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The effect of rotation accelerated shot peening on mechanical property and antimicrobial activity of pure copper
Surface & Coatings Technology 384 (2020) 125319
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The effect of rotation accelerated shot peening on mechanical property and antimicrobial activity of pure copper Yu Suna, Zixuan Zhanga, Yuan Qina, Xuran Xub, Sen Yanga,c,
T
⁎
a
School of Materials Science and Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China School of Chemical Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China c Sino-French Engineering School, Nanjing University of Science and Technology, No. 200 Xiaolingwei, Nanjing 210094, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Copper Rotation accelerated shot peening Surface nanocrystallizition Antimicrobial activity
In this work, we report the use of rotation accelerated shot peening (RASP) to achieve surface nanostructure on copper plate. To systematically investigate the microstructure, mechanical properties, antimicrobial ability and corrosion resistance of the specimens, two kinds of RASP specimens with processing time being 5 min (RASP-5) and 15 min (RASP-15) were employed in this study. A nanostructure layer was formed in the surface of copper specimen after RASP. With prolonging shot time, the size of surface grains decreased and the micro-hardness was significantly improved. The antimicrobial ability of the RASP specimen for E. coli was better than that of the base material, while no obvious difference existed for S. aureus. In electrochemical corrosion tests, it was confirmed that RASP treatment could effectively stimulate the copper ions release from the surface of specimen to increase antimicrobial activity.
1. Introduction In our daily life, causal agent reserving on environmental surfaces is one of the main paths for the acquisition of disease infection [1]. To solve this problem, many kinds of antimicrobial agents are used to kill bacteria and protect human health. Nevertheless, misuse or overuse of antibiotics would increase antimicrobial resistance and lead to the spread of life-threatening multi-drug-resistant (MDR) pathogens, such as carbapenems-resistant Acinetobacter baumannii, carbapenems-resistant Pseudomonas aeruginosa, vancomycin-resistant, and Enterococcus faecium [2,3]. Even though the antimicrobial resistance (AMR) can be divided into intrinsic and acquired drug resistance, the latter is more harmful, since the latter is the result of long-time interaction between bacteria and antibiotics and this interaction is almost impossible to defend. In hospital, on many touch surfaces like door handles, benches and toilets, amounts of living microbes exist that leading to a high possibility of cross infection [4]. Therefore, it is urgent to look for effectively metal inorganic antibacterial materials instead, such as copper, silver, etc. [5–9]. In general, antibiotic metal can be applied to keep these touch surfaces inhibiting the breeding of bacteria and avoid the development of AMR. Among them, copper receives growing attention as intrinsically antimicrobial material because of low cost, broad spectrum, eco-friendly, long service life and reusable [10–15].
Copper had been widely used before that antibiotic was discovered to tackle a plethora of infections ranging from tuberculosis to skin diseases [16]. In 2008, copper-based surfaces supported by many antibacterial performance tests were registered as solid antimicrobial surfaces by US Environmental Protection Agency (EPA) [17]. However, the poor mechanical properties of pure copper, like hardness and wear resistance, limit its application in real life, and the antimicrobial action mechanism of the pure copper is still not clear [18]. A lot of studies show that grain refinement can effectively increase mechanical properties of metal [19–22]. Generally speaking, a higher strength can be obtained with smaller grain size according to the classical Hall-Petch relationship [21]. As assessed by Yong et al. [20], the yield strength of Al can be significantly enhanced when its grain size decreases to 36 nm. Meanwhile, the ultimate tensile strength and yield stress of the CueZn alloy increases respectively to 386 MPa and 192 MPa after the refinement of the grains, as demonstrated by H. Puga et al. [22]. Up to now, many sever plastic deformation techniques are used to construct nanostructure on the bulk materials, such as equal channel angular pressing (ECAP) [23], high pressure and torsion (HPT) [24] and accumulative roll bonding (ARB) [25]. But these techniques have certain limitation in fabricating special size and shapes. It is well known that the most of premature failures of component, such as abrasion, fatigue fracture and erosion, are related to the
⁎ Corresponding author at: School of Materials Science and Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.surfcoat.2019.125319 Received 15 October 2019; Received in revised form 23 December 2019; Accepted 28 December 2019 Available online 31 December 2019 0257-8972/ © 2019 Elsevier B.V. All rights reserved.
Surface & Coatings Technology 384 (2020) 125319
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2.3. Bacterial strains and growth conditions
microstructure and its associating mechanical properties of material surface [20]. Therefore, surface modification of materials become an important route to improve various performances, such as wear, erosion, and corrosion resistance, and is more convenient and flexible at the same time. Shot peening is a well-known excellent surface treatment technique, by which nanostructured layer on base material can be designed. The previous researches show that the surface grain size of 1Cr18Ni9Ti stainless steel after shot peening can reach 18 nm [26], and the average surface grain size of Ti4Al2V processed by shot peening decreases to 35 nm [27]. Along with grain size decreasing, the mechanical properties of materials are significantly improved, for instance the maximum hardness of Ti6Al4V after shot peening reaches about 400 HV, which is much larger than that of 300 HV in base materials [28]. The fatigue strength of Cu-Ni-Si alloy after shot peening at 107 circles can even reach 280 MPa [29]. All these results claim the importance of shot peening technique in material modification. To further boost the practical application of this technique, the rotation of the impeller is applied to accelerate shots. And the specimen is spinning rapidly during processing, which makes both sides of the specimen be shot evenly. The aims of this study are using rotation accelerated shot peening (RASP) to enhance the surface performances of pure copper plate. The nanostructure on the surface and effect of antimicrobial capacity were systematically investigated.
E. coli CMCC 44102 and S. aureus CMCC 26003 were cultured aerobically on the LB solid medium (10 g Peptone, 10 g Yeast Extract, 5 g salt, 15 g Agar, 1000 mL distilled water) at 37 °C for 24 h. Then cells were transferred to 4 mL phosphate-buffered saline by inoculating loop. The average cell density was 1 × 108–3 × 108 cfu/mL. 2.4. Measurement of contact killing by wet plating Before contact killing experiments, all specimens were dipped in dilute hydrochloric acid solution and 100% ethanol, dried in clean bench and sterilized under UV light for 30 min. Briefly, 20 μL of cells suspended in phosphate buffer saline (PBS) were applied to specimens as a drop. After incubation with different durations (E. coli: 15, 60, 90 and 120 min; S. aureus: 30, 60, 120 and 180 min) in a water-saturated atmosphere, 10 μL bacterial suspension was withdrawn and serial dilutions in PBS were spread on LB solid medium. Following growth of bacteria for 24 h, colony forming units (cfu) were determined. 2.5. Differentiation between viable and dead cells To further observe the state of bacterial, the cells that exposed for different time durations (E. coli: 15 and 120 min; S. aureus: 30 and 180 min) to the surface of specimens were pipetted into centrifuge tubes. Then 2 μL green-fluorescent FDA (5 mg/mL Acetone solution of Fluorescein diacetate) and 5 μL red-fluorescent PI (1 mg/mL aqueous solution of Propidium Iodide) were added into 1 mL aliquots of cells in tube. And these tubes were kept in dark for 5 min. After that 50 μL aliquots were pipetted onto glass slides. The bacteria on the specimens were imaged by using confocal laser scanning microscope.
2. Materials and methods 2.1. Materials and specimen preparation In this experiment, the 99.97% bulk copper (TU1) plate with trace amounts of sulfur and iron was chose as study object, while AISI 304 stainless steel was used as the reference in antimicrobial test. A part of copper plates were processed by RASP after pre-rubbing. The process of RASP consisted of two steps. Firstly, 2 mm-diameter-pills were used to make specimens obtain much plastic deformation, and then 0.5 mmdiameter-pills were used to make the surfaces of specimens smoother. For comparison, the polished base materials were named as BM. All the specimens with dimensions of 10 × 10 × 3 mm3 were ultrasonic cleaned in acetone and 100% ethanol for 15 min respectively. The RASP parameters were list in Table 1.
2.6. Bacteria morphology and the internal structure The specimens for Scanning electron microscope (SEM) were prepared after 90 min immediately after killing experiments. Firstly, the specimens with bacteria were stored in glutaraldehyde solution (2.5% in PBS medium) at 4 °C more than 2 h. Secondly, they were rinsed in PBS solution and dehydrated in water-ethanol mixtures (30%, 50% 70%, 90%, 95%, 100%). Last, Au/PD was sputtered on dried specimens. The specimens were imaged using the scanning electron microscope (SEM). To observe the internal structure of damaged bacteria, SEM with Dual Beam microscope was used. Before cut by ion beam, the bacteria were locally sputtered in the SEM with an additional platinum protective layer to prevent damage during the FIB preparation. The FIB preparation was performed by applying a gradual reduction of the beam current up to a final value of 5 pA [19].
2.2. Materials characterization The grain size of base materials was measured by the electron backscattering diffraction (EBSD) with a 1.5 μm step size. The surface topography of specimens was tested by White light confocal microscope (Axio CSM 700).The three-dimensional view and surface roughness were tested by confocal three-dimensional profilometer. The surface grain size of RASP specimen was measured by the transmission electron microscope (TEM, Tecnai G2 20 LaB6). The micro-hardness was measured with a load of 100 g and loading time of 10s by the Vickers microsclerometer (HMV-G21), and three times of measurements were made at each depth.
2.7. Ion release measurements During the contact killing experiment, copper ions released from the surface of specimens to PBS solution. 20 μL aliquots were taken at specified time instants (15, 60, 90 and 120 min), diluted 500-fold (15, 60 and 90 min) and 5000-fold (120 min) with 0.065% HNO3. Then the copper content was quantified by inductively coupled plasma mass spectrometry (ICP-MS, NexION 350, PerkinElmer, America).
Table 1 Parameters of RASP.
2.8. Electrochemical corrosion
Step 1
RASP-5 RASP-15
The corrosion resistance of the copper specimens was evaluated by potentiodynamic polarization test. The corrosion potential was calculated through Tafel region extrapolation. The electrochemistry tests were performed in PBS solution at room temperature. The specimen acted as work electrode, a platinum electrode and a saturated calomel acted as counter electrode and reference electrode (SCE), respectively,
Step2
Pill size
Velocity
Time
Pill size
Velocity
Time
2 mm 2 mm
40 m/s 40 m/s
5 min 15 min
0.5 mm 0.5 mm
40 m/s 40 m/s
5 min 15 min
2
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grains are randomly distributed. The average surface grain size of RASP-5 is about 20 nm, while that of RASP-15 is 12 nm. As we demonstrated above, the refinement of microstructure come from plastic deformation induced by consecutive and high speed balls hitting on the specimen surface. Previous studies have showed the deformation twinning plays an important role in refining grains to the nanometer scale via plastic deformation [30,31]. Due to the interaction or connect of dislocations and large amounts of twin boundaries, the coarse grain is refined to a lot of twin lamellae firstly and the twin lamella is further refined to equiaxed nano-grains [32,33]. 3.2. Micro-hardness after RASP Fig. 5 shows the variations of micro-hardness from the top surface to the substrate of all specimens. There is an obvious tendency that microhardness of RASP specimens decrease rapidly within the top 200 μm thick layer. When the depth is more than 200 μm, the decrease becomes slow and then disappears. The continuously decreasing in hardness with depth is related to the increasing grain size, which means a gradient structure is formed from surface to matrix. Besides, this result confirms that surface grains absorb the majority of impact energy of shots and the residual compressive stress is produced on the surface. At the same time, the hardness curve of RASP-15 is much gentler than that of RASP-5. It implies that with prolonging shot time, more grains get refined, namely that the hardened layer thickens. It is observed that the surface hardness of RASP specimens reach 118HV0.1 and 121HV0.1 respectively and both of them are significantly higher than that of BM.
Fig. 1. Orientation distribution map of the base materials by EBSD.
forming the three-electrode cell. Three parallel tests were performed on each kind of specimens. 3. Results and discussion 3.1. Surface roughness and grain size
3.3. Antimicrobial activity
Fig. 1 shows the orientation distribution map of the base materials. As we can see, the majority of grains present coarse and equiaxed with some twin lamellae. The average grain size is about 85 μm figured out by EBSD. With RASP modifying the surface, the surface morphology and microstructure of the specimens have been changed as shown in Fig. 2. It can be found that the surface becomes rough, and many hemispheric concaves form. This is owing to the serious plastic deformation resulted from the repeated peening at high strain rates. Here, it is worth to note that we vary the RASP time for further optimize its effect. RASP-5 and RASP-15 represent the duration time of RASP being 5 min and 15 min, respectively. Fig. 3 shows the threedimensional images of the specimen surfaces after RASP. The surface of RASP-5 is obviously rougher than that of RASP-15 with the average roughness (Ra) being 26.8 μm and 15.9 μm for RASP-5 and RASP-15, respectively. The reason can be attributed to that with prolonging of the treatment time, much more plastic deformation is produced in the specimen and concaves that formed on the surfaces by shots overlap and override each other. Plastic deformation was generated on the top surface layer of RASP specimens, where grains were too fine to be observed directly with optical microscope. Fig. 4(a) and (b) show the TEM bright field images of the surface layers of RASP specimens. A large number of nanometersized grains can be observed and crystallographic orientations of these
Fig. 6 shows the comparison of bacteria viability on different specimen surfaces. Two kinds of bacteria were used, one was E. coli (Gramnegative) and the other was S. aureus(Gram-positive). Under wet conditions, there is no significant difference of the killing efficiency between RASP-5 and RASP-15 to E. coli (Fig. 6a). The concentration of bacteria was set as 1 × 108–3 × 108 cfu/mL. After 120 min, the highest killing efficiency with 3 logs was achieved on the RASP specimens. Meanwhile, the antimicrobial ability of BM specimen was relatively poorer with only 2 logs reduction of cells over 120 min. As control, the stainless steel exhibited no significant antimicrobial effect in the experiment. As stated in several papers [8,11,34,35], like silver ions, the copper ions have ability to destroy the structure of cell membranes and affect the membrane permeability. And the change of membrane permeability can cause the leakage of cytoplasm. Here, we believe the better antimicrobial ability of RASP specimens can be assigned to the high activity of surface atoms. The surface nanostructrue makes these atoms more active and it means that copper ions are more easily released into solution during the same time. Fig. 6(b) shows the survival of S. aureus on all the specimens. Obviously, the viability of S.aureus was stronger compared to E. coli. The killing efficiency of RASP specimen to S. aureus is low with 2 logs after
Fig. 2. The surface topography of a) RASP-5 and b) RASP-15. 3
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Fig. 3. Three-dimensional view and surface roughness (Ra) of (a) RASP-5 and (b) RASP-15 were obtained by confocal three-dimensional profilometer. Scans were carried out in an area of 4 × 4 mm.
180 min. At the same time, the number of live bacteria on BM specimen is similar to that on specimens treated by RASP. Here, one of antimicrobial mechanisms is proposed as follow to illustrate the difference. Cu ions enter the cell through cell wall and change the conformational structure of nucleic acids and proteins. As is known, the cell wall of S. aureus (20–80 nm) is much thicker than that of E. coli (10–15 nm). With the same Cu ion release rate and the same contact area (PBS solution with cells/specimen surface), S. aureus is more resistant than E. coli. As a consequence, although the activity of copper atoms on the surfaces of BM specimens and RASP specimens is different, the difference is not enough to have a greatly impact on the survival of S. aureus on the specimen surfaces. The different colors of live and dead cells are showed in Figs. 7 and 8. The live cells fluoresce green due to FDA, which has the ability to get into both live and dead bacteria. After it is catalyzed by non-specific esterase into fluorescein in cells, green fluorescence of 530 nm can be inspired by laser at 480 nm. The dead cells whose membranes are not intact fluoresce red due to PI, which can just enter the bacteria with incomplete membranes. Because when the membrane is not intact, FDA will spread beyond the cell while PI can combine with DNA. When it is excited by laser at 488 nm, red fluorescence will be inspired. In Fig. 7, cells of E. coli are mostly stained green after 15 min of exposure on specimens and a lot of cells are dyed red while being exposed for 120 min, especially for exposed to RASP-5 and RASP-15. The red cells exposed on the surface of BM are distinctly less than that on other specimens. Combining the analysis of Fig. 6, the results state adequately that the activity of E. coli on RASP specimens is much poorer than that on BM specimens. Fig. 8 shows that most cells of S. aureus are dyed green after 30 min and only a part of cells are dyed red after 180 min. Besides, the numbers of red cells in (b), (d) and (f) are almost the same. Since incomplete membrane is the premise that PI can get into a
Fig. 5. Variation curves of micro-hardness of RASP-5, RASP-15 and BM with distance from topmost to substrate.
cell, the result of the staining experiment indicates that contacting between bacteria and copper can denature the cell walls and damage the integrity of membranes.
3.4. The effect of copper ions on bacteria morphology and internal structure Fig. 9 shows the morphologies of live and dead bacteria on the copper specimens. In Fig. 9(a), the middle of the cell that is marked in red box presents cave and it means that the cell has died. In Fig. 9(b), the integrity of the bacteria structure which is marked in red box is
Fig. 4. (a) TEM graph of surface grains of RASP-5 and (b) RASP-15. 4
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Fig. 6. Survival of cells given in colony forming units (cfu) is measured by wet plating on surfaces of all specimens. (a) E. coli cells and (b) S. aureus cells.
cytoplasm can be drained out of broken membrane. These results state that membrane-damaging of cells caused by copper ions is an important mechanism in contact killing. Thus the release of copper ion requires a special focus.
damaged and cytoplasm escapes. The reason is that the cell wall and membrane are broken by copper ions and cell contents escape. The redox properties of copper ions can lead to depletion of sulfhydryls which exist in some kinds of amino acids of membrane [36]. Besides, reactive hydroxyl radicals are generated in redox reaction and can further take part in the oxidation of proteins. Fig. 10 shows the internal structures of bacteria on copper and stainless steel specimens. Compared with the intact structure of the bacteria on stainless steel in Fig. 10(b), the damage of bacteria on copper is apparent with hollowed out. This indicates that much of
3.5. Release of copper ion The concentration of cupric ions/cuprous ions keeps increasing when copper contacts with bacterial suspension until the dynamic equilibrium is reached [35]. This process could be pertinent under the
Fig. 7. Cells of E.coli were respectively exposed to (a)(b)BM, (c)(d)RASP-5 and (e)(f)RASP-15 copper plates and then stained by FDA and PI. Images were taken with a fluorescent microscope. Live bacteria with integrated membranes fluoresced green and bacteria with broken membranes fluoresced red. Cells were exposed for (a) (c)(e) 15 min and (b)(d)(f)120 min. 5
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Fig. 8. Cells of S. aureus were respectively exposed (a)(b) to BM, (c)(d)RASP-5 and (e)(f)RASP-15 copper plates and then stained by FDA and PI. Images were taken with a fluorescent microscope. Live bacteria with integrated membranes fluoresced green and bacteria with broken membranes fluoresced red. Cells were exposed for (a)(c)(e)30 min and (b)(d)(f)180 min.
surfaces of RASP specimens corresponds to the higher antimicrobial activity.
wet plating conditions to evaluate contact killing. Therefore, copper ion release measurement was performed. In the measurements, PBS buffer solution was used instead of bacterial suspension for instructions of the equipment. As shown in Fig. 11, the concentration of copper ions in PBS buffer solution on BM is lower than others after 10 min later. The concentration of copper ions of RASP-5 is basically in line with that of RASP-15. The relatively higher concentration of copper ions means that copper after RASP is more easily to corrode under the conditions of free corrosion as used in antimicrobial experiments. It is obvious that the nano-structure surface layers on RASP-5 and RASP-15 specimens have important impact. The overall faster release of copper ions from the
3.6. Electrochemical corrosion The release of copper ions is related to surface corrosion properties. In order to investigate the effect of RASP treatment on the corrosion behavior of pure copper, the corrosion resistance properties were assessed by Electrochemical tests. Fig. 12 shows the potentiodynamic polarization curves of specimens in 0.01 M PBS solution at room temperature. All specimens displayed a typical active–passive–transpassive
Fig. 9. SEM graph of (a) E. coli cells and (b) S. aureus cells on the pure copper after 90 and 120 min of killing experiment respectively. 6
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Fig. 10. Internal structure of E. coli bacteria on: (a) BM specimen after 90 min and (b) stainless steel specimen after 90 min.
behavior. As listed in Table 2, the corrosion potential (Ecorr) of RASP specimen shifts negatively with grain size decreasing. Since the corrosion resistance is mostly related to the size of grains on specimen surface, RASP treatment realizing the nano-sized grains make it more effective. The number of grain boundaries on surface becomes greater with grain size decreasing, and the higher energy of atoms at grain boundaries makes the atoms unstable. As shown in Fig. 12, the passive regions appear on the anodic branches. This means that the passive films formed on specimens immersed in PBS solution. As seen in Table 2, the specimen-to-specimen variations in the passivation current densities are noticeable. Meanwhile, the BM specimen exhibits wider passive regions than the RASP does. It suggests that the passive films forming on RASP specimens are broken more quickly and passive films have better protective ability to retard corrosion without RASP treatment. It is attributed to the surface atoms, especially intergranular atoms of the RASP specimens which have higher activity [37]. These results agree well with that of ion release test basically and support the concept that the release of copper ions is a pivotal parameter in contact killing.
Fig. 11. The concentration of copper ions in PBS buffer solution on different surfaces. A certain amount of liquid was removed respectively after 10 min, 60 min, 90 min and 120 min.
4. Conclusion In this study, pure copper was treated by rotation accelerated shot peening with two sets of parameters. Our findings clearly demonstrated the enhancement of mechanical and antimicrobial properties of copper could be achieved after the surface treatment. Besides, the other main conclusions are listed as follows: (1) Nano-structure surface formed on copper after RASP and the average grain size decreased from 85 μm to 20 nm (RASP-5) and 12 nm (RASP-15) respectively. The hardness of RASP-5 and RASP15 was gradually decreasing with depth. The max hardness of RASP-15 reached 121HV0.1, while that of BM was just 50HV0.1. Dislocations piling up at the grain boundary and sub-grain boundary which leads to dislocation motion hindered improve the hardness. (2) Under wet conditions, the activity of E. coli on RASP-5 and RASP-15 was poorer than that on BM, while the dead speed of S. aureus did not have much difference on all surfaces of specimens. The different activity of two kinds of bacteria on RASP specimens is due to the different structures of cell walls. Membrane-damaging by copper ions is an important mechanism in contact killing. (3) In the electrochemical measurement, RASP-5 and RASP-15 had more negative corrosion potential (Ecorr) and narrower passive regions than BM, Which meant grain refinement produced more unstable atoms to make corrosion resistance decrease.
Fig. 12. Typical potentiodynamic polarization curves of polished copper, shot peening treated RASP-5 and RASP-15 in PBS solution. Table 2 Electrochemical data for specimens. Specimen
Grain size
Ecorr (mV)
Ip(μA·cm−2)
Ef (mV)
Etr (mV)
BM RASP-5 RASP-15
38 μm 20 nm 12 nm
−95.69 −115.88 −124.35
2.23 6.93 20.69
−19.37 −7.88 4.38
381.41 169.47 133.38
Author contributions SEN YANG was in charge of project design and theoretical guidance. SUN YU was in charge of specific experimental design, most of experiments operation, data processing and paper writing. 7
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ZIXUAN ZHANG was in charge of microhardness test. YUAN QIN provided revisions of the paper. XURAN XU was in charge of shooting fluorescence photos of germs.
[13] D.J. Weber, D. Anderson, W.A. Rutala, The role of the surface environment in healthcare-associated infections, Curr. Opin. Infect. Dis. 26 (4) (2013) 338–344. [14] L. Weaver, J.O. Noyce, H.T. Michels, et al., Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus, J. Appl. Microbiol. 109 (6) (2010) 2200–2205. [15] C.D. Salgado, K.A. Sepkowitz, J.F. John, et al., Copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit, Infect. Control Hosp. Epidemiol. 34 (5) (2013) 479–486. [16] V. Villapún, L. Dover, A. Cross, et al., Antibacterial metallic touch surfaces[J], Materials 9 (9) (2016) 736. [17] M. Rosenberg, H. Vija, A. Kahru, et al., Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces, Sci. Rep. 8 (1) (2018) 8172. [18] M. Hans, J.C. Támara, S. Mathews, et al., Laser cladding of stainless steel with a copper–silver alloy to generate surfaces of high antimicrobial activity, Appl. Surf. Sci. 320 (2014) 195–199. [19] R.Z. Valiev, A.P. Zhillyaev, T.G. Langdon, Grain refinement in metallic systems processed by ECAP, Bulk Nanostructured Materials: Fundamentals and Applications (2014) 239–288. [20] Y. Liu, B. Jin, J. Lu, Mechanical properties and thermal stability of nanocrystallized pure aluminum produced by surface mechanical attrition treatment, Mater. Sci. Eng. A 636 (2015) 446–451. [21] H.Y. Guo, M. Xia, Z.T. Wu, et al., Nanocrystalline-grained tungsten prepared by surface mechanical attrition treatment: microstructure and mechanical properties, J. Nucl. Mater. 480 (2016) 281–288. [22] H. Puga, J. Barbosa, J.M. Machado, et al., Ultrasonic grain refinement of die cast copper alloys, J. Mater. Process. Technol. 263 (2019) 336–342. [23] R.Z. Valiev, R.K. Islagaliev, I.V. Alexandrov, Bulk nanostructured materials from severe plastic deformation, Prog. Mater. Sci. 45 (2) (2000) 103–189. [24] I.V. Alexandrov, R. Valiev, X-ray analysis of bulk nanostructured metals, Mater. Sci. Forum 321 (2000) 577–582. [25] S. Roy, B.R. Nataraj, S. Suwas, et al., Accumulative roll bonding of aluminum alloys 2219/5086 laminates: microstructural evolution and tensile properties, Mater. Des. 36 (2012) 529–539. [26] T. Wang, J. Yu, B. Dong, Surface nanocrystallization induced by shot peening and its effect on corrosion resistance of 1Cr18Ni9Ti stainless steel, Surf. Coat. Technol. 200 (16–17) (2006) 4777–4781. [27] J. Han, S. GM, H. GX, Nanostructured surface layer of Ti–4Al–2V by means of high energy shot peening, ISIJ Int. 48 (2) (2008) 218–223. [28] Q. Yang, W. Zhou, Z. Niu, et al., Effect of different surface asperities and surface hardness induced by shot-peening on the fretting wear behavior of Ti-6Al-4V, Surf. Coat. Technol. 349 (2018) 1098–1106. [29] O. Stav, H. Kasem, R. Akhvlediani, et al., Simultaneous shot-peening of hard and soft particles for friction reduction in reciprocal sliding, Tribol. Int. 130 (2019) 19–26. [30] K. Wang, N.R. Tao, G. Liu, et al., Plastic strain-induced grain refinement at the nanometer scale in copper, Acta Mater. 54 (19) (2006) 5281–5291. [31] Y.S. Li, N.R. Tao, K. Lu, Microstructural evolution and nanostructure formation in copper during dynamic plastic deformation at cryogenic temperatures, Acta Mater. 56 (2) (2008) 230–241. [32] A. Amanov, Y.S. Pyun, S. Sasaki, Effects of ultrasonic nanocrystalline surface modification (UNSM) technique on the tribological behavior of sintered Cu-based alloy, Tribol. Int. 72 (2014) 187–197. [33] W.L. Li, N.R. Tao, K. Lu, Fabrication of a gradient nano-micro-structured surface layer on bulk copper by means of a surface mechanical grinding treatment, Scr. Mater. 59 (5) (2008) 546–549. [34] G. Borkow, J. Gabbay, Copper as a biocidal tool, Curr. Med. Chem. 12 (18) (2005) 2163–2175. [35] M. Hans, A. Erbe, S. Mathews, et al., Role of copper oxides in contact killing of bacteria, Langmuir 29 (52) (2013) 16160–16166. [36] S.L. Warnes, C.W. Keevil, Inactivation of norovirus on dry copper alloy surfaces, PLoS One 8 (9) (2013) e75017. [37] W. Luo, C. Qian, X.J. Wu, et al., Electrochemical corrosion behavior of nanocrystalline copper bulk[J], Mater. Sci. Eng. A 452 (2007) 524–528.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This study was supported by the Fundamental Research Funds for the Central Universities (30919011412 and 30919011255), PAPD, and Jiangsu Key Lab of Micro-nano Materials and Technology. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. References [1] H. Palza, M. Nuñez, R. Bastías, K. Delgado, In situ antimicrobial behavior of materials with copper-based additives in a hospital environment, Int. J. Antimicrob. Agents 51 (6) (2018) 912–917. [2] B. Yousuf, J.J. Ahire, L.M.T. Dicks, Understanding the antimicrobial activity behind thin-and thick-rolled copper plates, Appl. Microbiol. Biotechnol. 100 (12) (2016) 5569–5580. [3] D. Hagiwara, K. Sato, M. Miyazaki, et al., The impact of earlier intervention by an antimicrobial stewardship team for specific antimicrobials in a single weekly intervention, Int. J. Infect. Dis. 77 (2018) 34–39. [4] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (2011) 1541–1547. [5] U. Brahma, R. Kothari, P. Sharma, et al., Antimicrobial and anti-biofilm activity of hexadentated macrocyclic complex of copper (II) derived from thiosemicarbazide against Staphylococcus aureus, Sci. Rep. 8 (1) (2018) 8050. [6] J.T. Ravensdale, D.T.W. Xian, C.M. Wei, et al., PCR screening of antimicrobial resistance genes in faecal specimens from Australian and Chinese children, Journal of Global Antimicrobial Resistance 14 (2018) 178–181. [7] C.L. Ventola, The antibiotic resistance crisis: part 1: causes and threats, Pharmacy and Therapeutics 40 (4) (2015) 277. [8] L. Xia, M. Xu, G. Cheng, et al., Facile construction of Ag nanoparticles encapsulated into carbon nanotubes with robust antibacterial activity[J], Carbon 130 (2018) 775–781. [9] L. Yang, F. Meng, X. Qu, et al., Multiple-twinned silver nanoparticles supported on mesoporous graphene with enhanced antibacterial activity[J], Carbon 155 (2019) 397–402. [10] B. Bagchi, S. Dey, S. Bhandary, et al., Antimicrobial efficacy and biocompatibility study of copper nanoparticle adsorbed mullite aggregates, Mater. Sci. Eng. C 32 (7) (2012) 1897–1905. [11] A.L. Casey, D. Adams, T.J. Karpanen, et al., Role of copper in reducing hospital environment contamination, J. Hosp. Infect. 74 (1) (2010) 72–77. [12] S. Niakan, M. Niakan, S. Hesaraki, et al., Comparison of the antibacterial effects of nanosilver with 18 antibiotics on multidrug resistance clinical isolates of Acinetobacter baumannii, Jundishapur Journal of Microbiology 6 (5) (2013).
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The effect of rotation accelerated shot peening on mechanical property and antimicrobial activity of pure copper Yu Suna, Zixuan Zhanga, Yuan Qina, Xuran Xub, Sen Yanga,c,
T
⁎
a
School of Materials Science and Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China School of Chemical Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China c Sino-French Engineering School, Nanjing University of Science and Technology, No. 200 Xiaolingwei, Nanjing 210094, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Copper Rotation accelerated shot peening Surface nanocrystallizition Antimicrobial activity
In this work, we report the use of rotation accelerated shot peening (RASP) to achieve surface nanostructure on copper plate. To systematically investigate the microstructure, mechanical properties, antimicrobial ability and corrosion resistance of the specimens, two kinds of RASP specimens with processing time being 5 min (RASP-5) and 15 min (RASP-15) were employed in this study. A nanostructure layer was formed in the surface of copper specimen after RASP. With prolonging shot time, the size of surface grains decreased and the micro-hardness was significantly improved. The antimicrobial ability of the RASP specimen for E. coli was better than that of the base material, while no obvious difference existed for S. aureus. In electrochemical corrosion tests, it was confirmed that RASP treatment could effectively stimulate the copper ions release from the surface of specimen to increase antimicrobial activity.
1. Introduction In our daily life, causal agent reserving on environmental surfaces is one of the main paths for the acquisition of disease infection [1]. To solve this problem, many kinds of antimicrobial agents are used to kill bacteria and protect human health. Nevertheless, misuse or overuse of antibiotics would increase antimicrobial resistance and lead to the spread of life-threatening multi-drug-resistant (MDR) pathogens, such as carbapenems-resistant Acinetobacter baumannii, carbapenems-resistant Pseudomonas aeruginosa, vancomycin-resistant, and Enterococcus faecium [2,3]. Even though the antimicrobial resistance (AMR) can be divided into intrinsic and acquired drug resistance, the latter is more harmful, since the latter is the result of long-time interaction between bacteria and antibiotics and this interaction is almost impossible to defend. In hospital, on many touch surfaces like door handles, benches and toilets, amounts of living microbes exist that leading to a high possibility of cross infection [4]. Therefore, it is urgent to look for effectively metal inorganic antibacterial materials instead, such as copper, silver, etc. [5–9]. In general, antibiotic metal can be applied to keep these touch surfaces inhibiting the breeding of bacteria and avoid the development of AMR. Among them, copper receives growing attention as intrinsically antimicrobial material because of low cost, broad spectrum, eco-friendly, long service life and reusable [10–15].
Copper had been widely used before that antibiotic was discovered to tackle a plethora of infections ranging from tuberculosis to skin diseases [16]. In 2008, copper-based surfaces supported by many antibacterial performance tests were registered as solid antimicrobial surfaces by US Environmental Protection Agency (EPA) [17]. However, the poor mechanical properties of pure copper, like hardness and wear resistance, limit its application in real life, and the antimicrobial action mechanism of the pure copper is still not clear [18]. A lot of studies show that grain refinement can effectively increase mechanical properties of metal [19–22]. Generally speaking, a higher strength can be obtained with smaller grain size according to the classical Hall-Petch relationship [21]. As assessed by Yong et al. [20], the yield strength of Al can be significantly enhanced when its grain size decreases to 36 nm. Meanwhile, the ultimate tensile strength and yield stress of the CueZn alloy increases respectively to 386 MPa and 192 MPa after the refinement of the grains, as demonstrated by H. Puga et al. [22]. Up to now, many sever plastic deformation techniques are used to construct nanostructure on the bulk materials, such as equal channel angular pressing (ECAP) [23], high pressure and torsion (HPT) [24] and accumulative roll bonding (ARB) [25]. But these techniques have certain limitation in fabricating special size and shapes. It is well known that the most of premature failures of component, such as abrasion, fatigue fracture and erosion, are related to the
⁎ Corresponding author at: School of Materials Science and Engineering, Nanjing University of Science and Technology, No.200 Xiaolingwei, Nanjing 210094, China. E-mail address: [email protected] (S. Yang).
https://doi.org/10.1016/j.surfcoat.2019.125319 Received 15 October 2019; Received in revised form 23 December 2019; Accepted 28 December 2019 Available online 31 December 2019 0257-8972/ © 2019 Elsevier B.V. All rights reserved.
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2.3. Bacterial strains and growth conditions
microstructure and its associating mechanical properties of material surface [20]. Therefore, surface modification of materials become an important route to improve various performances, such as wear, erosion, and corrosion resistance, and is more convenient and flexible at the same time. Shot peening is a well-known excellent surface treatment technique, by which nanostructured layer on base material can be designed. The previous researches show that the surface grain size of 1Cr18Ni9Ti stainless steel after shot peening can reach 18 nm [26], and the average surface grain size of Ti4Al2V processed by shot peening decreases to 35 nm [27]. Along with grain size decreasing, the mechanical properties of materials are significantly improved, for instance the maximum hardness of Ti6Al4V after shot peening reaches about 400 HV, which is much larger than that of 300 HV in base materials [28]. The fatigue strength of Cu-Ni-Si alloy after shot peening at 107 circles can even reach 280 MPa [29]. All these results claim the importance of shot peening technique in material modification. To further boost the practical application of this technique, the rotation of the impeller is applied to accelerate shots. And the specimen is spinning rapidly during processing, which makes both sides of the specimen be shot evenly. The aims of this study are using rotation accelerated shot peening (RASP) to enhance the surface performances of pure copper plate. The nanostructure on the surface and effect of antimicrobial capacity were systematically investigated.
E. coli CMCC 44102 and S. aureus CMCC 26003 were cultured aerobically on the LB solid medium (10 g Peptone, 10 g Yeast Extract, 5 g salt, 15 g Agar, 1000 mL distilled water) at 37 °C for 24 h. Then cells were transferred to 4 mL phosphate-buffered saline by inoculating loop. The average cell density was 1 × 108–3 × 108 cfu/mL. 2.4. Measurement of contact killing by wet plating Before contact killing experiments, all specimens were dipped in dilute hydrochloric acid solution and 100% ethanol, dried in clean bench and sterilized under UV light for 30 min. Briefly, 20 μL of cells suspended in phosphate buffer saline (PBS) were applied to specimens as a drop. After incubation with different durations (E. coli: 15, 60, 90 and 120 min; S. aureus: 30, 60, 120 and 180 min) in a water-saturated atmosphere, 10 μL bacterial suspension was withdrawn and serial dilutions in PBS were spread on LB solid medium. Following growth of bacteria for 24 h, colony forming units (cfu) were determined. 2.5. Differentiation between viable and dead cells To further observe the state of bacterial, the cells that exposed for different time durations (E. coli: 15 and 120 min; S. aureus: 30 and 180 min) to the surface of specimens were pipetted into centrifuge tubes. Then 2 μL green-fluorescent FDA (5 mg/mL Acetone solution of Fluorescein diacetate) and 5 μL red-fluorescent PI (1 mg/mL aqueous solution of Propidium Iodide) were added into 1 mL aliquots of cells in tube. And these tubes were kept in dark for 5 min. After that 50 μL aliquots were pipetted onto glass slides. The bacteria on the specimens were imaged by using confocal laser scanning microscope.
2. Materials and methods 2.1. Materials and specimen preparation In this experiment, the 99.97% bulk copper (TU1) plate with trace amounts of sulfur and iron was chose as study object, while AISI 304 stainless steel was used as the reference in antimicrobial test. A part of copper plates were processed by RASP after pre-rubbing. The process of RASP consisted of two steps. Firstly, 2 mm-diameter-pills were used to make specimens obtain much plastic deformation, and then 0.5 mmdiameter-pills were used to make the surfaces of specimens smoother. For comparison, the polished base materials were named as BM. All the specimens with dimensions of 10 × 10 × 3 mm3 were ultrasonic cleaned in acetone and 100% ethanol for 15 min respectively. The RASP parameters were list in Table 1.
2.6. Bacteria morphology and the internal structure The specimens for Scanning electron microscope (SEM) were prepared after 90 min immediately after killing experiments. Firstly, the specimens with bacteria were stored in glutaraldehyde solution (2.5% in PBS medium) at 4 °C more than 2 h. Secondly, they were rinsed in PBS solution and dehydrated in water-ethanol mixtures (30%, 50% 70%, 90%, 95%, 100%). Last, Au/PD was sputtered on dried specimens. The specimens were imaged using the scanning electron microscope (SEM). To observe the internal structure of damaged bacteria, SEM with Dual Beam microscope was used. Before cut by ion beam, the bacteria were locally sputtered in the SEM with an additional platinum protective layer to prevent damage during the FIB preparation. The FIB preparation was performed by applying a gradual reduction of the beam current up to a final value of 5 pA [19].
2.2. Materials characterization The grain size of base materials was measured by the electron backscattering diffraction (EBSD) with a 1.5 μm step size. The surface topography of specimens was tested by White light confocal microscope (Axio CSM 700).The three-dimensional view and surface roughness were tested by confocal three-dimensional profilometer. The surface grain size of RASP specimen was measured by the transmission electron microscope (TEM, Tecnai G2 20 LaB6). The micro-hardness was measured with a load of 100 g and loading time of 10s by the Vickers microsclerometer (HMV-G21), and three times of measurements were made at each depth.
2.7. Ion release measurements During the contact killing experiment, copper ions released from the surface of specimens to PBS solution. 20 μL aliquots were taken at specified time instants (15, 60, 90 and 120 min), diluted 500-fold (15, 60 and 90 min) and 5000-fold (120 min) with 0.065% HNO3. Then the copper content was quantified by inductively coupled plasma mass spectrometry (ICP-MS, NexION 350, PerkinElmer, America).
Table 1 Parameters of RASP.
2.8. Electrochemical corrosion
Step 1
RASP-5 RASP-15
The corrosion resistance of the copper specimens was evaluated by potentiodynamic polarization test. The corrosion potential was calculated through Tafel region extrapolation. The electrochemistry tests were performed in PBS solution at room temperature. The specimen acted as work electrode, a platinum electrode and a saturated calomel acted as counter electrode and reference electrode (SCE), respectively,
Step2
Pill size
Velocity
Time
Pill size
Velocity
Time
2 mm 2 mm
40 m/s 40 m/s
5 min 15 min
0.5 mm 0.5 mm
40 m/s 40 m/s
5 min 15 min
2
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grains are randomly distributed. The average surface grain size of RASP-5 is about 20 nm, while that of RASP-15 is 12 nm. As we demonstrated above, the refinement of microstructure come from plastic deformation induced by consecutive and high speed balls hitting on the specimen surface. Previous studies have showed the deformation twinning plays an important role in refining grains to the nanometer scale via plastic deformation [30,31]. Due to the interaction or connect of dislocations and large amounts of twin boundaries, the coarse grain is refined to a lot of twin lamellae firstly and the twin lamella is further refined to equiaxed nano-grains [32,33]. 3.2. Micro-hardness after RASP Fig. 5 shows the variations of micro-hardness from the top surface to the substrate of all specimens. There is an obvious tendency that microhardness of RASP specimens decrease rapidly within the top 200 μm thick layer. When the depth is more than 200 μm, the decrease becomes slow and then disappears. The continuously decreasing in hardness with depth is related to the increasing grain size, which means a gradient structure is formed from surface to matrix. Besides, this result confirms that surface grains absorb the majority of impact energy of shots and the residual compressive stress is produced on the surface. At the same time, the hardness curve of RASP-15 is much gentler than that of RASP-5. It implies that with prolonging shot time, more grains get refined, namely that the hardened layer thickens. It is observed that the surface hardness of RASP specimens reach 118HV0.1 and 121HV0.1 respectively and both of them are significantly higher than that of BM.
Fig. 1. Orientation distribution map of the base materials by EBSD.
forming the three-electrode cell. Three parallel tests were performed on each kind of specimens. 3. Results and discussion 3.1. Surface roughness and grain size
3.3. Antimicrobial activity
Fig. 1 shows the orientation distribution map of the base materials. As we can see, the majority of grains present coarse and equiaxed with some twin lamellae. The average grain size is about 85 μm figured out by EBSD. With RASP modifying the surface, the surface morphology and microstructure of the specimens have been changed as shown in Fig. 2. It can be found that the surface becomes rough, and many hemispheric concaves form. This is owing to the serious plastic deformation resulted from the repeated peening at high strain rates. Here, it is worth to note that we vary the RASP time for further optimize its effect. RASP-5 and RASP-15 represent the duration time of RASP being 5 min and 15 min, respectively. Fig. 3 shows the threedimensional images of the specimen surfaces after RASP. The surface of RASP-5 is obviously rougher than that of RASP-15 with the average roughness (Ra) being 26.8 μm and 15.9 μm for RASP-5 and RASP-15, respectively. The reason can be attributed to that with prolonging of the treatment time, much more plastic deformation is produced in the specimen and concaves that formed on the surfaces by shots overlap and override each other. Plastic deformation was generated on the top surface layer of RASP specimens, where grains were too fine to be observed directly with optical microscope. Fig. 4(a) and (b) show the TEM bright field images of the surface layers of RASP specimens. A large number of nanometersized grains can be observed and crystallographic orientations of these
Fig. 6 shows the comparison of bacteria viability on different specimen surfaces. Two kinds of bacteria were used, one was E. coli (Gramnegative) and the other was S. aureus(Gram-positive). Under wet conditions, there is no significant difference of the killing efficiency between RASP-5 and RASP-15 to E. coli (Fig. 6a). The concentration of bacteria was set as 1 × 108–3 × 108 cfu/mL. After 120 min, the highest killing efficiency with 3 logs was achieved on the RASP specimens. Meanwhile, the antimicrobial ability of BM specimen was relatively poorer with only 2 logs reduction of cells over 120 min. As control, the stainless steel exhibited no significant antimicrobial effect in the experiment. As stated in several papers [8,11,34,35], like silver ions, the copper ions have ability to destroy the structure of cell membranes and affect the membrane permeability. And the change of membrane permeability can cause the leakage of cytoplasm. Here, we believe the better antimicrobial ability of RASP specimens can be assigned to the high activity of surface atoms. The surface nanostructrue makes these atoms more active and it means that copper ions are more easily released into solution during the same time. Fig. 6(b) shows the survival of S. aureus on all the specimens. Obviously, the viability of S.aureus was stronger compared to E. coli. The killing efficiency of RASP specimen to S. aureus is low with 2 logs after
Fig. 2. The surface topography of a) RASP-5 and b) RASP-15. 3
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Fig. 3. Three-dimensional view and surface roughness (Ra) of (a) RASP-5 and (b) RASP-15 were obtained by confocal three-dimensional profilometer. Scans were carried out in an area of 4 × 4 mm.
180 min. At the same time, the number of live bacteria on BM specimen is similar to that on specimens treated by RASP. Here, one of antimicrobial mechanisms is proposed as follow to illustrate the difference. Cu ions enter the cell through cell wall and change the conformational structure of nucleic acids and proteins. As is known, the cell wall of S. aureus (20–80 nm) is much thicker than that of E. coli (10–15 nm). With the same Cu ion release rate and the same contact area (PBS solution with cells/specimen surface), S. aureus is more resistant than E. coli. As a consequence, although the activity of copper atoms on the surfaces of BM specimens and RASP specimens is different, the difference is not enough to have a greatly impact on the survival of S. aureus on the specimen surfaces. The different colors of live and dead cells are showed in Figs. 7 and 8. The live cells fluoresce green due to FDA, which has the ability to get into both live and dead bacteria. After it is catalyzed by non-specific esterase into fluorescein in cells, green fluorescence of 530 nm can be inspired by laser at 480 nm. The dead cells whose membranes are not intact fluoresce red due to PI, which can just enter the bacteria with incomplete membranes. Because when the membrane is not intact, FDA will spread beyond the cell while PI can combine with DNA. When it is excited by laser at 488 nm, red fluorescence will be inspired. In Fig. 7, cells of E. coli are mostly stained green after 15 min of exposure on specimens and a lot of cells are dyed red while being exposed for 120 min, especially for exposed to RASP-5 and RASP-15. The red cells exposed on the surface of BM are distinctly less than that on other specimens. Combining the analysis of Fig. 6, the results state adequately that the activity of E. coli on RASP specimens is much poorer than that on BM specimens. Fig. 8 shows that most cells of S. aureus are dyed green after 30 min and only a part of cells are dyed red after 180 min. Besides, the numbers of red cells in (b), (d) and (f) are almost the same. Since incomplete membrane is the premise that PI can get into a
Fig. 5. Variation curves of micro-hardness of RASP-5, RASP-15 and BM with distance from topmost to substrate.
cell, the result of the staining experiment indicates that contacting between bacteria and copper can denature the cell walls and damage the integrity of membranes.
3.4. The effect of copper ions on bacteria morphology and internal structure Fig. 9 shows the morphologies of live and dead bacteria on the copper specimens. In Fig. 9(a), the middle of the cell that is marked in red box presents cave and it means that the cell has died. In Fig. 9(b), the integrity of the bacteria structure which is marked in red box is
Fig. 4. (a) TEM graph of surface grains of RASP-5 and (b) RASP-15. 4
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Fig. 6. Survival of cells given in colony forming units (cfu) is measured by wet plating on surfaces of all specimens. (a) E. coli cells and (b) S. aureus cells.
cytoplasm can be drained out of broken membrane. These results state that membrane-damaging of cells caused by copper ions is an important mechanism in contact killing. Thus the release of copper ion requires a special focus.
damaged and cytoplasm escapes. The reason is that the cell wall and membrane are broken by copper ions and cell contents escape. The redox properties of copper ions can lead to depletion of sulfhydryls which exist in some kinds of amino acids of membrane [36]. Besides, reactive hydroxyl radicals are generated in redox reaction and can further take part in the oxidation of proteins. Fig. 10 shows the internal structures of bacteria on copper and stainless steel specimens. Compared with the intact structure of the bacteria on stainless steel in Fig. 10(b), the damage of bacteria on copper is apparent with hollowed out. This indicates that much of
3.5. Release of copper ion The concentration of cupric ions/cuprous ions keeps increasing when copper contacts with bacterial suspension until the dynamic equilibrium is reached [35]. This process could be pertinent under the
Fig. 7. Cells of E.coli were respectively exposed to (a)(b)BM, (c)(d)RASP-5 and (e)(f)RASP-15 copper plates and then stained by FDA and PI. Images were taken with a fluorescent microscope. Live bacteria with integrated membranes fluoresced green and bacteria with broken membranes fluoresced red. Cells were exposed for (a) (c)(e) 15 min and (b)(d)(f)120 min. 5
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Fig. 8. Cells of S. aureus were respectively exposed (a)(b) to BM, (c)(d)RASP-5 and (e)(f)RASP-15 copper plates and then stained by FDA and PI. Images were taken with a fluorescent microscope. Live bacteria with integrated membranes fluoresced green and bacteria with broken membranes fluoresced red. Cells were exposed for (a)(c)(e)30 min and (b)(d)(f)180 min.
surfaces of RASP specimens corresponds to the higher antimicrobial activity.
wet plating conditions to evaluate contact killing. Therefore, copper ion release measurement was performed. In the measurements, PBS buffer solution was used instead of bacterial suspension for instructions of the equipment. As shown in Fig. 11, the concentration of copper ions in PBS buffer solution on BM is lower than others after 10 min later. The concentration of copper ions of RASP-5 is basically in line with that of RASP-15. The relatively higher concentration of copper ions means that copper after RASP is more easily to corrode under the conditions of free corrosion as used in antimicrobial experiments. It is obvious that the nano-structure surface layers on RASP-5 and RASP-15 specimens have important impact. The overall faster release of copper ions from the
3.6. Electrochemical corrosion The release of copper ions is related to surface corrosion properties. In order to investigate the effect of RASP treatment on the corrosion behavior of pure copper, the corrosion resistance properties were assessed by Electrochemical tests. Fig. 12 shows the potentiodynamic polarization curves of specimens in 0.01 M PBS solution at room temperature. All specimens displayed a typical active–passive–transpassive
Fig. 9. SEM graph of (a) E. coli cells and (b) S. aureus cells on the pure copper after 90 and 120 min of killing experiment respectively. 6
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Fig. 10. Internal structure of E. coli bacteria on: (a) BM specimen after 90 min and (b) stainless steel specimen after 90 min.
behavior. As listed in Table 2, the corrosion potential (Ecorr) of RASP specimen shifts negatively with grain size decreasing. Since the corrosion resistance is mostly related to the size of grains on specimen surface, RASP treatment realizing the nano-sized grains make it more effective. The number of grain boundaries on surface becomes greater with grain size decreasing, and the higher energy of atoms at grain boundaries makes the atoms unstable. As shown in Fig. 12, the passive regions appear on the anodic branches. This means that the passive films formed on specimens immersed in PBS solution. As seen in Table 2, the specimen-to-specimen variations in the passivation current densities are noticeable. Meanwhile, the BM specimen exhibits wider passive regions than the RASP does. It suggests that the passive films forming on RASP specimens are broken more quickly and passive films have better protective ability to retard corrosion without RASP treatment. It is attributed to the surface atoms, especially intergranular atoms of the RASP specimens which have higher activity [37]. These results agree well with that of ion release test basically and support the concept that the release of copper ions is a pivotal parameter in contact killing.
Fig. 11. The concentration of copper ions in PBS buffer solution on different surfaces. A certain amount of liquid was removed respectively after 10 min, 60 min, 90 min and 120 min.
4. Conclusion In this study, pure copper was treated by rotation accelerated shot peening with two sets of parameters. Our findings clearly demonstrated the enhancement of mechanical and antimicrobial properties of copper could be achieved after the surface treatment. Besides, the other main conclusions are listed as follows: (1) Nano-structure surface formed on copper after RASP and the average grain size decreased from 85 μm to 20 nm (RASP-5) and 12 nm (RASP-15) respectively. The hardness of RASP-5 and RASP15 was gradually decreasing with depth. The max hardness of RASP-15 reached 121HV0.1, while that of BM was just 50HV0.1. Dislocations piling up at the grain boundary and sub-grain boundary which leads to dislocation motion hindered improve the hardness. (2) Under wet conditions, the activity of E. coli on RASP-5 and RASP-15 was poorer than that on BM, while the dead speed of S. aureus did not have much difference on all surfaces of specimens. The different activity of two kinds of bacteria on RASP specimens is due to the different structures of cell walls. Membrane-damaging by copper ions is an important mechanism in contact killing. (3) In the electrochemical measurement, RASP-5 and RASP-15 had more negative corrosion potential (Ecorr) and narrower passive regions than BM, Which meant grain refinement produced more unstable atoms to make corrosion resistance decrease.
Fig. 12. Typical potentiodynamic polarization curves of polished copper, shot peening treated RASP-5 and RASP-15 in PBS solution. Table 2 Electrochemical data for specimens. Specimen
Grain size
Ecorr (mV)
Ip(μA·cm−2)
Ef (mV)
Etr (mV)
BM RASP-5 RASP-15
38 μm 20 nm 12 nm
−95.69 −115.88 −124.35
2.23 6.93 20.69
−19.37 −7.88 4.38
381.41 169.47 133.38
Author contributions SEN YANG was in charge of project design and theoretical guidance. SUN YU was in charge of specific experimental design, most of experiments operation, data processing and paper writing. 7
Surface & Coatings Technology 384 (2020) 125319
Y. Sun, et al.
ZIXUAN ZHANG was in charge of microhardness test. YUAN QIN provided revisions of the paper. XURAN XU was in charge of shooting fluorescence photos of germs.
[13] D.J. Weber, D. Anderson, W.A. Rutala, The role of the surface environment in healthcare-associated infections, Curr. Opin. Infect. Dis. 26 (4) (2013) 338–344. [14] L. Weaver, J.O. Noyce, H.T. Michels, et al., Potential action of copper surfaces on meticillin-resistant Staphylococcus aureus, J. Appl. Microbiol. 109 (6) (2010) 2200–2205. [15] C.D. Salgado, K.A. Sepkowitz, J.F. John, et al., Copper surfaces reduce the rate of healthcare-acquired infections in the intensive care unit, Infect. Control Hosp. Epidemiol. 34 (5) (2013) 479–486. [16] V. Villapún, L. Dover, A. Cross, et al., Antibacterial metallic touch surfaces[J], Materials 9 (9) (2016) 736. [17] M. Rosenberg, H. Vija, A. Kahru, et al., Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces, Sci. Rep. 8 (1) (2018) 8172. [18] M. Hans, J.C. Támara, S. Mathews, et al., Laser cladding of stainless steel with a copper–silver alloy to generate surfaces of high antimicrobial activity, Appl. Surf. Sci. 320 (2014) 195–199. [19] R.Z. Valiev, A.P. Zhillyaev, T.G. Langdon, Grain refinement in metallic systems processed by ECAP, Bulk Nanostructured Materials: Fundamentals and Applications (2014) 239–288. [20] Y. Liu, B. Jin, J. Lu, Mechanical properties and thermal stability of nanocrystallized pure aluminum produced by surface mechanical attrition treatment, Mater. Sci. Eng. A 636 (2015) 446–451. [21] H.Y. Guo, M. Xia, Z.T. Wu, et al., Nanocrystalline-grained tungsten prepared by surface mechanical attrition treatment: microstructure and mechanical properties, J. Nucl. Mater. 480 (2016) 281–288. [22] H. Puga, J. Barbosa, J.M. Machado, et al., Ultrasonic grain refinement of die cast copper alloys, J. Mater. Process. Technol. 263 (2019) 336–342. [23] R.Z. Valiev, R.K. Islagaliev, I.V. Alexandrov, Bulk nanostructured materials from severe plastic deformation, Prog. Mater. Sci. 45 (2) (2000) 103–189. [24] I.V. Alexandrov, R. Valiev, X-ray analysis of bulk nanostructured metals, Mater. Sci. Forum 321 (2000) 577–582. [25] S. Roy, B.R. Nataraj, S. Suwas, et al., Accumulative roll bonding of aluminum alloys 2219/5086 laminates: microstructural evolution and tensile properties, Mater. Des. 36 (2012) 529–539. [26] T. Wang, J. Yu, B. Dong, Surface nanocrystallization induced by shot peening and its effect on corrosion resistance of 1Cr18Ni9Ti stainless steel, Surf. Coat. Technol. 200 (16–17) (2006) 4777–4781. [27] J. Han, S. GM, H. GX, Nanostructured surface layer of Ti–4Al–2V by means of high energy shot peening, ISIJ Int. 48 (2) (2008) 218–223. [28] Q. Yang, W. Zhou, Z. Niu, et al., Effect of different surface asperities and surface hardness induced by shot-peening on the fretting wear behavior of Ti-6Al-4V, Surf. Coat. Technol. 349 (2018) 1098–1106. [29] O. Stav, H. Kasem, R. Akhvlediani, et al., Simultaneous shot-peening of hard and soft particles for friction reduction in reciprocal sliding, Tribol. Int. 130 (2019) 19–26. [30] K. Wang, N.R. Tao, G. Liu, et al., Plastic strain-induced grain refinement at the nanometer scale in copper, Acta Mater. 54 (19) (2006) 5281–5291. [31] Y.S. Li, N.R. Tao, K. Lu, Microstructural evolution and nanostructure formation in copper during dynamic plastic deformation at cryogenic temperatures, Acta Mater. 56 (2) (2008) 230–241. [32] A. Amanov, Y.S. Pyun, S. Sasaki, Effects of ultrasonic nanocrystalline surface modification (UNSM) technique on the tribological behavior of sintered Cu-based alloy, Tribol. Int. 72 (2014) 187–197. [33] W.L. Li, N.R. Tao, K. Lu, Fabrication of a gradient nano-micro-structured surface layer on bulk copper by means of a surface mechanical grinding treatment, Scr. Mater. 59 (5) (2008) 546–549. [34] G. Borkow, J. Gabbay, Copper as a biocidal tool, Curr. Med. Chem. 12 (18) (2005) 2163–2175. [35] M. Hans, A. Erbe, S. Mathews, et al., Role of copper oxides in contact killing of bacteria, Langmuir 29 (52) (2013) 16160–16166. [36] S.L. Warnes, C.W. Keevil, Inactivation of norovirus on dry copper alloy surfaces, PLoS One 8 (9) (2013) e75017. [37] W. Luo, C. Qian, X.J. Wu, et al., Electrochemical corrosion behavior of nanocrystalline copper bulk[J], Mater. Sci. Eng. A 452 (2007) 524–528.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This study was supported by the Fundamental Research Funds for the Central Universities (30919011412 and 30919011255), PAPD, and Jiangsu Key Lab of Micro-nano Materials and Technology. Data availability The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations. References [1] H. Palza, M. Nuñez, R. Bastías, K. Delgado, In situ antimicrobial behavior of materials with copper-based additives in a hospital environment, Int. J. Antimicrob. Agents 51 (6) (2018) 912–917. [2] B. Yousuf, J.J. Ahire, L.M.T. Dicks, Understanding the antimicrobial activity behind thin-and thick-rolled copper plates, Appl. Microbiol. Biotechnol. 100 (12) (2016) 5569–5580. [3] D. Hagiwara, K. Sato, M. Miyazaki, et al., The impact of earlier intervention by an antimicrobial stewardship team for specific antimicrobials in a single weekly intervention, Int. J. Infect. Dis. 77 (2018) 34–39. [4] G. Grass, C. Rensing, M. Solioz, Metallic copper as an antimicrobial surface, Appl. Environ. Microbiol. 77 (2011) 1541–1547. [5] U. Brahma, R. Kothari, P. Sharma, et al., Antimicrobial and anti-biofilm activity of hexadentated macrocyclic complex of copper (II) derived from thiosemicarbazide against Staphylococcus aureus, Sci. Rep. 8 (1) (2018) 8050. [6] J.T. Ravensdale, D.T.W. Xian, C.M. Wei, et al., PCR screening of antimicrobial resistance genes in faecal specimens from Australian and Chinese children, Journal of Global Antimicrobial Resistance 14 (2018) 178–181. [7] C.L. Ventola, The antibiotic resistance crisis: part 1: causes and threats, Pharmacy and Therapeutics 40 (4) (2015) 277. [8] L. Xia, M. Xu, G. Cheng, et al., Facile construction of Ag nanoparticles encapsulated into carbon nanotubes with robust antibacterial activity[J], Carbon 130 (2018) 775–781. [9] L. Yang, F. Meng, X. Qu, et al., Multiple-twinned silver nanoparticles supported on mesoporous graphene with enhanced antibacterial activity[J], Carbon 155 (2019) 397–402. [10] B. Bagchi, S. Dey, S. Bhandary, et al., Antimicrobial efficacy and biocompatibility study of copper nanoparticle adsorbed mullite aggregates, Mater. Sci. Eng. C 32 (7) (2012) 1897–1905. [11] A.L. Casey, D. Adams, T.J. Karpanen, et al., Role of copper in reducing hospital environment contamination, J. Hosp. Infect. 74 (1) (2010) 72–77. [12] S. Niakan, M. Niakan, S. Hesaraki, et al., Comparison of the antibacterial effects of nanosilver with 18 antibiotics on multidrug resistance clinical isolates of Acinetobacter baumannii, Jundishapur Journal of Microbiology 6 (5) (2013).
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