key: cord-0825374-ic9b2qxb
authors: Ren, Guangyu; Huang, Lili; Hu, Kunling; Li, Tianxin; Lu, Yiping; Qiao, Dongxu; Zhang, Haitao; Xu, Dake; Wang, Tongmin; Li, Tingju; Liaw, Peter K.
title: Enhanced Antibacterial Behavior of a Novel Cu-bearing High-Entropy Alloy
date: 2022-02-08
journal: J Mater Sci Technol
DOI: 10.1016/j.jmst.2022.02.001
sha: 8ffc15a2e1f041f0820cbe90300ce8ca3241b394
doc_id: 825374
cord_uid: ic9b2qxb

Contact infection of bacteria and viruses has been a critical threat to human health. The worldwide outbreak of COVID-19 put forward urgent requirements for the research and development of the self-antibacterial materials, especially the antibacterial alloys. Based on the concept of high-entropy alloys, the present work designed and prepared a novel Co(0.4)FeCr(0.9)Cu(0.3) antibacterial high-entropy alloy with superior antibacterial properties without intricate or rigorous annealing processes, which outperform the antibacterial stainless steels. The antibacterial tests presented a 99.97% antibacterial rate against Escherichia coli and a 99.96% antibacterial rate against Staphylococcus aureus after 24 hours. In contrast, the classic antibacterial copper-bearing stainless steel only performed the 71.50% and 80.84% antibacterial rate, respectively. The results of the reactive oxygen species analysis indicated that the copper ion release and the immediate contact with copper-rich phase had a synergistic effect in enhancing antibacterial properties. Moreover, this alloy exhibited excellent corrosion resistance when compared with the classic antibacterial stainless steels, and the compression test indicated the yield strength of the alloy was 1015 MPa. These findings generate fresh insights into guiding the designs of structure-function-integrated antibacterial alloys.

antibacterial copper-bearing stainless steel only performed the 71.50% and 80.84% antibacterial rate, respectively. The results of the reactive oxygen species analysis indicated that the copper ion release and the immediate contact with copper-rich phase had a synergistic effect in enhancing antibacterial properties. Moreover, this alloy exhibited excellent corrosion resistance when compared with the classic antibacterial stainless steels, and the compression test indicated the yield strength of the alloy was 1015 MPa. These findings generate fresh insights into guiding the designs of structure-function-integrated antibacterial alloys.

Sessile microorganisms on abiotic surfaces and the microbiologically-influenced corrosion (MIC) have caused negative effects and represented considerable costs in different fields, such as health and medical-care occupations, food processing, and home-appliance industries [1] [2] [3] .

occupies the first place among human-related bacterial infections, and Staphylococcus aureus (S. aureus) takes the second position 4 . The most effective way to deal with the aforementioned challenge is to inhibit bacterial adherence and biofilm formation. In such a context, huge attention has been attached to antibacterial materials.

Copper (Cu) has been used for medical applications due to its essential cofactor of several enzymes and antibacterial activity 3 . Previous studies proved that the antimicrobial mechanisms of Cu ions included membrane damage 5 , oxidative stress 6 , and protein denaturation 7 .

Significantly, these were mainly metabolism-independent mechanisms that could minimize the development of bacterial resistance. Despite its strong bactericidal effect, the main disadvantage was that the pure Cu and Cu alloys might have a Cu-burst phenomenon at the early stage when used in implant materials, resulting in pain and excessive menstrual blood loss 8 . Therefore, several types of Cu-containing stainless steels (SS), such as 316L, 317L, and 304L [9] [10] [11] , and Ti-Cu 12-13 alloys have been designed, which have shown reliable antibacterial activity against common pathogens. However, the aforementioned alloys require complicated heat treatments to obtain the antibacterial property, and the physical, chemical, and mechanical behavior of Cucontaining SS and Ti-Cu alloys are susceptible to the Cu element. For example, the plasticity and corrosion resistance of Ti-Cu alloy decrease when the Cu element is added, and the plasticity of the Ti-10Cu alloy reported in the literature 14 is only 1%. Thus, the boundedness of traditional antibacterial alloys limits its development prospects.

Since 2004, high-entropy alloys (HEAs) [15] [16] have attracted considerable interest in different application scenarios due to their outstanding physical, chemical and mechanical properties [17] [18] [19] [20] .

Gradually, the integrating structures and functions of HEAs have been raised in recent years 20 The antibacterial property of Cu-HEAs on MIC applications and biomedical materials has been validated because of their higher Cu content and convenient preparation process. However, the mechanical properties and antibacterial mechanism of Cu-HEAs need to be improved and clarified.

In this study, Co 0.4 FeCr 0.9 Cu x (x = 0.3 and 0.5) Cu-HEAs (denoted as the Cu0.3 alloy and Cu0.5 alloy, respectively) were designed and prepared. The effect of the Cu element on the microstructures, mechanical properties, corrosion resistance, and antibacterial behavior was studied systematically, and the antibacterial mechanism of Cu in HEAs was explored. The mechanical properties and antibacterial behavior of Cu-HEAs in the as-cast condition were comprehensively evaluated to verify its potential to be applied to materials for civil household appliances and medical instruments.

The Cu-HEAs ingots were fabricated by vacuum-arc melting under a high-pure argon atmosphere. Nonequal molar ratios of commercially-pure Co, Cr, Cu (99.9 weight percent, wt.%), and Fe (99.5%, wt.%) were put into a water-cooled copper crucible. Each ingot was re-melted and flipped six times to ensure chemical homogeneity. The furnace chamber was first evacuated to 6 × 10 −3 Pa and then backfilled with purity argon gas to reach 0.05 MPa. Microstructures and chemical compositions of HEAs were analyzed, using a field emission electron probe micro analyzer (EPMA, JEOL JXA-8530F Plus) equipped with a wavelength-dispersive spectrometer (WDS). The phase constitution of the Cu-bearing HEAs was probed by an X-ray diffractometer (XRD, PANalytical Empyrean) with a Cu-Kα target and a scanning step of 4°/min.

Antibacterial properties of the HEAs were compared with the traditional 304 SS and 304-Cu SS (3.5 wt.% Cu). The specimens used for antibacterial testing were cut from the as-cast ingots of a size, 10 × 10 × 2 mm 3 . A schematic representation of the experiment can be viewed in the Supplementary Figure 1 . Metal coupons were subsequently put in an autoclave to sterilize at 121℃ for 20 min. Gram-negative E. coli and gram-positive S. aureus were activated by cultivation in a Luria-Bertani broth in advance at 37℃ for 18 h. Overnight cultures with a 10 5 colony-forming unit/mL (CFU/mL) concentration in the sterile phosphate-buffered saline (PBS) were prepared. First, 1 mL of the bacterial culture was co-cultured with three coupons for each metal tested in a sterilized 24-well culture plate. Cultures containing each metal were incubated at 37℃ for 2, 6, 12, 24, and 48 h. Then, the metals were taken out and suspended in 1 mL of sterile PBS. The adhered bacteria were obtained, followed by ultrasonication for 5 min. to isolate from metal surfaces. The planktonic bacteria were removed from the remaining solutions in the 24-well plate. Planktonic bacteria and adhered bacteria were serially diluted in the sterile physiological water (0.85 wt.% NaCl). Each dilution with a volume of 100 μL was cultured on the Luria-Bertani agar plates at 37℃ for 24 h. Then, the antibacterial rates (%) were calculated as follows:

where N ctrl represents the number of planktonic bacteria co-cultured with 304 SS or adhered bacteria on the 304 SS surface (control), and N anti denotes the number of planktonic bacteria cocultured with 304 SS or the number of adhered bacteria on the 304-Cu SS or HEA coupon surface.

The live and dead bacteria adhered to the surfaces of HEAs were stained, using a LIVE&DEAD Bacterial Staining Kit (Yisheng, Shanghai, China) in a dark environment at RT.

Live bacteria with intact cell membranes were stained with green-fluorescent DMAO, and dead bacteria with damaged cell membranes were stained with red-fluorescent EthD-Ⅲ

. DMAO and EthD-Ⅲ were mixed to observe the live (green) and dead (red) bacteria. Metals were removed from the E. coli and S. aureus suspensions after incubation at 37℃ for 6, 12, and 24 h and the samples were washed with sterile PBS to remove the nonadherent bacteria. The mixtures of DMAO and EthD-Ⅲ

were prepared with a volume ratio of 1:2, and then 10 μL of the mixture was added into the 24-well plate containing 1 mL of PBS and 100 μL of bacterial suspension in the dark for 15 min. Finally, a confocal laser scanning microscope (CLSM, OLYMPUS FV3000) was used to identify the live and dead bacteria 9 .

The 

The scanning electron microscope (SEM, Zeiss Supra 55) was used to observe the E. coli 

Room-temperature compression tests were performed, employing an Instron 5569 testing machine with a strain rate of 1 × 10 -3 s -1 at room temperature. The samples were cut into Ф5 × 10 mm directly from the ingot.

The data shown as the mean ± standard deviation (SD) was obtained by at least three independent experiments and analyzed by the GraphPad Prism software 2 . The statistical significance of observed differences was analyzed by two-way ANOVA test 2 . Fig. 1 shows the X-ray diffraction patterns of the Cu0.3 and Cu0.5 alloys. The XRD patterns of the as-cast Cu0.3 and Cu0.5 alloys show that both HEAs have a simple face-centered-cubic (FCC) and body-centered-cubic (BCC) structure. Fig. 2 (a)-(d) illustrate the backscattered electron (BSE) images of the two alloys. Both alloys had a matrix phase with dispersed precipitates (indicated in Fig. 2(b) ). Through the corresponding elemental distribution images (WDS mapping) of Cu0.3 alloys (Fig. 2(e) ), the regions with brighter contrast were Cu-rich phases. The Cu-rich phase and matrix contained 92 at.% and 3 at.% Cu, respectively, as presented in Supplementary Table 1 . On the contrary, Co, Cr, and Fe elements were distributed in the matrix homogeneously. 

The bacterial suspensions of E. coli and S. aureus were co-cultured with 304 SS, 304-Cu SS, Cu0.3, and Cu0.5 alloys for 2, 6, 12, 24, and 48 h. The results of live/dead cell staining were seen in Fig. 4 . The bacterial growth on the four different coupons after 6, 12, and 24 h was also used to study the antibacterial properties. Fig.   4 (a) shows that more live E. coli (green fluorescence) were observed on the 304 SS and 304-Cu after 24 h. Similar results for S. aureus indicated that both Cu0.3 and Cu0.5 alloys had an obvious bactericidal effect on these bacteria in the as-cast condition. The E. coli and S. aureus cultured on samples were observed, using an SEM (Fig. 5) . After 24 h of co-culture, 304 SS samples were covered by many rod-shaped and spherical bacteria, which presented as confluent colonies ( Fig. 5(a) , (e)). On the contrary, few colonies of E. coli and S. aureus were found on the Cu0.3 (Fig. 5(c) , (g)) and Cu0.5 (Fig. 5(d), (h) ) samples, and almost no complete bacterial cells were present on the aforementioned two alloys, which shows effective inhibition of the Cu0.3

and Cu0.5 alloys. The ion-release results of the 304-Cu SS, Cu0.3, and Cu0.5 alloys were investigated to explore whether Cu ions could be released to play a bactericidal role. Fig. 6 (a)-(c) presented the Based on the results that the Cu ions measured in the Cu0.5 alloy were all lower than that in the Cu0.3 alloy, the increased Cu element did not contribute to its release process. Fig. 6(b), (c) show that the Cu ions in the Cu0.3 and the Cu0.5 exceeded that in the 304-Cu SS at 6 h, and then reached twice as much as that in the 304-Cu SS at 12 h. The available data explained that the significant bactericidal behavior of the Cu0.3 and Cu0.5 was directly related to the concentration of Cu ions. Compared with the Cu-releasing rate in the DI water ( Fig. 6(a) ), the adequate supplies of Cu ions should result from the corrosive environments in the bacterial suspension, which ensured the continuous release and contributed to the long-term sterilization. displayed as mean ± SD and analyzed by the GraphPad Prism software 2 .

In addition, Cu ions catalyzed oxidases and produced ROS effectively 9 , which was proved to be highly toxic to most bacteria. According to Fig. 6(d) , the ROS contents produced when cocultured with 304-Cu SS, Cu0.3, and Cu0.5 alloys were 0.17, 0.62, and 0.38 μmol/mL, respectively.

In order to study the corrosion resistance of the Cu0.3 and Cu0.5 alloys, electrochemical measurements, immersion experiments and neutral salt spray tests were carried out, as shown in Fig. 7 . The potentiodynamic-polarization curves of the Cu-HEA samples indicated an increased corrosion potential after the Cu addition. Table 1 shows the corrosion potential (E corr ) values of Cu0.3 and Cu0.5 alloys were 115 mV and 111 mV, respectively, exhibiting a nobler corrosion potential than the 304 SS and 304-Cu SS (153 mV and 135 mV, respectively). Meanwhile, a similar corrosion current density (i corr ) when compared with 304 SS was seen in both Cu0.3 and Cu0.5 alloys. In addition, the corrosion rates calculated from the immersion tests were (0.0056 ± 0.0002), (0.0048 ± 0.0005), (0.0061 ± 0.0003), and (0.0058 ± 0.0003) mm/y for 304 SS, 304-Cu SS, Cu0.3, and Cu0.5 alloys, respectively. Hence, there was no significant difference in terms of the corrosion rate (as shown in Supplementary Figure 4 ). Fig. 8 shows the results of the neutral salt spray tests after 7 days, the images of the alloys all had a normal silver-grey lustre, and there were no obvious corrosion pits on the surfaces of the alloys. The aforementioned results amply proved that the corrosion resistance of the present work was similar to that of the 304 SS alloy. is still better than that of the traditional antibacterial stainless steel, as shown in Fig. 9 (b).

As mentioned earlier, the existence of Cu-rich precipitates and a large amount of Cu ions released in the microenvironment guaranteed the excellent bactericidal behavior of Cu-HEAs in the as-cast station. Bacterial adhesion and biofilm formation were strongly inhibited with the help of the antibacterial properties of Cu ions, and Nan et al. has confirmed the electrostatic forces of Cu 2+ can inhibit the adhesion force of bacteria directly, then the cell walls are damaged and contents in the cells leaked 39 . The possible antibacterial mechanism in this study was discussed (Fig. 10) . On the one hand, the Cu0.3 and Cu0.5 alloys were regarded as iron-based metals due to the influence of the composition element ratio. The higher standard electrode potential of Cu in the microenvironment 8 Moreover, the proton pump on the cell membrane continuously transfers H + produced in the respiratory chain, forming a potential difference of H + , which can stimulate the synthesis of adenosine triphosphate (ATP) to supply energy. Zhang et al. 41 supposed that the electron produced in the microenvironment consumes the H + , and the proton gradient will be destroyed.

So that the interruption of the ATP synthase ultimately kills the bacteria. Hence, as shown in Fig.   10 , the contact killing induced by Cu ions and the proton consumption exerted a synergistic antibacterial effect, and thus the excellent broad-spectrum antibacterial properties of Cu-HEAs were confirmed.

To conclude, the present work proposed and fabricated a novel Cu-bearing AHEA and the antibacterial behavior of the alloys were investigated. In this study, the as-cast Co 0.4 FeCr 0.9 Cu 0.3 alloy could achieve a high antibacterial rate without any intricate or rigorous annealing processes, which is more concise and efficient, and quite distinguished from the classic antibacterial stainless steels. With the help of Cu ions, Cu-bearing HEAs can be a promising alloy-type antimicrobial material because of the contact killing and the proton consumption mechanism, and have the potential to meet the needs of structure-function-integrated antibacterial alloys.

The authors declare no competing financial interests.

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

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: 

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