Yunhan Jiang,Yulong Fu,Xiaojie Xu,Xiaoguang Guo,Feiyu Wang,Xin Xu,Yao-Wei Huang,Jiyan Shi,Chaofeng Shen
Production of singlet oxygen from photosensitizer erythrosine for facile inactivation of coronavirus on mask
COVID-19,Disinfection,Erythrosine,Masks,Singlet oxygen,Transmissible gastroenteritis virus
Graphical abstract
Production of singlet oxygen from photosensitizer erythrosine for facile inactivation of coronavirus on mask



The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide and resulted in the global health crisis and tremendous economic distress (Callaway et al., 2020). As of 10 February 2023, the COVID-19 pandemic has circulated in more than 200 countries, with 755,385,709 confirmed cases, including 6,833,388 deaths (https://covid19.who.int/). It is known that SARS-CoV-2 can be transmitted through respiratory droplets, aerosols and close contact routes (Bao et al., 2020). As masks have been proven effective in protecting healthy individuals and cutting off transmission of the virus, the World Health Organization recommends them as part of prevention and control measures in response to the COVID-19 pandemic to block airborne transmission of SARS-CoV-2 (Narayanan et al., 2021). However, the widespread use of masks as “soldiers” in the fight against COVID-19 pandemic has put extreme pressure on the social production and ecological environment (Wang et al., 2021c). The surge in demand for personal protective equipment (PPE) such as masks, caused by the COVID-19 pandemic has led to shortages of PPE in hospitals and individual households, which is partly responsible for the high rates of infection and death due to COVID-19 (Ranney et al., 2020). According to statistics, about 129 billion masks are discarded into the environment every month, most of which are disposable products made of plastic microfibers, which undoubtedly adds more burden to the earth already burdened by plastic waste (Prata et al., 2020). The reuse of masks is considered the key to alleviating the risk of infection caused by the inadequate supply of masks and solving the above problems (Rubio-Romero et al., 2020). Disinfection of used masks is necessary to ensure their safe reuse, but improper decontamination may damage their structure and impair their protective effectiveness. At present, disinfection measures such as steam, dry heat and moist heat, ethanol, and ethylene oxide have been proved to be effective in the disinfection of masks (Liao et al., 2020, Christie-Holmes et al., 2021). However, most of these measures are highly destructive, which is not conducive to mask recycling (Fischer et al., 2020, Juang and Tsai, 2020). In addition, the lack of the indication of complete disinfection for these disinfection measures may lead to insufficient disinfection or excessive use of disinfectants. Therefore, there is an urgent need to develop an efficient and convenient disinfection method that has the indication of complete disinfection and does not affect the performance of masks, so as to realize the safe reuse of masks. Since 1930s, photodynamic technology has been widely used in cancer treatment and inactivation of pathogenic microorganisms due to its advantages of low damage and excellent inactivation effect (Heinemann et al., 2017, Kipshidze et al., 2020). During photodynamic inactivation, photosensitizer molecules are excited by visible light and interact with ground-state triplet oxygen molecules through Dexter energy transfer to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) and hydroxyl radicals (Wang et al., 2021b). ROS can destroy the structural integrity of viruses by peroxidation of biological molecules such as nucleic acids and proteins (Bhapkar et al., 2021, Willis et al., 2021). It’s worth noting that photodynamic inactivation is non-specific to the virus and can attack any part of the virus particle by producing ROS, which eliminates any possibility of virus tolerance and is also a better response to the high mutability of RNA viruses such as SARS-CoV-2 (Fekrazad, 2020). At present, a variety of organic dyes, such as methylene blue and emodin, have been proved to effectively inactivate viruses through 1O2 produced by photosensitization (Ding et al., 2020, Gendrot et al., 2020). Therefore, using simple photosensitizing dyes and the visible light to achieve the inactivation of virus and reuse of mask has a broad prospect. In this study, we used erythrosine (FD&C Red No. 3), a U.S. Food and Drug Administration-approved food dye, as a photosensitizer for photodynamic disinfection. Based on the photobleaching characteristics of erythrosine (Bergmann et al., 2021), the correlation between the inactivation effect of erythrosine against the virus and its color change was investigated, and the complete loss of color caused by photobleaching was taken as the safety indication of complete disinfection. With similar structural features to SARS-CoV-2, transmissible gastroenteritis virus (TGEV) has already been used as a surrogate for SARS-CoV-2 (Cantero et al., 2022). We investigated the inactivation and mechanism of erythrosine against TGEV in solution and disposable mask surface under visible light, as well as the photobleaching of erythrosine, aiming to provide the possibility of alleviating the pressure on social production and ecological environment caused by the extensive use and improper disposal of PPE.

Swine testis (ST) cells were used for 50% tissue culture infective dose (TCID50) assays and propagation of TGEV. The ST cell line (ATCC CRL-1746) was cultured in Dulbecco’s modified Eagle’s medium supplemented with 1% antibiotic–antimycotic (100 μg/mL of streptomycin, 100 units/mL of penicillin, and 0.25 μg/mL of amphotericin B) and 10% fetal bovine serum. All cells were grown at 37 °C with 5% CO2. The TGEV Purdue strain was used to mimic SARS-CoV-2, and produced in ST cells as previously described (Wang et al., 2018). The virus titer of TGEV was determined by TCID50 assay according to the method of Reed and Muench (Reed and Muench, 1938). In brief, host cells (ST cells) were inoculated into 48-well plates and incubated for 24 h, after which the non-adherent cells were removed. Then, varying virus diluents were added as endpoint diluents to the 48-well plates containing adherent host cells and incubated for 36 h. The cytopathic effects of the cells in the 48-well plates were observed using the XDS-1A Inverted Biological Microscope (Shanghai Optical Instrument Import & Export Co., Ltd, China), and the dilution at which 50% of the cell cultures were infected was determined and used to mathematically calculate a TCID50 value. Three replicates of each experiment were performed. Virus stocks were stored at −80 °C until use.

TGEV inactivation, photosensitized 1O2 production, and photobleaching were assessed in 150 mL glass bottles using a PLS-SXE300 xenon lamp (Beijing Trust-tech Co. Ltd., China) (wavelength range of 320 to 780 nm), where light intensity was adjusted to 200 mW/cm2. The solutions contained erythrosine (purity ≥ 95%) at varying concentrations (1, 5, and 10 μM), 1 × phosphate buffered saline (PBS, 1.0 mM, pH 7.2) or 90% heavy water (D2O), and furfuryl ethanol (FFA, 100 μM) or TGEV (∼106 TCID50/mL). Viruses were sampled every 5 min of illumination and then assayed immediately to avoid further inactivation. Three replicates were prepared for each concentration of erythrosine-treated group. The inactivation effect of the stable photooxidation products of erythrosine against the virus was also be investigated (Scheme S1 in Supplementary material). The above experiments were performed in a bio-safety level-2 (BSL-2) laboratory. All materials used in the experiments contacting viruses were autoclaved at 121 °C for 30 min. The TGEV was titrated by ST cells in 48-well plates with 6 wells per dilution of virus. The photobleaching of erythrosine was quantified by the loss of absorption at 526 nm using an UNICO UV-2100 spectrophotometer. The concentration of FFA was carried out on an Agilent 1260 high-performance liquid chromatography equipped with a C18 column and a photodiode array detector. The singlet oxygen steady-state concentration ([1O2]ss) was determined following the loss of FFA over time (Scheme S2 in Supplementary material). The virus regeneration test was carried out to further understand the photosensitized inactivation of erythrosine on virus. Specifically, 100 μL of virus solution treated with erythrosine for 30 min was added to the 48-well plate containing host cells (ST cells) (3 replicates, 6 wells each replicate) and incubated for 48 h in the dark, then the state of cells in the 48-well plate was observed. The viral RNA was extracted from the virus solution disinfected with erythrosine using the EasyPure Viral DNA/RNA kit (Beijing TransGen Biotech, Ltd., China) according to the manufacturer’s instructions. Viral RNA levels were determined by reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR of TGEV was performed on a Bio-Rad CFX96 Real-Time PCR Detection System using forward primer 5′-GGA TGA TCC TAA GAC TGG ACA AT-3′ and reverse primer 5′-GCA TCA GGT ACC ACA TCT TGC-3′. Serial dilution of the nucleocapsid protein-encoding gene (N) was utilized to generate a standard curve for quantification of viral gene. The relative abundance of genes was calculated by Ct value obtained by qPCR assay.

Ten μM erythrosine solution was selected as the target disinfectant and configured in the spray bottle. The disposable mask was cut with a sterile scissor to obtain 2 cm × 2 cm mask sheets. 100 μL of TGEV solution (∼2 × 107 TCID50/mL) was dropped on the surface of 2 cm × 2 cm disposable mask sheet placed in a glass Petri dish and left for 2 h to make the virus adhere to the mask sheet. And then, the erythrosine solution was sprayed on the surface of mask sheets inoculated with TGEV for different times (1, 2, 3, 5 times) to evaluate the disinfection effect of erythrosine on TGEV, and each spray is about 100 μL. Samples were irradiated with a xenon lamp at a light intensity of 200 mW/cm2 for 5, 10, 15, 20, 25, and 30 min, respectively, and then transferred to 2 mL of 1 × PBS and vortexed for 2 min to obtain the treated virus suspension. The virus recovery rate was > 95% (Fig. S1). The titer of TGEV was determined by TCID50 method. Photographs were taken to record the color changes on the surface of the mask sprayed with different times of erythrosine solution. Each experiment was conducted by triplicate.

The pristine non-woven masks were separated into three individual layers, and the middle layer made of melt-blown polypropylene microfibers, which is the key layer for protection (Rawal, 2020), was taken as the research object. The material of the mask is polypropylene, which has been confirmed by Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra. The effects of 10 cycles of erythrosine treatment on the fiber morphology and surface characteristics of masks were investigated and compared with those of commonly used disinfection measures, ozone (10 mg/L, 60 min) treatment and 75% ethanol treatment (spray 3 mL 75% ethanol evenly onto the mask and air dry). Scanning electron microscopy (SEM) images were collected using a Hitachi SU8010 SEM instrument with an accelerating voltage of 3 kV. The samples were sputtered with gold in order to reduce the tendency of electrostatic charging. X-ray photoelectron spectroscopy (XPS) experiments were performed using a Thermo Scientific K-Alpha XPS instrument with a monochrome X-ray source using Al Kα (1486.6 eV) radiation. For the O 1s and C 1s spectral regions, a pass energy of 50 eV and step size of 0.1 eV were used, with a minimum of 5 scans per region. The binding energies measured via XPS were corrected by referencing the C 1s line to 284.80 eV. ATR-FTIR spectra were recorded on a Nicolet iS50 FTIR (Thermo Scientific, USA) instrument in the wavenumber range 4000–400 cm−1 with a wavenumber resolution of 4 cm−1 and 30 scans at room temperature.

The filtration efficiency and surface electrostatic potential of masks after 10 cycles of erythrosine treatment was measured and compared with those of ozone (10 mg/L, 60 min) treatment and 75% ethanol treatment (spray 3 mL 75% ethanol evenly onto the mask and air dry). The experiment involving TGEV must be conducted in a BSL-2 laboratory, however, the measurement of the filtration efficiency of masks cannot be carried out in our BSL-2 laboratory due to the need for specific instruments. For safety reasons, when investigating the effect of disinfection treatment on the filtration efficiency of masks, the masks used were not inoculated with viruses. Instead, the entire pristine masks were directly treated with disinfection methods. Before measuring the filtration efficiency of masks, the masks were pretreated for (25 ± 1) h in a controlled ambiance with temperature of (38 ± 2.5) °C and relative humidity of (85 ± 5)%. The filtration efficiency of masks was measured using an Automated Filter Tester (TSI 8130) with NaCl as aerosol ((0.075 ± 0.020) μm count median diameter) and a flow rate of (85 ± 4) L/min. Each average measurement contains five individual sample measurements. A SIMCO FMX-004 Electrostatic Fieldmeter was used to determine the electrostatic potential on the mask surface before and after various disinfection treatments. Each experiment was conducted by triplicate.

The statistical analyses were conducted using SPSS software (Version 22.0). One-way ANOVA was used to determine the differences in virus titers and FFA degradation efficiency among different test groups. Student’s t-test was used to determine differences of filtration efficiency and surface electrostatic potential of masks between the treatment group and the control group. All means are expressed with standard deviation (±SD). Results were considered statistically significant if p < 0.05.

Before using erythrosine to inactivate coronavirus on the surface of masks, we first tested the photodynamic inactivation effect of erythrosine at different concentrations in 1 × PBS solution. In view of the high infectivity of SARS-CoV-2 to humans, TGEV was selected as its substitute (Cantero et al., 2022). As shown in Fig. 1 a, erythrosine has a dose-dependent effect on the photodynamic inactivation of virus. Compared with the light control group without erythrosine, the inactivation rate of the virus obviously increased even with a small amount of erythrosine (1 μM) (Fig. 1a). After 30 min of light irradiation, the virus titer of 1 μM and 5 μM erythrosine-treated groups were reduced by 1.42-log and 3.41-log, respectively. When the concentration of erythrosine was 10 μM, the virus titer was decreased by 4.92-log after exposure to light for 20 min (Fig. 1a). However, when the concentration of erythrosine was increased to 50 μM, the inactivation value of the virus did not increase much, which may be related to the decrease of light transmittance caused by the addition of a large amount of photosensitizer (Li et al., 2016). In addition, the virus titers in the dark control and light control groups remained almost unchanged after 30 min, which is consistent with the blank control group, indicating that erythrosine had no toxic effect on virus particles in the dark, and the virus was not inactivated by direct photolysis under light irradiation (Fig. 1a). The previous studies have confirmed that photosensitizers inactivate pathogenic microorganisms by generating 1O2 upon light irradiation (DeRosa and Crutchley, 2002). In this study, the generation of 1O2 is indicated by the degradation of FFA, which is a probe molecule that selectively reacts with 1O2. The 1O2 yield of erythrosine at different concentrations was positively correlated with its ability to inactivate virus by photosensitization (Fig. 1a, b). As shown in Table S1, the [1O2]ss of 1, 5, 10, and 50 µM erythrosine-treated groups were 9.17, 42.50, 75.00, and 96.67 pM, respectively. The good 1O2 generation performance of erythrosine was conducive to its inactivation of virus as a photosensitizer. As a visible light photosensitizer, erythrosine has conjugated carbon–carbon bonds networks that provide absorption at longer wavelengths (Bergmann et al., 2021). These conjugated networks are susceptible to 1O2 oxidation and direct photolysis, which destroys conjugation and eliminates absorbance in the visible spectrum, leading to color fading of the solution (photobleaching) and formation of photooxidation products (Beltukova et al., 2017, Kravchenko et al., 2022). In this study, the correlation between the inactivation effect of erythrosine against the virus and its color change was investigated (Fig. S2). By determining the appropriate combination of disinfection followed by color loss, the complete discoloration of erythrosine caused by photobleaching was taken as an indicator of the completion of virus inactivation. As shown in Fig. 1c, d, under the light irradiation, the erythrosine solutions with the concentrations of 1, 5, and 10 μM completely lost their color after 30, 50, and 60 min, respectively, while the 50 μM erythrosine solution still had a deep red color after 80 min of light irradiation. Although the 50 μM erythrosine-treated group achieved the desired inactivation effect against the virus, the persistent red color complicates the photobleaching safety indication. Considering the inactivation effect and photobleaching of different concentrations of erythrosine, 10 μM erythrosine solution, which ensures complete inactivation of the virus in the solution was achieved prior to complete color loss (Fig. S2), is selected as the target disinfectant for all subsequent experiments.

In view of the efficient inactivation ability of erythrosine to virus and the fading caused by photobleaching, which provides a possible indication for the complete disinfection, erythrosine is a potential disinfectant applied to the surface of masks. As shown in Fig. 2 a, the virus titers dropped below the detection limit (decreased by more than 99.999%) within 30, 30, 25, and 20 min of light exposure after 1, 2, 3, and 5 times spraying of the target disinfectant (corresponding doses of 100, 200, 300, and 500 μL), respectively. No dark inactivation was observed in the dark control group and there was no significant (p > 0.05) change in virus titer after exposure to light for 30 min in the light control group (Fig. 2a). As shown in Fig. 2b, the surface color of the mask sprayed with the target disinfectant for 1, 2, 3, and 5 times completely disappeared after 10, 15, 25, and 30 min of illumination, respectively. Since the virus titer only decreased by 99.44% and 99.97% at the time point corresponding to the complete fading of the disinfectant on the mask surface in the 1 time and 2 times spraying groups (Fig. 2a), that is, the virus was not completely inactivated, the target disinfectant sprayed once and twice does not have the safety indication ability based on photobleaching. Spraying for 3 times provides an appropriate disinfection combination, which can ensure that the complete loss of red color correctly indicates the sufficient disinfection of the mask, thus effectively improving the safety of reusing masks disinfected with erythrosine. In addition, compared with toxic dyes such as methylene blue, the amount of erythrosine used to treat a mask in this study (<0.12 mg) was converted well below its lowest approved acceptable daily intake (0.10 mg/kg body weight/day) (Ganesan et al., 2011), eliminating the possibility that using it to disinfect masks poses risks to personal health.

We investigated the effects of 10 cycles of erythrosine treatment on the fiber morphology and surface characteristics of masks, and compared them with the effects of ozone treatment and ethanol treatment. SEM images of the middle layer of the mask before and after treatment were obtained to investigate the fiber morphology evolution (Fig. 3 a). Compared with the control group, the fiber structure of middle layer was intact and dense after 10 cycles of erythrosine treatment, which indicated that this treatment did not induce significant structural degradation of the mask. However, both ozone treatment and ethanol treatment induced severe damage to the melt-blown polypropylene microfibers, and the defects have been circled in the SEM images, indicating the degradation of polypropylene microfibers. Although ozone treatment and ethanol treatment are effective methods to sterilize most pathogens, the structural degradation caused by them can seriously affect the protective performance of the mask. XPS was used to characterize the surface chemistry of the middle layer of the mask. The XPS spectra for C 1s and O 1s regions are shown in Fig. 3b and Fig. 3c, respectively. For the pristine material, the C 1s spectra showed that this material has a significant C—C/C—H bonding (Fig. 3b), which is consistent with the polypropylene composition (Wang et al., 2015). No C-O bonding was observed in the C 1s spectra. However, adventitious oxygen was observed in the O 1s spectra, which may be caused by molecular water absorption (Polzonetti et al., 1991). Due to the lower kinetic energy of the O 1s photoelectrons, the surface sensitivity of O 1s spectra is slightly higher than that of C 1s spectra, so there could be contributions from C—O and C PBM data was replaced with SVG by xgml2pxml: <glyph-data id="pc-E00C" format="PBM" resolution="300" x-size="12" y-size="9"> 000000000000 000000000000 000000000000 111111111111 000000000000 111111111111 000000000000 000000000000 000000000000 </glyph-data> <svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata> Created by potrace 1.16, written by Peter Selinger 2001-2019 </metadata><g transform="translate(1.000000,15.000000) scale(0.019444,-0.019444)" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg> O which are not seen in the C 1s data (Yan et al., 2021). Ten cycles of erythrosine treatment did not change the XPS spectra for C 1s and O 1s regions (Fig. 3b, c), indicating that the treatment did not change the surface chemistry of the middle layer. However, there was a noticeable intensity increase after the ethanol treatment in the O 1s spectra (Fig. 3c), which possibly suggested that ethanol treatment induced more physical adsorption of oxygenated species, such as H2O and O2, on the melt-blown polypropylene microfibers (Polzonetti et al., 1991). For the O 1s spectra of the ozone-treated group, additional peak is also observed at ca. 533–534 eV (Fig. 3c), assigned to the C-OH bond (Xie et al., 2020), which indicated that the ozone treatment may lead to the oxidation of mask middle layer (Massey et al., 2003). The chemical structure of the melt-blown layer was investigated by ATR-FTIR spectroscopy. According to the acquired ATR-FTIR spectra, there are absorption peaks of the middle layer of the mask at 2950, 2916, 2867, 2837, 1456, 1376, 1166, and 973 cm−1 (Fig. 3d), so it can be assigned to polypropylene material (Krylova and Dukstiene, 2013, Caban, 2022), which is consistent with the XPS spectra. The peaks of 2950, 2916, 2867, and 2837 cm−1 have been previously attributed to the C—H asymmetric stretch of —CH3, C—H asymmetric stretch of —CH2, C—H symmetric stretch of —CH3, and C—H symmetric stretch of —CH2, respectively. Following these, two intense peaks appear at 1456 and 1376 cm−1 which correspond to C—H bend of —CH2 and —CH3, respectively. In addition, the bands near 1166 and 973 cm−1 were originated from the rocking vibrations of the C—H bonds of —CH3. As shown in Fig. 3d, no significant difference was observed in ATR-FTIR spectra of all treatment groups, indicating that the chemical structure of the middle layer of the mask was relatively stable under the treatment of erythrosine, ozone, and ethanol. In general, compared with the ozone treatment and ethanol treatment, which are not conducive to the reuse of the mask due to the negative impact on the middle layer that plays a major role in the mask, the erythrosine treatment has no significant impact on the structural integrity and surface chemical characteristics of the mask within 10 disinfection cycles, and may be a promising disinfection measure that can be applied to the reuse of the mask.

The blocking efficacy of the mask is achieved by its mechanical filtration and electrostatic adsorption (Xiong et al., 2021, Yin and Liu, 2021). In this study, we measured the filtration efficiency and surface electrostatic potential of masks after 10 cycles of erythrosine treatment, and compared them with those treated with ozone and ethanol. As shown in Fig. 4 a, compared with the control group (96.12%), there was no significant (p > 0.05) change in the filtration efficiency of the mask after 10 cycles of erythrosine treatment (95.88%). This result indicated that even subjected to 10 cycles of erythrosine treatment, the mask still maintained excellent mechanical filtration capacity (>95.00%). However, ozone treatment and ethanol treatment decreased the filtration efficiency of masks by 4.00% and 8.32%, respectively (Fig. 4a), thus reducing their protective performance. Since the particle diameter of the droplets or dust carrying the virus is about 0.3 μm, it is difficult to filter them out by mechanical filtration alone, so the filtration of such particles must rely on electrostatic adsorption (Liu et al., 2018, Schmitt et al., 2021). In this study, ozone treatment and ethanol treatment caused the surface electrostatic charge attenuation of the mask, which reduced the surface electrostatic potential of the mask to 47.37% and 60.53% of the control group (1.52 kV), respectively (Fig. 4b). This decrease greatly reduced the blocking efficiency of the mask against virus, thereby increasing the risk of infection from reusing the masks. On the contrary, there was no significant (p > 0.05) change in the surface electrostatic potential (1.46 kV) of the mask after 10 cycles of erythrosine disinfection (Fig. 4b), which ensured the protective performance of the mask after treatment. In fact, previous studies have also investigated the applicability of commonly used disinfection measures for pathogenic microorganisms to mask disinfection and reuse (Peters et al., 2021, Seresirikachorn et al., 2021). Ultraviolet irradiation has the disadvantages of poor disinfection efficiency and incomplete disinfection (Ye et al., 2022), which may make the reuse of the mask after disinfection have a greater risk of infection (Fig. S3). Although the use of chlorine-based disinfectant and steam sterilization pose excellent disinfection ability, they have been proven to rapidly reduce the filtration efficiency of masks, which is not conducive to the reuse of masks (Ma et al., 2020). Fischer et al. (2020) confirmed that vaporized hydrogen peroxide exposure effectively inactivated SARS-CoV-2 and other pathogens in the mask and ensured its filtration performance in at least three disinfection cycles. However, this technology is not suitable for the general public because of the requirements of specific instruments and materials. Erythrosine treatment used in this study seems to be a promising disinfection method due to its excellent disinfection and recycling capabilities for masks. Ideally, safe reusable times for masks can easily be extended to at least 10 times by erythrosine treatment. This result shows that if this disinfection measure can be widely popularized in the current COVID-19 epidemic, the demand for mask production and disposal will be greatly reduced and tens of billions of masks will be saved every month (Prata et al., 2020). However, considering that the light intensity used in this study is 200 mW/cm2, which is higher than the intensity of sunlight and provided by the xenon lamp, the general public needs to be equipped with corresponding lamps in their daily lives to use this treatment method. Therefore, in order to make it more convenient for the public to use, it is necessary to study the inactivation effect of erythrosine against pathogenic microorganisms under sunlight or much lower light intensity in the future.

It was found that the virus inactivation induced by photosensitization is potentially caused by 1O2 generated from photosensitizer (DeRosa and Crutchley, 2002). Since the lifetime of 1O2 in D2O (67 μs) is much longer than that in water (3.6 μs) (Davis et al., 2018), the mechanism of virus inactivation induced by erythrosine was investigated by comparing the virus inactivation efficiency in 1 × PBS solution and 90% D2O solution. As shown in Fig. 5 a and Table S1, the [1O2]ss in D2O is 9.51 times higher than that in 1 × PBS. At the same time, compared with that in 1 × PBS, the inactivation efficiency of erythrosine in D2O was significantly improved (Fig. 5b). The virus titer decreased by 4.83-log after 15 min of illumination, while it took about 20 min in 1 × PBS to achieve the same inactivation effect. Therefore, the increase of steady-state 1O2 contributes to the improvement of virus inactivation efficiency. We also investigated the inactivation effect of the stable photooxidation products of erythrosine against the virus and found that erythrosine and its stable photooxidation products had no toxic effect on virus particles in the dark (Fig. S4). However, whether some unstable intermediates in the photooxidation of erythrosine can inactivate the virus needs further exploration. In addition, we extracted viral RNA from the virus solution disinfected with erythrosine and investigated the effect of photosensitizer on the genomic RNA of TGEV, as indicated by the copy number change of the N gene, which encodes the nucleocapsid protein (Zuniga et al., 2010), so as to determine the cause of viral inactivation from the genomic perspective. As shown in Fig. 6 , the copy number of the N gene continuously decreased with the extension of the irradiation time. After 30 min of illumination, the titer of the virus dropped below the detection limit (decreased by more than 99.999%) (Fig. 1a) and the copy number of the N gene reduced by 81.92% compared with that before treatment (Fig. 6), which confirmed that erythrosine can destroy the viral genomic RNA and achieve effective disinfection of the virus. The virus regeneration test was also carried out to further understand the photosensitized inactivation of erythrosine on virus. The results showed that no cytopathic changes occurred in all wells containing ST cells, which means that even after incubation in the dark for 48 h, no infectious virus could be detected in the virus solution after 30 min of erythrosine treatment, which indicated that virus had been completely inactivated, rather than simply inhibiting its growth and reproductive ability. Oppositely, ultraviolet disinfection has been proved to have the possibility of pathogen regeneration after disinfection (Wang et al., 2021a). The above results collectively reveal the inactivation mechanism of TGEV by erythrosine under light irradiation. Inactivation of the virus by photosensitization was attributed to a nonselective reaction result from light-induced 1O2 production. The oxidative damage caused by 1O2 makes the virus lose its infectivity, and directly destroys its genomic RNA, which eventually leads to the death of the virus without regeneration.

This study provides insights into the choice of appropriate disinfection and recycling strategies for masks to achieve high disinfection effect, clear indication of disinfection end point, and less degradation of materials, so as to maintain the overall function of masks. Our results showed that erythrosine treatment had the advantage of high accessibility and less damage to mask structure compared with the commonly used disinfection measures, and could be used in daily life. The complete color loss of erythrosine due to photobleaching can be used as an indicator of disinfection end point, and thus reducing the possibility of excessive use of disinfectants. At the same time, the nonselective inactivation of the virus induced by erythrosine also eliminate any possibility of virus tolerance, which is conducive to the thorough disinfection of the virus. In addition to the disinfection of masks, erythrosine can also be used for disinfection and recycling of various PPE, which can significantly alleviate the shortage of PPEs and the pressure of solid waste disposal caused by the large consumption of PPE during the pandemic. Meanwhile, the edibility of erythrosine also makes it have a wider application prospect than other disinfection measures, and it can be applied to the disinfection of pathogenic microorganisms on daily necessities and even food packaging.

Yunhan Jiang: Formal analysis, Investigation, Writing – original draft. Yulong Fu: Methodology, Writing – review & editing. Xiaojie Xu: Formal analysis. Xiaoguang Guo: Writing – review & editing. Feiyu Wang: Formal analysis. Xin Xu: Writing – review & editing. Yao-Wei Huang: Resources, Supervision. Jiyan Shi: Supervision. Chaofeng Shen: Conceptualization, Writing – review & editing, Supervision, Funding acquisition.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.