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Surface microstructures and antimicrobial properties of copper plasma alloyed stainless steel
Applied Surface Science 258 (2011) 1399–1404
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Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Surface microstructures and antimicrobial properties of copper plasma alloyed stainless steel Xiangyu Zhang, Xiaobo Huang, Li Jiang, Yong Ma, Ailan Fan, Bin Tang ∗ Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 22 May 2011 Received in revised form 19 September 2011 Accepted 20 September 2011 Available online 28 September 2011 Keywords: Stainless steel Plasma alloying Copper content Antibacterial property
a b s t r a c t Bacterial adhesion to stainless steel surfaces is one of the major reason causing the cross-contamination and infection in many practical applications. An approach to solve this problem is to enhance the antibacterial properties on the surface of stainless steel. In this paper, novel antibacterial stainless steel surfaces with different copper content have been prepared by a plasma surface alloying technique at various gas pressures. The microstructure of the alloyed surfaces was investigated using glow discharge optical emission spectroscopy (GDOES) and scanning electron microscopy (SEM). The viability of bacteria attached to the antibacterial surfaces was tested using the spread plate method. The antibacterial mechanism of the alloyed surfaces was studied by X-ray photoelectron spectroscopy (XPS). The results indicate that gas pressure has a great influence on the surface elements concentration and the depth of the alloyed layer. The maximum copper concentration in the alloyed surface obtained at the gas pressure of 60 Pa is about 7.1 wt.%. This alloyed surface exhibited very strong antibacterial ability, and an effective reduction of 98% of Escherichia coli (E. coli) within 1 h was achieved by contact with the alloyed surface. The maximum thickness of the copper alloyed layer obtained at 45 Pa is about 6.5 m. Although the rate of reduction for E. coli of this alloyed surface was slower than that of the alloyed surface with the copper content about 7.1 wt.% over the first 3 h, few were able to survive more than 12 h and the reduction reached over 99.9%. The XPS analysis results indicated that the copper ions were released when the copper alloyed stainless steel in contact with bacterial solution, which is an important factor for killing bacteria. Based on an overall consideration of bacterial killing rate and durability, the alloyed surface with the copper content of 2.5 wt.% and the thickness of about 6.5 m obtained at the gas pressure of 45 Pa is expected to be useful as antimicrobial materials that may have a promising future in antimicrobial applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction With the development of science technology and growth of living standard, public awareness on the sanitation and health of environments has been rapidly raised. However, microbial contamination is still one of the major threats to public health [1,2]. Bacterial have a strong ability to attach to solid surfaces, such as industrial equipment, food contact surfaces and work surfaces in hospital environment. Once bacterial attach to these surfaces, a multistep process starts leading to the formation of bacterial biofilm which is very difficult to treat clinically. This increases the risk of cross-contamination and infection. Although various methods can be adopted to control bacterial development on surfaces, one effective approach is to prevent bacterial adherence to the surfaces and avoid bacterial biofilms formation by means of surface
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (B. Tang). 0169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2011.09.091
engineering techniques to introduce antibacterial agents, such as copper (Cu), silver (Ag) into material surfaces [3–5]. It is well known that stainless steel is one of the most commonly used materials in daily life. Stainless steel surfaces with added antibacterial properties would get popular and be used in hospitals, food industries and kitchen appliances. Previous studies on preparing antibacterial stainless steel surfaces mainly by means of coating, ion implantation and chemical synthesis process [6–8]. However, it is difficult to obtain enough thick antibacterial modified layers to maintain the long-lasting antibacterial effect. The thicknesses of the antibacterial modified layers prepared by these methods are mostly less than 1 m. On the other hand, it is difficult to maintain antibacterial effect simultaneously with good wear and corrosion resistance. Therefore, an efficient and long-term antimicrobial method to prevent bacterial adherence on stainless steel surface is needed. Double glow plasma surface alloying technology [9,10] has been successfully used to produce surface alloys to improve wear resistance, corrosion resistance and anti-oxidation performance of
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X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
materials such as on steel, iron, titanium alloy and intermetallic compound. This technology can probably be used to introduce the desired alloying elements sputtered from the source electrode by bombardment of the ion produced in the second glow discharge into the surface of the work piece to form a surface alloy. It can probably be used to introduce copper into stainless steel surface to form an antibacterial alloyed layer. However, if use pure copper as a source electrode directly, a copper deposited layer would be produced on stainless steel surface since copper have a limited solid solubility in stainless steel. Considering that nickel is one of the major elements in AISI304 stainless steel and easily diffuses into stainless steel, a Ni–Cu alloy was selected as source target in this paper to prepare a high quality copper alloyed layer on stainless steel surface. The objective of this work is to study the influence of the gas pressure on the surface microstructures and antimicrobial properties of copper plasma alloyed stainless steel. The antibacterial mechanism of the copper containing stainless steel surfaces is also discussed in this paper.
The antibacterial activity of the samples was determined using Gram-negative Escherichia coli (E. coli) ATCC10536. The plain-plate dilute method was used to measure the number of live bacteria and to calculate the antibacterial rate of each specimen in this study. The broth for culturing the bacteria was made by dissolving 5.0 g flesh extract, 5.0 g NaCl and 10.0 g peptone into 1000 ml of distilled water with the pH adjusted to 7.0–7.2. All samples were sterilized by autoclave for 20 min. 0.4 ml of solution of bacteria with a concentration of 105 CFU/ml was dripped onto the surfaces of the samples, which had been horizontally placed into a sterilized Petri-dish. In order to monitor the relation between percentage of cells killed and contact time (reduction rate), the bacteria on the sample surface were incubated for 1 h, 3 h, 12 h at a temperature of 37 ◦ C. After the incubation, the bacteria solution on the sample surface was diluted with 0.6 ml of 0.85 wt pct NaCl solution, and 0.1 ml of the bacterial solution was added into a Petri-dish. The numbers of viable bacteria colonies were enumerated after incubation for 18 h at 37 ◦ C. The percentage reduction was calculated according to the formula Reduction % =
2. Experimental A Ni–Cu alloy (the composition is Ni60, Cu40 in weight percent) plate (70 mm × 60 mm × 5 mm) was used as the source target for supplying the alloying elements. AISI304 stainless steel with a size of 20 × 5 mm was used as the substrate material. The samples were abraded with SiC emery paper and polished to smooth surfaces (Ra < 0.01 m) with alumina paste and then cleaned in acetone. The plasma surface alloying was performed in a double glow plasma alloying furnace. In this furnace, there are three electrodes: an anode, a negative cathode (sample) and a sputtering source cathode (Ni–Cu alloy). Glow discharge was initiated created on the samples to sputter clean the surface, then the discharge on the anode and source cathode was also created. Copper atoms and nickel atoms were sputtered out from the source electrode by bombardment of the ion, and they travel to and diffuse into the surface of the work piece which was heated to a high temperature at the meantime to form a surface alloy. Gas pressure is one of the most important parameters for the double glow plasma alloying technology. In this paper, to discuss the effect of gas pressure on the surface composition and depth of the modified layer, a series of pressure were set as 30, 45 and 60 Pa, respectively. Other plasma surface alloying parameters with copper were conducted under the following conditions: source electrode voltage −500 to −700 V, substrate voltage −300 to −500 V, working gas Ar, temperature 950 ◦ C and treatment time 3 h. The copper alloyed samples obtained at 30, 45 and 60 Pa are indicated by the symbols A1, A2 and A3, respectively. The detailed process parameters are listed in Table 1. Major element percentage and depth profiling were observed using a Spectro GDA750 glow discharge optical emission spectroscopy (GDOES) spectrometer. The surface microstructures were investigated using LEO-1450 type scanning electron microscopy (SEM). The phases were identified by Rigaku DyMax2500 X-ray diffraction spectrometer. The chemical state of copper in the modified layer surface was investigated by VG ESCALAB Mark II X-ray photoelectron spectroscopy (XPS) with Al K␣ X-ray as the radiation source.
( − ) t 0 0
× 100%,
where t is the number of viable bacteria for the treated sample after a designated contact time and 0 is the number of viable bacteria for the untreated sample after incubated 12 h. 3. Results and discussion 3.1. Macrostructures Gradient alloying layer are formed on the samples after plasma surface alloying treatment. The content distribution of alloying elements in the surface alloying layer formed on stainless steel obtained from the different gas pressure are shown in Fig. 1. As shown, there are large differences in the surface elements concentration and the depth of the alloyed layer. The copper concentration increases continuously from about 1.2% to about 7.1% with increasing of gas pressure from 30 Pa to 60 Pa. However, the depth of Cu-alloyed layer is not increased constantly with the gas pressure gone up. The maximum thickness of the Cu-alloyed layer obtained at 45 Pa is about 6.5 m. Results indicated that the gas pressure has a great influence on the surface elements concentration and the depth of the alloyed layer. The detailed copper content of A1, A2 and A3 in the surfaces and the depth of the alloyed layers are listed in Table 1. The plasma has two basic functions in double glow plasma surface alloying process. Firstly, it makes the copper and nickel element sputtered from the source electrode, as well as stimulates and activates the carriage gas which carrying the alloying elements. Secondly, the surface of the stainless steel was sputtered by bombardment of the plasma, which affects the depth and the surface concentration of the alloyed layer directly. In the vacuum chamber, working pressure corresponds to the plasma density when other parameters are fixed. Thus, the plasma density will increase with the increase of gas pressure in the double glow process, which probably make the sputtered loss of the source electrode increase and improve the deposit velocity of the alloying elements on the surface of the substrate. On the other hand, pressure has an inverse relation
Table 1 Plasma surface alloying processes, copper content in the surfaces and the depth of the alloyed layers. Sample code
Gas pressure (Pa)
Source voltage (V)
Substrate voltage (V)
Temperature (◦ C)
Process time (h)
Copper content (wt.%)
Alloyed layer depth (m)
Al A2 A3
30 45 60
−500 to −700 −500 to −700 −500 to −700
−300 to −500 −300 to −500 −300 to −500
950 950 950
3 3 3
1.2 2.5 7.1
2.5 6.5 2.5
X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
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Fig. 1. The alloy elements distribution of the Cu-alloyed layer on different gas pressure: (a) A1, (b) A2 and (c) A3.
with the transport mean free path (MFP) of gaseous particles. The gas pressure increased, the MFP of gaseous particles decreased. The sputtered particles are constrained adjacent to the sputtering target because of frequent collisions with other particles due to small MFP and part of them may even redeposit back onto the target [11]. As a result, gas pressure can affect the formation of the copper alloyed layer and the copper content. Typical scanning electron surface morphologies of the treated stainless steel at gas pressure of 30, 45 and 60 Pa, are shown in Fig. 2a–c. It can be seen that the surface demonstrates nodular units’ morphology composed of many crystal grains and appears as a fine, dense and compact structure [12]. However, the unit cell dimension is not very uniform in relation to its nucleating and growing up procedures probably. It is evident that the surface becomes rougher with a increase of gas pressure. This is because the sputtering of the substrate increased as the gas pressure increased.
completely covered with bacteria colonies. The results indicate that the Ni-modified stainless steel do not exhibit antimicrobial activity. The results of antimicrobial test of A1, A2 and A3 are shown in Fig. 5 and Table 2. It can be seen that the antibacterial rate gradually rises with increase of the contact time. The antibacterial rates of all samples reach over 98% after 12 h. It is also observed that the bactericidal ability increases with copper content effectively. In our present study, A3 with copper content about 7.1% in the surface has an excellent antibacterial ability. The viable bacteria colonies were rapidly reduced after only 1 h and 99.9% of the bacteria were killed after 3 h. Although the rate of reduction for E. coli of A2 was slower than that of A3 over the first 3 h, few were able to survive more than 12 h and the reduction reached over 99.9%. In contrast with A2 and A3, A1 exhibits a relatively weak antibacterial ability. The experimental results indicated that the plasma surface alloyed austenitic stainless steel with Cu–Ni alloyed target showed excellent antibacterial properties; and the comparison results between control (untreated and Ni-modified stainless steel) and Cu-alloyed
3.2. Antibacterial test E. coli is used as the testing bacterial in this paper. The samples with E. coli were incubated at 37 ◦ C for a designed contact time. The antibacterial rate was measured by the plain-plate dilute method. The untreated stainless steel and Nimodified stainless steel serve as control for comparison. The microstructures of Ni-modified stainless steel are shown in Fig. 3. Fig. 4 shows the results of antimicrobial test of the untreated stainless steel and Ni-modified stainless steel when in contact with E. coli for 12 h. The two Petri dishes were almost
Table 2 Calculated bacterial reduction rate of viable bacterial on the surfaces of Cu-alloyed stainless steel. Sample code
Al A2 A3
Bacterial reduction rate lh
3h
12 h
20% 25% 98%
51% 80% 99.9%
85% 99.9% 100%
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Fig. 2. Surface morphologies of the Cu-alloyed samples: (a) A1, (b) A2, and (c) A3.
stainless steel suggest that copper within the alloyed layer plays a key role in killing the bacteria. Copper has long been known to exhibit a strong toxicity to a wide range of micro-organisms; for this reason Cu-based compounds have been used extensively in many bactericidal applications [13]. The mechanism of copper toxicity to bacterial remains to be fully understood, but has been reported to be associated with its interaction with the thiol groups of bacterial
proteins and enzymes. Li et al. proposed that the dissolved copper ions lead to the collapse of some lipopolysaccharide patches of the cell surface, and consequently alters the permeability and functionality of the outer cell membrane [14]. Ruparelia et al. that copper ions released subsequently may bind with DNA molecules and lead to disorder of the helical structure by cross-linking within and between the nucleic acid strands [15].
Fig. 3. (a) Elements distribution and (b) X-ray diffraction patterns of Ni-modified stainless steel.
X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
Fig. 4. Photos of colony forming units of viable E. coli after contact for 12 h of untreated and Ni-modified stainless steel.
X-ray photoelectron spectroscopy (XPS) was utilized to investigate the chemical state of copper on the surface of A2 before and after action between the Cu-alloyed samples and bacteria. Fig. 6 shows a high resolution spectrum of Cu 2p 3/2 for A2 before and after contact with the bacteria. For the Cu-alloyed samples before contacting the bacteria, only a binding energy component observed close to 932.3 eV is related to the presence of Cu0 . However, after contacting the bacteria, a binding energy component at 932.3 and 933.3 eV is appreciable for the treated stainless steel, which might be attributed to the presence of Cu0 and Cu2+ . The XPS results indicated that the copper ions were released when the Cu-alloyed stainless steel in contact with bacterial solution. According to the mechanism of bactericidal action of copper
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ions discussed above, the bactericidal effect of the Cu-alloyed stainless steel is caused by the release of Cu2+ when alloyed surfaces are contacted with the bacterial solution. The phenomenon that the antibacterial rate gradually raises with increase of the contact time can be explained by the release amount of copper ions. The copper ions release amount from surface of the treated stainless steel is relatively low at the early time and the antibacterial capability through the copper ions does not behave enough, only exhibiting some antibacterial activity by inhibiting the bacterial reproduction. With increase of the time, the action between the copper ions and the bacteria is enhanced due to the increase of copper ions leaking and the antibacterial rate is greatly enhanced. On the other hand, higher copper concentrations may be more enhanced copper release and more striking biological activity. This is why A3 with the highest copper surface concentration exhibits the best antibacterial ability, and A1 exhibits relatively weak antibacterial properties. The ideal antibacterial stainless steel surfaces for application should have excellent antibacterial properties and adequate antibacterial durability. In the present results, both A2 and A3 exhibit good antibacterial property against E. coli. However, the thickness of Cu-containing layer of A2 is reach to 6.5 m while the thickness of A3 is only 2.5 m. On the other hand, according to Hong and Koo [16], the pitting potential of the 304 after aging declines as the copper content increase and when the copper content exceeds 3.5 wt.%, the pitting potential declines more obviously. The maximum copper concentration in the surface layer of A3 is 10 wt.%, which probably makes the pitting potential decline markedly. Based on the comprehensive characterization of microstructure and compositions and antibacterial tests, it can be concluded around 45 Pa is the optimized gas pressure for generating an antibacterial surface by plasma surface alloying technique.
Fig. 5. Photos of colony forming units of viable E. coli after contact for 1–12 h with the surfaces of Cu-alloyed stainless steel.
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Fig. 6. XPS spectra of Cu2p 3/2 for A2 before (a) and after (b) contacting the bacterial.
4. Conclusions
References
The antibacterial stainless steel surfaces with various copper contents have been prepared by a plasma surface alloying technique at various gas pressures in this study. The antibacterial test results indicate that the antibacterial rate of the Cu-alloyed surfaces gradually increased to the maximum with increasing time of contacting the bacterial and the higher the amount of surface copper, the better is the antibacterial efficacy. The antibacterial mechanism of the alloyed surfaces is that Cu-ions could be dissolved from the Cu-alloyed surface of the stainless steel, killing the bacteria on the surface of steel. For alloyed surface obtained at the gas pressure of 45 Pa, a combination of sufficient thickness and good antibacterial properties are observed, which make them to be promising candidates applied on the surface of stainless steel in practical antimicrobial applications.
[1] V.B. Preethi Sudha, K. Ojit Singh, S.R. Prasad, P. Venkatasubramanian, Trans. R. Soc. Trop. Med. Health 103 (8) (2009) 819–822. [2] J.O. Noyce, H. Michels, C.W. Keevil, J. Hosp. Infect. 63 (3) (2006) 289–297. [3] W. Zhang, Y.H. Zhang, J.H. Ji, J. Zhao, Q. Yan, P.K. Chu, Polymer 47 (21) (2006) 7441–7445. [4] A. Daniel, C. Le Pen, C. Archambeau, F. Reniers, Appl. Surf. Sci. 256 (3) (2009) S82–S85. [5] Y.Z. Wan, S. Raman, F. He, Y. Huang, Vacuum 81 (9) (2007) 1114–1118. [6] X.B. Tian, Z.M. Wang, S.Q. Yang, Z.J. Luo, K.Y. Ricky, Fu Paul, K. Chu, Surf. Coat. Technol. 201 (19–20) (2007) 8606–8609. [7] Z.G. Dan, H.W. Ni, B.F. Xu, J. Xiong, P.Y. Xiong, Thin Solid Films 492 (1–2) (2005) 93–100. [8] L.M. Chen, L. Zheng, Y.H. Lv, H. Liu, G.C. Wang, N. Ren, D. Liu, J.Y. Wang, R.I. Boughton, Surf. Coat. Technol. 204 (23) (2010) 3871–3875. [9] Z. Xu, X. Liu, P. Zhang, Y. Zhang, G. Zhang, Z. He, Surf. Coat. Technol. 201 (9–11) (2007) 4822–4825. [10] W. Cong, Z.J. Yao, X.L. Zhu, Wear 268 (5–6) (2010) 790–796. [11] Z.Y. He, Z.X. Wang, W.B. Wang, A.L. Fan, Z. Xu, Surf. Coat. Technol. 201 (9–11) (2007) 5705–5709. [12] X.Y. Kui, J.Z. Wang, Y.P. Liu, J.Y. Xu, Y. Gao, Z. Xu, Surf. Coat. Technol. 201 (9–11) (2007) 5302–5305. [13] S. Mehtar, I. Wiid, S.D. Todorov, J. Hosp. Infect. 68 (1) (2008) 45–51. [14] N. Li, Y.Q. Liu, M.Q. Lv, K. Yang, J. Mater. Sci.: Mater. Med. 19 (9) (2008) 3057–3062. [15] J.P. Ruparelia, A.K. Chatterjee, S.P. Duttagupta, S. Mukherji, Acta Biomater. 4 (3) (2008) 707–716. [16] I.T. Hong, C.H. Koo, Mater. Sci. Eng. A 393 (1–2) (2005) 213–222.
Acknowledgements This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IR0972), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi, Graduate Innovation Project of Shanxi Province (No. 20103024).
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Surface microstructures and antimicrobial properties of copper plasma alloyed stainless steel Xiangyu Zhang, Xiaobo Huang, Li Jiang, Yong Ma, Ailan Fan, Bin Tang ∗ Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024, China
a r t i c l e
i n f o
Article history: Received 22 May 2011 Received in revised form 19 September 2011 Accepted 20 September 2011 Available online 28 September 2011 Keywords: Stainless steel Plasma alloying Copper content Antibacterial property
a b s t r a c t Bacterial adhesion to stainless steel surfaces is one of the major reason causing the cross-contamination and infection in many practical applications. An approach to solve this problem is to enhance the antibacterial properties on the surface of stainless steel. In this paper, novel antibacterial stainless steel surfaces with different copper content have been prepared by a plasma surface alloying technique at various gas pressures. The microstructure of the alloyed surfaces was investigated using glow discharge optical emission spectroscopy (GDOES) and scanning electron microscopy (SEM). The viability of bacteria attached to the antibacterial surfaces was tested using the spread plate method. The antibacterial mechanism of the alloyed surfaces was studied by X-ray photoelectron spectroscopy (XPS). The results indicate that gas pressure has a great influence on the surface elements concentration and the depth of the alloyed layer. The maximum copper concentration in the alloyed surface obtained at the gas pressure of 60 Pa is about 7.1 wt.%. This alloyed surface exhibited very strong antibacterial ability, and an effective reduction of 98% of Escherichia coli (E. coli) within 1 h was achieved by contact with the alloyed surface. The maximum thickness of the copper alloyed layer obtained at 45 Pa is about 6.5 m. Although the rate of reduction for E. coli of this alloyed surface was slower than that of the alloyed surface with the copper content about 7.1 wt.% over the first 3 h, few were able to survive more than 12 h and the reduction reached over 99.9%. The XPS analysis results indicated that the copper ions were released when the copper alloyed stainless steel in contact with bacterial solution, which is an important factor for killing bacteria. Based on an overall consideration of bacterial killing rate and durability, the alloyed surface with the copper content of 2.5 wt.% and the thickness of about 6.5 m obtained at the gas pressure of 45 Pa is expected to be useful as antimicrobial materials that may have a promising future in antimicrobial applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction With the development of science technology and growth of living standard, public awareness on the sanitation and health of environments has been rapidly raised. However, microbial contamination is still one of the major threats to public health [1,2]. Bacterial have a strong ability to attach to solid surfaces, such as industrial equipment, food contact surfaces and work surfaces in hospital environment. Once bacterial attach to these surfaces, a multistep process starts leading to the formation of bacterial biofilm which is very difficult to treat clinically. This increases the risk of cross-contamination and infection. Although various methods can be adopted to control bacterial development on surfaces, one effective approach is to prevent bacterial adherence to the surfaces and avoid bacterial biofilms formation by means of surface
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (B. Tang). 0169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2011.09.091
engineering techniques to introduce antibacterial agents, such as copper (Cu), silver (Ag) into material surfaces [3–5]. It is well known that stainless steel is one of the most commonly used materials in daily life. Stainless steel surfaces with added antibacterial properties would get popular and be used in hospitals, food industries and kitchen appliances. Previous studies on preparing antibacterial stainless steel surfaces mainly by means of coating, ion implantation and chemical synthesis process [6–8]. However, it is difficult to obtain enough thick antibacterial modified layers to maintain the long-lasting antibacterial effect. The thicknesses of the antibacterial modified layers prepared by these methods are mostly less than 1 m. On the other hand, it is difficult to maintain antibacterial effect simultaneously with good wear and corrosion resistance. Therefore, an efficient and long-term antimicrobial method to prevent bacterial adherence on stainless steel surface is needed. Double glow plasma surface alloying technology [9,10] has been successfully used to produce surface alloys to improve wear resistance, corrosion resistance and anti-oxidation performance of
1400
X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
materials such as on steel, iron, titanium alloy and intermetallic compound. This technology can probably be used to introduce the desired alloying elements sputtered from the source electrode by bombardment of the ion produced in the second glow discharge into the surface of the work piece to form a surface alloy. It can probably be used to introduce copper into stainless steel surface to form an antibacterial alloyed layer. However, if use pure copper as a source electrode directly, a copper deposited layer would be produced on stainless steel surface since copper have a limited solid solubility in stainless steel. Considering that nickel is one of the major elements in AISI304 stainless steel and easily diffuses into stainless steel, a Ni–Cu alloy was selected as source target in this paper to prepare a high quality copper alloyed layer on stainless steel surface. The objective of this work is to study the influence of the gas pressure on the surface microstructures and antimicrobial properties of copper plasma alloyed stainless steel. The antibacterial mechanism of the copper containing stainless steel surfaces is also discussed in this paper.
The antibacterial activity of the samples was determined using Gram-negative Escherichia coli (E. coli) ATCC10536. The plain-plate dilute method was used to measure the number of live bacteria and to calculate the antibacterial rate of each specimen in this study. The broth for culturing the bacteria was made by dissolving 5.0 g flesh extract, 5.0 g NaCl and 10.0 g peptone into 1000 ml of distilled water with the pH adjusted to 7.0–7.2. All samples were sterilized by autoclave for 20 min. 0.4 ml of solution of bacteria with a concentration of 105 CFU/ml was dripped onto the surfaces of the samples, which had been horizontally placed into a sterilized Petri-dish. In order to monitor the relation between percentage of cells killed and contact time (reduction rate), the bacteria on the sample surface were incubated for 1 h, 3 h, 12 h at a temperature of 37 ◦ C. After the incubation, the bacteria solution on the sample surface was diluted with 0.6 ml of 0.85 wt pct NaCl solution, and 0.1 ml of the bacterial solution was added into a Petri-dish. The numbers of viable bacteria colonies were enumerated after incubation for 18 h at 37 ◦ C. The percentage reduction was calculated according to the formula Reduction % =
2. Experimental A Ni–Cu alloy (the composition is Ni60, Cu40 in weight percent) plate (70 mm × 60 mm × 5 mm) was used as the source target for supplying the alloying elements. AISI304 stainless steel with a size of 20 × 5 mm was used as the substrate material. The samples were abraded with SiC emery paper and polished to smooth surfaces (Ra < 0.01 m) with alumina paste and then cleaned in acetone. The plasma surface alloying was performed in a double glow plasma alloying furnace. In this furnace, there are three electrodes: an anode, a negative cathode (sample) and a sputtering source cathode (Ni–Cu alloy). Glow discharge was initiated created on the samples to sputter clean the surface, then the discharge on the anode and source cathode was also created. Copper atoms and nickel atoms were sputtered out from the source electrode by bombardment of the ion, and they travel to and diffuse into the surface of the work piece which was heated to a high temperature at the meantime to form a surface alloy. Gas pressure is one of the most important parameters for the double glow plasma alloying technology. In this paper, to discuss the effect of gas pressure on the surface composition and depth of the modified layer, a series of pressure were set as 30, 45 and 60 Pa, respectively. Other plasma surface alloying parameters with copper were conducted under the following conditions: source electrode voltage −500 to −700 V, substrate voltage −300 to −500 V, working gas Ar, temperature 950 ◦ C and treatment time 3 h. The copper alloyed samples obtained at 30, 45 and 60 Pa are indicated by the symbols A1, A2 and A3, respectively. The detailed process parameters are listed in Table 1. Major element percentage and depth profiling were observed using a Spectro GDA750 glow discharge optical emission spectroscopy (GDOES) spectrometer. The surface microstructures were investigated using LEO-1450 type scanning electron microscopy (SEM). The phases were identified by Rigaku DyMax2500 X-ray diffraction spectrometer. The chemical state of copper in the modified layer surface was investigated by VG ESCALAB Mark II X-ray photoelectron spectroscopy (XPS) with Al K␣ X-ray as the radiation source.
( − ) t 0 0
× 100%,
where t is the number of viable bacteria for the treated sample after a designated contact time and 0 is the number of viable bacteria for the untreated sample after incubated 12 h. 3. Results and discussion 3.1. Macrostructures Gradient alloying layer are formed on the samples after plasma surface alloying treatment. The content distribution of alloying elements in the surface alloying layer formed on stainless steel obtained from the different gas pressure are shown in Fig. 1. As shown, there are large differences in the surface elements concentration and the depth of the alloyed layer. The copper concentration increases continuously from about 1.2% to about 7.1% with increasing of gas pressure from 30 Pa to 60 Pa. However, the depth of Cu-alloyed layer is not increased constantly with the gas pressure gone up. The maximum thickness of the Cu-alloyed layer obtained at 45 Pa is about 6.5 m. Results indicated that the gas pressure has a great influence on the surface elements concentration and the depth of the alloyed layer. The detailed copper content of A1, A2 and A3 in the surfaces and the depth of the alloyed layers are listed in Table 1. The plasma has two basic functions in double glow plasma surface alloying process. Firstly, it makes the copper and nickel element sputtered from the source electrode, as well as stimulates and activates the carriage gas which carrying the alloying elements. Secondly, the surface of the stainless steel was sputtered by bombardment of the plasma, which affects the depth and the surface concentration of the alloyed layer directly. In the vacuum chamber, working pressure corresponds to the plasma density when other parameters are fixed. Thus, the plasma density will increase with the increase of gas pressure in the double glow process, which probably make the sputtered loss of the source electrode increase and improve the deposit velocity of the alloying elements on the surface of the substrate. On the other hand, pressure has an inverse relation
Table 1 Plasma surface alloying processes, copper content in the surfaces and the depth of the alloyed layers. Sample code
Gas pressure (Pa)
Source voltage (V)
Substrate voltage (V)
Temperature (◦ C)
Process time (h)
Copper content (wt.%)
Alloyed layer depth (m)
Al A2 A3
30 45 60
−500 to −700 −500 to −700 −500 to −700
−300 to −500 −300 to −500 −300 to −500
950 950 950
3 3 3
1.2 2.5 7.1
2.5 6.5 2.5
X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
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Fig. 1. The alloy elements distribution of the Cu-alloyed layer on different gas pressure: (a) A1, (b) A2 and (c) A3.
with the transport mean free path (MFP) of gaseous particles. The gas pressure increased, the MFP of gaseous particles decreased. The sputtered particles are constrained adjacent to the sputtering target because of frequent collisions with other particles due to small MFP and part of them may even redeposit back onto the target [11]. As a result, gas pressure can affect the formation of the copper alloyed layer and the copper content. Typical scanning electron surface morphologies of the treated stainless steel at gas pressure of 30, 45 and 60 Pa, are shown in Fig. 2a–c. It can be seen that the surface demonstrates nodular units’ morphology composed of many crystal grains and appears as a fine, dense and compact structure [12]. However, the unit cell dimension is not very uniform in relation to its nucleating and growing up procedures probably. It is evident that the surface becomes rougher with a increase of gas pressure. This is because the sputtering of the substrate increased as the gas pressure increased.
completely covered with bacteria colonies. The results indicate that the Ni-modified stainless steel do not exhibit antimicrobial activity. The results of antimicrobial test of A1, A2 and A3 are shown in Fig. 5 and Table 2. It can be seen that the antibacterial rate gradually rises with increase of the contact time. The antibacterial rates of all samples reach over 98% after 12 h. It is also observed that the bactericidal ability increases with copper content effectively. In our present study, A3 with copper content about 7.1% in the surface has an excellent antibacterial ability. The viable bacteria colonies were rapidly reduced after only 1 h and 99.9% of the bacteria were killed after 3 h. Although the rate of reduction for E. coli of A2 was slower than that of A3 over the first 3 h, few were able to survive more than 12 h and the reduction reached over 99.9%. In contrast with A2 and A3, A1 exhibits a relatively weak antibacterial ability. The experimental results indicated that the plasma surface alloyed austenitic stainless steel with Cu–Ni alloyed target showed excellent antibacterial properties; and the comparison results between control (untreated and Ni-modified stainless steel) and Cu-alloyed
3.2. Antibacterial test E. coli is used as the testing bacterial in this paper. The samples with E. coli were incubated at 37 ◦ C for a designed contact time. The antibacterial rate was measured by the plain-plate dilute method. The untreated stainless steel and Nimodified stainless steel serve as control for comparison. The microstructures of Ni-modified stainless steel are shown in Fig. 3. Fig. 4 shows the results of antimicrobial test of the untreated stainless steel and Ni-modified stainless steel when in contact with E. coli for 12 h. The two Petri dishes were almost
Table 2 Calculated bacterial reduction rate of viable bacterial on the surfaces of Cu-alloyed stainless steel. Sample code
Al A2 A3
Bacterial reduction rate lh
3h
12 h
20% 25% 98%
51% 80% 99.9%
85% 99.9% 100%
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Fig. 2. Surface morphologies of the Cu-alloyed samples: (a) A1, (b) A2, and (c) A3.
stainless steel suggest that copper within the alloyed layer plays a key role in killing the bacteria. Copper has long been known to exhibit a strong toxicity to a wide range of micro-organisms; for this reason Cu-based compounds have been used extensively in many bactericidal applications [13]. The mechanism of copper toxicity to bacterial remains to be fully understood, but has been reported to be associated with its interaction with the thiol groups of bacterial
proteins and enzymes. Li et al. proposed that the dissolved copper ions lead to the collapse of some lipopolysaccharide patches of the cell surface, and consequently alters the permeability and functionality of the outer cell membrane [14]. Ruparelia et al. that copper ions released subsequently may bind with DNA molecules and lead to disorder of the helical structure by cross-linking within and between the nucleic acid strands [15].
Fig. 3. (a) Elements distribution and (b) X-ray diffraction patterns of Ni-modified stainless steel.
X. Zhang et al. / Applied Surface Science 258 (2011) 1399–1404
Fig. 4. Photos of colony forming units of viable E. coli after contact for 12 h of untreated and Ni-modified stainless steel.
X-ray photoelectron spectroscopy (XPS) was utilized to investigate the chemical state of copper on the surface of A2 before and after action between the Cu-alloyed samples and bacteria. Fig. 6 shows a high resolution spectrum of Cu 2p 3/2 for A2 before and after contact with the bacteria. For the Cu-alloyed samples before contacting the bacteria, only a binding energy component observed close to 932.3 eV is related to the presence of Cu0 . However, after contacting the bacteria, a binding energy component at 932.3 and 933.3 eV is appreciable for the treated stainless steel, which might be attributed to the presence of Cu0 and Cu2+ . The XPS results indicated that the copper ions were released when the Cu-alloyed stainless steel in contact with bacterial solution. According to the mechanism of bactericidal action of copper
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ions discussed above, the bactericidal effect of the Cu-alloyed stainless steel is caused by the release of Cu2+ when alloyed surfaces are contacted with the bacterial solution. The phenomenon that the antibacterial rate gradually raises with increase of the contact time can be explained by the release amount of copper ions. The copper ions release amount from surface of the treated stainless steel is relatively low at the early time and the antibacterial capability through the copper ions does not behave enough, only exhibiting some antibacterial activity by inhibiting the bacterial reproduction. With increase of the time, the action between the copper ions and the bacteria is enhanced due to the increase of copper ions leaking and the antibacterial rate is greatly enhanced. On the other hand, higher copper concentrations may be more enhanced copper release and more striking biological activity. This is why A3 with the highest copper surface concentration exhibits the best antibacterial ability, and A1 exhibits relatively weak antibacterial properties. The ideal antibacterial stainless steel surfaces for application should have excellent antibacterial properties and adequate antibacterial durability. In the present results, both A2 and A3 exhibit good antibacterial property against E. coli. However, the thickness of Cu-containing layer of A2 is reach to 6.5 m while the thickness of A3 is only 2.5 m. On the other hand, according to Hong and Koo [16], the pitting potential of the 304 after aging declines as the copper content increase and when the copper content exceeds 3.5 wt.%, the pitting potential declines more obviously. The maximum copper concentration in the surface layer of A3 is 10 wt.%, which probably makes the pitting potential decline markedly. Based on the comprehensive characterization of microstructure and compositions and antibacterial tests, it can be concluded around 45 Pa is the optimized gas pressure for generating an antibacterial surface by plasma surface alloying technique.
Fig. 5. Photos of colony forming units of viable E. coli after contact for 1–12 h with the surfaces of Cu-alloyed stainless steel.
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Fig. 6. XPS spectra of Cu2p 3/2 for A2 before (a) and after (b) contacting the bacterial.
4. Conclusions
References
The antibacterial stainless steel surfaces with various copper contents have been prepared by a plasma surface alloying technique at various gas pressures in this study. The antibacterial test results indicate that the antibacterial rate of the Cu-alloyed surfaces gradually increased to the maximum with increasing time of contacting the bacterial and the higher the amount of surface copper, the better is the antibacterial efficacy. The antibacterial mechanism of the alloyed surfaces is that Cu-ions could be dissolved from the Cu-alloyed surface of the stainless steel, killing the bacteria on the surface of steel. For alloyed surface obtained at the gas pressure of 45 Pa, a combination of sufficient thickness and good antibacterial properties are observed, which make them to be promising candidates applied on the surface of stainless steel in practical antimicrobial applications.
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Acknowledgements This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IR0972), the Program for the Top Young and Middle-aged Innovative Talents of Higher Learning Institutions of Shanxi, Graduate Innovation Project of Shanxi Province (No. 20103024).