Plasma Disinfection: Cold Atmospheric Pressure Plasma Attenuates SARS-CoV-2 Spike Protein Binding to ACE2 Protein and the RNA

Cold atmospheric pressure (CAP) plasma has a profound effect on protein-protein interactions. In this work, we have highlighted the deactivation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein by CAP plasma treatment. Complete deactivation of spike protein binding to the human ACE2 protein was observed within exposure time of 5 minutes and which is correlated to the higher concentration of hydrogen peroxide formation due to the interaction with the reactive oxygen species present in the plasma. On the other hand, it is established that CAP plasma is also capable of degrading RNA of SARS-CoV-2 virus which is also linked to hydrogen peroxide concentration. Results of this work could be useful in designing plasma-based disinfection systems over those based on environmentally hazardous chemicals-based disinfections.


Introduction
The novel coronavirus disease  is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is a positive sense single stranded RNA virus 1 . The genome of the virus is packed in an envelope comprising of three proteins namely, membrane, spike, and envelope proteins. It is reported that the sustained interactions in SARS-CoV-2 and the major infection spread is caused by the binding of spike protein with the human angiotensin converting enzyme 2 (hACE2) protein for the virus entry through the receptor binding domain (RBD) region 2,3 . The major cause of the spread is air-borne wherein the virus spreads through the respiratory droplets of infected patients. On entry into the nasal cavity, the spike protein of the virus binds to the ACE2 protein in the cell wall to make way for the virus entry 4 . The second wave has hit hard in most of the developing countries especially India (especially due to the delta variant of the SARS-CoV-2). With the rapid increase in the Covid-19 infections worldwide and deaths reaching 4.2 million as of August 2021 and it is therefore necessary to look for additional approaches to minimize the risk of SARS-CoV-2 transmission.
The interaction of cold atmospheric pressure (CAP) plasma with biological materials like living tissues, cells, bacteria, viruses is an emerging interdisciplinary eld of research [5][6][7] . The CAP plasma contains various active species such as electrons, ions, reactive oxygen and nitrogen species (RONS) and UVphotons making it useful in biomedical eld specially for deactivation of bacteria, fungi, spores and viruses [8][9][10][11] . CAP plasma has also been found effective for inactivation or modi cation of several proteins such as lysozyme 12 , myoglobin and haemoglobin 13 , NADPH oxidase (NOX) enzymes 14 and RNAse 15 . Recently, few reports have shown that CAP plasma is effective in inactivation of SARS-CoV-2 virus 16,17 . Plasma activated water has been used to study the spike protein deactivation 17 , however, there is storage issue of activated water for a longer time.
SARS-CoV-2 virus remains stable as aerosols for longer periods of time and, to stop their spread, it is crucial to treat not only surfaces but also the air inside hospital/laboratory as well as living/o ce rooms.
Traditional chemical disinfectants used in fogging have serious limitations due to its toxicity for human as well as material body. UV based disinfection can effectively deactivate the genetic material of the virus, however, has several associated issues with respect to affecting the human skin 18 . In view of environmental protection, novel environmentally friendly decontamination methods are a need of the hour. In this work, we explored the process of deactivation of the spike protein of the Covid-19 virus using cold atmospheric pressure plasma and its effect on the RNA of the virus.

Materials And Methods
SARS-CoV-2 (2019-nCoV) Spike (S1+S2 ECD-His Recombinant) protein, Anti-His tag HRP Antibody, human ACE2 protein (mFc Tag), TMB highly Sensitive HRP Substrate were purchased from Krishgen Biosystems, India and used without further puri cation. BSA, materials for buffer preparation were purchased from Sigma Aldrich, India. The reagents and buffers were prepared in Millipore water. RNA samples of SARS-CoV-2 virus extracted from samples of Covid-19 positive infected patients for Covid-19 testing (RT-PCR) were used. RNA extraction was done following standard protocols. Abundance of the viral RNA before and after CAP treatment was is evaluated in RT-PCR.

Experimental Setup
A cold atmospheric pressure plasma jet is used for the experiment. It is a modi ed form of the plasma jet described elsewhere with a modi ed electrode arrangement as shown in gure 1 (a) 19 . It consists of a glass tube with an inner diameter of 3 mm and an outer diameter of 7 mm. The glass tube acts as the dielectric between the two electrodes. The glass tube and the live electrode assembly is tted into a Te on housing with a gas inlet. Plasma is generated in between the electrodes applying sinusoidal voltages (2.5 KV and 5.0 KV) at frequency (30 KHz). Helium and a mixture of helium with compressed air have been used as the plasma forming gases. The gas ow has been measured using a variable area owmeter (Cole Parmer). For He plasma, a helium gas ow of 1000-3000 sccm (standard cubic cm per minute) has been maintained to obtain a stable plasma. For He-air plasma, air ow of 50-200 sccm is mixed with helium. The plasma produced between the two electrodes comes out into the open air with the gas ow. The sample for plasma treatment is placed underneath (as shown in Figure 1) for different exposure times.
To investigate the various reactive species, present in the plasma, an emission spectrum of the plasma jet was obtained by using an optical emission spectrometer (Andor-SR-303i-A). The light emitted from the plasma was collected by an optical ber tted to the spectrometer equipped with three gratings consisting of 300, 1200 and 2400 grooves/mm and the slit width can be varied from 10-2500 µm. In this experiment, a slit width of 50 µm was used to achieve a fair resolution along with su cient intensity. The survey spectrum was recorded using 1200 groves/mm grating in the wavelength range 250-900 nm, whereas the OH emission is recorded using 2400 groves/mm grating in the wavelength range 306-310 nm to achieve high resolution. The spectrometer's wavelength calibration is performed using a standard NIST traceable calibration lamp (StellarNet Inc.).
Enzyme-linked immunosorbent assay (ELISA): The hACE2 protein was diluted with a coating buffer PBS (136.9 mM NaCl, 10.1 mM Na 2 HPO 4 , 2.7 mM KCl, 1.8 mM KH 2 PO 4 , 0.2 µm ltered) adjusted to pH 7.4 to 2 µg/ml and added to the wells of 96-well plates (100 µl per well). The plates were incubated at 4°C overnight and washed with water. Then, the wells were blocked with blocking buffer PBS-T (0.05% Tween20 in PBS). containing 2% BSA for 60 min and washed. Next, a series of diluted SARS-CoV-2 (2019-nCoV) Spike protein from 20 µg/ml was prepared in the diluting buffer (0.1% BSA in PBS-T) and incubated for 60 min in the well plates. To study the binding of spike proteins with hACE2, various concentrations of the spike protein viz., 2.5, 5, 10, 12.5, 15, 17.5, 20 µg/ml were prepared and subjected to plasma treatment with two different feed gases He and He-air mixture. The wells were washed and incubated with 100 µl of HRP detection antibody for 60 min. After washing, TMB substrate (Thermo Fisher) and 2 M H 2 SO 4 were sequentially added to the wells.
Then, the optical density was measured using a microplate reader (Thermo Scienti c Varioskan Flash) at 450 nm.

Protein carbonyl content estimation
The carbonyl content of the native and plasma treated spike protein was determined by 2,4dinitrophenylhydrazine (DNPH) assay 20 . Brie y, 200 µl protein samples were properly mixed with 800 µl of freshly prepared 0.1% 2,4-dinitrophenylhydrazine in 2.5 M HCL and incubated for 1.5 h at room temperature. Protein was precipitated by addition of 1000 µl of 20% chilled trichloroacetic acid (TCA) followed by centrifugation at 4° C. The pellet was then re-suspended and washed with chilled 10% TCA followed by washing twice with ice-cooled 50:50 (v/v) acetate and ethanol. Finally, the pellet was resuspended in 500 µl of 6 M guanidium chloride and absorption spectra were recorded at 370 nm with a molar absorptivity of 22000 M −1 cm −1 .

RNA extraction
RNA was extracted from nasopharyngeal and oropharyngeal swab samples using Qiagen QIAmp® Viral RNA Mini Kit (250) (Cat no. 52906; Qiagen India Pvt. Ltd, New Delhi) as per manufacturer's instruction.
The swab samples were subjected to extraction with an elution volume of 50 µl 21 . Extracted RNA was used for plasma treatment in a Bio Safety Level (BSL) -II facility and a 10 µl of RNA sample from each treated and untreated samples was used in each SARS-CoV-2 detection assay.
SARS-CoV-2 detection assays. Written informed consents were taken for using the RNA samples. All the experiments were performed in an Indian Council of Medical Association (ICMR) approved biosafety level 2 (BSL-2) Covid-19 testing and research laboratory and by following the ethical and biosafety guidelines approved by IASST. Real-time RT-PCR assay was performed using the CoviPath TM COVID-19 multiplex kit (Cat no. A50780; Thermo sher Scienti c India Pvt. Ltd, Mumbai) as per manufacturer's instruction. RT-PCR was performed on Agilent AriaMx real-time PCR system with the parameters: 2 mins at 25°C for incubation, 10 min at 53°C for reverse transcription and 2 mins at 95°C for activation followed by 40 cycles of 3 s at 95°C and 30 s at 60°C 22,23 . The relative abundance of the viral RNA was calculated using 2 −ΔΔCT method 24,25 . Abundance of the N and ORF-genes were normalized to that of human RnaseP gene found in the swab samples and presented as the ΔCT value (CT of target gene -CT of RnaseP). The relative abundance SARS-CoV-2 genes with respect to untreated controls were calculated as 2 ΔΔCT where ΔΔCT = Control (ΔCT value) -test (ΔCT value).

Determination of OH, H 2 O 2 and Nitrates through assays
Hydrogen peroxide was determined using the assay kit by Sigma Aldrich (Catalog Number MAK165).
Hydroxyl radicals were quanti ed by treating 20 mM TA and comparing with a standard curve with hydroxyl-terephthalic acid. Nitrate/Nitrite was determined using the detection kit by Cayman Chemicals, USA.

Results And Discussion
Optical Diagnostics and Gas Temperature of the CAP Plasma Figure 2 shows emission spectra obtained from the He and He-air plasma jet. The spectrum covers wavelength from 250 nm to 900 nm. The spectral lines are identi ed using NIST atomic spectra database 26 and reference 27 . The presence of various reactive species in the plasma jet can be seen from the spectrum, which includes molecular bands such as OH (A-X), N 2 (C-B), N 2 + (B-X) and atomic lines of helium and singlet oxygen etc. The presence of the molecular bands in the UV region of the emission spectrum indicates the high energetic UV emission from the plasma jet. In Figure 2 (a), the OES spectrum corresponding to the He plasma jet is shown where very weak emission lines from reactive oxygen and nitrogen species are seen (enlarged in the inset). In He-air plasma, the concentrations of reactive oxygen and nitrogen species are signi cantly increased. As a result, we observed a manifold increase in the RONS concentrations in the He-air plasma jet spectrum as shown in Figure 2 (b). Slight enhancement in UV radiation has also been observed. Some of the helium and hydrogen lines are barely visible in the Heair plasma jet spectrum. This is due to the overlapping of second order lines with the primary spectrum in Figure 2(b).
It is essential to know the gas temperature in a cold atmospheric plasma jet accurately specially for biological applications. One of the most popular gas temperature determination methods in low temperature humid atmospheric plasmas is from the measurement of rotational temperature from OH (A-X) emission band around 306 -310 nm. The rotational temperature can be determined from the comparison of the experimental spectrum with a simulated theoretical spectrum obtained from spectral simulation software like LIFBASE 28 and SPECAIR 29 . The models assume Boltzmann distribution of rotational levels. The rotational temperature obtained from them is a good measure of gas temperature since the translational and rotational degrees of freedom remain in equilibrium in atmospheric pressure plasmas due to large number of collisions 30,31 . Figure 2c shows the best t the OH molecular band between the recorded and the simulated spectrum from the software LIFBASE. The temperature of both He plasma jet and He-air plasma jet is found to be similar and estimated to be 330 ± 20 K in the active region of plasma between the electrodes. While at the point of application, where the plasma plume comes out into open air, the gas temperature remains at room temperature. This can also be veri ed by the fact that the temperature of the sample remains unchanged after plasma treatment.

ELISA characterization of the spike protein binding to the ACE2 proteins
We performed the ELISA assay to characterize the binding of the ACE2 proteins to the Spike proteins before and after subjecting to the plasma exposure, and the results are presented in Figure 3. As mentioned earlier, CAP plasma was produced with two different feed gases, He and He-Air mixed gas with different discharge voltages (2.5 kV and 5 kV, 30 KHz) and very short interaction times of 1 min and 2 min, which will be relevant for practical applications in large scale.
The binding of ACE2 protein to the spike protein is the most important factor behind the Covid -19 infection. To model such binding, in our study, we had taken various concentration of the spike protein from 2.5 µg/ml to 20 µg/ml (Figure 3 (a)). The binding e ciency of the various concentration of the spike proteins with ACE2 was evaluated after treating the protein to the plasma treatment. ACE2 binds to the spike protein almost with ~100% e ciency (without plasma treatment). He plasma reduces the binding e ciency to about 70% and 60% respectively for the voltages of 2.5 KV and 5 KV within 1 min (Figure 3 (a)). The binding reduces to 40% with He-air plasma for same treatment time (1 min). Further increase in treatment time to 2 min reduces the binding e ciency more effectively and maximum reduction to ~ 18% has been estimated for He-air plasma treatment (Figure 3 (b)).
The superior effect of the He-air mixed feed gas plasma is attributed to the increase in the RONS production as observed from the optical emission spectra.
A similar treatment of the spike protein with the CAP plasma using both Helium and He-air mixed feed gas, for a particular concentration 10 µg/ml is presented in Figure 3 (c). Complete deactivation of the spike protein with the longer exposure time of 5 minutes is clearly evident for He-air plasma.
Further, the role of the reactive oxygen species (ROS) in the deactivation of the spike protein was determined using the DNPH assay. ROS can have major interactions with some amino acid residues such as Pro, Arg, Lys, and Thr leading to an oxidative cleavage of the protein backbone 32,33 . Protein-bound carbonyls are the most often used as markers to estimate protein oxidation. Earlier reports have shown that plasma treatment can modify or oxidize a variety of proteins, including horse radish peroxidase 34 and such interactions can be tuned by adjusting the RONS 35 . These protein-bound carbonyls for the plasma treated spike proteins increased from a 2.1% for the control to 17.2% and 36.1% for the He and He-air mixed plasma for the 2 min plasma exposure time. The various modi cations of the protein backbone due to the increased carbonyl content in the protein could be majorly responsible for the deactivation of the protein.
Additionally, we performed RT-PCR analysis of the extracted RNA after plasma treatment. The relative abundance of the nucleocapsid protein encoding N-gene and the open reading frame (ORF1ab) gene of the plasma treated samples were compared with the control and presented in Figure 4. The N-gene encodes the structural proteins in SARS-CoV-2, whereas the ORF gene encodes the nonstructural proteins 36 . It is reported that by detecting the sole N-gene, a positive rate similar to that of detecting both N-gene and ORF1ab gene can be obtained 37 . From Figure 4, we observed that the effects of the He and He-air plasma were different on the abundance of the N-gene and ORF-gene. Treatment with He plasma caused increase in the relative abundance of both the genes with respect to the untreated controls (239% for ORF1ab gene and 442% for N-gene; p< 0.05). On the other hand, treatment with He-air plasma caused a signi cant decrease in the abundance of the both the genes (23% for ORF1ab gene and 35% for N-gene; p< 0.05). This could be due to the non-interacting neutrals present in the plasma. The contrasting effects of the He and He-air plasma could be due to their differential effects on protein and RNA. This is possible if the plasma treatment causes degradation/deformation of structural proteins of the virus particles releasing RNA. While He-air Plasma treatment as explained earlier in Figure 2, induces more OH radicals which can interfere with the RNA structure causing the reduction in abundance of the gene. However, to have a clear understanding e we have quanti ed the role of different radicals produced in the He and He-Air plasma.
To verify the role of the RONS in the deactivation of the spike protein as well as the on the gene abundance, we quanti ed the radicals OH, H 2 O 2 and Nitrates using different assays ( Table 1). The striking feature we observed in this work is that with He Plasma we had a high concentration of H 2 O 2 for a treatment time of 1 min, which decreases nearly 5 times when treated for 2 min. This change is re ected in the increase of OH radicals (8 µM to 100 µM in 2 min) which could be due to the dissociation of the H 2 O 2 to OH radicals upon interacting with higher energy electrons obtained from the He Plasma 38 .
In the case of He-Air plasma, we observe that the concentration of H 2 O 2 increases from 28.6 µM to 37.2 µM in 2 min. The concentration of nitrate remains almost the same for He-Air plasma whereas the nitrite concentration increases for both He and He-Air plasma with the treatment time. The presence of nitrite and its increasing concentration with treatment time may be responsible for the interaction of these nitrites with the spike protein for further reactions. Such conversion of nitrites to nitrogen is reported for several amino acids and proteins 39 .  40,41 . We also see differential peroxide production in different plasma such as He and He-Air Plasma which could lead to the protein deactivation ( Figure 3) as well as the change in the gene abundance ( Figure 4).

Conclusions
In this study we have conducted a systematic evaluation of CAP plasma-based decontamination of SARS-Cov-2 virus. We observed complete inactivation of the spike protein in the presence of the He-air mixed feed gas plasma compared to the He-plasma. DNPH assay suggests the carbonyl modi cation of the spike protein due to the reactive oxygen species (ROS) induced in the plasma which inhibits the binding with hACE2 protein. On the other hand, direct exposure of the extracted RNA to the He and He-air plasma has shown evidence on modi cation on the gene abundance in the RT-PCR experiment. We have shown the hydrogen peroxide from the CAP mediates the deactivation of the spike protein and in uences the gene abundance. Additionally, CAP plasma disinfection is de nitely a better alternative compared to the current chemical-based decontamination which causes several environmental hazards and can be utilized for medical devices, surfaces and in air puri cation. Hence, cold atmospheric pressure plasma with optimal tuning of the reactive species could be a synergistic method to be used for SARS-CoV-2 virus inactivation.

Declarations
Written informed consents were taken for using the RNA samples. All the experiments were performed in an Indian Council of Medical Association (ICMR) approved biosafety level 2 (BSL-2) Covid-19 testing and research laboratory and by following the ethical and biosafety guidelines approved by IASST.