, the main sample is a simple-flower shape, which is composed of three intercrossing rhomboic sheets (Fig. 2b). Further increasing the amount of PAA to 2 g, the obtained sample is also flower-like, consisting of several sheets, and some sheets transit from rhombus to hexagon (Fig. 2c). Hence, varying the PAA amount could induce a change in the shape of Cu/PAA composite.
c
Fig. 2. SEM images of Cu/PAA samples prepared with at 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O and different amount of PAA. (a) 0.1 g; (b) 1 g; (c) 2 g.
3.3. Effects of N2H4⋅H2O Fig. 3 gives the SEM images of samples obtained with 0. 2 mmol Cu(OH)2 and 0.5 g PAA with different amount of N2H4⋅H2O. When the amount of N2H4⋅ H2O is 0.3 mL, the sample is spherical in shape and has an average diameter of 350 nm (Fig. 3a). Increasing the amount of N2H4⋅ H2O to 0.6 mL, the sample consists of intercrossing sheets, with small particles on the surface of sheet (Fig. 3b). With increasing the amount of N2 H4 ⋅ H2 O to 1.2 mL, uniformly thin sheet is obtained (Fig. 3c). It is clear that high amount of N2H4⋅ H2O is propitious to Cu/PAA sheet. 3.4. The optical properties of Cu/PAA samples Fig. 4 displays UV–Vis absorption spectra and photos (insert) of Cu/PAA solution obtained with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O, 0.2 g PAA and different reaction time. When the reaction time is 5 min, there appears a typical absorption peak at 575 nm, and the
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
absorption peak can be attributed to the excitation of plasmon resonance or interband transition, which is a characteristic property of the metallic nature of nanosized copper [30]. With extending the reaction time, the absorption peak exhibits slightly blue-shift, which indicates the stability of nanosized Cu in Cu/PAA composite. The appearance of this absorption peak further indicates the reduction of copper ions into nanosized copper under our synthetic conditions.
a
0.6 0.5
3.5. Formation mechanism According to the above analysis, the possible formation mechanism of Cu/PAA composite might be deduced. Cu(OH)2 would provide low concentration of Cu2+ ions in aqueous environments [Ksp (Cu(OH)2), 2.2 × 10− 20]. PAA molecule is soluble long-chain compound with plenty of carboxyl (\COOH) groups, which can coordinate Cu2+ ions in solution. When Cu2 + ions binding in PAA chains are reduced to form Cu NPs, the PAA molecules can coat in situ on the surface of Cu NPs and hinder the growth of Cu NPs. Meanwhile, the binding Cu NPs would link PAA chains together and affect the movement of PAA chains. Adjusting the amount of N2H4⋅ H2O or PAA would affect the size of Cu
0.7
Absorbance
208
0.4
5 min 10 min 30 min 1h 2h
0.3 0.2 0.1 0.0
400
500
600
700
800
Wavelength/nm
b
a
Fig. 4. (a) UV–Vis absorption spectra and (b) photograph of Cu/PAA samples prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅H2O, 0.2 g PAA and different reaction time.
NPs and PAA flexibility. PAA chains with moderate flexibility would form regular aggregates in solution and proper crosslinking is conducive to the formation of flower-like structure. Therefore, the reaction conditions had an important effect on the morphology of Cu/PAA composite.
b
c
3.6. Antibacterial activities Table 1 gives the MIC and MBC values of Cu/PAA flower prepared with 0.2 mmol Cu(OH)2, 1.2 mL N2H4⋅ H2O and 0.2 g PAA. According to the reactant amounts, the mass ratio of Cu:Cu/PAA is 0.4: 1. Here, the concentration is the equivalent copper content in Cu/PAA flower. It is clear that Cu/PAA flower exhibits excellent antibacterial activity against the tested bacteria, and MIC value is different for each strain. The MIC values for S. aureus, B. subtilis, E. coli and P. aeruginosa are 2.34 μg/mL, 0.29 μg/mL, 0.59 μg/mL and 0.59 μg/mL, respectively, which indicate that Cu/PAA flower can effectively release active Cu2+ or Cu particles. Interestingly, Cu/PAA sample presents more effective antibacterial activity against B. subtilis than S. aureus. For example, the MIC value against B. subtilis is 8 times less than that against S. aureus. The MBC values indicate that Cu/PAA flower could more effectively prevent growth of P. aeruginosa. For both E. coli and S. aureus, the sample shows identical bactericidal abilities. However, there is no bactericidal ability against B. subtilis within the test concentration, indicating that B. subtilis may be a copper-tolerant bacterium.
Table 1 The values of MIC and MBC of Cu/PAA flower against four strains.
Fig. 3. SEM images of Cu/PAA samples prepared with at 0.2 mmol Cu(OH)2, 0.5 g PAA and different amount of N2H4⋅H2O. (a) 0.3 mL; (b) 0.6 mL; (c) 1.2 mL.
Strains
MIC (μg/mL)
MBC (μg/mL)
S. aureus B. subtilis E. coli P. aeruginosa
2.34 0.29 0.59 0.59
9.38 – 9.38 4.69
represents no bactericidal activity.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
209
Fig. 5. Antibacterial tests of Cu/PAA flower against four strains measured by cup diffusion method.
The antibacterial activity of Cu/PAA flower against tested strains was also measured by cup diffusion method. Here, the concentrations of sample are 0.15 μg/mL 0.29 μg/mL, 0.59 μg/mL, 1.17 μg/mL, 2.34 μg/mL, 4.69 μg/mL, 9.38 μg/mL, 18.75 μg/mL, 37.5 μg/mL, 75 μg/mL, 150 μg/mL and 300 μg/mL, corresponding to the number from one to twelve, respectively. For S. aureus, the antibacterial activity is not observed when the concentration is less than 18.75 μg/mL (Fig. 5 a2). When the concentration is increased to 37.5 μg/mL, there appears a transparent growth inhibition ring with the diameter of 17.8 mm. It is clear that the concentration is higher and the inhibition ring is bigger (Fig. 5 a3). For B. subtilis, E. coli and P. aeruginosa, the inhibition rings could be observed at a relatively lower concentration of 4.69 μg/mL. Similarly, with increasing the concentration of sample, the inhibition ring becomes bigger. These results indicate that Cu/PAA flower has excellent antibacterial effect against tested strains. The antibacterial test was also performed by the bacterial growth curve. The time-dependent changes in the quantity of bacterial growth were monitored by measuring OD600. Fig. 6 gives the growth curve of tested bacteria with different concentration of Cu/PAA flower. It is clear that Cu/PAA at all tested concentrations has strong suppression of proliferation of tested strains. For S. aureus, when the concentration is 2.34 μg/mL or 4.69 μg/mL, the sample could inhibit completely the
growth of S. aureus during the whole 48 h (Fig. 6a). However, when the concentration is below MIC, for example, 1.17 μg/mL, it couldn't completely inhibit the growth of S. aureus within 48 h, causing a growth delay of S. aureus. For B. subtilis, the solution of 0.29 μg/mL or 0.59 μg/mL could completely inhibit the growth of bacteria. Similarly, the growth curve of B. subtilis at the concentration of 0.15 μg/mL also shows a lag phase (Fig. 6b). Similar phenomena are also observed in the case of Gram-negative E. coli and P. aeruginosa (Fig. 6c and d). In order to investigate the interaction between bacteria and Cu/PAA, SEM microscopy was used to evaluate the change in morphology of treated bacteria. Fig. 7 gives the SEM images of both native and treated bacteria. It is clear that the morphology of native S. aureus is spherical and has smooth surface, in contrast, the treated S. aureus has rough surface (Fig. 7a). Similarly, native E. coli, B. subtilis and P. aeruginosa are rodlike shape and have smooth surface (Fig. 7 b1, c1 and d1), whereas the treated bacterial cells are significantly changed and show major damage, which are characterized by coarse surfaces, pits or broken cells (Fig. 7b2, c2 and d2). These results can be attributed to the death of bacteria owing to the inhibition of bacteria division by being treated with the samples. In addition, we also have performed the antibacterial test of CuSO4 and CuCl2 solution (shown in SI). Free Cu2+ ions display identical MIC
210
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
0.7
0.40
a
0.6
0.30
0.5 control 1.17 g/mL 2.34 g/mL 4.69 g/mL
0.4 0.3
OD600
OD600
b
0.35
0.25 0.20 control 0.15 g/mL 0.29 g/mL 0.59 g/mL
0.15
0.2
0.10
0.1
0.05
0.0
0.00 0
6
12
18
24
30
36
42
48
0
6
12
18
Time/h 0.7
0.40
c
0.6
30
36
42
48
d
0.35 0.30
0.5 0.4
control 0.29 g/mL 0.59 g/mL 1.17 g/mL
0.3
OD600
OD600
24
Time/h
0.25 0.20 control 0.29 g/mL 0.59 g/mL 1.17 g/mL
0.15
0.2
0.10
0.1
0.05
0.0
0.00 0
6
12
18
24
30
36
42
48
Time/h
0
6
12
18
24
30
36
42
48
Time/h
Fig. 6. Bacterial growth curve in liquid media. Different concentrations of Cu/PAA flower were added to S. aureus (a), B. subtilis (b), E. coli (c) and P. aeruginosa (d) culture. The growth of the bacteria was monitored measuring the OD600. Each data point represents a minimum of three independent experiments shown with standard error of the mean.
values for S. aureus, B. subtilis and P. aeruginosa strains and different MBC values for four tested strains, which are different from that of Cu/PAA flower. The antibacterial mechanism of Cu/PAA flower might be similar to that of Cu ions. First, Cu NPs embedded in Cu/PAA flower could diffuse to the surface of composite. Then the exposed Cu NPs would release slowly Cu2+ ions by oxidization to environment medium and the release effect of Cu ions is significant. The concentration of Cu2+ ions is depended on the release and oxidation of Cu NPs and the morphology of composite would affect the release of Cu NPs. Therefore, the antibacterial activity of Cu/PAA flower is different from that of free Cu2+ ions. 4. Conclusion In this paper, we reported a simple route to prepare water-soluble Cu/PAA composite, which presented flower-like architecture, consisting of several intercrossing sheets. Their morphologies could be tuned by adjusting reaction condition, and increasing amount of PAA and reductant is advantageous to obtain regular sample. The Cu/PAA composite is selective in its antibacterial action, and displays effective antibacterial and bactericidal activity against S. aureus, E. coli and P. aeruginosa. Acknowledgment Financial support of this work from the National Science Foundation (no. 21271062) and the Construction Fund of Henan province and Department of Education (no. SBGJ090515) is gratefully acknowledged. Appendix A. Supplementary Data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2013.11.006. References [1] B. Krajewska, P. Wydro, A. Jańczyk, Biomacromolecules 12 (2011) 4144–4152.
[2] H.-S. Lee, D.M. Eckmann, D. Lee, N.J. Hickok, R.J. Composto, Langmuir 27 (2011) 12458–12465. [3] W.-L. Du, S.-S. Niu, Y.-L. Xu, Z.-R. Xu, C.-L. Fan, Carbohydr. Polym. 75 (2009) 385–389. [4] Y. Li, L. Yang, Y. Zhao, B. Li, L. Sun, H. Luo, Mater. Sci. Eng. C 33 (2013) 1808–1812. [5] S. Ghosh, A. Saraswathi, S. Indi, S. Hoti, H. Vasan, Langmuir 28 (2012) 8550–8561. [6] M. Li, L. Zhu, D. Lin, Environ. Sci. Technol. 45 (2011) 1977–1983. [7] G.A. Sotiriou, S.E. Pratsinis, Environ. Sci. Technol. 44 (2010) 5649–5654. [8] M. Li, M.E. Noriega-Trevino, N. Nino-Martinez, C. Marambio-Jones, J. Wang, R. Damoiseaux, F. Ruiz, E.M.V. Hoek, Environ. Sci. Technol. 45 (2011) 8989–8995. [9] M. Lv, S. Su, Y. He, Q. Huang, W. Hu, D. Li, C. Fan, S.-T. Lee, Adv. Mater. 22 (2010) 5463–5467. [10] K.R. Raghupathi, R.T. Koodali, A.C. Manna, Langmuir 27 (2011) 4020–4028. [11] B. Yu, K.M. Leung, Q. Guo, W.M. Lau, J. Yang, Nanotechnology 22 (2011) 115603–115611. [12] Z. Xiu, Q. Zhang, H.L. Puppala, V.L. Colvin, P.J. Alvarez, Nano Lett. 12 (2012) 4271–4275. [13] M. Gao, L. Sun, Z. Wang, Y. Zhao, Mater. Sci. Eng. C 33 (2013) 397–404. [14] M. Liong, B. France, K.A. Bradley, J.I. Zink, Adv. Mater. 21 (2009) 1–6. [15] A. Taglietti, Y.A.D. Fernandez, E. Amato, L. Cucca, G. Dacarro, P. Grisoli, V. Necchi, P. Pallavicini, L. Pasotti, M. Patrini, Langmuir 28 (2012) 8140–8148. [16] C.-N. Lok, C.-M. Ho, R. Chen, Q.-Y. He, W.-Y. Yu, H. Sun, P.K.-H. Tam, J.-F. Chiu, C.-M. Che, J. Biol. Inorg. Chem. 12 (2007) 527–534. [17] M.C. Fung, D.L. Bowen, Clin. Toxicol. 34 (1996) 119–126. [18] N. Cioffi, L. Torsi, N. Ditaranto, L. Sabbatini, P.G. Zambonin, G. Tantillo, Appl. Phys. Lett. 85 (2004) 2417–2419. [19] M. Raffi, S. Mehrwan, T.M. Bhatti, J.I. Akhter, A. Hameed, W. Yawar, M.M.ul. Hasan, Ann. Microbiol. 60 (2010) 75–80. [20] W.-L. Du, Y.-L. Xu, Z.-R. Xu, C.-L. Fan, Nanotechnology 19 (2008) 085707–085711. [21] Y. Wei, S. Chen, B. Kowalczyk, S. Huda, T.P. Gray, B.A. Grzybowski, J. Phys. Chem. C 114 (2010) 15612–15616. [22] Feng Gao, Huan Pang, Shuoping Xu, Qingyi Lu, Chem. Commun. (2009) 3571–3573. [23] A.K. Chatterjee, R.K. Sarkar, A.P. Chattopadhyay, P. Aich, R. Chakraborty, T. Basu, Nanotechnology 23 (2012) 085103–085113. [24] S. Mallick, S. Sharma, M. Banerjee, S.S. Ghosh, A. Chattopadhyay, A. Paul, ACS Appl. Mater. Interfaces 4 (2012) 1313–1323. [25] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Chem. Mater. 17 (2005) 5255–5262. [26] Y.H. Kim, D.K. Lee, H.G. Cha, C.W. Kim, Y.C. Kang, Y.S. Kang, J. Phys. Chem. B 110 (2006) 24923–24928. [27] B. Jia, Y. Mei, L. Cheng, J. Zhou, L. Zhang, ACS Appl. Mater. Interfaces 4 (2012) 2897–2902. [28] Y. Wang, A.V. Biradar, G. Wang, K.K. Sharma, C.T. Duncan, S. Rangan, T. Asefa, Chem. Eur. J. 16 (2010) 10735–10743. [29] Y. Wang, T. Asefa, Langmuir 26 (2010) 7469–7474. [30] A. Yanase, H. Komiyama, Surf. Sci. 248 (1991) 11–19.
B. Li et al. / Materials Science and Engineering C 35 (2014) 205–211
211
Fig. 7. SEM images of S. aureus, B. subtilis, E. coli and P. aeruginosa before (left) and after (right) treated with Cu/PAA flower solution. (a) S. aureus; (b) B. subtilis; (c) E. coli; (d) P. aeruginosa.
