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[SupplementaryMaterials2_final.pdf](https://mdr.nims.go.jp/filesets/67b4465f-9e14-45f7-9d4f-6d1c61de1df7/download)

## Creator

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

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[Creative Commons BY Attribution 4.0 International](https://creativecommons.org/licenses/by/4.0/)

## Other metadata

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

## Fulltext

Supporting Information for Enhancement of copper antiviral activity with glutathione treatment by Akiko Yamamoto1*, Masanori Kikuchi2, and Yasushi Suetsugu21Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan2Bioceramics group, Research Center for Macromolecules and Biomaterials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan*Corresponding author: e-mail YAMAMOTO.Akiko@nims.go.jpSupplementary procedure 1: Determination of corrosion parameter 1/Zdiff based on electrochemical impedance spectroscopy resultsSupplementary procedure 2: Determination of oxide thickness based on the results of chronopotentiometric measurementSupplementary Table S1: Chemical compositions of the testing materials (wt.%)Supplementary Table S2: The statistical analysis applied to the pair of the results in different treatment solutions and materials obtained by antiviral assaySupplementary Figure S1: Schematic explanation of the specimen for the electrochemical impedance measurement.Supplementary Figure S2: Schematic illustration of the antiviral assay using bacteriophage QβSupplementary Figure S3: Equivalent circuit model used for data analysis of electrochemical impedance spectroscopy.Supplementary Figure S4: Effect of the concentrations of glutathione on antiviral activity of C1020Supplementary Figure S5: Effect of the leaving time after glutathione application on the enhancement of antiviral activity of C1020Supplementary Figure S6: Results of electrochemical impedance measurement on C1020 under controlled humiditySupplementary Figure S7: Correlation between MICT, a parameter of antiviral activity and 1/Zdiff, a parameter of corrosion rate for copper and its alloys (a), and the correlation of enhancement level in antiviral activity and in corrosion rate by glutathione application (b)Supplementary Figure S8: H2O2 generation after 24 h of immersion into 1/500 NB with/without 1mM glutathione (Re-arranged from ref. 35)S-1Determination of corrosion parameter 1/Zdiff based on EIS resultsThe EIS data were analyzed with an equivalent circuit shown in supplementary Figure S3 [20].Capacitance components approach to zero as negligible at high frequency whereas those approachto infinity at low frequency. When we define the impedance at high frequency range (20 kHz) andat low frequency range (10 mHz) as Zhigh and Zlow, respectively, these can be expressed as followingequations;Zhigh = RsZlow = 2Rc + Rswhere Rc and Rs indicate charge transfer resistance and the sum of electric resistance of electrolyteand EIS measurement system, respectively. When we consider the difference between theseimpedances as Zdiff, it can be written as follows;Zdiff = Zlow − Zhigh = 2RcSince the corrosion rate, Icorr is proportional to the reciprocal number of Rc, 1/Zdiff is employed as acorrosion parameter to monitor the change in the corrosion rate asIcorr = k / Rc = k’ / Zdiffwhere k and k’ are constants. EIS measurements were performed in triplicate.S-2Determination of oxide thickness based on the results of chronopotentiometric measurementDuring the chronopotentiometric measurement, the copper cation in the oxide is reduced to themetal form, and calculated using the Faraday’s low of electrolysis as follows [22]:mCu−Cu(OH)2 = (MCu× I× tCu(OH)2) / 2FmCu−CuO = (MCu× I× tCuO) / 2FmCu−Cu2O = (MCu× I× tCu2O) / Fwhere mCu−Cu(OH)2, mCu−CuO and mCu−Cu2O indicate the weight (g) of reduced Cu in Cu(OH)2, CuOand Cu2O layer, respectively. MCu, F, and I represent the molecular weight (g/mol) of Cu, Faradayconstant (9.6485 × 104 C/mol), and current density (0.11 mA/cm2), respectively. tCu(OH)2, tCuO andtCu2O indicate the reduction time of Cu(OH)2, CuO and Cu2O, respectively. The amounts (weight) ofthe oxides and hydroxide were calculated by the following equations:mCu(OH)2 = mCu−Cu(OH)2× (MCu(OH)2 / MCu)mCuO = mCu−CuO× (MCuO / MCu)mCu2O = mCu−Cu2O× (MCu2O / 2MCu)where MCu(OH)2, MCuO and MCu2O represent the molecular weight of Cu(OH)2, CuO and Cu2O (97.6,79.6, 143.1 g/mol), respectively. The thicknesses (cm) of the oxide layers were decided using thefollowing equations:yCu(OH)2 = mCu(OH)2 / (S× dCu(OH)2)yCuO = mCuO / (S× dCuO)yCu2O = mCu2O / (S× dCu2O)where S, dCu(OH)2, dCuO and dCu2O indicate the surface area of the working electrode (0.899 cm2) andthe densities of Cu(OH)2, CuO and Cu2O (3.37, 6.31 and 6.0 g/cm3), respectively.S-3Cu Ni Fe Mn Pb Zn Sn P Si C Al SC1020 >99.99 － － － － － － － － － － －C5191 Rem. － 0.003 － 0.002 0.001 6.55 0.096 － － － －C7150 Rem. 30.11 0.28 0.770 0.001 0.001 － － － － － －C2680 65.0 － 0.00 － 0.00 Rem. － － － － － －Constantan Rem. 44.6 － 0.98 － － － － － － － －MONEL400 Rem. 64.7 0.03 1.56 － － － － 0.34 0.004 0.001 0.001Table S1. Chemical compositions of testing materials (wt.%)S-4Figs. Material/Treatment soln. A Material/Treatment soln. BAnalytical Methodp values1(a) C1020+4mM GSH in 99%EtOH C1020+99%EtOH Parallelism p < 0.001 for slopeC1020+4mM GSH in 99%EtOH C1020 Parallelism p < 0.001 for slopeC1020+4mM GSH in 99%EtOH Glass+4mM GSH in 99%EtOH Parallelism p < 0.001 for interceptC1020+4mM GSH in 99%EtOH Glass+99%EtOH Parallelism p < 0.001 for intercept1(b) C1020+4mM GSH in 99%EtOH C1020+4mM GSH in 95%EtOH Parallelism p<0.001 for slopeC1020+4mM GSH in 99%EtOH C1020+4mM GSH in 80%EtOH Parallelism p<0.001 for slopeC1020 C1020+4mM GSH in 95%EtOH Parallelism p<0.001 for slopeC1020 C1020+4mM GSH in 80%EtOH Parallelism p<0.05 for slope2 C1020+4mM GSH in 99%EtOH C1020 Parallelism p<0.001 for slopeC5191+4mM GSH in 99%EtOH C5191 Parallelism p<0.001 for slopeConstantan+4mM GSH in 99%EtOHConstantan Parallelism p<0.001 for slopeC2680+4mM GSH in 99%EtOH C2680 Parallelism p < 0.001 for interceptMONEL+4mM GSH in 99%EtOH MONEL Parallelism p < 0.001 for interceptC7150+4mM GSH in 99%EtOH C7150 NAS4 C1020+4mM GSH in 99%EtOH C1020+2mM GSH in 99%EtOH Parallelism p<0.001 for slopeC1020+4mM GSH in 99%EtOH C1020+1mM GSH in 99%EtOH Parallelism p<0.001 for slopeC1020 C1020+2mM GSH in 99%EtOH Parallelism p < 0.001 for interceptC1020 C1020+1mM GSH in 99%EtOH Parallelism p < 0.001 for interceptS5C1020+4mM GSH in 99%EtOH @5minC1020@5min t-test p < 0.01C1020+4mM GSH in 99%EtOH @10minC1020@10min t-test p < 0.01Table S2. The statistical analysis applied to the pair of the results in different treatment solutions and materials obtained by antiviral assay.C1020, C5191, C7150, C2680, Constantan, MONEL: testing materials. See Table S1.GSH: glutathioneEtOH: ethanolParallelism: Parallelism test for 2 regression lines.NA: Not applicable.t-test: Student’s t-test.S-5Fig.S1 Schematic explanation of the specimen for electrochemical impedancemeasurement100µmresinInsulating tape(0.5mm in height)10mmcopper foilgap between the foils (100µm)S-6Bacteriophage Qβ50µL of 2x107pfu/mLIncubatefor 5~30minRinse 3 times with 1mL of SCDLP mediumSerial dilutionPlaque assayAir-dry @r.t. for 1-168hApplication of glutathione soln.Polyethylene filmCollectX10       x102 x103 x104Inoculation onto the host bacteria Fig.S2 Schematic illustration of antiviral assay using bacteriophage QβS-7Fig. S3 Equivalent circuit model used for data analysis of electrochemical impedancespectroscopyRs: the sum of the electric resistance of electrolyte and the EIS system used for themeasurementRc: charge transfer resistanceCdl: double-layer capacitanceRcRsRcCdlCdlS-81.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+080 5 10 15 20 25Virus infectivity titer, N/pfu∙mL-1Contact time, t/minC1020C1020_1mM GSH_24hC1020_2mM GSH_24hC1020_4mM GSH_24h播種ファージ11Fig.S4 Effect of the concentrations of GSH on antiviral activity of C1020 (n=2, mean ± s.d.).By parallelism tests of 2 regression lines, C1020 treated with 4mM GSH in 99vol.% EtOH has astatistically different slope from that treated with 2 mM or 1 mM GSH in 99vol.% EtOH (p<0.001).Non-treated C1020 has a statistically different intercept from that treated with 2 mM or 1 mMGSH in 99vol.% EtOH (p<0.001).108107106105104103102101100C1020C1020_1mM GSH_99%EtOHC1020_2mM GSH_99%EtOHC1020_4mM GSH_99%EtOHInoculated phages24h after application S-9Fig.S5 Effect of the leaving time after GSH treatment on the enhancement of antiviral activity of C1020 (n=2, mean ± s.d.)The virus infectivity titer after 5 or 10 min of contact to the specimen surface was plottedagainst the time after GSH treatment. Statistical significance (p<0.01) was observedbetween the pair of the values with/without GSH treatment of a certain leaving time (1, 3,24, and 168 h) at the same contact time as 5 or 10 min by Student’s t-test.1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081 10 100 1000Virus infectivity titer, N/pfu∙mL-1Time after application, Tapp /hC1020_処理なし_5minC1020_処理なし_10minC1020_処理あり_5minC1020_処理あり_10minphage108107106105104103102101100C1020C1020_no application@5minC1020_no application@10minC1020_4mM GSH@5minC1020_4mM GSH@10minInoculated phages**** **p<0.01************S-10Fig.S6 Typical examples for the results of electrochemical impedance measurement on C1020 under controlled humidity.1.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.E+091.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04Impedance, ǀZǀ/ Ω·cm2Frequency, F/HzSoln.(100%)30%RH60%RH75%RH90%RH0.7M Na2SO4C10201.E+001.E+011.E+021.E+031.E+041.E+051.E+061.E+071.E+081.E+091.E-02 1.E-01 1.E+00 1.E+01 1.E+02 1.E+03 1.E+04Impedance, ǀZǀ/ Ω·cm2Frequency, F/HzSoln.(100%)30%RH60%RH75%RH90%RH0.7M Na2SO4, C102024h after GSH treatment109 10810710610510410310210110010-110-2 10-1 100 101 102 103 104109 10810710610510410310210110010-110-2 10-1 100 101 102 103 104(a)(b)S-11y = 0.9838x - 0.4566R² = 0.927401234560 1 2 3 4 5 6 7GSH(-)/GSH:MICTGSH/GSH(-):1/Zdiff@100%C5191y = 9E-05x-1.316R² = 0.97141101001000100000.00001 0.0001 0.001MICT(min)1/Zdiff@100%GSH(-)4mM GSHC5191MONELFig.S7 Correlation between MICT, a parameter of antiviral activity and 1/Zdiff, aparameter of corrosion rate for copper and its alloys (a), and the correlation ofenhancement level in antiviral activity and in corrosion rate by GSH treatmentdescribed by the ratio of GSH(-)/GSH(+) for MICT and GSH(+)/GSH(-) for 1/ Zdiff (b)GSH(+) and GSH(-) indicate with/without GSH treatment. The regression line shown in (a) wascalculated for 5 data with GSH treatment except MONEL. The regression line shown in (b) wascalculated for 5 data except C5191. The error bars in X and Y axes indicate standard deviations and95% confidential intervals, respectively.(a)(b)No application24h after GSH treatmentr2r2Corrosion parameter, (1/Zdiff) @ 100%RH/ (1/Ω·cm2)Antiviral activity parameter,MITC/ minTreatment effect on antiviral activity, GSH(-)/GSH(+) for MITCTreatment effect on corrosion,GSH(+)/GSH(-) for 1/Zdiff @100%RHS-12Fig. S8 H2O2 generation after 24 h of immersion into 1/500 NB with/without 1mM GSH(n=3, mean ± s.d.).NB: Nutrient Broth, GSH: glutathione, CBRA: CLEANBRASS® (Cu-27.4wt%Zn-2.0wt%Ni-0.5wt%Sn),CBRI: CLEANBRIGHT® (Cu-34.7wt%Zn-10.9wt%Ni-0.4wt%Sn), ABSS: antibacterial stainless steelNISSAM3 (Fe-18.1wt%Cr-9.4wt%Ni-3.8wt%Cu-1.4wt%Mn-0.6wt%Si). Re-arranged data in ref. 29.Five hundred times dilution of NB is specified as a suspension medium for antibacterial tests byISO22196:2011 Measurement of antibacterial activity on plastics and other non-porous surfaces. Thespecimen having 0.95 cm2 of an exposure area was immersed into 5 mL of 1/500 NB with/without 1mM GSH for 24h. Then, the H2O2 in the supernatant was measured using Hydrogen Peroxide Assay Kit(CL-204, National Diagnostics, Atlanta, USA) following the protocols supplied. The H2O2 generationwas decided as the difference in concentrations with/without addition of catalase (0.5 mg/mL, frombovine liver, for biochemistry, FUJIFILM Wako Pure Chemical).S-13051015C1020 C6932 CBRA C2680 CBRI ABSSGeneration of H2O2, C/µM1/500 NB1/500 NB+1mM GSHmaterials スライド 1 スライド 2 スライド 3 スライド 4 スライド 5 スライド 6 スライド 7 スライド 8 スライド 9 スライド 10 スライド 11 スライド 12 スライド 13