Accepted Manuscript

Title: Rapid and Ultrasensitive Detection of Biogenic Amines

with Colorimetric Sensor Array

Authors: Xianhua Zhong, Danqun Huo, Huanbao Fa,

Xiaogang Luo, You Wang, Yanan Zhao, Changjun Hou

PII: S0925-4005(18)31376-5
DOI: https://doi.org/10.1016/j.snb.2018.07.129
Reference: SNB 25098

To appear in: Sensors and Actuators B

Received date: 30-10-2017

Revised date: 28-6-2018
Accepted date: 27-7-2018

Please cite this article as: Zhong X, Huo D, Fa H, Luo X, Wang Y,

Zhao Y, Hou C, Rapid and Ultrasensitive Detection of Biogenic Amines with
Colorimetric Sensor Array, Sensors and amp; Actuators: B. Chemical (2018),
https://doi.org/10.1016/j.snb.2018.07.129

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Rapid and Ultrasensitive Detection of Biogenic Amines with
Colorimetric Sensor Array

Xianhua Zhong,† Danqun Huo,†* Huanbao Fa,‡ Xiaogang Luo,† You Wang,† Yanan
Zhao,† Changjun Hou†*

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†
Key Laboratory of Biorheology Science and Technology, Ministry of Education,

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College of Bioengineering, Chongqing University, Chongqing 400044, P.R. China

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‡
College of Chemistry and Chemical Engineering, Chongqing University, Chongqing

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400044, P.R. China
*
Corresponding author. Tel.: +862365112673; fax: +862365102507；
E-mail: houcj@cqu.edu.cn or huodq@cqu.edu.cn.
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Graphical abstract
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Abstract Graphic: (I) The reaction between the array fabricated from nanocomposites and TMA; (II)

The generation of difference map; (III) Ultrasensitive detection of TMA among the concentrations from

5 ppb to 50 ppm.
Highlights
 A new colorimetric sensor array was proposed and constructed with composites
of nanomaterials and chemo-sensitive dyes.
 Eight biogenic amines were easily differentiated with ≥92.6% accuracy.
 A good linear quantitative relationship was obtained for trimethylamine over a
wide range of concentrations (5 ppb – 50 ppm).
 An impressive LOD of 1.3 ppb was achieved for trimethylamine.


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Good stability against humidity was achieved by using hydrophobic substrate.
 Good reproducibility against temperature and a two-month shelf life indicate a

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wide applications.

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 The results demonstrate that amine gases can be differentiated simply based on
the difference of alkalinity.

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 We attempted to explore the complex interactions on cross-reactive arrays via
distinct compounds and particularly designed arrays (with one or two dominant
interactions).
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ABSTRACT:
Biogenic amine (BA) is an appropriate indicator of food spoilage. Thus, it is important
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to develop simple, portable and highly sensitive amine sensors for food safety. Here,
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detection of biogenic amines was investigated using a colorimetric sensor array

fabricated from complexes of pH-sensitive dyes and nanomaterials. The Brønsted-
Lowry acid-base reaction between the complex and BAs forms different amine salts.
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Variation in the alkalinity of these amines causes significant and distinct color changes
in the pH-sensitive dyes. Eight common BAs can be differentiated from visual
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observation of difference maps alone. In conjunction with pattern recognition, the

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sensor array clearly differentiates eight gaseous amines with ≥92.6% accuracy and
allows rapid quantification of trimethylamine (TMA) vapors with an impressive limit
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of detection (LOD) of 1.3 ppb. It also shows excellent chemical stability against
changes in temperature and humidity. This sensor array for amines can be used as an
alternative and promising tool for food monitoring.
KEY WORDS
Biogenic amines, Rapid recognition, Colorimetric array, GO, Brønsted-Lowry
reaction

INTRODUCTION
Inappropriate storage of meat can lead to the formation of biogenic amines (BAs)
(e.g., trimethylamine (TMA)) via microbial enzymatic processes, such as the

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decarboxylation of amino acids[1] and amination of carbonyl-containing organic

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compounds.[2] The accumulation of these amines can serve as an appropriate indicator

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of food spoilage.[3-5] A majority of BAs are malodorous, toxic, and carcinogenic,

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which may cause headaches, cardiac palpitations, mucosal burns, and irritation to the
eyes and the respiratory system.[6] The National Institute for Occupational Safety and

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Health (NIOSH) has established the maximum recommended exposure limit of amines
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as 10 ppm for long-term exposure of 10 h and 15 ppm for short-term exposure of 15
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min.[7] Furthermore, an excessive accumulation of TMA in breath, sweat, and urine
may indicate the occurrence of trimethylaminuria (TMAU), a metabolic disorder.[8-11]
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Therefore, there is a need to develop sensors for the sensitive detection of amines at
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low concentrations for both health and economic reasons.

Different strategies have been developed and investigated for amine detection,
including chromatography,[12, 13] absorption spectrometry,[14-18] fluorescence
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spectrometry,[19-24] circular dichroism spectroscopy,[25-27] electrochemistry,[28, 29]

and quartz crystal microbalance (QCM).[30] All these methods could be successfully
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employed to detect and discriminate between different BAs at low ppm concentrations.
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However, all of these strategies suffer from either one or more drawbacks such as
extensive sample preparation procedures, high instrumentation costs, cumbersome
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instrumentation requirements, requirement of specially trained professionals, and low

resistance against environmental disturbances. Therefore, these methods have not been
widely employed for the in-situ detection of amines. With further improvements in
living conditions, even more strict requirements have been put forward for monitoring
harmful chemicals in food. However, the detection of amines at ppb concentrations has
not been widely investigated to date. Therefore, it is still a challenge to develop
ultrasensitive sensors for amine detection.
In comparison, colorimetric sensor arrays[31-39] show good environmental
tolerance and high selectivity. A broad response enables them to recognize numerous
analytes. They also have advantages such as simple sampling methods, visual sensing
mechanism, and facile miniaturization. All these properties make the colorimetric
sensor arrays an ideal method for the detection of amines as the results can be obtained

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within minutes.[40-42] As traditional colorimetric arrays are generally based on weaker

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interactions (e.g., van der Waals interactions), they are only capable of a limited

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sensitivity towards different analytes. Additionally, a few arrays are particularly

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designed for sensing a specific chemical family. Therefore, in order to improve
sensitivity, it is necessary to fabricate colorimetric sensor arrays based on strong

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interactions such as electrostatic ion-ion and Brønsted-Lowry acid-base interactions
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particularly for detection of amines, known as volatile alkalis.
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Apart from utilizing a variety of strong interactions, another method of improving
sensitivity involves the functionalization of devices with appropriate nanomaterials.
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Recently, a variety of nanomaterials such as TiO2 nanoporous films,[35] single-walled

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carbon nanotube/metalloporphyrin composites,[28] and organic nanoparticles[43] have

been prepared and used for a wide range of amine-sensing applications, achieving
impressive limits of detection (LODs). Interestingly, gold nanoparticles (GNPs) can be
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used as a direct and sensitive probe for histamine with a LOD of 0.6 M.[24] The
intriguing qualities of GNPs have enabled their use as signal amplification tags in
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diverse biosensors.[44, 45] A graphene oxide (GO)/chitosan nanocomposite has also

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been used for fabricating amine sensors with a LOD below 3 ppm.[30] Abundant
surface functional groups such as carboxyl, hydroxyl, and epoxides together with a high
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surface-to-volume ratio contribute to the reactivity and adsorption of amines on such

nanomaterials.[46] Furthermore, the porous structure of organically modified silicates
(ormosils) is also conducive to the pre-concentration of amines.[32] Thus,
nanomaterials have proven to be promising candidates for improving the sensitivity of
amine sensors.
 In this work, eight amines were differentiated and ultrasensitive quantification of
TMA over a wide range of concentrations was accomplished by using a nanomaterial-
based colorimetric array. The sensor design is mainly based on the Brønsted-Lowry
acid-base reaction between BAs and nanocomposites comprised of GNPs, GO, a porous
silicone hydrogel, and pH-sensitive dyes (Figure S1 and Table S1). The unique
properties of citrate-coated GNPs, GO, and ormosils play an important role in their
interactions with amines and thus contribute to the extremely high sensitivity of the

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array. The BAs demonstrate different alkalinity (Scheme 1a) depending on the electron-

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donating capacity and steric hindrance of the substituent groups surrounding the amine.

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The differences in alkalinity can be probed by using our appropriately designed

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colorimetric array, in which the pH-sensitive dye correspondingly showed significant
and distinct color changes (Scheme 1b and c). A typical difference map is obtained by

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digital subtraction, pixel by pixel, of the image of the array before and after exposure
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to the BA (Scheme 1d). In this manner, eight amines and several different
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concentrations of TMA have been identified based on the color difference values with
extremely high dimensions, in conjunction with pattern recognition. Finally, the
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reproducibility, stability, and reversibility of the sensor have been discussed.

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EXPERIMENTAL SECTION
Materials and apparatus. The chemo-responsive dyes were bought from Sigma-
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Aldrich, Shanghai, China. Porphyrins derivatives were purchased from Frontier

Scientific Co., USA. Graphite was bought from Alfa Aesar. Sodium nitrate,
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concentrated sulfuric acid, potassium permanganate, tetrachloroauric acid, sodium

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citrate, methanol, tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMS) and

so on were purchased from Aladdin, Shanghai, China. Deionized water was prepared
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by a Molecular system (Chongqing, China). The PVDF membrane (Millipore

IPVH00010) is purchased from Millipore, Co., USA. The pure amines chemicals were
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All reagents
were analytical-reagent grade and used as received. Tedlar® PVF bags and PVF tubes
were obtained from Delin Co. Ltd. (Dalian, China). TEM (LIBRA 200, Germany), SEM
(FEI Nova 400, USA), FT-IR (Nicolet iS5, USA) and ZETA potential analyzer
(Malvern ZS 90, UK) were used to characterize the prepared nanomaterials. A LZB-3
flowmeter (FM) and a HTC-2 hygrometer were purchased from Shuangbo Instrument
Co. Ltd. (Changzhou, China). Precision syringes (maximum volume: 500 L, 1 mL, 2
mL, 5 mL, 10 mL and 20 mL) were bought from Jinta Instrument Co. Ltd. (Shanghai,
China). A GMPA-3 peristaltic pump was purchased from Jinshou development and
production center (Beijing, China). A flatbed scanner (Epson Perfection V10, China)

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was employed to capture the images of the array.

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Array preparation. Gold nanoparticles, graphene oxide and silicone hydrogel

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were prepared according to previously reported methods.[24, 30, 47, 48] [32] The

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formula of each spot is elaborated in Table S1 along with a color-coded legend
indicating the color of the spots printed on the array. Detailed preparation methods for

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these nanomaterials and the detailed composition of each spot are shown in supporting
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information (SI). Under a nitrogen atmosphere, the 6×6 colorimetric sensor arrays was
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artificially printed on a square polyvinylidene fluoride (PVDF) film (side length: 25
mm) with glass capillaries (inner diameter: 0.3 mm). For each spot, about 200 nL
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solution of composite was transferred to the corresponding site on the film. The organic
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solvent-dominated sensor solutions can diffuse well on the hydrophobic film. Hence,
there is no need to adapt the sensor solutions. Once printed, the arrays were stored in a
watch-glass sealed with plastic wrap, and underwent a 5-min nitrogen purging to take
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away the volatilized solvents. Then the watch-glass is filled with nitrogen, thoroughly
wrapped up with plastic wrap and tinfoil, and finally transferred into a sealed drying
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chamber which is also filled with nitrogen and kept in dark place. The arrays were aged
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for at least three days before any sensing application to maximally weaken baseline
drifts. A coded image of the actual array is shown in Figure S1.
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Analytes generation. All gases at their selected concentrations were prepared by

mixing the analyte gas with dry and/or wet nitrogen gas. Figure S2 shows the schematic
illustration of gas mixing device. TMA is used as example to introduce the preparation
of analyte gas samples. The saturated vapor pressure of TMA at 298 K is 2118421 ppm
(see Table 1). To obtain 10 L 50 ppm TMA gas sample, through calculation, 236 L
(i.e., (50 ppm×10 L)/2118421 ppm) TMA saturated vapor at 298 K is need. In practical
operation, 10 L pure nitrogen was slowly transferred into a 10 L-Tedlar® PVF bag. At
same time, 236 L TMA saturated vapor at 298 K was injected into the bag with a 500
L-syringe. Under the flowing of nitrogen, the TMA saturated vapor was fully
dispersed in the mixed gas sample. During this procedure, the flowmeter and the
hygrometer were adopted to ensure gas volume and relative humidity of the sample

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respectively. Once prepared, the sample was kept in a dark place under 30 °C for 10

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minutes, then it was immediately analyzed with the array. So far, 10 L 50 ppm TMA

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gas sample has been prepared. Other gas samples were prepared in a similar procedure.

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Data obtained on flatbed scanner. Figure 1 shows the Schematic illustration of

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acquisition system of difference maps, which was developed by us in a previous work.
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Under the driving of peristaltic pump, analyte gas sample circulated (flow rate:
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6mL/min) to the transparent cell in which the array was placed, then react with the
sensitive materials on the array for 2 minutes. The flatbed scanner was employed to
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capture the images of the array before and after analyte exposure. Then a distinct
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difference map is easily generated by subtracting the image before exposure from the
image after exposure, namely each spot’s red value after exposure minus corresponding
spot’s red value before, green minus green, blue minus blue. The difference map is the
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visualized result of a 108-dimensional difference vector from the digital subtraction,

and it is, as molecular fingerprint, applied to fast identification of analytes.
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Database analysis. The statistical analysis was performed based on 108-

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dimensional difference vectors representing an amine using the SPSS 18.0 software;
“Ward’s Method” was used as criterion for hierarchical cluster analysis (HCA),
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principal component analysis (PCA), and linear discriminant analysis (LDA) in all
cases. Of these, the dendrogram from HCA was completed with MATLAB 2007, while
the histogram and fitting curves were plotted using the Origin 8.0 software.

RESULTS AND DISCUSSION

 The prepared nanomaterials were characterized by means of TEM or SEM and
FT−IR. Figure S3a shows a typical TEM image of the GNPs which are monodisperse
nanocrystals in a narrow distribution of 17 nm diameter (Figure S4a). ZETA potential
determination indicates negative potential (about -50mV) of GNPs, which suggests that
the prepared GNPs are very likely covered with a layer of citrate radicals (Figure S4b).
This electronegativity can obviously improve the affinity to electropositive radicals or
molecules such as amino or amines. Figure 3b presents the typical SEM image of the

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GO monolayer. Figure S4c shows the FT-IR spectrum of the GO monolayer. The

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absorption peak at 3422 cm−1, 1718 cm−1, 1636 cm−1 and 1095 cm−1 is assigned to the

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−OH stretching vibration, the C=O stretching vibration, the C−OH bending vibration

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and C−O−C vibration, respectively[32]. These strong absorptions imply abundant
hydroxyl (−OH), carbonyl (−C=O), carboxyl (−COOH), and epoxy groups (C−O−C)

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anchoring at the surface of GO monolayer which, as a result, presents higher reactivity
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to alkaline chemicals such as trimethylamine. Not surprisingly, SEM image of silica
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hydrogel-functionalized PVDF film indeed shows a nanoporous structure (Figure S3d).
A longer shelf time can be achieved by immobilizing the dyes in the nanoporous
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structure. Additionally, the porosity contributes to the analytes diffusing rapidly

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through the matrix to the indicators, and consequently shorten the response time[17].
Based on the composites of these nanomaterials and dyes, the particularly designed
array was constructed for subsequent sensing applications.
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Figure S5 shows the response of the array to 10 ppm TMA gas as a function of time,
indicating that the time needed to reach equilibrium is less than 2 min. The data from
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the sensor array had incredibly high dimensionality (in total 36 (number of colorants)
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 3 (changes in the red, green, and blue values)  108 dimensionalities), therefore,
hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear
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discriminant analysis (LDA) were performed to evaluate the ability of the sensor array
in the recognition and monitoring of BAs. This section demonstrates the differentiation
of eight BAs and rapid quantitation of TMA at extremely low concentrations using our
colorimetric sensor array.
 Classification of BAs. The developed sensor array functions similarly to other
colorimetric sensors, obtaining distinct visual pattern for different amines. Eight
common BAs (i.e., ethylenediamine (EDA); trimethylamine (TMA); dimethylamine
(DMA); methylamine (MA); hydrazine (HHA); ammonia (NH3); formamide (FMA);
aniline (PA)), can be differentiated from visual observation of the patterns alone (Figure
2). The response of the sensor array to the eight BAs decreased gradually, as shown in

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Figure 2, which is highly consistent with the alkalinity of the various amines. Pearson

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correlation coefficient between visualized response and pKb of different amines is -

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0.847, indirectly confirming the consistence (Table S2 and Table S3). The visual

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response to EDA is of interest as it was stronger than that to either MA or DMA.
However, the alkalinity of EDA (pKb = 4.07/7.15) is weaker than that of both MA (pKb

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= 3.38) and DMA (pKb = 3.27) (Scheme 1a). Nevertheless, EDA is a diamine with a
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linear chain structure. When regarded as “monoamine”, EDA has comparable alkalinity
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to MA and DMA. Additionally, EDA is less sterically hindered, and therefore, it garners
a stronger response. However, steric hindrance was found to be the dominant factor that
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affects the response for both PA and FMA. The large benzene ring impeded the transport
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of aniline across the porous substrate (Figure S3), which led to a weaker response to PA
than FMA, despite the higher pKb value of the former. A consistent result was observed
using HCA (Figure 3). The eight amines were categorized into two groups. The first
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group contained amines with higher alkalinity, such as MA, DMA, and TMA, typically
representing the common features of methyl amines, the diamine EDA was
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subsequently added to this group. The second group consisted of amines with weaker
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alkalinity, and significantly affected by steric hindrance, such as NH3, HHA, and FMA,
also known as small molecular amines. PA was placed in the second group. Hydrazine,
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one of the eight BAs investigated, was primordially paired with ammonia into a cluster.
This indicates the similar properties of ammonia and hydrazine. As the design of the
sensor array allowed the discrimination of amines based on alkalinity and molecule size,
FMA took precedence over aniline in the clustering. In general, no uncertainties or
errors in clustering were observed over 56 trials.
 To evaluate the capacity of the sensor array in discriminating the amines, the
relationship between cumulative variance and the number of principle components (PC)
was investigated. Each principle component represents a chemical reactivity. Twenty
dimensions are required to capture 95% of the total variance, which indicates a
considerable “chemical reactivity space” for the recognition of numerous amines
(Figure S6a). Although the first two principle components captured 60.3% of the total
variance (Figure S6b), a PCA score plot based on the first and third dimensionality

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(52.7% of the total variance) presented a more distinct discrimination of the eight

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amines at 50 ppm (Figure 4a). In the direction of PC1, the eight amines were divided

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into six groups (blue dashed lines in Figure 4a) and their marshalling sequence, from

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EDA to PA, also conformed to the trend of decreasing alkalinity as we expected. This
is in agreement with the result of the difference maps (Figure 2 and Table S3) and HCA

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(Figure 3). However, owing to high similarity in alkalinity and structure, two pairs of
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amines: TMA and DMA, NH3 and HHA, are partially overlapped in first two
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dimensions of PCA.
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To obtain optimal discrimination, a supervised LDA was performed and optimized

classifiers were obtained. In contrast to the PCA, the first two classifiers (factor 1 and
factor 2) from the LDA, captured 71% of the total variance, allowing a more distinct
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discrimination among the eight amines with no overlapping (Figure 4b).

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In the direction of factor 1, the rule that the distribution of the eight amines changed
with alkalinity was also observed. To ensure the classification accuracy of the array, 57%
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of the data of each amine were randomly selected for a training set, whilst the remaining
43% of the data were omitted as blind samples (i.e., validation set). The validation set
was recognized by classifiers generated from the training set, to identify which amine
of the eight BAs were present. Subsequently, the results were determined as either true
positive (TP), false positive (FP), true negative (TN), or false negative (FN). For
example, TMA represented positive results, and the remaining seven amines and
controls were treated as negative results when the array was used to recognize TMA.
The classification accuracy was evaluated by calculating the sensitivity (TP/TP + FN),
specificity (TN/TN + FP), and accuracy (TP + TN/sample size) of the constructed
model (Table 2). The lowest accuracy of recognition was observed for DMA and HHA
(92.6%), while that of other amines was 100%.

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Rapid quantification of TMA. TMA was selected as a sample for rapid and

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sensitive quantification of BAs due to the salient correlation of TMA concentration with

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food spoilage and trimethylaminuria. The visual responses were observed to gradually

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increase in correlation with the increasing concentration of TMA, as shown in Figure
5a, which was further illuminated by classification with HCA. Septuplicate trials using

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the same concentration were correctly clustered into a separate group without any
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uncertainties or errors (Figure 5b). In the dendrogram for TMA at different
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concentrations, the two nearest-neighbor concentrations were initially paired into a
cluster, e.g., 5 ppb and 50 ppb, 1 ppm and 5 ppm, 10 ppm and 50 ppm, and then
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subsequently paired with the other nearest-neighbor concentration. The cluster analysis
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essentially confirmed and clarified the resemblance between two nearest-neighbor

concentrations. Interestingly, seven concentrations of TMA were clustered into two
groups, consisting of low concentrations and the other of higher concentrations.
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Surprisingly, a deflection in the distribution of different concentrations was also

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observed at 1 ppm in the two dimensional PCA score plot (from red arrow to blue arrow,
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Figure 6a). In fact, the response of the array to TMA at 1 ppm is up to 66.3% of that
observed at 50 ppm, which is precisely in the range of inflection point of the response
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curve (red arrow, Figure S7b). Therefore, it is reasonable for the dendrogram from HCA
to generally sort into two clusters, and for the deflection to occur in PCA. Although the
discrimination based on LDA did not present a similar and regular distribution, TMA
at different concentrations could be clearly distinguished and the septuplicate trials of
every concentration were accurately classified with no uncertainties or errors (Figure
S7a). The difference between the distribution of concentrations from LDA and PCA is
possibly resulted from the fact that the classifiers used for LDA are optimized imaginary
dimensions, which are essentially different from the principal components of PCA that
represent various real chemical reactivities or interactions.
The construction of linear quantitative correlativity based on the responses from the
array strategy or the colorimetric method is challenging as they differ from specific
recognition. Many researchers in this field assert a characteristic “semi-quantitation”

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for array or colorimetric sensors.[40-42, 49-51] Accordingly, little effort was made to

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solve this difficulty. Chen et al. applied the signal ratio of the green and red channel to

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quantify the concentration of Cr3+ in aqueous solutions.[52] Suslick et al. extrapolated

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the limits of detection (LODs) for linear quantification of specific chemicals.[31, 53]
In this work, multiple linear fitting was primordially considered. However, the

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significant collinearity between the multichannel signals made it unsuitable for linear
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quantification applying the multiple linear fitting to the issues of sensor array or
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colorimetric method. Additionally, the Euclidean distance of the response of the sensor
array did not correlate linearly with TMA over a wide range of concentrations (i.e.,
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from 5 ppb to 50 ppm), but presented the correlation of a saturation curve (Figure S7b).
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Therefore, a linear fitting of Euclidean distance of the response of the sensor array on
the logarithm of the concentration of TMA was proposed (Figure 4b). An excellent
linearity was obtained from 5 ppb to 50 ppm (R = 0.994). The limit of detection (LOD)
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is defined as the concentration required to obtain 3S/N versus background for the largest
response among the 108 color difference vectors. The calculated LOD for TMA is 1.3
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ppb, and an obvious response was still observed when the concentration was as low as
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5 ppb, which is comparable to the threshold of human olfactory receptors (2.5 ppb).
This LOD is also well below the NIOSH/OSHA permissible exposure limit (PEL) of
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TMA, i.e., 10 ppm for long-term exposure.

Reproducibility, Stability, and Reversibility. Septuplicate trials were conducted

for every amine or concentration, and excellent reproducibility was observed with the
largest relative standard deviation (RSD) of 10.3% for TMA at 5 ppb. The RSD of PA
and controls were 7% and 9.2%, respectively, whilst that of other amines and
concentrations were all below 5% (Table S5). Furthermore, the array presents good
long-term stability. After a month of storage, the response of the array only decreased
to 91% of that of the freshly prepared sensor array (Figure S8). Excellent thermal
stability was obtained, whilst the change of humidity slightly interfered with the result
when the sensor array responded to amines (Figure S9). However, owing to the super-
hydrophobicity of polyvinylidene fluoride used as substrate, the interference had no

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effect on the differentiation of different BAs or concentrations (Figure S9b). Only a 3%

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decrease in response was observed in the presence of different relative humidity, which

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was result from the competition between the affinity of water molecular and the

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preponderant interactions of sensitive nanocomposites to amines, and from the
repulsion of hydrophobic substrate to the small amount of amine hydrates. The only

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disadvantage of the sensor array was the irreversibility of the chemical reaction between
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the amines and the nanocomposites (Figure S10). And about two hours were required
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to recover in natural condition. The second exposure only accounted for 68.6% of the
initial response, whilst the third exposure accounted for 81.4%. Therefore, the array is
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not suitable for repeated detection. However, it is inexpensive to produce and thus
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suitable for disposable applications.

To clearly recognize the advantages/disadvantages of the colorimetric sensor array,

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we have compared it with other reported amine sensors, as summarized in Table 3. The
linear detection ranges and the limits of detection obtained in present work were better
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than the results from those reported analytical studies. Additionally, a shorter response
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time was also achieved. Such excellent sensing performance might be attributed to
strong affinity of acidic groups on the surface of nanomaterials to amines and pre-
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concentration of amine vapors induced by nanoporous structure of nanomaterial/dye

complex or substrate.

Real sample analysis. In order to illustrate the reliability of the developed sensor
array for detection in real samples, a standard spike recovery test was carried out. As
shown in Table 4, the recoveries of TMA in exhaled gas samples were observed in the
range from 94.9% to 104.9% and the relative standard deviation (RSD) (n = 3) was less
than 6%, suggesting the successful applicability of the particularly designed
colorimetric sensor array to determination of TMA vapor in the real samples.

CONCLUSIONS
In conclusion, a simple, disposable colorimetric sensor has been developed for the

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rapid differentiation of eight amines and detection of different concentrations of

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trimethylamine. 90% of the whole response can be achieved within 30 sec. The distinct

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color differences allow for a facile identification of the eight amines and varied

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concentrations of trimethylamine, even by visual observation alone. Hierarchical
cluster analysis, principal component analysis, and linear discriminant analysis all show

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excellent discriminatory ability for amines and different concentrations of TMA, with
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a high correlation to the alkalinity of amines, affirming our original design. The
accuracy for the identification of the eight amines was ≥92.6%. An excellent linear fit
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was obtained over a wide range of concentrations of TMA (from 5 ppb to 50 ppm), with
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a calculated LOD of 1.3 ppb, which is well below the NIOSH/OSHA permissible
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exposure limit. The only disadvantage is that the sensor could not be reused for strong
reactions. A long-term shelf life and stability against temperature and humidity changes
would allow the sensor to be used as a promising and powerful tool for monitoring
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different harmful amines involved in food safety and diagnosis of diseases.

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ACKNOWLEDGMENTS
The authors acknowledge the National Natural Science Foundation of China (NSFC)
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(No. 81772290, 81271930), Chongqing Graduate Student Research Innovation Project

(No. CYB17037), Chongqing University Innovation Training Program for College
students (No. 201710611100), the workstation in Sichuan Province GY2015-01 and the
sharing fund of Chongqing University’s large equipment for financial support. We also
thank Dr. Yang Yehong for helpful statistical analysis.
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Biography

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Professor Chanjun Hou finished his PhD in biomedical engineering in 2004 at
Chongqing University. After a period of research at the University of Illinois at Urbana-

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Champaign as a visiting scholar, he returned to China and got tenure at the college of
bioengineering in Chongqing University. In 2010, he became a full professor in

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biomedical engineering. In 2013, he did research in Durham University as a visiting
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scholar. His scientific interests involve nanomaterials and nanotechnologies, the design
and construction of biochemical sensing systems, food safety monitoring, controlled
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drug delivery system, and other related fields.
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Figure 1. Schematic illustration of acquisition system of colorful difference maps.

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Figure 2. The visual fingerprints for eight amines at a concentration of 50ppm obtained
from the colorimetric array. EDA: ethylenediamine; MA: methylamine; TMA:
trimethylamine; DMA: dimethylamine; HHA: hydrazine; NH3: ammonia; FMA:

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formamide; PA: aniline. The responses decrease gradually from left to right.

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Figure 3. The dendrogram from hierarchical cluster analysis (HCA) of eight common
amines at a concentration of 50ppm. EDA: ethylenediamine; TMA: trimethylamine;
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DMA: dimethylamine; MA: methylamine; HHA: hydrazine; NH3: ammonia; FMA:

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formamide; PA: aniline. All experiments were performed in septuplicate; no

uncertainties or errors in clustering were observed in 56 trials.
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Figure 4. a) PCA score plot based on the first and the third dimension and b)
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Discrimination based on LDA for septuplicate trials of eight amines at 50 ppm.

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Figure 5. a) The visual pattern from responses of the array and b) the dendrogram of
hierarchical cluster analysis for TMA at different concentrations. All of the
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concentrations were clearly discriminable from each other.

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Figure 6. a) Two dimensional PCA score plot for septuplicate trials of TMA at different
concentrations. b) Linear fitting of Euclidean distance on logarithms of concentrations
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of TMA. The concentration of TMA varied from 5 ppb to 50 ppm.

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Scheme 1. Diagram of partial reactions involved in the array and achievement of visual
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fingerprints. a) Alkalinity of trimethylamine (TMA) and pKb values of eight biogenic

amines (BAs). Color changes of complex of dyes and b) gold nanoparticles (GNPs) and
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c) graphene oxide (GO) when responded to BAs. d) Achievement of visual fingerprints

through digital subtraction, pixel by pixel, of the image of the array before and after
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BA exposure.
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Table 1. Saturated vapor pressure (SVP) at specific temperature (Temp.) for each amine.
Compound Abbr. Temp./K SVP/ppm
Ethylenediamine EDA 293 14112.7
Methylamine MA 298 1999963
Dimethylamine DMA 283 1999962.5
Trimethylamine TMA 298 2118421
Ammonia NH3 298 9896683

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Hydrazine HHA 298 6612.3

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Formamide FMA 293 79998.5
Aniline PA 350 19738.1

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Table 2. The specificity, sensitivity, and accuracy of array for every amine.
Specificity Sensitivity Accuracy
BAs TP FP TN FN
/% /% /%

TMA 3 0 24 0 100 100 100

MA 3 0 24 0 100 100 100

DMA 2 1 23 1 95.8 66.7 92.6

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NH3 3 0 24 0 100 100 100

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EDA 3 0 24 0 100 100 100

FMA 3 0 24 0 100 100 100

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HHA 2 1 23 1 95.8 66.7 92.6

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PA 3 0 24 0 100 100 100

Control 3 0 24 0 100 100 100

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Table 3. Comparison with other reported sensors for amine detection.
Linear range Detection
Sensors Analyte(s) tres Ref.
(ppm) limit
Dyes Array TMA 0.1~10 ppm 50 ppb 2 min [31]
Dyes Array NH3 0.2~20 ppm 0.2 ppm 2 min [32]
Porphyrins Array 12 amines 0.6~60 ppm 0.6 ppm --- [33]
Dyes/nanoporous TiO2 Array TMA 0.06~10 ppm 60 ppb 9 min [35]
Poly(thiophene)s-metal ion 7 diamines --- 5 mM 0~12 [38]
composites hours

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Organic nanoparticles Spermidine 0~2.2 M 35 nM 1 min [43]
SWCNTs/Porphyrins NH3 0.5~20 ppm 0.5 ppm 30 s [28]

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Dyes/nanomaterials Array TMA 0.005~50 ppm 1.3 ppb 2 min This work

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Table 4. Determination of TMA vapor in exhaled gas samples.
Sample Added (ppb) Found (ppb) Recovery (%) RSD (%)
1 0 2.96 --- ---
2 10 10.49 104.9 4.9
3 50 51.81 103.6 3.6
4 1500 1422.94 94.9 5.1
5 7000 7193.65 102.8 2.8
6 35000 34108.45 97.5 2.5

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