Essential oil mediated synthesis and application of highly stable copper nanoparticles as coatings on textiles and surfaces for rapid and sustained disinfection of microorganisms

Rampant pathogenesis induced by communicable microbes has necessitated development of technologies for rapid and sustained disinfection of surfaces. Copper nanoparticles (CuNPs) have been widely reported for their antimicrobial properties. However, nanostructured copper is prone to oxidative dissolution in the oil phase limiting its sustained use on surfaces and coatings. The current study reports a systematic investigation of a simple synthesis protocol using fatty acid stabilizers (particularly essential oils) for synthesis of copper nanoparticles in the oil phase. Of the various formulations synthesized, rosemary oil stabilized copper nanoparticles (RMO CuNPs) were noted to have the best inactivation kinetics and were also most stable. Upon morphological characterization by TEM and EELS, these were found to be monodispersed (φ 5–8 nm) with copper coexisting in all three oxidation states on the surface of the nanoparticles. The nanoparticles were drop cast on woven fabric of around 500 threads per inch and exposed to gram positive bacteria (Staphylococcus aureus), gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa), enveloped RNA virus (phi6), non-enveloped RNA virus (MS2) and non-enveloped DNA virus (T4) to encompass the commonly encountered groups of pathogens. It was possible to completely disinfect 107 copies of all microorganisms within 40 min of exposure. Further, this formulation was incorporated with polyurethane as thinners and used to coat non-woven fabrics. These also exhibited antimicrobial properties. Sustained disinfection with less than 9% cumulative copper loss for upto 14 washes with soap water was observed while the antioxidant activity was also preserved. Based on the studies conducted, RMO CuNP in oil phase was found to have excellent potential of integration on surface coatings, paints and polymers for rapid and sustained disinfection of microbes on surfaces.

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Introduction
Indiscriminate anthropogenic activities for societal development [1], hygiene and at times just for exotic leisure [2] are causing spontaneous mutations in the environmental microbial fauna.Some of these have the potential of inflicting lethal pathogenesis on humans.
A visible outcome is the increased number of novel waterborne and airborne pathogens, such as the SARS-CoV-2 [3], Zika Virus [4], Ebola Virus [5], multi drug resistant bacteria [6] and multi drug resistant fungi [7] to name a few.The roots can be curbed only through effective implementation of environmental stewardship policies to preserve ecological balances.
Devising and implementing these measures may be beyond the scope of pure science.Moreover, with the ever-increasing rise in the severity of disease caused by microbes, researchers are forced to focus on combating the aftereffects.These include development of novel diagnostics [7], therapeutics [8], vaccines [9], drugs [10] and disinfectants [11].
Antimicrobial and antiviral properties of copper nanocomposites have been extensively studied and documented, however, its extensive use in the nano form for disinfection purposes is limited due its poor oxidative stability in both aqueous and oil phases.Over the years, strategies have been devised to increase the stability of the nanoparticles in both aqueous and oil phases with the use of stabilizers.Stabilizers such as, polyphenols [12,13], gelatin [14,15] or polysaccharides [16,17] have been used for CuNPs synthesis in the aqueous phase, while, for synthesis in oil phase, stabilizers such as fatty acids [18][19][20], surfactants [21] or glycols [22,23] have been reported.In the oil phase particularly, it is difficult to synthesize stable nanoparticles due to the gradual oxidative dissolution of the CuNPs [18].Hence, it is desirable that the capping agent has antioxidative properties.The presence of these antioxidants further enhances the antimicrobial potency of the nanoparticles.Through this study, we report a method of synthesis and phase transfer of CuNPs to the organic phase for integration with commercial polymers, as well as adsorptive retention in natural woven fabrics.Fatty acid stabilizers, such as oleic acid, stearic acid, lauric acid, and fatty acid con-taining essential oils, such as, cinnamon oil and rosemary oil were explored for attaining optimal stability of the CuNPs in the organic phase.Rosemary oil stabilized nanoparticles were found to be the most stable and efficient for the disinfection of surfaces.The antiviral and antimicrobial action of these CuNPs were analyzed with bacteriophages MS2, SUSP2, and Phi6 to encompass non-enveloped RNA, nonenveloped DNA and enveloped RNA viruses respectively, and, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa to encompass classes of gram-positive and gramnegative bacteria.The nanoparticles were characterized by HR-TEM and copper leaching from cloth surfaces was analyzed with ICP-AES.The results revealed that the optimized nanoparticle formulations, 5-8 nm in size could inactivate 10 7 copies of bacteria and viruses within 90 and 50 min respectively.Since the nanoparticles are in an organic (toluene) base, they can easily be incorporated in paints, coatings and textile manufacturing industries.
Most studies investigating the antibacterial and antiviral effects of novel formulations focus merely on disk susceptibility tests to determine the zone of inhibition, or the minimum lethal dose (LC 50 ) as summarized in table 1.This is one of the few studies which investigates in detail the microbial inactivation kinetics of both bacteria and virus comprehensively along with the synthesis and integration of copper nanoparticles as neat, or with polymers, for rapid and sustained disinfection of surfaces, with minimal leaching.A comparative analysis of the antimicrobial activity of the synthesized rosemary oil stabilized copper nanoparticles (RMO CuNPs) with previously reported glycols, surfactants and fatty acids stabilized nanoparticles, has been presented in table 1.The parameters compared were the concentration of the metal precursor used for synthesis of the nanoparticles; antimicrobial activity in terms of zone of inhibition or minimum inhibitory concentration or copies of microbes inactivated from an initial titer; and the duration of inhibition/inactivation. Analysis of the literature revealed that with a minimum concentration of metal precursor, RMO-CuNPs could inactivate an initial titer of 10 8 copies of bacteria/bacteriophage in the least time.

Reagents
Analytical grade toluene, tetraoctylammonium bromide (TOAB), dodecanethiol (DDT), 2,2-diphenyl-1picrylhydrazyl (DPPH), copper sulfate, oleic acid, stearic acid and lauric acid were procured from Merck, India.Deionized (type II) water was used for preparing all aqueous solutions.Rosemary oil and cinnamon oil were procured from a local retailer in Mumbai.Woven and non-woven fabrics were procured from a local retailer.Commercial grade polyurethane used for coating of the non-woven fabrics was procured from a bio-safe apparel manufacturer in Surat, India.

Synthesis protocol
In a typical synthesis protocol 25 mM equivalent of TOAB (phase transfer agent) and 25 mM of the chosen fatty acid (cinnamon oil/rosemary oil/oleic acid/stearic acid/lauric acid), as stabilizers, were dispersed in 25 ml of toluene in a conical flask kept in a bath sonication at 50 • C. The mixture was transferred to a hotplate stirrer at 50 • C at 800 RPM.DDT (5 µl) was added to this mixture as an additional stabilizer.To this, 25 ml of 5 mM copper sulfate solution was added.The solution was stoppered and stirred for about 30 min for homogenization of all ligands.Finally, 10 mM equivalent of freshly prepared sodium borohydride was added and the stirring was continued for another 30 min.
Complete phase transfer of the CuNPs was observed as the bottom aqueous layer became transparent while the toluene layer turned black to dark coffee brown to reddish brown (figure 1(b)).The organic phase containing the copper nanoparticles was carefully decanted and stored in an airtight glass container at 4 • C.

Preparation of CuNPs coated cloth
Antibacterial and antiviral efficacies of the nanoparticles were tested with CuNPs coated woven cloth.Cotton cloth pieces of approximately 500 threads per inch and surface area 16 cm 2 were incubated with 1 ml of the synthesized nanoparticles and air dried.The cloth pieces were then rinsed with deionized water twice, to remove excess and unadsorbed reagents, dried at room temperature, and used for further studies.Antiviral disinfection kinetics of the nanoparticles were also studied on CuNPs coated non-woven cloth.

Microorganism cultures and buffers
All bacteria were maintained as glycerol stocks at −80 • C. E. coli MTCC 443 and P. aeruginosa were cultured in Luria Broth, S. aureus was cultured in nutrient broth while E. coli C3000 was cultured in 271B media (reconstituted as per standard CLSI guidelines).Each week, the primary culture was grown from glycerol stock in the respective bacterial media and refrigerated at 4 • C. Just before initiating an experiment, a secondary culture was made by inoculating 1% of the primary culture and grown until the OD was between 0.7 and 1.The bacterial culture was reconstituted to the desired concentration (corresponding to 10 6 CFU per 10 microliters).Serial dilutions of the cultures were plated to obtain the count of bacteria in CFU ml −1 .
Bacteriophage SUSP2 was purified by inoculating the phage in a fresh E. coli C3000 bacterial culture, grown to an OD of 0.7 with a multiplicancy of infection between 3 and 5 (Implying the total count of phages in the suspension would be three to five times higher than that of the bacteria).The mixture was kept in a shaker at 37 • C. In about four hours, the turbidity decreased significantly indicating bacterial lysis and release of phages.The suspension was ultra-centrifuged at 70 000 g for 45 min.The supernatant was discarded and the sediment reconstituted in phosphate buffer comprising 1 mM MgCl 2 and 10 µg ml −1 DNAse.A few drops of chloroform were added to the mixture to induce complete bacterial lysis and release of phages.The DNAse in the solution would digest off the bacterial DNA.The mixture was kept in a shaker at 37 • C for about 45 min after which it was centrifuged at 20 000 g.This time the sediment was discarded (as it comprised bacterial debris) and the supernatant was collected.The supernatant was syringe filtered using Whatman™ 0.22 µm sterile syringe filter and centrifuged at 100 000 g for 90 min.The pellet comprising of purified bacteriophage was reconstituted in SM buffer.Dilutions of the purified phages were made in SM buffer and the counts were determined using standard plaque assay protocols.The same protocol was followed for propagation of MS2 and phi6 with their respective hosts.Viral samples of 10 8 PFU ml −1 were used for experiments with suitable controls as illustrated in the figures

Protocol for antioxidant assay of the CuNP coated substrates
Antioxidant assay was conducted on both porous and nonporous substrates modified with RMO CuNPs, by multiple wash-dry cycles using deionized (DI) water with 1.2% sodium dodecyl sulphate (SDS) by DPPH assay [33].Typically, 0.1 mM solution of DPPH was prepared in methanol and recorded for absorbance using UV-visible spectrometer.6 ppm of ascorbic acid (AA), equivalent to the copper content on the cloth was used as a positive control.Both porous and nonporous substrates were coated with 1 ml of synthesized CuNPs (6 ppm) and air dried as described earlier.Further, the substrates were washed successively with 5 ml of 1.2% SDS in deionized water at room temperature (25 • -30 • centigrade) at 300 RPM on a stirrer for 20 min.After the requisite wash, the substrate was first dried and subject to DPPH analysis solution with the same settings as earlier.The spectral analysis of the solution post incubation was performed with a UV−Vis spectrophotometer.Absorbance spectra of the obtained elutes were recorded (SI figure 1) and the reduction of DPPH was quantified, as shown in figure 5 and SI table 2. All the spectrophotometric measurements were done on separate porous and nonporous substrates with three replicates for each measurement.

Protocol for bacterial and viral inactivation assay
Antiviral and antibacterial studies were carried out on CuNPs coated natural woven and non-woven fabrics.It has been reported that a typical activity of sneeze and cough approximately releases 10 3 -10 6 copies of bacteria or virus.To simulate such a scenario, typical experiment involved dispensing of 10 droplets of viral or bacterial suspensions of microbial titer 10 7 per droplet and volume 5 µl on one such cloth piece and an unmodified cloth piece served as control.At each time point, the microbes were eluted by rinsing the cloth piece using a suitable buffer and performing a plaque/microbial assay by plating method.Microbial counts from such cloth pieces was studied with five cloth pieces (three for measurement and two for control).The variation in the microbial counts is indicated using standard deviations (figure 3).Such experiments with a particular microorganism were sequentially carried out on woven as well as non-woven fabric.

Characterization of the CuNPs
The rosemary oil stabilized CuNPs were seen as spherical nanoparticles of diameter 5-8 nm in FEGTEM analysis (figure 2(a)).Copper was found to co-exist in all three oxidation states Cu(I), Cu (II) and Cu on the surface of the nanoparticles through SAED analysis (ICDD PDF Card-01-070-3038_Cu, PDF Card-00-034-1354_Cu 2 O, PDF Card-00-041-0254_CuO).
FTIR analysis of the CuNPs was done to confirm presence of rosemary oil as a stabilizing agent and is depicted in figure 2(b).Of particular interest are peaks 1414 cm −1 , 1078 cm −1 and 781 cm −1 corresponding to C=C aromatic, C=O stretching of ester and tertiary alcohol, C-O-C symmetrical stretch, and C-H bending of isoprenoids.These aromatic functional groups are likely to have originated from 1,8 cyneole, alpha pinene and camphine with particular free radical scavenging properties [34].Dhouibi et al estimated the Gibbs free energy of adsorption of RMO on copper as −18.87 kJmol −1 elucidating spontaneous adsorption of these on the phase transferred nanoparticle [35].The capping blocks off most cathode active sites on the copper, substantially improving the stability of the RMO-CuNP.
XRD analysis of RMO-CuNPs with k beta filter, 0.01 step size, and 7 kW power revealed peaks corresponding to Cu, CuO, and Cu 2 O (ICDD PDF Card-01-070-3038_Cu, PDF Card-00-034-1354_Cu 2 O, PDF Card-00-041-0254_CuO) which confirmed the presence of copper in all three oxidation states.XRD analysis before and after washing demonstrates negligible changes in the ratio of phases indicating good stability of the prepared formulations (SI table 3).
Leaching of copper from the woven fabric was characterized with inductively coupled plasma atomic emission spectroscopy (ICP-AES), Spectro, Arcos, GmbH.

Effect of fatty acids
Fatty acids and essential oil containing fatty acids, with known antioxidant properties such as cinnamon oil (CNO), rosemary oil (RMO), oleic acid [36], stearic acid (SA), and lauric acid (LA) were used as stabilizers for the synthesis of copper nanoparticles as per the synthesis protocol laid out earlier.An instantaneous color change from colorless to black/dark brown was observed in the organic phase after the addition of a reducing agent to all fatty acid-stabilized suspensions.However, the solution soon turned colorless within a day after synthesis in most cases except for synthesis using when cinnamon oil (CNO), rosemary oil (RMO), and oleic acid [36].This is possibly due to the rapid oxidative dissolution during synthesis confirming the stability of nanoparticles (supplementary information section S1).Only these stable nanoparticle suspensions, that is, CNO, RMO, and OA CuNPs dissolution of the synthesized nanoparticles in the oil phase [18].HR-TEM images of RMO-CuNPs taken after one month were used for the evaluation of antiviral efficacy (figure 3).It was found that RMO CuNPs exhibited the best viral inactivation kinetics and hence this formulation was shortlisted for all further studies.

Effect of wash-dry cycles
Woven cloths loaded with RMO CuNP, as per the protocols mentioned in section 2.3, were subjected to 14 consecutive wash-dry cycles of fixed volumes of (a) deionized (type II) and (b) 1.2% SDS containing deionized water.Individual cloth pieces were incubated in 5 ml of (a) and (b) separately and stirred at 300 RPM for 20 min at room temperature (25 • -30 • centigrade).After each wash event, the eluate was collected and analyzed by ICP-AES for copper content.To get the average initial copper content in the cloth piece, before subjecting it to any wash, three cloth pieces were digested and analyzed by ICP-AES and were found to contain 6.3 ppm of Cu.The cumulative percentage of copper loss after each wash was then calculated with respect to the initial concentration and the results are summarized in figure 4. It was observed that in the initial three washes, the cumulative copper leachate was the maximum at around 2.7% in DI water and 5.2% in 1.2% SDS solution, after which, by 14 wash-dry cycles, the leaching of CuNPs decreased and almost stabilized, indicating excellent absorptive retentivity of the CuNPs on the woven fabrics.The cumulative copper leachate was less than 5%

Antioxidant activity of CuNPs on porous and non-porous substrates
The effect of CuNP antioxidant activity on substrates with varying porosity was assessed by 2,2-diphenyl-1picrylhydrazyl (DPPH) assay [33] after each consecutive wash-dry cycle for 22 consecutive cycles with 5 ml of deionized water with 1.2% SDS mimicking soap wash.Antioxidant activity was tested on a woven fabric, a porous substrate of 16 cm 2 area, and a non-woven fabric which is a nonporous substrate with similar dimensions, and calculated by the equation (1) [37] where, where Abs control is the absorbance value of the unmodified fabric washed with DPPH assay solution while Abs test implies the absorbance value of the respective fabric modified with CuNP.Ascorbic acid (6 ppm, equivalent to Cu content on the fabrics) was used as a positive control Antioxidantactivity (%) = Abs control − Abs test Abs control X100.(1) Decrease of absorbance at 518 nm was recorded (figure 5(c)).The instantaneous color change of 0.1 mM DPPH solution added to the nanoparticle deposited substrates was quantified to assess the cumulative loss of anti-oxidant activity of the substrate, after each wash-dry cycle, as shown in figure 5.
The effect of RMO as a stabilizer CuNP was also quantified by DPPH assay on both woven and non-woven cloth substrates.The initial five washes of woven substrate showed a 8.84% loss of antioxidant activity where RMO was used as a stabilizer in the synthesis of CuNP that was coated, and a 12.56% loss of antioxidant activity where the coated CuNPs were synthesized without RMO.After 22 wash-dry cycles, a cumulative activity loss of 16.55% for CuNPs synthesized without RMO while 12.85% with CuNPs with RMO as a stabilizer was noted.These results clearly demonstrate that RMO enhances the stability as well as the antioxidant activity of the CuNPs.The woven (porous) substrates coated with RMO-CuNP retained 83.33% of antioxidant activity while  non-woven (non-porous) substrates retained 78.23% of antioxidant activity against a positive control of 6 ppm ascorbic acid, which when dropcast on woven fabrics showed 97.2 ± 1.71% antioxidant activity, while the same in the case of woven substrates while 95.2 ± 3.79% antioxidant activity was seen on non-woven fabric.The results clearly illustrate that the RMO-CuNP retain their antioxidant activity even after 22 washes and hence are capable of sustained disinfection of microorganisms.Similar observations were noted for non-woven substrates and are illustrated in figure 5(b) while the raw spectroscopic data is presented in SI figure 1 and SI table 2. These results are also in synergy with the ICP-AES results (figure 4 and SI table 1), where the overall loss of CuNPs from the substrates may lead to a loss of the overall antioxidant activity as seen on the surface.

Antiviral assays on non-woven fabric
Non-woven fabric and commercial polyurethane [15] coating was procured from a local mill.The coating was thinned with the RMO CuNP solution prepared earlier and an equivalent amount of copper was coated on the 20 cm 2 area of non-woven fabrics.The inactivation kinetics are depicted in (figure 6).It was noted that the droplets remained intact for over 120 min of incubation and hence absolute inactivation was not seen even after three hours.It was also observed that active viral particles were better retained on coated non-woven fabrics than the uncoated woven fabrics.This is probably because of the hydrophobic nature of the non-woven fabric, allowing for prolonged activity in a relatively higher hydration.These observations are in line with the theoretical studies by Bhardwaj and Agrawal [38].The lower inactivation in case of CuNP-coated nonwoven fabrics may also be attributed to a relatively lower surface availability of the RMO CuNP due to embedding in the PU.

Discussion
Copper nanostructures were synthesized in the oil phase using a modified version of the Brust-Schiffrin method [39].Various combination of fatty acids and dodecanethiol were tested for synthesis of stable copper nanoparticles.Contrary to the conventional Brust-Schiffrin method where phase transfer of soft acid ions precedes its reduction in the oil phase, it appeared that on addition of reducing agent, copper ions were reduced to form copper nanoparticles and simultaneously migrated to the oil phase.This was ascertained as the aqueous phase retained a tinge of blue until the addition of sodium borohydride when the oil phase changed color from colorless to black to reddish brown.The plausible mechanisms of the CuNPs adsorbing and adhering on the woven fabric are adsorption on porous cellulose threads [40], hydrophobic interactions of rosemary oil (capping agent) with cellulose [41], and hydrogen bonding of phenolic components of rosemary oil with cellulose [40].Rosemary oil typically comprises of compounds that may be classified as diterpenes (carnosic acid), flavonoids (apigenin and luteolin) and phenolic acids (Rosmarinic acid).Each of these compounds have aromatic -OH, -COOH and C=C aromatic along with C=O stretching, with the exception of carnosic acid which has -COOH and -OH groups only.These groups would typically have strong dipole-dipole interactions with the linearly stacked glucose structures and glycosidic linkages [42].Electrostatically driven CO-π and CH-π interactions of the flavonoids and phenolic acids with cellulose [43] also possibly explain the strong adsorption of the RMO stabilized copper nanoparticles on the cellulose and minimal leaching in aqueous (polar) solvents.In the case of non-woven fabrics, the formulation is integrated in PU which polymerizes on the cloth.The results of the antioxidant analysis clearly demonstrate better sustained antioxidant action as well as better retentivity of the RMO stabilized CuNPs.
The two primary ingredients of rosemary oil are 1,8 cyneole and α pinene both of which are proven to have excellent antioxidant as well as antibacterial properties [44,45].These thus act as stabilizers preventing oxidative dissolution of the nanoparticles in the oil phase as well as enhances the antimicrobial characteristics of the formulations.Rosemary oil coated cloth pieces itself led to inactivation of 4 orders of virus Phi6.The synergistic action of rosemary oil and copper nanoparticles led to complete disinfection of Phi6 within 25 min.
The antimicrobial and antiviral action of CuNPs can be attributed to the affinity of copper of Cu(I) and Cu(II) ions to sulfur and phosphorus, present abundantly in proteins, cell membrane and nucleic acids of bacteria and virus.Complexation of copper ions with proteins causes a loss in protein functionality leading to an increase of reactive oxygen species [4] in the cell, oxidative stress and eventually cell death.Complexation of Cu(II) ions with nucleic acids leads to loss in cell division potency.Further, the cycling of copper between the three oxidation sates possibly increases oxidative stress in the microorganism.The physiosorbed/chemisorbed or complexed ions and nanoparticles eventually limit the multiplication of the microorganism even in a favorable host or environment eventually leading to cell inactivation and/or death [46].
It was observed that inactivation of bacteriophage MS2 was the slowest while that of phi6 was the fastest.This is probably due to higher vulnerability of the viral lipid envelope to the formulations developed.Envelope is not present in MS2 whereas Phi6 is an enveloped RNA bacteriophage.Hence, we believe that these formulations shall have a higher efficacy towards enveloped viruses such as the SARS-CoV-2 and can be used for sustained disinfection of the same.Also S. aureus, a gram positive bacteria, was observed to be inactivated faster than E. coli or P. aeruginosa (gram negative bacteria).Copper has a higher affinity to proteins than lipids.Hence, gram positive bacteria, which has a thicker peptidoglycan layer and more abundance of proteins, are inactivated faster when compared to gram negative bacteria.The results are in in-line with that reported by Tiwari et al [47].Cytotoxicity analysis of the RMO-CuNPs was also done and presented in the supplementary information.
Minimal leaching of copper from the cloth pieces when subject to aqueous solutions was observed in aqueous media (figure 4).Hence, it may be ascertained that the antimicrobial and antiviral action of the RMO-CuNP loaded fabrics is primarily by contact killing as investigated in [48].Sustained disinfection was further investigated by subjecting the same cloth piece to 10 6 CFU ml −1 of bacteria (E.coli) for 60 min on a weekly basis with the protocol as described earlier.Complete disinfection was observed for eight consecutive weeks.Similar results were seen for virus (T4) for eight consecutive weeks where complete disinfection implies that the order of the bacteria or virus was between 10 5 and 10 6 where the initial count was 10 6 .Hence, the formulation immobilized on fabrics is most suitable for sustained use.

Conclusion
Antimicrobial properties of CuNPs synthesized in the aqueous phase have been extensively studied.However, their use in polymeric coatings for sustained disinfection remains limited due to their low oxidative stability in organic solvents.This article presents a method of synthesis of copper nanoparticles in the oil phase.Systematic analysis of various formulations revealed rosemary oil, cinnamon oil and oleic acid stabilized copper nanoparticles as the most stable formulation, of which RMO-CuNPs were found to have the fastest antimicrobial activity.These CuNPs could effectively inactivate 10 7 counts of bacteria and virus within a contact time of 90 min and 50 min respectively.Also, the demonstrated potential integration of these into polymers such as polyurethane lays out prospects of their use in sustained disinfection when added to paints, plastics, hospital surfaces and natural fabrics.Most studies investigating the antibacterial and antiviral effects of novel formulations focus merely on disk susceptibility tests to determine the zone of inhibition, or the minimum lethal dose  E. coli [28] Tween 80 synthesized Fe    (LC 50 ) as summarized in table 1.This is one of the few studies which investigates in detail the microbial inactivation kinetics of both bacteria and virus comprehensively.The usability of our proposed formulation for bacterial and viral inactivation with diverse textiles and surfaces stands comprehensively established.

Figure 1 .
Figure 1.(a) Schematic representation of the method of synthesis of rosemary oil stabilized nanoparticles.(b) Step by step method for synthesis of RMO-CuNPs.(c) Mechanism of antibacterial and antiviral action of the synthesized CuNPs.

Figure 2 .
Figure 2. (a).FEG-TEM images and diffraction pattern of CuNPs with rosemary oil (RMO) as the stabilizing agent (b) FTIR (KBr) analysis of RMO CuNPs (c) XRD characterization shows that copper coexist in all three oxidation states Cu(I), Cu (II) and Cu on the nanoparticles.

Figure 3 .
Figure 3. (a) Disinfection kinetics of coliphage MS2 with RMO CuNPs, CNO CuNPs and OA CuNPs, studied on woven cloth as microdroplet assays.Measurement at each time frame was done in triplicates.(b) Inactivation kinetics of bacteria Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa with RMO CuNPs.(c) Inactivation kinetics of bacteriophages Phi6, MS2 and SUSP2 with RMO CuNPs.

Figure 4 .
Figure 4. Accumulated copper loss from RMO-CuNP coated cloth pieces, washed multiple times with deionized water and deionized water with 1.2% SDS.

Figure 5 .
Figure 5. Cumulative anti-oxidant activity loss of (a) non-woven, non-porous fabric, and (b) woven, porous fabric, coated with CuNPs and RMO-CuNPs with multiple wash-dry cycles; (c) absorbance spectra of the elutes obtained from RMO-CuNP coated woven fabric for 22 successive wash-dry cycles.

Table 1 .
Comparison of antiviral and antimicrobial action of RMO CuNP with oil phase nanocomposites previously reported.