key: cord-0879966-6x1302h0
authors: Li, Wei; Wang, Yixin; Tang, Xin; Yuen, Terrence Tsz-Tai; Han, Xing; Li, Jiaqian; Huang, Nan; Chan, Jasper Fuk-Woo; Chu, Hin; Wang, Liqiu
title: Liquid Repellency Enabled Anti-Pathogen Coatings
date: 2021-10-07
journal: Mater Today Bio
DOI: 10.1016/j.mtbio.2021.100145
sha: b53c180e5f2adfc2ea3a494ebf04b7fd402bad11
doc_id: 879966
cord_uid: 6x1302h0

Currently, Coronavirus Disease 2019 (COVID-19) – a respiratory contagion spreading through expiratory droplets – has evolved into a global pandemic, severely impacting the public health. Importantly, the emerging of immune evasion SARS-CoV-2 variants and the limited effect of current antivirals against SARS-CoV-2 in clinical trials suggested that alternative strategies in addition to the conventional vaccines and antivirals is required to successfully control the COVID-19 pandemic. Here, we propose to use liquid-repellent coatings to prevent the spread of disease in the absence of effective vaccines, anti-microbial agents or therapeutics, wherein the deposition and penetration of pathogen droplets are prohibited. We use SARS-CoV-2 as a model pathogen and find that SARS-CoV-2 remnants are reduced by seven orders of magnitude on coated surfaces, yielding a repelling efficacy far outperforming the inactivation rate of disinfectants. The SARS-CoV-2 remnant scales exponentially with the liquid/solid adhesion, uncovering the mechanism and effective means for minimizing pathogen attachment. The anti-pathogen coating that both repels and inactivates pathogens is demonstrated by incorporating the super-liquid-repellent coating with anti-pathogen additives. Together with its versatility over a wide range of substrates and pathogens, the novel anti-pathogen coating is of considerable value for infection control in everyday life as well as during pandemics.

promote interfacial detachments [31] [32] [33] . By introducing re-entrant micro-/nano-structures and special low-surface-energy polymers, the surface can be made repellent to both water and oil, termed superamphiphobicity (SAP), or even nearly all types of liquids that include alcohol in addition to water and oil, termed superomniphobicity (SOP) [34] [35] [36] [37] [38] [39] . Upon impact, liquid penetration attempt is thwarted by the nanoscopic porosity, building a shield towards the pathogenbearing droplets [40] . By further incorporating anti-pathogen additives such as silver nanoparticles into the coating, the trace amount of residual pathogens could be further inactivated. Our preliminary study shows that superhydrophobic surface demonstrates super repellency to droplets loaded with SARS-CoV-2 [41] . However, the exact repelling efficacy of other liquid-repellent surfaces, including superamphiphobic and superomniphobic surfaces, against SARS-CoV-2 remains unexamined. Moreover, the anti-viral efficacy of common metals, including silver and copper nanoparticles, towards SARS-CoV-2 remains unknown.

To study the repellent repertoire, we fabricate six types of non-wetting surfaces on a large materials gamut, including glass, fabric, steel, copper, mask, nitrile glove, and paper. We use SARS-CoV-2 as a model pathogen and find that they have super repellency to droplets loaded with SARS-CoV-2. We traced viral residue along droplets' rolling trails and find that SARS-CoV-2 remnants are reduced by seven orders of magnitude, a value outperforms the inactivation rate of disinfection procedure and the filtration efficiency of mask. More importantly, we uncover that the amount of SARS-CoV-2 remnant grows exponentially with the increasing of liquid/solid adhesion, capillary force parameterized by pinned fractions ϕ (depending on physical textures) and receding contact angles θrec (depending on surface chemistry). Furthermore, silver nanoparticles are found to exhibit remarkable anti-SARS-CoV-2 effect and the composite anti-pathogen coating is proposed by incorporating silver nanoparticles into super-liquid-repellent coatings. Such pathogen J o u r n a l P r e -p r o o f repelling strategy can be widely applied and would be effective for various pathogens such as bacterium, fungus in addition to virus, thus being valuable for infection control in daily life as well as during epidemics. The anti-pathogen coating will also be useful in preventing the spread of disease in the absence of specific vaccines, anti-microbial agents or therapeutics.

1H,1H,2H,2H-perfluorodecyltrichlorosilane (PFDTS) (97%) was purchased from Gelest.

1H,1H,2H,2H-perfluorodecyltriethoxysilane (>98.0%) and tris(hydroxymethyl)aminomethane (>99.0%) were purchased from Tokyo Chemical Industry Corporation. 1H,1H,2H,2Hperfluorooctyltriethoxysilane (98%), 1H,1H,2H,2H-perfluorodecanethiol (97%), silver nitrate (≥99.0%), tetraethyl orthosilicate (TEOS) (≥99%), dichloromethane (≥99.5%), and fluorescein isothiocyanate (FITC)-BSA (FITC-BSA) were purchased from Sigma-Aldrich. Sodium hydroxide (97%) and potassium persulfate (≥99.0%) were purchased from J&K Scientific. Ammonium hydroxide (28 to 30% in water) and hydrochloric acid (37% in water) were purchased from Acros.

Silica nanoparticles (15 nm) was purchased from Shanghai Maikun Chemical Co., Ltd., China.

Titanium oxide nanoparticles (99.8%, anatase, ∼100 nm) was purchased from Macklin. Nano silver solution (1000 ppm in water) was purchased from Aladdin. Sylgard 184 silicone elastomer kit was purchased from Dow Corning. Ethanol (absolute) was purchased from VWR International. Deionized water was produced by a deionized water system (DINEC, Hong Kong). Commercial glass slide, polyester fabrics, steel sheet, copper sheet, mask, nitrile gloves and printing paper are cut into ~2cm×2cm.

J o u r n a l P r e -p r o o f

SARS-CoV-2 HKU-001a was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed COVID-19 patient in Hong Kong as we previously described [42] . The virus was suspended in in Dulbecco's Modified Eagle Medium (DMEM) and was titered in VeroE6 cells with plaque assays as we previously described [43] . All experiments involving live SARS-CoV-2 followed the approved standard operating procedures of the Biosafety Level 3 facility at the Department of Microbiology, University of Hong Kong. The inactivated SARS-CoV-2 virus solution was prepared by incubating the virus in 4% paraformaldehyde for 24 hours. The lack of infectious titer was confirmed with median tissue culture infectious dose (TCID50) assays on VeroE6 cells. 50 µl of droplet with viable SARS-CoV-2 at a concentration of 1×10 7 PFU ml -1 in DMEM only medium was applied to bare or coated surface. The DMEM medium consists of water and proteins, which is designed and manufactured to simulate the characteristic of body fluid [44, 45] . Therefore, the DMEM medium can at least in part represent the body fluid generated during talking/coughing/sneezing. After 30 seconds waiting, surfaces were held aloft and 90 degree-tilted to allow free falling of the virus droplet. The droplet is pinned on bare non-permeable surface and wets the bare permeable surface, while it quickly rolls off the coated surfaces. Each surface was then submerged in 5 ml of phosphate-buffered saline (PBS) and was incubated for 10 minutes at room temperature. After incubation, 100 µl of the virus-containing PBS was aspirated and mixed with 400 µl of AVL buffer for further RNA extraction sampling following the QIAamp® Viral RNA Mini Handbook.

We perform RNA extraction instead of viability assays for two reasons. First, RNA extraction detects the amount of total viral genome while the viability assay (plaque assay) detects J o u r n a l P r e -p r o o f only the infectious virus particle. In this regard, the results from RNA extraction will provide a more complete picture for the virus adhesion test. Second, for many coated surfaces that we tested, only trace amount of virus is retained on the substrates. In this scenario, RNA extraction, which has a better sensitivity than viability assays (plaque assays), will have a higher probability of detecting the signals. 9×10 7 virus gene copy is obtained when 50 µl of virus at 1×10 7 PFU ml -1 is submerged in 5ml PBS, incubated for 10 minutes at room temperature and 100 µl of the solution is subsequently extracted. As a control, 3.33% to 100% recoveries are obtained from the bare surfaces, ranging from 3×10 6 (fabric) to 9×10 7 (mask) virus gene copy.

100 µl of virus-containing PBS solution was lyzed with 400 µl of AVL buffer and was subsequently extracted for total RNA with the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany). Quantitative real-time one-step qRT-PCR was used for quantitation of residual SARS-CoV-2 using the QuantiNova Probe RT-PCR kit (Qiagen) with a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland) as we previously described [46] . The primers and probe sequences were previously reported [47] and were against the RNA-dependent RNA 

In order to test the anti-viral effect of copper and silver nanoparticles, 500 µl of SARS- 

In the abrasion test, the super-repellent surfaces were placed facedown to the sandpaper (Standard glasspaper, 2000 cw) [48] . The surfaces were longitudinally abraded for 2 cm by the sandpaper under a pressure of ~0.2 kPa and then abraded backward for another 2 cm. This process is defined as 1 cycle. The virus adhesion test was conducted after abrasion for 50 cycles and 100 cycles. A solid with mass of 8 g is placed on top of the samples (2cm×2cm) to maintain a constant pressure of ~0.2 kPa over the 50/100 abrasion cycles. 

To fabricate SHP SiO2 coated surfaces, commercial spray, Glaco (Soft99), was used to render substrate to be superhydrophobic by spray coating. Coated surface was then baked at 80 °C for 30 min to enhance inter-particle binding.

To fabricate SAP SiO2 coated surfaces, the suspension of polysiloxane/silica was prepared The SOP SiO2 surface was prepared by modifying the previously reported superamphiphobic surface based on candle soot [31, 49] . The glass slide (Luoyang Tengjing glass Co. Ltd) was first coated with candle soot, and then placed in a desiccator together with 1 ml of tetraethoxysilane and 1 ml of ammonia hydroxide. The desiccator was closed, and the vacuum was maintained for 18 hours. Then, the carbon soot core was removed by annealing at 550 °C for 3

hours in an oven. The annealed sample was treated with air plasma for 5 min using a plasma cleaner To prepare the SHP TiO2 solution, 1.00 g of 1H,1H,2H,2H-perfluorooctyltriethoxysilane was placed into 99 g of absolute ethanol, and the solution was mechanically stirred for 2 hours. To the resulting solution, 6 g of titanium oxide nanoparticles and 6 g of Degussa P25 titanium oxide were added to make a paint-like suspension. In the following experiments, we used simply a syringe needle to paint the substrate, and the paint was then dried in air for 20 min.

To fabricate SHP CuO coated surfaces, the copper sheet was ultrasonically cleaned in ethanol and deionized water for 10 min, respectively, followed by washing with diluted To fabricate PDMS micropost surface, the negative mold consisting of the hole array (diameter ~80 µm, depth ~60 µm, pitch ~230 µm) was first fabricated on a silicon wafer (<100> type) with a thickness of 420 ± 5 µm by standard photolithography. PDMS precursor containing 10 weight % (wt %) curing agent (Sylgard 184 silicone elastomer kit) was thoroughly stirred and vacuumed for 1 hour to remove internal gas. The prepared PDMS was then casted on the negative mold and vacuumed for 1 hour, followed by curing at 80 ºC for 1 hour. The PDMS micropost surface was obtained after peeling the PDMS out from the mold. The nano-textured PDMS micropost surface was prepared by spray-coating the SAP SiO2 solution onto the PDMS micropost surface.

The physical structures of the coatings were imaged using a Hitachi S4800 scanning electron microscope. Energy-dispersive X-ray scattering was used to obtain the elemental mapping 

To fabricate SAP SiO2 coated glove and mask, the suspension of polysiloxane/silica (20 ml) was sprayed onto vertically placed substrate using an airbrush (Paasche H-SET) with 0.2 MPa nitrogen.

To fabricate SHP SiO2 coated button, doorknob and clothing, 0.5 ml of commercial spray, Glaco Soft99, was sprayed onto the substrates. Coated surface was then baked at 80 °C for 30 min to enhance inter-particle binding.

We use viable SARS-CoV-2 for virus adhesion test (see Materials and methods section for details). As the tiny droplets and the resulting virus aerosol are highly risky to the operator even in Biosafety Level 3 facility, we deposit a 50 µl virus-laden droplet, containing 1×10 7 PFU ml -1 viable SARS-CoV-2, on bare and coated substrates to perform the virus adhesion test (Movie S1). Unlike the firmly pinned droplets on pristine glass (Figure 1a) , on coated surface, the virus solution minimizes the contact by forming a spherical droplet and rolls off the repellent slope in a frictionless manner, leaving behind no observable liquid residues (Figure 1b and Movie S2) [31] .

As shown in Figure 1c , d, we quantify the retention of SARS-CoV-2 Ni (virus gene copy) on different coating-substrate pair and detect a substantial reduction as high as seven orders of magnitude compared with that of the bare substrate N0, generating a repelling rate (N0-Ni)/N0 as high as 99.99999%, a value surpassing the filtration rate of the N95 mask (e.g. 95%) and

inactivation rate of the chemical disinfectant (e.g. 99.9% in 1 hour) [53] . For the evaluated materials, superior repelling performance generally conserves except for nitrile glove overlaid with SHP SiO2 and SHP TiO2. Such failure is potentially caused by the coating exfoliation, a consequence of the poor interfacial bonding between the rigid coating and elastic plastic sheet ( Figure S1 ). The SARS-CoV-2 repellency has some levels of variation across different substrates, as observed that the performance of the SHP SiO2 coating peaks for paper but degrades on other substrates, signalling a pairwise optimization between the coating and substrates properties such as the textures and flexibility.

As shown in Figure 2a , the scanning electron microscopy (SEM) image delineates the nanoscale topography whose geometry and interfacial chemistry work in concert to reduce the liquid/solid adhesion and consequently the liquid retention ( Figure S2 ). As shown in Figure 2b , the nano-textures effectively withstand the impact of a SARS-CoV-2 droplet (Weber number = J o u r n a l P r e -p r o o f 5.5), as observed that the droplet rebounds for more than six times before its final rest, acting as a soft elastic object (Movie S3). For different coating-substrate pairs, the virus-bearing droplets all exhibit a large contact angle (> 150˚) and a low roll-off angle (< 5˚), minimizing the liquid/solid contact and maximizing the in-plane liquid mobility (Figure 2c , Figure S3 and Table S1 ). , implying a rapid growth rate of N as F  increases. Given the fact that the transmission of SARS-CoV-2 is highly efficient, it is key to reduce the interfacial adhesion towards SARS-CoV-2-laden droplets to minimize the infection risk.

Such interfacial adhesion originates in the capillary bridges formed atop the nanotextures [54, 55] . To sidestep the structural irregularities, we assume that the coating caps isotropic nanopost array on the substrate. Along the apparent contact line, nano-capillary bridges form (Figure 3a) . 

We then test the utility by simulating an interaction between a sneeze and the repellent coating (Movie S6). As shown in Figure 4a , inactivated-SARS-CoV-2-laden droplets of sizes ranging from ~10 to ~2000 µm are sprayed towards a glass slide coated with the SHP TiO2 coating through an airbrush. A large SARS-CoV-2 droplet with a diameter of 1021 µm impinges the surface at 4.8 ms and immediately rebounds at 10.6 ms. At the end of the violent spraying (105.2 ms), a diminutive SARS-CoV-2 droplet with a diameter of ~18 µm can still be effectively shed off, maintaining the hygiene and cleanness of the surface (Figure 4b ). For mostly mild-to-moderate cases [58] , the virus fraction is less than 0.01% for droplets below 18 µm. Therefore, more than 99.99% of the droplets with diameter being below 18 µm will not carry any virus [59] . Thus, the SHP TiO2 coating is effective for sneeze shielding as droplets of diameter being above 18 µm can be quickly shed off. After sneeze impact, the fluorescence intensity and fraction area on TiO2 coated glass are only 0.5% and 0.6% of those on the pristine glass (Figure 4c, d) , confirming the function of the coating.

Anti-microbial ingredients can be also incorporated into the super-liquid-repellent coatings to form composite anti-pathogen coatings that both repel the pathogens and inactivate the trace amount of residual pathogens left on the coating. Silver nanoparticle (nano Ag) and copper nanoparticles (nano Cu) have been exploited as antimicrobial agents [26] . However, their effects against SARS-CoV-2 are mostly unknown. We then first examine the anti-viral efficiency of nano Ag (~15 nm) and nano Cu (~60 nm) towards SARS-CoV-2 (see Experimental Section for details).

As shown in Figure 5a , both nano Ag and nano Cu demonstrate remarkable anti-SARS-CoV-2 effect as evidenced by the significantly reduced infectious SARS-CoV-2 virus particles, which is determined by TCID50 assays. Specifically, nano Cu demonstrated a 10-fold decrease in infectious SARS-CoV-2 titer and nano Ag demonstrated a 426-fold decrease in infectious SARS-CoV-2 titer.

Because of the outstanding anti-SARS-CoV-2 property of nano Ag and super-repellency of SAP SiO2 coating, we then evaluate the virus repellent effect of nano Ag doped SAP SiO2 coating (SAP SiO2+1% nano Ag) by performing virus adhesion test. As shown in Figure 5b , the size of nano Ag ranges from ~3 nm to ~42 nm. Adding 1%wt nano Ag into the SAP SiO2 coating J o u r n a l P r e -p r o o f has no effect on the morphology of coating (Figure 5c ) and the nano Ag is homogeneously distributed in the composite coating ( Figure 5d ). As shown in Figure 5e , f, the 1% nano Ag doped SiO2 coating largely retains its SARS-CoV-2 repellent capacity on diverse substrates except for nitrile glove. Such failure may be caused by the coating exfoliation as the hydrophilic nano Ag may weaken the interfacial bonding between SAP SiO2 coating and elastic plastic sheet. Though future work is still required to examine the SARS-CoV-2 activity on the composite anti-viral coating, our results demonstrate that nano Ag is an effective anti-viral agent against SARS-CoV-2 and it is feasible to introduce anti-viral additives like nano Ag into the super-liquid-repellent coating to form a composite anti-viral coating that both repels pathogens and inactivates the residual pathogens.

The mechanical durability of the coatings is tested by performing virus adhesion test after sandpaper abrasion. The result shows that the micro-textures of the coatings are largely retained after the sandpaper abrasion ( Figure S6 ), being in agreement with the result reported in the literature [48] , and thus maintain their super repellency to droplets loaded with SARS-CoV-2 ( Figure S7 ). We then perform virus adhesion test after different abrasion cycles and find that the coatings' virus-repellent performance can essentially maintain up to 100 abrasion cycles ( Figure   6a ). It becomes counter-intuitive as some coating such as SAP SiO2 on fabric becomes more virally-repellent after abrasion. Such effect is potentially caused by additional micro-scale roughness introduced by the abrasion which further improve the liquid repellency ( Figure S6 ).

In real-life situations, once the pathogen-laden droplets move off the coated frequently touched surfaces, they subsequently deposit on the ground or other non-frequently touched surfaces, where the infection risk is greatly reduced. Moreover, disinfection is only required for the ground and non-coated surfaces rather than all surfaces, thus reducing the usage of disinfectants and lowering their harm to human body [60] . We then treat personal protection equipment (PPE) and frequently touched objects such as glove, mask, button, doorknob, and clothing with the repellent coating (Figure 6b and Movie S7). Coated objects exhibit excellent repellency to inactivated SARS-CoV-2 droplets which confirms that the coating can adapt to various substrates with contrasting composition, texture, and geometry, showing its potential for wide applications.

Aside from high performances of these coatings, the large-area and low-cost fabrication [50, 61] , increased mechanical robustness [49, 62] and long-term stability [63] will pave the way for their practical applications for infection control.

Infection prevention and control of pandemic are critical to human being, considering the fact that our society is constantly battered by widespread infectious diseases, for example, the 

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. 

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The authors wish to thank Prof. B.P. Chan for equipment support. The financial support from the Research Grants Council of Hong Kong (GRF 17204420, 17123920, 17210319,