Delta SARS-CoV-2 inactivation and bactericidal performance of cotton wipes decorated with TiO2/Ag nanoparticles like Brazilian heavy-fruited Myrciaria cauliflora.

The current pandemic of Coronavirus Disease 2019 (COVID-19) raised several concerns about using conventional textiles for manufacturing personal protective equipment without self-disinfecting properties since the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is transmitted mainly by aerosols that can transpose cotton masks. Therefore, developing new cotton fibers with high self-disinfecting ability is essential to avoid a new pandemic due to new SARS-CoV-2 variants. Herein, we developed cotton wipes (CFs) with fibers coated by Ag, TiO2, and Ag/TiO2 hybrid nanoparticles like Brazilian heavy-fruited Myrciaria cauliflora by a sonochemical approach. Moreover, the coated CFs present high antimicrobial performance against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), being able to inactivate infectious SARS-CoV-2 (Delta variant) by the destruction of the spike, membrane, and nucleocapsid proteins while the viral RNA is not significantly affected, according to the molecular biological findings.

Introduction

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been causing a worldwide collapse of the health systems due to the global Coronavirus Disease 2019 (COVID-19) pandemic. Beyond the epidemiological crisis, humanity faced socioeconomic issues and several changes in all human living aspects [1], [2]. This coronavirus is transmitted from person to person mainly via respiratory droplets dispersed in the air (aerosol), but direct and indirect contact routes also represent transmission pathways. With the identification of more than four SARS- CoV-2 variants since 2020, there are several concerns about the emergence of new pandemic waves associated with them, since more infectivity, transmissibility, reinfection risks, along with a limited potential efficacy of the current vaccines against these coronavirus variants due to multiple mutations in the spike protein [3], [4], [5], [6], [7].

The indirect coronaviruses transmission via fomites is epidemiologically worrisome because these viruses remain infectious for more than 24 h on the surface of different materials, including cotton [8], which are frequently used to make personal protective equipment (PPE) worn by citizens, as well as healthcare personnel [9]. For this reason, it is crucial to develop new materials able to destroy the SARS-CoV-2 particles quickly, providing effective and safe protection against the spread of coronavirus disease.

Synthetic and natural materials are fundamental in the manufacture of textile fibers. Among them, cotton differentiates as a renewable natural resource, proper to fabricate low-cost yarns for the confection of clothes and PPE, such as masks, with softness and breathability features [10]. These characteristics of cotton are highly appreciated allowing its widespread use in several wearable textile products. However, cotton does not present self-disinfection capability. Consequently, the cotton’s surfaces may contribute to the proliferation of microorganisms as it cannot deactivate viruses [11].

The cotton fiber’s surface modification with antimicrobial agents has been the main technological method to impart bactericidal properties to cotton textiles [12]. Copper (Cu), copper oxide II (CuO), gold (Au), platinum (Pt), Silver (Ag), silicon dioxide (SiO2), titanium dioxide (TiO2), and zinc oxide (ZnO) [13], [14], [15], [16] are the major antimicrobial agents playing this role. These inorganic materials can provoke several irreversible damages to virions, bacteria and fungi cells due to releasing metal ions and the generation of reactive oxygen species (ROS) [15], [17], [18]. In addition to antimicrobial properties, silver has exciting properties and has shown high potential in technological areas, such as catalysis and electronics [19]. The anatase phase of TiO2 stands out as a non-toxic and low price antimicrobial compound. Also, this oxide displays high photochemical activity and stability [20], [21]. The photocatalytic and self-disinfecting performance of TiO2 can be improved by doping this semiconductor with silver and other noble metals, such as Au, Pt, and Pd. These transition metals cause interfacial charge transfer that delays the recombination of electron-hole pairs, acting as electron trapping agents [22], which improves the photocatalytic and antimicrobial efficiencies of TiO2. In addition, the surface plasmon resonance of Ag nanoparticles (AgNPs) can affect the electron transfer from metallic silver to TiO2, resulting in charge separation activated by visible light, widening the excitation electromagnetic spectrum range, thus improving the self-disinfecting capability [23].

The sonochemical method is an alternative route to synthesize these antimicrobial nanoparticles, suitable to be attached onto cotton fibers' surface by using binders, imparting bactericidal and/or anti-virus functionalities [11], [24] to multifunctional textiles. The sonochemical route is considered sustainable and not time-consuming for coating routes of solid surfaces, as well as the synthesis of metal and oxide nanoparticles (NPs). The ultrasonic irradiation in aqueous media can provoke acoustic cavitation, local heating (> 5000 K), and local pressurization (> 1000 atm), leading to chemical reactions responsible for the reduction of metal ions of metal/oxide precursors dispersed in water [25], [26], [27], [28], [29]. For this purpose, the ultrasound waves must have a relatively high frequency, typically from 20 kHz to 1 MHz [30]. Ramezani et al. [31] successfully synthesized Ag-based NPs via sonochemistry for anticancer applications. Zhanjiang et al. [19] obtained AgNPs with diameters less than 10 nm using ultrasound irradiation and silver nitrate as a precursor agent. Weiße et al. [30] coated textile surfaces with ZnO and TiO2 nanoparticles by a sonochemical approach.

Different studies have investigated the photocatalytic [32], [33], [34], [35] and antimicrobial [36], [37], [38], [39] activities of Ag/TiO2 nanocomposites. Also, several metallic and oxide intrinsic antimicrobial agents have been applied to manufacturing smart self-disinfecting textiles [40], [41], [42], [43], [44], [45]. However, the sonochemical approach for making NPs of these hybrid materials aiming at surface modification of cotton textiles has not been reported. In this work, we synthesized TiO2/Ag hybrid nanoparticles (TiO2/Ag-NPs) by sonochemical treatment of TiO2-NPs with different silver contents. Moreover, we evaluate the bactericidal and antiviral performances of cotton wipes coated with these hybrid nanoparticles. This research demonstrates that silver improves the antimicrobial activity of the CFs coated with TiO2-NPs against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), being able to damage the partially external structures of Delta SARS-CoV-2 virions. Besides that, the reduction of methylene blue was used to investigate the photocatalytic activity of the cotton wipes coated with TiO2/Ag nanocomposites. Using our proposed coating method, we obtained cotton fibers decorated with spherical micro and nanoparticles that resemble the peculiar and interesting heavy-fruited branches shown by the Brazilian Myrciaria cauliflora.

Experimental section

Materials

The cotton wipes (CFs) were purchased from a local fabric store (São Paulo, Brazil), based on woven fibers constituted predominantly by cellulose [40]. Anatase TiO2-NPs were acquired from Sigma-Aldrich Inc. (São Paulo, Brazil). Phenol, neutral detergent, ethanol, L-ascorbic acid, trisodium citrate, and silver nitrate were supplied by Labsynth (São Paulo, Brazil). The water-based acrylic binder (Acrylic Resin 180 W) was purchased from Redelease (São Paulo, Brazil). Nutrient agar culture medium was purchased from Himedia. Methylene blue (MB) was purchased from Merck. Anhydrous gallic acid was obtained from InLab.

Synthesis of TiO2/Ag-NPs

Trisodium citrate (1 g) was dissolved in 100 mL of an aqueous suspension of TiO2-NPs (2000 ppm) containing several concentrations of AgNO3 (0, 250, 500, 750, and 1000 ppm). Then the suspension was ultrasonicated for 3 min, using a tip probe (Vibra Cell VCX 500, Sonics, USA) with an amplitude of 40%.

Coating procedure

The binder (1 g) was added to the TiO2/Ag-NPs dispersion (100 mL) and mixed. Then, CF (prewashed with aqueous detergent solution, 6.7 g L-1) (dimension 10 cm × 10 cm) was immersed in the dispersion and left for 30 min. After that, CF was heated at 120 ± 5 °C (20 min), washed and dried at 120 ± 5 °C (20 min). The coated cotton wipes (CFs) were named CF-TiAgX, where X represents the concentration of the silver ions applied in the synthesis of TiO2/Ag-NPs suspensions.

Characterization of the nanoparticles

Dynamic light scattering

The hydrodynamic diameter (DH) distributions of the nanoparticles were obtained by dynamic light scattering (DLS) technique with a 90° scattering angle. Zetasizer Nano-ZS (Malvern Panalytical Ltd., Malvern, U.K.) was the equipment utilized for the DLS characterization. Aliquots of the suspensions (100 µL) were suspended in distilled water (2 mL), and each sample was analyzed in duplicate.

Zeta potential (ζ)

The zeta potential was measured using Zetasizer Nano-ZS (Malvern Instruments) and Smoluchowski model. For this purpose, aliquots of the TiO2/Ag-NPs suspensions (100 µL) were suspended in distilled water (2 mL), and each sample was analyzed in duplicate. The surface charge density () was calculated from the ζ and DH data using Eq. (1) [46].where states for the vacuum permittivity,, the relative permittivity of the suspension medium, , the Boltzmann constant, , the absolute temperature, , the ion valence, , the fundamental electron charge, and , the Debye-Huckel parameter determined by Eq. (2), using the apparent viscosity of the suspension medium ().

UV-Visible spectroscopy

The absorption spectra from the aqueous suspensions with the synthesized nanoparticles were collected by UV–vis spectrophotometer (Varian Cary Model 50) at the wavelength range of 200–800 nm.

The optical bandgap (Eg) of the nanoparticles was obtained from the UV-Vis spectra using the graphical method: plotting (αhν) versus hν and extrapolating the linear region on the radiation energy axis (hν) [47]. Where h is Planck's constant, ν is the frequency of electromagnetic radiation, n depends on the nature of the transition (n equal to 2 for indirect transition and equal to ½ for direct transition), and α is the absorption coefficient. In this theoretical approximation, it is assumed that the refraction index of the semiconductors is constant in the energy range involving the electronic transitions [48], [49].

Characterization of the coated cotton wipes

Scanning Electron Microscopy (SEM) and Energy-dispersive X-ray spectroscopy (EDS)

Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) was performed in the JEOL JSM-6010LA scanning electron microscope equipped with a chemical microanalysis module (EDS). The SEM micrographs and EDS spectra were obtained using a working distance of 14 mm and an electron acceleration voltage of 10 keV. The samples were previously coated with a 20 nm thick gold layer, using Sputtering Leica EM ACE 200 (Leica Microsystems, São Paulo, Brazil).

Fourier-transform infrared spectroscopy

Samples were characterized using Fourier-transform infrared spectroscopy (FTIR) with attenuated total reflectance (ATR) of a diamond crystal (Frontier 94942 equipment, PerkinElmer Inc., Massachusetts, USA). The spectra were collected at 4 cm-1 spectral resolution, after 64 scans from 4000 to 600 cm-1.

Photocatalytic activity

The photocatalytic activity was evaluated by monitoring the photodecomposition of methylene blue (MB). The CF (1 cm × 1 cm) were immersed in 5.75 mL of aqueous solutions of MB (37.5 mg mL-1, pH = 7). To start the photocatalytic reactions, the solutions with the cotton samples were exposed to constant illumination of a blue lamp (440–490 nm, 100 W m-2). Then, the solution was analyzed at regular intervals using a UV–Vis spectrophotometer (UV-M51, LAB 1000) by measuring the absorption at 665 nm [50]. The photodegradation efficiency () of methylene blue (MB) was calculated by Eq. (3).where A0 is the initial absorbance of MB solution; At is the absorbance of the MB solution after illumination at time t.

The photocatalytic activity was evaluated by the pseudo-first-order kinetic Langmuir-Hinshelwood model, Eq. (4), which is suitable for photocatalytic cases with low dye concentration and involves a predominant effect of the adsorption step [20].where C0 is the initial concentration of MB solution; Ct is the MB concentration in the solution after t time illumination; is the rate constant of the photocatalytic reaction (min-1). The relative concentration () can be calculated by the relative absorbance () [51], [52].

Identification of Reactive Oxygen Species (ROS)

These experiments are identical to the photocatalytic degradation assay of MB except that one radical scavenger, each time, is added to the system. The ROS, active species, generated by the cotton wipes coated with TiO2/Ag-NPs were evaluated by the photodegradation of MB in the presence of trapping agents at 1 mmol L-1. Phenol, ethanol, and L-ascorbic acid were added to the MB photodegradation to catch hydroxyl radical (OH), holes (h+), and hydroperoxyl radical (H2O) [51], [53], [54], [55], respectively. Gallic acid was applied as a trapping agent of both OH, H2O, and hydrogen peroxide (H2O2) deactivator [56], [57].

Bactericidal assay

The bactericidal assays are performed using diffusion tests in agar. In this way, the CF samples (1 cm × 1 cm) were placed in contact with the sterilized Petri dishes with nutrient agar culture medium and inoculated with bacteria suspension (Escherichia coli, E. coli ATCC 25922, or Staphylococcus aureus, S. aureus ATCC 6548) at 105 CFU mL-1, which were incubated at 36 ± 1 °C and 85% humidity. The viability of the bacteria inoculums was confirmed via a spread-plate method on agar nutrient as the positive control. The inhibition zone from the bactericidal activity of the CFs was evaluated after 48 h of incubation.

SARS-CoV-2 inoculum

SARS-CoV-2 inoculums were prepared using oropharyngeal and nasopharyngeal swabs (sterile swabs) from patients (anonymous – unidentified) diagnosed with COVID-19 from the laboratory of clinical analysis and molecular biology of the Faculty of Medicine of ABC (FMABC). The patients were contaminated with the Delta SARS-CoV-2 variant. The Delta strain was identified by genetic sequencing with Nanopore's MinION sequencer, using the ARTIC protocol. The contaminated swabs were placed in sterile saline solution (NaCl, 0.9%) in a Falcon tube (approximately 5 mL) and frozen at − 20 ºC until molecular biology assays. Five biological samples were selected, with a Ct value equal to 18 ± 2 (RT-qPCR previously performed for diagnosis in the FMABC laboratory) as a parameter used for selection. These samples were mixed to have a viable uniform viral inoculum. To assess the antiviral efficiency of the CFs, three exposure times were selected: 5, 10, and 15 min. We put 50 µL of the viral inoculum (COVID+) in each Eppendorf tube with a pre-established CF (10 variations) at room temperature for the incubation period previously fixed. After the incubation period, we added 300 µL of lysis solution for virus inactivation (Lysis Buffer – PureLink Invitrogen kit). These samples submitted to the incubation process with the CF samples (previously sterilized with UV radiation) were applied on antigen-detecting rapid test and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) for SARS-CoV-2.

SARS-CoV-2 antigen-detecting rapid diagnostic tests

We used the SARS-CoV-2 Rapid Antigen Test kit (Roche) (reference 9901-ncov-01 g) to detect the presence of antigens in the samples studied. We added 3 drops of the viral inoculum in the place indicated on the cassette using the sampler supplied with the kit. We wait 15 min as indicated by the manufacturer. The rest of the SARS-CoV-2 inoculum not used in the rapid test was frozen and stored at –20º C until the moment of RT-qPCR experiments. The rapid tests are chromatographic immunoassays for the qualitative detection of specific SARS-CoV-2 antigens. Then, the reduction of the number of infectious coronaviruses on the media (antiviral activity) was estimated by the red and white color intensity of the “test line” in rapid diagnostic tests using ImageJ software. The antigen detection limits of rapid tests for SARS-CoV-2 are: Quantity of stock of the titrant in the tests is: 1 × 106.2 TCID50/mL (maximum red intensity); Minimum detected viral load is 3.12 × 102.2 TCID50/mL (maximum white color intensity). The red and white color intensities were normalized to estimate the antigen TCID50/mL percentual reduction from SARS-CoV-2 antigen-detecting rapid tests.

Quantitative reverse transcription-polymerase chain reaction (RT-qPCR)

The frozen SARS-CoV-2 inoculums were vortexed for 30 s after defrosting. Then, the extraction process was started in a commercial kit (PureLink™ Viral RNA Mini Kit - Invitrogen™) using 350 µL of ethanol (70%) and homogenization. Next, 700 µL of the sample was transferred to the column and centrifuged for 1 min at 12,00 rpm, discarding the flow-through. The next step was washing with 700 µL of Wash Buffer I. Centrifugation for 1 min at 12,000 rpm. Discard the flow-through. 500 µL of Wash Buffer II was added. Centrifugation for 1 min at 12,000 rpm and this procedure was repeated, washing the sample twice with this same buffer. After the second wash, the filtrate was discarded, and centrifugation was carried out for 2 min at 12,000 rpm with the column dry. The column was transferred to a 1.5 mL microtube, and 50 µL of ultrapure water was added; the sample was then incubated for 1 min at room temperature and centrifuged for 2 min at 12,000 rpm. RNA was stored at − 80 ºC until detection of SARS-CoV-2 by RT-qPCR. Verification of the presence of SARS-CoV-2 genetic material was performed by PCR assays using the 2019-nCoV TaqMan RT-PCR kit (Norgen, Cat. TM67120). Manufacturers' guidelines were followed to operate the kit and the equipment used (CFX Opus Real-Time PCR Systems – Bio-Rad). The mix for the reactions was planned to reach a final volume of 20 µL, using in its composition: 5 µL of extracted RNA, 10 µL of Master Mix 2x, 1.5 µL of Mix (Primer and Probe in amounts proportional to those indicated for the assay) and 3.5 µL of ultrapure water, always in duplicate samples. The programmed reaction cycle was: cycle 1 – 50 °C for 30 min; cycle 2 – 95 °C for 3 min; cycle 3–45 × 95 °C for 3 s and 55 °C for 30 s. A negative control (water + reaction mix) and a set of positive controls in serial dilution (104, 105, 106, 107) we used to compose a standard curve for the selected targets (genes N1 and N2). Calibration curve data: N1 (y = −3.491x + 43.065; R2 = 0.996; Efficiency = 93.4%), N2 (y = −3.723 x + 44.750; R2 = 0.998; Efficiency = 85.6%) The limit of detection (LOD) was 10 copies of the genome for: Ct of 39.28 ± 0.05 for N1 and Ct of 39.77 ± 0.58 for N2.

Statistical analysis

Variance analysis (one-way and two-way ANOVA) and Tukey's test (T-test) were applied to statistically evaluate the significant differences between the samples' responses using the GraphPad Prism 7 and a 95% confidence level.

Results and discussion

TiO2/Ag hybrid nanoparticles

The TiO2-NPs modified with different silver contents were prepared by a one-step sonochemical method, using AgNO3 as a silver ion precursor and trisodium citrate as a reductant agent. Fig. 1a presents a schematic illustration of the nanoparticles’ synthesis and the CF coating procedure with these TiO2/Ag-NPs. The TiO2/Ag-NPs size distributions are shown in Fig. 1b. The ultrasound waves are the energetic source responsible for initiating the reduction of silver cations due to the generation of different reductant species [27], [58]: generation of free radicals via acoustic cavitation and pressure, according to Eq. (5); and generation of secondary radicals by the proton abstraction of organic molecules, as citrates, are described by (6), (7). All these radicals acts as chemical reductants bring on the formation of the AgNPs due to the reduction of Ag- to Ag0, according to (8), (9). Moreover, the oxidation of the citrate anions due to heating caused by the ultrasonic irradiation cause the silver reduction, according to Eq. (10) [27], [40]. The citrate anions can stabilize metallic nanoparticles, avoiding their aggregation or overgrowth via capping coordination [40].

Fig. 1

(a) Schematic representation of the sonochemical synthesis of the TiO2/Ag-NPs and surface coating of the cotton wipes. (b) The hydrodynamic diameter of the TiO2-NPs modified with several contents of silver. (c) Alteration of the direct optical bandgap (Eg) of the TiO2-NPs after sonochemical treatment with different Ag contents.

The DH distributions of the TiO2-NPs modified with silver are shown in Fig. 1b. The TiO2-NPs present a monomodal size distribution, with an average hydrodynamic diameter of 0.7 ± 0.3 nm. After sonochemical treatment with silver, the average DH of the TiO2-NPs is increased ( Table 1). At the same time, the DH distributions remain monomodal, indicating that a metallic silver layer covered the TiO2-NPs, forming a TiO2/Ag-NPs hybridous nanocomposite with diameters in the range of 2–220 nm depending on the silver ion content utilized in the sonochemical treatment. The ζ and results are also detailed in Table 1. TiO2-NPs and TiO2/Ag-NPs present ζ values out of the ± 15 mV range, indicating the absence of agglomerates in the suspension and effective repulsive forces among the electrically charged particles [59], [60]. According to the Stern model, the negative ζ value associated with the diffuse layer on the surface of the TiO2-NPs suggests a positive charge surface with a predominant presence of TiOH2 + groups rather than TiO- [61]; since the ionic charge of the electric double layer (EDL), including the diffusive layer, in the electrolyte medium has equal and signal opposite to the electronic surface charge () of the nanoparticles.

Table 1

Hydrodynamic diameter (DH), zeta potential (ζ), and surface charge density () of the TiO2-NPs after sonochemical treatment with different Ag contents.

Sample	DH distribution	Average DH (nm)	ζ (mV)	δ (C nm-2)
TiO2/Ag(0 ppm)	Monomodal	0.7 ± 0.3	-20.1 ± 4.0	+ 3.2 ± 0.5
TiO2/Ag(250 ppm)	Monomodal	220.5 ± 20.9	-28.4 ± 11.2	+ 0.2 ± 0.1
TiO2/Ag(500 ppm)	Monomodal	32.5 ± 7.8	-42.5 ± 4.6	+ 0.7 ± 0.1
TiO2/Ag(750 ppm)	Monomodal	3.7 ± 1.9	-38.5 ± 2.8	+ 1.9 ± 0.2
TiO2/Ag(1000 ppm)	Monomodal	15.6 ± 3.5	-34.6 ± 7.6	+ 0.8 ± 0.2

For the TiO2/Ag-NPs suspensions, the average ζ magnitude enhancement occurs due to the charges relative to the COOH- groups of the citrate anions capping the nanoparticles, electrically stabilizing them in the suspension. TiO2-NPs suspension does not have trisodium citrate. This increase of the average ζ magnitudes for the TiO2-NPs due to Ag modification is accompanied by a diminution of the average values, confirming the effective stabilization of the TiO2/Ag-NPs even with a low electrostatic thickness of citrate anions adsorbed at their surfaces. Also, the tendency to increase the ζ to even more negative values shows no significant effects of hydrophobic or steric molecular interactions of citrate molecules on the physicochemical stability of TiO2/Ag-NPs suspension [62].

The Eg of the anatase TiO2-NPs determined by the Tauc plot (Fig. 1S) equals 3.1 eV (Fig. 1c), similar to data reported in the literature [63], was abruptly reduced to 2.6 ± 0.2 eV after modification with silver as expected due to interfacial charge transfers occasioned by plasmon resonance effects. This optical bandgap reduction is attractive because the wide bandgap for anatase TiO2 limits its photocatalytic applications to absorption of electromagnetic radiation shorter than 380 nm, corresponding to UV light [37].

Coated cotton wipes

SEM and EDS

According to SEM micrographs ( Fig. 2), all compositions of TiO2-NPs and TiO2/Ag-NPs here evaluated successfully covered the CFs with microparticles (diameters of 1.5 – 8.5 µm), forming interesting structures that evoke the heavy-fruited branches of the Brazilian Myrciaria cauliflora. The EDS spectra (Fig. 2S) confirm that Ti (4.5 and 4.9 keV) and Ag (2.95, 3.2 and 3.4 keV) constitute these particles that are formed from NPs nucleation and growth due to condensation reactions of carboxylic acids (COOH) from the acrylic binder with Ti-OH groups from the TiO2-NPs and OH groups at the surface of silver [64], along with carboxylate bonds during the CF coating. The CF is mainly composed of carbon (0.26 keV) and oxygen (0.53 keV) from cellulose, lignin, and hemicellulose macromolecules, which FTIR characteristic signals are shown in Fig. 3a and detailed in Table 1S. Reduction of the relative intensities of the bands at 980, 1000, 1030, 1055, 1115, and 1160 cm-1 (Fig. 3b) is ascribed to the reaction between OH groups in cellulose and COOH groups in the acrylic binder/NPs aggregates, as well as merely due to the covering of the cotton fibers by nanoparticles.

Fig. 2

SEM micrographs of the cotton wipes (CFs) before and after surface modification with TiO2 -NPs and TiO2/Ag-NPs. CFs coated: CF-TiAgX, where X represents the silver concentration (in ppm) applied in the sonochemical synthesis of the TiO2/Ag-NPs. Scale bars = 50 µm.

Fig. 3

(a) FTIR spectra of the cotton wipes (CFs) before and after surface modification with TiO2/Ag-NPs. CFs coated: CF-TiAgX, where X represents the silver concentration (in ppm) applied in the sonochemical synthesis of the TiO2/Ag-NPs. (b) Zoom in the region 800–1200 cm-1 of the FTIR spectra. (c) Inhibition zone diameter determined from the bacterial plaque assay against E.coli (d) and S. aureus (e).

CF coated with TiO2-NPs (CF-TiAg0 sample) is not able to inhibit the bacterial colony growth of E. coli (Gram-negative bacteria) and S. aureus (Gram-positive bacteria), according to the inhibitory plaque assays (Figs. 3c, 3d and 3e). CFs coated with TiO2/Ag-NPs present inhibition zone diameters from 10 to 13 mm against E. coli and an even more pronounced bactericidal effect against S. aureus (inhibition zone diameters from 15 to 16 mm). The bactericidal action is due to irreversible damage to the bacterial cells caused by oxidative (ROS generation) and non-oxidative (electrostatic attraction of Ag+ ions with negatively charged microbial cells and biological molecules) mechanisms [15], [65], [66], [67]. Ag+ ions can also generate ROS (O2 , H2O2, 1O2, H2O, and OH), which incite cleavage of proteins, lipids, DNA, and RNA and contribute to the bacteria cells' oxidative stress [15], [65], [66], [67].

The photocatalytic properties of the coated CFs relative to ROS were accessed following the concentration decay of methylene blue (MB). Up to 1000 min of irradiation time ( Fig. 4a), CF-TiAg0 presented the lowest photocatalytic efficiency (), which indicates a limited bactericidal performance of the CF coated solely with TiO2-NPs. Similar curve profiles for photodegradation efficiency () in the function of irradiation time were observed for all samples. However, only those modified with TiO2/Ag-NPs have approached at least 90% of MB decomposition after long irradiation times (> 5000 min). In the experimental conditions evaluated, the two-way ANOVA indicates that the Ag content significantly influences the photodegradation efficiency of the cotton fabrics for long periods of exposure to light, involving an interaction effect of the trapping agent with this parameter utilized in the synthesis of TiO2/Ag-NPs.The ROS scavenging experiments (Fig. 4b) suggest that h+ and H2O2 play a critical role in the photocatalytic activity of CF coated with TiO2-NPs and their inhibition by the trapping agents leads to an evident reduction of the results. On the other hand, OH and H2O radicals also seem to constitute a significant fraction of the ROS, leading to the MB photodegradation in the CF coated with TiO2/Ag-NPs. The photodegradation kinetics profile of ln() vs. time in Fig. 4c was utilized to calculate the photocatalytic rate constant (). The plot of in the function of Ag content used in the modification of the TiO2-NPs is shown in Fig. 4d. The addition of silver in the TiO2-NPs incremented from 3.3 ± 0.1 (10-4 min-1) to values higher than 3.9 ± 0.1 (10-4 min-1) due to the reduction of the Eg of the TiO2-NPs (by Ag-TiO2 interface plasmon-induced charge transference) combined with the Schottky barrier at the Ag-TiO2 interface, delaying the recombination of electron-hole photogenerated pairs [23], [32].

Fig. 4

(a) Photodegradation efficiency () of the cotton wipes (CFs) coated with TiO2/Ag-NPs. CFs coated: CF-TiAgX, where X represents the silver concentration (in ppm) applied in the sonochemical synthesis of the TiO2/Ag-NPs. (b) Trapping experiment data. (c) Kinetics profile. (d) Rate constant () of the photocatalytic degradation of MB dye by the CF coated with Ag/TiO2-NPs under blue light irradiation (450 nm). * The data are significantly different (p < 0.05) according to Turkey's test using a 95% reliability level.

The photogeneration of electrons-holes (e-−h+) pairs, ROS, and MB photodegradation by the TiO2/Ag-NPs onto CF surface are associated with several reactions [32], [37], [68], as illustrated in Fig. 5. First, electron-hole pairs are photogenerated on the TiO2 surface, depending on the photon energy of the incident light (Fig. 5). The photogenerated e- can be transferred to the Ag, while O2 adsorbed at the AgNPs or TiO2-NPs surface is reduced to O2 . h+ can oxidize H2O adsorbed at the surface of TiO2, leading to the formation of hydroxyl radicals OH. OH is also formed by the decomposition of H2O2 that results from reactions among O2 , H+, and H2O. HO2 is generated by the reaction of H2O with O2 in the aqueous media. The MB dye is degraded in water by ROS species and, then, carbon dioxide is released as a reaction product [32], [34], [37]. The uncoated CF sample indicates photobleaching of methylene blue under irradiation, involving 60% photodegradation efficiency of the MB dye due to the auto-generation of singlet oxygen [69], [70].

Fig. 5

Schematic illustration of the reactions involving ROS photogeneration by the TiO2/Ag-NPs fixed on the surfaces of the cotton wipes (CFs). ROS species are responsible for MB photodegradation and SARS-CoV-2 inactivation. Silver cations released from TiO2/Ag-NPs also contribute to the inactivation of the virions. Illustration out of scale.

SARS-CoV-2 antiviral tests

The antiviral activities of the CFs before and after surface coating with TiO2-NPs (CF-TiAg0 sample), AgNPs (CF-Ag500 sample), and TiO2/Ag-NPs (CF-TiAg1000 sample) are shown in Fig. 6. Although the CF-TiAg0 sample does not present bactericidal activity against E. coli and S. aureus, it can inactivate 18% of the infectious SARS-CoV-2 after the first 5 min of direct contact with the virus inoculum, while around 5% of this virus is inactivated when in contact with the uncoated CF at the same time. After 15 min, this sample inactivates 27% of the infectious virus particles. The CFs coated with AgNPs and TiO2/Ag-NPs are more successful in reducing infectious SARS-CoV-2. CF-TiAg1000 sample inactivates 26%, 27%, and 32% of virions after 5, 10, and 15 min of CF/inoculum contact, respectively. It is important to highlight that the CF-TiAg1000 sample corresponds to the CF coated with TiO2-NPs modified with the highest content of silver (1000 ppm) via sonochemical synthesis. CF-TiAg1000 sample also presented the highest bactericidal activity against S. aureus. The quantification of SARS-CoV-2 by RT-qPCR ( Fig. 7) searched two regions of the nucleocapsid gene (N1 and N2). The quantities of viral RNA in the inoculum samples are suitable to perform the RT-qPCR properly since the cycle threshold (Ct) values are lower than 30. Ct is the number of amplifying cycles required for the RT-qPCR equipment to detect the amplified genetic virus material using a specific qPCR fluorescent probe.

Fig. 6

Antiviral activity of the cotton wipes (CFs) before and after surface coating with TiO2-NPs (CF-TiAg0 sample), AgNPs (CF-Ag500 sample), and TiO2/Ag-NPs (CF-TiAg1000 sample) against SARS-CoV-2 after 5, 10, and 15 min of cotton-virus exposition. CFs coated: CF-TiAgX, where X represents the silver concentration (in ppm) applied in the sonochemical synthesis of the TiO2/Ag-NPs. According to Turkey's test using a 95% reliability level, the results do not display significant differences (ns). * The data are significantly different (p < 0.05).

Fig. 7

Amplified RNA and Ct values determined by RT-qPCR for SARS-CoV-2 using the N1 (superior frame) and N2 (inferior frame) gene targets. CF-Ag500 sample is the CF coated with AgNPs prepared with 500 ppm of silver without TiO2. According to Turkey's test using a 95% reliability level, all amplified RNA and Ct results do not display significant differences (ns).

ANOVA indicates no significant differences between the amplified RNA by PCR of the inoculums after 5, 10, and 15 min of direct contact with the CFs decorated with TiO2-NPs, AgNPs, and TiO2/Ag-NPs. Therefore, the genetic material of the infectious SARS-CoV-2 is considered intact or unaffected by ROS oxidative stress. Besides disrupting viral integrity via ROS, the improvement of the antiviral effect due to the Ag addition in the TiO2-NPs can also be attributed to the Ag+ releasing, which inhibits the detection of SARS-CoV-2 surface proteins (specific SARS-CoV-2 antigens), according to the chromatographic immunoassay rapid test [71], [72]. In both cases, the SARS-CoV-2 antigens that undergo ROS disruption or Ag complexation do not bound to the nitrocellulose chromatographic membrane coated with monoclonal anti‑SARS‑CoV‑2 antibodies, which is necessary to form an antigen‑antibody color particle complex [73], leading to a reduction of the red color intensity of the “test line” in rapid diagnostic tests. Therefore, the antiviral activities of these antimicrobial cotton wipes are due to the damage to the outer proteic structures at the SARS-CoV-2 surface that, basically, are spike, membrane, and viral capsid proteins, as illustrated in Fig. 5.

Conclusions

TiO2/Ag-NPs nanocomposites were successfully obtained via sonochemistry and fixed on the surface of cotton wipes (CFs). Ag decreases the direct optical bandgap and increases the size of the TiO2-NPs. After surface coating, CFs surprisingly present fibers decorated by TiO2/Ag-NPs and TiO2-NPs similar to the heavy-fruited branches of a Brazilian Myrciaria cauliflora. Silver further improves the photocatalytic efficiency and MB photodegradation rate of the CFs coated with the nanoparticles. These physicochemical properties of the TiO2/ Ag-NPs lead to the high bactericidal activity of the CFs against E. coli and S aureus. The antiviral activity of the coated CFs against infectious SARS-CoV-2 seems to be linked to damage to the structural proteins at the surface of the viral particles. However, the coated CFs do not guarantee total and rapid SARS-CoV-2 inactivation to prevent the transmission of COVID-19 effectively.

Funding

This research was funded by (2019/16301-6), (305819/2017-8), CAPES-Pandemias (88881.504639/2020-01) and Brazilian National Council of Scientific and Technological Development () in partnership with Ministry of Science, Technology, Innovations and Communications (), and Ministry of Health (), Secretariat of Science, Technology, Innovation and Strategic Inputs – Decit/SCTIE 07/2020 (Research to cope with COVID-19, its consequences and other severe acute respiratory syndromes – No. 402432/2020-7).

CRediT authorship contribution statement

Daniel José da Silva: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Adriana Duran: Conceptualization, Methodology, Validation, Formal analysis. Aline D. Cabral: Conceptualization, Methodology, Validation, Formal analysis, Review. Fernando L. A. Fonseca: Project administration, Resources, Funding acquisition. Rodrigo F. Bueno: Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition. Shu Hui Wang: Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Derval S. Rosa: Resources, Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of Competing Interest

There are no conflicts of interest.