By Ilan Arvelo1*, Ernest R. Blatchley III2, William P. Bahnfleth3, Phil Arnold1, Ashley Fry1, Maria Topete4, Ling Zhou4, William Palmer5, Patrick J. Piper6, Jianping Zhang4, W. Andrew Dexter7, Nilson Palma1, Nicholas J. Heredia 6
1. SafeTraces Inc., 2. Purdue University, 3. The Pennsylvania State University, 4. Bolb Inc., 5. AeroMed Inc., 6. Far UV Technologies Inc., 7. Aerosol Research and Engineering Laboratories Inc. * Corresponding author
Editor’s Note: Part 1 was published in Issue 1 2025 of UV Solutions.
Transmission of respiratory pathogens occurs primarily in indoor settings. Engineering interventions that are known to reduce the risk of transmission of respiratory pathogens include increases in outdoor air introduction, filtration and inactivation by technologies including ultraviolet germicidal irradiation (UVGI). However, testing and validation of these interventions are challenging, particularly in actual applications. This study introduces an aerosol tracer system utilizing DNA as the tracer molecule, aimed at quantitative characterization of the performance of indoor air cleaning systems. Two DNA tracers, one designed to be relatively UV-resistant and another designed to be relatively UV-sensitive, were employed to assess air quality changes related to filtration and ventilation systems and the unique contributions of UVGI fixtures in various built environments.
Results
UV Sensitivity on Surfaces
The results of the UV exposure experiments on foil coupons, focusing on the UV-sensitive and resistant tags, are presented in Figure 3. The investigation covered UV wavelengths of 222 nm, 254 nm and 282 nm, with doses ranging from 0 to 100 mJ/cm². The y-axis represents the total DNA copies measured using ddPCR, while the x-axis plots the dose in mJ/cm².
Table 5 summarizes results used to create a linear model of DNA-tag decay based on the dose applied on coupon surfaces. Doses of UV at the three wavelengths tested ranged from 0 to 100 mJ/cm2 for DNA-tagged tracer D1 and 0 to 50 mJ/cm2 for DNA-tagged tracer LM4 in the linear range of response for tracer decay (see Figure 3). A total of 14-18 observations (N) were made for the data sets in Table 5; the range of copies of DNA quantified from the extraction of the coupons is reported as minimum and maximum values in the table.
Distinct patterns were revealed in the UV dose response of the UV-sensitive tag (Tag-LM4, triangles on Figure 3) at each wavelength. At 222 nm, a linear dose response was observed over the entire range of doses up to 100 mJ/cm², showing a reduction in DNA copies with increasing UV dose. For 254 nm and 282 nm, the UV-sensitive tag demonstrated a linear response up to 50 mJ/cm², beyond which the reduction in signal became non-linear (gray triangles, removed for statistical analysis). For Tag-LM4 using 222 nm, one observation at 100 mJ/cm2 was noted to be highly influential with a Cooks’ distance of 1.24. It therefore was removed from the analysis before model estimations. 25,28 Separately, in the case of Tag-LM4 and the wavelength 254 nm and 282 nm, the group of values removed was because their presence showed a non-linear trend (lack of fit due to no random errors) in the residuals values when plotted against dose as the independent variable, or subsequently showed high influence in the decay rate estimations. For Tag-LM4 and the wavelengths 222 nm, 254 nm and 282 nm, the coefficient of determination (r2) was improved to 88%, 92% and 98% respectively from 80%, 87% and 59% when the points that did not fit well with the log-linear models were removed.
Notably, the UV-resistant tag exhibited substantially less reduction in signal across all UV wavelengths and doses (red dots and lines, Tag-D1), indicating its resistance to the effects of UV exposure. The foil coupons exhibit large changes with the dose exposures, and it should be noted that the coupons were hand spotted and air-dried, which can contribute to some noise in the data.
Decay rates were estimated between 0 and 50 mJ/cm2 for Tag-LM4, and between 0 and 100 mJ/cm2 for Tag-D1. The UV dose (mJ/cm2) per 1 Log reduction of MS2 (Phage) ATCC 15597 is reported in the literature around 19 to 20 mJ/cm2 (see Table 6). DNA-tag LM4 is close to the reported bacteriophage MS2 with a dose ranging between 24.0-78.1 mJ/cm2 for 1 Log10 reduction in the range of wavelengths tested. DNA-tag D1 requires at least one order of magnitude of more dose to be equivalent in response to the bacteriophage MS2, which is why it is considered more resistant to UV than DNA-tag LM4. These findings underscore the differential impact, detected by the DNA-tagged tracers, of UV exposure and UV-induced DNA damage.
The observed reduction in DNA copies on a UV dose basis highlights the efficacy of UV in diminishing the amplifiable DNA content, establishing a relationship between DNA UV-induced damage assessment and its potential application in UV-exposed environments.
UV Sensitivity of DNA-Tagged Aerosols and MS2 Bacteriophage
As expected, the bacteriophage MS2 sensitivity to UVGI showed a great range of variation, with reduction differences of orders of magnitude among devices. A summary of the range of values observed is presented in Table 7. The highest reduction in concentration for an individual sample was observed in the Lexus 2.1 device as measured in eACH (see Table 7). However, in terms of rate (see Table 8), the SUVOS-25 showed a higher sensitivity than the Lexus 2.1, with estimated values of 9.97 eACH and 9.27 eACH respectively. The AB24 and Krypton devices followed with decay rates equivalent to 5.48 eACH and 2.30 eACH respectively. When the UVGI devices were used, all estimated decay rates were on average higher than the natural decay observed in the chamber, which was estimated at 1.53 eACH (see Figure 4).
For the DNA-tagged aerosol tracers, the maximum observed reduction for an individual sample was 2.40 eACH and 4.02 eACH for Tag-D1 and Tag-LM4 respectively (see Table 9), both happening for exposure to the device SUVOS-25. There was a clear increase in the estimated eACH given the presence of the UVGI devices, and as expected, Tag-LM4 showed higher decay rate with more sensitivity than Tag-D1. It is important to note that for SUVOS-25 the results followed a log-linear trend for 25 minutes. After this period the values did not follow the same rate of reduction and were therefore omitted from the analysis.
Scaling Factor
Using the linear regression estimates of eACH for each DNA-tag and the MS2 bacteriophage, a relationship between their decay magnitudes was calculated (see Table 10). The relationship shown in the following table can be used to estimate the bacteriophage reduction when only the DNA-tag decay rate is measured in a space.
Commercial Building Application of DNA-Tracer Tag
To assess the sensitivity of the DNA-tagged tracers under different UVGI fixture conditions in a commercial building setting, experiments were conducted in a conference room. The dimensions of the room were 4.87 m by 4.57 m and floor to ceiling height 2.74 m, resulting in a volume of 61.16 m3 (2160 ft3). A square ceiling diffuser supplies roughly 42 L/s of air to the space, which is exhausted via a ceiling return grill diagonally opposite the supply. The supply air flow rate corresponds to an air change rate of approximately 2.5 ACH. The return vented directly into the ceiling, and a portion of the air likely was picked up again at some point in the system for recirculation, the exact amount is unknown.
The baseline condition, with UVGI fixtures off, provided a reference point for comparison and to determine natural loss. Subsequent experiments involved testing each UVGI fixture individually, with the tracer aerosolized in the room. The results, depicted in Figure 5 and Figure 6, illustrate the tracer’s responsiveness to the diverse UVGI devices employed in the study. The varying levels of sensitivity observed in response to individual UVGI fixtures highlight the nuanced interactions between the tracer and different UV technologies, offering insights into the performance of these fixtures in commercial building indoor environments (see Table 11 and Table 12).
Discussion
The study aimed to assess the feasibility of using DNA-tagged aerosol tracers for in situ testing in commercial building settings, allowing for the differentiation of UVGI fixture contributions into air disinfection. These tracers, exemplified by the UV-sensitive LM4, proved effective in occupied spaces and could scale with known airborne viral pathogen surrogates like MS2 bacteriophage.
Despite limitations, such as the inability to aerosolize MS2 bacteriophage in the conference room setting, due to practicality, and the weak responsiveness of MS2 bacteriophage to 222 nm UVGI fixtures, the study demonstrated strong support for scaling and implementation in field testing. Furthermore, while DNA-tagged tracers currently model DNA damage only, they showed promising responsiveness to a broad range of UV-C wavelengths, including 222 nm wavelength exposure, suggesting potential for future testing with more susceptible viral surrogates sensitive to Far UV-C wavelengths and protein damage.
The implementation of a DNA-tracer-based approach, as demonstrated in this study, offers a tool for evaluating and optimizing indoor air quality in commercial building settings by providing a way to measure liquid aerosols reduction by ventilation, and UVGI contributions in units of equivalent ventilation (as VACS) as shown in Table 13. The UV-sensitive tracers, when aerosolized in conjunction with the MS2 phage under controlled test chamber conditions, provide an understanding of the dynamics of these surrogate airborne particles under ideal test conditions: zero air changes per hour, well mixed chamber via mixing fans and allowing time for the aerosols to reach homogeneity.
This controlled testing enabled scaling of the log reductions between the bacteriophage and the DNA-tagged tracers when exposed to UVGI fixture wavelengths. This scaling permits an assessment of the contributions of UVGI fixtures to the sampling points under study and correlates the DNA-tagged tracer data to MS2 bacteriophage UV inactivation behavior. This is possible because the controlled test chamber experiments under ideal conditions demonstrated high reproducibility, and because of the log linear trend of bacteriophage MS2 and DNA-tagged tracer decay rates.
This was true for the UV off conditions as well as the UV on. Importantly, in the UV off condition, the MS2 bacteriophage and the DNA-tagged tracers’ decay rates in the test chamber are similar, which is attributable to the matching of the chemical properties of the solutions for the bacteriophage and the DNA-tagged tracer. This allowed for similar particle sizes generated from the pneumatic nebulizers.
The composition of the DNA-tagged tracers has been developed to mimic the chemical composition of human upper respiratory excretions. The same pneumatic nebulizers were used in the commercial conference room testing as the controlled test chamber and thus provide confidence in correlating the results from the DNA-tagged tracers aerosolized in the conference room with a scaling to MS2 bacteriophage, which the authors have consolidated in terms of eACH. A general limitation of in situ testing is that it is not easy to understand the air mixing behavior of the room under study. In situ scenarios typically are not well mixed due to unique geometries, occupancy, furniture, variable locations of HVAC supplies and returns, and other unique situational factors.
By aerosolizing the DNA-tagged tracers and measuring the response from a selected origin point for aerosol emission, along with an informed selection of the location of the air samplers, it is possible to get a representative view of what is happening at the location of the air samplers. In the case of the conference room, the air samplers were placed on the conference room table, a position that has a high chance of being occupied in such a scenario. This technology gives a valuable snapshot of the airflow and UVGI dynamics at select, highly pertinent points in the building, and the ability to scale those results with how MS2 bacteriophage, a viral surrogate, would be expected to respond to ventilation, filtration and any UVGI installed in that space.
Ventilation Effectiveness, Guidelines and Standards
Aligning with recent CDC guidelines, advocating for a minimum of five air changes per hour (residing in the CDC COVID-19 guidelines), this study contributes a practical and quantitative means of evaluating devices that can provide equivalent ventilation effectiveness. 32 Correlating log reductions of the DNA tracer and MS2 phage with a scaling factor, as detailed in these findings, provides insights essential for establishing healthier indoor environments. Such a scaling then could be correlated to known log reductions of airborne pathogens to UV exposure from published literature, such as Kowalski. 10 This information can aid in determining whether a space meets or exceeds the recommended air changes per hour.
These results represent the level of risk protection in terms of equivalent air changes per hour (eACH) and alternatively, equivalent clean airflow rate in CFM (VACS). This is the estimation of the same pathogen decay rate that can be provided by different mechanisms, such as ventilation and filtration, now parsed out in this case as germicidal UV-light contributions to healthier indoor environments. 33 ASHRAE Standard 241 outlines comprehensive specifications for employing filtration and air cleaning systems to efficiently and economically attain the necessary clean airflow requirements in a safe and effective manner.
Conclusions
This research introduces a practical method for measuring UV effectiveness and optimizing indoor air quality in diverse settings. The comprehensive results from controlled experiments and commercial building scenarios highlight the versatility and adaptability of the authors’ DNA-based tracer system. This system is chemically inert and considered non-hazardous, which enables UV testing in occupied spaces. Precise measurements can be made on UV-sensitive DNA-tagged tracers and their response to UVGI fixtures by employing the highly quantitative droplet digital PCR methodology. By offering a nuanced understanding of the impact of UVGI fixtures on airborne particles and scaling the results to viral surrogates like bacteriophage MS2, this method allows a detailed understanding of UV contributions to public health and safety considerations in the built environment.
The correlation between log reductions of the DNA tracer and the MS2 bacteriophage, along with the scaling factor, provides a quantitative means of evaluating a room’s unique ventilation and pathogen disinfection effectiveness. This aligns with recent CDC guidelines recommending a minimum of five air changes per hour to reduce the risk of exposure to pathogens, by providing a new technology that can be used to verify the performance of air cleaning interventions.
Our approach allows for a practical assessment of whether a space meets or exceeds the recommended air changes per hour, contributing to the establishment of healthier indoor environments. These findings have broader implications for public health, emphasizing the importance of precise monitoring and understanding of complex airflow dynamics in diverse building settings.
Funding/Disclosures
This research project was funded by SafeTraces, Inc.
Acknowledgements
The authors acknowledge Carlos E. Carpio from the Department of Agricultural and Applied Economics of Texas Tech University, Lubbock, Texas, for his guidance in the data analysis process and the interpretation of results. We would also like to thank Ulrike Hodges for project management assistance. These acknowledgements do not mean a formal agreement with all the findings and statements made in this publication.
Additional discussion and references
Foil Coupon Testing
Our investigation commenced utilizing foil coupons with dried DNA-tagged tracers to elucidate the responses of the tracers to UV exposure at different wavelengths of UV radiation. This format permitted a streamlined workflow to assess UV dose response of the tracers tested. These experiments not only describe the wavelength-dependence of the intrinsic kinetics of the photochemical reactions that are responsible for DNA detection decay but also inform the interpretation of data from testing of the effects of UVGI fixtures in actual use settings. The results of these experiments illustrated LM4’s heightened sensitivity to 282 nm radiation, relative to radiation at shorter wavelengths, a phenomenon potentially influenced by DNA absorption characteristics. Importantly, LM4 is capable of detecting UV response across all three wavelengths tested. It is important to note also that the DNA-tagged tracers are synthetic DNA and are not encapsulated in a viral envelope or proteins. This is a limitation of the DNA tracer because UV damaging effects on proteins are not simulated by the DNA-tagged tracers, and only nucleic acid damage is simulated by the tracers. Nonetheless, the ability to accurately quantify the effects of UV exposure through a safe, inert aerosol that can be used in occupied spaces provides insights into commercial building applications, heretofore, not possible with other technologies like radiometric readings and inert gas release studies. Future work in developing tracers may include estimating protein damage to the aerosol tracers. Another important finding with the UV-sensitive tracer, LM4, is that there is a point in the UV dose response behavior (at doses above 50 mJ/cm2), where there is a non-linearity in LM4 response, not seen with tracer D1. This nonlinearity appears as a “hockey stick” shape, with a leveling out of the decay response at these higher doses (Figure 3). One possible explanation for this behavior is that the UV photodamage tracer LM4 has reached an equilibrium in the forward and reverse reaction of creation and undoing of photodamage, such as thymine dimer formation30. UV-induced photoproducts do not necessarily break the DNA backbone, and thus it is possible that at this energy regime, an equilibrium condition could develop. Only the linear portion of the dose response was considered in the subsequent analysis of the decay rates for LM4.
Aerosolized Testing in a 30 Cubic Meter Test Chamber
Transitioning to aerosolized testing in a 30 cubic meter controlled test chamber at 0 ACH conditions, the effectiveness of the UV-sensitive DNA-tagged tracer LM4 in responding to UV doses at different UVGI fixture wavelengths from different devices was assessed. The UV-resistant DNA-tagged tracer D1, exhibits a smaller and less accurate change of response for UV on/off compared to LM4, as expected for a more UV-resistant DNA tracer. This difference underscores the benefit of LM4 being sensitive to UV exposure.
Despite challenges in measuring the Krypton 222 nm UVGI device’s reduction response with aerosolized bacteriophage MS2 in the test chamber, DNA-tagged tracer LM4 aerosolized in the same test chamber is able to detect the effect of 222 nm exposure. In this example of wavelength (222 nm) and DNA tracer Tag-LM4, it is interesting that the tracer outperforms the bacteriophage viral surrogate in sensitivity to UV doses in the Far UVGI fixture spectrum. Simultaneously releasing MS2 phage and aerosolized DNA-tracers Tag-D1 and Tag-LM4 in these tests, Figure 4 – Figure 6 underscore Tag-LM4’s superior sensitivity to the UVGI fixture at 222 nm, in comparison to the MS2 bacteriophage’s response, serving as a human viral pathogenic surrogate, in the Far UVGI fixture spectrum (222 nm). These results imply that bacteriophage MS2 may not be a good surrogate for Far UV germicidal fixtures. Bacteriophage MS2 was chosen in this study for the reason that it best fit the guidelines of standards such as the recently published ASHRAE Standard 241-2023. 26 As there was no large change in bacteriophage MS2 with the 222 nm device, the scaling factor is probably not entirely reliable for this regime of UVGI fixture, and in future studies, a more sensitive, viral surrogate might be considered for demonstrating the efficacy of Far UV technology with our UV-sensitive DNA-tagged tracers. Ideal challenge agents will demonstrate UV dose-response behavior that is slightly conservative, as compared to target pathogens31.
It is important to note that the LED-based SUVOS-25 produced a powerful effect in the test chamber in terms of log reduction over time, as seen in Figure 6 and Table 10 and 11 with tag LM-4, which was log-linear only within 25 minutes. After 25 minutes, the values did not follow the same reduction rate. There is a “hockey-stick” effect in the data after 25 minutes for SUVOS-25, similar to the “hockey-stick” non-linearity seen with Tag-LM4 at higher wavelengths and UV doses on foil coupon exposure (Figure 3). This effect could imply and contribute to inaccuracy when scaling between surrogate bacteriophage and DNA-tagged tracers of very high powered UVGI devices. This is why we chose to scale only in the linear response range, the first 25 minutes, for Tag-LM4 with this device in the test chamber.
Comparison of Scaled MS2 Results in Test Chamber
A scaling factor, derived by converting logarithmic reductions to equivalent air changes per hour, facilitated the assessment of UVGI fixture efficacy. The scaling factor, calculated on a per-fixture basis, represents the ratio of the delta in eACH between UV off and on conditions for both the bacteriophage and the tracer. This approach provides an understanding of the impact of UVGI fixtures on the DNA tracers and a facile way to scale to a pathogen surrogate, in this case MS2 bacteriophage. Figure 10 provides a quick view on how the scaling factor between MS2 and DNA-Tag is kept constant if it is calculated as reduction rates on eACH unit, which is a logarithmic scale. This implies that, if the eACH for the DNA-Tag is known, the eACH for MS2 can be calculated. It is important to highlight that this is only an accurate estimation if the decay rate of the DNA-Tag and MS2 are log-linear during the whole period of measurement, therefore, the data collected during experimentation should always be inspected for this assumption.
Figure 10. Scaling factor for MS2 and DNA-Tag explanation.
Table 12 outlines the scaling of the responses to the four UVGI fixtures and their UV exposures in the controlled test chamber of the DNA-tagged tracers scaled to the same UV exposures of bacteriophage MS2. Table 10 – Table 11 and Figure 5 – Figure 6 illustrate the response of both DNA-tagged tracers to exposure to UV light from the four test fixtures tested in the controlled aerosol test chamber. For Tag-D1, its resistance to UV-C decay significantly affects the accuracy of the scaling relationship, mathematically as the delta of the DNA-tagged tracer’s response under UV on vs UV off conditions approaches zero, the scaling factor goes to infinity, emphasizing the unsuitability of Tag-D1 as a surrogate due to its greater variability and lower sensitivity, compared with DNA-tagged tracer Tag-LM4. While comparing the scaled MS2 results for Tag-LM4 and Tag-D1 in Table 15, the proximity of their outcomes shows that Tag-D1 results are potentially useful, but in most of the cases, given the high scale relationship they could end in higher equivalent reduction rates. For the previous reason our study supports the idea that Tag-LM4 provides a more accurate representation, in addition to its wider range of measurement of change given its sensitivity.
Commercial Building Conference Room Testing
Expanding our investigation beyond controlled environments, experiments in a commercial building conference room demonstrate the adaptability of the DNA-tracer system. In the context of our findings, the differences between AB24 (a UVGI troffer fixture) in the commercial building application and controlled test chamber conditions were unexpected, with AB24 performing much better than SUVOS-25 in the commercial building setting. This highlights the importance of validating results in practical settings. The unique airflow geometries of the conference room, distinct from the continuous fans in the test chamber, likely contributed to these variations, as well as a troffer having its own fan at approximately 125 CFM (212.4 m3/h). Another possibility is that the SUVOS-25 device might be better scaled with Tag-D1, as it was linear over the full hour in the test chamber with Tag-D1 vs Tag-LM4 (Figure 5 and Figure 6). If this is the case, then the SUVOS-25 eACH is probably more accurately 6.22 vs. 4.68 for Tag-D1 and Tag-LM4 respectively, scaling with bacteriophage MS2 (Table 15). The test chamber experimentation was designed to provide the most accurate effect of the tracers to UV exposure under controlled conditions, with exceptional mixing and 0 ACH by sealing the chamber. The conference room, by contrast, has its own unique geometry from the room design, HVAC supply and return positions, tables, chairs, and equipment. The conference room air sampler positions were chosen to be between the supply and return and situated on the conference room table, where people would most likely be seated at any given time while it is in use.
The same four UVGI fixtures used in the controlled chamber testing, were then applied to the conference room to assess the DNA-tagged tracers’ responses to the fixtures’ UV output over one hour. Table 15 yielded the final conversion of the conference room results scaled to MS2 bacteriophage in this scenario. The baseline eACH for the room, as determined by the no UV condition with Tag-D1 and Tag-LM4, was 2.27 eACH and 2.54 eACH respectively. All of the UVGI devices tested contributed to an increased eACH and when scaled to MS2 bacteriophage reduction, the total eACH of the room ranged from 2.83 to 7.87 eACH based on the MS2 scaling with Tag-LM4 tracer (Table 15). These were significant improvements to the conference room’s overall eACH, when translating to percentages, three devices Lexus, SUVOS-25, and AB24 increased the estimated MS2 decay rate to more than 99% reduction per hour (equivalent of 4.6 eACH total).
The air cleaning system equivalent clean airflow rate in CFM (VACS) was also calculated in Table 15, using equation 3 in the methods. This calculates, according to ASHRAE Standard 241, the amount of equivalent clean airflow rate, at the air sampler positions, that air cleaning systems, in this case UVGI fixtures, are producing. The amount of equivalent clean air delivered to the air sampler locations on the conference room table ranged from 10.44 to 191.88 CFM after being scaled to bacteriophage MS2 (with Tag-LM4). In the ASHRAE 241 Standard, the recommended Equivalent Clean Air delivered to the space per person (ECAi) is 30 CFM for an office type space. Using this metric, the fixtures are adding enough CFM of equivalent clean air, at the conference room table, for the equivalent of an additional, approximate 0.4 to 6.4 people to be at the table. It is important to note that this is after scaling the DNA tracer response to bacteriophage MS2. The Krypton 222 nm device is likely underrepresented here when scaling to bacteriophage MS2 in terms of its effectiveness (0.4 additional people at the table), since Far UV-C wavelengths likely should be scaled to more sensitive surrogate viruses. Nonetheless, the non-Far UV-C fixtures, when scaled to bacteriophage MS2, yield 2.6 (SUVOS-25), 4.6 (AB24) and 6.4 (Lexus 2.1) additional people to be at the conference room table based on the VACS calculations for DNA Tag-LM4 scaled to bacteriophage MS2. This is a significant improvement to the room occupancy capability.
