sensors
ArticleBiochemical Methanol Gas Sensor (MeOH Bio-Sniffer) for
Non-Invasive Assessment of Intestinal Flora from
Breath Methanol
Koji Toma 1 , Kanako Iwasaki 2 , Geng Zhang 2 , Kenta Iitani 1 , Takahiro Arakawa 1 , Yasuhiko Iwasaki 3
and Kohji Mitsubayashi 1,2, *
1

2

3

*

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Citation: Toma, K.; Iwasaki, K.;
Zhang, G.; Iitani, K.; Arakawa, T.;
Iwasaki, Y.; Mitsubayashi, K.
Biochemical Methanol Gas Sensor(MeOH Bio-Sniffer) for Non-Invasive
Assessment of Intestinal Flora from
Breath Methanol. Sensors 2021, 21,4897. https://doi.org/10.3390/
s21144897
Academic Editor: Roberto
Pizzoferrato
Received: 17 June 2021
Accepted: 16 July 2021

Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering,Tokyo Medical and Dental University (TMDU), 2-3-10 Kanda-Surugadai, Chiyoda-ku,
Tokyo 101-0062, Japan; toma.bdi@tmd.ac.jp (K.T.); i.bdi@tmd.ac.jp (K.I.); arakawa.bdi@tmd.ac.jp (T.A.)
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU),
1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; ma170016@outlook.jp (K.I.); ma201102@tmd.ac.jp (G.Z.)
Faculty of Chemistry, Materials and Bioengineering, Kansai University, Osaka 564-8680, Japan;
yasu.bmt@kansai-u.ac.jp
Correspondence: m.bdi@tmd.ac.jp

Abstract: Methanol (MeOH) in exhaled breath has potential for non-invasive assessment of intestinal
flora. In this study, we have developed a biochemical gas sensor (bio-sniffer) for MeOH in the gas
phase using fluorometry and a cascade reaction with two enzymes, alcohol oxidase (AOD) andformaldehyde dehydrogenase (FALDH). In the cascade reaction, oxidation of MeOH was initially catalyzed by AOD to produce formaldehyde, and then this formaldehyde was successively oxidized via
FALDH catalysis together with reduction of oxidized form of β-nicotinamide adenine dinucleotide(NAD+ ). As a result of the cascade reaction, reduced form of NAD (NADH) was produced, and
MeOH vapor was measured by detecting autofluorescence of NADH. In the development of the
MeOH bio-sniffer, three conditions were optimized: selecting a suitable FALDH for better discrimination of MeOH from ethanol in the cascade reaction; buffer pH that maximizes the cascade reaction;and materials and methods to prevent leaking of NAD+ solution from an AOD-FALDH membrane.
The dynamic range of the constructed MeOH bio-sniffer was 0.32–20 ppm, which encompassed the
MeOH concentration in exhaled breath of healthy people. The measurement of exhaled breath of ahealthy subject showed a similar sensorgram to the standard MeOH vapor. These results suggest that
the MeOH bio-sniffer exploiting the cascade reaction will become a powerful tool for the non-invasive
intestinal flora testing.
Keywords: intestinal flora; methanol; gas-phase biosensor; enzymatic cascade reaction; fluorescence

Published: 19 July 2021

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1. Introduction
Intestinal flora has been gaining its attention year by year after several reports weremade regarding influences of the intestinal flora to general health conditions [1,2]. In
particular, two dominant bacteria, the Bacteroidetes and the Firmicutes [3–6], play importantroles as their composition in intestinal bacteria, and metabolites affect the host’s immune
function, obesity, drug metabolism, and absorption [7–9].
Currently, there are two main methodologies to assess the status of intestinal flora:
culture method and genome analysis. In culture method, bacteria extracted from stool
sample are cultured, and the resultant morphology and color tone of the colonies areobserved [10,11]. This method is inexpensive, but one needs a long time to have results.
Furthermore, it is unable to observe more than 90% of intestinal microorganisms becauseof lack of growth substrates and difficulties in maintaining the culture environment suchas temperature, pH, and osmotic pressure, which are varied for different bacteria [12].
For example, it is hard for obligatory anaerobic bacteria such as Bacteroidetes to survive

Sensors 2021, 21, 4897. https://doi.org/10.3390/s21144897

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Sensors 2021, 21, 4897

2 of 11and grow in healthy conditions if the stool sample is exposed in ambient air for a longtime. Therefore, this method is suitable for detecting the presence of bacteria of interestin a stool sample that one can grow, but not for reflecting actual intestinal flora nor for
quantitative assessment.
On the other hand, in genome analysis with next generation sequencing, it is possible
to obtain nucleotide sequence information rapidly along with high-throughput without theculturing. This method identifies sequential information of 16S ribosomal RNA (rRNA),
which is amplified using polymerase chain reaction (PCR). The obtained sequential information allows phylogenetic classification and identification of bacteria. A report said
93% of 1822 16S rRNA sequences from intestine bacteria that are detectable by the genome
analysis cannot be identified by the cultured method [13]. The disadvantage of this method
is the cost through molecular phylogenetic processes [14].
Methanol (MeOH) is produced from the degradation of dietary fiber, pectin, and byintestinal bacteria, including Bacteroidetes [15–17]. This produced MeOH is transferredto the blood stream, and a fragment of the blood MeOH is released through exhaled
breath. The study of Laakso et al. showed a good correlation of the MeOH concentration
between exhaled breath and blood [18]. There are some reports demonstrating increasein MeOH concentration in exhaled breath after intaking pectin. For example, a study by
Dorokhov et al. observed the increase of the blood MeOH concentration up to 1.5 times after
eating vegetables [19]. According to a study by Lindinger et al., eating pectin-contained
fruits, such as apples, lead to the increase in breath MeOH concentration [20]. These
reports suggest the potential for non-invasive assessment of intestinal bacteria activity bymeasuring MeOH in exhaled breath.
MeOH in exhaled breath has been analyzed by various methodologies, such as gasliquid chromatography [21], proton-transfer-reaction mass spectrometry (PTR-MS) [20],and selected-ion flow-tube mass spectrometry (SIFT-MS) [22,23]. Despite their capabilityof identifying MeOH in gas mixture, the size and complexity of the system are drawbacks
for the on-site MeOH assessment.
On the other hand, sensors are expected to be used closer to the site than lab-based
analytical devices. Therefore, it is required that sensors be simple to operate and compact insize along with sufficient sensitivity, selectivity, and capability of continuous measurement
for some occasions [24–29]. However, generally, there is a trade-off between being compact
in size and having high sensitivity, and it is very challenging to meet both requirements. To
overcome these challenges, a lot of efforts have been devoted to improving the sensitivity
and selectivity of MeOH gas sensors. For example, Li et al. synthesized a Pt-SnO2 nanoparticles composite for a chemoresistive gas sensor [30]. An alumina tube surface was coatedwith this nanocomposite to fabricate a sensor, and MeOH vapor was measured by changes
in the resistance of the sensor. As a result, the sensor using Pt-SnO2 showed about 10-foldbetter sensor output to 100 ppm methanol vapor at 120 ◦ C lower operating temperaturethan those with pristine SnO2 microspheres (220 ◦ C). As an another SnO2 -based chemoresistive gas sensor, Chen et al. developed a lanthanum-doped SnO2 nanocomposite. Similar
to Li’s study, the lanthanum-doped SnO2 nanocomposite was coated on a ceramic tube, and
MeOH vapor was measured by changes in the resistance. The sensor output was improvedby a factor of 7 with the lanthanum-doped SnO2 compared to the sensor with only SnO2
when measuring 75 ppm MeOH vapor at the operating temperature of 220 ◦ C [31]. A
MeOH gas sensor employing metal-organic framework was reported by Andrés et al. [32].
The sensor was fabricated by depositing MIL-96(Al) nanoparticles on a Si/SiO2 substratewith interdigitated electrodes. It showed different sensor outputs to water and MeOH
vapor from other VOCs, which will be useful to identify these two components out of gas
mixture. There are also reports regarding handheld MeOH gas sensors for immediate use
in the field [18,33,34]. Broek et al. introduced a MeOH sensor that was composed of aseparation column and Pd-doped SnO2 chemoresistive sensor. This combination made thesize compact and made it possible to measure MeOH vapor even in the presence of muchhigher concentration of ethanol within 2 min [33]. As another approach, recent advances inSensors 2021, 21, 4897

3 of 11

machine learning were applied to an electronic nose (e-nose) with a pattern recognition
algorithm. Due to their high technological affinity, the e-nose using graphene FET was able
to classify water, MeOH, and ethanol with high accuracy [35].
Although there has been great advancement of such sensors, it is not yet easy to
selectively measure MeOH. Recently, we have developed a liquid phase biosensor for
MeOH (the AOD-FALDH fluorosensor) that employed two-enzyme cascade reaction withalcohol oxidase (AOD), formaldehyde dehydrogenase (FALDH), and fluorometry [36].
The cascade reaction begins with oxidation of MeOH via AOD to produce formaldehyde,
and then produced formaldehyde is oxidized via FALDH together with reduction of acoenzyme, which is an oxidized form of β-nicotinamide adenine dinucleotide (NAD+ ).
MeOH was measured by detecting autofluorescence from a reduced form of NAD (NADH).
Due to the cascade reaction, the AOD-FALDH fluorosensor was able to measure MeOH
with high selectivity, especially from other aliphatic alcohols, including ethanol. Basedon these results, it is expected that the AOD-FALDH fluorescence is applicable to the
non-invasive measurement of MeOH in such body fluids as urine or saliva, but it would
be quicker and easier if the same assessment could be made with exhaled breath. As
mentioned above, the measurement of gas phase samples is more challenging than liquid
phase samples because gas phase samples are usually invisible and more rapidly diffuse.
In particular, exhaled breath contains a considerable portion of water with the relative
humidity of 65–91%, according to the study by Mansour et al. [37]. Besides, the MeOH
concentration in breath is approximately 3000-times lower than that in blood [18]. Therefore,
a sensor for MeOH in exhaled breath needs to be highly sensitive to MeOH as wellas insensitive to humidity. Our biochemical gas sensor (bio-sniffer) has shown thoserequired characteristics in previous studies for ethanol, acetaldehyde, isopropanol, andacetone [38–41], and in this study, we combined the advantages of the bio-sniffer and
AOD-FALDH cascade reaction to measure MeOH in exhaled breath.
2. Materials and Methods
2.1. Materials and Reagents
Alcohol oxidase (AOD, A2404-1KU, from Pichia pastoris) was obtained from SigmaAldrich Japan (Tokyo, Japan). Formaldehyde dehydrogenase (FALDH, from Pseudomonassp.) was from two different manufacturers, Funakoshi (Tokyo, Japan) and Toyobo (Osaka, Japan). An oxidized form of β-nicotinamide adenine dinucleotide (NAD+ ) was
purchased from Oriental Yeast (Tokyo, Japan). A hydrophilic polytetrafluoroethylene(H-PTFE) membrane (thickness: 80 µm, porosity: 80%, pore size: 0.2 µm) was from
Millipore (Burlington, MA, USA). Poly [2-methacryloyloxyethyl phosphorylcholine (MPC)co-2-ethylhexyl methacrylate (EHMA)] (PMEH) was synthesized in house by the free
radical-polymerization method [42]. Polyethyleneimine (PEI, average molecular weightof 10,000, 164-17821) and glutaraldehyde (GA, 079-00533) were purchased from Fujifilm
Wako Pure Chemical (Osaka, Japan).
All reagents for the following buffer solutions were obtained from Fujifilm Wako
Pure Chemical (Osaka, Japan). For the phosphate buffer (PB, 100 mM) solution, we added
potassium dihydrogen phosphate solution (100 mM in ultrapure water) to disodiumhydrogen phosphate solution (100 mM in ultrapure water) to buffer the solution pH
from 6.5 to 8.5. For the Tris-HCl solution (100 mM), we added HCl solution to 100 mM
trimethylolaminomethane solution to buffer the pH from 8.5 to 10.5.
2.2. Construction of MeOH Bio-Sniffer
The MeOH bio-sniffer exploited a cascade reaction with AOD and FALDH that is
described elsewhere (Figure 1a) [36]. The reaction begins with oxidation of MeOH via AOD
catalysis, which produces formaldehyde. Then, oxidation of the produced formaldehyde
occurs via FALDH in conjunction with the reduction of NAD+ , in which formic acidand NADH are produced. We measured MeOH vapor indirectly by autofluorescence ofNADH, whose emission λem and excitation λex wavelengths were 490 nm and 340 nm,

Sensors 2021, 21, 4897

methacrylate was attached. An AOD-FALDH membrane was attached on the flow-celland worked as a gas-liquid diaphragm (Figure 1b). In the flow-cell, NAD+ solution flowed
over the AOD-FALDH membrane at the flow rate of 1.5 mL/min using a peristatic pump(MP-1000-H, Tokyo Rikakikai, Tokyo, Japan). When MeOH vapor reached and diffused
into the membrane, the enzymatic reaction described in Figure 1a occurred, which re4 of 11
sulted in producing NADH in the solution. The excitation UV light, which originated fromthe LED and traveled through the bifurcated optical fiber and the optical fiber probe, wasirradiated to NADH in the solution, and then emitted fluorescence from NADH was collectedby the opticalfiber. Afterthe
beingfiltered bya bandpass
filter if(BPF,
λ = 490 ± 5 nm,
respectively.
In this principle,
fluorescenceintensity
increased
the concentrationof
Asahi
Spectra,Tokyo,
Japan),
thefluorescence
light
was
detected
by
the
PMT.
MeOH increased.

Figure1.1.(a)(a)Measurement
Measurementprinciple,
principle,andand(b)(b)aaschematic
schematicillustration
illustrationof
ofthe
theMeOH
MeOHbio-sniffer.
bio-sniffer.
FigureThe device
configuration Membraneof the MeOH bio-sniffer was similar to the ones in our
2.3. Preparationof AOD-FALDH
previous studies except for the membrane [39,43,44]. Briefly, it consisted of a bifurcatedFirst, waterproofing was made on the gas phase side of the membrane, which was
optical fiber (PVSMA2-2 STU600-STUH190S, Mitsubishi Cable Industries, Tokyo, Japan),
opposite to the side of AOD and FALDH being immobilized, because the AOD-FALDH
an optical fiber probe (F1000-900, Ocean Insight, Orlando, FL, USA), a UV light emitting
membrane was hydrophilic, and thus, the NAD+ solution in the flow-cell had leaked into
diode (UV-LED, UF4LU-0GD01, DOWA, Tokyo, Japan), and a photomultiplier tube (PMT,C9692-11, Hamamatsu Photonics, Shizuoka, Japan). At the end of the optical fiber probethat was connected to the bifurcated optical fiber, a flow-cell made from poly methyl
methacrylate was attached. An AOD-FALDH membrane was attached on the flow-celland worked as a gas-liquid diaphragm (Figure 1b). In the flow-cell, NAD+ solution flowed
over the AOD-FALDH membrane at the flow rate of 1.5 mL/min using a peristatic pump(MP-1000-H, Tokyo Rikakikai, Tokyo, Japan). When MeOH vapor reached and diffused into
the membrane, the enzymatic reaction described in Figure 1a occurred, which resulted in
producing NADH in the solution. The excitation UV light, which originated from the LED
and traveled through the bifurcated optical fiber and the optical fiber probe, was irradiatedto NADH in the solution, and then emitted fluorescence from NADH was collected by the
optical fiber. After being filtered by a bandpass filter (BPF, λ = 490 ± 5 nm, Asahi Spectra,
Tokyo, Japan), the fluorescence light was detected by the PMT.
2.3. Preparation of AOD-FALDH MembraneFirst, waterproofing was made on the gas phase side of the membrane, which was
opposite to the side of AOD and FALDH being immobilized, because the AOD-FALDH
membrane was hydrophilic, and thus, the NAD+ solution in the flow-cell had leaked into
the gas phase (Figure 2). In order to avoid both sides of a H-PTFE membrane becominghydrophobic, the H-PTFE membrane was moderately wetted with ultrapure water before
the treatment. Then, a surface of the H-PTFE membrane was coated with a hydrophobic
material. After the coating, the H-PTFE membrane was dried out, and the membrane beinghydrophobic on onside and hydrophilic on the other side was obtained. In the waterproof
treatment, three different materials, including PMEH, a fluorine resin (Amedas, Columbus,
Tokyo, Japan), and a silicone resin (NeverWet, Lancaster, PA, USA), were compared.

Sensors 2021, 21, 4897

from the membrane with 300 μL PB for three times. Afterwards, we dropped 300 μL GA
solution (2.5v/v% in PB) to the membrane and gently shook it for 5 min to crosslink PEI
and GA. After rinsing excessive GA from the membrane with PB for three times, a mixed
solution of AOD (2.0 unit/cm2) and FALDH (1.0 unit/cm2) was casted over the membrane
and left for 15 min in a dark cold place (4 °C) to immobilize them. Finally, Tris-HCl solu5 of 11
tion was used to passivate the residual aldehyde group of GA, which would cause nonspecific binding of NADH to GA.

FigureFigure2.2.Preparation
Preparationprocesses
processesof
ofthe
theAOD-FALDH
AOD-FALDH membrane.
membrane.

2.4. Measurement
Standard
MeOH orVapor
and Exhaled
MeOH
PMEH was ofeitherspin-coatedhand-coated.
InBreaththe caseof spin-coating, a 2 × 2 cm2
H-PTFEwettedwithbio-sniffer
ultrapure water
was fixed onwith
a silicon
wafer,and thenFirst,membrane
the developed
MeOH
was characterized
standard
MeOH
vait
was
set
on
a
spin-coater(1H-D7,
Tokyo,
Mikasa,Japan).
After
addingPMEH
solution
por. The standard MeOH released from a gas generator (PD-1B-2, Gastec, Kanagawa, Ja(10 w/w%
in ethanol)to themembrane,it wasspin-coatedat spinrate
pan)
was pumped
to dropwise
the flow-cellof H-PTFE
the bio-sniffer
at theregulated
flow rateof 200
of
4000
rpm.
In
the
hand-coating,the
PMEH
solution
appliedtothe
H-PTFE
membranemL/min by a mass flow controller (FD-C1, Keyence, Osaka, Japan). The measurement bewaswith
spreadout wellbybaselinea hand.ofThewith thefluorineresinsiliconeresin wasganacquiringthethecoating
fluorescenceintensity
from
airor
cleanedthroughancarriedcarbon
out byfilter
spraying
a resin toGLa H-PTFE
for 5 sandandadrying
it for30 min.
active(3001-17201,
Sciences,membrane
Tokyo, Japan)
silica gel
filter(3001AOD and
FALDH
were
immobilizedon usedthis waterproofed
H-PTFE
membrane
17111,Next,
GL Sciences,
Japan).
Thisfiltered
air was also
as a carrier gas
for MeOH
vapor.
with the similar protocol that has been described in detail elsewhere [36]. AOD and
FALDH were crosslinked to PEI using GA on the hydrophilic side of a H-PTFE membranein the following procedures. We first dropped 300 µL PEI solution (1 w/w% in PB) onto a
2 × 2 cm2 H-PTFE membrane and gently shook it for 5 min. Then, we rinsed excessive PEI
from the membrane with 300 µL PB for three times. Afterwards, we dropped 300 µL GA
solution (2.5 v/v% in PB) to the membrane and gently shook it for 5 min to crosslink PEI
and GA. After rinsing excessive GA from the membrane with PB for three times, a mixed
solution of AOD (2.0 unit/cm2 ) and FALDH (1.0 unit/cm2 ) was casted over the membrane
and left for 15 min in a dark cold place (4 ◦ C) to immobilize them. Finally, Tris-HCl solution
was used to passivate the residual aldehyde group of GA, which would cause non-specificbinding of NADH to GA.
2.4. Measurement of Standard MeOH Vapor and Exhaled Breath MeOHFirst, the developed MeOH bio-sniffer was characterized with standard MeOH vapor.
The standard MeOH released from a gas generator (PD-1B-2, Gastec, Kanagawa, Japan)
was pumped to the flow-cell of the bio-sniffer at the regulated flow rate of 200 mL/minby a mass flow controller (FD-C1, Keyence, Osaka, Japan). The measurement began withacquiring the baseline of the fluorescence intensity from air cleaned through an active
carbon filter (3001-17201, GL Sciences, Tokyo, Japan) and a silica gel filter (3001-17111,
GL Sciences, Japan). This filtered air was also used as a carrier gas for MeOH vapor.
Then, the gas line was switched to the gas generator, and MeOH vapor flowed to thebio-sniffer for 5 min. Finally, the gas line was switched back to the filtered air to initialize
the measurement.
After the series of characterizations, the bio-sniffer was applied to the measurementof MeOH in exhaled breath of a healthy subject. This experiment was approved by the
ethics committee of Institute of Biomaterials and Bioengineering, Tokyo Medical and
Dental University (approval number: B2014-001), and was performed in accordance withthe guidelines and regulations after informed consent had been obtained from a subject.
Exhaled breath was collected from a subject who had been fasting for 2 h before theSensors 2021, 21, 4897

6 of 11experiment and had administered no alcohol and medicine for three days prior to theexperiment. The exhaled breath was sampled through the end-expiratory sampling. A
subject drew in a deep breath though the nose and stopped for 10 s. Then, the subject
breathed out through a mouth gently for 3 s to discard the dead space, followed by exhaling
into a gas sampling bag. In the breath measurement, we pumped the breath sample to the
MeOH bio-sniffer at the flow rate of 200 mL/min using a diaphragm pump (DSA-2-12,
Denso Sangyo, Tokyo, Japan).
3. Results and Discussion
3.1. Optimization of AOD-FALDH MembraneFirst, FALDH from different manufacturers were compared by the AOD-FALDH
fluorosensor. The details of the AOD-FALDH fluorescence was described in the previous
study [36]. Briefly, the AOD-FALDH membrane was attached at the end of the opticalfiber probe that was connected to a bifurcated optical fiber (BIF600-UV/VIS, Ocean Optics,
USA). Two ends of the bifurcated fiber were connected to the same UV-LED and PMT with
the MeOH bio-sniffer. The fiber probe with the AOD-FALDH membrane was immersed in
NAD+ solution (20 mM in pH8.5 PB) to excite NADH that was produced from the cascade
reaction of MeOH with AOD and FALDH. Please note that the AOD-FALDH membraneused in this liquid phase fluorosensor had no waterproof treatment.
Figure 3a shows a comparison of the sensor output of the AOD-FALDH fluorosensor
to 1 mM MeOH or ethanol solution. For the AOD-FALDH membrane using FALDH
from Funakoshi, the relative sensor output to ethanol solution was about 3 times lower
than that from Toyobo, indicating that higher selectivity to MeOH can be obtained using
FALDH from Funakoshi. This difference may be attributed to variations of bacteria andmanufacturing conditions between two manufacturers. Although both FALDH werefrom Pseudomonas species, at present, there are more than 180 validly named species of
Pseudomonas [45]. Therefore, it is supposed that activity and specificity of enzymes from
Sensors 2021, 21, x FOR PEER REVIEW
7 of 12different manufacturers are different. Based on this result, we decided to use FALDH from
Funakoshi to prepare the membrane in subsequent experiments.

Figure 3. (a) Comparison of sensor output to MeOH and ethanol solutions using two different
Figure 3. (a) Comparison of sensor output to MeOH and ethanol solutions using two different
FALDH.(b)(b)Influences
Influencesofofbuffer
buffersolution
solutionpH
pHon
onthe
theactivity
activityofofthe
theAOD-FALDH
AOD-FALDHmembrane.
membrane.Both
Both if
FALDH.
theexperiments
werecarriedout
bythe
AOD-FALDH
fluorosensor
intheliquidphase.
if the experiments were carried out by the AOD-FALDH fluorosensor in the liquid phase.

Next, the optimum pH for the AOD-FALDH membrane was investigated using the
The influences of waterproof materials on the AOD-FALDH membrane+ were invesAOD-FALDH fluorosensor. The pH of the buffer solutions containing NAD was changed
tigated. Except for the membrane coated with fluorine resin, no water leakage occurred.
from 6.5 to 10.5 using PB and Tris-HCl (Figure 3b). MeOH solution was dropped into
Figure 4 shows sensor responses to 4.8 ppm MeOH vapor by the MeOH bio-sniffer using
the buffer solution to make 1 mM MeOH solution while the probe of the AOD-FALDH
differently waterproofed AOD-FALD membranes. All types of membranes resulted in the
fluorosensor was immersed in the buffer solution. As a result of three trials, the fluorescenceincrease in the fluorescence from NADH upon applying MeOH vapor. Among them, theoutput from 1 mM MeOH solution increased with increasing pH and became the maximum
membrane waterproofed by spin-coating PMEH showed the largest sensor output alongwith a superior response represented by the time to reach 90% of the stable value (T90).
The membrane prepared by spin-coated PMEH exhibited T90 of 91 sec, which was morethan twice better than the others: casted PMEH, 229 sec; fluorine resin, 295 sec; silicone
resin, 195 sec. Based on the all abovementioned aspects, we decided to waterproof the gas-Sensors 2021, 21, 4897

7 of 11

with pH 8.5 PB solution. Although the fluorescence output from Tris-HCl solution at pH
Figure(a) Comparison
sensoroutputto MeOHethanol solutions
differentwith
8.5 was3. lower
than thatofwith
PB,
it showed
theandmaximumat pH 8.5using
andtwodecreased
FALDH.
(b) pH.
Influences
buffer indicatedsolution pHthaton theactivityof the AOD-FALDH
membrane.
Bothincreasing
Theseofresults
using
PB solution
at pH 8.5 would
allow
for
if
the experiments
were carriedout bythe AOD-FALDH
AOD-FALDH fluorosensor
thetherefore,liquid phase.
maximizing
the catalytic
activityof themembrane, inand
we decided
to use pH 8.5 PB solution in subsequent experiments.
The
The influences
influences ofof waterproof
waterproof materials
materials onon thethe AOD-FALDH
AOD-FALDH membrane
membrane were
were invesinvestigated.
Exceptforthemembranecoatedwithfluorine
resin,
nowater
leakagetigated. Except for the membrane coated with fluorine resin, no water leakage occurred.
occurred.
Figureto 4.84.8 ppm
ppm MeOH
MeOH vapor
vapor byby thethe MeOH
MeOH bio-sniffer
bio-sniffer using
using
Figure 44 shows
shows sensor
sensor responses
responses todifferently
waterproofed
AOD-FALD
membranes.
All
types
of
membranes
resultedinthedifferently waterproofed AOD-FALD membranes. All types of membranes resulted in theincreaseinthefluorescence
from
NADH
upon
applying
MeOH
vapor.
Among
them,
the
increase in the fluorescence from NADH upon applying MeOH vapor. Among them, themembranespin-coating PMEH
showed thethe largest
largest sensor
sensor output
output along
along
membrane waterproofed
waterproofed byby spin-coating
PMEH showedwith aa superior
superiorresponse
responserepresented
represented
time
to reachof stable
the stable
90).
with
byby
thethetime
to reach90%90%of thevaluevalue(T90 ).(T
The
90
of
91
sec,
which
was
more
The
membranepreparedbyspin-coated
PMEH
exhibitedT
membrane prepared by spin-coated PMEH exhibited T90 of 91 sec, which was more than
thanthanothers:
the others:
PMEH,
229fluorine
sec; fluorine
295silicone
sec; silicone
twicetwice
betterbetter
than thecastedcasted
PMEH,
229 sec;
resin, resin,
295 sec;
resin,
resin,
195
sec.
Based
on
the
all
abovementioned
aspects,
wedecidedtowaterproof
the gas195 sec. Based on the all abovementioned aspects, we decided to waterproof the gas-phase
phaseof the AOD-FALDH
membraneby spin-coatingside ofside
the AOD-FALDH
membraneby spin-coating
PMEH.PMEH.

Figure 4.
4. Sensor
Sensor responses
responses to
to4.8
4.8ppmppmMeOH
MeOHvaporvaporfromfromthetheMeOH
MeOHbio-sniffer,bio-sniffer,which
whichemployedemployed
Figurefour
different
waterproof
treatments(spin-coating
PMEH,
casting
PMEH,
andspraying
siliconeor
four different waterproof treatments (spin-coating PMEH, casting PMEH, and spraying silicone or
fluorine resins) on the gas-phase side of the AOD-FALDH
AOD-FALDH membrane.
membrane.

3.2. Sensitivity to MeOH Vapor
3.2. Sensitivity to MeOH Vapor
The sensitivity of the MeOH bio-sniffer to MeOH vapor was investigated. The sensorThe sensitivity of the MeOH bio-sniffer to MeOH vapor was investigated. The sensorgrams of the bio-sniffer to various concentrations of MeOH vapor are shown in Figure 5a.
grams of the bio-sniffer to various concentrations of MeOH vapor are shown in Figure 5a.
Following the stable baseline in air, the fluorescence intensity increased as a response to
Following the stable baseline in air, the fluorescence intensity increased as a response to
MeOH vapor. The increment in the fluorescence intensity was different depending on
the concentration of MeOH vapor, suggesting the sensor output and the MeOH vapor
concentration have a correlation. After demonstrating the measurement three times foreach concentration, we summarized the sensor output, which was obtained by averaging
fluorescence intensity for the last 30 s of the MeOH vapor application, as a function of theconcentration of MeOH vapor in Figure 5b. A fitting curve of the plot was made by thefollowing equation:∆FI (cps) = A [MeOH vapor (ppm)])B(1)
where A = 1.23 × 104 and B = 1.02 are the coefficients with a correlation coefficient R of
0.991; [MeOH vapor] is the concentration of MeOH vapor in ppm. This calibration curve
allowed us to determine the dynamic range of the MeOH bio-sniffer as 0.32–20 ppm. Here,the limit of quantification (LOQ) was calculated from ten times the standard deviation of
the sensor baseline signal and Equation (1). The obtained dynamic range was found toencompass MeOH concentration in exhaled breath (0.10–2.3 ppm) [46], and therefore, themeasurement of MeOH in exhaled breath by the MeOH bio-sniffer was highly possible.

Sensors 2021, 21, 4897

where A = 1.23 × 104 and B = 1.02 are the coefficients with a correlation coefficient R of
0.991; [MeOH vapor] is the concentration of MeOH vapor in ppm. This calibration curve
allowed us to determine the dynamic range of the MeOH bio-sniffer as 0.32–20 ppm. Here,the limit of quantification (LOQ) was calculated from ten times the standard deviation of
the sensor baseline signal and Equation (1). The obtained dynamic range was found to
8 of 11encompass MeOH concentration in exhaled breath (0.10–2.3 ppm) [46], and therefore, themeasurement of MeOH in exhaled breath by the MeOH bio-sniffer was highly possible.

Figure5.5.(a)(a)Sensor
Sensoroutputs
outputstotovarious
variousconcentrations
concentrationsof
of MeOHMeOH vapor.
vapor. (b)(b) AA calibration
calibration curve
Figurecurve ofof thebio-sniffer
to
MeOH
vapor.
the bio-sniffer to MeOH vapor.

characteristicscomparison
comparison ofof thethe MeOH
MeOH bio-sniffer
AAcharacteristics
bio-sniffer withwithvarious
variousreported
reportedsensors
sensorsis
shown in Table 1. It shows the high sensitivity of the bio-sniffer among the state-of-the-art
is shown in Table 1. It shows the high sensitivity of the bio-sniffer among the state-of-theMeOH gas sensors.
art MeOH gas sensors.
Table1.1.Comparison
Comparisonofofthe
theproposed
proposedMeOH
MeOHbio-sniffer
bio-snifferwith
withvarious
variousreported
reportedsensors.
sensors.
Table
Technology
Material
DynamicRange
Range Operating
OperatingTemp.
Temp.
Ref.
Technology
Material
Dynamic
Ref.
Pd-SnO2
Chemoresistive
1–1000350[33]◦C
Chemoresistive Pd-SnO2 nanoparticles
1–1000ppm
ppm
350°C
[33]
nanoparticles
Substrate
Substrate
integrated
Polyindole
5–500
RT
[47]
integrated
Polyindole
5–500ppm
ppm
RT
[47]
waveguide
waveguide
Carbonnanotube
CarbonnanotubeSplit
ringresonator
10–300ppm
ppm
RT
[48]
10–300
RT
[48]
Split
ring
resonator
coatedcarbonfiber
coated carbon fiber
Ce-doped In2 O3
Chemoresistive
2–1000 ppm
320 ◦ C
[49]
Ce-doped In2O
3
porous nanospheres
Chemoresistive
2–1000 ppm
320 °C[49]
porousnanospheres
Platinumdecorated
Amperometric
10–300 ppm
RT[50]
Platinumdecoratedmesoporous
TiN
Amperometric
10–300ppm
RT[50]
Biofluorescence
AOD-FALDH
0.32–20 ppm
RT
This work
mesoporous
TiN
Biofluorescence
AOD-FALDH
0.32–20 ppm
RT
This work
3.3. Measurement of MeOH in Exhaled Breath

The measurement of MeOH in exhaled breath of a healthy subject was carried out
using the MeOH bio-sniffer. The sensorgram during the measurement is presented in
Figure 6. The observed sensorgram to the exhaled breath was similar to that of the standard
MeOH vapor (see Figure 5a). The fluorescence intensity increased upon pumping thebreath sample to the bio-sniffer; then it returned to the baseline when changing breath
sample to the filtered air. The concentration of MeOH in the exhaled breath sample wascalculated from the average fluorescence intensity of the last 30 s of the breath sample
application and equation 1, and it was of 0.78 ppm. The consistency of this value with theconcentration range reported by other studies (0.10–2.3 ppm) [46] supported the validity ofthe measurement.

Sensors 2021, 21, 4897

cade reaction with AOD and FALDH allowed for selective discrimination of methanolfrom other aliphatic alcohols including ethanol, 1-propanol, 1-butanol, and 2-propanol[36]. On the other hand, it was also observed that use of FALDH lead to reaction withaldehydes such as acetaldehyde and formaldehyde. However, according to the reports byČáp et al., concentrations of those aldehydes in healthy subject’s breath (acetaldehyde: 7
9 of 11
ppb; formaldehyde: 1 ppb) are much lower than that of MeOH (189 ppb) [46]. Therefore,
we have concluded that the signal observed in this experiment was derived from MeOH.

Figure 6.
6. Sensor
Sensor response
response toto exhaled
exhaled breath
breath fromfrom aa healthy
healthy subject.
subject.
FigureAccording to our previous study regarding the AOD-FALDH fluorosensor, the cascade
4. Conclusions
reaction with AOD and FALDH allowed for selective discrimination of methanol fromthis study,
we havedevelopeda biochemicalgas sensorand(bio-sniffer)for[36].
MeOH
otherInaliphatic
alcohols
includingethanol,
1-propanol, 1-butanol,
2-propanol
On
using
a
cascadereaction
with
AOD
and
FALDH
for
the
non-invasive
assessmentofintesthe other hand, it was also observed that use of FALDH lead to reaction with aldehydes
tinal
After a seriesof formaldehyde.
optimizations including
FALDH,
pH ofsolution,andsuch flora.
as acetaldehyde
andHowever,
accordingto buffer
the reportsby Čáp
methods
toprevent
water
leak
from
the
AOD-FALDH
membrane,thedynamic
rangeof
et al., concentrations of those aldehydes in healthy subject’s breath (acetaldehyde: 7 ppb;
the
MeOH bio-sniffer
found lower
to be 0.32–20
ppm.
This highwith theformaldehyde:
1 ppb)wasare muchthan thatof MeOH(189sensitivityppb) [46]. along
Therefore,
weknown
superior that
selectivity
to MeOH
withreactiontheMeOH.
possibility
have concluded
the signal
observedin thethiscascade
experiment
was suggested
derived fromof breath MeOH measurement. In the measurement of exhaled breath sample from a
4. Conclusions
healthysubject, a sensorgram similar to that with standard MeOH vapor was observed.
The calculated
MeOH
concentration
the exhaledgas
breathwas(bio-sniffer)
0.78 ppm. The
consistency
In this study,
we havedeveloped ina biochemicalsensorfor MeOH
usingofthis value
to the reported
concentration
the validity ofthe measurement
anda cascadereactionwith AOD
and FALDHsupported
for the non-invasive
assessmentof intestinal
thecapabilityof theofMeOH
bio-sniffer.
The characterizations
and asolution,
preliminary
demonflora.
After a series
optimizations
including
FALDH, pH of buffer
and methods
strationwithexhaled
MeOH
this study indicate
the utility
and feasibility
bioto prevent
water
leak fromthein
AOD-FALDH
membrane,the dynamic
range of ofthetheMeOH
snifferin thebreathMeOH
futurestudy,
we willalong
perform
deeperstatisbio-sniffer
was
found
to bemeasurement.
0.32–20 ppm.InThis
highsensitivity
withthe known
tical
analysisby increasingthenumberof subjects
andsuggested
investigate
correlationof exsuperior
selectivity
to MeOH
withthe cascade
reactionthethepossibilityof breath
MeOHmethanol
measurement.
In the measurement
exhaledbreath bio-sniffer
sample fromhealthythe
subject,
haledand intestinal
flora status ofusing
the MeOH
to aexplore
posa sensorgram
similar toassessmentthat with standard
MeOH
was observed.
sibilityof non-invasiveof intestinal
floravapor
by exhaled
MeOH. The calculated
MeOH concentration in the exhaled breath was 0.78 ppm. The consistency of this value tothe reported concentration supported the validity of the measurement and the capability of
the MeOH bio-sniffer. The characterizations and a preliminary demonstration with exhaled
MeOH in this study indicate the utility and feasibility of the bio-sniffer in the breath MeOH
measurement. In future study, we will perform deeper statistical analysis by increasing thenumber of subjects and investigate the correlation of exhaled methanol and intestinal flora
status using the MeOH bio-sniffer to explore the possibility of non-invasive assessment ofintestinal flora by exhaled MeOH.
Author Contributions: Conceptualization, K.T., K.I. (Kenta Iitani), T.A. and K.M.; methodology,
K.I. (Kanako Iwasaki), G.Z. and Y.I.; validation, K.T., K.I. (Kanako Iwasaki), G.Z. and K.I. (Kenta
Iitani); investigation, K.I. (Kanako Iwasaki); T.A., Y.I. and K.M.; data curation, K.I. (Kanako Iwasaki),G.Z.; writing—original draft preparation, K.T. and K.I. (Kanako Iwasaki); visualization, K.T. and
K.I. (Kanako Iwasaki); supervision, K.T., K.I. (Kenta Iitani), T.A. and K.M.; project administration,
K.T., K.I. (Kenta Iitani), T.A. and K.M.; funding acquisition, T.A. and K.M. All authors have read andagreed to the published version of the manuscript.
Funding: This work was partly supported by Japan Society for the Promotion of Science (JSPS, Japan)
KAKENHI Grant Number JP 17H01759, 16KK0143 and 15H04013; a grant from Japan IDDM network;
the Ministry of Education, Culture, Sports, Science and Technology (MEXT, Japan) Special Funds for“Cooperative Research Project of Research Center for Biomedical Engineering”.

Sensors 2021, 21, 4897

10 of 11

Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Ethics Committee of Institute of Biomaterials andBioengineering, Tokyo Medical and Dental University (approval number: B2014-001).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Conflicts of Interest: The authors declare no conflict of interest.

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