Abstract

The antimicrobial properties of copper are well-known but maintaining a low oxidation state of Cu in particles is difficult. Herein, antimicrobial CuxP particles were synthesized through phosphorization of Cu(OH)2, to lock copper in its monovalent state (as Cu3P). We found that the phosphorization could be achieved at temperatures as low as 200°C, with stable surface presence of Cu(I) on the resulting CuxP particles. Cu(I) can act as a one-electron reducing agent for molecular oxygen, to generate the highly reactive hydroxyl radical. In this study, CuxP displayed antibacterial activities on the Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with minimum inhibitory concentrations of 32 mg/L for the highest temperature particles (350°C) on both model bacteria. The evident membrane damage is consistent with the intended hydroxyl radical bacterial targeting mechanism. Low-temperature CuxP, although exhibiting lower antibacterial efficacies than those of the higher temperature variant, still showed competitive growth inhibiting activities when compared to other reported antimicrobial copper-based particles. The present work showcases advancements in particle technology that can lead to the development of a more robust antimicrobial agent, presenting a potent additive for self-disinfection applications.

1. Introduction

The world is now facing a pandemic-level infection, with pathogens capable of spreading via inanimate objects and surfaces. Research efforts have been increasingly focussed to address this so-called fomite transmission, in particular on the development of self-disinfecting materials. The application of antimicrobial agents on surfaces can reduce the risk of fomite transmission of pathogens in household and hospital settings, with the present work focusing on copper (Cu)-based antimicrobial particles. When compared to other metals, copper-based particles are cheaper to produce, with faster leaching rates, to release the toxic copper ions, including in biological systems [13]. Various copper-based antimicrobial particles have been developed, showing both in vitro and in vivo antibacterial activities [1]. For instance, metallic copper particles have been shown to exhibit bacterial-killing activities, with studies observing the particle apparent physical interactions with bacterial membranes, compromising their integrity [4]. Copper oxide (CuO) particles, on the other hand, have been indicated to exert different antibacterial mechanism, with the so-called Trojan horse-type toxicity. The mechanism involves intracellular leaching of copper ions following particle penetration into cells, leading to cell death and/or growth inhibition [47]. Copper-based particles have shown efficacies on Gram-positive and Gram-negative bacteria, both on their free-living planktonic and surface-attached biofilm forms of growth [47]. Research inquiries have further described the effects of particle characteristics on their antibacterial activities. Applerot et al. [5] found that smaller CuO particles (∼2 nm) were associated with stimulation of a more intense oxidative stress, and therefore, a more effective antibacterial with higher extent of cell killing when compared to the larger particles (∼30 nm). Studying the particle shapes, Laha et al. [8] reported higher cell-killing effects of nanosized spherical CuO on Gram-negative bacteria (Proteus vulgaris and Escherichia coli), whereas nanosheets of CuO were more effective on Gram-positive bacteria (Bacillus subtilis and Micrococcus luteous).

Copper has also been combined with other metals to form antimicrobial alloys [9]. Zhou et al. [10] reported the cell-killing activities of Cu2O–ZrP hybrid nanosheets on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus. The antibacterial effects were shown to correlate with the indicated oxygen radical targeting of bacterial membranes [10]. Likewise, Shalom et al. [7] reported the cell-killing activities of Zn-doped CuO nanoparticles (deposited on catheters) on urinary tract infection-causing bacterial pathogens (E. coli, S. aureus, and P. mirabilis). Combining Cu with silver has shown synergistic antibacterial effects; whereby presence of silver was indicated to increase the bacterial membrane permeability, increasing the growth inhibitory activities of the alloy to up to eight fold (on E. coli and B. subtilis), when compared to copper alone [11]. Metal ions have been indicated to interact with electronegative groups that are present in membrane phospholipids, with the interactions being linked, at least in part, to increasing membrane permeability and in turn, influx of the ions into bacteria to further disrupt cellular functions, including DNA synthesis [11]. A recent study by Tomina et al. [12] reported a significant increase in the antibacterial (on both Gram-positive and Gram-negative bacteria) and antifungal activities of mono- and bifunctional silica microspheres following doping with Cu(II), which are suggested to correlate with the oxygen radical-generating Fenton-like copper redox cycling (see below), leading to oxidative attack on membranes. Similarly, in a study by Naz et al. [13] Cu(II)-doped O-Carboxymethyl chitosan (OCMC) showed higher antibacterial activity on E. coli and B. subtilis in comparison to the OCMC alone. Another study by Wilks et al. [14] observed higher cell-killing rates (on E. coli) with increasing copper content in alloys with nickel, brass and steel, indicating major antimicrobial contribution from copper.

Copper antimicrobial applications, up to this stage, have explored the use of mainly Cu(II) and Cu(0)-based materials. For instance, quite recent work from our group found that a Cu(II) complex embedded within a poly(vinyl chloride) matrix could effectively inhibit the growth of surface-attached biofilms of nitrifying bacteria. The copper complex system generated nitric oxide, a quorum sensing inhibitor, via Cu(II)/Cu(I) redox cycling reactions when in the presence of nitrite and ascorbic acid (note that quorum sensing is a cell-to-cell communication signalling that allow controls of specific biological processes, including biofilm formation and adaptation to external stressors) [15]. Another example is a 3D printed Cu(0)-based self-disinfecting surfaces [16]. For the latter, a study by Champagne and Helfritch [3] demonstrated the cell-killing activities of three copper-based surfaces; developed by the deposition of Cu(0) using plasma spray, wire arc spray, and cold spray; on MRSA [3]. The cold spray technique, which results in the development of copper microstructure morphologies with enhanced diffusion of the toxic copper ions (more specifically, the high-velocity particle impact with the cold spray technique led to high grain dislocations density within the copper deposit, which in turn, increases copper ion diffusion in the metal), displayed the highest antibacterial activities [3]. Another study by Noyce et al. [17] observed a complete killing of MRSA suspensions (107 colony forming unit per mL) when exposed for 90 min at 22°C room temperature to Cu(0) surfaces. Although studies have reported the antimicrobial activities of Cu(I)-based materials [1, 2, 1820], the use of Cu(I) however, is often hindered by its labile nature [15]. Cu(I)-containing antimicrobial systems have rarely been explored without an additional matrix support. Herein, the present work aims to develop a hydroxyl radical-generating antimicrobial copper particles, exploiting the relatively high redox potential of Cu(I). We synthesized Cu(I)-rich CuxP particles via facile phosphorization to help stabilize the surface presence of Cu(I) for a redox-based hydroxyl radical generation and studied their antimicrobial effects on model Gram-positive and Gram-negative bacteria. With a lower redox potential, Cu(I) species (Cu(I)/Cu(II) of 0.153 V) is more favorable than Cu(0) species (Cu(0)/Cu(II) of 0.342 V, Cu(0)/Cu(I) of 0.521 V) to induce the one-electron reduction of molecular oxygen (O2) to form oxygen radicals [19]. The reactions generate superoxide radical from O2 (non-Fenton, reaction 1). The then undergoes a proton-coupled electron transfer reaction to form H2O2 (Reaction 2), and ultimately, the Fenton-like reaction to form the highly reactive hydroxyl radical (Reaction 3) [21, 22].

Molecular oxygen reduction reactions:

Phosphorization of copper particles allows the locking of Cu in its monovalent state, potentially maintaining a high concentration of Cu(I) on the particle surface [23]. Cu(OH)2 particles were first synthesized (by precipitation from a saturated ammonia solution), followed by phosphorization at different calcination temperatures. The phosphorization process led to the formation of Cu3P (Cu(I) state) and CuP2 (Cu(II) state), the latter more prevalent at higher calcination temperatures (≥300°C). Our study found that the CuxP particles exhibit competitive growth inhibiting activities when compared to other copper-based particles on the model bacteria. Further, we present studies elucidating the origins of the CuxP antibacterial activities, including the hydroxyl radical-mediated targeting.

2. Results

2.1. Synthesis and Characterisation of CuxP Particles

Phosphorization of Cu(OH)2 was carried out in the presence of NaH2PO2 (under Ar atmosphere, NaH2PO2 decomposes to PH3(g) at ≥200°C) at different calcination temperatures of 150–350°C to obtain CuO150, CuP200, CuP250, CuP300, and CuP350 particles. The extent of phosphorization was examined with X-ray diffraction (XRD, Figure 1(a)). The XRD patterns of the calcined particles showed gradual shifts in chemical composition with increasing temperatures. At 150°C, the samples existed as CuO and Cu2O, suggesting the dehydration and partial reduction of Cu(OH)2. The oxides formation most likely resulted from direct redox reaction of Cu(OH)2 with NaH2PO2, as the calcination temperature (150°C) was not sufficiently high for the NaH2PO2-to-PH3 decomposition. At 200 and 250°C, the CuO and Cu2O transformed to Cu3P (Cu(I) state), along with CuP2, the latter in small amounts (Figure 1(b)). The eutectic formation of CuP2 became significant at ≥300°C (eutectic formation herein refers to the formation of CuP2 as part of a solid-solution phase change of Cu3P/CuP2 mixture), with 30% of the calcined particles present as CuP2 at 300°C, and ∼45% at 350°C. It is worth noting that the formation of CuP2 (Cu(II) state) is undesirable as it cannot act as a reducing agent for the intended formation of oxygen radicals.

Using X-ray photoelectron spectroscopy (XPS), we next analyzed the chemical states of Cu of the prepared particles (6 months after synthesis). The particle surface analysis was to also assess for the stable presence of the redox-active Cu(I) species. The Cu LMM spectra of the calcined particles (Figure 1(c)) showed shifts in the binding energies of the peaks with increasing temperatures, corresponding to the complex transitions of Cu(OH)2 to CuO, Cu3P, and CuP2 (as well as Cu3(PO4)2) (identified according to the work of Biesinger [24]). The presence of Cu LMM peak at 919.2 eV for CuO150 corresponds the surface presence of CuO, whereas the peaks at 917.6 eV for CuP200 to CuP350 calcined particles most likely correspond to the surface presence of the Cu(I) state Cu3P species (Figure 1(c)). Note that no zero-valent Cu peak was seen in the Cu LMM spectra of the CuP200 to CuP350 particles, which indicates that the occurrence of Cu 2p peaks at 932.8 eV for the particles correspond solely to Cu(I) state (Figure S1). The observations indicate stable Cu(I) surface presence in these particles. For the higher temperature CuP300 and CuP350 calcined particles, the Cu LMM peaks at 916.5 eV most likely correspond to the surface presence of CuP2 species (Figure 1(c)), which is consistent with the occurrence of the Cu(II) state peaks at 934.7 eV with the Cu 2p spectra (Figure S1). The (surface) detection of Cu3P and CuP2 are consistent with the appearance of symmetric P 2p peaks at 129.5 eV and 133.3 eV for the CuP200 to CuP350 particles (Figure S2). Further analysis also showed that a fraction of the (surface) Cu(II) species was present as Cu3(PO4)2 for the CuP350 particle, as indicated by the occurrence of asymmetric P 2p peaks at 133.7 eV and 129.8 eV (Figure S2e). Also note that at lower calcination temperatures (≤250°C), some of the (surface) Cu was still present as Cu(OH)2, as indicated by the presence of O 1 s peak at 533.1 eV (Figure S3).

To further understand the phosphorization process, we examined the particle size, morphologies, and aggregation. The electron transmission micrographs of CuO150 (with bulk composition of CuO, Cu2O as well as Cu(OH)2) showed spherical particles of <10 nm primary size (Figure 1(d)). Following the calcination at higher temperatures (≥200°C), sintering of the particles occurred with significant (∼100 nm increments) increase in primary size, with the elevated temperatures, with the particles no longer retaining the spherical morphologies (the sintering was a result of reaction between CuO and PH3(g), the latter was from NaH2PO2 decomposition). The measured surface area corroborated with the TEM micrographs of the particles (Figure 1(i)). The specific surface area of CuO150 was determined at 129 m2/g, and after phosphorization, gradually decreasing to 30 m2/g with the increasing temperatures (CuP200 to CuP350). The phosphorization, however, only caused minimal change in the particle “overall” aggregate size, with the increasing temperatures. The CuP200 to CuP350 particles (with bulk composition of mainly Cu3P and CuP2) fused, forming large aggregates (1,000–2,000 nm, Figure 1(e)1(h)). Next, the hydrodynamic size of the calcined particles was studied via dynamic light scattering (DLS). As shown in Table 1, the average sizes of the particles are all within ∼350–550 nm range (excluding CuO150 with ∼100 nm size) when dispersed in water, with polydispersity indexes of 0.40–0.65. Again, we observed no significant impact of the calcination temperature on the hydrodynamic aggregate sizes. The colloidal stability of the CuP300 was further examined in PBS or tris–HCl, the latter used for the antibacterial studies. The average hydrodynamic sizes of CuP300 increased to 2.2 µm and 1.2 µm, respectively, in PBS and tris–HCl, indicating further aggregation and wider size distribution (0.29 (PBS) and 0.31 (tris–HCl) polydispersity index) in the buffer solutions, when compared to the water system. The aggregation was most likely attributed to the presence of relatively high concentrations of anions and counter cations in the buffer solutions, creating a charge shielding effect which neutralises long-range electrostatic interactions, in turn facilitating inter-particle interactions [25]. Smaller CuP300 aggregates were also likely to form in the tris–HCl system (relative to those in the PBS), due to the steric hindrance effects imposed by adsorbed tris molecules [26]. The zeta potential measurement showed a net negative surface charge of the CuxP (CuP200 to CuP350) particles in PBS (−20.8 to −27.1 mV). The different calcination temperatures did not seem to affect the zeta potential.

2.2. Oxygen Radical Formation by CuxP Particles

The coumarin test was used to assess the hydroxyl radical () generation of the CuO150 and CuxP (CuP200, CuP250, CuP300, CuP350) particles. Coumarin reacts with to form the fluorescent 7-OH-coumarin (450 nm) [27]. As shown in Figure 2(a), the CuO150 particle did not generate radicals, with essentially no detection of the fluorescence signal. This is consistent with the XRD and XPS data for CuO150 (Figure 1), showing that the particle is entirely composed of CuO, Cu2O (and Cu(OH)2), with no surface presence of Cu(I) species. The CuxP (Cu3P and CuP2) particles CuP200, CuP250, and CuP300 generated radicals, and at comparable extent, as shown by the overlapping 7-OH-coumarin fluorescence intensity detected over time (Figure 2(a)). The observations are in line with the similar ∼50% surface Cu(I) molar ratios (relative to Cu(II)) being estimated for CuP200, CuP250, and CuP300 from the XPS Cu 2p spectra (Figure 2(b), Figure S1). The surface Cu(I) molar ratio decreased to 30% for CuP350. Interestingly, the coumarin response for CuP350 was apparently the highest when compared to the lower temperature CuxP particles, indicating the most extensive generation. It is still unclear at this stage however, as with the underlying reasons for the highest extent of the radical generation for the CuP350 particles.

2.3. Antibacterial Efficacies of CuxP Particles and Mechanistic Studies

The antibacterial effects of the CuO and CuxP particles (CuO150, CuP200, 250, 300, 350) were assessed on a Gram-positive model bacterium S. aureus and a Gram-negative model bacterium E. coli. As shown in Figure 3, all the CuO and CuxP particles exhibited dose-dependent toxicity on S. aureus and E. coli. For example, exposures of S. aureus to increasing 8–256 mg/L CuP200 concentrations inhibited the growth of the bacterium, from ∼80% extent of biomass growth (relative to the cell-only control growth) at 8 mg/L particle exposure to ∼20% growth at 32 mg/L particle concentration, then ultimately to <5% growth at 256 mg/L concentration. Comparable trends were observed with E. coli, with ∼95% growth at 8 mg/L CuP200 exposure to <5% growth at 256 mg/L particle concentration. Among the particles, CuP350 showed the highest growth inhibition effects with minimum inhibitory concentration (MIC, for ≥95% growth inhibition) of 32 mg/L for both S. aureus and E. coli, whereas CuO150 showed the lowest effects (MIC of >256 mg/L) (Figure 3). For CuP200, CuP250, and CuP300, the MICs were determined at 64 mg/L for both S. aureus and E. coli. These CuO and CuxP MICs are lower when compared to the previously studied copper-based antibacterial particles, although the much larger sizes of the particles (∼100–2000 nm CuO and CuxP aggregates, Figure 1(d)1(i), Table 1). Sharma and Kumar [28] reported an MIC of 391 mg/L with CuO particles (d = 5–9 nm) on E. coli, whereas Gunawan et al. [29] reported a significantly higher CuO MIC of 900 mg/L (d = 14 nm) on E. coli. In another study, Argueta-Figueroa et al. [30] reported a 100 mg/L MIC with metallic Cu0 particles (d = 4 nm) on S. aureus and E. coli. The apparent higher extent of growth-inhibiting activity observed in the present study is thought to result, at least in part, from the unique antibacterial mechanisms of the particles, as next described.

The antibacterial activities of our copper particles are most likely to primarily originate from the earlier described redox generation of hydroxyl radical (). The levels of the growth-inhibiting activities are consistent with the extent of the radical generation. CuP350 with the lowest MICs (32 mg/L) on S. aureus and E. coli, produced the highest amount of radical, followed by CuP200, CuP250, and CuP300 with less formation and evidently, higher MICs (64 mg/L) on the bacteria (Figures 2(a) and 3). CuO150 with undetectable formation seemed to only “reach” MICs for both bacteria at >256 mg/L concentration. Hydroxyl radical is the most reactive oxygen radical, with research inquiries already establishing its reactivity on biomolecules. The one-electron oxidant has been known to cause peroxidation of phospholipids in bacterial cell envelopes (present in the inner membrane of S. aureus, and in the outer and inner membranes of E. coli) [31]. Initiated by abstraction of an allylic hydrogen atom, the peroxidation modifies lipids into lipid hydroperoxides. The radical can also cleave phosphate esters in phospholipids [31, 32]. These radical attacks on phospholipids, in many cases, have been known to result in leaky membranes [33, 34]. Herein, we stained the particle-exposed E. coli samples with AM1-43 fluorescent dye to probe the cell membranes phospholipid moieties [35, 36]. Indeed, leaky membranes were observed in the E. coli population for all tested particles (Figure 4). Note the less fluorescent (green) membranes of the particle-exposed bacterial samples when compared to the cell-only control. The leaky membrane is also indicated by an influx of the dye into cells, with the evident green fluorescence mass inside the particle-exposed samples, which is absent in the cell-only control [37, 38].

The phospholipid fluorescence staining was also performed on the particle-exposed S. aureus samples, however, with inconclusive results (Figure S4). This rather expected observation is likely to result from the presence of relatively thick, outermost peptidoglycan layer in the Gram-positive cell envelope, hence limiting the dye penetrability to reach the inner membrane. Regardless, earlier nanoparticle studies have shown that peptidoglycan is also prone to hydroxyl radical attack [31]. The radical can damage amide bonds that are present in the glycan strands (more specifically, in the amino sugars and peptide moieties) of peptidoglycan. Studies have also indicated hydroxyl radical attack on teichoic acid, another major cell envelope component of Gram-positive bacteria; with the radical targeting the C=O ester, phosphate ester, and amide bonds in the molecule [31]. The known hydroxyl radical targeting on Gram-positive cell envelope components could explain the similar antibacterial activities herein observed between the Gram-positive and Gram-negative models. Taken together, the indicative observations of hydroxyl-radical mediated cell envelope damages, shown with membrane phospholipids in the present work, are in agreement with the growth-inhibiting effects of the CuP350, along with the less potent CuP200, CuP250, and CuP300 particles. The radical is also known to target base pairing in DNA and RNA, as well as introducing covalent modifications in amino acids, such as the sulfur-containing cysteine and methionine, in turn inactivating functional proteins [3943].

A more detailed investigation revealed other sources for the antibacterial activities, apart from the redox hydroxyl radical generation. This is particularly apparent with the dose-dependent growth inhibition effects seen with the nonhydroxyl radical-producing CuO150 particle (Figure 3(a)). Our leaching studies found that the particle almost completely dissolved (∼80% relative to total copper content, measured in MHB culture medium, pH 6), releasing soluble copper into the exposure systems (Figure S5). The CuO150 is composed of CuxO with mainly CuO on its surface (Figure 1). Studies have reported relatively high extent of soluble copper leaching from CuO particles in organic-containing media due to complexation-mediated leaching [29]. Previous work on copper-based particles have established the antibacterial roles of the leached soluble copper, which stimulate oxidative stress in cells, being linked to Cu(II) ion targeting of proteins [29, 43]. A borderline Lewis acid, the ion has high affinities to donor groups in amino acid side chains, such as the –NH+ (in imidazole ring), –NH3+, and thiol (–S) groups in histidine, lysine, and cysteine, respectively, forming complexes with the amino acids in proteins [44]. The ion can also disrupt the iron–sulfur (cysteine) clusters that are present in many physiologically essential biosynthetic and catabolic enzymes, releasing the Fenton-active Fe(II) ion, with the latter further reacting with cellular H2O2 to form hydroxyl radical [45]. Cu(II) ion has also been indicated to target cell envelopes, disrupting functional groups in peptidoglycan (peptidoglycan is also present in Gram-negative bacteria, as thin layer in between the outer and inner membranes) and phospholipids, the latter affecting membrane permeability [46, 47]. The implied leached soluble Cu-mediated cellular hydroxyl radical generation and cell envelope targeting are consistent with the damaged membrane observation, herein also evident with the CuO150-exposed bacterial samples (Figure 4(b)).

The soluble copper leaching could also contribute to the growth-inhibiting effects of the higher temperature CuxP particles. The CuP350 (composed of ∼55% Cu3P and ∼45% CuP2, with surface presence of Cu3P, CuP2, and Cu3(PO4)2, Figure 1) and CuP250 (∼90% Cu3P and ∼10% CuP2, with surface presence of mainly Cu3P and Cu(OH)2, Figure 1) particles had similar extent of soluble copper leaching (∼7% relative to total copper, Figure S5), although much less when compared to CuO150 (∼80% leaching relative to total copper). In addition to the different particle surface composition, the lower leaching could result from the larger aggregate size of the higher temperature CuxP particles (Table 1) [5]. Finally, the data also suggest a potential antibacterial contribution from the solid particulates that remain after leaching. This is evident with the CuP300 bacterial exposures, with already ∼40%–60% growth inhibition effects manifesting on both S. aureus and E. coli at the lowest particle dosage (8 mg/L, Figure 3(d)), despite the only moderate redox hydroxyl radical generation (Figure 2(a)) and leaching of soluble copper (Figure S5). Research inquiries have reported that copper particles can adhere onto bacterial membranes through electrostatic interaction, which leads to membrane damage and in some case, penetration of the particles into the cytoplasm [4850].

3. Conclusion

Herein, we developed CuxP particles for antibacterial purposes, with stable surface presence of Cu(I) species intended for the redox generation of the highly reactive hydroxyl radical. The particles were synthesized via a temperature-dependent phosphorization of Cu(OH)2, with particle surface analysis confirming the stable presence of the Cu(I) state Cu3P species. The phosphorization process, however, led to sintering effects and increased the particle size. Despite the larger particle size, our studies with Gram-positive and Gram-negative bacteria models showed a significantly higher extent of antimicrobial activities when compared to other copper-based particles. The CuxP particles were found to generate hydroxyl radical, most likely involving one-electron reduction of molecular oxygen by the surface Cu(I), leading to the observed inhibition effects on bacterial growth, with further evidence of cell membrane targeting. In summary, the CuxP particles with their ability to use molecular oxygen to generate radicals, as well as, apparently, their relatively low extent of copper leaching, present a promising particle technology for alternative antimicrobial applications, in particular for the development of self-disinfecting surfaces, to slow down the fomite transmission of pathogens.

Data Availability

The data used to support the findings of this study are included within the article and supplementary information file.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Funding

Open access publishing facilitated by University of Technology Sydney, as part of the Wiley - University of Technology Sydney agreement via the Council of Australian University Librarians.

Acknowledgments

The work was supported by the Australian Research Council Discovery Project DP200103587 (Z. Gu) and DP180100474 (C. Gunawan). The authors would also like to acknowledge the use of the facilities within the UNSW Mark Wainwright Analytical Centre and the UoW Electron Microscopy Centre.

Supplementary Materials

Figure S1: Cu 2p XPS spectra of (a) CuO150 and phosphorized CuxP: (b) CuP200, (c) CuP250, (d) CuP300, and (e) CuP350. Figure S2: P 2p XPS spectra of (a) CuO150 and phosphorized CuxP: (b) CuP200, (c) CuP250, (d) CuP300, and, (e) CuP350. Figure S3: O 1s XPS spectra of (a) CuO150 and phosphorized CuxP: (b) CuP200, (c) CuP250, (d) CuP300, and (e) CuP350. Figure S4: Fluorescence micrographs of fluorescent dye (FM4-64) stained S. aureus after exposure to CuxP particles: (a) Blank, (b) CuO150, (c) CuP250, and (d) CuP350. Figure S5: Solubility of CuO and CuxP particles in (a) MHB buffer solution at pH 6 and (b) deionized water. (Supplementary Materials)