Imidazolyl-based zwitterionic polymers for dual antiviral and antibacterial functions

To reduce the serious health risks and enormous economic impact posed by the spread of SARS-CoV-2, an imidazolyl-based zwitterionic polymer was designed, synthesized, and demonstrated to achieve contact loss of human coronaviruses under dry ambient conditions.
Live, this environmental condition is similar to fomite propagation
.

Zwitterionic polymers further showed excellent antifouling properties, reducing the adhesion of coronaviruses and the formation of bacterial biofilms
.
The polymer was synthesized using a substrate-independent solvent-free process using an all-dry, i.
e.
, induced chemical vapor deposition (iCVD) technique
.
The broad applicability of the method is demonstrated by applying the polymer to a range of substrates that are curved and/or have high aspect ratio nano/microporous structures, which remain intact after coating treatment
.
The zwitterionic polymers and synthetic methods reported here provide an effective solution for reducing viral transmission without the need for manual disinfection, reducing the health and economic impact of the ongoing epidemic
.

Introduction

The ongoing coronavirus disease 2019 (COVID-19) pandemic is one of the most dangerous epidemics in human history, killing millions and changing the way people live
.

Formette transmission, the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through contaminated surfaces (1) that is, Formette surface, which has long been considered a cause of community sprawl (2), which in turn had a huge economic impact (three)
.

With global surface disinfectant sales reaching $4.

5).

However, a number of materials have been found to inactivate viruses upon contact [eg, metals and inorganic materials based on their toxicity and/or ability to generate reactive oxygen species] (7–9),Polyelectrolyte(10,11) and photosensitizers (12,13)] they used bacteriophages or viruses similar to the SARS-COV2 virus (14) Therefore, their reported antiviral effects may not be extrapolated to SARS-CoV-2 due to the unique structure of coronaviruses (6).

(iii) Inactivation of virus without incubation with culture medium [e.

15,16), recent studies have further highlighted various molecular interactions between zwitterionic structures and aromatic-rich amino acids such as phenylalanine, tyrosine, and tryptophan (17,18), via cation-π interactions or polar interactions (19), causing proteins (for example, the spike glycoproteins that line the surface of SARS-CoV-2) to denature when exposed to zwitterionic moieties

To demonstrate the broad-spectrum antifouling effect of imidazolyl-based zwitterionic polymers, we quantifiedPseudomonas aeruginosaBiofilm formation on imidazoline-based zwitterionic polymers was reduced to 15% of that on polyvinyl alcohol (PVC) surfaces

or with high aspect ratio nanopores (i.

figure 1) In the first step, iCVD deposition of vinylimidazole (VI; monomer) and divinylbenzene (DVB; crosslinker) was performed

29) by adjusting the flow of VI and DVB to control the composition of the copolymer (figure 1), which in turn determines a key synthesis parameter,P^rice/P^sitThat is, the ratio of the partial pressure of the monomer to its saturation pressure at the stage temperature

The XPS measurement scans confirmed the essential composition of O, N and C in the deposited polymer films (Fig.

34) assuming the same bond oscillator strength for all polymers synthesized here, the area under the double absorption peaks at 1228 and 1284 cm^?1(imidazole ring) and absorption at 2871 cm^?1The concentrations of unreacted vinyl and vinyl copolymer were calculated separately

Figure 1BThe VI content in the copolymers was between 17% and 55% due to the low reactivity of VI

The temperature of the derivatization reaction is variable to achieve a balance between high conversion at higher temperatures and good reaction conditions to ensure that the method is applicable to a wide range of substrates, some of which may have limited Thermal stability

Derivatization temperatures of 40°C, 60°C, and 100°C were used, and 100°C was used as the control, where complete conversion was expected

Figure 1E) new peak at 1037 cm^?1considered to be the result of symmetrical stretching_three^?group(Figure 1D) indicates the formation of the zwitterionic structure (38).

Figure 2Bfigs
.
S3 to S5)
.
The peak at 401.
5ev corresponds to the N(1s) in the imidazole ring (atFigure 2B) while the peaks at 399.
5 and 400.
6 eV correspond to the two unreacted nitrogen atoms in PVI (orange and blue, respectivelyFigure 2B) (39,40)
.
When increasing the derivatization temperature from 40°C to 60°C, the conversion slightly increased from 51.
5% to 70.
2%, but further increasing to 100°C did not improve the conversion further (78.
9%) (
Figure 2B)C(1s) and S(2p) high-resolution scans further confirmed this composition of the film surface
.
The coiled carbon signal shown in Figure S3 is attributed to three types of chemical environments (i.
e.
carbon between two nitrogen atoms in the imidazole ring, carbon next to nitrogen or sulfur, or carbon surrounded by carbon) whose composition is related to the N(1s) scan The results are consistent
.
S(2p) high-resolution scans confirm SO_three^?section
.
Therefore, the derivatization temperature of 60°C was chosen for the subsequent experiments because higher conversions were achieved at this temperature, while remaining below the aforementioned common softening point
.

The water contact angle (CA) also measures the macroscopic hydrophilicity of the treated films, which reflects the conversion of the derivatization step and correlates with the potential enthalpy of fouling adhesion, which determines the antifouling performance (

Figure 2A) The CA of PVI and PDVB were 16.
9°±1.
2° and 87.
3°±0.
3°, respectively
.
After the copolymerization of the two components, the CA value of CP55 was 55.
3°±2.
3°.
Due to the higher DVB content in CP26 and CP17, the CA values ​​were 79.
5°±0.
5° and 78.
9°±0.
9°, respectively, which were close to the CA values ​​of PDVB (Figure 2A).
Static CA images obtained during these measurements are included in Fig.
S6
.
The water CA values ​​of the films treated with 1,3-propynone water vapor were significantly reduced
.
The CA values ​​of the three precursor polymers generally decreased with increasing derivatization temperature
.
Among them, CP55-60 and CP55-100 exhibited superhydrophilicity (i.
e.
, CA values ​​below 10°), despite the high DVB content of 45%, the CA values ​​of CP55-60 and CP55-100 were 9.
9°±2.
1° and 9.
9°±2.
1°, respectively.
7.
5°±0.
7°
.
This superhydrophilicity is attributed to the concentrated zwitterionic moieties on the surface, as demonstrated with high-resolution XPS
.
Through the depth distribution of imidazole and imidazole content, diffusion-limited derivatives spontaneously formed a concentration gradient from the coating surface to the bulk film, achieving the highest conversion at the topmost surface (Fig.
S7) (41).

We further demonstrate that the iCVD coating preserves the topography of the substrate, i.
e.
the surface roughness obtained by atomic force microscopy (AFM) remains unchanged before and after the iCVD process and derivatization (

Figure 2C) compared with the surface roughness [0.
11±0.
01nm root mean square (RMS) roughness] of the uncoated silicon wafers, the surface roughness values ​​of the silicon wafers coated with CP55 were 0.
51±0.
06 and 0.
44±0.
08nm, respectively
.
The superior slipperiness also ensures minimal exposure of available binding sites for viral or bacterial attachment
.

Inactivation of human coronavirus HCoV-OC43 by imidazolyl zwitterionic polymers

The antiviral activity of imidazolyl-based zwitterionic polymers was determined using human HCoV-OC43

BetakonovirusIt belongs to the same genus as SARS-CoV-2, but with lower lethality (42).
This choice was made to ensure that the results reported here are applicable to pandemic-causing pathogens, while minimizing the risks of the experiments described below
.
To test antiviral activity, we compared the repulsion and deactivation efficiencies of imidazolyl-based zwitterionic polymers with glass, PVC, and Cu, which represent a range of inorganic, plastic, and metal surfaces commonly used in public, medical, and manufacturing facilities
.
The antiviral activity of these surfaces is described by two methods: (i) inactivation of viruses under dry environmental conditions, as described below; and (ii) repulsion of viruses under underwater conditions, discussed in the next section
.

Virus inactivation was assessed using a process we developed to simulate drying of virus-containing liquids on surfaces under dry ambient conditions
.

The HCL-8 cell suspension culture protocol was established with HCL-8 as the host (43)] were applied to the aforementioned surfaces [i.
e.
glass, PVC, Cu and coating CP55-60 (applied on glass slides)] and air-dried for about 30 minutes in a laboratory setting
.
Once the absence of visible liquid was confirmed, the surface was subsequently incubated at 34°C, 50% relative humidity for 24 hours, and finally,
Viruses were collected by vigorous washing with phosphate-buffered saline (PBS) and assessed for infectivity
.
Again, HCT-8 cells were used as host cells for infectivity assays
.
HCoV-OC43 suspended in PBS solution was inoculated into HCT-8 cells at a multiple of infection (MOI) of 0.
05, and the virus culture was quantified 36 h after infection
.
Subsequently, HCT-8 and HCoV-OC43 were stained with Hoechst 33358 and primary anti-HCoV-OC43 S antibody and Alexa-Fluor 568-labeled goat anti-rabbit immunoglobulin G (IgG), respectively, for imaging
.

as shown

Figure 3A, distinguish HCT-8 and HCoV-OC43 with different colors, where the nucleus is blue and the virus is stained red
.
The virus has a large number of blue particles around the nucleus, indicating that the virus is proliferating around the nucleus
.
Such cells are labeled as infected cells
.
The infectivity of the inoculated virus can be indirectly known by the statistics of the percentage of cells infected with the virus
.

as shown

Figure 3BUnder the same conditions, the virus infection rate on the surface of CP55-60 was the lowest (i.
e.
, the proportion of infected cells to the total number of cells) was 13.
4%, while the virus infection rates on glass, PVC and copper surfaces were 28.
0% under the same conditions, respectively , 31.
5% and 51.
5%
.
The inactivation ability of CP55-60 against coronaviruses under dry ambient conditions is attributed to various molecular interactions between imidazolyl-based zwitterionic polymers and aromatic-rich amino acids through cation-π interactions or polar interactions ,Causes protein denaturation (i.
e.
, spiking glycoproteins on the surface of SARS-CoV-2) to denature upon contact with the amphiphilic moiety
.
Theoretically, this interaction is stronger for imidazoline zwitterionic polymers because the carbon atom in the C2 position of imidazoline carries a considerable positive charge
.
This charge makes its hydrogen an excellent hydrogen-bond donor, giving it enhanced interactions with amino acids
.

Reduced adhesion of human coronavirus HCoV-OC43 on imidazoline-based zwitterionic polymers

The repulsion of underwater viruses was also quantified to assess the ability of imidazolyl-based zwitterionic polymers to resist viral adhesion under physiologically relevant conditions
.

The above surfaces were incubated with the HCoV-OC43 virus suspension, using the median tissue culture infectious dose (TCID₅₀, a measure of viral titer) 10^7.
5/ml is the stock solution with the highest concentration, allowing the virus to adhere for 30 min at room temperature
.
The virus attaches to the surface by physical adsorption, which has little correlation with the incubation time (44) We therefore chose a relatively short incubation time to capture the potential adhesion of the virus
.

Characterization of the adhesion density of virus particles by scanning electron microscopy (SEM) images (

Figure 3C).
To ensure that our results were statistically representative, we took four non-overlapping SEM images on each surface with a field of view of 25 μm × 15 μm (see Figure S8 and data analysis in Supplementary Information for details)
.
The size of this field of view was chosen to be large enough to capture the statistical average of viral adhesion without losing the resolution required to correctly identify viral nanoparticles, with an average of only 100 to 200 nm, spherical (Fig.
S9)
.

as shown

Figure 3COf all surfaces tested, CP55-60 exhibited the lowest amount of viral adhesion at the end of the incubation period
.
The surface virus adhesion density of the control group was 8.
1×10⁶/centimeter²(glass), 1.
4×10⁶/centimeter²(PVC), 4.
3×10⁵/centimeter²After (Cu) treatment, the adhesion density on the surface of the CP55-60 coating decreased by 97.
4% to 2.
1 × 10⁵/centimeter²compared to glass surfaces
.

Previous studies have shown that zwitterionic polymers synthesized by a two-step gas-phase method can be slightly negatively charged (e.
g.
zeta potential in the vicinity of

?13 mV, pyridyl zwitterionic polymer in 100 mM NaCl standard solution, pH 7) (26)
.
Although a negative surface charge is desirable, since virions are also considered negatively charged (
45), we do not believe that electrostatic repulsion is the main reason for the reduction in viral adhesion, since charge neutrality is a prerequisite for the anti-biofouling behavior demonstrated by imidazoline zwitterionic polymers
.
We attribute this reduction in viral adhesion to the strong hydration and charge neutrality of CP55-60, which minimizes nonspecific binding between viral particles and surfaces (15,46).

Biofilm formation and siderophore generation of imidazolyl zwitterionic polymers

In addition to the excellent antiviral properties, imidazolyl-based zwitterionic polymers can reduce biofilm formation, which is comparable to the antifouling properties of methacrylic acid or pyridyl zwitterionic polymers reported so far (

47).
To characterize the dirt thermal resistance of CP55-60,P.
aeruginosaStrain PAO1 was selected as a model organism due to its ability to rapidly produce biofilms (48) huge number of hospitalizations due to PAO1 each year (49).

Biofilm growth was quantified using the O'Toole protocol (

50) have been used to characterize the antifouling properties of flat substrates and coated surfaces (43).
Compared with PVC materials commonly used in health care facilities, CP55-60 coating exhibited reduced biofilm formation, and the amount of biofilm on the coating surface was 16% of that of PVC, as measured by crystal violet staining (50) In contrast, underivatized CP55 or PDVB induced biofilm growth comparable to PVC (Figure 4A) Furthermore, the reduction in biofilm formation on CP55-60 was not due to its antibacterial effect, as liquid cultures incubated with all surfaces showed similar stable optical densities at 600 nm (OD)₆₀₀) (Fig.
S10)
.
The difference between imidazole-based zwitterionic coatings and existing antibacterial coatings is that the bacteria on the zwitterionic surface are not inactivated, which is worthless
.
The strong hydration of the zwitterionic surface inhibits cell adhesion without killing the cells
.
Therefore, zwitterionic surfaces generally have a longer-lasting effect of inhibiting biofilm formation than antimicrobial surfaces (51) SEM images of PAO1 biofilms grown on four surfaces were captured to gain further insight into the impact of surface chemistry on biofilm physiology (Figure 4B)PVC, PDVB, and CP55 displayed a thick biofilm containing dense extracellular polymers (EPS), whereas biofilms grown on CP55-60 exhibited sparse linear EPS
.

Rampant PAO1 biofilms continuously secrete virulence factors, such as pyridoxone, which remove a large amount of iron ions from the host, causing severe toxicity to mammalian cells, such as mitochondrial damage, electron transfer, and reduced production of adenosine 5′-triphosphate, and eventually lead to the renewal of mitochondria (

52)
.
Therefore, inhibition of microbial pyoverdine production has the potential to alleviate
P.
aeruginosa(twenty three) To demonstrate that imidazoline-based zwitterionic polymers can reduce the generation of pyrrolidine, the content of pyrrolidine in the supernatant was measured by the fluorescence intensity at 460 nm, and then normalized with OD₆₀₀to offset potential changes in culture conditions
.
Compared to CP55, PDVB or PVC surfaces, CP55-60 significantly reduced the generation of pyrrolidine (Figure 4C) due to limited biofilm formation
.

Substrate independence and conformality of solvent-free synthetic methods

The all-dry synthesis method described above was used to prepare pyridyl zwitterionic coatings on (i) substrates curved with centimeter-scale curvature (i.
e.
, 96-well plates;

Figure 5A), (ii) micropores with curled 3D structures (i.
e.
glass fiber filters;Figure 5B), and (iii) nanopores with aspect ratios up to 165 (i.
e.
polycarbonate membranes with 800 nm pores;Figure 5C) with a 600 nm thick coating on a 96-well plate, a 200 nm coating on a glass fiber, and a 10 nm coating on a polycarbonate membrane, the substrate topography was well obtained save
.
The thickness of each coating is less than 10% of its characteristic length to maintain its original form
.

Scanning electron microscopy (SEM) energy dispersive X-ray analysis (EDX) was used to demonstrate the successful synthesis of CP55-60 coatings on various substrates and the consistency of the coatings, where elemental sulfur (existing only in CP55-60 coatings) and not present in any matrix) elemental mapping indicates the presence of a coating
.

Furthermore, the distribution of sulfur completely overlaps with the nano- and micro-structure of the underlying layer (Figure 5, B and C) on coated substrates, which means that the CP55-60 coating has good consistency
.
The combination of conformability and substrate independence of this synthetic method indicates the broad application of imidazolyl zwitterionic polymers in numerous industries such as healthcare and manufacturing.
And promises its reproducible synthesis and consistent antiviral and antifouling properties to mitigate public health threats
.

discuss

We developed an imidazoline-based zwitterionic polymer that effectively inhibits contact inactivation and anti-adhesion of the human coronavirus HCoV-OC43
.

The design based on imidazolyl zwitterionic chemistry is based on the known antifouling properties of zwitterionic chemistry, as well as the interaction between the zwitterionic moiety and aromatic amino acids, which are ubiquitous in the spike glycoprotein of coronaviruses, leading to viral loss live
.

To simulate the spread of the coronavirus, droplets containing HCoV-OC43 (representing an aerosol) were applied to imidazoline-based zwitterionic polymers, copper surfaces, glass, and PVC, and prior to quantifying the infectivity of HCoV-OC43 The droplets were allowed to evaporate to demonstrate the effect of contact inactivation
.

This method showed that exposure to imidazolyl-based zwitterionic polymers alone reduced the infectivity of HCoV-OC43 to 13.
4%, a 74% reduction in infectivity compared to copper surfaces thought to be antimicrobial (6)
.
The virus is naturally inactivated on the surface due to dehydration
.

The reported half-life of the virus on untreated common surfaces is 10 to 32 hours at 30°C (1) of which Formite transmission poses a serious threat to human health
.
On the CP55-60 surface, this inactivation is accelerated due to a putative interaction between imidazoline and virus
.
Due to the combined effect of imidazoline chemistry and viral particle dehydration, longer virus surface contact times may lead to further virus inactivation
.

The imidazolyl-based zwitterionic polymers also exhibit excellent antifouling properties under humid conditions
.

Anti-adhesion was assessed by incubating the polymer with HCoV-OC43 suspension and then quantifying the number of virions adhering to the surface
.
The imidazole-based zwitterionic polymer showed a 97.
4% reduction in viral adhesion compared to widely used glass surfaces
.
The polymer further reduced PAO1 biofilm formation by 84% compared to standard PVC surfaces and reduced the production of the biofilm-secreted virulence factor pyoverdine by 33%, demonstrating its antifouling properties and also suggesting Reduced toxicity of biofilms grown on imidazolyl-based zwitterionic polymers
.

A substrate-independent conformal synthesis method was developed to enable the facile deployment of the zwitterionic coatings reported here in a wide range of devices
.

Various substrates with curved surfaces and/or high-aspect-ratio nano/microporous structures were successfully coated with imidazoline-based zwitterionic polymers by the iCVD method
.
While the two-step synthesis process may increase the complexity of process design during scale-up, both steps can be accomplished in a single vacuum chamber, improving its compatibility with industrial manufacturing configurations
.
The latest breakthrough in roll-to-roll manufacturing using iCVD (53,54) further supports the potential for vacuum process scale-up to achieve conformal and uniform coatings on 20-meter flexible substrates²Thus, vacuum-based processing promises rapid deployment of materials and synthetic methods to protect public health and reduce economic losses associated with controlling the spread of Formite
.

Imidazolyl-based zwitterionic polymers have excellent antiviral and antifouling properties
.

Although further increasing the content of imidazoline-based zwitterionic moieties, for example, beyond the 55% content achieved here, can improve their antiviral effect, the low reactivity of VI (55) limits its binding to copolymers
.
An important future research goal is to increase the VI content by optimizing the crosslinker or replacing VI with other more reactive imidazoline-enabled monomers
.
Furthermore, despite the C2-mediated antiviral mechanism and demonstrated antiviral effect, the underlying mechanism of viral inactivation on imidazoline-based zwitterionic polymers remains unclear, a key focus of our current and future studies
.
In addition, although the thickness of CP55-60 has little effect on its antiviral and antifouling properties due to its high degree of crosslinking and smooth surface morphology, the effect of film thickness on its long-term stability and performance is still our future.
focus of the research
.
However, antiviral zwitterionic polymers represent a major advance in the design of anticoronavirus materials
.
The non-substrate application of this polymer as a coating implies a broad and easy application, which will reduce the economic and health impacts of the COVID-19 pandemic
.

Materials and methods

Initiated chemical vapor deposition

All polymer coatings were fabricated using iCVD technology in a custom cylindrical vacuum reactor (Sharon vacuum Co.
, Brockton, MA, USA)
.

Thermal excitation of the initiator was provided by heating a 0.
5 mm nickel/chromium filament (80% nickel/20% chromium, Goodfellow) mounted on a parallel filament array
.
Filament temperature is controlled by a feedback loop that reads from a thermocouple attached to one of the filaments
.
The filament holder spans the deposition stage, which uses a cooler to maintain the desired substrate temperature
.
The vertical distance between the filament array and the stage is about 2 cm
.
Deposition was performed on various substrates: silicon wafers (P/Bo<100>, PureWave), 96-well microplates (2797, Corning), glass slides (Thermo Fisher Scientific), Petri dishes (Thermo Fisher Scientific), copper Foil (MTI Corporation), PVC sheet (McMaster-Carr), glass fiber filters, and polycarbonate membrane filters (Sigma-Aldrich)
.
Cooling of the microplates is further enhanced by a custom aluminium bracket
.
originator[third party-Butyl peroxide (TBPO) (98%; Sigma-Aldrich)] and monomers [VI (99%; Sigma-Aldrich) and DVB (Sigma-Aldrich, 80%) were used without further purification
.
During iCVD deposition, TBPO and argon sheet flows were fed to the reactor at room temperature through a mass flow controller at 1.
0 standard cubic centimeters/min and desired flow rates, respectively
.
The VI was heated to 70 °C in a glass jar to generate sufficient pressure to drive the steam flow
.
Films were deposited at a filament temperature of 230°C
.
The total pressure of the chamber is controlled by a butterfly valve
.

In situ interferometry was performed on the growth of thin films on silicon substrates using a HeNe laser light source (wavelength 633 nm, JDS single-phase)
.

derivatization

1 g of 1,3-propynyl sulfone (98%; Sigma-Aldrich) was added to the crystallization dish (VWR) to immobilize it in the crystallization dish (VWR)
.

The crystallization dish was placed in a vacuum oven maintained at the desired temperature for 24 hours to allow the 1,3-propynone vapor to react with the PVI-limited company-DVB coating
.

Polymer Film Characterization

Transmission FTIR measurements were performed on a Bruker-Vertex V80v vacuum FTIR system
.

Deuterated sulfate triglycine KBr detector, detection range is 400 ~ 4000cm^?1Resolution passed at 4 cm^?1.
Measurement is averaged over 64 scans to obtain adequate signal-to-noise ratio
.
All spectra were baseline corrected by subtracting the background spectrum of silicon
.

During the XPS process, the samples were analyzed using a Surface Science Instruments SSX-100esca spectrometer with a working pressure of approximately 1 × 10

^?9Thor
.
In a region with a diameter of 800 μm, monochromatic alkα x-rays (1486.
6 eV) with photoelectrons were collected
.
Photoelectrons were collected at an emission angle of 55° with a source-analyzer angle of 70°
.
The hemispherical analyzer measures electron kinetic energy with a pass energy of 150 eV for wide/measure scans and 50 eV for high-resolution scans
.
Charge neutralization of non-conductive samples with a flood gun
.
Data analysis was performed by CasaXPS with Shirley in the background
.
All samples were subjected to XPS analysis after one week of vacuum storage at room temperature
.
To measure the depth distribution of imidazole and imidazole content, we prepared CP55-60 films on silicon wafers by ion milling
.
Corrosion depths of 20, 40, 60 and 100 nm were sampled using XPS measurement scans
.
The content of imidazole and imidazole groups was calculated by analyzing the content of sulfur (atomic ratio)
.
The content of unreacted imidazole was calculated as a supplement to the imidazole content
.

CA measurements were performed with a Rame Hart model 500 goniometer equipped with an automatic water dispenser
.

2μl
。

MFP-3D-BIO AFM
。

，1.
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5μm×2.
5μm
。

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。

，
。iphone12pro
。

HCT-8[American Type Culture Collection（ATCC）CCL-244]H-CoV-OC43（ATCC VR1558）G.
Whittaker
。

HCT-8DulbeccoEagle（DMEM）（），10%（FBS）（Gibco，USA）1%/（Gibco，USA）
。37°C5%CO₂.
H-CoV-OC4334°C5%COHCT-8
₂2%FBS1%/DMEM
。8，
。，，?80℃，10^7.
5
TCID₅₀/
。

（、、PVC600 nm CP55-60）1 cm×1 cm
。

To attach the virus to the surface, the substrate was immersed in the virus suspension and incubated for 30 min at room temperature
.
The substrate was then gently washed with increasing concentrations of 50%, 70%, 85%, 95% and 100% ethanol
.
The number of attached viruses was counted from SEM images
.
SEM images were acquired with a Zeiss gemini500 at an accelerating voltage of 3 kV
.
A 2 nm layer of gold was sputtered on all samples prior to imaging
.

To test for surface inactivation, 10 μl of virus stock was spread over samples measuring 1.
5 cm × 1.
5 cm
.

Samples were air-dried at room temperature and then transferred to an incubator maintained at 34°C and 50% humidity for 24 hours
.
The samples were then washed repeatedly with a total of 1 ml of PBS to transfer the virus into the solution
.
Detection of HCT-8 cells by immunofluorescence method (5×10)⁴cells per well) were cultured in 3.
5 mm glass bottom dishes (Cellvis, USA), then infected with HCoV-OC43 at an MOI of 0.
05 and incubated at 34 °C for 36 h
.
After 36 hours, HCT-8 cells were fixed with 4% paraformaldehyde
.
Fixed cells were washed twice with PBS and permeabilized with 0.
5% tritonx-100 for 13 min at room temperature
.

Subsequently, samples were blocked in 5% goat serum in PBS for 45 min at room temperature and treated with primary anti-HCoV-OC43 S antibody (Cusabio) diluted 1:500 in 3% goat in PBS at 4°C Incubate overnight in serum
.
Subsequently, Alexa Fluor 568-labeled goat anti-rabbit IgG (Thermo Fisher Scientific) was diluted 1:1000 in PBS and incubated for 1 hr
.
Nuclei were stained with Hoechst33258 (Thermo Fisher Scientific) diluted 1:10,000 in PBS
.
After immunostaining, cells were washed with 0.
5% Tween-20 in PBS
.

Finally, stained cultures were mounted onto glass slides in Extended Diamond Antifade Medium (Thermo Fisher Scientific) and stored at 4°C
.

Infectivity (%) is calculated by the following formula
.

For each sample, count cells from five different fields

$\%$

?number of infected cells=optocoupler?43?Number of positive cellsTotal number of cells?(Hearst)×100

biofilm formation

PAO1 from

?80°C frozen stock was streaked on a Trichostatin a plate
.
Place the dish in an incubator (37 °C) overnight until a single colony forms
.
A single colony was inoculated in lysogenic broth (LB) medium
.
The inoculated medium was incubated overnight at 37 °C, keeping it still in a shaker (225 rpm)
.
The overnight suspension was diluted 100-fold in fresh LB medium and incubated at 37 °C on a shaker (225 rpm) until the optical density reached 0.
2
.

150 μl of the bacterial suspension was then added to each well of a surface-modified 96-well microplate
.
Incubate the microplates for 24 hours (37°C) to ensure biofilm maturation
.
Then transfer the liquid to a new microplate
.
The relative concentration of pyrrolididin can be obtained by exposing the culture to excitation light at 405 nm and recording the fluorescence intensity at 460 nm
.
Rinse each well three to four times vigorously with deionized water (dH) to remove remaining liquid culture and loosely attached bacteria₂O) a
.
Biofilms were then stained with 175 μl dH₂O was soaked in 0.
1 wt% crystal violet solution for 10 minutes
.
Then, remove the crystal violet solution by washing each well three to four times until the liquid in each well becomes a clear solution
.

The microplates were air-dried for 24 hours at room temperature to remove residual water in each well
.
Biofilms then form
.
Add 200 μl of acetic acid solution (30 v/v%) to each well to release the absorbed crystal violet and quantify the relative amount of absorbed crystal violet spectrophotometrically by measuring OD₅₇₀A microplate reader (Infinite M1000 Pro, Tecan) was used
.

To obtain SEM images of biofilms, microplates were fixed with 0.
05M calcium carbonate buffer containing 2% glutaraldehyde and 1% osmium tetroxide after 24 hours of incubation
.

It is then dehydrated by critical point drying
.
SEM images were acquired with a Zeiss gemini500 at an accelerating voltage of 3 kV
.
All samples were sputter-coated with gold prior to imaging
.

Statistical Analysis

At least three statistical analyses were performed for the experiment
.

All data are presented as mean ± standard deviation
.
Statistical significance by paired studentsttest
.