Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316L stainless steel

Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316L stainless steel

Accepted Manuscript Title: Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316 L stainless steel Auth...

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Accepted Manuscript Title: Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316 L stainless steel Author: Jinlong Zhao Dake Xu M.Babar Shahzad Qiang Kang Ying Sun Ziqing Sun Shuyuan Zhang Ling Ren Chunguang Yang Ke Yang PII: DOI: Reference:

S0169-4332(16)31263-6 http://dx.doi.org/doi:10.1016/j.apsusc.2016.06.036 APSUSC 33406

To appear in:

APSUSC

Received date: Revised date: Accepted date:

14-3-2016 22-5-2016 7-6-2016

Please cite this article as: Jinlong Zhao, Dake Xu, M.Babar Shahzad, Qiang Kang, Ying Sun, Ziqing Sun, Shuyuan Zhang, Ling Ren, Chunguang Yang, Ke Yang, Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316L stainless steel, Applied Surface Science http://dx.doi.org/10.1016/j.apsusc.2016.06.036 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of surface passivation on corrosion resistance and antibacterial properties of Cu-bearing 316L stainless steel Jinlong Zhao1†, Dake Xu1†, M. Babar Shahzad1, Qiang Kang1, Ying Sun1, Ziqing Sun1, Shuyuan Zhang1, Ling Ren1, Chunguang Yang1*, Ke Yang1

1

Institute of Metal Research, Chinese Academy of Sciences, Shenyang, 110016, China

†Jinlong Zhao and Dake Xu equally contributed to this work. *

Corresponding author

E-mail: [email protected]

Highlights 

A new passivation solution that adding proper concentration of Cu2+ ions into nitric acid solution was proposed.



The new passivation method can simultaneously guarantee the better corrosion resistance and stable antibacterial property for 316L-Cu SS.



The results of XPS analysis and Cu2+ ions release explained the antibacterial mechanism.



The 316L-Cu SS after antibacterial passivation determined by RTCA assay was completely biocompatible.

Abstract The resistance for pitting corrosion, passive film stability and antibacterial performance of 316L-Cu SS passivated by nitric acid solution containing certain concentration of copper sulfate, were studied by electrochemical cyclic polarization, electrochemical impedance spectroscopy (EIS) and co-culture with bacteria. Inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the Cu2+ ions release from 316L-Cu SS surface. XPS analysis proved that the enrichment of CuO, Cr2O3 and Cr(OH)3 on the surface of specimen could simultaneously guarantee a better corrosion resistance and stable antibacterial properties. The biocompatibility evaluation determined by RTCA assay also indicated that the 316L-Cu SS after antibacterial passivation was completely biocompatible.

Keywords:

passivation;

biocompatibility

corrosion

resistance;

antibacterial

performance;

1. Introduction In many fields, such as dentistry, orthopedics, and other health related industries, stainless steel (SS) is widely applied as a kind of high performance biomaterial for nearly 100 years. Its versatile properties render it as a promising material for various biomedical applications, but unfortunately, it also owns a major drawback in the form of easy bacterial survival and biofilm formation on the surface of SS [1-5]. The bacterial biofilm can speed up the corrosion and eventually lead to severe fracture and bacterial infection. Serious postoperative complications in medical implants and surgical instruments have been reported by many researchers [6-9]. For example, type 316L austenitic SS is widely used as implant material because of its better balance of mechanical strength, corrosion resistance and biocompatibility [10, 11]. However, SS is susceptible to the microbiologically influenced corrosion (MIC) [12-14], which can lead to an accelerated corrosion process in the human body, leading to further infection in the health of infected host [15]. According to the literature, Cu-bearing ions solution used as an antimicrobial agent has a long history of more than 200 years [16, 17]. The antibacterial properties with Cu-bearing SS have also been widely explored. The Cu-bearing antibacterial SS was developed by adding certain amount of Cu into conventional SS during metallurgical process, followed by a subsequent heat treatment, to form nano-sized Cu-rich precipitate in the steel matrix. When the surface of antibacterial SS comes in contact with solution, Cu2+ ions can be easily released into the environment to participate in the process of killing bacteria. Based on the above principle, the Cu-bearing SS endowed the materials broad-spectrum antibacterial performance

[18-21]. Zhang et al. have proved that the Cu-bearing stainless steel (SS) showed excellent antibacterial performance against gram-positive Staphylococcus aureus and garm-negative Escherichia coli [22]. However, Cu-bearing stainless steel is more prone to the pitting corrosion which may induce the release of more heavy metal ions including Cr3+, Cr6+, Ni2+ and Cu2+ to the environment, and increases the risk to cause health problem [23]. Oguzie et al. [24] also found that Cu in SS had negative effect on the corrosion resistance in the deaerated sulfuric acid solution, and further explained that addition of Cu in SS could destroy the continuity of passivation film, due to the potential difference between Cu and Fe, which is a easy way to galvanic corrosion. So as a future implant material, to prevent possible cytotoxicity due to release of metal ions during the corrosion process, it is necessary to improve the corrosion resistance of 316L-Cu SS. In this regard, the surface passivation technique is considered as an effective method to improve the corrosion resistance of the stainless steel. Most studies [25-27] showed that surface passivation can effectively improve the corrosion resistance of medical SS, and surface passivation is often used as the last treatment of implant devices before implantation into bodies. The nitric acid passivation is a traditional passivation method for SS. The main role of nitric acid is to remove the inclusions from the alloy surface and promote the growth of the dense oxide film, its thickness is usually few times thicker than that of natural passivation film [28]. However, the antibacterial performance of Cu-bearing SS after surface passivation treatment maybe diminished or inhibited. The uniform and continuous passive film may hinder the release of Cu2+ ions from the surface of SS through

passivation film, so it seems to be very difficult to balance the contradiction between the antibacterial performance and corrosion resistance. Therefore, it is significant to explore a new passivation method which can simultaneously satisfy the antibacterial performance, corrosion resistance and quite importantly, should ensure the biocompatibility of SS after surface passivation. In the aforementioned context, incorporating the appropriate content of Cu-rich ions solution into traditional passivation solution during the process of surface passivation of 316L-Cu SS, the structure of passivation film can contain both antibacterial Cu element and Cr-rich oxide. In other words, when passivated 316L-Cu SS is soaked in the simulated body fluid, the antibacterial Cu2+ ions in passivation film can dissolve into the solution to exert the antibacterial function, meanwhile, Cu-bearing passivation film can also provide the corrosion resistance due to the presence of Cr-rich oxide. Under this hypothesis, the aim of the present study was to investigate the influence of nitric acid passivation solution containing certain amount of Cu ions on both corrosion resistance and antibacterial performance of 316L-Cu SS. The concentration of Cu2+ ions released from the surface of 316L-Cu SS before and after passivation in a 0.9 wt. % NaCl solution was compared by using ICP-MS measurement to understand the related mechanism of passivation on corrosion resistance and antibacterial performance, and X-ray photoelectron spectroscopy (XPS) technique was used to characterize the structure of passivation film formed on the surface of 316L-Cu SS. The cytotoxicity test was also employed to assess biocompatibility of the 316L-Cu SS after the antibacterial passivation treatment [29].

2. Experimental 2.1 Materials and methods The chemical compositions of the 316L SS and Cu-bearing 316L austenitic stainless steel (316L-Cu SS) are listed in Table 1. The steels were melted by a 25 kg vacuum induction furnace. The casting ingots were hot forged into slabs with a thickness of 10mm after reheated to 1100 ºC for 30 minutes (min). The antibacterial heat treatment was conducted at 700 ºC for 6 hours (h) to precipitate enough Cu-rich phases before quenching, which was the main reason to realize antibacterial performance, and then 316L-Cu SS was cold rolled to final thickness of 4 mm. The 316L SS was served as a control for comparison. The samples with size of Φ10 × 4 mm were used for experiment. The passivation solution containing 30 wt. % nitric acid with two concentration gradient of anhydrous cupric sulfate, including Cu2+ ions concentration of 20 ppb and 10 ppm, were chosen to passivate the 316L-Cu SS samples surface at 60 ºC for 1 h. Then samples were immersed in solution of 1 wt. % NaOH for 1-2 min, followed by rinsing with distilled water for 1 min, finally all the samples were dried in a stream of warm air and immediately stored in a desiccator for later characterization.

Table 1 Chemical composition (wt. %) of the experimental SS SS

Cr

Ni

Mo

Mn

C

Si

P

S

Cu

Fe

316L 316L-Cu

17.85 18.31

13.9 14.1

2.7 2.75

0.68 0.71

0.02 0.014

0.62 0.61

0.007 0.008

0.001 0.001

4.46

Balance Balance

2.2 Electrochemical measurements Open circuit potential (OCP), electrochemical cyclic polarization and electrochemical impedance spectroscopy (EIS) were performed on Autolab potentiostat/galvanostat (Reference 600TM, Gamry Instruments, Inc., USA) to study the corrosion resistance of 316L-Cu SS before and after passivation treatment. The electrochemical measurements were made using a three-electrode cell while samples were immersed in 0.9 wt. % NaCl solution at 37 ± 2 ºC. A saturated calomel electrode (SCE) and platinum sheet were used as the reference and counter electrode, respectively. The samples of 316L SS and 316L-Cu SS before and after passivation, embedded in epoxy resin with an exposure area of 0.785 cm2, acted as the working electrodes. Before the experiment, the sample surface was mechanically ground up to 1200 grit, rinsed with ethanol and dried in warm air. Before EIS test, OCP was measured in order to keep chemical stability of samples in the 0.9 wt. % NaCl solution, then EIS was performed under a sinusoidal ac perturbation of 5 mV vs OCP applied to the electrode in a frequency range of 0.01 to 100,000 Hz. Electrochemical cyclic polarization was conducted at a scan rate of 0.5 mV/s after 1 h of OCP test, from the corrosion potential (Ecorr) to 1100 mV (vs. SCE), and then scan was reversed to the starting potential when the current density reached a value of 10-4A·cm-2. Corrosion current density (icorr) was generally obtained by the extrapolation of the

cathodic and anodic slopes between 50 and 100 mV away from Ecorr. Passive film breakdown potential (Eb) and protective potential (Ep) were obtained from the cyclic polarization curves, where Eb is the potential when the stable pitting formed. The irreversible process led to a rapid increase of current density while the intersection of scanning curve in negative direction and anode polarization curve provided the Ep. Each sample measurement was repeated for three times in order to ensure the reproducibility of the tests. 2.3 Antibacterial test Plate count measurements were applied to assess the antibacterial performance after antibacterial passivation [30]. Gram-negative E.coli ATCC 25922 was assumed as the main pathogenic microbe responsible for implant-induced infection in human body. The Luria-Bertani (LB) medium was made by dissolving 5.0 g beef extract, 10.0 g peptone, 5.0 g NaCl and 20.0 g agar into 1000 mL of distilled water with pH value of 7.2 ± 0.1 [31]. Before the antibacterial test, all the experiment instruments were sterilized by autoclaving at 121 ± 2 ºC for 20 min, and the samples were ultrasonically cleaned in the ethanol for 20 min. 50 μL bacterial suspension with a concentration of 105 CFU/mL was dripped on the surfaces of the sterile samples, and then incubated in an incubator at 37 ±2 ºC for 24, 72 and 120 h under a humidity of 95%, respectively. After the incubation, 100 μL of bacterial suspension after washed off the surfaces of samples was first diluted 40 times, and then spread on the total area of Petri dish filled with solid culture medium, finally the Petri dishes were incubated at 37 ± 2 ºC for 24 h under a humidity of 95% before counting the bacterial colonies.

The antibacterial rate was calculated according to the formula (1): K%=

(1)

where K stands for the antibacterial rate, α0 is the number of bacterial colonies for the 316L SS, and correspondingly, αt is the number of bacterial colonies before and after passivation for 316L-Cu SS. Besides, three parallel samples were prepared for each test. 2.4 ICP-MS analysis Passivation solution containing 10 ppm Cu2+ ions was used to analyze the released content and rate of Cu2+ ions for 316L-Cu SS before and after passivation. The samples were placed into 6 centrifuge tubes, where each tube contained 6 samples. Then all the samples were placed in an incubator at 37 ± 2 ºC while immersed in 0.9 wt. % NaCl solution for 24, 72 and 120 h, respectively. At each time point, the samples were removed from the corresponding tubes and the solutions in the tubes were stored in a fridge at 4 ºC. After achieving soaking time of 120 h, all the extracted solutions and a control of 0.9 wt.% NaCl solution were collected, and the content of Cu, Cr, Ni and Mn elements in the solutions were analyzed by ICP-MS (iCAP Q, Thermo, USA). The ICP-MS test for Cu2+ ions release was based on the standard test method of ASTM D6800-12 [32]. The value obtained using ICP was determined by the average value of three repeated tests. 2.5 X-ray photoelectron spectroscopy (XPS) analysis The surface structure of passivation film before and after passivation was characterized by XPS. The XPS analysis was performed using an ESCALAB250

surface analysis system (Thermo VG, USA). Samples surfaces were irradiated with AlKα radiation (1486.6 eV) in an UHV chamber (10-10 mbar). The C1s peak at 284.6 eV was used as the reference to correct the charging shifts in this study. Analyses were carried out at 90° angle between the samples surface and the analyzer. Etching speed was 0.2 nm/s. Before this measurement, all the samples of 316L-Cu SS were subjected to a passivation treatment in nitric acid solution with different Cu2+ ions concentration (20 ppb, 10 ppm). 2.6 RTCA cytotoxicity assay Real-time cell analyzer (RTCA) is a method to assess the biocompatibility of biomaterial. In this experiment, MC3T3-E1 mouse cells at the concentration of 1000 cells/ml were seeded in a 96-well electronic plate (E-Plate View 96; ACEA Biosciences Inc., San Diego, CA, USA). The xCELLigence DP system (ACEA Biosciences Inc., San Diego, CA, USA) was used to monitor cells in the E-plate at 5 min intervals. After 18 ± 1 h of RTCA profiling, the assay was paused, and the E-plate was removed from the xCELLigence DP system. The existing liquid in each well was removed, and replaced with a 0.9 wt.% NaCl solution which was exposed to 316L SS before and after passivation in nitric acid solution with different Cu2+ ions concentration (10 ppm and 20 ppb) for 24 h and 72 h, respectively. The 96-well E-plate was placed back into the xCELLigence system and cells were monitored every 5 min for 100 h. Each sample was analyzed by three duplicates. RTCA plot was generated using RTCA software (Version 2.0; ACEA Biosciences Inc., San Diego, CA, USA). The data of normalized cell index (NCI) was exported to Microsoft Excel

software for mathematical analysis. The data was normalized to a starting CI of 1.0 at the time point immediately prior to the solution switch and addition of samples. 3. Results and discussion 3.1 Corrosion resistance of 316L-Cu SS after passivation Fig. 1a shows the electrochemical cyclic polarization curves of 316L-Cu SS before and after passivation. The pitting corrosion potential (Eb), the corrosion current density (icorr) and the protective potential (Ep) were calculated by Tafel linear extrapolation method from the curves in Fig.1a, and the corresponding values are listed in Table 2. As shown in Fig.1a and Table 2, the 316L-Cu SS before and after passivation immersed in 0.9 wt.% NaCl solution shows typical active-passive-pitting of corrosion behavior, and different Cu2+ ions concentration of passivation treatment significantly increased the Eb of 316L-Cu SS. The Eb of 316L-Cu SS without passivation was 534 mV, whereas those of 316L-Cu SS after passivation in nitric acid solution containing 20 ppb and 10 ppm Cu2+ were 807 mV and 832 mV, respectively. In general, Eb is considered to be the breakdown potential, and when potential is above Eb, the passivation film is damaged to form stable corrosion pitting. So Eb is a key parameter to characterize the corrosion resistance of SS. In all tests, the passivated 316L-Cu SS in the passivation solution containing 10 ppm Cu2+ ions concentration possessed the best pitting corrosion resistance. The icorr value of passivated 316L-Cu SS in solution containing 10 ppm Cu2+ ions was slightly lower than that of the non-passivated 316L-Cu SS. The icorr generally represents the uniform corrosion rate of material, and a higher value of icorr represents a faster uniform

corrosion rate. This demonstrated that the uniform corrosion rate of 316L-Cu SS after passivation in nitric acid solution containing 10 ppm Cu2+ ions did not significantly decreased. After passivation treatment, the Ep value of the non-passivated 316L-Cu SS was 1.5 mV, whereas that of 316L-Cu SS passivated in 20 ppb and 10 ppm Cu2+ ions solutions were 160.6 mV and 29.2 mV, respectively. The Ep represented the self-repairing and self-passivated ability of passivation film after pitting corrosion occurred, and as the Ep value increased, the passivation film became less destructible and can be easily formatted. It is well known that, due to chemical stability difference of Fe and Cr ions, the transport rate of Fe3+ was eight times higher than that of Cr3+ in the passive film [33], so passive film were consisted of inner dense Cr-rich oxide and outer loose Fe-rich oxide. The results of Ep showd that passivation treatment effectively strengthened the stability of passivation film, but Cu deposition may possibly influence the compactness of passive film, precisely, Cu increased the electron transfer in passive film, decreased the compactness of passive film to some degree. With increase of Cu concentration in passive film, the flux of electron transfer correspondingly increase, which can directly weaken self-repairing ability of passive film. With the increased concentration of Cu2+ ions from 20 ppb to 10 ppm in passivation solution, the passivation film of passivated 316L-Cu SS was more and more difficult to be self-repaired, as shown in Table 2. EIS is a nondestructive technique to characterize electrochemical reactions at corrosion interface [34]. Fig.1b shows the typical Nyquist diagrams of 316L-Cu SS in

the 0.9 wt.% NaCl solution before and after passivation by nitric acid solution containing various concentration of Cu2+ ions. As shown in Fig.1b, only one time constant was observed in the Nyquist plots. The radius of the capacitive loop for passivated 316L-Cu SS kept almost same, and it can be clearly found that the radius of the capacitive loop was greater than that of the non-passivated 316L-Cu SS. The radius of capacitive loop reflected the corrosion behavior at interface of between passive film and electrolyte, and a larger radius indicated a better corrosion resistance. Fig. 2 shows the physical model and the corresponding equivalent circuits used for fitting the impedance spectra of 316L-Cu SS before and after passivation. The equivalent electrical circuit in Fig. 2b which was based on the amount of time constant includes the following elements: the electrolyte resistance Rs, the capacitance parameter QCPE and the charge transfer resistance Rct of the interface between the passive film and electrolyte. The data of electrochemical impedance parameters for 316L-Cu SS before and after passivation are listed in Table 3. The Rct of 316L-Cu SS passivated in nitric acid solution containing 20 ppb and 10 ppm Cu2+ ions were 498 and 504 kΩ cm2, respectively, higher than that of the non-passivated 316L-Cu SS. The larger impedance indicated that the corrosion resistance was obviously improved, which could explain the better corrosion resistance of 316L-Cu SS after passivation. The above results demonstrated that improved corrosion resistance of the passivated 316L-Cu SS may be attributed to the formation of stable and dense oxidation film as it prevented the penetration of electrolyte into the surface of 316L-Cu SS. The pitting potential increased after passivation, representing a

protective behavior occurred by the formation of passivation layer, which blocked the electrolyte passage through the passive film to corrode the surface of 316L-Cu SS. It has also been proved that the pitting corrosion resistance of 316F SS was clearly improved via soaking the samples in Cu2+ containing solution [35], and the increase in pitting potential was attributed to the newly Cu2-δS thin film which prevented the MnS from dissolution. The results of Wu et al. [36] also demonstrated that the corrosion resistance did not reduce due to the introduction of Cu in the modified passivation film. The corrosion resistance of SS was increased due to passivation film with addition of Cu. However, the observation also indicated that Cu2+ ions in passivation film had no obvious influence on stability of passivation film.

Table 2 The electrochemical corrosion parameters determined from the cyclic polarization curves of the 316L-Cu SS

Type of passivation

Eb /mV

icorr/μA cm-2

Ep/mV

No passivation

534±8.2

0.266±0.15

1.5±1.3

20 ppb Cu2+

807±7.9

0.158±0.08

160.6±3.5

10 ppm Cu2+

832±9.1

0.259±0.12

29.2±2.5

Table 3 Electrochemical impedance parameters of 316L-Cu SS before and after passivation based on the physical model

Type of passivation

Rs(Ω cm2)

Rct (kΩ cm2)

QCPE(Ω-1 Sn cm-2)

n

No passivation

17.60±1.21

159±0.015

5.119±0.211

0.89±0.02

20 ppb Cu2+

20.67±1.12

498±0.012

4.686±0.325

0.88±0.02

10 ppm Cu2+

20.98±1.21

504±0.017

4.176±0.355

0.91±0.01

1.2

a)

140000

No Passivation

1.0

20ppb Cu

2+

10ppm Cu

0.8

b)

120000

2+

2

-Z``(ohm.cm )

Potential,E/VSCE

100000

0.6 0.4 0.2 0.0

80000 60000

20000

-0.2

0

-0.4 -10

No Passivation 20ppb 10ppm

40000

-9

-8

-7

-6

-5

-4

-3

-2

0

20000

40000

60000

80000

100000

120000

140000

2

-2

Z`(ohm.cm )

Log (i/A cm )

Fig.1. The electrochemical cyclic polarization curves (a) and Nyquist plots (b) of 316L-Cu SS in 0.9 wt.% NaCl solution at 37 ºC before and after passivation in nitric acid solution containing 20

ppb and 10 ppm Cu2+ ions, respectively.

a)

b)

Rs(QCPERct) Fig.2. Physical model (a) and the corresponding equivalent circuits (b) used for fitting the impedance spectra of 316L-Cu SS before and after passivation.

3.2 In vitro antibacterial test The plate count measurement was employed to investigate the antibacterial properties of the 316L-Cu SS before and after passivation. Fig. 3 shows the images of the bacterial colonies in petri dishes after immersion of samples in E.coli suspension for 24, 72 and 120 h. After incubating for 24 h, the petri dish of 316L SS was almost completely covered by bacterial colonies (Fig. 3 a1) whereas, few bacterial colonies were present for 316L-Cu SS before and after passivation (Fig. 3 b1,c1 and d1). After

72 h and 120 h, the same tendency can be observed in Fig. 3 (a2, b2 ,d2) and Fig. 3 (a3, b3, d3) indicating that the passivated 316L-Cu SS inhibited bacterial colonization. As listed in Table 4, after 24 h of co-culture of the samples and bacterial suspension, the numbers of E. coli on the surface of 316L-Cu SS before and after passivation were all much lower than that of 316L SS. According to the formula (1), with the increase of Cu2+ ions concentration, the antibacterial rates of 316L-Cu SS against E. coli are 91.5±1.2%, 92.7±1.1% and 96.9±0.3%, respectively. To study the effect of culture time for the samples and bacterial suspension on the antibacterial rate, 316L-Cu SS passivated by 10 ppm Cu2+ ions concentration of nitric acid solution was selected to immerse in bacterial suspension at 37 ºC for 72 h and 120 h. After 72 h, the amount of E. coli for 316L SS increased to 9800±300 CFU/mL, on the contrary, the number of bacteria for 316L-Cu SS before and after passivation rapidly decreased to 908±80 and 4±1 CFU/mL, with antibacterial rates of 90.7±1.8% and 99.9±0.1%, respectively. After prolonging the co-cultured time to 120 h, the antibacterial rates of 316L-Cu SS reached to 92.5±0.9% and 99.9±0.01%, respectively. These results indicated that 316L-Cu SS treated by antibacterial passivation had a strong antibacterial ability against E. coli. 316L-Cu SS passivated by concentration of 10 ppm Cu2+ had a long lasting antibacterial property as the antibacterial rate was still remained at 99.9±0.01% after 120 h. Therefore it can be reasonably deduced that the 316L-Cu SS exhibited strong antibacterial ability against E.coli with increasing Cu2+ ions concentration in nitric acid solution.

(a1)

(b1)

(c1)

(d1)

d1)

(a2)

(b2)

(d2)

(a3)

(b3)

(d3)

Fig. 3. Photos of bacterial colonies for 316L SS and 316L-Cu SS in E.coli suspension at 37 ºC. The alphabet a , b, c and d represent different state of materials: (a) 316L SS, (b) non-passivated 316L-Cu SS, (c) passivated 316L-Cu SS in 20 ppb Cu2+ ions of solution, (d) passivated 316L-Cu SS in 10 ppm Cu2+ ions of solution, respectively. The subscript 1, 2 and 3 represent the co-culture

time of 24, 72 and 120 h, respectively.

Table 4 Bacterial numbers (CFU per milliliter) after incubation of samples and E.coli suspension at 37 ºC for 24, 72, 120 h, using the standard plate-count method. 316L-Cu SS, CFU mL-1

316L SS, CFU mL-1

No passivation

20 ppb Cu2+

10 ppm Cu2+

Blank control

24

6000±220

512±55

440±45

184±15

6000±121

72

9800±300

908±80

-

4±1

6000±150

120

10000±330

750±70

-

8±1

6000±135

Time, h

3.3 Cu2+ ions release rate in passive film Since the passivated 316L-Cu SS in the solution containing 10 ppm Cu2+ ions have a long lasting antibacterial performance, correspondingly, the release rate of Cu2+ ions was detected to clarify the reason of its antibacterial persistence. The concentrations of Cu2+ ions released from the passive film on 316L-Cu SS in 0.9% NaCl solution at 24, 72 and 120 h are shown in Fig. 4. Compared with the concentration of Cu2+ ions released from non-passivated 316L-Cu SS in the solution, ranging in 0.0113-0.0265 mg/L, Cu2+ ions concentration released from passivated 316L-Cu SS, ranging in 0.0149-0.0293 mg/L, was obviously higher, and increased with prolonging time from 24 to 120 h. It is well known that Cu element is an excellent antibacterial agent [37]. The passivation treatment with nitric acid solution containing Cu2+ ions on the surface of 316L-Cu SS endowed it excellent antibacterial

property as shown in Fig. 3.

0.0350

No passivation 2+ 10 ppm Cu

2+

Concentration of Cu /mg L

-1

0.0315 0.0280 0.0245 0.0210 0.0175 0.0140 0.0105 0.0070

0

24

48

72

96

120

144

Time,t/h

Fig.4. The released Cu2+ ions concentration from the surface of 316L-Cu SS before and after passivation.

3.4 Surface composition of passivated 316L-Cu SS The results of XPS in Fig. 5 show the composition variation of Cu and Cr on the surface of 316L SS and 316L-Cu SS before and after passivation by nitric acid solution containing 20 ppb and 10 ppm Cu2+ ions with increase of the etching time at an interval of 10 seconds. It can be noted from Fig. 5(a) that the content change tendency of Cu and Cr in 316L SS and 316L-Cu SS before and after passivation with identical etching time was similar For the passive films of 316L-Cu before and after passivation, the concentration of Cu and Cr in the passivated 316L-Cu SS was obviously higher than that of the

non-passivated 316L-Cu, as shown in Fig. 5(a) and (b). It is worth noting that the atomic percentages of Cr and Cu in 316L SS and 316L-Cu SS at 0 s of etching time were lower than those in the inner layer of passive films. This was related to the bi-layer structure of passive film, where the outer layer was enriched in iron oxide and the inner layer was enriched in chromium oxide [38]. Lin et al. observed the similar phenomenon for Cu-bearing SS (Fe-18Cr-8Ni-xCu) passivated in sulfuric acid solution [39]. In general, Cr enrichment in passive film was well known as the process of passivation promoting the redox reaction of Cr on surface of SS [40-42]. After the antibacterial passivation, Cr in passive film of 316L-Cu SS was still one of the main composition elements, which guaranteed the sufficient corrosion resistance for SS. Cu enrichment in passive film was also observed after adding Cu2+ ions to nitric acid solution, which implied that Cu could be absorbed into the passive film. Therefore, when Cu2+ ions contained passive film immersed in the 0.9% NaCl solution, Cu2+ ions in passive film should be released to exert the expected antibacterial function.

5.0

a)

15

4.5

14

4.0

Atomic Percent/%

Atomic Percent/%

16

13

316L No passivation

12 11

b)Cu 20ppb

2+

10ppmCu

2+

b)

3.5 3.0

No passivation 2.5

20ppb Cu

2+

10ppm Cu

2.0

2+

10

1.5 9 -20

0

20

40

60

80

100

120

140

160

Etch time, t/s

1.0 -20

0

20

40

60

80

100

120

140

160

Etch time, t/s

Fig. 5. XPS depth concentration profiles of the passive film formed on surface of 316L SS and 316L-Cu SS: (a) Cr element (b) Cu element.

To understand the characteristics of the Cu-bearing passive films, the chemical

compositions on surface of 316L-Cu SS before and after passivation were further analyzed by XPS. Typical XPS spectra of Cr 2p at etching time of 15 s are shown in Fig. 6. The standard procedures were used to calculate the decomposition and fitting of the spectra [43, 44]. For the passive film on 316L-Cu SS, several peaks around 576.6 eV and 577.2 eV in the XPS spectra were observed, which were distinguished as Cr2O3 and Cr(OH)3, respectively. Moreover, the peaks corresponding to Cr in the form of simple substance was also observed. However, the Cr peak was relatively weaker on the passivated surface of 316L-Cu SS. The most noticeable difference after passivation was the increased contents of Cr oxide and hydroxide. Table 5 shows that the total contents of Cr oxide and hydroxide in the passive film on 316L-Cu SS after 20 ppb Cu2+ ions and 10 ppm Cu2+ ions of passivation were 5.75% and 5.84%, respectively, which were higher than that of non-passivated 316L-Cu SS. Ju et al. [45] found that when 316L SS was coupled with Pd electrode, the passive film of 316L SS showed much better corrosion resistance than the passive film formed in air. The reasons could be attributed to the enrichment of Cr, and the more contents of Cr2O3 and Cr(OH)3, the less defects were in the passive film. Other studies also proved that the improved corrosion resistance of SS mainly resulted in the form of Cr oxide and hydroxide in the passive film [24-25]. So it is easy to deduce that the corrosion resistance of passivated 316L-Cu SS in this work was markedly enhanced.

Table 5 Parameters for decomposition of Cr 2p spectra at 15 s etching time by XPS analysis

Cr

Cr2O3

Type of

Cr(OH)3 Binding

Binding

Binding passivation

Area,%

Area ,%

energy,

Area ,%

energy,

energy, eV eV

eV

No passivation

8.55

574.2

3.56

576.6

1.43

577.1

20 ppb Cu2+

8.49

574.2

4.51

576.6

1.24

577.2

10 ppm Cu2+

8.94

574.2

4.24

576.6

1.60

577.2

50000

55000

a)

Cr Intensity(a.u.)

Intensity(a.u.)

35000

Cr(OH)3

30000

25000

Cr2O3

40000

Cr(OH)3

35000 30000 25000

20000

15000 582

Cr

45000

Cr2O3

40000

b)

50000

45000

20000

580

578

576

574

572

15000 582

570

580

578

576

574

572

570

Binding energy,Eb/eV

Binding energy,Eb/eV

60000 55000

c) Cr

Intensity(a.u.)

50000

Cr2O3

45000 40000 35000

Cr(OH)3

30000 25000 20000 582

580

578

576

574

572

570

Binding energy,Eb/eV

Fig. 6. The surface XPS spectra of Cr 2p detected in the passive films of 316L-Cu SS before and after passivation at 15 s etching time. (a) No passivation, (b) 20 ppb Cu2, (c) 10 ppm Cu2+ +.

Fig. 7 shows the XPS spectra of Cu 2p at etching time of 15 s, the data were analyzed to investigate the effect of Cu addition on the structure of passive film. For the passive film, it can be seen that there were two peaks at 932.4 eV and 933.5 eV in the Cu 2p spectra, which were defined as metallic Cu and CuO, respectively. And the peaks of Cu in the form of simple substance were dominant compared with the peak of CuO. It suggested that the state of Cu in the passive film of 316L-Cu SS before and after passivation were mainly composed of metallic Cu and CuO. Parameters for decomposition of Cu 2p spectra by XPS are listed in Table 6. The Cu content in the passivation film of passivated 316L-Cu SS was almost the same as that of

non-passivated 316L-Cu SS. Whereas when 316L-Cu SS were treated with 20 ppb or 10 ppm Cu2+ ions, the CuO content after passivation generally increased than that of non-passivated 316L-Cu SS, reaching to 0.99% and 0.94%, respectively. CuO can be formed from either simple substance such as metallic Cu or monovalent Cu2O [46]. In our case, CuO was formed via a chemical reaction of Cu2+ ions and aqueous solution, according to the formula (2): (2) Therefore, after the passivation treatment by Cu2+ ions contained solution, the CuO content in the passive film on 316L-Cu SS was increased. According to the results of antibacterial tests and corrosion resistance, it can be concluded that the valence state of Cu provided two kinds of contribution towards the antibacterial property and corrosion resistance. In this regard, metallic Cu was the main valence state responsible for the excellent antibacterial performance. When metallic Cu containing passive film immersed in 0.9% NaCl solution, it can easily lose two electrons converting itself to divalent Cu2+ ions, and the released Cu2+ ions can capture and kill the bacteria. But the corrosion resistance of passivated 316L-Cu SS was weakened in some extent due to existence of metallic Cu, so the value of icorr increased. Secondly, enough Cu2+ ions in passivation solution participate in the reaction given in equation (2), so more CuO could be formed in passivation film. CuO and Cr-rich oxide guaranteed the pitting corrosion resistance. According to the previous report, Cu-rich passive film can effectively repair the pitting damage because of Cr depletion [35]. In addition, as the reaction in equation (2) was reversible, the CuO in passive film can

also transfer to Cu2+ ions providing long lasting antibacterial performance for the passivated 316L-Cu SS.

Table 6 Parameters for decomposition of Cu 2p spectra at 15 s etching time by XPS analysis Cu

CuO

Type of passivation

Binding energy,

Binding energy,

Area,%

Area,% eV

eV

No passivation

3.67

932.4

0.52

933.6

20 ppb Cu2+

3.76

932.5

0.99

933.6

10 ppm Cu2+

3.56

932.4

0.94

933.4

170000

165000

b)

a) 160000

160000

Intensity(a.u.)

Intensity(a.u.)

155000

Cu

150000 145000

CuO

140000 135000

150000

Cu 140000

CuO

130000

130000 120000

125000 938

936

934

932

930

938

928

936

934

932

930

928

Binding energy,Eb/eV

Binding energy,Eb/eV

170000 165000

c)

160000

Intensity(a.u.)

155000 150000

Cu

145000

CuO

140000 135000 130000 125000 938

936

934

932

930

928

Binding energy,Eb/eV

Fig. 7. The surface XPS spectra of Cu 2p in the passive film of 316L-Cu SS before and after passivation, (a) No passivation, (b) 20 ppb Cu2+, (c) 10 ppm Cu2+.

3.5 Cytotoxicity of passivated 316L-Cu SS Many laboratory report [47-50] demonstrated that the RTCA method reproducibly provided accurate results for evaluation of in vitro cytotoxicity. Fig. 8

shows the RTCA growth profile of MC3T3-E1 mouse cells exposed to extracted test samples. From the overall trend of RTCA plot, since MC3T3-E1 mouse cells grew as control in healthy state, so the test of RTCA was trustable. After 24 and 72 h, the normalized cell index (NCI) of 316L-Cu SS passivated by nitric acid solution containing 20 ppb and 10 ppm Cu2+ ions were universally higher than those of non-passivated 316L SS and 316L-Cu SS, respectively. Compared to 316L SS and non-passivated 316L-Cu SS, the passivated 316L-Cu SS stimulated the cell growth until 24 h. After 72 h, cell apoptosis started and the cell growth rate began to decrease. It was found that the grade of cytotoxicity increased with the elevated Cu concentrations [51]. In addition, the results of Chai et al. [52] proposed that the expression of insulin-like growth factor-1 (IGF-1) in osteoblasts around 316L-Cu SS screws tracts increased compared with 316L SS control. This can be reasonably explained that the NCI of 316l-Cu SS before and after passivation were higher within 72 h, compared with that of 316L SS. So a certain concentration of Cu2+ ions leads to the MC3T3E1 detachment from the electrodes resulting in a change in NCI. Here, 316L SS was used as a control material, and the NCI for 316L SS was assigned a standard which represented no cytotoxicity. A cytotoxicity score of any material that was more than that of 316L SS was considered non-cytotoxic. The results in the present study suggested that two kinds of passivated 316L-Cu SS did not introduce cytotoxicity to the mouse cells within experimental time. Therefore, the preliminary results indicated that 316L-Cu SS passivated by nitric acid solution containing 20 ppb Cu2+ ions, which shows better biocompatibility compared to the 316L-Cu SS

passivated by nitric acid solution containing 10 ppm Cu2+ ions.

11 10

Normalized cell index

9

72h

8

addition of extracted test samples

7

Control

6 5 4

2

20ppb Cu2+ 10ppm Cu2+

1

316L-Cu

24h

3

0 -1

316L 0

18

36

54

72

90

108

126

Time, t/h

Fig. 8. RTCA profiles of various extracted test samples for mouse MC3T3-E1 cells with 1,000 MC3T3-E1 cells per well.

4. Conclusions (1) The antibacterial passivation effectively improved the pitting corrosion resistance and the protective potential of 316L-Cu SS, but did not affect its corrosion current density. (2) The passivated 316L-Cu SS had slightly higher antibacterial rate than non-passivated 316L-Cu SS. The results of Cu2+ release rate proved that passivated 316L-Cu SS did not prevent Cu2+ release from the surface of stainless steel, which guarantees an excellent antibacterial performance. (3) The passive film thickness of the passivated 316L-Cu SS was measured to be

about 6 nm that was identical with that of the non passivated 316L-Cu SS. The chemical structures of passive film on 316L-Cu SS after passivation consisted of Cr2O3, Cr(OH)3, CuO and metallic Cr and Cu. Cu-rich and Cr-rich passive film on surface of 316L-Cu SS provided reliable corrosion resistance and antibacterial property. (4) The results of RTCA test proved that the passivated 316L-Cu SS has better biocompatibility than the non-passivated 316L-Cu SS.

Acknowledgement This work was financially supported by the National Natural Science Foundation of China (Grant No. 51371168) and (Grant No. 51501188), the National Natural Science Foundation of China Research Fund for International Young Scientists (Grant No. 51450110439) the National Basic Research Program of China (Grant No.2012CB619101), Shenyang National Lab for Materials Science, and the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials.

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