Microorganisms: Impacts of Surface Characteristics and Dew Point On The Blue-Light (BL) Inactivation of Viruses

microorganisms
Article
Impacts of Surface Characteristics and Dew Point
on the Blue-Light (BL405) Inactivation of Viruses
Castine Bernardy and James Malley *
Department of Civil and Environmental Engineering, College of Engineering & Physical Sciences,
University of New Hampshire, Durham, NH 03824, USA; castine.bernardy@unh.edu
* Correspondence: jim.malley@unh.edu; Tel.: +1-(603)-862-1449
Abstract: The increased prevalence of multidrug-resistant organisms (MDROs), healthcare associated

infections (HAIs), and the recent COVID-19 pandemic has caused the photoinactivation industry
to explore alternative wavelengths. Blue light (BL405 ) has gained significant interest as it is much
less harmful to the skin and eyes than traditional germicidal wavelengths; therefore, in theory,
it can be used continuously with human exposure. At present, the viricidal effects of BL405 are
largely unknown as the literature predominately addresses bacterial disinfection performed with
this wavelength. This work provides novel findings to the industry, reporting on the virucidal effects
of BL405 on surfaces. This research utilizes three surfaces: ceramic, PTFE, and stainless steel. The
efficacy of BL405 inactivation varied by surface type, which was due to surface characteristics, such
as the contact angle, porosity, zeta potential, and reflectivity. Additionally, the effect of the dew
point on BL405 inactivation efficacy was determined. This research is the first to study the effects
of the dew point on the virucidal effectiveness of BL405 surface inactivation. The effects of the dew
point were significant for all surfaces and the control experiments. The high-dew-point conditions
(18 ◦ C) yielded higher levels of BL405 inactivation and viral degradation for the experiments and
controls, respectively.
Keywords: BL405 ; blue light; surface; dew point; inactivation; MS2; dose–response; ceramic; PTFE;
stainless steel
Citation: Bernardy, C.; Malley, J.

Impacts of Surface Characteristics
and Dew Point on the Blue-Light 1. Introduction
(BL405 ) Inactivation of Viruses.
The COVID-19 pandemic highlighted the need for more robust surface disinfection
Microorganisms 2023, 11, 2638.
techniques. The germicidal properties of UV254 are well understood [1–3], and new devel-
https://doi.org/10.3390/
opments in the photonics industry suggest that other wavelengths may have germicidal
microorganisms11112638
effects [4]. Visible light, specifically blue light at 405 nm, is a promising new disinfection
Academic Editor: Hideharu Shintani technology [5,6].
The healthcare industry has taken an interest in the germicidal properties of blue light,
Received: 21 September 2023
Revised: 23 October 2023
as hospitals continue to struggle with millions of cases of hospital-associated infections
Accepted: 24 October 2023
(HAIs) [7]. These technologies can provide another barrier of protection for hospital
Published: 26 October 2023 patients and may also assist in addressing the rising threat of multidrug-resistant organisms
(MDROs) [8]. Additionally, exposure to 405 nm is considered safe for human contact [5];
therefore, blue-light technologies have the potential to provide a continuous low level
of disinfection.
Copyright: © 2023 by the authors. Unlike UV wavelengths, which are known to cause erythema and photokeratitis [2,9,10],
Licensee MDPI, Basel, Switzerland. this visible-light wavelength is considered safe for human skin and eye exposure [11].
This article is an open access article The 405 nm range does not share the harmful effects of BL440 and BL480, which are known
distributed under the terms and
to cause photo retinitis and the disruption of the circadian rhythm, respectively [5].
conditions of the Creative Commons
The relative safety of blue light was compared to the traditional wavelength, UV254 ,
Attribution (CC BY) license (https://
by the American Conference of Governmental Industrial Hygienists [12]. Their 2021
creativecommons.org/licenses/by/
publication released threshold limit values (TLVs) at wavelengths between 180–400 nm.
4.0/).
Microorganisms 2023, 11, 2638. https://doi.org/10.3390/microorganisms11112638 https://www.mdpi.com/journal/microorganisms

Microorganisms 2023, 11, 2638 2 of 23
The TLV for 405 nm was extrapolated from the data and compared to 254 nm. The TLVs
set for 254 and 405 nm were 6.0 mJ/cm2 and 1.2 × 105 mJ/cm2 , respectively. Exposures
up to these TLVs are considered safe for human eyes and skin, without experiencing
complications due to erythema and photokeratitis. The TLV value for 405 nm is five
orders of magnitude higher than the TLV for 254 nm, providing sufficient evidence that
405 nm wavelengths are safer for human skin and eye exposure than 254 nm [12]. BL405
is particularly advantageous as this wavelength can provide a continuous, low level of
disinfection, as exposure to people at doses lower than the TLV in public places is not a
health concern.
Applications of BL405 disinfection in the literature [5,13–15] predominantly focus on
bacterial and fungal studies. There is a consensus that the BL405 inactivation of bacteria
and fungi is caused by interactions with porphyrin molecules within the fungal or bacterial
cells. The blue-light wavelength is absorbed by the porphyrin molecules, which become
photo excited. The photo-excited porphyrins then react with other cellular components or
oxygen, leading to the creation of reactive oxygen species (ROS). The ROS leads directly
to cell death via oxidative damage to the cell membrane (proteins and lipids) [5,13–15].
ROS-induced cell damage occurs via two pathways: type 1, the creation of superoxide ions
via electron transfer, or type 2, the production of singlet oxygen. Both pathways cause
sufficient damage to the cellular components and ultimately lead to cell death [16].
Studies on the effectiveness of 405 nm technologies for viral inactivation are limited.
It was previously thought that, due to the lack of porphyrins in viruses, this technology
would not be effective for viruses without the use of an added photosensitizer. The industry
calls for more research on the viricidal effects of blue light to determine its effectiveness
and potential applications [13,14].
Previous work [17–20] has revealed that surface type and characteristics greatly influ-
ence the efficacy of photoinactivation of the virus. Factors, such as shadowing and porosity,
were studied by Moore et al. [18] and Tomas et al. [20], respectively. Bernardy et al. [19]
studied UV254 inactivation efficacy on five surface types: aluminum, ceramic, Formica lam-
inate, PTFE, and stainless steel. Their work investigated the effects of viral recovery from
these surfaces as a function of the contact angle, surface roughness, porosity, reflectivity,
and zeta potential. These results reinforce that surface type/characteristics greatly impact
the efficacy of line-of-sight inactivation technologies, such as UV254 or BL405 .
The effects of ambient environmental conditions on the photoinactivation of viruses
have been studied in the literature [2,21,22] for UV254 , which may provide some insights
into the effects on BL405 . Kowalski et al. [2] tabulated UV254 viral inactivation data at
varying relative humidities. These findings revealed that the effect of relative humidity on
viral species was largely species dependent and found that general conclusions regarding
the effect of relative humidity on the UV254 inactivation of viruses could not be assessed.
However, the research exploring the effect of temperature, relative humidity, or dew
point have not been conducted previously with BL405 technologies. Therefore, in this paper,
the data are developed to elucidate the effects of high and low-dew-point conditions, which
consider the interaction of temperature and relative humidity, for the BL405 inactivation of
the virus.
The objective of this study is to address the many research gaps in the literature
on BL405 inactivation. This work enhances the body of knowledge of BL405 viricidal
effects. The effects of surface characteristics are explored using ceramic, polytetrafluo-
roethylene (PTFE), and stainless-steel disks. The inactivation of MS2 bacteriophage, a
+ssRNA (positive-sense single-stranded ribonucleic acid) virus commonly studied in pho-
toinactivation research, is tested on these surfaces. The experiments were repeated at high
(18 ◦ C) and low (−4 ◦ C) dew points to also determine if these environmental conditions had
an impact on the BL405 inactivation efficacy. This novel work explores the viricidal impacts
of the surface characteristics and dew point for BL405 inactivation applications, which
have not been studied in the literature. This research provides significant contributions to
the BL405 inactivation industry, which will ultimately lead to the greater protection of the
public’s health.
2. Materials and Methods

2.1. Surfaces
The surfaces evaluated for this work included ceramic, PTFE, and stainless steel.
Table 1 below displays the details of each surface type utilized in the experiments.
Table 1. Surfaces utilized for experimentation.
Surface Type Manufacturer/Description

Ceramic Ceramic Solutions (Conroe, TX, USA): 99.8% Alumina Discs
Thorlabs (Newton, NJ, USA): Fabricated from sintered, crystalline, fused,
PTFE
and skived virgin PTFE
Stainless steel MetalsDepot (Winchester, KY, USA): 304 Stainless with Mirror Finish (#8)
Table 1 displays the specifications and manufacturer information for each of the
materials utilized for the experimentation.
The surface porosity, zeta potential, and contact angle of these surfaces were quantified.
The exact methods used to quantify these characteristics were described in a previous
publication (Bernardy et al.) [19].
2.2. Experimental Conditions

This research utilized a controlled environmental chamber to conduct the experi-
ments. Table 2 below displays the temperature and relative humidity conditions in the
controlled chamber.
Table 2. Temperature and relative humidity conditions in controlled chamber.
Dew Point (◦ C) Temperature (◦ C) Relative Humidity (%)

−4 12 32
18 28 55
Table 2 displays the dew-point conditions maintained in the experimental chamber.

These dew-point conditions were selected as they were representative of the average
annual high and low dew points in the United States reported by the National Oceanic and
Atmospheric Administration [23]. The relative humidity and temperature were carefully
monitored during the experimentation using EXTECH Instruments, Humidity Alert II. The
environmental chamber controlled the temperature, while humidifiers and dehumidifiers
controlled the relative humidity.
2.3. BL405 Irradiance and Dose

To determine the range of BL405 doses needed for this work, a literature review
was conducted. The results of the review were tabulated and are shown in the
Supplementary Materials (Figure S1). The characteristics of each virus studied in the
literature [14,24–26] were compared to the characteristics of the MS2 bacteriophage surro-
gate used in this study and are shown in Table S1. The doses selected for experimentation
were 0, 50, 100, and 200 J/cm2 , based on the data from viruses that were structurally similar
to MS2.
These experiments utilized a collimated LED manufactured by Thorlabs, model
M405L4C1—405 nm. To accurately quantify the BL405 dose, the average irradiance received
by the surfaces was determined using similar methods to those of Linden and Bolton [27].
A 10 by 10 cm grid was created and the irradiance values were recorded with a calibrated
NIST traceable IL1700 radiometer every 2 cm in the X and Y directions. The radiometer
was calibrated in compliance with National Institute of Standards and Technology (NIST)
Microorganisms 2023, 11, x FOR PEER REVIEW 4 of 23
a calibrated NIST traceable IL1700 radiometer every 2 cm in the X and Y directions. The
radiometer was calibrated in compliance with National Institute of Standards and
practices recommended in the NIST Handbook 150-2E [28]. Figure 1 displays the spread
Technology (NIST) practices recommended in the NIST Handbook 150-2E [28]. Figure 1
of irradiance emitted by the Thorlabs collimated beam unit and was used to determine a
displays the spread of irradiance emitted by the Thorlabs collimated beam unit and was
weighted average of the irradiance that the surfaces received.
used to determine a weighted average of the irradiance that the surfaces received.
BL405 Irradiance Map

10 cm
8 cm
6 cm
Y Coordinates
4 cm
2 cm
0 cm
0 cm 2 cm 4 cm 6 cm 8 cm 10 cm
X Coordinates
0-5 5-10 10-15 15-20
All irradiance values are reported in mW/cm2
Figure 1. BL405 irradiance map emitted by the Thorlabs collimated beam. The collimated beam was
Figure 1. BL405 irradiance map emitted by the Thorlabs collimated beam. The collimated beam was
positioned 34 cm above the radiometer for these measurements. The weighted average irradiance
positioned 34 cm
at this height wasabove the radiometer
13 mW/cm for black
2 inside of the these circle,
measurements. The weighted
which represents average
the outline of theirradiance
surfaces. at
this height was 13 mW/cm2 inside of the black circle, which represents the outline of the surfaces.
The average irradiance received by the surfaces (13 mW/cm2) 2was determined by the
The average irradiance received by the surfaces (13 mW/cm ) was determined by the
black outline displayed in the irradiance map shown in Figure 1. The collimated beam
black outline
was positioneddisplayed in the
34 cm above theirradiance
radiometer,map shown
as this heightinproduced
Figure 1.the
The collimated
most beam
even spread
was positioned
of irradiance. 34 cm above the radiometer, as this height produced the most even spread
of irradiance.
To determine the BL405 doses received during the experimentation, the following
To determine
equation the BL405 doses received during the experimentation, the following
was utilized:
equation was utilized:
BL405 Dose (mJ/cm2) = BL405 Irradiance (mW/cm2) × Surface Exposure Time (Seconds)
BL405The
Dose (mJ/cm2 ) = BL
corresponding 405 Irradiance
surface exposure(mW/cm
2
times were) × Surface Exposure
determined Time
by dividing the(Seconds)
target
BL405 dose by the average irradiance value and are shown in Table 3.
The corresponding surface exposure times were determined by dividing the target
BL
Tabledose
405 bydoses
3. BL405 the average irradiance
and exposure times. value and are shown in Table 3.
Table 3. BLDoses (J/cm

405 doses and2)exposure times.
0 50 100 200
Exposure time (hour, min) 0 1 h 4 min 2 h 8 min 4 h 16 min
Doses (J/cm2 ) 0 50 100 200
Exposure time (hour, min) 0 1 h 4 min 2 h 8 min 4 h 16 min
Table 3 displays the blue-light doses and corresponding exposure times used for
the experimentation.
The BL405 doses were delivered using the times shown in Table 3 and corresponded
to 0, 50, 100, and 200 J/cm2 . These were referred to as the ‘BL405 Light On’ data points.
Additionally, due to the long times required to achieve adequate doses of BL405 , the effect
of viral degradation over time was quantified for each surface using control runs that were
referred to as ‘405 Light Off’ data points.
2.4. Rate Constants (k Values)

Two rate constants (k) were determined for each surface: non-viricidal and viri-
cidal. These constants represented the viricidal effects of BL405 and viral degradation
(non-viricidal) due to the long exposure times of these experiments. The k values were
calculated from polynomial equations, as they helped to illustrate the different regions of
the dose–response curves (shoulder, exponential, and tailing), which was consistent with
the industry’s guidance [29]. The EPA does not advise the use of extrapolation from these
equations outside of the tested dose range. Mattle et al. [30] studied the effects of tailing
during the UV254 inactivation of MS2 bacteriophage. The authors found that after exponen-
tial decay, the inactivation rate decreased due to clumping (aggregation) and recombination.
Bernardy et al. [19] suggested that polynomial fits were the best to incorporate the effects of
the surface characteristics on the dose–response curve. Furthermore, readers are cautioned
against extrapolating outside of the tested data range.
2.4.1. Non-Viricidal Rate Constants

The non-viricidal rate constants were calculated to determine the viral degradation
over time at the high- and low-dew-point conditions. These values represent the exper-
iments conducted without turning on the BL405 collimated beam and are shown in the
dose response curved as ‘405 Light Off’ data points. The slope of the line at each interval
was used to find the k values. These values were averaged to find the rate of change of
the polynomial.
2.4.2. Viricidal Rate Constants

The viricidal rate constant was calculated using similar methods as described for the
time exposure-rate constants. The viricidal rate constant represents the true viricidal effect
of BL405 on MS2 by surface type and dew point. This rate constant (k value) excluded the
losses due to viral degradation over time to achieve an understanding of the effects solely
due to BL405 inactivation.
The equation to determine the viricidal k value is shown below:
Viricidal k (1/h) = Total Loss k (1/h) − Viral Degradation k (1/h)
The viricidal k value was converted to units of 1/s and then divided by the BL405
irradiance value (W/cm2 ) to produce a k value with units that were consistent with the
literature, cm2 /J.
2.5. MS2 Bacteriophage

The surrogate used for experimentation was the MS2 bacteriophage. For detailed
information regarding the contract laboratory used for the analysis of the MS2 samples,
please refer to a previous publication [19].
2.6. Experimental Procedure

The surfaces were inoculated with the MS2 bacteriophage using a cotton swab. The
swab was submerged in a stock MS2 solution and then applied to the respective surface.
A 5 cm-diameter stencil was placed over the surfaces prior to swabbing to keep the inocula-
tion area consistent between the surfaces. This method provided the most reproducible
results of all the inoculation methods tested. Previous methods that were tested included
using a spray bottle and nebulizer.
After receiving their respective BL405 doses, the surfaces were rinsed with 50 mL of
sterile phosphate-buffered solution (PBS) using a pointed spray nozzle. A clamp was used
to hold the surface above a beaker, which collected the PBS rinse water. Each experiment
(surface type and dew-point condition) was run in triplicate. The samples of PBS rinse
water were sent to a contracted lab (GAPLAB, London, ON, Canada) for a plaque assay
analysis to determine the remaining MS2 bacteriophage infectivity for each experimental
condition. The contracted lab split the samples into duplicates for QA/QC purposes;
therefore, each blue-light dose had a total of six data points.
All materials used in the laboratory were sterilized to prevent contamination. The
stainless steel, glassware, and phosphate-buffered solution were autoclaved prior to exper-
imentation. The ceramic, PTFE, plastic stencils, and clamps were disinfected in a bleach
solution. These materials received a 20 min contact time in the bleach solution, after which
they were thoroughly rinsed with tap water, followed by RO water. These items were then
set out to dry before the subsequent experiment.
Negative-control experiments were conducted in our laboratory on bleached and
autoclaved materials. The MS2 bacteriophage recovery was below the detection limit for
all the materials tested. These data can be found in the compiled data DOI listed at the
bottom of this manuscript. In addition, MS2 concentration checks were performed every
experimental day to ensure the health of the viral stock prior to analysis.
2.7. Statistical Analysis

Statistical analyses were conducted to determine if the surface type and dew point
significantly affected BL405 inactivation and viral degradation over time. These tests
were conducted using the Fit Model platform in JMP, version 16.1. The effects with
p-values less than 0.05 were considered as significant. For more information, refer to
the Supplementary Materials Section.
3. Results
Figures 2–6 display the BL405 inactivation data for the MS2 bacteriophage on ceramic,
PTFE, and stainless-steel surfaces. The results show the effect of blue-light inactivation
at high (18 ◦ C) and low (−4 ◦ C) dew points on these three surfaces. These figures are
displayed as a function of time. Two trendlines exist, representing the log loss of MS2 with
the BL405 on and off.
The results of the ceramic high- and low-dew-point experiments are shown below.
3.1. Ceramic
3.1.1. High Dew Point (18 ◦ C)
Figure 2 displays the log MS2 bacteriophage loss as a function of time (hours). Two
trendlines are shown, representing the loss of MS2 with (blue circle) and without (black
triangle) exposure to BL405 irradiance. The BL405 doses corresponding to the time (hours)
shown for the ‘405 Light On’ data are 0, 50, 100, and 200 J/cm2 , respectively. Three trials
were conducted for each BL405 dose; the figure displays the average of these trials and the
standard deviation as error bars.
Additionally, the figure displays the MS2 degradation as a function of time without
exposure to BL405 irradiance. These data indicate the significant effect of time, with an
increase in the log loss as the elapsed time increased.
Both trendlines were adjusted to account for the retention of MS2 on the ceramic disks.
The area between these two trendlines (shaded blue) represents the viricidal effect of BL405 .
This area excludes any losses due to viral degradation on the surface.
Microorganisms2023,
Microorganisms 11,x2638
2023,11, FOR PEER REVIEW 7 7ofof23
23
Figure
Figure2.2.Log
Logloss
lossofofMS2
MS2bacteriophage
bacteriophageas asaafunction
functionof
oftime
timeon
onceramic
ceramicdisks
disksin inaahigh-dew-point
high-dew-point
environment.
environment.Time Timeininhours
hoursis shown on on
is shown thethe
x axis andand
x axis log loss of MS2
log loss is shown
of MS2 on theony axis.
is shown the yTwo
axis.
trendlines are displayed,
Two trendlines indicating
are displayed, the log
indicating the loss of MS2
log loss as as
of MS2 a function
a functionofoftime
timewith
withand
andwithout
without
exposure
exposureto toBL
BL405 irradiance. The shaded region between the two trendlines represents the viricidal
405 irradiance. The shaded region between the two trendlines represents the viricidal
effect of BL405 on ceramic disks in high-dew-point environments. The microbial detection limit for
effect of BL405 on ceramic disks in high-dew-point environments. The microbial detection limit for
these experiments is shown as a line at the top of the figure.
3.1.2.Additionally,
Low Dew Point the figure
(−4 ◦ C)displays the MS2 degradation as a function of time without
exposure to BL405 irradiance. These data indicate the significant effect of time, with an
Figure 3 displays the log MS2 bacteriophage loss as function of time (hours). Two
increase
trendlines in the
are log loss as
shown, the elapsedthe
representing time increased.
loss of MS2 with (blue circle) and without (black
Both trendlines were adjusted to account
triangle) exposure to BL405 irradiance in a low-dew-pointfor the retention of MS2 The
environment. on the ceramic
BL405 doses
disks. The area between these two trendlines (shaded blue) represents the
corresponding to the time (hours) shown for the ‘405 Light On’ data are 0, 50, 100, viricidal effect
and
of BLJ/cm
200 405. This area excludes
2 , respectively. anytrials
Three losses dueconducted
were to viral degradation
for each BL on dose;
the surface.
the figure displays
405
the average of these trials and the standard deviation as error bars. When compared to
3.1.2. Low Dew Pointdata,
the high-dew-point (−4 °C)
the effect of BL405 was reduced in the low-dew-point experimen-
Figure 3
tal environment.displays the log MS2 bacteriophage loss as function of time (hours). Two
trendlines are shown,
Additionally, therepresenting
black trendlinethe loss of MS2
displays thewith (blueofcircle)
log loss MS2 and withoutof
as function (black
time,
triangle)
without exposure to BL405 405irradiance
irradiance.inThe
a low-dew-point
effect of viral environment.
degradation over The BL
time doses
405 on the
corresponding
ceramic disks to theless
was timein(hours) shown for theenvironment
the low-dew-point ‘405 Light On’compared
data are 0,to50,the
100, and 200
high-dew-
J/cm
point2, respectively.
environment.Three trials were conducted for each BL405 dose; the figure displays the
average oftrendlines
Both were
these trials andadjusted to account
the standard for theasretention
deviation of MS2
error bars. When on compared
the ceramictodisks.
the
The area between
high-dew-point these
data, two
the trendlines
effect of BL405(shaded blue) represents
was reduced the viricidal effect
in the low-dew-point of BL405 .
experimental
This area excludes any losses due viral degradation over time on the surface. To determine
environment.
the viricidal effects of BL405 without the confounding effects of viral degradation over time,
the k value for the ‘405 Light Off’ was subtracted from the ‘405 Light On’ k value. These
data are shown in Table 4.
2023, 11,
Microorganisms 2023, 11, 2638
x FOR PEER REVIEW 88of
of 23
Figure 3.
Figure 3. Log
Log loss
lossofofthe
theMS2
MS2bacteriophage
bacteriophage as as a function
a function of time
of time on ceramic
on ceramic disksdisks in a low-dew-
in a low-dew-point
point environment. Time in hours is shown on the x axis and the log loss of MS2
environment. Time in hours is shown on the x axis and the log loss of MS2 is shown on the is shown onythe y
axis.
axis. Two trendlines are displayed, indicating the log loss of MS2 as a function of time with and
Two trendlines are displayed, indicating the log loss of MS2 as a function of time with and without
without exposure to BL405 irradiance. The shaded region between the two trendlines represents the
exposure to BL405 irradiance. The shaded region between the two trendlines represents the viricidal
viricidal effect of BL405 on ceramic disks in low-dew-point environments. The microbial detection
effect of BL
limit for on ceramic disks
405 experiments
these in low-dew-point
is shown as a line at theenvironments. The microbial detection limit for
top of the figure.
Additionally, the black trendline displays the log loss of MS2 as function of time,
The viricidal k value for BL405 in high-dew-point environments was slightly higher
without exposure to BL405 irradiance. The effect of viral degradation over time on the
than the viricidal k value in low-dew-point environments. The high-dew-point experiments
ceramic disks was less in the low-dew-point environment compared to the high-dew-
experienced a higher log loss of MS2 at each BL405 dose, when compared to the low-dew-
point environment.
point experiments. The viral loss over time was much higher at the high dew point;
Both trendlines were adjusted to account for the retention of MS2 on the ceramic
therefore, the true viricidal effects of BL405 in each environmental condition were similar.
disks. The area between these two trendlines (shaded blue) represents the viricidal effect
of BL
3.2. 405. This area excludes any losses due viral degradation over time on the surface. To
PTFE
determine the viricidal effects of BL405 without the confounding effects of viral
The results of the high- and low-dew-point experiments on PTFE disks are shown below.
degradation over time, the k value for the ‘405 Light Off’ was subtracted from the ‘405
Light High
3.2.1. On’ kDewvalue. These data are shown in Table 4.
Point
The viricidal k value for BL405 in high-dew-point environments was slightly higher
than the are
trendlines viricidal
shown,k representing
value in low-dew-point
the loss of MS2 environments. The
with (blue circle) andhigh-dew-point
without (black
experiments experienced a higher log loss of MS2 at each BL 405 dose, when compared to
triangle) exposure to BL405 irradiance. The irradiance corresponding to the ‘405 Light On’
the low-dew-point
trendline experiments.
was 13 mW/cm The viral
2 ; therefore, it isloss over time was
representative muchdoses
of BL higherofat0,the
50,high
100, dew
and
405
point; therefore,
2 the true viricidal effects of BL405 in each environmental condition were
200 J/cm . Three trials were conducted for each BL405 dose; the figure displays the average
similar.
of these trials and the standard deviation as error bars. The highest level of inactivation
(4.96 log) was achieved at a BL405 dose of 200 J/cm2 .
3.2. PTFE
Additionally, the figure displays the MS2 loss as a function of time without exposure to
BL405The results of
irradiance. Thethe
loghigh- and low-dew-point
MS2 degradation experiments
varied over on PTFE
time, plateauing disks
after are shown
approximately
2below.
h. The peak degradation of MS2 was 1.5 log for these time exposure experiments.
trendlines are shown, representing the loss of MS2 with (blue circle) and without (black
triangle) exposure to BL405 irradiance. The irradiance corresponding to the ‘405 Light On’
trendline was 13 mW/cm2; therefore, it is representative of BL405 doses of 0, 50, 100, and
200 J/cm2. Three trials were conducted for each BL405 dose; the figure displays the average
of these trials and the standard deviation as error bars. The highest level of inactivation
(4.96 log) was achieved at a BL405 dose of 200 J/cm .
2
Figure 4.
Figure 4. Log
Log loss
loss of
of the
the MS2
MS2 bacteriophage
bacteriophage as
as aa function
function of
of time
time onon PTFE
PTFE disks
disks in
in aa high-dew-point
high-dew-point
environment. Time in hours is shown on the x axis and the log loss of MS2 is shown on
environment. Time in hours is shown on the x axis and the log loss of MS2 is shown on the
the yy axis.
axis.
Two trendlines are displayed, indicating the log loss of MS2 as a function of time with
Two trendlines are displayed, indicating the log loss of MS2 as a function of time with and without and without
effect of BL405 on PTFE disks in high-dew-point environments. The microbial detection limit for these
effect of BL405 on PTFE disks in high-dew-point environments. The microbial detection limit for these
experiments is shown as a line at the top of the figure.
Additionally, the figure displays the MS2 loss as a function of time without exposure
Both trendlines were adjusted to account for the retention of MS2 on the PTFE disks.
to BL405 irradiance. The log MS2 degradation varied over time, plateauing after
approximately 2 h. The peak degradation of MS2 was 1.5 log for these time exposure
This area excludes any losses due to viral degradation over time on the surface.
experiments.
3.2.2.Both
Lowtrendlines
Dew Pointwere ◦ C)
(−4 adjusted to account for the retention of MS2 on the PTFE disks.
The area between these two trendlines
The loss of the MS2 bacteriophage (shaded
over blue)
time represents
(hours) onthe viricidal
PTFE diskseffect of BL405
is shown in.
This area excludes any losses due to viral degradation over time on the surface.
Figure 5. This figure displays two trendlines, representing the loss of MS2 with (blue circle)
and without (black triangle) exposure to BL405 irradiance. The irradiance corresponding to
3.2.2.
the Low
‘405 Dew
Light Point
On’ (−4 °C)was 13 mW/cm2 ; therefore, it is representative of BL doses
trendline 405
of 0, The loss and
50, 100, of the MS2
200 J/cm 2 . Three trials
bacteriophage over time
were (hours) onfor
conducted PTFE disks
each is shown
BL405 in Figure
dose; the figure
5. This figure
displays displaysoftwo
the average trendlines,
these trials andrepresenting
the standardthe loss of MS2
deviation withbars.
as error (blueThe
circle) and
highest
level of inactivation (1.91 log) was achieved at a BL405 dose of 200 J/cm2 . The ‘405 Light
On’ data points displayed very little variation; therefore, the error bars on the trendline
were hidden.
exposure to BL405 irradiance. It appears that viral degradation was not significant over
time. This trendline displays an essentially flat line, indicating no effect of time.
without (black triangle) exposure to BL405 irradiance. The irradiance corresponding to the
‘405 Light On’ trendline was 13 mW/cm2; therefore, it is representative of BL405 doses of 0,
50, 100, and 200 J/cm2. Three trials were conducted for each BL405 dose; the figure displays
the average of these trials and the standard deviation as error bars. The highest level of
inactivation (1.91 log) was achieved at a BL405 dose of 200 J/cm2. The ‘405 Light On’ data
points displayed very little variation; therefore, the error bars on the trendline10wereof 23
hidden.
Figure
Figure 5.
5. Log
Log loss
loss of
of MS2
MS2 bacteriophage
bacteriophage asas aa function
function of
of time
time on
on PTFE
PTFE disks
disks in a low-dew-point
in a low-dew-point
environment. Time in hours is shown on the x axis and the log loss of MS2 is shown on the y axis.
environment. Time in hours is shown on the x axis and the log loss of MS2 is shown on the y axis.
exposure
effect to405BL
of BL 405
on irradiance.
PTFE disks inThe shaded region
low-dew-point between the The
environments. two microbial
trendlinesdetection
represents the for
limit viricidal
these
effect of BL405ison
experiments PTFEasdisks
shown inat
a line low-dew-point
the top of theenvironments.
figure. The microbial detection limit for these
Both to
exposure trendlines were adjusted
BL405 irradiance. to account
It appears for the
that viral retention ofwas
degradation MS2 onsignificant
not the PTFE disks.
over
time. This trendline displays an essentially flat line, indicating no effect of time.
Both trendlines were adjusted to account for the retention of MS2 on the PTFE disks.
BL was more effective on the PTFE surfaces in the high-dew-point conditions. Al-
The area405
between these two trendlines (shaded blue) represents the viricidal effect of BL405.
though the high dew point yielded greater MS2 loss over time, the total loss (‘405 Light On’)
was significantly higher than the total loss for the low-dew-point experiments. Essentially,
BL405 was more effective on the PTFE surfaces in the high-dew-point conditions.
no viral degradation due to time exposure was observed in the low-dew-point conditions
Although the high dew point yielded greater MS2 loss over time, the total loss (‘405 Light
on the PTFE surface.
On’) was significantly higher than the total loss for the low-dew-point experiments.
Essentially,
3.3. Stainlessno viral degradation due to time exposure was observed in the low-dew-point
Steel
conditions on the PTFE surface.
The results of the high-dew-point experimental conditions for the stainless-steel disks
are shown in Figure 6.
3.3. Stainless Steel
HighThe
Dewresults ◦ C) high-dew-point experimental conditions for the stainless-steel
of the
Point (18
disksFigure
are shown in Figure
6 displays 6. loss of the MS2 bacteriophage as a function of time, measured
the log
in hours. This figure shows two trendlines, representing the loss of MS2 with (blue
circle) and without (black triangle) exposure to BL405 irradiance. The BL405 irradiance
corresponding to the ‘405 Light On’ trendline was 13 mW/cm2 ; therefore, it is representative
of BL405 doses of 0, 50, 100, and 200 J/cm2 . Three trials were conducted for each BL405 dose;
Figure 6 displays the average of these trials and the standard deviation as error bars. The
highest level of inactivation on the stainless-steel disks (3.76 log) was achieved at a BL405
dose of 200 J/cm2 .
Microorganisms 2023,
Microorganisms 2023, 11,
11, 2638
x FOR PEER REVIEW 11
11 of
of 23
Figure 6.
Figure 6. Log
Log loss
loss of
of MS2
MS2 bacteriophage
bacteriophage asas aa function
function of
of time
time on
on stainless-steel
stainless-steel disks
disks in
in aa high-dew-
high-dew-
point environment. Time in hours is shown on the x axis and the log loss of MS2 is shownononthe
point environment. Time in hours is shown on the x axis and the log loss of MS2 is shown y
the
axis. Two trendlines are displayed, indicating the log loss of MS2 as a function of time
y axis. Two trendlines are displayed, indicating the log loss of MS2 as a function of time with and with and
viricidal effect of BL405 on the stainless-steel disks in high-dew-point environments. The microbial
viricidal effect of BL405 on the stainless-steel disks in high-dew-point environments. The microbial
detection limit for these experiments is shown as a line at the top of the figure.
detection limit for these experiments is shown as a line at the top of the figure.
High Dew Point (18 °C)
Additionally, the black trendline displays the effect of time on MS2 degradation. This
Figureexperiments
represents 6 displays conducted
the log losswithout of the exposure
MS2 bacteriophage as a function
to BL405 irradiance, measuringof time,
the
measured
effects in hours.
of viral This figure
degradation shows
over time. Astwo trendlines,
the time representing
increased, the loss of MS2
the viral degradation with
increased.
(bluehighest
The circle) viral
and without
loss, 1.72 (black
log, wastriangle) exposure
observed after to
4.3BL
h.405 irradiance. The BL405 irradiance
corresponding
Both trendlines were adjusted to account for the viral 13
to the ‘405 Light On’ trendline was mW/cm
retention on2;the
therefore, it is
stainless-steel
representative of BL doses of 0, 50, 100, and 200 J/cm 2. Three trials were conducted for
disks. The area between these two trendlines (shaded blue) represents the viricidal effect of
405
each
BL 405BL 405 dose;
. This area Figure
excludes 6 displays
any losses thedue
average of these
to viral trials and
degradation on the
the standard
surface. deviation as
errorThese
bars. experiments
The highest were level repeated
of inactivation
on theon the stainless-steel
stainless-steel disks indisks (3.76 log) was
the low-dew-point
achieved at a BL
environment. No405 dose of 200
significant J/cm
effect of2.BL405 inactivation occurred on the stainless-steel disks
Additionally,conditions;
in low-dew-point the black trendline displaysin
this is displayed thethe
effect of time on MS2
Supplementary degradation.
Materials (FigureThis
S2).
represents experiments conducted without exposure to BL405 irradiance, measuring the
3.4. k Values
effects for Each
of viral Surface byover
degradation Dew Point
time. As the time increased, the viral degradation
increased.
Figures The2–6highest
show thatviralBLloss,
405 1.72 log, wasefficacy
inactivation observed and after 4.3degradation
viral h. over time are
affected
Both bytrendlines
surface type were and dew point.
adjusted The viricidal
to account for the kviral
values from each
retention surface
on the type and
stainless-steel
dew-point
disks. The conditions
area between were tabulated
these and are shown
two trendlines (shaded in blue)
Table represents
4. The valuesthewere calculated
viricidal effect
by subtracting
of BL the excludes
405. This area total loss anyof ‘405
lossesLight
dueOn’
to from the ‘405 Lighton
viral degradation Off’
theksurface.
values for the true
viricidal
These k values. The k values
experiments from the on
were repeated trendlines were divided
the stainless-steel by the
disks average
in the irradiance
low-dew-point
to report units of cm 2 /J.
environment. No significant effect of BL405 inactivation occurred on the stainless-steel
disks in low-dew-point conditions; this is displayed in the Supplementary Materials
(Figure S2).
Table 4. Viricidal k values for each surface by dew point.
Surface High-Dew-Point k (cm2 /J) Low-Dew-Point k (cm2 /J)

Ceramic 0.0093 0.0087
PTFE 0.0385 0.0210
Stainless steel 0.0193 No Data
Table 4 displays the viricidal k values for each surface at high and low dew points.
As shown in Table 4, the high-dew-point k values (cm2 /J) are higher than the low-
dew-point k values, indicating that BL405 inactivation is greater in the high-dew-point
environment. The data for the stainless steel at the low dew point was inconclusive,
therefore was not included in this data set. The PTFE surfaces achieved the highest
level of inactivation at both dew-point conditions, whereas the ceramic surfaces exhibited
the lowest.
Figures 2–6 demonstrate a trend where viral degradation increases in the high-dew-
point conditions for each surface type. Table 5 displays the variation, or increase, in
viral degradation in the high-dew-point environment, compared to the low dew point
environment. The viral degradation rates are shown as a function of time (1/h). These data
were tabulated from the ‘405 Light Off’ trendlines shown in Figures 2–6 and are displayed
in Table 5 as non-viricidal k values.
Table 5. Non-viricidal k values for each surface by dew point.
Surface High-Dew-Point k (1/h) Low-Dew-Point k (1/h)

Ceramic 0.8550 0.1221
PTFE 0.4109 No Data
Stainless steel 0.4492 0.2076
Table 5 displays the non-viricidal k values for each surface at the high- and low-dew-
point conditions.
As shown in Table 5, the non-viricidal k values are higher for each surface in the
high-dew-point conditions compared to the low-dew-point conditions. These data are
shown in Figure 7. Figure 7 displays the total loss (405 Light On) values compared to the
non-viricidal data (405 Light Off) shown in Table 5. These data are displayed in units of 1/h.
The data in Figure 7 display the non-viricidal and total loss k values for each surface
in both dew-point conditions. The total loss k values represent the ‘405 Light On’ trendlines
and the non-viricidal k values represent the ‘405 Light Off’ trendlines in Figures 2–6. The
high-dew-point conditions yielded higher total loss and non-viricidal k values for all
surface types.
The ceramic surface displayed the greatest variation between the non-viricidal effects
of dew points with k values of 0.855 and 0.122 1/h for the high and low dew points,
respectively. The variations of the total loss for the high and low dew points were 1.09
and 0.341 1/h, respectively. The effects of the dew points were significant for the total and
non-viricidal losses on the ceramic surface.
The PTFE disks displayed the highest total loss k values (1.38 and 0.523 1/h) for the
high and low dew points, respectively. The non-viricidal losses were low compared to the
total losses on the PTFE surface.
The stainless-steel disks displayed a moderate variation between the non-viricidal
and total loss k values, 0.935 and 0.461 1/h, respectively, for the high-dew-point conditions.
Interestingly, the surface displayed the lowest variation between the total losses and non-
viricidal k values in the low-dew-point environment, indicating a minimal effect of BL405
in these conditions.
x FOR PEER REVIEW 1313of
of 23
Figure
Figure 7.
7. Comparison
Comparison of of the
thetotal
totalloss
loss(405
(405‘Light
‘LightOn’)
On’)and
and non-viricidal
non-viricidal k values
k values (405
(405 ‘Light
‘Light Off’)
Off’) for
for each surface type. The surface type and dew point are on the x axis. The k values (1/h) are shown
each surface type. The surface type and dew point are on the x axis. The k values (1/h) are shown on
on the y axis. The blue and orange bars represent the non-viricidal and total losses, respectively, for
the y axis. The blue and orange bars represent the non-viricidal and total losses, respectively, for each
each surface in the high-dew-point conditions. The gray and yellow bars represent the non-viricidal
surface
and totalinlosses,
the high-dew-point conditions.
respectively, in The gray conditions.
the low-dew-point and yellow bars represent the non-viricidal and
total losses, respectively, in the low-dew-point conditions.
The data in Figure 7 display the non-viricidal and total loss k values for each surface
Statistical testing was conducted on these results and the values are shown in
in both dew-point conditions. The total loss k values represent the ‘405 Light On’
Figures S3 and S4 in the Supplementary Materials. These tests revealed that time expo-
trendlines and the non-viricidal k values represent the ‘405 Light Off’ trendlines in Figures
sure, dew point, and blue light were highly significant. Several interactions between these
2–6. The
factors werehigh-dew-point
also significant,conditions
including theyielded
effecthigher
of dewtotal
pointlossoverand non-viricidal
time, k values
surface type and blue-
for all surface types.
light inactivation, blue-light dose, and the effect of the dew point on blue-light inactivation.
The ceramic
For more detailedsurface displayed
information on thethe greatestoutputs,
statistical variationseebetween the non-viricidal
the Supplementary effects
Materials.
of dew points with k values of 0.855 and 0.122 1/h for the high and low
The results of these studies suggest that viral loss on ceramic, PTFE, and stainless-steel dew points,
respectively.
surfaces is a The resultvariations
of BL405 of the total loss
inactivation forviral
and the high and low dew
degradation due topoints wereexposure
the long 1.09 and
0.341
times required to reach the desired dose. These novel results highlight that surface non-
1/h, respectively. The effects of the dew points were significant for the total and type
viricidal losses on the
and environmental ceramic surface.
conditions, such as how the dew point greatly affects the efficacy of
BL405The PTFE disks
inactivation displayed the
technologies. Thehighest total loss
mechanisms ofkthevalues (1.38 and
variations 0.523 1/h)
observed for the
by surface
high and low dew points, respectively.
type and dew point are addressed below. The non-viricidal losses were low compared to the
total losses on the PTFE surface.
4. Discussion
The stainless-steel disks displayed a moderate variation between the non-viricidal
and The
totalresults
loss kofvalues,
this work0.935 and 0.461
revealed many1/h, respectively,
important findings.for the
Thesehigh-dew-point
studies assist
conditions.
in explaining the mechanisms of BL405 viral inactivation and provide evidencethe
Interestingly, the surface displayed the lowest variation between total
that its
losses and non-viricidal k values in the low-dew-point environment,
efficacy is impacted by environmental conditions (dew point), prolonged time exposure, indicating a minimal
effect of BL405type.
and surface in these
Theconditions.
viricidal k values reported for this work were significantly lower
than Statistical
the viricidal testing was conducted
k values for the MS2 onbacteriophage
these results andwiththe UVvalues are shown
254 [19,31]; in Figures
this was due to
S3
theand S4 in inactivation
different the Supplementary Materials.
mechanisms Thesewith
associated teststhese
revealed that time exposure, dew
wavelengths.
point,Theandviricidal
blue light were highly
k values significant.
determined in thisSeveral interactions
work (Table between kthese
4) fell between valuesfactors
that
were tabulated
also significant, including the effect of dew point over time, surface
from the literature [13,14,24,32], ranging from 0.0013–0.1762 cm /J. These ktype2 and blue-
light
valuesinactivation,
ranged two orders blue-light dose, andas the
of magnitude theyeffect of the dew
were calculated point
for five on blue-light
different viruses
in many different media conditions. Rathnasinghe et al. [14] studied 405 inactivation of
SARS-CoV-2, Influenza A, and encephalomyocarditis virus in liquid PBS. Tomb et al. [13]
experimented with several media, including liquid PBS, artificial saliva, plasma, fetal bovine
serum, and artificial feces. Lau et al. [24] and Vatter et al. [32] studied bovine coronavirus
and Phi-6 bacteriophage, respectively, in liquid PBS. It should be noted that these k values
were calculated for the experiments conducted in water; therefore, direct comparisons
could not be made to the surface inactivation k values reported in these studies.
This work revealed many novel findings related to viral inactivation mechanisms
and the impact of surface characteristics and the dew point. It was discovered that all the
surface types achieved greater inactivation at the high dew points, compared to the low dew
point. Additionally, the PTFE surface achieved the highest level of inactivation, whereas the
ceramic surface achieved the lowest. This was thought to be due to the reflectivity, porosity,
zeta potential, and contact angles of these surfaces. The effects of surface characteristics
also impacted viral degradation over time; these findings are discussed in detail below.
Additionally, the surfaces were not allowed an inoculum drying period prior to
experimentation. The literature [33–35] reports several studies on the effects of inoculum
drying time before photoinactivation. SARS-CoV-2 studies suggest that the inactivation
rate of SARS-CoV-2 is unaffected by drying prior to UV treatment [33,34]. This was likely
due to the short drying times and UV dosing times utilized for these experiments. A study
on SARS-CoV-2 and MS2 Bacteriophage using pulsed UV for inactivation reported varying
results. The SARS-CoV-2 achieved higher inactivation rates with a wet inoculum, yet no
effect of drying time was observed for the MS2 results [35]. These studies indicate that the
inoculum drying time deserves consideration as it may influence the photoinactivation results.
4.1. Viral Inactivation Mechanisms

This work shows that BL405 has virucidal properties on ceramic, PTFE, and stainless-steel
surfaces. Although the research in this field is sparse, some researchers [13,24,25,32] believe the
dominant viral inactivation mechanism by 405 nm light exposure is by oxidative stress on
the viral proteins. This damage can occur on both non-enveloped and enveloped viruses via
damage to the viral capsid or viral envelope, respectively. It is thought that photosensitizers,
or materials with porphyrin-like structures, must be present in the immediate surrounding
environment and absorb the 405 nm wavelength. This interaction creates reactive oxygen
species (ROS) that create stress on the proteins that comprise the viral capsid, or the viral
envelope, leading to viral inactivation [13,24,25,32]. Viral envelopes consist of a lipid
bilayer that encapsulates the viral capsid. The exterior of the bilayer is coated with a
layer of glycoproteins [36]. Therefore, the extrapolation of the ROS mechanism from a
non-enveloped virus to an enveloped virus is valid. These mechanisms may not transfer to
viruses without protein coats, such as viroids [37] and Hepatitis D [38]. Further studies on
viruses without protein coats are recommended.
Tomb et al. [13] conducted a comprehensive study on BL405 to observed the inac-
tivation of feline calicivirus (non-enveloped), exploring many possible mechanisms for
viral degradation during experimentation. The first experiments suspended the virus
in a phosphate-buffered solution and exposed the samples to doses of 0, 561, 1683, and
2804 J/cm2 . The highest 405 nm dose (2804 J/cm2 ) achieved a 3.9 log inactivation of feline
calicivirus, with a calculated k value of 0.0013 cm2 /J [13].
Additional experiments were conducted with the virus suspended in various organi-
cally rich media, such as artificial saliva, blood plasma, and artificial feces. The experiments
using a virus suspended in an artificial saliva solution achieved a 2.25 log reduction at
280 J/cm2 , with a corresponding k value of 0.0129 cm2 /J. When suspended in a blood plasma
solution, the virus achieved a 4.8 log inactivation at 561 J/cm2 (k value = 0.0087 cm2 /J).
Finally, when suspended in an artificial feces solution, the feline calicivirus achieved a
4.5 log inactivation at 1402 J/cm2 , with a k value of 0.0032 cm2 /J [13].
From these results, the inactivation of the feline calicivirus is greater when suspended
in organically rich mediums. The highest inactivation level was observed using the artificial
saliva solution. Tomb et al. [13] reported that the organic material in these solutions may
have been sensitive to 405 nm, absorbing this wavelength and producing reactive oxidative
species (ROS). The creation of ROS caused oxidative stress on the viral proteins, rendering
it inactivated [13].
Another paper [32] studied the viricidal effects of BL405 on the Phi-6 bacteriophage, a
surrogate for SARS-CoV-2. The Phi-6 bacteriophage is an enveloped dsRNA virus and is
selected as a viral surrogate as the blue-light inactivation mechanisms of bacteriophages
and mammalian viruses are thought to be similar. The goal of this study was to inten-
tionally exclude photosensitizers to reveal the effects of BL405 without enhancement from
extraneous material in the solution. The Phi-6 bacteriophage was suspended in phosphate-
buffered saline (PBS) and a saline magnesium gelatin buffer. After exposure to a blue-light
dose of 1300 J/cm2 , a 3 log inactivation of the virus was observed in both solutions. When
suspended in PBS, the calculated k value for the Phi-6 inactivation was 0.0026 cm2 /J. The
author suggested that the mechanism responsible for the inactivation of this bacteriophage
was the material from the host cell cultures. The host cell for the Phi-6 bacteriophage
is Pseudomonas syringae, whereas the host cell for the MS2 bacteriophage is E. coli. Both
bacterial species were grown in tryptic soy broth. These cells had photosensitizers similar
to porphyrins and may have been responsible for the inactivation observed [32].
The results of Vatter et al. [32] and Tomb et al. [13] relate to the findings of this research.
E. coli, a bacterium and host cell for the MS2 bacteriophage, may have porphyrins that
react to blue-light wavelengths. Portions of the E. coli cells and culture media were likely
present in the MS2 solution applied to the surfaces. Figure S5 in the Supplementary
Materials displays the correlation between the total organic carbon (TOC) of MS2 growth
media at varying concentrations and its corresponding absorbance at 405 nm. The TOC
concentration in the solution used to inoculate the surfaces was 130 mg/L, corresponding
to 0.0231 cm−1 at a 405 nm absorbance. It is likely that portions of the E. coli cells and
organic material from the MS2 broth solution acted as photosensitizers upon the interaction
with 405 nm wavelengths.
Another mechanism proposed by Tomb et al. [13] suggests that the inactivation of
the feline calicivirus in the PBS solution may have been due to inactivation from ex-
traneous wavelengths outside of the 405 nm band. The typical spectral distribution of
wavelengths for commercially purchased blue-light collimate beams commonly seen in the
literature [13,14] falls between 390–430 nm, with the peak irradiance output at 405 nm. It has
been reported that 390 nm causes oxidative stress to the viral capsid and other proteins [39].
Wavelengths between 420–430 nm were found to cause the inactivation of the murine
leukemia virus (enveloped), due to damage to the virion-associated reverse-transcription
complex [40]. Therefore, over the long exposure times associated with these experiments,
these wavelengths may have caused the inactivation observed [13]. Additionally, a 2021
paper tested the effects of a 405 nm viral inactivation on SARS-CoV-2 (enveloped) and
Influenza A Virus (enveloped) without the use of any photosensitizers and confirmed that
inactivation by UVA (~390 nm) and 420–430 nm contributed to viral losses [14].
This provides a further insight into an additional inactivation mechanism respon-
sible for the degradation of the MS2 bacteriophage on our surfaces. As shown in the
Supplementary Materials in Figure S6, 8.3% of the energy emitted by the Thorlabs col-
limated LED fall in the UVA range. A total of 91.5% of the irradiance fell within the
blue-light range, with 10.3% falling between 420–430 nm. Regardless of the presence of
photosensitizers in the inoculum solution, these wavelengths may have contributed to
all or part of the inactivation of the MS2 bacteriophage observed on ceramic, PTFE, and
stainless-steel surfaces.
4.2. Surface Characteristics

The viricidal effects of BL405 and non-viricidal viral degradation also varied by surface
type. Several surface characteristics were explored to explain the causal mechanisms behind
the observed variations.
The MS2 inactivation was significantly higher on the PTFE surface for both the high
and low dew points, compared to the ceramic and stainless-steel surfaces. The reflectivity
of PTFE was studied for wavelengths ranging from 250–500 nm and was reported to
be approximately 97% [41]. For comparison, the visible-light reflectivity for stainless
steel was approximately 50% [42]. These high-reflectivity values allowed the wavelength
to continuously reflect as it contacted the surface, and therefore could move across the
surface and into pore spaces more easily. This phenomenon has been studied in the UV254
literature [2,19,43,44] and is reported to increase the contact between UV light and viral
particles, dramatically increasing inactivation.
The ceramic surface achieved the lowest viricidal effect of BL405 for both the high- and
low-dew-point experiments. This was likely due to the surface porosity of this material.
Figure S7 in the Supplementary Materials display the porosity values of each surface. The
ceramic surface is the most porous of the three surfaces, with a surface porosity of 17.3%,
compared to 1.6% and 2.9% for PTFE and stainless steel, respectively. The literature [45]
concerning UV254 studies indicates that surfaces with a higher surface porosity yield
lower the photoinactivation levels better than non-porous surfaces. This phenomenon
has been named the ‘Canyon wall effect’ [45] due to the relative size of the virus particle,
approximately 27 nm [46], compared to the larger surface pores, which allow the viral
particles to penetrate deeper into the surface. The pore spaces shield viral particles from
UV wavelengths, therefore reducing the level of photoinactivation that can occur on the
surface [19,45]. As shown in Table 4, this phenomenon is pertinent to the BL405 data as well.
Additionally, as shown in Figures 2, 3 and 7, non-viricidal viral degradation over time
is higher on the ceramic surface compared to the PTFE and stainless steel. The literature
suggests that non-viricidal viral degradation over time increases on porous surfaces [47,48].
It has been found that coronaviruses are less stable on porous surfaces than non-porous
surfaces over time. This is thought to be due to adsorption onto the surface, electrostatic
interactions, and intermolecular effects, such as van der Waals forces [47]. Figure S8 in the
Supplementary Materials display a correlation between the viral degradation and porosity,
such that as the porosity increases, the viral degradation increases.
Figure 7 shows that these findings are exaggerated in high-dew-point environments,
specifically for the ceramic surface. As shown by this work, viral degradation increases as
the temperature increases. This phenomenon may be amplified by porous surfaces. As the
temperature increases, the viscosity of the virus inoculum decreases. Therefore, it follows
that higher-temperature environments promote the further penetration of the inoculum
into the porous surface. This likely heightened the viral degradation due to the forces
mentioned above, as the virus had a greater surface area for interactions with the surface.
Additionally, van der Waals forces have been reported to become stronger due to increasing
long-range molecular interactions as the temperature increases [49], resulting is a stronger
viral attachment to the surface.
The contact angles of the ceramic, PTFE, and stainless-steel samples are shown in
Figures S9–S11 in the Supplementary Materials. The contact angle is a measure of hy-
drophilicity/hydrophobicity. Surfaces with lower contact angles are more hydrophilic,
whereas surfaces with higher contact angles are more hydrophobic [50]. Figure S9 displays
the correlation for high-dew-point conditions between the viricidal k value and contact
angle of each surface, such that as the contact angle increases, the viricidal effectiveness
increases. This can be explained by the literature [51] as surfaces with lower contact angles
are conducive to an increased penetration of the inoculum and viral attachment, whereas
surfaces with higher contact angles may retain the inoculum as a droplet on the surface.
The surfaces with the higher contact angles retained a greater portion of the virus and
inoculum on the top of the surface, therefore allowing the increased exposure of the inocu-
lum and virus to the BL405 wavelengths, creating more ROS, thus leading to higher levels
of inactivation. This is consistent with the high-dew-point results presented in this paper,
as the stainless-steel and PTFE surfaces had higher contact angles and achieved higher
viricidal inactivation values.
Figure S10 in the Supplementary Materials displays viral degradation as a function

of the contact angle. This figure shows that, as the contact angle increases, the viral
degradation decreases. The literature reveals that the contact angle is affected by the
porosity of the material [52–54]. Figure S11 in the Supplementary Materials shows a strong
correlation between the contact angle and porosity (R2 = 0.996), such that as the contact
angle increases, the porosity decreases. Therefore, after understanding this relationship,
Figure S11 reinforces the findings of Figure S8 and the literature stating that more porous
surfaces yield lower viral stability results over time.
The zeta-potential data are shown in Figures S12 and S13 in the Supplementary Materials.
Figure S12 displays a strong correlation (R2 = 0.999) between the viricidal k values and
zeta potential, such that as the zeta potential increases, viricidal k decreases. This follows
as the literature [54] suggests that surfaces with more positive zeta potentials will have
higher negative viral depositions on the surface. The MS2 bacteriophage is a negatively
charged virus [55]; therefore, surfaces with more negative zeta potentials, such as PTFE,
will have a weaker attraction of negative viral particles to the surface. Consequently, the
virus and inoculum are more exposed to BL405 wavelengths. Increased exposure to BL405
increases the production of ROS, thus increasing the inactivation of the MS2 bacteriophage
on the surface.
Finally, Figure S13 displays the correlation between the zeta potential and the non-
viricidal k value. The figure shows that as the surface charge becomes less negative, the
non-viricidal k value increases. This follows as the negatively charged MS2 virus is more
likely to adsorb/bond with a surface with a less negative charge. Increased adsorption on
the surface yields lower recovery values, thus increasing the non-viricidal k value.
4.3. Increased Inactivation in High-Dew-Point Conditions

Another significant finding of this research revealed the importance of the dew point
regarding BL405 inactivation efficacy. This work demonstrates that BL405 inactivation
technologies are more effective in high-dew-point environments. This phenomenon was
observed with ceramic, PTFE, and stainless-steel surfaces. This finding supports the pro-
posed dominant inactivation mechanism of BL405 and is confirmed by atmospheric studies
in the literature that demonstrate that reactive oxidant species increase as the moisture
content increases [56,57]. The research conducted by Lespade et al. [58] demonstrated the
importance of water molecules in the oxidation of organic molecules by a superoxide anion.
This work demonstrated that the first layer of surrounding water molecules was essential
for reactivity, as it decreased the energy of the excited states of the hydrated superoxide
anion and facilitated the reaction with organic molecules [58]. At high relative humidities,
there was an increase in the soluble oxygen available for the reaction with blue light.
Three studies in other disciplines [56,57,59] support the theory that as the relative
humidity increases, the creation of reactive oxygen species increases. This research revealed
increased virucidal k values in high-dew-point environments. These findings are consis-
tent with the literature [13,24,25,32] that suggests that ROS is the dominant mechanism
responsible for the BL405 inactivation of viruses.
4.4. Viral Degradation over Time: Temperature and Relative Humidity

This work highlighted the importance of conducting controls, or time exposure tests,
for BL405 experiments. These experiments required long exposure times (in the order of
hours), due to the high dose needed and the limited irradiance that could be provided
by BL405 sources. Previous studies [14,24,32] reported that viral degradation over time
was not a significant source of viral loss, although our work suggested otherwise. The
studies mentioned were conducted in liquid media, which likely contributed to the dis-
parity in the results of the control experiments on surfaces. Additionally, the irradiance
values used by Lau et al. [24] (144.4 mW/cm2 ) and Vatter et al. [32] (78.6 mW/cm2 ) were
significantly higher than the 13 mW/cm2 BL405 irradiance output used for our studies. The
high-irradiance values decreased the required exposure time for these experiments, likely
limiting the viral degradation.
This paper demonstrated that significant viral degradation over time in the absence
of BL405 occurred on all surfaces and was exacerbated by the high-dew-point conditions.
The effects of viral degradation as a function of temperature and dew point are reported
in the literature [22,60–62]. In this research, high-dew-point experiments were conducted
at 28 ◦ C. A 2018 study [62] quantified the effects of relative humidity and temperature on
Phi-6 bacteriophage recovery and found similar results to those reported in this paper. The
highest recovery was found with relative humidities below 60% or above 80%. When the
relative humidity was set at 75% and the temperature increased from 19 ◦ C to 25◦ C, the
infectivity of Phi-6 decreased by two orders of magnitude [62].
Another study [22] using SARS-CoV-2 supported the results found in this paper.
This study tested varying relative humidity and temperature conditions on polypropylene
plastic surfaces. The results of this study found that viral decay increased as the temperature
increased, regardless of the relative humidity. The relative humidity had a lesser effect
on viral decay compared to temperature. The effect of relative humidity appeared to be
U-shaped, in that viral decay was the highest at a 65% relative humidity and lower at
40% and 85% relative humidity levels [22]. The author suggested that the low and high
relative humidity conditions hindered the ionic equilibrium, which directly impacted the
evaporation rate of the droplet [22].
Temperature effects have also been observed with the MS2 bacteriophage. A 2012
study [61] looked at the structural integrity of MS2 after various inactivation techniques.
These experiments were conducted in a PCR thermocycler for 2 min. An 8.5 log inactivation
of the MS2 bacteriophage was observed and was found to exist due to structural damage to
the proteins responsible for binding to the host cell. The virus remained infectious outside
of the host cell but could not replicate to produce progeny [61].
Additionally, viral samples sent to the partnering laboratory for this work, GAPLAB,
in Ontario, Canada, must comply with their QA/QC requirements for temperature. The
samples must arrive at the laboratory between 2–10 ◦ C; samples outside that range cannot
be analyzed due to sample non-conformance. This requirement reiterates that the MS2
bacteriophage is sensitive to increased temperatures. As the temperature increases, MS2
stability decreases [29,63].
The data gathered from the work of Morris et al. [22], Wigginton et al. [61],
Prussin et al. [62], and EPA [29,63] strongly suggest that the higher degradation of MS2 in
the high-dew-point experiments is likely due to the higher temperature, 28 ◦ C as compared
to 10 ◦ C in the low-dew-point conditions. The viral proteins likely degraded due to the
high temperature and long exposure times of these experiments, rendering a portion of the
MS2 bacteriophage unable to infect the host E. coli. The low-dew-point environments may
have caused the desiccation of the MS2 virus [64]; yet, these effects were highly variable.
Finally, this research contributed the necessary data to the photoactivation industry
regarding the effects of surface characteristics and dew points on BL405 inactivation efficacy.
When properly designed, BL405 technologies may provide an extra layer of protection
against viral pathogens. However, the long exposure times required for adequate viral
inactivation (2–3 log) may suggest that these technologies cannot keep up with the viral
shedding of an infected individual. The approximate viral shedding rate of a person
infected with SARS-CoV-2 is 1800 pathogens/h [65]. Depending on the surface type
and environmental conditions, it may be beneficial to utilize an additional inactivation
technology concurrently with BL405 for the optimum protection of human health.
The following citations can be found in the Supplementary Materials [14,19,24–26,66–75].
5. Conclusions
This work uncovered many important findings regarding the efficacy of BL405 surface
inactivation. The major findings are listed below:
• This research provides sufficient evidence that BL405 has a viricidal effect on the
MS2 bacteriophage.
• The high-dew-point conditions yield higher levels of inactivation of the MS2 bacterio-
phage on all the surfaces tested.
• Conducting time exposure controls, represented by the ‘405 Light Off’ data, is prudent
for determining the true viricidal effect of BL405 . Due to the long exposure times
required, viral degradation occurs on the surface without exposure to BL405 . These
effects increase in the high-dew-point conditions.
• The highest level of inactivation due to BL405 (3.9 log) is observed on the PTFE surface,
in the high-dew-point conditions.
• The characteristics (zeta potential, contact angle, and porosity) of each surface impact
BL405 inactivation efficacy and viral degradation over time.
# As the zeta potential increased, the viricidal effects of BL405 decreased, and
viral degradation increased in the high-dew-point conditions.
# As the contact angle increased, the viricidal effects of BL405 increased, and viral
degradation decreased in the high-dew-point conditions.
# As the porosity increased, viral degradation increased in the high-dew-
point conditions.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/microorganisms11112638/s1. Figure S1: displays the BL405
dose response curve for six viral species. BL405 dose (0–260 J/cm2 ) is shown on the x-axis and log
inactivation is displayed on the y axis; Figure S2: displays the log loss of MS2 bacteriophage as a
function of time on stainless steel disks in a low dew point environment. Time in hours is shown
on the x axis and log loss of MS2 is shown on the y axis. Two trendlines are displayed, indicating
the log loss of MS2 as a function of time with and without exposure to BL405 irradiance. The shaded
region between the two trendlines represents the viricidal effect of BL405 on stainless steel disks in
low dew point environments. The microbial detection limit for these experiments is shown as a line
on the top of the figure; Figure S3: displays the results of a full factorial analysis conducted in JMP;
Figure S4: displays the results of the statistical analysis performed in JMP. Statistical significance was
determined by p values greater than 0.05; Figure S5: displays the concentration of total organic carbon
(TOC) in mg/L versus the BL405 absorbance (cm−1 ). The TOC concentration is on the x axis and the
BL405 absorbance is on the y axis; Figure S6: displays the irradiance distribution of the Thorlab 405 nm
collimated beam. The irradiance measurements were recorded with an ILT960 Spectroradiometer;
Figure S7: displays the surface porosity (measured with SEM) of ceramic, PTFE and stainless steel;
Figure S8: displays the surface porosity of ceramic, PTFE, and stainless steel as a function of their
non-viricidal k values in high dew point environments; Figure S9: displays the viricidal k value as a
function of the contact angle of ceramic, stainless steel and PTFE; Figure S10: displays the correlation
between the contact angle of ceramic, stainless steel, and PTFE and non- viricidal k values in high
dew point environments; Figure S11: displays contact angle versus porosity for aluminum, ceramic,
Formica laminate, PTFE, and stainless steel. Contact angle (◦ ) is shown on the x axis. Percent surface
porosity is shown on the y axis; Figure S12: displays the zeta potential (mV) of PTFE, stainless steel,
and ceramic versus the viricidal k values in the high dew point environments; Figure S13: displays
the zeta potential (mV) for PTFE, stainless steel, and ceramic as a function of the non-viricidal k
values in the high dew point conditions; Table S1: displays the characteristics of the viruses shown in
Figure S1. The characteristics of MS2 bacteriophage were included in this table for comparison.
Author Contributions: Conceptualization, C.B. and J.M.; methodology, C.B. and J.M.; software, C.B.;
validation, C.B. formal analysis, C.B. and J.M.; investigation, C.B.; resources, J.M.; data curation, C.B.;
writing—original draft preparation, C.B.; writing—review and editing, C.B. and J.M.; visualization,
C.B. and J.M.; supervision, J.M.; funding acquisition, J.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This doctoral research project was funded by CoRE (1DD092), UIC (1DDJM2), and the
Business and Industry Consortia (14b411) grants provided to the University of New Hampshire.
Data Availability Statement: For access to the file containing all raw data collected during experi-
mentation, please visit https://dx.doi.org/10.34051/d/2023.2.
Acknowledgments: The authors would like to thank many organizations and individuals for their
assistance with this work. We are grateful for the support of this research by the Civil and Envi-
ronmental Engineering Department at the University of New Hampshire. We thank our funding
organizations for their financial support, UNH CoRE (1DD092), UIC (1DDJM2), and the Business
and Industry Consortia (14B411). We greatly appreciate the laboratory collaboration with GAPLAB
(Ontario, Canada), specifically all the guidance from Shawn Verhoeven. Several employees from the
University Instrumentation Center (UIC) were tremendously helpful in determining the porosity and
SEM imaging. These individuals include Nancy Cherim, Shawn Banker, and Mark Townley. Darcy
Silver and Scott Campbell from the Technical Service Center assisted in determining the contact
angles and cutting the surfaces to size. We thank Emeritus R. Faust for his assistance with the surface
zeta-potential analysis. We appreciate the help of Nathan Daigle from the John Olson Advanced
Manufacturing Center for further assistance in cutting materials for the SEM processes. Finally, we
thank all the individuals that assisted with the laboratory work and its analysis, including Kellen
Sawyer and Paula Mouser.
Conflicts of Interest: The authors declare no conflict of interest.
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Microorganisms: Impacts of Surface Characteristics and Dew Point On The Blue-Light (BL) Inactivation of Viruses

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Microorganisms: Impacts of Surface Characteristics and Dew Point On The Blue-Light (BL) Inactivation of Viruses

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microorganisms

Abstract: The increased prevalence of multidrug-resistant organisms (MDROs), healthcare associated

Citation: Bernardy, C.; Malley, J.

Microorganisms 2023, 11, 2638. https://doi.org/10.3390/microorganisms11112638 https://www.mdpi.com/journal/microorganisms

2. Materials and Methods

Table 1. Surfaces utilized for experimentation.

Surface Type Manufacturer/Description

2.2. Experimental Conditions

Table 2. Temperature and relative humidity conditions in controlled chamber.

Dew Point (◦ C) Temperature (◦ C) Relative Humidity (%)

Table 2 displays the dew-point conditions maintained in the experimental chamber.

2.3. BL405 Irradiance and Dose

Microorganisms 2023, 11, 2638 4 of 23

BL405 Irradiance Map

0-5 5-10 10-15 15-20

All irradiance values are reported in mW/cm2

Table 3. BLDoses (J/cm

2.4. Rate Constants (k Values)

2.4.1. Non-Viricidal Rate Constants

2.4.2. Viricidal Rate Constants

Viricidal k (1/h) = Total Loss k (1/h) − Viral Degradation k (1/h)

2.5. MS2 Bacteriophage

2.6. Experimental Procedure

2.7. Statistical Analysis

Table 4. Viricidal k values for each surface by dew point.

Surface High-Dew-Point k (cm2 /J) Low-Dew-Point k (cm2 /J)

Table 5. Non-viricidal k values for each surface by dew point.

Surface High-Dew-Point k (1/h) Low-Dew-Point k (1/h)

4.1. Viral Inactivation Mechanisms

4.2. Surface Characteristics

Figure S10 in the Supplementary Materials displays viral degradation as a function

4.3. Increased Inactivation in High-Dew-Point Conditions

4.4. Viral Degradation over Time: Temperature and Relative Humidity

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