sensors
ArticleGas-Phase Biosensors (Bio-Sniffers) for Measurement of
2-Nonenal, the Causative Volatile Molecule of Human
Aging-Related Body Odor
Kenta Iitani 1 , Hidehisa Mori 2 , Kenta Ichikawa 1 , Koji Toma 1,3 , Takahiro Arakawa 1,4 , Yasuhiko Iwasaki 5
and Kohji Mitsubayashi 1,2, *
1

2

3

4
5

*

Citation: Iitani, K.; Mori, H.;
Ichikawa, K.; Toma, K.; Arakawa, T.;
Iwasaki, Y.; Mitsubayashi, K.
Gas-Phase Biosensors (Bio-Sniffers)
for Measurement of 2-Nonenal, theCausative Volatile Molecule of
Human Aging-Related Body Odor.
Sensors 2023, 23, 5857. https://doi.org/10.3390/s23135857
Academic Editor: Jesus Lozano

Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo
Medical and Dental University (TMDU), Tokyo 101-0062, Japan; i.bdi@tmd.ac.jp (K.I.);
ichikawa.kenta@tmd.ac.jp (K.I.); k-toma@shibaura-it.ac.jp (K.T.); arakawath@stf.teu.ac.jp (T.A.)
Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU),Tokyo 113-8510, Japan; morihidehisa@gmail.com
Department of Electronic Engineering, College of Engineering, Shibaura Institute of Technology,
Tokyo 135-8548, Japan
Department of Electric and Electronic Engineering, Tokyo University of Technology, Tokyo 192-0982, Japan
Faculty of Chemistry, Materials and Bioengineering, Kansai University, Osaka 564-8680, Japan;
yasu.bmt@kansai-u.ac.jp
Correspondence: m.bdi@tmd.ac.jp; Tel.: +81-3-5280-8091; Fax: +81-3-5280-8094

Abstract: The molecule 2-nonenal is renowned as the origin of unpleasant human aging-related bodyodor that can potentially indicate age-related metabolic changes. Most 2-nonenal measurements
rely on chromatographic analytical systems, which pose challenges in terms of daily usage and theability to track changes in concentration over time. In this study, we have developed liquid- andgas-phase biosensors (bio-sniffers) with the aim of enabling facile and continuous measurementof trans-2-nonenal vapor. Initially, we compared two types of nicotinamide adenine dinucleotide(phosphate) [NAD(P)]-dependent enzymes that have the catalytic ability of trans-2-nonenal: aldehyde dehydrogenase (ALDH) and enone reductase 1 (ER1). The developed sensor quantified thetrans-2-nonanal concentration by measuring fluorescence (excitation: 340 nm, emission: 490 nm)
emitted from NAD(P)H that was generated or consumed by ALDH or ER1. The ALDH biosensor
reacted to a variety of aldehydes including trans-2-nonenal, whereas the ER1 biosensor showed highselectivity. In contrast, the ALDH bio-sniffer showed quantitative characteristics for trans-2-nonenal
vapor at a concentration range of 0.4–7.5 ppm (with a theoretical limit of detection (LOD) and limitof quantification (LOQ) of 0.23 and 0.26 ppm, respectively), including a reported concentration(0.85–4.35 ppm), whereas the ER1 bio-sniffer detected only 0.4 and 0.8 ppm. Based on these findings,
headspace gas of skin-wiped alcohol-absorbed cotton collected from study participants in their 20s
and 50s was measured by the ALDH bio-sniffer. Consequently, age-related differences in signals
were observed, suggesting the potential for measuring trans-2-nonenal vapor.

Received: 1 May 2023
Revised: 15 June 2023

Keywords: gas-phase biosensor; trans-2-nonenal; body odor; aging; fluorescence

Accepted: 21 June 2023
Published: 24 June 2023

1. Introduction
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Humans release a diverse array of volatile organic compounds (VOCs) [1]. Since someof those human-borne VOCs (H-VOCs) include metabolites produced in human biological activities, studies of them have been actively conducted to realize disease screening
and metabolic monitoring [2–4]. While the majority of the H-VOCs are devoid of odor,
there are specific odorous chemicals within H-VOCs that contribute to body odor [5–7].
Among those components are those associated with aging [8,9]; 2-nonenal, with an odor
described as greasy and grassy, is typical. This odorous molecule is known as one of thecausatives of unpleasant body odor that is emitted by people over around 40 years old,

Sensors 2023, 23, 5857. https://doi.org/10.3390/s23135857

https://www.mdpi.com/journal/sensors

Sensors 2023, 23, 5857

2 of 13and its concentration is reported to increase with aging [10]. In brief, the generation of 2nonenal is influenced by age-related metabolic changes through the following mechanisms.
Initially, the formation of lipid peroxides occurs when unsaturated fatty acids react withreactive oxygen species (ROS) present in skin surface lipids [11]. As individuals age, thequantity of ROS increases due to alterations in intracellular metabolism and mitochondrial
function [12]; simultaneously, the concentration of antioxidant enzymes and molecules inthe body decreases [13]. This combination makes it difficult to suppress the formation oflipid peroxide by ROS. Consequently, these lipid peroxides cause oxidative degradation ofω7-monounsaturated fatty acids such as palmitoleic acid and vaccenic acid in skin surface
lipids, ultimately leading to the production of 2-nonenal. Additionally, along with the lipid
peroxides, the levels of ω7-monounsaturated fatty acids also increase in skin surface lipids
as individuals age [14]. Therefore, 2-nonenal serves as a discernible odorous marker of
the aging process. Although the precise mechanism underlying the age-related increase inω7-monounsaturated fatty acids remains unclear, another study [15] supports the findingsof Haze et al. [10] by reporting similar ager-related increases in these fatty acids.
As previously mentioned, 2-nonenal possesses a potent odor, and individuals often utilize deodorants to maintain positive social interactions [16–18]. However, self-assessmentof one’s own body odor can be challenging due to olfactory adaptation [19]. In addition
to the interpersonal aspect of body odor, there is the potential for evaluating changes in
the metabolic capacity associated with aging based on the concentration of 2-nonenal.
Therefore, methods to quantitatively measure its concentration are required. Most of themethods use gas chromatography–mass spectrometry and high-performance liquid chromatography. While numerous studies have focused on the measurement of 2-nonenal infood products [20–25], there has been relatively less research conducted specifically onhuman-borne 2-nonenal [26,27]. Given that laboratory-grade analytical instruments are
unsuitable for daily monitoring of body odor and metabolic status due to their size, cost,and complexity, there is a demand for small and facile operative 2-nonenal gas sensors.
Sensor devices resembling electronic noses, comprising multiple gas sensors [28–32] aswell as plasmonic sensors [33], have been developed and commercialized for evaluation ofbody odor. However, to the best of our knowledge, there is currently no continuous gas
sensor available that can selectively and quantitatively measure 2-nonenal.
We have developed a fiber-optic biochemical gas sensor (bio-sniffer) using nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase. In this particular study,
our initial investigation involved comparing different enzymes capable of catalyzing trans-2nonenal in the liquid phase. Subsequently, a bio-sniffer for trans-2-nonenal was developed,
and the basic characteristics were evaluated. Furthermore, we employed the ALDH biosniffer to measure the headspace gas emitted from cotton wipes used on various body areasof study participants in their 20s and 50s.
2. Materials and Methods
2.1. Materials and Reagents
Aldehyde dehydrogenase (ALDH, cat# 10171832001, from yeast, EC 1.2.1.5, with a
specific activity of 20 units/mg protein, Roche, Switzerland) and enone reductase 1 (ER1,
Lot# AE-001, with a specific activity of 1.3 units/mg powder, Daicel, Tokyo, Japan) [34]were utilized without additional purification. The oxidized form of nicotinamide adenine dinucleotide (NAD+ ) and reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Oriental Yeast, Japan. A copolymer consisting of
2-methacryloyloxy ethyl phosphorylcholine (MPC) and 2-Ethylhexyl methacrylate (EHMA),
known as PMEH [35], was synthesized within the laboratory and employed for enzyme
immobilization [36]. Disodium hydrogen phosphate and potassium dihydrogen phosphate
were used to prepare potassium buffer (PB). The pH of buffer was adjusted by a pH meter(D-25, Horiba, Kyoto, Japan). For sensor characterization, trans-2-nonenal (#255653-5G,
Sigma-Aldrich, Burlington, MA, USA), acetaldehyde (#015-09576, FUJIFILM Wako, Osaka,
Japan), hexanal (#085-02633, FUJIFILM Wako, Osaka, Japan), heptanal (#084-00143, FUJI-

Sensors 2023, 23, 5857

3 of 13

FILM Wako, Osaka, Japan), octanal (#150-00053, FUJIFILM Wako, Osaka, Japan), nonanal(#149-06002, FUJIFILM Wako, Osaka, Japan), decanal (#045-21532, FUJIFILM Wako, Osaka, Japan), ethyl vinyl ketone (#L06829.06, Thermofisher, Waltham, MA, USA), ethanol(#14033-00, Kanto Chemical, Tokyo, Japan), and acetone (#016-00346, FUJIFILM Wako,
Osaka, Japan) were employed.
2.2. trans-2-Nonenal Biosensor Using NAD(P)H-Dependent Enzymes
Figure 1A illustrates the enzymatic reactions of ALDH and ER1 with trans-2-nonenal
as a substrate, based on the literature [37,38]. Both enzymatic reactions used NAD(P) as
an electron carrier. In the reactions, NAD(P) can exist in the oxidized form NAD(P)+ andreduced form NAD(P)H. NAD(P)H absorbs UV light at 340 nm and emits fluorescence at
490 nm. The fluorescence intensity of NAD(P)H varies with its concentration, allowing
enzymatic reactions to be monitored by measuring the fluorescence intensity of NAD(P)H.
In the ALDH-catalyzed reaction, NADH was produced through the oxidation of trans2-nonenal, which takes place by recognizing the formyl group. On the other hand, in
the ER1-catalyzed reaction, NADPH is utilized during the reduction of trans-2-nonenal,
which proceeds by recognizing the α-β double bond. Consequently, trans-2-nonenal can bedetected by an increase in fluorescence intensity with ALDH and a decrease in fluorescenceintensity with ER1. Figure 1B illustrates the fluorescence detection system used in this study
as well as the previous studies [39,40]. The system consists of UV-LED (λ = 335 nm, Sensor
Electronic Technology, Newbury Park, CA, USA), an excitation band-pass filter (BPFEX ,340 ± 5 nm, Asahi spectra, Tokyo, Japan), an optical fiber probe (F1000-900 PROBE, Ocean
Insight, Orlando, FL, USA), a fluorescence band-pass filter (BPFFL , 490 ± 5 nm, Asahi
spectra, Tokyo, Japan), and a photomultiplier tube (PMT, C9692, Hamamatsu photonics,
Tokyo, Japan). These components are connected using a bifurcated Y-shape optical fiber(BIF600-UV/VIS, Ocean Insight, Orlando, FL, USA). The PMT was connected to a PC withphoton-counting mode to measure NAD(P)H fluorescence emitted at the tip of the opticalSensors 2023, 23, x FOR PEER REVIEW
4 of 14
fiber probe. Based on the fluorescence detection system, liquid and vapor phase biosensors
for trans-2-nonenal were developed.

Figure1. (A)
Detection
principleofoftrans-2-nonenaltrans-2-nonenal based
based onon theandFigure1. (A)
Detection
principle
the catalytic
catalyticreaction
reactionofofALDH
ALDH
and ER1.
ER1. (B) Schematic diagram of the fiber-optic fluorescence measurement system of NAD(P)H. (C)(B) Schematic
diagramof the fiber-optic
fluorescencemeasurement
system of
NAD(P)H.
(C) Detection
Detection schemeof trans-2-nonenalin liquidphase with
ALDH-immobilized
membrane
combinedwith the fiber-optic system. (D) Detection scheme of trans-2-nonenal vapor with the flow cell. (E)Measurement scheme of the human sample with bio-sniﬀer.

The trans-2-nonenal biosensors of the liquid and vapor phase used enzymeimmobilized membranes located on the tip of the optical fiber probe (Figure 1C) or theflow cell (Figure 1D), respectively. The ALDH-immobilized membrane was prepared as

Sensors 2023, 23, 5857

4 of 13scheme of trans-2-nonenal in liquid phase with ALDH-immobilized membrane combined with thefiber-optic system. (D) Detection scheme of trans-2-nonenal vapor with the flow cell. (E) Measurement
scheme of the human sample with bio-sniffer.

The trans-2-nonenal biosensors of the liquid and vapor phase used enzyme-immobilized
membranes located on the tip of the optical fiber probe (Figure 1C) or the flow cell
(Figure 1D), respectively. The ALDH-immobilized membrane was prepared as follows(see Figure S1). First, 2 cm × 2 cm hydrophilic polytetrafluoroethylene membrane (HPTFE, thickness 80 µm, pore size 0.2 µm, Merck Millipore, St. Louis, MO, USA) was
rinsed with ultrapure water and dried. Then, a mixture of ALDH solution and PMEH
(10 w/w% in ethanol, 10 µL/cm2 ) was spread on an H-PTFE membrane by spatula, withthe enzyme quantity being 2.1 units/cm2 . Then, the prepared membrane was dried for
3 h at 4 ◦ C. Afterward, the unentrapped ALDH was rinsed with phosphate buffer (PB).
Similarly, the ER1-immobilized membrane was prepared following the same procedure as
the ALDH-immobilized membrane. The quantity of ER1 used was 1.3 units/cm2 .
2.3. Evaluation of ALDH or ER1 Biosensors on Trans-2-Nonenal Solution
As shown in Figure 1C, ALDH or ER1 membrane was attached to the tip of the opticalfiber probe by using a silicone O-ring and then dipped in the coenzyme solution. In theevaluation of the ALDH biosensor, 10 mM NAD+ solution (in 300 µL of PB at pH 8.0,
100 mM) was poured into a cuvette, and trans-2-nonenal solution diluted with 80% of
ethanol diluted by PB was added dropwise every 5 min to reach a final concentration of
1 nM–10 mM. The ER1 biosensor was evaluated in the same way as the ALDH biosensor,
except that 100 µM NADPH (in 300 µL of PB at pH 7.0, 100 mM) solution was used as thecoenzyme solution in the cuvette.
To assess the selectivity of the ALDH and ER1 biosensors, coenzyme solutions compatible with each enzyme were employed. trans-2-nonenal, along with various aldehydes(acetaldehyde, hexanal, heptanal, octanal, nonanal, and decanal), were prepared at a concentration of 10 µM in 80% ethanol solution. Additionally, ethyl vinyl ketone (which is theprimary substrate of ER1), ethanol (employed as a solvent), and acetone (a typical skin gas
component) were likewise evaluated at 10 µM each.
2.4. Measurement of trans-2-Nonenal Vapor
As depicted in Figure 1D, a gas-liquid flow cell was employed in the trans-2-nonenal
bio-sniffer by attaching it to the optical fiber probe. The space between an enzymeimmobilized membrane and the opening of the optical fiber probe served as a liquidchannel for the coenzyme solution. The flow cell was fabricated by machining an acrylicrod (O.D. 12 mm) with a 1.0 mm diameter hole on the side. The gap between the probe end
and the enzyme-immobilized membrane was set at 0.4 mm. Figure 1D provides a schematic
representation of the flow cell featuring the ALDH-immobilized membrane. NADH was
generated on the ALDH-immobilized membrane by loading the trans-2-nonenal vapor,and then NADH was immediately discharged by the delivery of fresh coenzyme solution.
Due to the constant flow rate of the coenzyme solution, the higher concentration of trans2-nonenal vapor resulted in an accelerated NADH generation rate, thereby yielding anelevated fluorescence intensity. Conversely, in the case of the bio-sniffer with ER1 membrane, the fluorescence intensity diminished with increasing trans-2-nonenal concentration.
The flow cell effectively maintained the moisture of the enzyme membrane, rendering it lesssusceptible to samples with high humidity, such as skin gases. Previous iterations of biosniffers demonstrated accurate quantification of molecule concentrations in exhaled breath,
employing a calibration curve derived from dry standard gas [41]. This substantiates thelimited influence of humidity in the bio-sniffer.
The trans-2-nonenal vapor was prepared by using polyvinylidene difluoride (PVDF)
sampling bags. In the experiment, a pure trans-2-nonenal solution was injected into a PVDF
bag using a micro syringe (7000.5 KH, Hamilton, Reno, NV, USA) to adjust a final gas
concentration of 0.4 ppm to 10 ppm and then heated in a water bath at 60 ◦ C for 20 min toSensors 2023, 23, 5857

5 of 13

facilitate vaporization of the trans-2-nonenal and obtain the standard vapor. The injection
volume of trans-2-nonenal solution was based on the calculation outlined in Equation (1),taking into consideration the principles of the ideal gas law:C=

D ×Vinj
273+ T
1013
3
MW × 273 × P × 22.4 × 10

(1)

Vdilu

where C is concentration (ppm), MW is molecular weight (g/mol), D is density (g/mL),
Vinj is injection volume (µL), T is temperature (◦ C), P is atmospheric pressure (hPa), andVdilu is dilution gas volume (L).
The gas measurement setup is illustrated in Figure S2. A peristaltic pump (MP-1000-H,
Tokyo Rikakikai, Tokyo, Japan) was used to flow over the coenzyme solution at 1.4 mL/min.
The coenzyme solution was changed depending on the enzyme as follows: 1 mM NAD+
solution (in PB at pH 8.0, 100 mM) for ALDH and 50 µM NADPH solution (in PB at
pH 7.0, 100 mM) for ER1. The trans-2-nonenal vapor in the PVDF bag was delivered tothe bio-sniffer at a flow rate of 140 mL/min using a diaphragm pump (DSA-3-12, Denso
Sangyo, Kobe, Japan). PTFE tubing with low adsorption characteristics was used for thegas piping. The response of the bio-sniffer was evaluated by first loading dry clean air, thenswitching to trans-2-nonenal vapor, and then switching back to dry clean air to observe
temporal changes in fluorescence intensity.
2.5. Measurement of Human Samples
The experiment was conducted with the approval of the ethics review committee atTokyo Medical and Dental University (TMDU) in accordance with the latest version of the
Declaration of Helsinki (approval number 2012-06). Prior to the experiment, participants
were provided with a detailed explanation regarding the methods and significance of thestudy, and written informed consent was obtained from participants. The study included a
total of six participants, consisting of two individuals in their 50s and four individuals intheir 20s. Initially, participants were requested to complete a questionnaire regarding theirgender, age, and body mass index (BMI). To collect samples, the nape, axilla, back of theear, and shoulder of each participant were gently wiped using cotton gauze soaked in a
500 µL solution of 50% ethanol. The wiped samples were sealed in 9 mL screw bottles andplaced in a dark location for one day to prepare headspace gas. The human samples anda blank sample for one participant were measured by the ALDH bio-sniffer, as shown in
Figure 1E. The measurement was performed with a sample application time of 10 min, and10 min was taken after application without sample gas.
3. Results and Discussion
3.1. Responses of ALDH and ER Biosensors
Figure 2A shows the response of the ALDH biosensor against the different concentrations of trans-2-nonenal solution. An increase in fluorescence intensity from the baseline (fluorescence intensity of 0–5 min) was observed by adding trans-2-nonenal solution,
attributed to the catalytic reaction facilitated by ALDH. Furthermore, a convergence towards a constant fluorescence intensity corresponding to trans-2-nonenal concentration
was observed. Subsequently, the output ∆I was defined as the difference between the
average fluorescence intensity at 4–5 min after injection of trans-2-nonenal and the baseline.
Figure 2B depicts a correlation between ∆I and trans-2-nonenal concentration. The dynamic
range of the ALDH biosensor against trans-2-nonenal was 1–500 µM with the calibration
curve (R2 = 0.998) shown in Equation (2).
∆I (cps) =315.5 − 65.3 × 103
1 + ([trans-2-nonenal conc. (µM)]/384.3)0.85+ 65.3 × 103(2)

Sensors
23, x FOR PEER REVIEW
Sensors2023,2023,
23, 5857

7 of 14
6 of 13

Figure 2. (A) Time course of fluorescence intensity with applying diﬀerent concentrations of trans-

Figure2. (A)solution
Time course
fluorescenceintensity
applying
different
concentrationsof trans-22-nonenal
againstofan
ALDH biosensor.
Thewith
diﬀerence
betweenbaseline
and after applying
nonenalsolution against
an ALDH biosensor.
Theto
difference
between baseline
after applying
each concentrationof trans-2-nonenal
was definedΔI. (B) Calibration
curve of andtrans-2-nonenal
biosensor. (C)Responseof an
ER1 biosensor
diﬀerent
solutionwith the ALDH
each
concentrationof trans-2-nonenal
wasdefinedcurves
to ∆I. (B)Calibration
curveagainstof trans-2-nonenal
concentrationsof trans-2-nonenal.
Slope(C)of thefluorescencechangedefinedto S. (D)Calibration
solutionwith theALDH biosensor.
Response
curvesof anwasER1
biosensor
againstdifferent
curveoftrans-2-nonenal
solution
with
the
ER1
biosensor.
concentrations of trans-2-nonenal. Slope of the fluorescence change was defined to S. (D) Calibration
curve of trans-2-nonenal solution with the ER1 biosensor.

Figure 2C displays the temporal changes in fluorescence upon the addition of trans2-nonenal
to thecuvettecontaining
theinimmersed
ER1 uponbiosensor.
Althoughofthe
Figure solution
2C displays
thetemporalchangesfluorescence
the additiontransactual fluorescence intensity decreases with the introduction of trans-2-nonenal, the graph2-nonenal solution to the cuvette containing the immersed ER1 biosensor. Although thepresents an inverted representation of the amount of change in fluorescence intensity. In
actual fluorescence intensity decreases with the introduction of trans-2-nonenal, the graphcomparison to the ALDH biosensor, the ER1 biosensor exhibited a lower responsivenesspresents an inverted representation of the amount of change in fluorescence intensity. In
to trans-2-nonenal and did not exhibit a constant fluorescence intensity. Consequently, thecomparison to the ALDH biosensor, the ER1 biosensor exhibited a lower responsivenessslope of Δintensity S was employed to evaluate the quantitative characteristic. As a result,to trans-2-nonenal and did not exhibit a constant fluorescence intensity. Consequently,
the dynamic range of the ER1 biosensor was 10 µM–1 mM, supported by the calibrationtheslope of ∆intensity S was employed to evaluate the quantitative characteristic. As
curve (R2 = 0.996) outlined in Equation (3). A potential factor contributing to this behavior
a could
result,bethe
dynamicenzyme
range ofthe ER1
biosensor
10 µM–1ofmM,
supported
diminishedactivity
resultingfrom wasthe presence
80% ethanol
usedbyas the2calibration(R = 0.996) outlinedin Equation(3). A potential
contributing
the solventcurvefor trans-2-nonenal.
The tolerance
of enzymes
to organicfactor
solvents
can vary to
thisbehavior
couldbediminishedenzymeactivity
resultingfromthepresenceof 80%
significantly depending on their type and origin. Generally, exposure to organic solvents
ethanolusedasthesolvent
fortrans-2-nonenal.
The
toleranceof
enzymestoorganic
leads to permanent conformational changes in enzymes, resulting in a loss of activity.
solvents
canvary significantly
dependingtheiralterations
type and origin.
Generally,
exposurePrevious
studieshave demonstrated
that onnotablein protein
conformation
tocommenceorganic solventsleads
topermanent
conformationalchangesinenzymes,resultingat ethanol concentrations above 20%, with the most pronounced eﬀects ina loss of activity. Previous studies have demonstrated that notable alterations in protein
conformation commence at ethanol concentrations above 20%, with the most pronounced

Sensors 2023, 23, x FOR PEER REVIEW

Sensors 2023, 23, 5857

8 of 14

7 of 13
observed at 50% ethanol [42,43,44,45]. However, this hypothesis has not been verified
andis a future issue.
0.03 14.2
𝑆 cps/sat 50% ethanol [42–45]. However, this hypothesis has
14.2(3)
effects observed
notbeen verified
1
trans-2-nonenal conc. (µM) ⁄69.2 .

and is a future issue.

−0.03 − 14.2
3.2. Selectivity ofS ALDH
= ER Biosensors(cps/s)and1 + ([trans-2-nonenal conc. (µM)]/69.2)0.80+ 14.2(3)

Figure 3A,B presents the selectivity against trans-2-nonenal in comparison to various
aldehydes
and othermolecules.
The graphs display the relative output values of each
3.2. Selectivityof ALDHand ER Biosensors
biosensor,
withtrans-2-nonenalsettingagainst
the reference
at 100%.
In the caseof the ALDH
Figure3A,B
presents the selectivity
trans-2-nonenalin comparison
to various
biosensor,
a andhighersignalwasobserved
for hexanal,
heptanal,
aldehydes
otherfluorescence
molecules. The
graphsdisplay
the relative
output valuesof eachoctanal,
biosensor,
with trans-2-nonenalsetting thereferenceat 100%. In the caseof thereported
ALDH that
nonanal,acetaldehyde,
and decanalthanfor trans-2-nonenal.
It wasbiosensor,ahigherfluorescencesignalwasobservedforhexanal,heptanal,
octanal,nonanal,relatively high concentrations of hexanal, heptanal, octanal, nonanal, acetaldehyde, andacetaldehyde,
and decanalwere
than contained
for trans-2-nonenal.
It wasreported thathigh [46].
decanalamong aldehydes
in the VOC
compositionof relatively
healthy people
concentrations of hexanal, heptanal, octanal, nonanal, acetaldehyde, and decanal among
Therefore, while the ALDH biosensor could be used to detect trans-2-nonenal, thealdehydes were contained in the VOC composition of healthy people [46]. Therefore, whilemeasurementof humansampleswaslikelyto be influenced
by otherofaldehydes.
In
the ALDH biosensor
could
be used todetecttrans-2-nonenal,
the measurement
human
the
relativevaluescontrast,
theER1
biosensor
also
showedoutputfor
otheraldehydes,
butsamples was likely to be influenced by other aldehydes. In contrast, the ER1 biosensor also
wereless than13%
eachcomponent,
which
resulted
selectivity
substrate
showedoutputforforotheraldehydes,
but therelativevalues inwereless than based
13% foron
eachcomponent,
which
resultedinselectivity
basedon
substrate
specificity.
specificity.

Figure3. Results
ofofselectivityof(A)(A)ALDH
ALDH
biosensor(B) biosensor
ER1 biosensor
Figure3. Results
selectivity test
test ofbiosensor
andand(B) ER1
againstagainst
various various
molecules.
molecules.
3.3. Quantitative Characteristics of ALDH Bio-Sniffer against trans-2-Nonenal

3.3. Quantitative Characteristics of ALDH Bio‐Sniﬀer against trans‐2‐Nonenal

Figure 4A shows the response curves of the ALDH bio-sniffer to various concentrations
4A shows
the Aresponse
curves andof stable
the value
ALDH
bio-sniﬀeroutput
to various
ofFigure
the trans-2-nonenal
vapor.
significant increasein fluorescenceconcentrationsof the trans-2-nonenal
vapor.
A significant
stable valueinwith trans-2-nonenal
vapor and a recovery
to theinitial value increase
when theandgas supplyisfluorescence
outputwithtrans-2-nonenal
vapor
and
a
recoverytotheinitialvalue
whenstopped were observed. It can be observed that the ∆intensity has not completely convergeda steady-statevalue in the
1.1 observed.
ppm and 2.6Itppm
trans-2-nonenal
the togas
supply is stopped
were
can ofbethe
observedthat themeasurements.
Δintensity has not
Therefore,
in
order
to
obtainvalues
as
close
to
the
convergencevalue
aspossibleevencompletely converged to a steady-state value in the 1.1 ppm and 2.6 ppm oftheattrans-2theseconcentrations,wecalculatedtheaverage∆intensity
value
at
8–9
min
after
the
start
of
nonenal measurements. Therefore, in order to obtain values as close to the convergence
the sample application and used those values for evaluation of quantitative characteristics
value as possible even at these concentrations, we calculated the average Δintensity value(see Figure 4B). The dynamic range of the ALDH bio-sniffer was 0.4–7.5 ppm, which
at 8–9min after
the start ofrange
the sample
and±used
forasevaluation
includeda concentrationof 2.6 ±application
3.5 ppm (meanSD) thosethat is values
reported
theof quantitative
characteristics(see
Figure4B).
The
dynamic
range
of
the
ALDH
bio-sniﬀer
2-nonenal concentration range emitted from 40–70-year-old subjects [10]. The calibration
2 = 0.994)
shown in a
Equation(4). Therange
theoreticalofppm
detection(LOD)
wasequation
0.4–7.5 (Rppm,
whichisincluded
concentrationof 2.6limit
± 3.5
(mean
± SD) thatis reported as the 2-nonenal concentration range emitted from 40–70-year-old subjects[10]. The calibration equation (R2 = 0.994) is shown in Equation (4). The theoretical limit ofdetection (LOD) and quantification (LOQ), calculated from 3 and 10 times the standard

ensors 2023, 23, x FOR PEER REVIEW
Sensors 2023, 23, 5857

9 of
8 of 13deviation
values of(LOQ),
baselinevalues,to be
0.23 values
and of0.26 ppm
and quantification
calculated
from 3were
and 10determined
times the standard
deviationbaseline values, were determined to be 0.23 and 0.26 ppm, respectively.
respectively.

Figure4. (A)
Time
courseof
offluorescence
fluorescence intensity
by applying
trans-2-nonenal
vapor against
Figure4. (A)
Time
courseintensity
by applying
trans-2-nonenal
vaporALDH
against ALD
bio-sniffer.
(B)
The
calibration
curve
based
on
the
plateauvalue
offluorescencechangeat eachbio-sniﬀer. (B) The calibration curve based on the plateau value of fluorescence change
at each trantrans-2-nonenal concentration.
2-nonenal concentration.

The ER1 biosensor was also employed to measure trans-2-nonanal vapor; however,The
ER1wasbiosensor
was also
employed to ofmeasuretrans-2-nonanal
vapor;
thesignalonly observable
at concentrations
0.4 and 0.8
ppm (refer to FigureS3).howeveThis
limitationislikelyattributed
to
an
issue
with
the
ER1
immobilization
method(PMEH
the signal was only observable at concentrations of 0.4 and 0.8 ppm (refer to Figure S3
achievequantitativewiththeimmobilization
highly selective ER1
bio- (PME
Thisentrapment).
limitation isTo
likelyattributed
to anmeasurements
issue with the
ER1
methodsniffer, optimization of the immobilization method will be pursued in future studies.
entrapment). To achieve quantitative measurements with the highly selective ER1 bi
Note that ALDH and ER1 bio-sniffers could be used continuously for at least 3 h. On
inusablefuturestudies.
sniﬀer,optimizationof theexperiments
immobilization
methodwill bethepursued
the otherhand, long-term
are needed
to determine
maximumtime.

Note that ALDH and ER1 bio-sniﬀers could be used continuously for at least 3 h. O
avg. long-term∆intensity (cps(4) usab) = 8449.6 ×are[trans-2-nonenal(ppm)]0.7624
the other hand,experiments
needed to conc.
determine
the maximum
time.3.4. Signal of Headspace Gases Acquired from Skin-Wiped Cotton at Different Body Sites.
Figure5 shows
the signalscps
obtainedfrom the ALDH
bio-sniffer using
samplescollected
𝑎𝑣𝑔.
𝛥𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦
8449.6trans-2-nonenal
conc.
(ppm)
(
from various sites (A: nape, B: back of the ear, C: axilla, and D: shoulder) of a maleparticipant in their 20s. The participant refrained from bathing and washing his body for
3 days prior
to the sample
a result,
fluorescentsignals
were observed
at
3.4. Signalof Headspace
Gasescollection.
Acquired As
fromSkin‐Wiped
Cottonat Diﬀerent
Body Sites
sites other than the shoulder. However, it is important to note that these signals cannot be
Figure 5 shows the signals obtained from the ALDH bio-sniﬀer using sampl
attributed solely to trans-2-nonenal due to the broad catalytic capacity of ALDH for various
collected
fromConsideringvarious sites(A: nape,
B: backof theC: axilla,
and D:
ofaldehydes.
that hexanal,
nonanal,octanal,andear,decanalare detectedwithshoulder)highmalefrequency
participant
in
their
20s.
The
participant
refrainedfrombathingandwashinghisbodregardless of age [9,46], the experimental results in Figure 5 may be defined asbaselinebiosensor
for humanfor 3thedays
priorfortothe
theALDH
samplecollection.
As asamples.
result, fluorescent signals were observedsites other than the shoulder. However, it is important to note that these signals cannot b
attributed solely to trans-2-nonenal due to the broad catalytic capacity of ALDH fvarious aldehydes. Considering that hexanal, nonanal, octanal, and decanal are detectewith high frequency regardless of age [9,46], the experimental results in Figure 5 may b
defined as the baseline for the ALDH biosensor for human samples.

Sensors 2023, 23, x FOR PEER REVIEW
Sensors 2023, 23, 5857

9 of 13

10 of 14

FigureComparisonsof
of ALDH
ALDH biosensor
againstheadspacegas sample
obtainedfrom
Figure5. 5.
Comparisonsbiosensorsignals
signalsagainstheadspacegas sample
obtainedfromalcohol-absorbedcotton
cottonwiped
wiped on
(B)(B)
backof ear,(C) axilla,and (D)shoulderofalcohol-absorbedon thethe(A)(A)nape,nape,backof ear,(C) axilla,and(D) shoulderofparticipants
their20s.
20s. O.R.
O.R. means
wasoutoutof rangein theincalibration
curve. curve.
participants
inintheirmeansthethesignal
signalwasof range
the calibration

3.5. Difference of Signal between Participants in Their 20s and 50s

3.5. Diﬀerence of Signal between Participants in Their 20s and 50s

In this comparison, participants in their 20s and 50s were instructed to refrain fromIn thistheircomparison,in their
20stheand50s collection.
were instructed
to shows
refrain fromwashingbodies afterparticipantsbathing the night
beforesampleFigure 6A
washing
theirbodiesfrom
afterthebathingnight before
samplecollection.
Figurethe signal
obtained
ALDHthebio-sniffer
using thesamplescollected
from thenape6Aof shows
in theirfrom
20s and
Additionally,a blank
samplethat iscollected
headspacefrom
gas from
theparticipantssignal obtained
the50s.
ALDH
bio-sniﬀer
using
samplestheanape of
cottongauze
soakedin50%
ethanol
was
includedformeasurement.
The
results
revealed
participants in their 20s and 50s. Additionally, a blank sample that is headspace gas froma twofold higher fluorescence output from samples obtained from participants in theira cottongauze soaked in 50% ethanol was included for measurement. The results revealed
50s. Previous studies have indicated that among aldehydes, the detection frequency ofa twofoldfluorescencesamples
obtainedfrom
2-nonenalhigher
significantly
increases output
with agefrom[10], andthe nape
region has
beenparticipants
reported to bein their
50s.a Previous
have
indicatedthat
among aldehydes,
the detection
frequency of 2site where studies
2-nonenal
is morelikely to
be generated[26]. Therefore,
the age-dependent
nonenalsignificantly
increaseswithagemay[10],
the nape
region hasreported
increasein output observedin thisstudy
beand
attributed
to 2-nonenal.
It isbeen
important
to to benotewherethat in2-nonenal
the previousissection,
participants
their 20s refrained
from bodythe
washingfora sitemore likely
to be ingenerated[26]. Therefore,
age-dependent
a period
3 days,observed
and no significant
differencecomparedto the resultsofincrease
in ofoutputin this study
maywasbe observed
attributed
to 2-nonenal.
It is important
thisexperiment
where
the
prohibitionofbodywashingwaslimitedtoone
day.
Further
to note that in the previous section, participants in their 20s refrained from body washingexperiments involving participants in their 50s are deemed necessary to investigate changes
for a period of 3 days, and no significant diﬀerence was observed compared to the resultsin trans-2-nonenal concentration due to sebum accumulation.

of this experiment where the prohibition of body washing was limited to one day. Further
experiments involving participants in their 50s are deemed necessary to investigatechanges in trans-2-nonenal concentration due to sebum accumulation.

Sensors
23, x FOR PEER REVIEW
Sensors2023,2023,
23, 5857

11 of1014of 13

Figure(A)
Comparisonof
ofALDH
ALDH biosensor
biosensor signals
gas
sampleobtainedfrom
Figure
6. 6.
(A)Comparison
signalsagainst
againstheadspaceheadspacegas
sampleobtainedfromnape-wiped alcohol-absorbed cotton of participants in their 20s and 50s. Blank refers to the signal
nape-wiped alcohol-absorbed cotton of participants in their 20s and 50s. Blank refers to the signal
from headspace gas samples of alcohol-absorbed cotton without body wiping. (B) Comparison offromheadspacegas samples
of alcohol-absorbed
body
wiping.
(B) Comparison
cottoncotton
wipedwithout
on the backof earof participantsin their of
signalsof headspacegas of alcohol-absorbed
signalsofheadspacegasofalcohol-absorbed
cotton
wiped
on
the
back
of
ear
of
participantsin their
50s and 20s. O.R. means the signal was out of range in the calibration curve.
50s and 20s. O.R. means the signal was out of range in the calibration curve.

Figure 6B compares the signal of the ALDH bio-sniﬀer obtained using headspace gas
Figurecollected
6B compares
the back
signalbio-sniffer
obtainedusing
headspacesamplesfrom theof ofthetheearALDH
in several
participants.
Similarage-relatedgas
samples collectedfrom theof theseveralSimilardiﬀerenceswere observed
on back
the back
ofear
theinear,whichparticipants.
is characterizedby age-related
a higherdifferences
observedon thebackof theear, secretion,
which is characterized
by a highermade
numbernumber ofwere
sebaceousglandsandactivesebum
akin to the observationsof on
sebaceousthe napeglands
region.and active sebum secretion, akin to the observations made on the nape

region.
3.6. Limitations and Advantages of the Developed Sensors

3.6. Limitations
and Advantagesof theSensors exhibited limited selectivity for
As previously
mentioned,
theDeveloped
ALDH bio-sniﬀer
As previouslyTherefore,
mentioned,
ALDHtobio-sniffer
exhibited limited
for transtrans-2-nonenal.
it theis crucial
further investigate
whetherselectivity
the age-related
output diﬀerence
observed
was specificallyto trans-2-nonenal
by considering
2-nonenal.
Therefore,
it is crucial
to furtherattributed
investigate
whether the age-related
outputthe outputs
from othersuchattributed
as ER1. Furthermore,
the examinationof temporal
difference
observed
was sensors,
specificallyto trans-2-nonenal
by considering
the outchanges
was notachieved
inas
this
study
as headspace
was utilized.
However, changes
with
puts
from othersensors,
such
ER1.
Furthermore,thegasexaminationof temporalenhancementsinsensorsensitivity,
thebio-sniﬀer
could
beemployedforcontinuous
was not achieved in this study as headspace gas was utilized. However, with enhancementsof skin thegas bio-sniffer
componentscould
usingbea employedskin gas collection
cell, as demonstrated
in monitoring
sensor sensitivity,
for continuous
monitoring ofinskin
previous
research
[47].
This
would
enableamorecomprehensive
understandingof the[47].
gas components using a skin gas collection cell, as demonstrated in previous research
dynamics
and variationsin skin gas composition
over time.
Thiswould enable
a more comprehensive
understandingof the dynamics and variations inskin gas composition over time.
4. Conclusions

In this study, our focus was on developing a gas-phase biosensor for the continuous
4. Conclusions
measurementof trans-2-nonenal
vapor.
Initially, awecomparedbiosensor
two NAD(P)H-dependent
In this study,
our focus was on
developing
gas-phasefor the continuous
enzymes,
ALDH
and
ER1,
which
exhibitedcatalytic
activity
towardstrans-2-nonenal inmeasurement of trans-2-nonenal vapor. Initially, we compared two NAD(P)H-dependentboth liquid
andandgasER1,phases.
The
ALDH catalytic
biosensoractivity
and bio-sniﬀer
demonstrated highenzymes,
ALDH
which
exhibited
towards trans-2-nonenalin bothsensitivity but low selectivity for trans-2-nonenal, while ER1 displayed the opposite
liquid and gas phases. The ALDH biosensor and bio-sniffer demonstrated high sensitivity
characteristics to ALDH, being more selective but less sensitive.
but low selectivity for trans-2-nonenal, while ER1 displayed the opposite characteristics to
The dynamic range of the ALDH bio-sniﬀer for trans-2-nonenal was determined to
ALDH, being more selective but less sensitive.
be 0.4–7.5 ppm, which encompasses the range of 2-nonenal concentration reported in the
The dynamic range of the ALDH bio-sniffer for trans-2-nonenal was determined toprevious study [10]. When comparing the outputs of headspace gas samples collected
be 0.4–7.5 ppm, which encompasses the range of 2-nonenal concentration reported in thefrom participants of diﬀerent ages using the ALDH bio-sniﬀer, significant diﬀerences
previous
study [10].
Whencomparing
outputs ofgas50s.
samples
were observed
betweensamplesfrom theparticipantsin headspace
their 20s andIt hascollected
been
from
participantsofdifferent
ages
using
the
ALDH
bio-sniffer,significant
differenceswere
reported that in terms of detection rate, 2-nonenal is the only aldehyde component thatobserved between samples from participants in their 20s and 50s. It has been reported
that in terms of detection rate, 2-nonenal is the only aldehyde component that undergoes
significant changes with age [10], suggesting that the observed output difference may be
attributed to 2-nonenal. However, a quantitative analysis of each aldehyde concentration

Sensors 2023, 23, 5857

11 of 13

was not conducted, and further investigation is required to confirm whether the observed
output is specifically derived from 2-nonenal. One potential solution is to utilize the logicalproduct (AND) of the outputs from the ALDH and ER1 bio-sniffers. Statistical analysis methods, including deep learning approaches, could also be employed to effectively
combine data from multiple sensors [48].
Bio-sniffer technology provides us with the capability to measure concentration
changes over time, and we aim to expand its application to the continuous measurement ofvarious skin components, including trans-2-nonenal, in future studies.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/s23135857/s1, Figure S1: A preparation method of an ALDHimmobilized membrane; Figure S2: Experimental setup for measurement of trans-2-nonenal vapor;
Figure S3: Time course of fluorescence intensity by applying trans-2-nonenal vapor against ER1
bio-sniffer.
Author Contributions: Conceptualization, K.I. (Kenta Iitani), H.M., K.T., T.A. and K.M.; methodology, K.I. (Kenta Iitani), H.M. and Y.I.; validation, K.I. (Kenta Iitani) and H.M.; investigation, K.I.
(Kenta Iitani), H.M. and Y.I.; data curation, K.I. (Kenta Iitani), H.M., K.T., T.A. and K.M.; writing—original draft preparation, K.I. (Kenta Iitani) and H.M.; writing—review and editing, K.I. (Kenta
Ichikawa), K.T., T.A., Y.I. and K.M.; visualization, K.I. (Kenta Iitani), H.M., K.T., T.A. and K.M.;
supervision, K.T., T.A. and K.M.; project administration, K.I. (Kenta Iitani), K.I. (Kenta Ichikawa),
K.T., T.A. and K.M.; funding acquisition, K.I. (Kenta Iitani), T.A. and K.M. All authors have read andagreed to the published version of the manuscript.
Funding: This work was supported by the Japan Society for the Promotion of Science (JSPS) Grantsin-Aid for Scientific Research (KAKENHI) grant numbers 22K14267, 21H04888, and 26280053; andthe Ministry of Education, Culture, Sports, Science and Technology (MEXT) Special Funds for“Cooperative Research Project of Research Center for Biomedical Engineering”.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the Ethics Committee of Tokyo Medical and Dental
University (approval number: 2012-06).
Informed Consent Statement: Written informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: The data that support the findings of this study are available from thecorresponding author (K.M.) upon reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

De Lacy Costello, B.; Amann, A.; Al-Kateb, H.; Flynn, C.; Filipiak, W.; Khalid, T.; Osborne, D.; Ratcliffe, N.M. A Review of theVolatiles from the Healthy Human Body. J. Breath Res. 2014, 8, 014001. [CrossRef] [PubMed]
Vasilescu, A.; Hrinczenko, B.; Swain, G.M.; Peteu, S.F. Exhaled Breath Biomarker Sensing. Biosens. Bioelectron. 2021, 182, 113193.
[CrossRef] [PubMed]
Broza, Y.Y.; Vishinkin, R.; Barash, O.; Nakhleh, M.K.; Haick, H. Synergy between Nanomaterials and Volatile Organic Compounds
for Non-Invasive Medical Evaluation. Chem. Soc. Rev. 2018, 47, 4781–4859. [CrossRef] [PubMed]
Jalal, A.H.; Alam, F.; Roychoudhury, S.; Umasankar, Y.; Pala, N.; Bhansali, S. Prospects and Challenges of Volatile Organic
Compound Sensors in Human Healthcare. ACS Sens. 2018, 3, 1246–1263. [CrossRef]
Kanlayavattanakul, M.; Lourith, N. Body Malodours and Their Topical Treatment Agents: Body Malodours and Treatment
Agents. Int. J. Cosmet. Sci. 2011, 33, 298–311. [CrossRef]
Penn, D.J.; Oberzaucher, E.; Grammer, K.; Fischer, G.; Soini, H.A.; Wiesler, D.; Novotny, M.V.; Dixon, S.J.; Xu, Y.; Brereton, R.G.
Individual and Gender Fingerprints in Human Body Odour. J. R. Soc. Interface 2007, 4, 331–340. [CrossRef]
Pandey, S.K.; Kim, K.-H. Human Body-Odor Components and Their Determination. TrAC Trends Anal. Chem. 2011, 30, 784–796.
[CrossRef]
Gallagher, M.; Wysocki, C.J.; Leyden, J.J.; Spielman, A.I.; Sun, X.; Preti, G. Analyses of Volatile Organic Compounds from Human
Skin. Br. J. Dermatol. 2008, 159, 780–791. [CrossRef]
Yamazaki, S.; Hoshino, K.; Kusuhara, M. Odor Associated with Aging. Anti-Aging Med. 2010, 7, 60–65. [CrossRef]
Haze, S.; Gozu, Y.; Nakamura, S.; Kohno, Y.; Sawano, K.; Ohta, H.; Yamazaki, K. 2-Nonenal Newly Found in Human Body Odor
Tends to Increase with Aging. J. Investig. Dermatol. 2001, 116, 520–524. [CrossRef]

Sensors 2023, 23, 5857

11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

21.

22.
23.
24.
25.
26.
27.

28.
29.
30.

31.
32.

33.
34.
35.

36.

37.

38.

12 of 13

Halliwell, B.; Gutteridge, J.M.C. Free Radicals in Biology and Medicine, 5th ed.; Oxford University Press: Oxford, UK, 2015;
ISBN 978-0-19-871747-8.
Balaban, R.S.; Nemoto, S.; Finkel, T. Mitochondria, Oxidants, and Aging. Cell 2005, 120, 483–495. [CrossRef]
Finkel, T.; Holbrook, N.J. Oxidants, Oxidative Stress and the Biology of Ageing. Nature 2000, 408, 239–247. [CrossRef]
Praticò, D. Lipid Peroxidation and the Aging Process. Sci. Aging Knowl. Environ. 2002, 50, re5. [CrossRef] [PubMed]
Nazzaro-Porro, M.; Passi, S.; Boniforti, L.; Belsito, F. Effects of Aging on Fatty Acids in Skin Surface Lipids. J. Investig. Dermatol.
1979, 73, 112–117. [CrossRef]
Hara, K.; Mikuni, K.; Hara, K.; Hashimoto, H. Effects of Cyclodextrins on Deodoration of “Aging Odor”. J. Incl. Phenom. 2002, 44,
241–246. [CrossRef]
Ham, J.-S.; Kim, H.-Y.; Lim, S.-T. Antioxidant and Deodorizing Activities of Phenolic Components in Chestnut Inner Shell Extracts.
Ind. Crops Prod. 2015, 73, 99–105. [CrossRef]
Lee, S.; Kim, J.H.; Nguyen, N.-T.; Park, R.-M.; Lee, S.-M.; Bang, S.H.; Jeon, G.; Lee, J.; Kim, S.; Cho, B.-K.; et al. Removal ofTrans-2-Nonenal Using Hen Egg White Lysosomal-Related Enzymes. Mol. Biotechnol. 2020, 62, 380–386. [CrossRef]
Dalton, P. Psychophysical and Behavioral Characteristics of Olfactory Adaptation. Chem. Senses 2000, 25, 487–492. [CrossRef]
Verhagen, L.C.; Strating, J.; Tjaden, U.R. Analysis of E-2-Nonenal in Beer at the Ultra Trace Level by High-Performance Liquid
Chromatography Using Precolumn Derivatization and Column Switching Techniques. J. Chromatogr. A 1987, 393, 85–96.
[CrossRef]
Guido, L.F.; Carneiro, J.R.; Santos, J.R.; Almeida, P.J.; Rodrigues, J.A.; Barros, A.A. Simultaneous Determination of E-2-Nonenaland β-Damascenone in Beer by Reversed-Phase Liquid Chromatography with UV Detection. J. Chromatogr. A 2004, 1032, 17–22.
[CrossRef]
Svoboda, Z.; Mikulíková, R.; Běláková, S.; Benešová, K.; Márová, I.; Nesvadba, Z. Optimization of Modern Analytical SPME andSPDE Methods for Determination of Trans-2-Nonenal in Barley, Malt and Beer. Chromatographia 2011, 73, 157–161. [CrossRef]
Palma-Harris, C.; McFeeters, R.F.; Fleming, H.P. Solid-Phase Microextraction (SPME) Technique for Measurement of Generationof Fresh Cucumber Flavor Compounds. J. Agric. Food Chem. 2001, 49, 4203–4207. [CrossRef] [PubMed]
Lam, H.S.; Proctor, A. Milled Rice Oxidation Volatiles and Odor Development. J. Food Sci. 2003, 68, 2676–2681. [CrossRef]
Yazawa, M.; Takahashi, K.; Uchida, H.; Hasegawa, Y. Development of LB Film Taste Sensor for Beer Degradation Evaluation and
Its Physical Properties. Electron. Comm. Jpn. 2019, 102, 47–54. [CrossRef]
Kimura, K.; Sekine, Y.; Furukawa, S.; Takahashi, M.; Oikawa, D. Measurement of 2-Nonenal and Diacetyl Emanating from Human
Skin Surface Employing Passive Flux Sampler—GCMS System. J. Chromatogr. B 2016, 1028, 181–185. [CrossRef]
Saito, K.; Tokorodani, Y.; Sakamoto, C.; Kataoka, H. Headspace Solid-Phase Microextraction/Gas Chromatography–Mass
Spectrometry for the Determination of 2-Nonenal and Its Application to Body Odor Analysis. Molecules 2021, 26, 5739. [CrossRef][PubMed]Mussinan, C.J.; Morello, M.J. (Eds.) Flavor Analysis: Developments in Isolation and Characterization; ACS Symposium Series;
American Chemical Society: Washington, DC, USA, 1998; Volume 705, ISBN 978-0-8412-3578-6.
Kohl, D.; Heinert, L.; Bock, J.; Hofmann, T.; Schieberle, P. Gas Sensors for Food Aroma during Baking and Roasting Processes
Based on Selective Odorant Measurements by an Array. Thin Solid Film. 2001, 391, 303–307. [CrossRef]
Itoh, T.; Koyama, Y.; Shin, W.; Akamatsu, T.; Tsuruta, A.; Masuda, Y.; Uchiyama, K. Selective Detection of Target Volatile Organic
Compounds in Contaminated Air Using Sensor Array with Machine Learning: Aging Notes and Mold Smells in Simulated
Automobile Interior Contaminant Gases. Sensors 2020, 20, 2687. [CrossRef]
Liu, C.; Maeshima, K.; Ge, L.; Hayashi, K. A Flexible and Printable Chemiresistor Sensor Array for Detection and Recognition ofAging-Associated Human Body Odor. Meet. Abstr. 2020, MA2020-01, 2011. [CrossRef]
Gaggiotti, S.; Hurot, C.; Weerakkody, J.S.; Mathey, R.; Buhot, A.; Mascini, M.; Hou, Y.; Compagnone, D. Development of an
Optoelectronic Nose Based on Surface Plasmon Resonance Imaging with Peptide and Hairpin DNA for Sensing Volatile Organic
Compounds. Sens. Actuators B Chem. 2020, 303, 127188. [CrossRef]
Endo, T.; Ryuuno, Y.; Yanagida, Y.; Hatsuzawa, T. Development of Plasmonic Chemical Sensor for Detection of Aldehyde
Compounds. IEEJ Trans. SM 2013, 133, 372–373. [CrossRef]
Yamamoto, H.; Kimoto, N. Novel Enone Reductases, Methods for Producing Same, and Methods for Selectively Reducing a
Carbon-Carbon Double Bond of an Alpha, Beta-Unsaturated Ketone Using the Reductases. US 20040253695A1, 29 June 2004.
Ishihara, K.; Tanaka, S.; Furukawa, N.; Nakabayashi, N.; Kurita, K. Improved Blood Compatibility of Segmented Polyurethanesby Polymeric Additives Having Phospholipid Polar Groups. I. Molecular Design of Polymeric Additives and Their Functions. J.
Biomed. Mater. Res. 1996, 32, 391–399. [CrossRef]
Chu, M.; Kudo, H.; Shirai, T.; Miyajima, K.; Saito, H.; Morimoto, N.; Yano, K.; Iwasaki, Y.; Akiyoshi, K.; Mitsubayashi, K. A Softand Flexible Biosensor Using a Phospholipid Polymer for Continuous Glucose Monitoring. Biomed. Microdevices 2009, 11, 837–842.
[CrossRef]
Stiti, N.; Podgórska, K.; Bartels, D. Aldehyde Dehydrogenase Enzyme ALDH3H1 from Arabidopsis Thaliana: Identificationof Amino Acid Residues Critical for Cofactor Specificity. Biochim. Biophys. Acta (BBA)-Proteins Proteom. 2014, 1844, 681–693.
[CrossRef] [PubMed]
Gao, X.; Ren, J.; Wu, Q.; Zhu, D. Biochemical Characterization and Substrate Profiling of a New NADH-Dependent Enoate
Reductase from Lactobacillus Casei. Enzym. Microb. Technol. 2012, 51, 26–34. [CrossRef]

Sensors 2023, 23, 5857

39.
40.

41.
42.
43.

44.
45.

46.
47.

48.

13 of 13Toma, 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. [CrossRef] [PubMed]
Iitani, K.; Chien, P.-J.; Suzuki, T.; Toma, K.; Arakawa, T.; Iwasaki, Y.; Mitsubayashi, K. Improved Sensitivity of Acetaldehyde
Biosensor by Detecting ADH Reverse Reaction-Mediated NADH Fluoro-Quenching for Wine Evaluation. ACS Sens. 2017, 2,
940–946. [CrossRef]
Iitani, K.; Chien, P.-J.; Suzuki, T.; Toma, K.; Arakawa, T.; Iwasaki, Y.; Mitsubayashi, K. Fiber-Optic Bio-Sniffer (Biochemical Gas
Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde. ACS Sens. 2018, 3, 425–431. [CrossRef]
Buck, M. Trifluoroethanol and Colleagues: Cosolvents Come of Age. Recent Studies with Peptides and Proteins. Quart. Rev.
Biophys. 1998, 31, 297–355. [CrossRef]
Nikolaidis, A.; Andreadis, M.; Moschakis, T. Effect of Heat, PH, Ultrasonication and Ethanol on the Denaturation of Whey Protein
Isolate Using a Newly Developed Approach in the Analysis of Difference-UV Spectra. Food Chem. 2017, 232, 425–433. [CrossRef][PubMed]Shiraki, K.; Nishikawa, K.; Goto, Y. Trifluoroethanol-Induced Stabilization of the a-Helical Structure of b-Lactoglobulin: Implication for Non-Hierarchical Protein Folding. J. Mol. Biol. 1995, 245, 180–194. [CrossRef] [PubMed]
Uversky, V.N.; Narizhneva, N.V.; Kirschstein, S.O.; Winter, S.; Löber, G. Conformational Transitions Provoked by Organic Solventsin β-Lactoglobulin: Can a Molten Globule like Intermediate Be Induced by the Decrease in Dielectric Constant? Fold. Des. 1997,
1997, 163–172. [CrossRef] [PubMed]
Mochalski, P.; King, J.; Unterkofler, K.; Hinterhuber, H.; Amann, A. Emission Rates of Selected Volatile Organic Compounds from
Skin of Healthy Volunteers. J. Chromatogr. B 2014, 959, 62–70. [CrossRef] [PubMed]
Arakawa, T.; Suzuki, T.; Tsujii, M.; Iitani, K.; Chien, P.-J.; Ye, M.; Toma, K.; Iwasaki, Y.; Mitsubayashi, K. Real-Time Monitoringof Skin Ethanol Gas by a High-Sensitivity Gas Phase Biosensor (Bio-Sniffer) for the Non-Invasive Evaluation of Volatile Blood
Compounds. Biosens. Bioelectron. 2019, 129, 245–253. [CrossRef] [PubMed]
Molinara, M.; Cancelliere, R.; Di Tinno, A.; Ferrigno, L.; Shuba, M.; Kuzhir, P.; Maffucci, A.; Micheli, L. A Deep Learning Approachto Organic Pollutants Classification Using Voltammetry. Sensors 2022, 22, 8032. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.