Bio-aerosols can serve as a transmission vehicle for numerous and diverse types of non-pathogenic or pathogenic bio-particles including virus, bacteria, and fungi (II et al., 2017; Alonso et al., 2014; Cowling et al., 2013; Li et al., 2004). Pathogenic bio-particles are associated with a wide range of adverse public health impact and are mainly infected through respiratory tract (Douwes et al., 2003). Thus, it is important to monitor the bio-aerosols of the respiratory aerosol fraction that may contain pathogens in real-time to reduce the damage of life and property. However, traditional approaches involving culture and genetic technologies are not appropriate because they require days or complex procedures to get the results (Denoya, 2016). There have been many efforts to overcome the disadvantages of traditional approaches by adopting optical instruments (Denoya, 2016; Wei et al., 2016; Choi et al., 2014; Huffman et al., 2009). One of them is the laser-induced fluorescence (LIF) instrument. The principle is that bio-particles fluoresce when subjected to ultraviolet light (UV), which is not common response for non-biological particles, resulting in discrimination of bio-particles against non bio-particles in real time. The fluorescence of bio-particles is due to intrinsic auto-fluorophores. The predominant auto-fluorophores found in bio-particles are the amino acids, which are the fundamental building blocks of peptides and proteins. Of the 20 candidate amino acids, only tyrosine, phenylalanine, and tryptophan are fluorescent (due to their aromatic components), and have absorption maxima between 260-280 nm and fluorescence maxima between 280-360 nm (Fennelly et al., 2018). Coenzymes are further category of the auto-fluorophores that contribute to atmospherically relevant bio-aerosol fluorescence. Coenzymes, such as NADH (Nicotinamide Adenine Dinucleotide, reduced) and NADPH (Nicotinamide Adenine Dinucleotide Phosphate, reduced), are typically present in many bio-particles. These coenzymes absorb maxima between 340-360 nm and fluoresce maxima between 440-470 nm (Fennelly et al., 2018). Riboflavin is also an important contributor to the autofluorescence of bio-particles. Its absorption and fluorescence maxima are between 450-488 nm and between 520-560 nm, respectively (Fennelly et al., 2018; Pan, 2015). Depending on the type of microorganisms such as bacteria, fungal spores and viruses, the composition of auto-fluorophores is different. The common auto-fluorophores of these bio-particles are amino acids like tryptophan and tyrosine, while NADH and riboflavin are absent in individual virus and almost negligible in fungal spore (Hill et al., 2013). Nonetheless, one reported that the tested viruses fluoresce between 475-500 nm when excited at 405 nm, which was considered to be influenced by the culture medium (Pan, 2015). This means that the proliferation and release processes of virus particles, as well as their innate auto-fluorophores composition, also have a significant impact on fluorescence monitoring, especially for airborne bio-particles. Others also demonstrated the instrument-specific such as laser intensity and signal collection efficiency as well as the excitation wavelength in fluorescence detection (Saari et al., 2014). The most well-known real-time LIF instrument is UVAPS (Ultraviolet Aerodynamic Particle Sizer^TM, TSI Inc., St. Paul, MN, USA) The instrument uses a 355 nm laser source and detects the fluorescence of aerosol particles within the size range of 0.5-15 μm. The performance of the UVAPS against bacteria, fungal spores, and auto-fluorophores (NADH and riboflavin) has been reported (Agranovski et al., 2004; Agranovski et al., 2003). Another commercial version of the instrument is BioScout (Environics Ltd, FI-50101, Mikkeli, Finland) which has 405 nm laser source and the size range of 0.5-10 μm. Although its excitation wavelength are different from that of UVAPS, it has been reported that the performance against bacteria, fungal spore, and auto-fluorophores is virtually no difference from UVAPS (Saari et al., 2014). Bacteria or fungal spores are mostly used to assess the performance of the LIF instrument for bio-aerosol monitoring, however, virus particles are rarely used, despite being easily infected through the air. The study on the applicability of the LIF instrument with 405 nm to monitoring bio-aerosols including virus particles has not been done well. In the study, we demonstrate the real-time monitoring capability of the LIF instrument with 405 nm using aerosolized bacteria and virus particles. For the purpose, we used the LIF instrument with 405 nm, BDS (Bio-aerosol Detection System) (Yoon et al., 2018). Monitoring was performed within the two kinds of aerosol chambers: One is for the fluorescence efficiencies and monitoring ranges, the other for real-time monitoring. To our knowledge, this represents the first study on the performance of the LIF instrument with 405 nm by monitoring aerosolized bio-particles including virus vaccines or the bacteriophage MS2, as well as bacteria.

In order to evaluate the LIF-based BDS with 405 nm on real-time monitoring of aerosol particles including virus particles as well as bacteria, we have performed experiments in aerosol chambers using Bacillus subtilis, two kinds of virus vaccine (ND, IB) and bacteriophage MS2. First, we investigated the fluorescence response of the BDS on particle sizes of auto-fluorophores including NADH, riboflavin, tryptophan and tyrosine. As shown in Fig. 4, NADH and riboflavin exhibited fluorescence in the size ranges of 1 to 15 μm. On the other hands, tryptophan and tyrosine showed almost no fluorescence in all observed particle size ranges as expected. This is due to the nature of the auto-fluorophores, which rarely absorbs light at 405 nm (Pan, 2015). The absorption peaks of NADH generally ranges from 340 nm to 360 nm (Fennelly et al., 2018). Nonetheless, in this study, NADH fluoresced by 405 nm. One reason is that NADH may absorb some wavelength of the incident light (405 nm) even though 405 nm is not within the absorption peak range of NADH (Hill et al., 2013). Some also observed the fluorescence of NADH using the LIF-based BioScout with 405 nm, and reported that fluorescence efficiency is not only specific for biomolecules or biological particles, but also depends on equipment details such as the excitation source and the detection band (Saari et al., 2014). As shown in Fig. 5B, comparing the fluorescence efficiencies of auto-fluorophores depending on the particle size ranges, in the range of 1 to 2 μm, NADH was about 4 times higher in the SWR than in the LWR, and riboflavin was about 300 times higher in the LWR than in the SWR. Meanwhile, in the range of >2 to 15 μm, NADH was about 1.1 times higher in the SWR than in the LWR, and riboflavin was about 4 times higher in the LWR than in the SWR. Overall, NADH was higher in the SWR than in the LWR and riboflavin was reversed. These results implies that the fluorescence monitoring of bio-particles containing NADH and riboflavin, which contribute to atmospherically relevant bio-aerosol fluorescence (Fennelly et al., 2018), is possible by the LIF-based instrument with 405 nm. Moreover, the BDS measures them as bio-particles when the fluorescence signals from two wavelength ranges occur simultaneously so that it increases the accuracy of bio-particles rather than using one kind of fluorescence signal. However, individual viruses, which typically is not associated with NADH or riboflavin, commonly contain one or more of the fluorescing amino acids (tyrosine, tryptophan) will not fluoresce by 405 nm. Nonetheless, it may be assumed that the atmospheric virus particles, unlike individual viruses, may also fluoresce by 405 nm due to auto-fluorophores from a complex mixture of host respiratory track fluids, because they are discharged through the respiratory tract of their host (Reynolds and Chrétien, 1984). One reported the virus particles [VEE (Venezuelan equine encephalitis TC83), MS2] fluoresce by 405 nm, the reason is probably dominated by the fluorescence of the lysate of the host cells (Vero cells in the case of VEE, E. coli in the case of MS2) (Pan, 2015). Most virus vaccines are also dominated by the allantoic fluid of embryonated chicken eggs, the host used for the proliferation process (Brauer and Chen, 2015). Therefore, MS2 or virus vaccine is considered to be more a suitable sample than individual virus particles for experiments on airborne virus particles. Thus, next, the possibility of monitoring airborne-virus particles using 405 nm was examined by using virus vaccines and bacteriophage MS2, virus simulant. As shown in Fig. 6, the aerosolized particle distribution pattern varied somewhat for each sample. But the number distribution of the fluorescence particles was similar, it ranged from approximately 2 to 15 μm in the tested samples, while the fluorescence efficiencies were different depending on the test samples. The fluorescence efficiencies were 70% at 3 μm for ND and BS, and 70% and 50% at 5 μm for IB and MS2, respectively. Thus, It was higher in ND and BS rather than in IB and MS2 (Fig. 7). The difference in the fluorescence efficiency may be due to differences in the constituents from each sample production process. In the study, the aerosolized virus particle capable of fluorescence monitoring by the BDS ranges from 2 to 15 μm, which is considerably larger than individual virus particle ranging from several nm to several tens of nm. One demonstrated that more than one-half of the viral particles detected by PCR were within the respirable aerosol fraction (diameter, <4 μm) (Blachere et al., 2009). Moreover, the viability of viruses is reported to be particle size dependent. Some found that the viable IAV (Influenza A Virus) and PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) can only be isolated from particles larger than 2.1 μm (Alonso et al., 2015), and also demonstrated 93% recovery on aerosolized MS2 of 2-5 μm using a novel growth tube collector (Pan et al., 2016). This suggests that virus particles in the size range detectable (2-15 μm) by the LIF-based BDS using 405 nm may have high viability. Finally, for real-time monitoring, the samples was dispersed in the aerosol chamber. The results in Table 1 show the total fluorescence efficiency by indicating the concentration of the total fluorescence particles and the total scattering particles passing through the monitoring module without dividing the particle size ranges. The total fluorescence efficiency was no significant difference depending on the concentration, but there was significant difference depending on the samples by showing about 3 times higher in ND than in IB. This is thought to be a difference between the two vaccine components as mentioned above. Overall, despite the insensitivity of the BDS to tyrosine and tryptophan, and given the observed fluorescence efficiencies of bio-particles including virus particles with the BDS using 405 nm excitation light, it appears that this excitation wavelength may prove interesting in the continued development of instrumentation for the real-time monitoring of airborne-virus particles. In particular, it is not easy to predict the appearance of virus particles in the ambient environment. This is because it is not virus-specific and not easy to distinguish when the concentration of background bio-particles is high. Taken together, along with the fluorescence monitoring function of the LIF instrument with 405 nm, further development of algorithms to distinguish virus epidemic situation from normal background concentration will improve the capability of real-time monitoring of the BDS as the first responder in the field.