key: cord-0851602-oob5sdkr
authors: Piri, Amin; Kim, Hyeong Rae; Park, Dae Hoon; Hwang, Jungho
title: Increased survivability of coronavirus and H1N1 influenza virus under electrostatic aerosol-to-hydrosol sampling
date: 2021-02-12
journal: J Hazard Mater
DOI: 10.1016/j.jhazmat.2021.125417
sha: 1b319d19b810a8674cb66bdbe66301e6522293ca
doc_id: 851602
cord_uid: oob5sdkr

Airborne virus susceptibility is an underlying cause of severe respiratory diseases, raising pandemic alerts worldwide. Following the first reports of the novel severe acute respiratory syndrome coronavirus-2 in 2019 and its rapid spread worldwide and the outbreak of a new highly variable strain of influenza A virus (H1N1) in 2009, developing quick, accurate monitoring and diagnostic approaches for emerging infections is considered critical. Efficient air sampling of coronaviruses and the H1N1 virus allows swift, real-time identification, triggering early adjuvant interventions. Electrostatic precipitation is an efficient method for sampling bio-aerosols as hydrosols; however, sampling conditions critically impact this method. Corona discharge ionizes surrounding air, generating reactive oxygen species (ROS), which may impair virus structural components, leading to RNA and/or protein damage and preventing virus detection. Herein, ascorbic acid (AA) dissolved in phosphate-buffered saline (PBS) was used as the sampling solution of an electrostatic sampler to counteract virus particle impairment, increasing virus survivability throughout sampling. The findings of this study indicate that the use of PBS+AA is effective in reducing the ROS damage of viral RNA by 95%, viral protein by 45% and virus yield by 60%.

Viral pandemics can cause major concern worldwide, being some of the greatest global health challenges experienced by humanity. In the last 110 years, five pandemics have emerged, all originating from different subtypes of the influenza A virus (Saunders-Hastings and Krewski, 2016) , such as " Spanish flu, 1918 ," "Asian flu, 1957 ," and "Hong Kong flu, 1968 ." More recently, "Swine flu, 2009," where society faced a new, highly variable strain of the influenza A virus (H1N1), resulted in a global threat, and even today, the "COVID-19, 2019" pandemic caused by the novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has presented itself as another great uncertainty of this century.

The influenza virus is an enveloped virus with a diameter of approximately 100 nm. The envelope of influenza A consists of a lipid bilayer and contains two major surface proteins, hemagglutinin (HA) and neuraminidase (NA), as well as glycoproteins (Skehel and Wiley, 2000) .

The influenza virus is a highly contagious and rapidly spreading respiratory virus (Ladhani et al., 2017) . Several influenza transmission events are suspected to occur via viruses in the air (Richard and Fouchier, 2015) . Similarly, coronaviruses (CoVs) are a large family of enveloped zoonotic RNA viruses. Their genome is a positive single-stranded RNA capable of causing respiratory, digestive, hepatic, and neurological disorders in a wide range of animal species (Marra et al., 2003) . CoVs can also be transmitted from animals to humans, followed by subsequent person-to-person transmission (Zumla et al., 2016) . The subfamily Coronavirinae J o u r n a l P r e -p r o o f contains four genera: alpha-, beta-, gamma-, and deltacoronaviruses (Van Der Hoek et al., 2004 ).

An example of an alphacoronavirus is human coronavirus 229E (HCoV-229E), which was identified in the 1960s and results in a mild respiratory tract illness (Marra et al., 2003) .

In contrast, betacoronaviruses mainly infect mammalian species and have attracted great interest in recent years as they are the leading cause of Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV) (Meo et al., 2020; Van Der Hoek et al., 2004) . In 2019, an atypical pneumonia outbreak, caused by a novel betacoronavirus, later known as SARS-CoV-2 was detected in China. The diameter of this virus varies from 60 to 140 nm (Liu et al., 2020; Zhu et al., 2020) . SARS-CoV-2 contains major proteins such as spike proteins, nucleocapsid proteins, membrane proteins, and envelope proteins, which are needed to produce a structurally complete virus particle (Cohen and Normile, 2020; Dömling and Gao, 2020) . SARS-CoV-2 has diverse biological and epidemiological characteristics, making it more contagious than MERS-CoV and SARS-CoV (Khan et al., 2020; Meo et al., 2020) . Furthermore, the spread of the H1N1 influenza virus and SARS-CoV-2 occurs primarily through respiratory droplets that arise from individuals harboring the virus. Recently it is reported that small particles, droplet nuclei, and aerosol mode should not be ignored, especially in enclosed and indoor space (CDC, 2020) . Hence, it is essential to monitor and detect airborne viruses. As in airborne bacterial monitoring, the first step is the air sampling of virus particles (Ladhani et al., 2017) .

After the virus is successfully sampled, a wide array of emerging molecular technologies and biosensors can be used for detection (Dalal et al., 2020; Hideshima et al., 2013; Nidzworski et al., 2014) . Immunofluorescence staining and flow cytometry have been frequently used as they allow the enrichment, quantification, and identification of certain pathogens (Schloter et al., J o u r n a l P r e -p r o o f 1995). For virus specific detection, quantitative real-time polymerase chain reaction (qRT-PCR) cab be used (Van Elden et al., 2001) . Correspondingly, protein-based detection is also viable. For instance, enzyme-linked immunosorbent assay (ELISA) is a plate-based assay technique designed for detecting and quantifying substances such as proteins, antibodies, and hormones (Crowther, 2009 ).

Air sampling is the most critical part of any bio-aerosol study. Currently, there are various sampling methods available. Among these, electrostatic precipitation (EP) is an effective method, attributable to causing less damage and having higher relative recovery of sensitive bio-particles as well as its lower pressure drop (Mainelis, 1999; Mainelis et al., 2006) . Moreover, a wide range of airborne bio-aerosols of different sizes can be collected using corona discharge (Hong et al., 2016) . Furthermore, since most bio-detection methods need the bio-particles to be in a liquid suspended state, the use of electrostatics for aerosol-to-hydrosol (ATH) sampling is increasing (Piri et al., 2020; Han et al., 2010; Park et al., 2016; Yao et al., 2009 ). However, recent findings have shown that applying EP may have inhibitory effects on airborne particles because EP results in the generation of oxygen/nitrogen-derived reactants (Mainelis, 1999; Yao et al., 2009) . In particular, corona discharge can ionize the air, and result in reactive nitrogen species (RNS) and reactive oxygen species (ROS) production, which can be dissolved in the liquid underneath the discharge (Kurake et al., 2017; Lukes et al., 2014; Shimizu et al., 2011; Winter et al., 2014 (Aboubakr et al., 2015; Wende et al., 2015) . Consistently, ROS J o u r n a l P r e -p r o o f such as 1 O 2 and OH• have a stronger destructive effect on microorganisms than other ROS (Laroussi and Leipold, 2004; Lukes et al., 2014; Pavlovich et al., 2013; Sekimoto et al., 2015) .

During the ATH sampling process, the sampled virus particles are exposed to corona discharge, which can lead to protein oxidation and deformation (Boys et al., 2009 ) If either the RNA or protein is damaged throughout the collection process, the PCR or protein-based detections cannot occur. Therefore, it is crucial to avert damage during the sampling process.

Similarly, the polarity and intensity of the applied current, voltage, type of liquid and the liquid flow rate (LFR) applied can have a significant impact on both the collection efficiency and survival state of the sampled viruses. To minimize the damage caused by these ROS, a phosphate-buffered saline (PBS) and ascorbic acid (AA) solution (PBS+AA) can be used as an effective strategy to prevent the ROS damage. PBS+AA protects the bacteria from OH•, O 2 -•, and 1 O 2 and prevents the formation of harmful compounds such as H 2 O 2 and O=NOOH, resulting in improved bacterial viability (Ke et al., 2017) . Moreover, AA is an ROS-scavenger that reacts with singlet oxygen, superoxides, and OH radicals (Surai, 2002) . PBS+AA can terminate ROS-induced chain radical reactions through electron transfer owing to the resonance stabilized structure of AA, and therefore, can deactivate the free radicals produced during corona discharge exposure. After the electron transfer with AA, the resulted semidehydroascorbate is oxidized to dehydroascorbic acid, which is relatively unreactive and does not interfere with the cellular mechanisms (Alsayed et al., 2018; Saini et al., 2016) .

Since virus particles are smaller and more sensitive than bacteria and have different physical and structural characteristics, it is necessary to investigate the degree of damage caused to virus particles and whether PBS+AA can exert any protective effects during ATH EP sampling of virus particles. Therefore, in this study, we investigated the application of PBS+AA as the J o u r n a l P r e -p r o o f sampling solution in our ATH EP air sampler; and evaluated the protective effect of PBS+AA when sampling airborne viruses such as H1N1 influenza virus and CoVs.

All experiments presented in this study were performed at a biosafety level 2 approved to the flask. The flasks were placed at 37 °C for 48 h in 5% CO 2 and 95% relative humidity condition. After 2 days, the culture media containing the newly produced H1N1 viruses were collected and centrifuged at 1,200 rpm at 4 °C (for 10 min). The clarified supernatant was placed at −80 °C for further analysis. A final H1N1 virus concentration of 1.5 10 7 pfu/mL was obtained by performing a plaque assay (see "Plaque assay" section).

Frozen H1N1 virus and HCoV-229E stocks were thawed until they reached 25 °C. One 

In this study, ELISA analysis was performed only for H1N1 influenza virus. HA is the outermost and most antigenic surface protein of the influenza virus (Petrova and Russell, 2018) .

In principle, detection is made by estimating the conjugated enzyme activity via incubation with a substrate (Lequin, 2005 

For experiments with the H1N1 influenza virus, on day one, 5.0 × 10 5 MDCK cells cultivated at 37 °C in 5% CO 2 and 95% relative humidity atmosphere until they reached 90%

confluence. On day 1, 1.0 × 10 6 cells suspended in 1 mL of MEM/EBSS culture media were seeded on 6-well plates. The plates were incubated overnight at 37 °C in an atmosphere of 5% CO 2 and 95% relative humidity. On day 2, confluent monolayers of MRC-5 cells were washed twice with 1× PBS buffer and then infected with 800 µL of serial 10-fold dilutions of the HCoV-

incubation at 37 °C for 1 h with frequent agitation. After infection, the virus inoculums were aspirated, and the monolayers were washed once with 1× PBS buffer. Then, the monolayers were overlaid with 3 mL of agar overlay medium, which consisted of 2x DMEM, 7.4 g/L sodium bicarbonate, 1% antibiotic-antimycotic solution, 2.5% HEPES 1M, and 1% agar. The plates were incubated at 25 °C for 10-15 min until the agar was solidified and incubated at 37 °C in an atmosphere of 5% CO 2 and 95% relative humidity for 1-2 weeks (until plaques were observed).

Agar-covered monolayers were then fixed in 10% formalin (Daejung, Korea). The agar was J o u r n a l P r e -p r o o f removed using a spatula, and the fixed cells were stained using 0.5% crystal violet solution (Sigma-Aldrich, USA), then, the plaques were counted under transmitted light. After counting the plaques, the concentration of the virus suspension in pfu/mL was calculated using the following equation (Eq. 1):

where N p is the average number of plaques, D is the number of dilutions, and V is the volume of diluted virus added to the plate.

The sampling process continued for 20 min. Throughout the sampling, the number concentration of H1N1 influenza virus and HCoV-229E aerosols were assessed using a scanning The sampler's collection efficiency (η) was determined based on the following equation (Eq.

2), in which and represent the number concentrations of virus particles exiting the sampler when the power to the sampler is switched on and off, respectively.

The total concentration of virus particles sampled inside the liquid (N l ) was calculated based on the following equation (Eq. 3), where N is the virus particle concentration in the air (N= ), Q a is the air flow rate, and Q l is the liquid flow rate.

All experiments were performed in triplicate, as specified in the figure legends. Data are expressed as the mean ± standard error of the mean (SEM). The statistical significance of normally distributed data was determined using an unpaired t-test or one-way analysis of variance. Multiple comparisons were made, and the p value was corrected using Tukey's correction. Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, USA). Significant differences are represented by *p < 0.05, **p < 0.01, and ***p < 0.001. Fig. 3 shows the PCR result analysis for the prepared DI and PBS+AA virus suspensions (10 mM AA concentration). Amplification curves were obtained for the H1N1 influenza virus in the multiplex PCR reaction for the DI (Fig. 3a) and PBS+AA suspensions (Fig. 3b) , respectively.

Similarly, amplification curves for HCoV-229E in the multiplex PCR reaction for the DI (Fig. 3d) and PBS+AA suspensions (Fig. 3e) are shown. C t values (X-axis) are displayed in relation to the fluorescence obtained (Y-axis). Consistently, the DI solutions showed persistent viral damage with a decrease in the liquid flow rate, which is directly proportional to an increased C t for the H1N1 influenza virus and for HCoV-229E. In the case of H1N1 virus, the C t value for the nonexposed (NE) DI and PBS+AA samples was 15 (Fig. 3c) . However, as the LFR decreased, due to PBS+AA sample, it increased only 0.05-fold when the LFR was 20 μL/min. In contrast, for

HCoV-229E, the PBS+AA protective effect was more noticeable. The PCR cycle number for the NE DI and PBS+AA samples was 23; similar to the case of the H1N1 virus, as the LFR decreased, the PCR cycle number increased. In the DI samples, the PCR cycle number increased 0.6-fold, whereas it increased only 0.04-fold in the PBS+AA samples when the LFR was 20 μL/min (Fig. 3f) . Collectively, the results indicate that PBS+AA displays a robust and consistent sigmoidal function with a higher endpoint relative fluorescence unit (RFU) value, representing a more efficient PCR and hence showing lower virus impairment than DI. Hence, PBS+AA displayed a significantly lower C t value compared to DI, indicating that PBS+AA prevents viral RNA from being damaged.

The H1N1-HA antibody concentration in DI diminished progressively in a liquid flow ratedependent manner (Fig. 4a) . When the LFR was 20 µL/min, the DI sample showed a significant reduction (0.6-fold) (Fig. 4a) . Notably, after corona exposure, the H1N1-HA antibody content in PBS+AA was greater than that in DI, despite the decrease in the LFR. Hence, the results indicate the potential use of PBS+AA as a protective agent when sampling the H1N1 influenza virus or similar viruses.

To further evaluate H1N1 influenza virus and HCoV-229E infectivity upon corona discharge exposure, plaque assays were performed. In principle, the plaque forming ability of a single infectious virus on a cell-culture monolayer can be measured by this assay. When replication takes place, the host cell dies. Virus titers were determined by counting the number of pfu. The results are shown in Fig. 4b (H1N1 influenza virus) and Fig. 4c (HCoV-229E) . Compared to the DI samples, PBS+AA was able to preserve the virus yield, showing a higher number of plaques.

In case of H1N1 influenza virus, compared to the NE samples, PBS+AA was showed a 0.6-fold decrease, whereas the DI sample showed a 0.9-fold reduction at 20 µL/min. Similarly, HCoV-229E plaque titrations assay showed a 0.4-fold decrease in the PBS+AA sample, whereas the DI sample showed a 0.8-fold reduction at 20 µL/min. The protective effect of PBS+AA on H1N1 virus plaque formation is tightly correlated with the LFR (corona discharge exposure time).

infectivity between the DI and PBS+AA hydrosols after corona discharge exposure. Fig. 5a shows the size distributions of aerosolized H1N1 influenza virus when the sampler was on and off. The collection efficiency of the sampler was 75% (Eq. 2). The PCR analysis revealed a substantial decrease in cycle number when PBS+AA was used as the sampling liquid.

The Ct value for the DI sample was 31, whereas the Ct value was 23 for the PBS+AA sample; this 0.8-fold increase of Ct value in the DI sample was correlated with the viral RNA impairment induced upon corona exposure ( Fig. 5b and 5c) . Moreover, the ELISA results for the PBS+AA sample showed a 1.1-fold increase in the H1N1-HA antibody concentration than for the DI sample (Fig. 5d) . Similarly, virus titers were determined by counting the number of plaques.

Compared to the DI solution, the PBS+AA solution was able to preserve virus yields to a greater extent (1.3-fold) (Fig. 5e) . The size distributions of aerosolized HCoV-229E, when the sampler was on and off, are shown in Fig. 5f . In line with results of test with H1N1 influenza virus,

HCoV-229E experiments also revealed a lower Ct value (Ct = 29) for the PBS+AA sample than for the DI sample (Ct = 34). This increase of Ct value in the DI sample was correlated with the viral RNA impairment, induced upon corona discharge exposure ( Fig. 5g and 5h) . The virus J o u r n a l P r e -p r o o f titers determined that PBS+AA solution was able to preserve virus yields to a greater extent (2.1fold) (Fig. 5i) , when compared to DI samples. The correlation between PCR and virus viability results are shown in Fig. 5j . Collectively, these results imply that PBS+AA preserves virus plaque formation by exerting protective effects during corona exposure. 

The development of standardized methods to minimize sampling damage is one of the significant scientific challenges in the field of bio-aerosols. Bio-aerosol sampling through EP may result in the collection of non-viable particles due to the electric charge applied to bioparticles (Mainelis, 2020) . Several approaches for the sampling evaluation of bio-aerosols have been developed (Sirikanchana et al., 2008) . To determine viral concentrations, virus cultivation is commonly used. However, most bio-aerosol sampling techniques affect virus infectivity, making virus cultivation inadequate for calculating the concentrations of airborne viruses (Verreault et al., 2008) . Another approach is the employment of qRT-PCR to confirm the presence of amplifiable virus genomes, which can be correlated with undamaged virus samples.

Thus, for effective amplification, the sample preparation and quality of collection for PCR should be improved. Similarly, ELISA was used to evaluate the effect of ROS damage on viral proteins. Proteins are less sensitive to heat and high salinity and therefore have higher stability than RNA and DNA (Forterre, 2005) . However, oxidative damage due to corona discharge generates ROS, which can cause loss of protein functionality (Berlett and Stadtman, 1997; Stadtman and Levine, 2003) . By performing ELISA, we consistently discerned between healthy and damaged H1N1 influenza HA proteins since the virus surface contains host binding antigens that are responsible for binding with host cells and aid the virus in infecting the host cell. If these surface proteins are damaged, the virus becomes inactivated and loses its capability of causing an infection. This may hold positive outcomes for the general population; however, for virus analysis, quantification, isolation, and vaccine-development, this represents a challenge since it is imperative that the sampled virus retains its infectivity and replication properties. Similarly, the ability of viruses to form plaques was investigated through plaque assay analysis by examining the infectability of the virus after corona discharge exposure and the sampling process. Generally, a low pfu/mL value is correlated to a high PCR cycle number while a high pfu/mL value is correlated to a low J o u r n a l P r e -p r o o f PCR cycle number (Zhang et al., 2013) . The plaque assay results suggested that the pfu/mL values for the samples in PBS+AA were higher than those for the samples in DI. The results for plaque assay analysis are also in line with the obtained PCR results, indicating that PBS+AA is significantly effective in reducing the in preventing the loss of virus infection due to ROS damage during sampling process. Compared to the NE samples, the use of PBS+AA reduced the corona discharge-generated ROS damage of H1N1 viral RNA by 95% and preserved viral protein and virus yield by 45% and 60%, respectively. The results presented in this study provide sufficient evidence that PBS+AA plays a crucial role in protecting small and sensitive viruses such as influenza and coronaviruses and can significantly contribute to enhancing current bio-aerosol monitoring systems, resulting in the prevention of future virus pandemics such as the "COVID-19, 2019" pandemic. HCoV-229E suspensions. The results represent the mean ± SEM of three independent assays.

Significant differences are represented by *p < 0.05, **p < 0.01, and ***p < 0.001. DI, deionized water; PBS+AA, phosphate-buffered saline and ascorbic acid solution; NE, non-exposed; RFU, relative fluorescence units.

J o u r n a l P r e -p r o o f Significant differences are represented by *p < 0.05, **p < 0.01, and ***p < 0.001. DI, deionized water; PBS+AA, phosphate-buffered saline and ascorbic acid solution; NE, non-exposed.

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

Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus

Antioxidant activity of vitamins against free radicals

Protein oxidation in aging, disease, and oxidative stress

Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack?

New SARS-like virus in China triggers alarm

Systems in ELISA. The ELISA Guidebook

Detection methods for influenza A H1N1 virus with special reference to biosensors: a review

Chemistry and Biology of SARS-CoV-2

The two ages of the RNA world, and the transition to the DNA world: a story of viruses and cells

Performance of an Electrostatic Precipitator with Superhydrophobic Surface when Collecting Airborne Bacteria

Attomolar detection of influenza A virus hemagglutinin human H1 and avian H5 using glycan-blotted field effect transistor biosensor

Gentle sampling of submicrometer airborne virus particles using a personal electrostatic particle concentrator

Effect of N2/O2 composition on inactivation efficiency of Escherichia coli by discharge plasma at the gas-solution interface

Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach

Non-thermal plasma-activated medium modified metabolomic profiles in the glycolysis of U251SP glioblastoma

Sampling and detection of airborne influenza virus towards point-of-care applications

Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure

Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)

Viral architecture of SARS-CoV-2 with post-fusion spike revealed by

Aqueous-phase chemistry and bactericidal effects from an air discharge plasma in contact with water: evidence for the formation of peroxynitrite through a pseudo-second-order post-discharge reaction of H2O2 and HNO2

Collection of Airborne Microorganisms by Electrostatic Precipitation

Bioaerosol sampling: Classical approaches, advances, and perspectives

Performance of a compact air-to-liquid aerosol collector with high concentration rate

The genome sequence of the SARSassociated coronavirus

Novel coronavirus 2019-nCoV: prevalence, biological and clinical characteristics comparison with SARS-CoV and MERS-CoV

Universal biosensor for detection of influenza virus

Continuous and real-time bioaerosol monitoring by combined aerosol-to-hydrosol sampling and ATP bioluminescence assay

Ozone correlates with antibacterial effects from indirect air dielectric barrier discharge treatment of water

The evolution of seasonal influenza viruses

Prevention of damage caused by corona dischargegenerated reactive oxygen species under electrostatic aerosol-to-hydrosol sampling

Influenza A virus transmission via respiratory aerosols or droplets as it relates to pandemic potential

Kinetics and mechanism of oxidation of Lascorbic acid by chromic acid in presence of hydrochloric acid

eviewing the history of pandemic influenza: understanding patterns of emergence and transmission

The use of immunological methods to detect and identify bacteria in the environment

Effects of H3O+, OH−, and NO x for Escherichia coli inactivation in atmospheric pressure DC corona discharges

Formation of thermal flow fields and chemical transport in air and water by atmospheric plasma

Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication

Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Natural Antioxidants in Avian Nutrition and Reproduction

Identification of a new human coronavirus

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR

Methods for sampling of airborne viruses. Microbiol

Identification of the biologically active liquid chemistry induced by a nonthermal atmospheric pressure plasma jet

Tracking plasma generated H2O2 from gas into liquid phase and revealing its dominant impact on human skin cells

Comparison of electrostatic collection and liquid impinging methods when collecting airborne house dust allergens, endotoxin and (1, 3)-βd-glucans

A disposable microfluidic virus concentration device based on evaporation and interfacial tension

A novel coronavirus from patients with pneumonia in China

Coronaviruses-drug discovery and therapeutic options

The size distribution of the aerosolized virus. (b) qRT-PCR amplification curves for the DI and PBS+AA samples. (c) The effect of liquid type on the PCR C t values in DI and PBS+AA

The size distribution of the aerosolized virus. (g) qRT-PCR amplification curves for the DI and PBS+AA samples. (h) The effect of liquid type on the PCR Ct values in DI and PBS+AA. (i) Plaque titrations assay. (j) The correlation between PCR and virus viability results. The results represent the mean ± SEM of three independent assays

RFU, relative fluorescence units

Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Validation, Visualization, Resources, Writing -review & editing. Hyeong Rae Kim: Validation

Dae Hoon Park: Validation, Visualization. Jungho Hwang: Supervision, Project administration, Writing -review & editing

This research was supported by the Bio Nano Health-Guard Research Center, funded by the Ministry of Science and ICT (MSIT) of Korea as a Global Frontier Project (HGUARD_2013M3A6B2078959). Additionally, Amin Piri would like to thank Dr. Adriana Rivera-Piza for her invaluable and insightful assistance.

■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.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: