key: cord-0316520-wbvdcg04
authors: Luescher, A. M.; Koch, J.; Stark, W. J.; Grass, R. N.
title: Silica-encapsulated DNA tracers for measuring aerosol distribution dynamics in real-world settings
date: 2021-05-20
journal: nan
DOI: 10.1101/2021.05.19.21257392
sha: 3909c4fe34cdc9386a27fbda5a87f1a7a37fd376
doc_id: 316520
cord_uid: wbvdcg04

Aerosolized particles play a significant role in human health and environmental risk management. The global importance of aerosol-related hazards, such as the circulation of pathogens and high levels of air pollutants, have led to a surging demand for suitable surrogate tracers to investigate the complex dynamics of airborne particles in real-world scenarios. In this study, we propose a novel approach using silica particles with encapsulated DNA (SPED) as a tracing agent for measuring aerosol distribution indoors. In a series of experiments with a portable setup, SPED were successfully aerosolized, re-captured and quantified using quantitative polymerase chain reaction (qPCR). Position-dependency and ventilation effects within a confined space could be shown in a quantitative fashion achieving detection limits below 0.1 ng particles per m3 of sampled air. In conclusion, SPED show promise for a flexible, cost-effective and low-impact characterization of aerosol dynamics in a wide range of settings.

Aerosol dynamics are an important factor when assessing the circulation of hazardous pollutants and pathogens with regard to human health and the environment. Viable bioaerosols are a wellknown cause for many infectious diseases such as tuberculosis 1 , measles 2 , Legionnaire's disease 3 , influenza 4 , gastroenteritis 5 and SARS-CoV-1 6 and SARS-CoV-2 [7] [8] [9] [10] . With the COVID-19 pandemic spreading globally, the importance of understanding the mechanisms of aerosol spreading is perhaps more evident than ever and discussions surrounding the topic have reached the mainstream. Specifically, the assessment and characterization of aerosol distribution properties within specific indoor spaces such as lecture halls, office spaces, hospitals, public transport vehicles or event venues is of high interest with regard to public and occupational health.

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The copyright holder for this preprint this version posted  https://doi.org/10.1101/2021.05. 19 .21257392 doi: medRxiv preprint 3 Many of the existing approaches to assess indoor environments regarding aerosol dynamics rely on computational models. However, such models are based on the pre-existing understanding of fluid dynamics and prone to numerical error 11 , while also depending on data for setting realistic parameters 12 . Therefore, predicting real-world behavior in complex, non-controlled environments is computationally challenging. For those reasons, physical tracing is still a vital field to assess aerosol-related properties in real-world settings to help validate and complement model-based studies. Table 1 shows a list of studies reporting tracing methods to characterize (bio-)aerosol distribution indoors. Carbon dioxide, for example, is a commonly used tracer gas, applied either alone or in combination with other tracers, and in conjunction with one or more CO2 detectors. For example, Knibbs et al. 13 employed CO2 to assess the effect of ventilation rates in various room settings, using the results to model virus-specific infection risks. While CO2 is widely accessible, non-toxic (in low concentrations), easily detected and self-clearing, it is also naturally present in ambient air.

Subtle effects may therefore be harder to detect and relatively high working concentrations are needed. Moreover, aerosol dynamics are complex and not necessarily approximated accurately by a non-particulate tracer gas.

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The copyright holder for this preprint this version posted n/a n/a n/a n/a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) 14 therefore combined CO2 with an aerosol tracer to characterize distribution in a large concert hall. Their combinatorial approach has the advantage of monitoring aerosol droplets with temporal and spatial resolution, with the limitation that the particle counters used for detection are not specific to the tracer aerosols and the resolution of detection is only in the µg/m 3 range.

Another approach, by Pyankov et al. 15 , uses Influenza A virus as a tracer and a personal sampler for detection. However, they focus on introducing a new method of detection rather than a routine tracer entity for room characterization. Any approach using live viruses is not routinely feasible in a real-world setting as it requires high safety precautions, well-trained personnel and extended experimental preparation time.

Further physical tracers 18-24 , typically used outdoors and for environmental tracing, exist, but they, too, face challenges related to either environmental background concentrations, hazard and toxicity or non-specificity of the detection method.

None of the reviewed tracing methods are suitable for complex multi-tracing or offer an extensive platform for flexible tuning of properties. There is still an unmet need for additional tracing methods allowing for direct and reliable experimental real-world characterization of indoor spaces regarding (bio-)aerosol distribution. Hence, novel tracing materials that are cost-effective, nontoxic and can be detected with a high sensitivity and specificity in a broad range of scenarios would be a valuable addition to existing tools.

We propose a new approach using aerosolized Silica Particles with Encapsulated DNA (SPED) to analyze aerosol dynamics. These sub-micron particles stand out in their ease of synthesis, durability and quantitative analysis with a low detection limit, as described by Paunescu et al. 25 Silica is . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint recognized as "safe" by the FDA with silica nanoparticles being used as food additive and in medicine (e.g. imaging). 26 The encapsulated artificial DNA, or "DNA barcode", is not present in the environment and can be detected with an extraordinarily high selectivity and sensitivity using real-time quantitative polymerase chain reaction (qPCR). This powerful detection method is known for its use in diagnostics to identify various pathogens, for example in food 27 , water 28 and also aerosols 15, 29, 30 . Likewise, SPED are already established as tracers for liquids and surfaces and have been used to characterize aquifers 31 , assess pesticide drift 32 , track and trace comodities 33, 34 and observe trophic interactions within food webs. 35 Moreover, they have been employed as surrogate tracers for bacteria in a hospital environment 36 . Similarly, bacteriophages, C. difficile spores and cauliflower mosaic DNA have been employed for surface tracing in various settings, sometimes in conjunction or compared with fluorescent markers [37] [38] [39] [40] [41] [42] . The DNA sequences are unique identifiers, offering a great advantage over other methods by allowing for specific detection. In the case of SPED, the sequences are synthetic, not bound to an organism, can be changed at will and are more robust through silica encapsulation. Since even a sequence length of just 60-100 nucleotides offers trillions of potential barcodes, a system with nearly unlimited multi-tracing capabilities is conceivable. Thus, the advantages of highly sensitive PCR detection can be implemented without the need for tracing organisms.

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The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint In this study, SPED are dispersed as aerosols and re-collected using commercial biosampler impingers, which offer a proven method to capture aerosols [43] [44] [45] . The DNA is released from its silica protection and quantitatively analyzed. The overall principle is summarized in Figure 1 . Two batches of SPED, S1 and S2, were used, which differ in the sequence of their DNA barcode.

Working with these unique identifiers has the advantage that they enable a wide variety of experiments, largely eliminating contamination between experiments and permitting scenarios such as simultaneous multi-source sampling.

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Microsynth AG, Balgach, Switzerland) was added to 200 mL ultrapure water (mQ; type 1, 18.2 MΩ·cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany). 0.4 g of the functionalized particles were added to the DNA solution and shaken for 10 seconds. Subsequently, 4 µL TMAPS were added, then the mix was shaken and sonicated for 20 seconds. Next, 62.5 µL of tetraethyl orthosilicate (TEOS) (≥99.0%; Sigma-Aldrich, St. Louis, Missouri, USA) were added, followed by 5 hours of shaking at 600 rpm on a mixer (Vibramax 100; Heidolph, Schwabach, Germany) with a universal clamping attachment (VX 8; IKA, Staufen, Germany). In a further step, 10 mL isopropanol and 5.9 mL TEOS were mixed with 484.1 mL mQ water and combined with the previous mixture. The batch was again stirred at 600 rpm for 4 days, before washing twice with water.

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The aerosol experiments were performed in a 277 m 3 laboratory running at a ventilation throughput of 1840 m 3 /h. A technical layout of the room can be found in the supplementary material, Figure   S2d . SPED-containing aerosols were generated by a commercial airbrush gun with a 0.35 mm nozzle (type AFC-101A; Conrad Electronic, Hirschau, Germany) using an air pressure of 1 bar, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint mounted to a height of 1.6 m on a mobile stand. Phase Doppler anemometry was used to characterize droplet size distribution as created by the airbrush gun used for aerosol generation.

The point of measurement was 2 cm in front of the nozzle, using a standard SPED suspension. For a more detailed description refer to the supplementary material, section S1.2.

For each sampling, 5 mL of a 0.2 mg/mL aqueous suspension of the respective SPED species were nebulized over the course of approx. one minute. For the treatment and wash protocol of SPED before dispersion, refer to the supplementary material, section S1.4. Table 2) were ordered in dry state from Microsynth AG (Balgach, Switzerland) and dissolved in PCR-grade . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Characterization of the two SPED species (S1 and S2) used for the following tracing studies revealed a DNA load of 23 µg and 26 µg dsDNA/mg for S1 and S2 particles, respectively. The median hydrodynamic size was determined to be 133.5 nm for S1 and 160.6 nm for S2, as shown in Figure 2b . This is further confirmed by electron microscopy (Figure 2a) , which shows the spherical shape and homogenous size distribution of the particles. The selected core particle size of 110 nm is similar to viruses like adenovirus 46 , influenza A 47 , or SARS-CoV-2 48 , all ranging between 90 and 120 nm in size.

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In addition to the hydrodynamic diameter of SPED in suspension, the droplet size generated by the airbrush gun was determined using phase Doppler anemometry (PDA), the results of which are shown in the supplementary material, section S1.2. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint which is close to a human's minute ventilation for a low-level activity, such as driving a car 49 .

Through the focused airflow, aerosols are trapped in the collection water, which can directly be used for further processing and subsequent PCR-analysis. The setup is mobile and only requires electricity from regular sockets and a small compressed air source for the airbrush gun. A batterypowered version of the same setup would also be feasible.

As a pilot test, a time-resolved experiment was conducted. For this experiment, two flow impingers were used, mounted 2 m and 6 m frontal to the origin of aerosol flow, respectively, and the test laboratory ran at regular ventilation output. 1 mg of SPED 1 was dispersed and during a continuous sampling time of 120 min, 200 µL samples were removed from the initial 20 mL solution at 14 time points and analyzed separately for their SPED concentration. Figure 4a shows the integrated aerosol levels measured in the two impingers over time. The first measurement, at t=0, was taken before dispersion and is thus the experimental negative control, marking the minimal limit of detection (MLD) of 4.5·10 -9 mg/mL in the sampling solution for this experiment. Assuming a conservative physical collection efficiency for the impingers of 10%, as estimated from literature data [50] [51] [52] , this corresponds to a particle concentration detection limit of 6·10 -2 ng/m 3 of sampled air.

Comparing this value to commercial monitoring systems for inorganic particulate matter 2.5 (PM2.5) 53, 54 the MLD of the present method is at least one order of magnitude lower. More information on the impinger efficiency, concentration measurements and how detection limits were determined can be found in the supplementary material, section S1.5.

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The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint Following the aerosol generation, the amount of SPED collected rises sharply in both impingers, prior to reaching a plateau load in the range of 3·10 -4 mg and 2·10 -4 mg, respectively. At position . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint 1, the maximum is reached within the first measurement increment after 3 minutes, whereas at a distance of 6 m, there is a distinguishable saturation curve reaching the plateau after an estimated 10-20 minutes, attributing to the fact that the aerosols reach the second sampling point with a delay due to further traveling distance. This position-dependent effect is also reflected in the total collection loads, which correspond to estimated position-specific dosages of 0.35% and 0.2% of the total amount of released SPED. Throughout the measurement time, there are some fluctuations; however, these deviations are within the expected range for PCR reactions, as discussed further below.

To further investigate position-dependency, the total load over 120 min was measured under otherwise equal sampling conditions. In addition to the two impingers used in the previous experiments, two locations with a 2 m offset relative to the airbrush gun's line of flight were sampled simultaneously (labelled positions 3 and 4 in Figure 3b ). The results of three separate runs -two with barcode S1 and one with S2 -are displayed in Figure 4b . To relate total load to the distance from the source, the data are always normalized to the 2 m frontal position, which is standing within the aerosol flow and therefore considered an experimental positive control. The data show that relative exposure is much lower at all three locations that are either further apart from the source, and/or not in the direct line of flight. Additionally, the three experiments qualitatively compare to each other, independent of the DNA barcode used.

In a next step we wanted to determine the influence of ventilation to aerosol distribution and clearance. We therefore repeated the previous measurements, but turned off the ventilation system with doors and windows remaining closed, as before. Figure 4c compares total load after 2 hours . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Interestingly though, the exposure is considerable in the ventilated scenario, but very low without ventilation. Based on this outcome, we hypothesized that ventilation dynamics could lead to a suction effect from the main room to the atrium. The layout of the ventilation piping (see supplementary material, Figure S2d ), which shows a connection between the two rooms, is compatible with this theory.

As these results were unexpected, we wanted to reconfirm them using another method. We therefore used a fog machine to visualize air dynamics and to add qualitative evidence to the previous results. The fog was generated at the same position as the previous source and a video camera was installed in the atrium. Indeed, with ventilation running, fog could be visually detected with a delay of ca. 5 min (see supplementary video file) seemingly coming from the ventilation pipe. The fog does not allow for quantification or localized detection and has a comparatively high detection limit, but it was sufficient to qualitatively confirm our measurements. This shows that . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint the method introduced here is suitable to detect unexpected effects in the distribution dynamics of aerosols in a real-world setting, as caused by a specific ventilation system. There are indications that ventilation systems and air-conditioning can contribute to the spreading of airborne diseases instead of preventing it 10 , which is why it is already recommended to replace air recirculation by increased inflow of outdoor air 55 . The results show that the described method is able to measure such effects and could for example be employed in finding appropriate ventilation settings to limit aerosol spread.

The present proof-of-concept shows that a simple setup can be used to reliably measure time-, position-, and ventilation-dependent relative aerosol loads indoors using different DNA barcodes.

Current limitations of the sampling and detection method are a direct result of PCR analysis, which requires normalization to achieve quantitative results. Furthermore, PCR data are logarithmic to the concentration levels. Consequently, result quantification requires a range of control experiments, and small concentration differences are more difficult to detect, requiring numerous sample replicas. Furthermore, the individual measurements were conducted in a real-world environment under similar conditions, but without perfect control of external parameters. Factors such as relative humidity, temperature and the presence and concentration of other particulate matter in the ambient air can influence aerosol dynamics and lead to a difference in absolute aerosol levels accessible to measurement.

In spite of the limitations mentioned above, the SPED-based method introduced here has several important benefits, the combination of which makes it novel and unique among aerosol tracers. Important features are the versatility, sensitivity and specificity towards the sampled barcode and . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint suitability to detect effects related to air circulation. The silica layer protects the DNA from physical and chemical damage 25 , which is an additional advantage over other DNA-based tracers and makes SPED suitable for indoor and outdoor use alike. The costs of SPED are estimated at 500 USD/g, which benchmarks the price of particles for a single experiment of the scale discussed here at less than 1 USD.

Even though flow impingers were used in this experimental setup, the use of SPED in combination with other means of aerosol capturing, including surface sampling 36 , is conceivable. And while the airbrush gun used in this pilot study presents a cost-effective, user-friendly solution, SPED could equally be used in combination with advanced dispersion systems to e.g. mimic human breathing or coughing 56 . Furthermore, it has been shown that SPED detection is possible at a single particle level in solution 57 . Thus, when limiting barcodes to single use and with a further focus on optimization of the respective sampling and measurement conditions, an additional improvement of the detection limit in the range one or two orders of magnitude is conceivable. Consequently, the flexible barcodes offer countless combination possibilities in future measurements, such as simultaneous surface and air sampling with multiple sources and detectors at extremely low detection limits.

In conclusion, this study presents a simple, cost-effective setup for investigating aerosol distribution using a novel tracing agent. The SPED-based platform offers a foundation for a range of real-world tracing scenarios at low detection limits using qPCR as method of analysis. Future applications are the study of aerosol flow in complicated architectural settings, as well as the . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 20, 2021. ; https://doi.org/10.1101/2021.05.19.21257392 doi: medRxiv preprint dynamics of free convection resulting from heating, daytime effects and door/window arrangements. In the near future, SPED could additionally be tuned in size, made biodegradable, or be designed to mimic specific pathogens or hazardous pollutants. These are ideal prerequisites for developing a robust platform to examine places of interest regarding potential health hazards and environmental risks. 

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We thank Prof. Ulrike Lohmann and Jörg Wieder for supplying us with additional biosampling devices, and Nikita Kobert for providing the laboratory space and technical support. This work was financially supported by ETH Zürich.

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