11 (2022) 100169

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Biosensors and Bioelectronics: X

journal homepage: www.journals.elsevier.com/biosensors-and-bioelectronics-x

Headset bio-sniffer with wireless CMOS camera for percutaneous ethanol

vapor from the ear canal
Takahiro Arakawa a, b, Riki Ishikawa c, Kenta Iitani a, Koji Toma a, Yasuhiko Iwasaki d,
Kohji Mitsubayashi a, c, *
a
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Kanda-surugadai 2-3-10, Chiyoda-ku, Tokyo, 101-0062, Japan
b
Department of Electric and Electronic Engineering, Tokyo University of Technology, 1404-1 Katakura, Hachioji City, Tokyo 192-0982, Japan
c
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan
d
Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita-Shi, Osaka, 564-0836, Japan

A B S T R A C T

Volatile organic compounds contained in human volatiles are related to metabolism and disease. Measurement of these components is expected to enable non-
invasive and simple metabolic evaluations and disease diagnoses. In particular, skin gas can be collected continuously without constraints, a useful property for
the next generation of wearable measurement devices. In this study, we develop a wearable biochemical gas sensor (biosniffer) for ethanol by constructing a headset-
type unrestrained ear canal gas measurement system integrated with a complementary metal-oxide semiconductor (CMOS) camera. This system measures ethanol gas
released by the skin by detecting the increase in fluorescence of NADH, which is produced during the oxidation of ethanol by alcohol dehydrogenase (ADH). In this
system, excitation light from a UV-LED irradiates an ADH enzyme-immobilized membrane through a band-pass filter, and the fluorescence of NADH is detected by a
small CMOS camera with Wi-Fi functionality. This system is integrated into an earmuff, forming a wearable headset. Fluorescence images shows an increased output
correlated with the ethanol gas concentration. When integral analysis is used to assess the increase in fluorescence, it is possible to measure ethanol gas at 11
ppb–444 ppm, a range that includes the ethanol concentration in the skin after alcohol administration. Furthermore, when this device is attached to a human
participant who is consuming alcohol, the increase and decrease output based on drinking and metabolism is confirmed, indicating the possibility of directly
measuring ethanol gas from the ear canal with minimal effect of perspiration.

1. Introduction
Human volatiles, such as exhaled breath and skin gases, contain
volatile organic compounds (VOCs) that emanate from blood compo­
nents related to metabolism and disease (Aliverti, 2017; Boots et al.,
2012; Lourenço and Turner, 2014; Majchrzak et al., 2018; Saidi et al.,
2018; van der Schee et al., 2015). There is an expectation that the
measurement of these chemical components will enable non-invasive
analyses, simple metabolic evaluations, and disease diagnoses (Kim
et al., 2016; Rydosz, 2018; Wang et al., 2014a). ‘Human volatiles’ is a
general term for gases emitted from humans, including exhalation,
halitosis, body odor, foot odor, and intestinal gas (LaurentDormont
et al., 2013; Jalal et al., 2018). These gases have been reported to
contain hundreds to thousands of chemical components. Clarification of
the release kinetics and the mechanisms generating these components
will provide meaningful biological information, similar to the informa­
tion that can currently be ascertained by an analysis of chemicals in the
blood (Mochalski et al., 2014a; Wang et al., 2014b).
Exhaled breath contains the gases exchanged in the alveoli and
expelled along with the outside air inhaled through the mouth or nose
(Anderson et al., 2003). The composition of the exhaled gas is 79% ni­
trogen, 16% oxygen, 4% carbon dioxide, and 1% other; the other 1%
contains more than 500 blood-derived VOCs (de LacyCostello et al.,
2014). The concentrations of these chemical substances are well-known
to vary depending on age, gender, metabolic state, and presence of
disease. The oxygen obtained through respiration is metabolized in the
tissues and exchanged with carbon dioxide for transport to the pulmo­
nary capillary beds. Chemical components derived from metabolism or
ingestion also circulate in the body in the blood and are transported from
the pulmonary arteries to the pulmonary capillary beds along with
carbon dioxide gas. There, an exchange takes place through the diffusion
of gas molecules, and like carbon dioxide, the chemical components in
the blood also move into a gaseous phase, causing VOCs to be released
from the body in the exhaled air (Manolis, 1983; van Oort et al., 2018).
Therefore, endogenous VOCs concentrations in exhaled air are corre­
lated with their concentrations in the blood, and exhaled air analysis is

* Corresponding author. Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental Uni­
versity, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo, 101-0062, Japan.
E-mail address: m.bdi@tmd.ac.jp (K. Mitsubayashi).

https://doi.org/10.1016/j.biosx.2022.100169
Received 18 February 2022; Received in revised form 15 May 2022; Accepted 16 May 2022
Available online 25 May 2022
2590-1370/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
T. Arakawa et al.
of great value as a rapid and non-invasive diagnostic technique. In
particular, it has been suggested that when a person suffers from a dis­
ease, compounds that are not normally detected are produced. There­
fore, these substances are indicators or biomarkers for understanding
the state of the body, and analysis of the components of exhaled gas is
expected to be used as a clinically meaningful biological test (Wang and
Sahay, 2009). Although many studies have been carried out on exhaled
gas, it is not suitable for continuous measurement because it requires
sampling of exhaled human breath.
In recent years, many researchers have focused on skin gas mea­
surement for long-term and non-invasive human monitoring, which can
be performed continuously (Mitsubayashi et al., 2022). Skin volatiles
are biological gases emitted from the human skin; they contain various
VOCs that are products of metabolism (Vishinkin et al., 2021; Wie­
senhofer et al., 2018). Tsuda et al. found several gases that emanate from
human skin, which they refer to as ‘skin gas’. As the chemical compo­
nents in breath and sweat relate to some diseases, skin gas components
also have a relationship with the state of body conditions (Naitoh et al.,
2000; Tsuda et al., 2011). Skin gases can be broadly classified into three
categories: first, the VOCs in the blood that are emitted through the skin,
second, the VOCs that are emitted from sweat gland secretions, and
third, the VOCs formed as coproducts produced when sweat, sebum,
keratin, and other substances are metabolized by indigenous bacteria on
the skin surface (Buono, 1999; Nose et al., 2005; Sheybani and Shukla,
2017; van Geffen et al., 2016).
Skin volatiles can be collected more easily than exhaled breath as
they can be measured continuously without constraint (L. Dormont
et al., 2013). Currently, various studies are being conducted on the
measurement, metabolic evaluation, and disease screening of various
VOCs contained in exhaled air and skin gas using gas
chromatography-mass spectrometry (GC-MS) and semiconductor sen­
sors (Giles et al., 1987; Shimouchi et al., 2010; Tsuda et al., 2011).
However, human volatiles contain a variety of chemical components,
and their concentrations tend to be affected by the state of the body and
time (Ebeler et al., 1997; Kalantar-Zadeh et al., 2018; Naitoh et al.,
2000; Pal et al., 2020). While the goal is to continuously measure target
chemical components in human volatiles and evaluate metabolism, it is
considered difficult to use existing measurement devices, as there are
issues with continuity and selectivity.
In addition, the measurement of trace amounts of VOCs in skin gases
needs to be improved due to the small sampling volume. For example,
the concentration of ethanol in the exhaled air of a healthy person at rest
is reported to be 37–207 ppb, however, the concentration of ethanol in
skin gas is extremely low (3.6–79.2 ppb/min), even when the entire
upper arm and hand are measured (Fenske and Paulson, 1999;
Mochalski et al., 2014b). The kinetics of skin gas emission are not well
defined. Therefore, it is important to consider the effects of sweat and
differences in the measurement site. Therefore, it is difficult to measure
skin gas with existing devices, and there is a need for a gas sensor with
high sensitivity and excellent gas selectivity.
In terms of sweat, VOCs emanated from the skin gas can be collected
continuously. Skin gas is hypothesized as a mixture of VOCs that are
transpired from blood directly, VOCs in sweat, and VOCs that are me­
tabolites of microorganisms on the skin (LaurentDormont et al., 2013;
Hara et al., 2014). In human volatiles measurement, the relationship
between skin gas and sweat is also important. Studies of gas measure­
ment near the ear, where sweat evaporation is low, have been reported
(Arakawa et al., 2022; Iitani et al., 2020).
In our previous study, we developed a “fiber optic gas sensor” (bio-
sniffer) with high selectivity using NADH-dependent dehydrogenase as
the detection element (Kudo et al., 2010; Toma et al., 2021). We re­
ported the detection of percutaneous ethanol gas after alcohol ingestion
(Arakawa et al., 2020). The bio-sniffer technology has also been applied
to achieve optical measurement of ethanol in exhaled breath and skin
gas after alcohol ingestion (Arakawa et al., 2015; Iitani et al., 2019).
However, both methods require large measurement systems and have
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Biosensors and Bioelectronics: X 11 (2022) 100169
poor portability, so are of limited use in daily life. For this reason, we
constructed a headset-type gas measurement system that is easy to
attach and detach. In this study, we constructed a wearable and potable
ethanol gas measurement system using a wireless complementary
metal-oxide semiconductor (CMOS) camera and applied it to the mea­
surement of ethanol gas in the ear canal after alcohol ingestion in order
to achieve unconstrained measurement of ethanol gas.
2. Experimental section
2.1. Skin ethanol gas measurement system using a wireless CMOS camera
Ethanol is oxidized to acetaldehyde by a reaction catalyzed by
alcohol dehydrogenase (ADH), which produces reduced NADH using
oxidized NAD (NAD+) as an electron donor. Equation (1) shows the
principle of ethanol gas measurement based on the fluorescence of co­
enzyme NADH. NADH is autofluorescent (ex: 340 nm, fl: 490 nm). By
combining an excitation light source and a wireless CMOS camera, we
could detect the NADH produced by the reduction reaction after gas
loading. In this way, we constructed a system to measure ethanol.
(1)
ADH
ethanol + NAD+ ̅→acetaldehyde + NADH + H +
To detect the concentration of ethanol gas, we used a wireless CMOS
camera to capture the change in fluorescence intensity, which increases
as NADH is produced due to the oxidation of ethanol gas via ADH. Fig. 1
shows a schematic of the ethanol gas measurement system using a
wireless CMOS camera. In this system, an ultraviolet light emitting
diode (UV-LED, λ = 340 nm, 340-FL-01-G01, DOWA Electronics Mate­
rials Co., Ltd.) was placed 31 mm from the ADH enzyme-immobilized
membrane at an angle of 32◦ , and a wireless CMOS camera was
placed 28 mm (center-to-center) from the enzyme membrane at an angle
of 42◦ to capture the fluorescence generated by the enzyme membrane.
The experimental system was optimized and designed to maximize
fluorescence intensity. A band-pass filter (BPF; λ = 340 ± 10 nm, Asahi
Spectra Co., Ltd.) was placed in front of the UV-LED, and a second BPF
(λ = 490 nm ± 10 nm, Asahi Spectra) was placed in front of the wireless
CMOS camera. Next, to perform ethanol gas measurement, we first had
to select a wireless CMOS camera to detect the fluorescence of NADH.
Three wireless CMOS cameras, C1 (k-camera-020, China), C2 (mc-
ut015, China), and C3 (ABC-108RB, Japan), were compared.
In the experiments, NADH solutions of various concentrations (0.1
μM–100 mM) were wetted onto hydrophilized polytetrafluoroethylene
(Omnipore™, H-PTFE membrane filters, 0.2 μm Merck Millipore Ltd.
Ireland) cut to 2 × 2 cm2. The quantitative properties of NADH detection
were examined for each camera. H-PTFE membranes soaked with 100
μM NADH were measured for 4 min to investigate the stability of the
fluorescence intensity of each camera during long-term imaging.
The ADH enzyme-immobilized membranes were prepared by
immobilizing 40 μL of ADH (E.C.1.1.1.1, 128 units/mg, Oriental Yeast
Co., Ltd. Japan) enzyme solution onto H-PTFE membrane (pore size: 0.2
μm, thickness: 80 μm, Merck Millipore Ltd. Ireland) carrier. A mixture of
enzyme and a copolymer (PMEH; 15% wt in ethanol (poly [MPC-co-
EHMA])) of 2-methacryloyloxyethyl phosphorylcholine (MPC) and 2-
ethylhexyl methacrylate (EHMA) was spread (60 units/cm2) on the H-
PTFE membrane, which was then dried in a cool dark place at 4 ◦ C for 3
h (Arakawa et al., 2019; Iwasaki and Ishihara, 2012). The
enzyme-immobilized membrane was soaked with 50 μL of 500 μM/l
NAD+ solution prepared in phosphate buffer (pH 8.0, 100 mM). For
ethanol gas measurement, standard ethanol gas (11 ppb–444 ppm) of
various concentrations prepared by a gas generator (Permeater,
custom-ordered low concentration gas generator and PD-1B-2, GASTEC.
Co. Japan). This gas generation system utilizes permeation and diffusion
tubes to generate the calibration gas by a compressed air as the dilution
gas. Ethanol gas was loaded on the enzyme membrane, and the fluo­
rescence intensity due to the reduction reaction was quantified to
T. Arakawa et al. Biosensors and Bioelectronics: X 11 (2022) 100169

Fig. 1. Schematic image of the portable human volatile measurement system for ear canal gases using headset gas sensor and a wireless CMOS camera. A UV-LED
was placed 31 mm from the ADH enzyme-immobilized membrane at an angle of 32◦ , and a wireless CMOS camera was placed 28 mm (center-to-center) from the
enzyme membrane at an angle of 42◦ to capture the fluorescence generated by the enzyme membrane.

evaluate the quantitative characteristics of this system. In the evaluation
of the selectivity of the system for ethanol gas, the main VOC compo­
nents in human volatiles (ethanol, methanol, 1-propanol, 2-propanol,
1-butanol, acetaldehyde, formaldehyde, acetone, and 2-butanone) at a
concentration of 1 ppm were loaded onto the sensor and the outputs
were compared.
In order to construct a battery-powered ear canal gas measurement
system, we fabricated a new operating circuit to drive the UV-LED with a
battery in order to downsize the DC stabilized power supply (GS200;
Yokogawa Test & Measurement Co., Ltd. Japan) used in the ethanol gas
measurement system. This circuit was composed of a lithium-ion battery
(3.7 V, 2500 mAh, P11-18650STD-D, SEnergy Co., Ltd. Japan), a DC/DC
converter (TPS61088), and a variable current source IC (LT3092EST,
Analog Devices Co., Ltd. USA), as shown in Supplementary Fig. 1. It was
designed to be rechargeable. The output voltage of the DC/DC converter
was set to 10 V for stable operation, since the voltage at both ends of the
UV-LEDs and the variable current source IC were 4.5 V and 3 V,
respectively, when operating at 100 mA for UV-LEDs irradiation. The
stability of the current flowing through the UV-LED and the output light
intensity is important for the measurement of low concentrations of
ethanol gas. In order to evaluate the stability of the current and the light
intensity output from the UV-LED, we measured the current and the light
intensity for 2 h using a power meter (7Z01550, Ophir Optronics
Solutions, Ltd. USA) and an ampere meter (2450, Tektronix, Inc. USA) as
shown in Supporting Fig. 2. The distance from the UV-LED to the light
collector of the power meter was the same as the distance between the
UV-LED and the ADH enzyme-immobilized film in the ethanol gas
measurement system. Next, ethanol gas measurements were conducted
using the UV-LED operating circuit fabricated as described above. For
the gas measurements, standard ethanol gas prepared by the permeator
was loaded as above, and the videos of the fluorescence intensity
changes captured by the wireless CMOS camera were quantified.
2.2. Construction and characterization of the ethanol measurement
system for ear canal gas
The ethanol gas measurement system was housed inside a headset-
type earmuff (X5A, 3M Co., Ltd. USA). The ear cups of the earmuffs
were made of ABS plastic, and the cushion in contact with the outer ear
was made of soft polyurethane foam covered with a soft vinyl chloride
sheet. The ethanol gas measurement system was installed in the earmuff
using a jig made by a 3D printer (MF-2500EPII, MUTOH INDUSTRIES
LTD. Japan).
Supplementary Fig. 3 shows the ethanol gas measurement system
situated in the headset ear canal gas measurement device. In this system,
an PTFE membrane (pore size: 5 μm, thickness: 100 μm, Sumitomo

Fig. 2. The UV-LED operating circuit and DC power supply (left), a photo of headset-type ear canal gas measurement device in use (center), and the ethanol gas
measurement system (right).

3
T. Arakawa et al.
Electric Industries, Japan) was attached to the tip of a funnel to simulate
ear canal gas, and a low-flow gas that mimicked skin gas was loaded
onto the ADH enzyme-immobilized membrane. In the experiment,
various concentrations of standard ethanol gas (11 ppb–444 ppm) were
loaded for 5 min, and the quantitative characteristics of the system were
evaluated by integrating the increase in fluorescence intensity due to the
reduction reaction.
2.3. Gaseous ethanol measurements from ear canal gas
Ethanol gas measurement in the ear canal gas during alcohol con­
sumption was conducted using the headset ear canal gas measurement
device we fabricated. This study was approved by the ethics committee
of Tokyo Medical and Dental University, School of Medicine (approval
number: M2018-160). For alcohol intake, body temperature, blood
pressure, and pulse rate were measured and confirmed in healthy adult
participants who had consented to the experiment. The purpose of the
experiment was explained beforehand and the participants were given a
questionnaire on diet and lifestyle. An alcohol patch test using ethanol
was conducted to determine ALDH2 [+] (active) and ALDH2 [-] (inac­
tive) type. Participants had not taken any medication or consumed any
alcohol within the 72 h prior to the test. They fasted for at least 4 h
before consuming the alcoholic beverages orally at a dose of 0.4 g of
alcohol per kg of body weight.
For measurements, the headset-type ear canal gas measurement de­
vice attached the enzyme membrane was worn to measure the ear canal
gas every 20 min after the alcohol was ingested. The concentration of
exhaled ethanol gas was also examined using a detector tube. In the
exhaled gas collection. A human subject first took a large breath in
through the nose, held it for 10 s, exhaled slowly for 3 s to remove the
Fig. 3. Characteristics of the ethanol gas measurement system situated in the headset ear canal gas measurement device (a) Fluorescence images after the loading of
each indicated concentration of ethanol gas. (b) Time dependence of fluorescence intensity on each ethanol gas concentration. (c) Quantitative characteristics of the
ethanol gas sensor.
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Biosensors and Bioelectronics: X 11 (2022) 100169
exhaled air that had accumulated in the physiologic dead space, and
then blew the rest of their exhaled air into a sample bag (1L, 1-7605-01,
AS ONE, Japan) at 36 ◦ C. The ethanol gas concentration was evaluated
using an ethanol detector tube (112L, GASTEC Co., Ltd. Japan).
3. Results and discussion
3.1. Measuring ethanol gas using a wireless CMOS camera
In order to select the best wireless CMOS camera, we conducted an
experiment to compare the performances of three cameras. First, the
quantitative characteristics (Supplementary Fig. 4 (a)) and signal-to-
noise ratio (Supplementary Fig. 4 (b)) for NADH were calculated for
the three wireless CMOS cameras. The results showed that C1 and C2
had similar detection sensitivities for NADH, and C1 had the best signal-
to-noise ratio. Next, we investigated the stability of the fluorescence
intensity of the image for each camera during long-term operation.
Supplementary Fig. 5 shows the change in fluorescence intensity for
each camera during 4 min of fluorescence captured from 100 μM NADH.
Stable fluorescence intensity was observed using C1 and C3. However, in
C2, periodic fluctuations in fluorescence intensity were observed. This
was caused by the automatic readjustment of the camera’s ISO sensi­
tivity due to continuous shooting in a dark environment. In terms of
sensitivity to NADH, signal-to-noise ratio, and stability of fluorescence
intensity over a long period of time, C1 was most suitable for NADH
measurements. Additionally, the operating time of C1 was 12 h, longer
than that of the other cameras. We decided to use C1 as the wireless
CMOS camera for subsequent evaluations and it was integrated into the
ethanol gas measurement system.
Next, standard ethanol gas of various concentrations was loaded on
T. Arakawa et al. Biosensors and Bioelectronics: X 11 (2022) 100169

the ADH enzyme-immobilized membrane to investigate the character­

istics of the measurement system. Fig. 3 shows the fluorescence images,
fluorescence intensity changes, and quantitative characteristics in
response to ethanol gas. The fluorescence of the images varied according
to the ethanol concentration. In comparison with the change in fluo­
rescence intensity for the carrier gas alone, a change in fluorescence
intensity was observed even for low concentrations of ethanol gas. Based
on the steady-state values of fluorescence intensity, we investigated the
quantitative characteristics of ethanol gas and determined it was
possible to quantify 11 ppb–129 ppm, a range that includes the skin
ethanol gas concentration after alcohol ingestion (70–110 ppb/cm2).
The selectivity was evaluated by loading representative VOCs into
the system. Supplementary Fig. 6 shows the relative output for other gas
components (1 ppm) when the output for ethanol gas was set to 100%.
The highest output was observed for ethanol. Since the exhaled con­
centration of 1-propanol (8.1 ppb) is sufficiently low compared to that of
ethanol (78 ppm or higher) after alcohol ingestion, the effect of 1-prop­
anol on the measurement of ethanol gas after drinking is considered to
be negligible. The selectivity of this sensor for ethanol based on the
enzyme-substrate specificity of ADH was confirmed.

3.2. Battery-powered ear canal gas measurement system

We measured the current flowing through the UV-LEDs and the

amount of light output from the UV-LEDs in a system with a battery-
powered operating circuit to investigate the operational stability of
the UV-LEDs. Supplementary Fig. 7 shows the measurement results of
the current flowing to the UV-LEDs and the light intensity. Immediately
after power-on, the UV-LED current was 100.9 mA. It stabilized to the set
value within 3 min. The peak-to-peak current flowing through the UV-
LEDs at the time of stabilization was Ipp = 28 μA, and the stability of
the light source was confirmed by measuring the light intensity of the
UV-LEDs. The increase in the current value immediately after the power
turned on was a transient phenomenon called rush current (a large
current that flows the moment voltage was applied), which was caused
by the capacitor connected to the DC/DC converter. However, the UV-
LED used in this system can deliver a current of up to 350 mA. The ef­
fect of the increase in current due to power-on was considered to be
small. In addition, the output current from the lithium-ion battery was
measured to be 310 mA. Since the nominal voltage of the battery used
was 3.7 V and capacity was 2500 mAh, the system could be operated
continuously for 8 h. Fig. 4. Comparison of the wearable human volatile measurement system using
either the DC power supply or a UV-LED drive circuit. (a) Fluorescence intensity
change to ethanol vapor. (b) Quantitative characteristics of each measure­
3.3. Characterization of the system for measuring ethanol
ment system.

We conducted ethanol gas measurements using the battery-powered

imaging system. Fig. 4 (a) shows the change in fluorescence intensity in
response to ethanol gas load, and (b) compares the fluorescence in­
tensity of the battery-powered UV-LED drive circuit with DC power
supply. We observed an increase in fluorescence intensity with gas load
and a stable fluorescence intensity according to the ethanol concentra­
tion. The same level of fluorescence intensity was obtained using the
battery-powered UV-LED drive circuit and the DC power supply. This
allowed us to measure ethanol gas using a battery-powered device.
Fig. 5 (a) shows the change in fluorescence intensity detected by the
wireless CMOS camera during a 5 min exposure to ethanol gas. We
observed an increase in fluorescence intensity along with increasing
ethanol concentration. However, when ethanol gas of 129 ppm or higher
was loaded, the increase in fluorescence intensity decreased over time.
This may be due to the corresponding decrease in the concentration of
available NAD+ on the enzyme membrane.
The time integration of the fluorescence intensity is shown in Fig. 5
(b). In order to obtain quantitative characteristics from this integration
analysis, the quantitative characteristics for ethanol gas were investi­
gated from the integrated values at t = 1, 2, 3, 4, and 5 min (Fig. 5 (c)).
The results showed that from 3, 4, and 5 min, ethanol gas could be
quantified at 11 ppb–444 ppm, which includes the skin gas concentra­
tion after alcohol is ingested (70–110 ppb/cm2). To evaluate variability,
the coefficient of variation (CV) values of the integrated values were
calculated. The CV values of the integrated values at 4 and 5 min were
lower than those of other times, and measurement with less variation
was possible (Fig. 5 (d)). Since the CV values of the integrated values at 4
and 5 min were similar, subsequent gas measurements were performed
using the integrated values at 4 min.
3.4. Characterization of ethanol measurements in the ear canal
The headset imaging system was used to measure the ear canal gas
during and after alcohol consumption. When this system was attached to
the auricle of ear, the ear canal gases were tested every 20 min after
alcohol consumption and an increase in the fluorescence intensity
derived from ethanol in the ear canal gas was confirmed. Supplementary
Video 1 shows the ear canal gas measurements from the device and the
actual output changes when the device was worn by a human partici­
pant. Fig. 6 shows the results of ethanol gas measurement in the ear
canal gas associated with alcohol consumption and the concentration of

5
T. Arakawa et al.
Fig. 5. Analysis of the concentration of ethanol gas detected by the headset ear canal gas measurement system. (a) Fluorescence intensity changes. (b) Integral
analysis results. (c) Quantitative characteristics. (d) Comparison of coefficient value of integral time.
Fig. 6. Changes over time in ethanol gas concentration in exhaled breath and
ear canal gas (assessed using the headset-type ear canal gas measurement sys­
tem) after alcohol ingestion.
ethanol gas in the exhaled air measured with a detector tube. The con­
centration of ethanol gas in the ear canal was defined as the concen­
tration (ppm/cm2) per 15.7 cm2, representing the inner area of the ear
pad of the headset.
Supplementary video related to this article can be found at https://
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Biosensors and Bioelectronics: X 11 (2022) 100169
doi.org/10.1016/j.biosx.2022.100169
The ethanol concentration in the ear canal gas reached its peak (2.4
ppm/cm2) 70 min after alcohol ingestion, and then gradually decreased
due to alcohol metabolism. A relatively similar change was observed in
the results of exhalation measurements using detector tubes conducted
at the same time. Compared to the ethanol concentration of exhaled
breath, the peak value of ear canal gas was delayed, which was in
agreement with results obtained in a previous report (Toma et al., 2021).
The ethanol concentration in the ear canal gas was higher than that
in skin gas (70–110 ppb/cm2) during alcohol consumption. This may be
due to the mastoid honeycomb and sweat glands in the ear canal, which
are responsible for the gas ventilation function of the middle ear cavity,
located deeper than the ear canal. The mastoid honeycomb (surface
area: about 89.1 cm2), like the alveoli, has a thin layer of squamous
epithelium directly above abundant capillaries, which may facilitate the
permeation of gas components from the blood compared to other skin
areas. In addition, the release of ethanol gas from the sweat glands in the
ear canal due to perspiration may also add to the high concentration of
ethanol in ear canal gas.
A possible reason for the delay is the difference in the thickness of the
layer through which the VOCs components penetrate. The VOCs
permeable layer (vascular air barrier) in the alveoli, where exhaled gas
and blood VOCs are exchanged, is extremely thin at 2.2 μm. On the other
hand, the tympanic membrane, which separates the middle ear cavity
from the ear canal, is 50–100 μm thick. Therefore, VOCs do not diffuse
through to the ear canal gas as easily as VOCs diffuse to exhaled air.
T. Arakawa et al.
4. Conclusion
In this study, we developed a biofluorescent gas imaging system for
ethanol using a wireless CMOS camera and constructed an unrestrained,
headset style “ear canal gas measurement system” for routine metabolic
monitoring. This system incorporates a wireless CMOS camera, an ADH
enzyme-immobilized membrane, and a UV-LED inside a headset style
earmuff, and is capable of quantifying 11 ppb–444 ppm ethanol, a range
that includes the ethanol gas concentration in the skin gas of a healthy
person after alcohol has been consumed. As an application of this system
to the measurement of human volatiles, we measured the gas in the ear
canal during and after alcohol ingestion, and were able to observe the
increase in ethanol-derived fluorescence in the ear canal. It was possible
to observe both the increase in ethanol concentration in the ear canal gas
with alcohol consumption and the gradual decrease in ethanol concen­
tration due to metabolism in the human body, confirming the usefulness
of this system. There is a responsiveness issue because the imaging is
based on an enzymatic reaction. Although it cannot adequately respond
to rapid changes in concentration, the effect on the measurement of skin
volatiles would be small.
Future improvements to enable continuous gas measurement would
allow for real-time measurement of human volatiles and non-invasive
metabolic sensing using ear canal gases.
CRediT authorship contribution statement
Takahiro Arakawa: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Resources, Data curation, Writing –
original draft, Visualization, Supervision, Funding acquisition. Riki
Ishikawa: Methodology, Software, Validation, Formal analysis, Inves­
tigation, Resources, Data curation. Kenta Iitani: Methodology, Valida­
tion, Formal analysis, Investigation, Resources, Data curation. Koji
Toma: Conceptualization, Methodology, Validation, Investigation.
Yasuhiko Iwasaki: Conceptualization, Investigation, Resources. Kohji
Mitsubayashi: Conceptualization, Investigation, Writing – review &
editing, Project administration, Funding acquisition, All authors have
read and agreed to the published version of the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by the JSPS KAKENHI grant numbers
JP19J01649, JP17H01759, JP16J09604, and JP15H04013; and the
Ministry of Education, Culture, Sports, Science and Technology (MEXT)
Special Funds for “Cooperative Research Project of Research Center for
Biomedical Engineering”.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biosx.2022.100169.
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