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[Akiko Yamamoto](https://orcid.org/0000-0002-9451-8147), [Yasushi Suetsugu](https://orcid.org/0000-0002-8161-1908)

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[Enhancement of copper antiviral activity with glutathione treatment](https://mdr.nims.go.jp/datasets/282642c6-0793-445e-ab71-213a55855e16)

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mt6c00520 1..12www.acsabm.org ArticleThis article is licensed under CC-BY 4.0Downloada NATL INST FOR MATLS SCIENCE (NIMS) on June 24, 2026 at 05:16:26 (UTC).See https:.acs.org/sharingguidelines for options on how to legitimately share published articles.Enhancement of Copper Antiviral Activity with Glutathione TreatmentAkiko Yamamoto,* Masanori Kikuchi, and Yasushi SuetsuguCite This: ACS Appl. Bio Mater. 2026, 9, 5237−5248 Read OnlineReceiRevisAccep© 2026 The Authors. Publishedby American Chemical SocietyPubli5237ACCESS Metrics & More Article Recommendationsved: March 18, 20ed: May 11, 202ted: May 26, 202shed:    June 3, 2026Supporting InformationABSTRACT: Copper and its alloys have antimicrobial activity effective for various pathogens. Their application to touch surfacessuccessfully reduced bioburden in hospital intensive care units but failed to completely prevent the transmission of pathogens derivedfrom the hospital room. Discoloration of pure copper with surface oxide growth is another issue to discourage its application to touchsurfaces. Common copper alloys have relatively high resistance to discoloration, but their antimicrobial activity is lower than that ofpure copper. Therefore, enhancement of the antimicrobial activity of copper alloys is beneficial for their touch surface application. Inthis study, glutathione was employed for surface treatment of copper and its alloys to enhance their antiviral activity. Treatment with4 mM glutathione in 99 vol % ethanol−1% H2O markedly enhanced copper and its alloys’ antiviral activities against bacteriophage Qβexcept MONEL, which has the lowest Cu content (33.4 wt %). Electrochemical impedance measurement under a thin electrolyte filmrevealed acceleration of copper and its alloys’ corrosion by the glutathione treatment. The antiviral activity of tested materials with theglutathione treatment correlated well with their corrosion rate, except MONEL. Potentiodynamic and chronopotentiometry measure-ments in 6 M KOH + 1 M LiOH demonstrated reduction in the thickness of the surface oxide layer by the glutathione treatment.These facts suggest that the glutathione treatment reduces the surface oxide layer, resulting in acceleration of corrosion with anincrease in Cu ion release, which enhances antiviral activity.KEYWORDS: antiviral tests, copper alloys, glutathione, electrochemical impedance spectroscopy, bacteriophage Qβed vi//pubs1. INTRODUCTION the application of copper and its alloys on hospital touch sur-2666Infection prevention and control is still one of the fundamentalissues for hospitals and healthcare facilities. A risk of pathogentransmission is reported from a previously occupying patientvia a hospital room.1 To reduce this risk, sterilization of theroom is attempted by irradiation of ultraviolet light or hydrogenperoxide vapor.2,3 They are effective to reduce the infection ofClostridioides difficile or Vancomycin resistant Enterococci(VRE) in some degree, but failed to perfectly eliminate theinfection derived from hospital rooms.2,4 Therefore, there is stilla need for the technologies to prevent the pathogen transmis-sion via touch surfaces or other hospital environments.Copper and its alloys are well-known to have antimicrobialactivity, the so-called “contact killing”, which is continuouslyeffective without energy supply for a variety of pathogens suchas Gram-positive/-negative bacteria, fungi, and enveloped andnonenveloped viruses.5−8 Therefore, many studies attemptedfaces, demonstrating their successful reduction on bioburdenin the hospital room as well as healthcare-associated infection(HAI).9−14 Nine studies reported a significant reduction in totalmicrobial burden on copper alloy surfaces from those on con-trol surfaces (37−100% in reduction), and the meta-analysisof 3 studies resulted in the reduction of HAI by 26% (44 to3% in a 95% confidential interval).10 Though many clinicalstudies reported its success on bioburden reduction, the applica-https://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://doi.org/10.1021/acsabm.6c00520https://pubs.acs.org/doi/10.1021/acsabm.6c00520?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsabm.6c00520?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acsabm.6c00520?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acsabm.6c00520?goto=supporting-info&ref=pdfhttp://www.acsabm.org?ref=pdfhttps://creativecommons.org/licenses/by/4.0https://creativecommons.org/licenses/by/4.0https://creativecommons.org/licenses/by/4.0https://acsopenscience.org/researchers/open-access/https://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org Articletion of copper and its alloys to high touch surfaces is still limitedin numbers. One of the reasons may be discoloration of copperin ambient conditions. The metallic copper surface is coveredby its oxides (a mixture of Cu2O and CuO), which keep grow-ing in thickness, loosening the metallic copper appearance.15Copper alloys have better resistance against discoloration, buttheir antimicrobial activity depends on copper content in thealloy and is lower than that of pure copper.6,16Another reason to discourage the application of copper andits alloys may be the reaction time of their antimicrobial activityin the real hospital environment. Generally, copper and itsalloys require half an hour to several days to kill the microbescontacting to their surface depending on the conditions andtypes of microbes.5 This is relatively faster than other antimicro-bial materials (such as commercially available antimicrobialproducts made of resin containing antimicrobial reagents).16However, it may be not quick enough to prevent pathogentransmission via touch surfaces. Enhancement of the antimicro-bial activity of copper and its alloys enables more efficient pre-vention of HAI via touch surfaces.For the process of “contact killing” on a copper surface, thefollowing 4 stages are proposed:5 (1) corrosion of the coppersurface with copper ion release, (2) depolarization and ruptureof the cell membrane, (3) generation of reactive oxygen species(ROS) induced by copper ions, and (4) deoxyribonucleic acid(DNA) degradation. The importance of corrosion reactionand copper ion release is confirmed by the uptake and accumu-lation of copper ions inside the bacterial cells with the very fastreaction when contacting the copper surface.17 Therefore, oneof the strategies to improve the antimicrobial activity of coppermaterials is to increase their corrosion rate.For the ROS generation involving copper ions, the follow-ing reactions (1−3) are suggested;5 Cu2+ reacts with a sulfhy-dryl group (RSH), such as in cysteine or glutathione,transforming into Cu+, which generates hydrogen peroxide(H2O2) and hydroxyl radicals (OH·) by reacting with oxygenand H2O2, respectively.2 Cu2+ + 2 RSH→2 Cu+ +RSSR + 2 H+ (1)2 Cu+ + 2 H+ +O2→2 Cu2+ +H2O2 (2)Cu+ +H2O2→Cu2+ +OH− +OH· (3)The generation of Cu+ will increase ROS generation, causingsevere damage to microbes. On the copper surface, Cu2O is ini-tially formed, followed by CuO at a longer exposure time.15,18This native oxide layer is not self-protective; oxidation con-tinues at room temperature.15 The water solubility of CuO islower than Cu2O, agreeing with the higher toxicity of the lat-ter.18,19 We hypothesized that an appropriate supply of the sulf-hydryl substrate to the reaction place of copper with microbesshould increase Cu+ to enhance antimicrobial activity.Glutathione (γ-glutamylcysteinylglycine, abbreviated asGSH), is a low-molecular-weight tripeptide containing sulfhy-dryl groups. It is one of the common antioxidants widely dis-tributed in plants, animals, and microbiomes.20 Theconcentration of GSH leaches as high as 0.5−10 mM in animalcells,20 suggesting its relatively low toxicity for humans.Therefore, we selected GSH as a sulfhydryl source.5238In the present study, we investigated the surface treatment ofpure copper and 5 kinds of copper alloys with GSH to enhancetheir antimicrobial activity focusing on the antiviral effect. Weselected an alcohol−water solvent system as the treatment solu-tion, emulating an alcohol disinfectant to be a simple and easyone, which enables repetitive application to copper high touchsurfaces as needed. We decided to focus on antiviral activityagainst nonenveloped virus, bacteriophage Qβ, which is notdestroyed by alcohol. Antiviral tests were performed followingthe Japanese Industrial Standard (JIS) R1706:2020,21 which isthe base document of updating ISO 18061:2014 “Fine Ceramics(Advanced Ceramics, Advanced Technical Ceramics)—Determination of antiviral activity of semiconducting photocata-lytic materials—Test method using bacteriophage Q-beta”, with-out ultraviolet (UV)-light irradiation.Electrochemical impedance measurement was also per-formed to investigate the effect of GSH treatment on corrosionof copper and its alloys. We selected this method to mimic thesimilar condition to bacteriophage Qβ application to the coppersurface, under a thin solution layer. Based on the obtainedresults, the correlation between corrosion acceleration and anti-viral activity enhancement by GSH treatment was discussed.2. MATERIALS AND METHODS2.1. Testing MaterialsMaterials used were oxygen-free copper (C1020) and 5 kinds of cop-per alloys (C5191, C7150, C2680, Constantan, and MONEL400).Their chemical compositions are described in Table S1. The foils(0.03−0.1 mm in thickness) of these materials were purchased fromthe Nilaco Corporation (Tokyo, Japan). All materials were cut into20 mm squares for an antiviral assay. Prior to the assay, the specimensurfaces were cleaned with a detergent (a mixed solution of sodiumα-dodecan-1-yl-ω-(sulfonatooxy)poly(oxyethylene) and fatty acid alka-nolamide), followed by rinsing thoroughly with running ultrapurewater and air-drying. The bottom surface of each specimen was cov-ered with a thin silicone film (0.1 mmt, AS ONE, Osaka, Japan) toavoid the contact to the collecting solution of surviving viruses. Thetop surface was cleaned with a lint-free wiper containing absolute eth-anol (abbreviated as EtOH, ethanol 99.5, guaranteed reagent,FUJIFILM Wako Pure Chemical, Osaka, Japan).For electrochemical impedance spectroscopy, the copper and alloyfoils of 0.1 mm thickness were cut into pieces of 10 mm width and15 mm length. One side of the foil was covered by a 50 μm-thick pol-yimide insulating tape (PIA220, 3 M Japan, Tokyo, Japan). Then, twopieces of the same material were layered with the covered side facinginward and were vertically embedded into low-viscosity resin(27−777, Refine Tech, Yokohama, Japan) using a round mold with a25.4 mm diameter. The two cross sections of 10 mm length and0.1 mm width appeared on the resin top surface in parallel with agap of 0.1 mm, as shown in Figure S1. The specimen surface waspolished by SiC paper up to #1200, followed by rinsing thoroughlywith running ultrapure water and air-drying. The periphery of the resinwas covered by the insulating tape making a rim of 0.5 mm in height.Prior to glutathione treatment (described in the following paragraph),the specimen top surface was plasma-treated with a hydrophilic treat-ment device (HDT-400, JEOL DATUM, Tokyo, Japan) for 120 s ata diamond knife mode to help the wetting of resin with a glutathionesolution.2.2. Surface Treatment with GlutathioneGlutathione (reduced form, Wako Special Grade, abbreviated as GSH)was purchased from FUJIFILM Wako Pure Chemical. A 0.123 g por-tion of GSH was dissolved with 1 mL of ultrapure water to prepare400 mM solution. Then, an appropriate portion of the 400 mMhttps://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org ArticleGSH solution was mixed with ethanol and ultrapure water to prepare 1,2, and 4 mM GSH in 1 vol % H2O−99 vol % EtOH or 4 mM GSH in99−80 vol % EtOH. Since GSH is not soluble into EtOH, 4mM is themaximum concentration in 99 vol % EtOH absent of precipitation. TheGSH solution was prepared on the same day of applying to the speci-men surface.Each of the specimens for the antiviral assay was placed on the bot-tom of a glass dish individually. Then, a 5 μL portion of the GSH treat-ment solution was poured and spread on the specimen surface using amicropipette tip. Since the specimen was a 20 mm square, the hypo-thetical thickness of the GSH solution spread on the specimen surfacewas calculated as 1.25 μm. Due to its high ethanol content, the appliedportion of the treatment solution was promptly dried within 5 min atambient temperature. Then, the specimen was placed in the safety cab-inet at ambient temperature for certain periods of time (1, 3, 24, and168 h) prior to the antiviral test.For electrochemical impedance measurement, a 13 μL portion ofthe 4 mM GSH in 99 vol % EtOH was poured and spread on eachspecimen (parallel electrodes embedded in resin) immediately afterits hydrophilic treatment using a micropipette tip. Since the specimendiameter was 25.4 mm, the hypothetical thickness of the GSH solutionspread on the specimen surface was calculated as 2.57 μm. The speci-men was air-dried at ambient temperature overnight prior to the elec-trochemical impedance measurement.2.3. Antiviral Assay Using Bacteriophage QβThe procedure of the antiviral assay is schematically shown inFigure S2. The assay is based on the JIS R1706:202021 without irradi-ation of UV light. Bacteriophage Qβ (NBRC20012) and its host bacte-rium, Escherichia coli (E. coli, NBRC106373), were obtained from theNational Institute of Technology and Evaluation (Tokyo, Japan) andprepared with the following protocols21 as briefly described below.Experiments were performed in duplicate.2.3.1. Preparation of Bacteriophage Stock Suspension. Aportion of stored E. coli on agar slants of Luria−Bertani (LB) broth(LB agar “DAIGO”, SHIOTANI M.S., Amagasaki, Japan) was addedto calcium-containing LB (CaLB), which contains 9.9 g/L peptone,5.0 g/L yeast extract, 9.9 g/L NaCl, and 0.29 g/L CaCl2·2H2O, pre-pared by addition of CaCl2·2H2O (guaranteed reagent, FUJIFILMWako Pure Chemical) to LB (LB broth “DAIGO”, SHIOTANIM.S.). The bacterial suspension was cultured at 37 °C with shakingat 110 ± 10 rpm for 18 ± 2 h. Then, a portion of this bacterial suspen-sion was transferred into the new batch of CaLB at the ratio of 1/1000and cultured at the same condition to obtain the bacterial density of2 × 108 cfu/mL (reaching an absorbance of ca. 0.12 at 650 nm witha 0.63 cm path length). Then, bacteriophage Qβ was added to thebacterial suspension to be roughly 1/10 of the bacteria, that is, ca.2 × 107 pfu/mL. It was cultured at the conditions previously describedfor 4 h. Then, the bacteriophage-infected bacterial suspension wastransferred and stored overnight in the fridge (4 °C). The solutionwas centrifuged at 10,000 g for 20 min at 4 °C. The supernatant wascollected and filtered through a membrane filter with a pore size of0.22 μm (DISMIC-25SS, ADVANTEC TOYO KAISHA, Tokyo,Japan) and stored at −80 °C. The virus infectivity titer was decidedprior to the antiviral assay.2.3.2. Preparation of E. coli for the Plaque Assay. A portionof stored E. coli was added into CaLB and cultured for 20 ± 4 h at37 °C. Then, this bacterial suspension was transferred into a new batchof CaLB at the ratio of 1/10 and cultured for 6−7 h at 37 °C to obtainthe bacterial density of 0.5−2.0 × 109 cfu/mL (reaching an absorbanceof ca. 0.4 at 650 nm with a 0.63 cm path length).2.3.3. Preparation of Bacteriophage Loading Suspensionand Application. The stock suspension of the bacteriophage Qβwas appropriately diluted with a 500-fold dilution of nutrient broth(NB “Eiken”, Eiken Chemical, Tokyo, Japan), which is abbreviated as1/500 NB thereafter, to be an expected infective titer of 0.67−2.6 ×107 pfu/mL. The 1/500 NB contained 0.006 g/L meat extract,52390.02 g/L peptone, and 0.01 g/L NaCl. Then, the loading suspensionwas stored at 0 °C and used within 2 h.A 50 μL portion of the bacteriophage suspension in 1/500 NB wasplaced onto a specimen surface and covered by a polyethylene film(0.04 mmt, UNIPACK, SEISANNIPPONSHA, Tokyo, Japan) of12 mm square, which was cleaned by EtOH in advance. A bottom ofa clean, sterile glass dish was used as a control surface. The specimenwas placed for certain time periods (5, 10, and 20 min) in the safetycabinet at ambient temperature. Then, the bacteriophages on the spec-imen surface were collected into 1 mL of soybean-casein digest brothwith lecithin and polyoxyethylene sorbitan monooleate (abbreviatedas SCDLP broth, SHIOTANI M.S.), which contained 17 g/L casein,3.0 g/L soybean peptone, 5.0 g/L NaCl, 2.5 g/L Na2HPO4, 2.5 g/Lglucose, 1.0 g/L lecithin, and 7.0 g/L nonionic surfactant, by pipetting.Then, the collected solution was stored at 0 °C before the quantifica-tion of virus infectivity titer by the plaque assay.2.3.4. Quantification of Virus Infective Titer by the PlaqueAssay. The collected solution was serially diluted by a 10-fold mannerwith physiological saline containing 0.1 wt % peptone (abbreviated aspepNaCl), which contains 1.0 g/L peptone (Kyokuto peptone,Kyokuto Pharmaceutical Industrial, Tokyo, Japan) and 8.5 g/L NaCl(guaranteed reagent, FUJIFILM Wako Pure Chemical). The aliquotsof collected and diluted solutions were individually added to 0.1 mLof E. coli prepared for the plaque assay, mixed, and warmed at 37 °Cfor 10 min. Then, the suspension containing bacteria and bacterio-phage was mixed with a 4 mL portion of CaLB soft agar (agar concen-tration of 0.5 wt %), which was maintained at 55 °C, well-pipetted, andlayered over a CaLB agar (agar concentration of 1.5 wt %) dishwarmed at 37 °C. After solidification at ambient temperature in a safetycabinet, the multilayered agar dishes were incubated for 18 ± 2 h at37 °C prior to counting plaques.The virus infectivity titer, N (pfu/mL), was decided by the followingequation:N =A×DF × ð1=VÞwhere A indicates the average number of plaques in the duplicateddishes at the same dilution and DF describes dilution factor. V repre-sents the volume (mL) of the collected or diluted bacteriophage solu-tions added to E. coli suspension.Two parameters for antiviral activity were introduced: T0.001 andminimum inhibitive contact time (MICT). The former is the contacttime to reduce N on the specimen surface (Nmat) to 1/1000 of thaton the control surface (Ncont). The latter is the contact time whenNmat becomes zero. T0.001 was determined by probit regression onNmat/Ncont against the logarithm of contact time. When probit regres-sion was not applicable, T0.001 was estimated by linear regression onthe logarithm of N of the inoculated phages (at contact time zero)and the logarithm of Nmat (=0) at contact time 5. MICT was estimatedby linear regression on the logarithm of Nmat against the logarithm ofcontact time.2.4. Electrochemical Impedance AnalysisIn order to investigate the effect of GSH treatment on the corrosion ofcopper and its alloys, electrochemical impedance spectroscopy (EIS)under a thin electrolyte layer was performed. This method enables usto mimic the contact condition of bacteriophage on the specimen sur-face in the antiviral assay; a small quantity (50 μL) of bacteriophagesuspension was placed on the specimen surface with the polyethylenefilm cover to form a thin layer. The ratio of the solution amount tospecimen surface area is one of the key factors to influence the corro-sion behavior of testing materials.The experimental procedures were described in our previousstudy,22 which uses a specimen having parallel electrodes of testingmaterials as described earlier (Figure S1). Briefly, 0.5 mL of 0.7 MNa2SO4 (guaranteed reagent, FUJIFILM Wako Pure Chemical) wasapplied on the specimen surface with/without GSH treatment, givinghttps://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org Articlea salt density of 9.8 mg/cm2. The specimen was placed in the incubator(25± 1 °C), and EIS measurement was immediately started with an ACamplitude of 10 mV in the frequency range of 1 × 10−2 to 2 × 104 Hzusing a potentiostat equipped with a frequency response analyzer(Interface 1010 T, Gamry Instruments, Warminster, USA). This resultin the electrolyte was indicated as 100% humidity. Then, the humidityin the incubator was controlled to be 30 ± 5% relative humidity (RH)using silica gel overnight to dry the moisture. EIS was performed thenext day, followed by the increases in the humidity to 60 ± 5, 75 ±5, and 90 ± 5% RH by controlling the open surface area of ultrapurewater in a vessel in the incubator. EIS measurement was started 0.5 hafter the relative humidity of the incubator reached to the designatedrange. The humidity in the incubator was monitored with a hygrometer(Weathecom II electronic thermos and hygrometer EX-502, EMPEXInstruments, Tokyo, Japan) through the measurement.The EIS data were analyzed with an equivalent circuit shown inFigure S3.23 The difference between the impedances at high(20 kHz) and low (10 mHz) frequency ranges was calculated as Zdiff,and its reciprocal number (1/Zdiff) was employed as a corrosionparameter. This parameter was introduced as a practical solution forcorrosion monitoring by impedance measurement under a thin electro-lyte layer without curve fitting.23,24 It gives a reasonable accuracy forthe relative comparison of the corrosion behavior of testing materialsin different conditions.23,24 Details of the calculation procedure and rel-evance of 1/Zdiff are described in the Supporting Information. EIS mea-surements were performed in triplicate.2.5. Electrochemical Analysis of the Oxide Layer Formedon the Copper SurfaceIn order to analyze the effect of GSH treatment on the oxide layer ofthe copper surface, potentiodynamic (PD) and chronopotentiometric(CP) measurements were carried out in the highly alkaline solution.25As a testing specimen, a thin plate of C1020 with a thickness of 0.3 mmwas cut into 15 mm squares and used as received. Prior to the measure-ment, 5.6 μL of 4 mM GSH in 99 vol % EtOH was applied to the spec-imen and kept for 24 h in the ambient condition, if necessary. Then,the testing specimen was placed at the bottom of an electrochemicalchamber with an exposed area of 0.899 cm2 as a working electrode.A platinum wire was set as a counter electrode, whereas a saturatedAg/AgCl (3 M NaCl) electrode was used as a reference electrode. A27 mL portion of 6 M KOH + 1 M LiOH,25 which was prepared byappropriate dilution of 8 M KOH and 4 M LiOH (both for volumetricanalysis, FUJIFILM Wako Pure Chemical), was employed as an elec-trolyte. For PD measurement, a potential was swept from the open-circuit potential (∼ −0.6 V vs Ag/AgCl) to that for hydrogen evolution(−1.6 V vs Ag/AgCl) at the rate of 1.0 mV/s. For CP measurement,the current density was set as 0.1 mA/cm2 while recording potentialagainst the reference electrode at the sampling rate of 0.1 s.The thickness of oxides [Cu(OH)2, Cu2O, and CuO] was deter-mined based on the results of CP measurement following the proce-dure in the literature,26 and the details are described in theSupporting Information. The PD and CP measurements were per-formed in triplicate.2.6. Statistical AnalysisFor the results of the antiviral assay, parallelism tests of 2 regressionlines were performed using a free version of statistical analysis software(Kyplot 6.0, KyensLab, Japan) except some cases; the pairs of the virusinfectivity titer with/without GSH treatment at a contact time of 5 or10 min were analyzed by Student’s t-test. The analysis methods appliedare listed for the combination of different surface treatment solutionsand different materials in Table S2.For the results of EIS measurement, the pairs of the 1/Zdiff values ofthe same material with/without GSH treatment were analyzed byStudent’s t-test. For the CP analysis, the pairs of the oxide thicknessfor the same materials with/without GSH treatment were analyzedby Student’s t-test.5240In all experiments, the rejection of the obtained values was decidedby Dixon’s Q-test at 90% confidence.3. RESULTS3.1. Effect of Glutathione Treatment on the AntiviralActivity of Copper and Its AlloysThe effect of GSH treatment on the antiviral activity ofC1020 is shown in Figure 1a. At 24 h after the treatment,the N of C1020 treated with 4 mM GSH in 99 vol % EtOH(abbreviated as 4 mM GSH_99 in the following) wasdecreased to zero just after 5 min of contact, whereas thatof nontreated C1020 was around 105 pfu/mL after 5 min ofcontact and was not decreased to zero even after 20 min ofcontact. C1020 treated only with 99 vol % EtOH (withoutGSH) had a similar result to that of nontreated C1020.These results clearly demonstrate the enhancement effect ofGSH treatment on the antiviral activity of C1020. Figure 1aalso shows that the N of glass treated with 4 mM GSH_99was reduced to 3.9 × 105 pfu/mL, which is slightly lower thanthat of glass treated only with 99 vol % EtOH. However, bothN values for glass treated with 99 vol % EtOH with/withoutGSH did not decrease further with an increase in contact time.This indicates that nontreated C1020 had higher antiviral activ-ity than glass with GSH treatment and that GSH treatment didnot work on glass in the similar level to that on C1020, suggest-ing the involvement of Cu ions in the enhancement process ofantiviral activity by GSH treatment. C1020 treated with 4 mMGSH_99 had a statistically different regression line from thoseof C1020 treated only with 99 vol % EtOH, nontreatedC1020, and glass treated with 99 vol % EtOH with/withoutGSH (p < 0.001).The effects of the EtOH/water ratio and GSH concentrationin the treatment solution on the enhancement of antiviral activ-ity of C1020 are shown in Figure 1b and Figure S4, respectively.At 24 h after the treatment, the reduction in EtOH concentra-tion in the treatment solution increased N, indicating the reduc-tion in antiviral activity (Figure 1b). The decrease in GSHconcentration increased N of C1020 (Figure S4), suggestingthe best treatment solution among those tested in this studyas 4 mM GSH_99. In both experiments, C1020 treated with4 mM GSH_99 has a statistically different regression line fromthat treated with 4 mM GSH in 95 vol % or 80 vol % EtOH(p < 0.001) and from that treated with 2 or 1 mM GSH in99 vol % EtOH (p < 0.001).The duration of the enhancing effect by GSH treatment wasevaluated using C1020 at the contact times of the phage suspen-sion as 5 and 10 min, as shown in Figure S5. At the contact timeof 5 min, the N of GSH-treated C1020 reached to zero at up to24 h after the treatment, but it increased to about 103 pfu/mL at168 h (=7 days) after the treatment. However, at the contacttime of 10 min, N was still zero even at 168 h after the treat-ment. These results indicated that the GSH treatment main-tained its maximum effectiveness up to 24 h after thetreatment and that its enhancement effect remained in a practi-cal level even at 168 h after the treatment. In both contacttimes, the N of nontreated C1020 was significantly higher thanthat of GSH-treated C1020 at all leaving time points after thetreatment (p < 0.01).Figure 2 indicates the effect of GSH treatment on antiviralactivity of copper alloys with various Cu contents. C7150, ahttps://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520Figure 1. Effect of GSH treatment (a) and ethanol concentration (b) on antiviral activity of C1020 at 24 h after the treatment (n = 2, mean ± s.d.).By parallelism tests of 2 regression lines, C1020 treated with 4 mM GSH in 99 vol % EtOH has a statistically different slope from that of C1020treated only with 99 vol % EtOH or nontreated one (p < 0.001). C1020 treated with 4 mM GSH has a statistically different intercept from thatof glass treated with 99 vol % EtOH with/without 4 mM GSH (p < 0.001). C1020 treated with 4 mM GSH in 99 vol % EtOH has a statisticallydifferent slope from that treated with 4 mM GSH in 95 vol % or in 80 vol % EtOH (p < 0.001). Nontreated C1020 has a statistically different slopefrom that treated with 4 mM GSH in 95 vol % or in 80 vol % EtOH (p < 0.001 or p < 0.05, respectively).ACS Applied Bio Materials www.acsabm.org ArticleCu-30 wt % Ni alloy, showed superior antiviral activity withouttreatment as N leached to zero at 10 min of contact. GSH treat-ment to C7150 resulted in a zero value of N at 5 min of contact,indicating the enhancement effect of the GSH treatment on itsantiviral activity. Nontreated C5191, C2680, and Constantanhad similar antiviral activity to that of C1020. GSH treatmentto these alloys decreased their N values, but not as low asGSH-treated C1020. GSH treatment to MONEL slightlyreduced N, especially at the shorter contact time, but the valuesare much higher than other copper alloys, indicating lesseffectiveness of the GSH treatment on antiviral activity enhance-ment. For C5191, C2680, Constantan, and MONEL, the GSHtreatment significantly changed their antiviral behavior, which isconfirmed by parallelism tests for 2 regression lines (p < 0.001for C5191, C2680, and Constantan, p < 0.01 for MONEL).Two parameters of the antiviral activity, T0.001 and MICT,were calculated for each testing material and are shownin Table 1. Without GSH treatment, the smallest T0.001 andMICT values were observed for C7150, followed by C1020.T0.001 and MICT values tended to increase with a decrease inCu content in the alloys except C7150. With GSH treatment,the trends in T0.001 and MICT were similar to those withoutGSH treatment; the smallest values were observed for C71505241and C1020, followed by C5191, and increased with a decreasein Cu content in the alloys.3.2. Effect of GSH Treatment on the Corrosion of Copperand Its AlloysThe examples of the electrochemical impedance spectra ofC1020 with/without GSH treatment under various humidityare shown in Figure S6. In both cases, the impedance increasedwith reduction in humidity. As a corrosion parameter, 1/Zdiffwas calculated and is plotted against humidity in Figure 3a. Itindicates the increasing trend in 1/Zdiff by GSH treatment atthe humidity range of over 75% RH.The 1/Zdiff values of all materials with/without GSH treat-ment at 100% and 90% RH are shown in Figures 3b and 3c,respectively. At 100%, 1/Zdiff of GSH-treated materials tendedto be higher than those of nontreated, indicating the increasein the corrosion rate by GSH treatment. In addition, 1/Zdiff alsotended to decrease with reduction in Cu content in the mate-rials. However, the difference in 1/Zdiff with/without GSHtreatment decreased with reduction in humidity (seeFigure 3a). Even at 90% RH, there is no difference in 1/Zdiffbetween materials and with/without GSH treatment exceptC1020 and C5191 (Figure 3c).https://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520Figure 2. Antiviral activity of common copper alloys with/withoutGSH treatment (n = 2, mean ± s.d.). By parallelism tests of 2 regres-sion lines, C1020, C5191, or Constantan treated with 4 mM GSH has astatistically different slope from the corresponding nontreated material(p < 0.001). C2680 or MONEL treated with 4 mM GSH has a statis-tically different intercept from the corresponding nontreated material(p < 0.001 or p < 0.01, respectively). The parallelism test was notapplicable to the data of C7150.ACS Applied Bio Materials www.acsabm.org Article3.3. Correlation between Antiviral Activity and Corrosionof Copper and Its AlloysIn order to investigate the correlation between the antiviralactivity and corrosion rate of testing materials, the T0.001 andMICT of the testing materials treated with/without GSH wereplotted against 1/Zdiff at 100% RH (in an electrolyte) in loga-rithmic scales as shown in Figure 4a. The 1/Zdiff of the non-treated materials showed no clear trend, but those of theTable 1. Time to Reduce the Infectivity Titer of the BacteriophageInhibitive Contacting Time (MICT, min) of Testing Materials wimaterial Cu content (wt %) Ni content (wt %) no treaC1020 100 11C5191 93.3 15C7150 68.8 30.1 6C2680 65.0 16Constantan 54.4 44.6 15MONEL 33.4 64.7 122aT0.001 was estimated by the linear regression of the logarithm of the virus inprobit method.5242GSH-treated ones indicated a good linear correlation to T0.001except MONEL (r2 = 0.934). The similar trend was observedfor the correlation between the 1/Zdiff and MICT for theGSH-treated materials except MONEL (r2 = 0.971,Figure S7a). These results suggest that the GSH treatment-induced higher corrosion rate in the electrolyte resulted in thehigher antiviral activity of the testing materials exceptMONEL, which has a relatively low Cu content (33.4 wt %).In order to investigate the enhancement level of antiviralactivity by the GSH treatment in relation to acceleration inthe corrosion rate, the ratio of the T0.001 of testing materialstreated with/without GSH [abbreviated as GSH(+) andGSH(−), respectively] were plotted against that of the 1/Zdiff.For the T0.001 and MICT, in which the smaller values indicatethe higher antiviral activity, the larger values of GSH(−)/GSH(+) indicate the higher levels in enhancement of the anti-viral activity. For the 1/Zdiff, in which the larger values indicatethe higher corrosion rate, the larger values of GSH(+)/GSH(−) indicate the higher levels of acceleration in the corro-sion rate. As shown in Figure 4b, the GSH(−)/GSH(+) of theT0.001 linearly increased with an increase in the GSH(+)/GSH(−) of the 1/Zdiff except C5191 (r2 = 0.889). The sametrend was also observed for MICT as r2 = 0.927(Figure S7b). These trends suggested that the enhancementin antiviral activity depended on the corrosion acceleration oftesting materials by the GSH treatment.3.4. Effect of Glutathione Treatment on the Oxide Layer ofthe Copper SurfaceIn order to investigate the effect of GSH treatment on the nat-urally formed oxide layer of the copper surface, PD and CPmeasurements were carried out under the condition previouslyreported as effective to identify Cu2O and CuO.25 The typicalexamples of the measurements for C1020 with/without GSHtreatment are displayed in Figure 5. In the results of PD mea-surement (Figure 5a), the C1020 specimen shows a sharp peakaround −0.7 V vs Ag/AgCl and a very small peak around−1.2 V vs Ag/AgCl, which can be assigned to Cu(OH)2 andCuO, respectively.27 This result corresponds well to the corro-sion process of the copper plate in the laboratory air;Cu(OH)2 is first formed and eventually converted to CuO.27Then, Cu2O is formed after a certain period of time(∼10 days),27 but it was not clearly observed in our specimens.The C1020 specimen with GSH treatment had a smaller peakaround −0.7 V vs Ag/AgCl and no peak around −1.2 V vsAg/AgCl, suggesting less oxide layer on the specimen surface.to 1/1000 of the Control Surface (T0.001, min) and Minimumth/without GSH TreatmentT0.001(min) MICT (min)tment24 h afterGSH treatment no treatment24 h afterGSH treatment.7 <2.88a 22.9 <5.2 4.05 28.1 12.6.48 <3.00a 10 <5.8 9.36 30.9 31.0.7 7.64 31.5 16.3230 5910b 9040bfectivity titer values against contact time. bMICT was estimated by thehttps://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520Figure 3. Results of electrochemical impedance measurement onC1020 under controlled humidity (a) and enhancement in the corro-sion rate of copper alloys by GSH treatment in an electrolyte(100% RH, b) and that under 90% RH (c) (n = 3, mean ± s.d.).The pairs of the 1/Zdiff values of nontreated and treated materials wereanalyzed by Student’s t-test. **p < 0.01, *p < 0.001.Figure 4. Correlation between the antiviral activity and corrosion rateof copper and its alloys (a) and correlation in the enhancement levelsof antiviral activity and those of the corrosion rate by GSH treatment(b). GSH(+) and GSH(−) indicate with/without GSH treatment.The regression line shown in (a) was calculated for the 5 data withGSH treatment except MONEL. The regression line shown in (b) wascalculated for the 5 data except C5191. The error bars in X and Y axesindicate standard deviations and 95% confidential intervals, respectively.ACS Applied Bio Materials www.acsabm.org ArticleThis can be attributed to the reduction activity of GSH appliedto the surface. This decrease in the surface oxide was also con-firmed by the CP measurement shown in Figure 5b. The non-treated C1020 surface had the retention of potentials around−0.7 and −1.2 V vs Ag/AgCl, corresponding to the reductionof Cu(OH)2 and CuO, respectively. It also showed a very smalladditional potential drop around −1.4 V vs Ag/AgCl assignedto the reduction of Cu2O. The GSH-treated C1020 had similarbehavior, with the potential retention around −0.7 and −1.2 Vvs Ag/AgCl, but their duration was shorter than that of non-treated C1020, indicating that the oxide layer on the GSH-treated C1020 was thinner than that on nontreated. Table 2shows the estimated thickness of Cu(OH)2, CuO, and Cu2O5243for C1020 with/without the GSH treatment based on theresults of CP measurement. This clearly indicated the reductionin Cu(OH)2 layer thickness after the GSH treatment (p < 0.1).The thicknesses of CuO and Cu2O were slightly increased anddecreased with the GSH treatment, respectively, but their differ-ences from those on C1020 without the treatment were not sta-tistically significant.4. DISCUSSIONCopper and its alloys are well-known to have antimicrobialactivity effective for a wide range of pathogens. It is ideal to testdeveloped materials with every kind of pathogen, but it is notpractical. Usually, representative ones are employed as thescreening tests of the developed materials. The general orderof resistance of microbes to disinfectants and sterilants is pro-posed as follows: prions > bacterial spores > parasitic oocysts> mycobacteria > nonenveloped viruses > fungal spores > veg-etative fungi > vegetative bacteria > enveloped viruses.28 It ispreferable to use the most resistant pathogen for the efficacytests, but it also gives the higher biological risk requiring thehigher level of safety equipment. In the present study, a none-nveloped virus, bacteriophage Qβ, which can be handled in aBSL1 laboratory, was employed for antiviral activity evaluationsince it is not destroyed by ethanol. As described earlier,https://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248http://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520Figure 5. Typical examples from the results of (a) potentiodynamicand (b) chronopotentiometric measurements of C1020 specimenswith/without GSH treatment.Table 2. Surface Oxide Thickness (nm) Estimated byChronopotentiometric Measurements of C1020 with/without GSH Treatment (n= 3, Mean ± s.d.)no treatment GSH treatmentCu(OH)2 11.31 ± 1.55a 8.86 ± 0.41aCuO 1.80 ± 0.38 2.87 ± 1.30Cu2O 5.17 ± 2.53 1.22 ± 0.03aA statistically significant difference was observed between “no treat-ment” and “GSH treatment” by Student’s t-test (p < 0.1).ACS Applied Bio Materials www.acsabm.org Articlebacteriophage Qβ is assigned as a testing virus for antiviraltests of fine ceramics by JIS R1706:2020,21 which is the basedocument of updating corresponding ISO 18061:2014.Nonenveloped viruses are not the highest pathogens in theresistance hierarchy but are higher than fungi, vegetativeGram-positive/negative bacteria, and enveloped viruses, expect-ing that the materials having the sufficient antiviral activityagainst nonenveloped viruses would be effective againstthese pathogens. This approach is acceptable for the screeningstage of antimicrobial materials, but it is necessary to test thematerials with target pathogens on the final stage of the devel-opment process.As shown in Figure 2, the GSH treatment enhanced the anti-viral activity of copper and its alloys. Although the enhancementwas unclear on MONEL having the lowest Cu content as33.7 wt %, it was clearly higher on C5191, C2680, andConstantan in comparison to nontreated C1020. The antimicro-bial activity of copper alloys generally depends on their Cu5244content.6,16 C1020, which has the highest antimicrobial activityamong copper materials, is most frequently studied on touch sur-face application, but its practical application is limited by discolor-ation. Common copper alloys such as C2680 have betterresistance against discoloration, but their antimicrobial activityis lower than that of C1020. Therefore, enhancement of theirantiviral activity by the simple GSH treatment would be a goodpractical solution to this issue.Most of the studies on enhancement of copper antimicrobialactivity use copper salts, oxides, nanoparticles (NPs), or nano-clusters (NCs) with a combination to a certain type of organiccompound.29−34 GSH is commonly employed for the synthesisof copper NPs and NCs as a capping agent since the thiolgroup in GSH has high affinity to copper,29 resulting in inhibi-tion of aggregation or growth of NPs/NCs. In some cases,GSH is also used as a reducing agent, but in other cases, typicalreducing agents such as ascorbic acid, chitosan, citrate, hydra-zine, and sodium borohydride are added.29 Since GSH cappinginfluences the sizes of synthesized NPs/NCs, no study com-pared the antimicrobial activity of GSH-capped NPs/NCs withthose of uncapped NPs/NCs. Furthermore, no study wasreported about GSH treatment on bulk copper and its alloysfor the enhancement of their antimicrobial activity exceptour approach.35Several research groups studied addition of a reducing agentto testing solution to enhance the antibacterial activity of cop-per salts or NPs.30,32,36 Ojaym et al. reported that the additionof extra ascorbic acid (VC) to VC-capped copper NPsenhanced their antibacterial activity against Staphylococcusaureus and Pseudomonas aeruginosa due to an increase in ROSgeneration inside the bacterial cells.30 GSH also has reducingactivity reacting with copper ions, which may result in increas-ing ROS generation as shown in eqs 1−3. When copper andits alloys were immersed in 1/500 NB containing 1 mM GSHfor 24 h, the generation of H2O2 was detected in all testedmaterials35 (Figure S8).In the present study, EIS of the specimen with/without GSHtreatment under a thin electrolyte was measured to investigatethe acceleration of corrosion by the GSH treatment at a condi-tion mimicking the bacteriophage contact in the antiviral assay.Figures 3 suggests the acceleration effect of GSH on corrosionat relatively high humidity such as 90% RH and in an electrolyte(100%). Figure 3b also suggests the trend in the effect of theGSH treatment depending on Cu content in the alloys; thehigher Cu content tends to have the higher enhancement effect.Figure 4a and Figure S7a revealed the correlation tendencybetween the corrosion rate (1/Zdiff) and antiviral activities(T0.001 and MICT) with the GSH treatment except MONEL,suggesting the importance of the corrosion rate on antiviralactivity. It is also confirmed by the correlation tendencybetween the enhancement levels in antiviral activity and thosein corrosion rates except C5191, as shown in Figure 4a andFigure S7b. Even with a few exceptions, these data suggest thedependent tendency of the antiviral activity of testing materialson their corrosion rates. Copper antimicrobial activity is mainlyattributed to released copper ions and related reactions such asROS generation.5,17,18 The involvement of copper ions in theirantibacterial activity is confirmed by Cu accumulation inside thebacterial cells in contact with the copper surface.37 Our previousstudy22 also reported the importance of the corrosion rate oncopper antibacterial activity; retardation in the corrosion rateunder a low-humidity environment contributed to reductionhttps://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org Articlein copper antimicrobial activity. For Cu−Ni alloys, the increasein Ni content decreases the corrosion rate of the alloy in neutralNaCl solution,38 which agrees with the results of this study;MONEL has a lower corrosion rate than C7150 andConstantan (Figure 3a). The surface analysis of MONELexposed in ambient conditions revealed the formation ofNi(OH)2 together with Cu2O;39 the former is the main compo-sition of the Ni passivity film.40 Cu−Ni alloys with higher Cucontent lose the benefit of Ni passivity, showing the corrosionbehavior similar to Cu.41 When Cu-66 wt % Ni-2 wt % Feand Cu−23 wt % Ni−12 wt % Zn−2.5 wt % Sn alloys wereimmersed into synthetic sweat, Ni compounds were found inthe corrosion products of the former, but not of the latter,42suggesting the Ni ion release from the high-Ni-content alloy.Ni has antimicrobial activity but is much lower than that ofCu.43 These will explain the lower corrosion acceleration ofMONEL by GSH treatment as well as its lower antiviral activitythan other copper alloys.The mechanism of how GSH accelerates copper corrosion canbe explained by the change in the surface oxide thickness. Asshown in Figure 5 and Table 2, the thickness of surface oxideon C1020 was slightly reduced after GSH treatment. The surfaceoxide formation on C1020 contributes to slowing down the dif-fusion of oxygen at the surface, retarding the corrosion reactionof Cu. Therefore, the thinning of the oxide layer reduces thisFigure 6. Schematic illustration of the effect of GSH treatment on the oxide lno treatment, copper corrosion occurs as shown in eq 4, forming Cu2O via Cand Cu2+, which contributes to form CuO via Cu(OH)2 (eqs 6 and 7). (b) Iwill go through the disproportionation and decrease oxide formation. Cu+ alspresence of H+ (eqs 2 and 3), which can give extra damage to viruses and mtheir lifetime is relatively short due to their decomposition (eqs 8 and 9). Atcopper surface is consumed by the reaction shown in eq 1; the thickness of theCu+ ions, resulting in a decrease in their antiviral/antibacterial activity.5245retardation, resulting in relatively faster corrosion. The GSH reac-tion with copper surface oxide may require H2O, as suggested byFigure 1b; the applied solution with lower H2O content is moreeffective for the enhancement of antiviral activity. Since GSH isinsoluble in EtOH, the treatment solution must contain H2Oto dissolve GSH. However, the presence of H2O helpsCu(OH)2 formation at the copper surface, which is relatively fastin a humid atmosphere.27 The GSH treatment solution with lowH2O content has two possible roles to minimize the Cu(OH)2formation; one is simple minimization of the Cu(OH)2 forma-tion by low H2O content, and the other is inhibition of theCu(OH)2 formation via reduction of Cu2+ to Cu+ by GSH asshown in eq 1. The latter role would be important to describethe results in the present study. C1020 treated with 99 vol %EtOH without GSH has slightly increased its antiviral activity ata short contact time in comparison to nontreated C1020 asshown in Figure 1a, while GSH-treated C1020 indicated betterantiviral activities than the nontreated one as shown inFigure 1b and Figure S4. These results cannot determine thedominance between the following processes for antiviral activity:ROS generation by Cu+ reaction with O2/H2O2 (eqs 2 and 3) orcorrosion acceleration by thinning of the Cu(OH)2 layer. Ineither of them, GSH contributed via reduction of Cu2+ to Cu+.The influence of GSH treatment on the copper surface isschematically described in Figure 6. The nontreated copperayer of the copper surface and its corrosion resistance. (a) In the case ofuOH (eq 5). In high concentrations, Cu+ will disproportionate into Cun the case of GSH treatment, GSH reduces Cu2+ to Cu+ (eq 1), whicho contributes to the generation of ROS such as H2O2 and %OH in theicrobiomes when they are challenged on the copper surface. However,the long exposure time after the treatment, all the GSH applied to thesurface oxide layer increases again. This decreases the release of Cu2+/https://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org Articlesurface is naturally oxidized via CuOH formation in the follow-ing equations:44Cu+14O2 +12H2O→Cu+ +OH− (4)2CuOH→Cu2O+H2O (5)With an increase in its concentration, Cu+ goes through dispro-portionation into Cu and Cu2+, in which the latter contributesto form CuO via Cu(OH)2 by the following equations.442Cu+→Cu+Cu2+ (6)Cu2+ + 2OH−→CuðOHÞ2→CuO+H2O (7)On the GSH-treated surface, besides the reactions describedabove, GSH reduces Cu2+ to Cu+ (eq 1), which goes throughthe disproportionation (eq 5), resulting in decrease of oxide for-mation. Cu+ also contributes to the generation of ROS such asH2O2 and •OH in the presence of H+ (eqs 2 and 3), which cangive extra damage to viruses and bacteria when they are in con-tact with the copper surface. However, the lifetime of ROS isrelatively short due to their decomposition.·OH+ ·OH→H2O2 (8)2H2O2→2H2O+O2 (9)This means that the only ROS generated “in situ”−very close tothe pathogens−can give damage to them. The Cu2+ reductionby GSH (eq 1) consumes GSH, which decreases the remainingGSH on the surface with an increase in exposure time.Therefore, at a long exposure after the treatment, all the GSHapplied to the copper surface is consumed by the reaction ofeq 1 and the thickness of the surface oxide layer increases again.This also decreases the release of Cu+ ions, resulting in adecrease in their antiviral/antibacterial activity. Figure S5 con-firms that the long exposure of 168 h after the treatmentdecreased the antiviral activity of the GSH-treated C1020 tothe same level to that of the nontreated one, though the contacttime of the nontreated one was doubled.The difference in the effectiveness of the GSH treatmentamong testing materials can be derived from the difference insurface oxide thickness and composition, resulting in differentinfluences on corrosion reaction. The corrosion acceleration ismore obvious on high-Cu-content alloys than low-Cu-contentalloys (Figure 3b), suggesting that the incorporation of alloyingelements in surface oxide influences the effect of the GSH treat-ment. Further investigation is necessary to clarify the roles ofalloying elements in surface oxide and the GSH treatment.The developed GSH treatment is simple and easily applicableto copper high touch surfaces when needed, in such a way as analcohol disinfectant. This treatment has the following advan-tages: (1) GSH is widely distributed in mammalian cells,expecting low toxicity to animals. (2) A water−ethanol systemis employed as a solvent, which can be applicable like otheralcoholic disinfectants. (3) There is no need for heat treatmentnor a specific equipment for application. Since we tested with asmall specimen, we just dropped a small portion of the treat-ment solution onto the specimen surface, but it is possible to5246apply by spraying to a large surface. This also allows the appli-cation to existing touch surfaces. (4) Since the GSH treatmentmainly works to reduce the natural oxide thickness, damage tothe copper materials is limited. Therefore, it enables repetitiveapplication. When the treated surface receives mechanical wearor chemical cleaning, it can be applied again to the surface.The limitation of this treatment is the duration of its effectshorter than 7 days, which requires a repetitive application.The exposure of the treated surface was performed in a labora-tory environment at ambient temperature and humidity around50% RH, which is a relatively mild, indoor atmosphere. A highertemperature and humidity may accelerate copper oxide forma-tion, which accelerates consumption of GSH, resulting inshorter duration of the treatment effect. Furthermore, the anti-viral assay is performed in laboratory conditions; bacteriophageQβ suspension is applied to the specimen surface with a poly-ethylene film, making a thin suspension layer, keeping it moistthrough the contact time. In practical conditions, however,pathogen contact to high touch surfaces is mainly by a droplet,which dries relatively fast under ambient conditions. The GSHtreatment accelerates copper corrosion markedly at 100% RH,but it decreases in low-humidity conditions. This suggests thatGSH requires H2O for thinning the surface oxide layer and thatthe improvement in the antimicrobial effect by this treatmentmay be reduced in a practical, less humid environment than thatobtained in a laboratory. In this study, the GSH influence onthe oxide layer of the copper surface was investigated by electro-chemical methods. Further investigation with detailed analysisof the oxide layer by X-ray photoelectron spectroscopy or othermethods is necessary to deeply understand the durability andefficacy of the GSH treatment with different copper materials.5. CONCLUSIONSWe attempted a surface treatment of copper to enhance its anti-viral activity, which is evaluated using bacteriophage Qβ, anonenveloped virus. Treatment of copper and its alloy surfacewith 4 mM GSH in 99 vol % EtOH distinctly enhanced theirantiviral activity except MONEL, the Ni-33.4 wt % Cu alloy.Electrochemical impedance measurement of the testing mate-rials revealed the significant acceleration in their corrosion ratesby the GSH treatment for C1020, C7150, and Constantan, whilethe increase in those of C5191 and C2680 was not statistically sig-nificant. The antiviral activity of testing materials with the treat-ment correlates well to their corrosion rate with the treatmentexcept MONEL, suggesting the involvement of Ni passivity.The enhancement level in their antiviral activity by the treatmenttends to correlate to the acceleration level in their corrosion rates.Potentiodynamic and chronopotentiometric measurement con-firmed the reduction of copper surface oxide by the treatment,suggesting its contribution to the acceleration of corrosion.Most of copper alloys have higher stability against discolor-ation than C1020, but their antibacterial/antiviral activity islower than C1020. The GSH treatment improved the antiviralactivity of C5191, C2680, and Constantan to the level superiorto nontreated C1020, which may encourage the touch surfaceapplication of copper alloys.■ ASSOCIATED CONTENTSupporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acsabm.6c00520.https://doi.org/10.1021/acsabm.6c00520ACS Appl. Bio Mater. 2026, 9, 5237−5248https://pubs.acs.org/doi/suppl/10.1021/acsabm.6c00520/suppl_file/mt6c00520_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acsabm.6c00520?goto=supporting-infohttp://www.acsabm.org?ref=pdfhttps://doi.org/10.1021/acsabm.6c00520ACS Applied Bio Materials www.acsabm.org Article(Table S1) Chemical compositions of testing materials,(Table S2) statistical analysis applied to the pair of theresults in different treatment solutions and materialsobtained by the antiviral assay, (Figure S1) schematicexplanation of the specimen for electrochemical imped-ance measurement, (Figure S2) schematic illustration ofthe antiviral assay, (Figure S3) equivalent circuit model,(Figure S4) effect of the concentrations of GSH,(Figure S5) effect of the leaving time, (Figure S6) typicalexamples for the results of electrochemical impedancemeasurement, (Figure S7) correlation between MICTand 1/Zdiff, and (Figure S8) H2O2 generation after 24 hof immersion (PDF)■ AUTHOR INFORMATIONCorresponding AuthorAkiko Yamamoto – Research Center for Macromolecules andBiomaterials, National Institute for Materials Science, 1-1Namiki, Tsukuba, Ibaraki 305-0044, Japan; 0000-0002-9182-4886; Email: YAMAMOTO.Akiko@nims.go.jp.Tel.: +81-29-860-4169. Fax: +81-29-860-4626AuthorsMasanori Kikuchi – Bioceramics Group, Research Center forMacromolecules and Biomaterials, National Institute forMaterials Science, 1-1 Namiki, Tsukuba, Ibaraki305-0044, JapanYasushi Suetsugu – Bioceramics Group, Research Center forMacromolecules and Biomaterials, National Institute forMaterials Science, 1-1 Namiki, Tsukuba, Ibaraki305-0044, JapanComplete contact information is available at:https://pubs.acs.org/doi/10.1021/acsabm.6c00520Author ContributionsConceptualization, A.Y.; data curation, A.Y.; formal analysis,A.Y.; funding acquisition, A.Y. and M.K.; methodologies, A.Y.;project administration, A.Y. and M.K.; resources, A.Y. andM.K.; supervision, A.Y. and M.K.; validation, Y.S. and M.K.;visualization, A.Y.; writing-original draft, A.Y.; writing-reviewand editing, Y.S. and M.K. All authors have given approval tothe final version of the manuscript.FundingThis work was partially supported by JST A-STEP (GrantNumber JPMJTM20KSPS), Japan.NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThe authors appreciate Ms. Masuko Tsuda and Ms. AkemiKikuta from NIMS for their technical assistance on antiviraltests and electrochemical impedance measurements.■ ABBREVIATIONSVRE vancomycin-resistant EnterococciHAI healthcare-associated infectionICU intensive care unit5247DNA deoxyribonucleic acidUV ultravioletJIS Japanese Industrial StandardC1020 oxygen-free copperC5190 Cu-6.5 wt % Sn alloyC7150 Cu-30 wt % Ni alloyC2680 Cu-35% Zn alloyMONEL MONEL400, Ni-30 wt % Cu alloyGSH glutathioneEtOH ethanolLB Luria−Bertani brothCaLB LB-supplemented calcium saltpfu plaque-forming unitNB nutrient brothSCDLP soybean-casein digest broth with lecithin and polyox-yethylene sorbitan monooleateN virus infectivity titerA average number of plaques in the duplicated dishes atthe same dilutionDF dilution factorV volume of the bacteriophage suspension added to theE. coli suspensionNmat N on the specimen surfaceNcont N on the control surfaceT0.001 contact time to reduce Nmat to 1/1000 of NcontMICT minimum inhibitive contact timeEIS electrochemical impedance spectroscopyRH relative humidityZhigh the impedance at high frequency rangeZlow the impedance at low frequency rangeRs the sum of electric resistance of the electrolyteRc charge transfer resistanceZdiff the difference between Zhigh and ZlowIcorr corrosion rate (corrosion current density)ka constantk’a constantPD potentiodynamicCP chronopotentiometricGSH_99 4 mM GSH in 99 vol % EtOH + 1 vol % H2O■ REFERENCES(1) Mitchell, B. 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