Food Chemistry 396 (2022) 133654

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Hydrogen sulfide gas sensing toward on-site monitoring of chilled meat

spoilage based on ratio-type fluorescent probe
Xiaowei Huang a, Wei Sun a, Zhihua Li a, *, Jiyong Shi a, *, Ning Zhang a, Yang Zhang a,
Xiaodong Zhai a, Xuetao Hu a, Xiaobo Zou a, b, *
a
School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd, 212013 Zhenjiang, Jiangsu, China
b
International Joint Research Laboratory of Intelligent Agriculture and Agri-products Processing (Jiangsu University), Jiangsu Education Department, China

A R T I C L E I N F O A B S T R A C T

Keywords: In this study, a fluorescence sensing platform for visual detection of hydrogen sulfide (H2S) based on ratiometric
Ratio fluorescence fluorescent substances was developed to real-time monitor meat spoilage. The copper nanoclusters (CuNCs) and
Hydrogen sulfide gas nitrogen-doped carbon quantum dots (CNQDs) were used as dual emission fluorescence materials. The fluo­
Visual analysis
rescence ratio of the two wavelengths decreased in the sulphide concentration range of 0–3 μmolL(exp)− 1, with a
Chilled meat
detection limit of 62.7 nmolL(exp)− 1. In order to capture hydrogen sulfide gas in the air, the ratio fluorescent
Spoilage
Paper-based sensors material is loaded on the paper base. By processing the RGB value of the photo under UV light, the detection limit
of the sensor is 4.35 ppt in the range of 0 ~ 45.2 ppt H2S gas concentration. This portable visual analysis greatly
simplifies the steps of H2S gas detection while ensuring sensor stability and sensitivity. It also provides a new
method for H2S detection during the meat storage process.

1. Introduction
Meat and meat products are the main supply of basic nutrition for
people because of rich protein and trace elements. However, the rapid
growth of microorganisms consuming protein makes meat and meat
products prone to spoilage. The advent of a cold chain could slow this
down considerably. Chilled meat has become the mainstream of the
meat market. Nevertheless, the cold chain does not inhibite all microbial
reproduction such as Lactobacillus, Salmonella et al. remain microbial
activity at 0 ~ 4℃. They cause meat spoilage and produce hydrogen
sulphide (H2S) when growing on chilled meat. hydrogen sulfide is a
colorless, sweet, odorous egg-flavored irritating asphyxiating gas (Borch
& Agerhem, 1992). In addition, Sulphurous compounds other than
hydrogen sulphide have been implicated in a type of spoilage described
as putrid, particularly associated with meat of a high pH-value (Mourão,
Rebelo, Ribeiro, Peixe, Novais, & Antunes, 2020). So hydrogen sulfide
can be used as an indicator of meat freshness especially of chilled meat
(Zhai et al., 2019). For evaluation of meat freshness, traditional chem­
ical methods, such as the determination of total volatile base nitrogen
(TVB-N), played a crucial role in meat spoilage evaluation due to their
relatively precise results, but they need to be conducted in specialized
laboratories with time-consuming processes. Compare with TVB-N, H2S
is a pure gas rather than a mixture and more suitable for no on-contact
rapid nondestructive testing method. As meat spoilage is highly time-
dependent, it is important to determine real-time meat spoilage (Lin,
Yan, Lin, & Chen, 2014; Mourão et al., 2020). Therefore, it is of great
significance to prepare a highly sensitive selective probe for on-line
detecting gaseous phase hydrogen sulfide in chilled meat. The elec­
tronic nose technology (Li et al., 2019; Shu wang, Xiao wei, Zhen zhen,
Tong tong, & Shuli, 2019; Sinju et al., 2022; Tian, Cao, Chen, Liu, Wang,
& Gao, 2019; Zhang et al., 2019) are common rapid nondestructive
testing techniques for gaseous samples. Metal-oxide–semiconductor-
based electronic noses have high detection sensitivity, but cannot be
used to detect H2S generated from chilled meat due to high operating
temperature and large size. Visible light colorimetric technology is a
particularly intuitive approach but it cannot avoid the error caused by
environment light disturbance (Ahn, Han, & Lee, 2021; Kaushik, Ghosh,
Singh, & Jose, 2018; Kim, Lee, Ahn, & Lee, 2019; J. Liu, Chen, Zhang,
Gao, Zhang, & Hou, 2018; Paul, Sarkar, Sarkar, Barui, & Sarkar, 2020).
The infrared spectroscopy is susceptible to ambient humidity (Tian
et al., 2019). They are not suit for complex storage, transportation,
processing and marketing environment of chilled meat. Among spec­
troscopy technology, fluorescent probe method is favored by researchers
because of its high sensitivity and selectivity (Chen et al., 2021; El-

* Corresponding authors at: School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Rd, 212013 Zhenjiang, Jiangsu, China.
E-mail addresses: lizh@ujs.edu.cn (Z. Li), shi_jiyong@ujs.edu.cn (J. Shi), zou_xiaobo@ujs.edu.cn (X. Zou).

https://doi.org/10.1016/j.foodchem.2022.133654
Received 10 April 2022; Received in revised form 13 May 2022; Accepted 5 July 2022
Available online 9 July 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
X. Huang et al.
Fig. 1. Composite schematics of CuNCs, CNQDs, CuNCs/CNQDs nanocomposites, and paper-based sensors.
Shamy, 2021; Ghosh & Biswas, 2021). Especially, ratio fluorescence
probes are highly anti-jamming compared to single-emission signal-
based fluorescent probes because they provide built-in self-calibration
by calculating the strength ratio of the two fluorescent signals, providing
more accurate quantification (Hu, Cao, Dong, & Tang, 2021). Although
some organic probes have good detection characteristics, but its own
biotoxicity has always been a food safety test must be considered (Li,
Guo, & Lu, 2015). As a new type of fluorescent material, copper cluster
has good prospects in the field of food safety testing because of its good
emission signal, low biotoxicity, high selectivity and high sensitivity
(Ling, Wu, Gao, Li, & Luo, 2015).Ma and his colleagues developed a low-
toxic, simple, and inexpensive fluorescent probe using the unique opti­
cal properties of copper nanoclusters and good chemical stability (Ma,
Chen, & Chang, 2014). Xue et al prepared a copper cluster, which
showed that the ethanol solution could affect the interaction between
the synthetic copper clusters. With the change of order between copper
clusters, the fluorescence signal is different, and a simple ratio fluores­
cence sensing is obtained by using this phenomenon (Hu et al., 2021).
However, the detection based on copper clusters and nitrogen-doped
carbon points is mostly detected in the liquid phase and is not suitable
for instant detection (Li, Yang, Wang, & Huang, 2021). This greatly
limits the application of fluorescent probes, and portable fluorescent test
paper can overcome these shortcomings due to its portability and simple
preparation process (Wen et al., 2019).
In this study, a dual-emission fluorescent paper-based sensor was
designed for ratio detection of H2S (Fig. 1). D(-)-Penicillamine (DPA)
was used as a template to synthesize copper nanoclusters (CuNCs),
which combine with carbon-doped nitrogen quantum dots（CNQDs)to
form a CuNCs/CNQDs complex through electrostatic interaction. When
the complex is exposed to hydrogen sulfide gas, the red fluorescent
emission is extinguished due to the production of the Cu-S bond, while
the blue fluorescent emission remains unchanged. Therefore, as the
concentration of hydrogen sulfide gas increases, the fluorescence of the
probe changes from red to blue. The probe was loaded the onto a paper
base and prepared a portable fluorescent filter paper to detect hydrogen
sulfide gas concentrations in meat spoilage.
2
Food Chemistry 396 (2022) 133654
2. Materials and methods
2.1. Chemicals and materials
D-penicillamine (DPA) purchased from Aladdin Industrial Corpora­
tion (Beijing, China), Copper(ii) nitrate hydrate (Cu(NO3)2⋅3H2O), So­
dium sulfide (Na2S⋅9H2O), Concentrated sulfuric acid (H2SO4), Sodium
hydroxide (NaOH), PBS Buffer (1 × ) were purchased by Sinopharm
Chemical Reagents Co., Ltd. (Shanghai, China). Ultrapure Milli-Q water
(Millipore) was used throughout the experiment. All chemicals were at
least of analytical grade and used as received without further
purification.
2.2. Instruments and measurements
Powder X-ray diffraction patterns (PXRD) were recorded with a
Bruker D8 diffractometer system (Germany). The emission spectra of the
synthesized materials were measured using the F–98 spectrofluorometer
(Shanghai Leng Guang technology Co. Ltd, China). FT–IR spectrum was
recorded with a Nicolet iS50 Fourier Transform Infrared Spectroscopy
(Thermo Scientific, USA). Transmission electron microscopy (TEM)
images were obtained using a transmission electron microscope (JEM-
2100(HR), JEOL, Japan). X-ray photoelectron spectroscopies (XPS) were
obtained by Thermo ESCALAB 250XI (USA).
2.3. Preparation of CuNCs/CNQDs nanocomposites
CNQDs are synthesized as follows: sodium citrate (0.1 g), ammonium
chloride (0.53 g) and 5 ml water are mixed and transferred to Teflon
stainless steel autoclave, which is placed in an oven at 180℃ for 4 h, and
then placed at room temperature to cool naturally. The cooled mixture is
purified with a dialysis film of 1000 Da to remove unresponsive pre­
cursors. The resulting product is centrifuged for 5 min at 7104 g to
remove insoluble large particulate matter, and the purified CNQDs so­
lution is kept in a 4℃ environment.
CuNCs/CNQDs are synthesized on the basis of previous studies
(Ghosh & Biswas, 2021): under intense stirring, the copper nitrate so­
lution (40 μl, 100 mmolL(exp)− 1) was mixed with the 10 mmolL(exp)− 1
X. Huang et al.
Fig. 2. (a) the fluorescence spectrum of CNQDs and the dual emission of CuNCs/CNQDs; (b)TEM image of CuNCs/CNQDs nanocomposites; (c) Zeta potential of the
CuNCs, CNQDs, CuNCs/CNQDs; (d) The X-ray photoelectron spectroscopy(XPS) spectrum of the composites; (e) XPS spectrum of Cu 2p electrons; f, FTIR spectra of
CuNCs/CNQDs, CuNCs, and CNQDs.
penicillin solution with the purified 4 ml CNQDs, stirred for 10 min,
resulted in white precipitation, red light under ultraviolet light, and
stirred the mixture at 20℃ for 35 min. The resulting product is washed
with water 3 times after centrifugation 10 min at 8000 rpm, then vac­
uum freeze-dried and stored at − 20℃ for a long time.
2.4. Detection of sulfide by nanocomposites
Considering the instability of sulphides in the solution, a series of
different concentrations of sodium sulfide solution are provided with
PBS buffer solution. The 1 ml nanocomposite is mixed with a sulphide
solution of 50 μl at different concentrations, and the fluorescence in­
tensity of the different mixtures at 440 nm and 645 nm is recorded at the
xenonex s 365 nm. And all relevant measurements are made three times.
2.5. Visual detection of hydrogen sulfide gas by composite paper-based
sensor
The CuNCs/CNQDs methanol solution (5 mg/ml) of 10 μl is dripped
on the filter paper (d = 1 cm, d is the side length of the filter paper) and
then dried on the paper base down with nitrogen. The paper base is
placed in a custom-made acrylic containers for a transparent and sealed
H2S gas generator. In order to generate gaseous H2S, different concen­
trations of sodium sulfide solution are added to the sample pool, and
excess dilute sulfuric acid is injected into the sample pool by Micro sy­
ringe. Paper-based colors are monitored by Four-purpose UV analyzer
and recorded on a smartphone. The concentration of hydrogen sulfide
produced by the reaction process of sulfur ions and excess dilute sulfuric
acid was calculated and the distribution of hydrogen sulfide gas in the
gas phase and liquid phase was studied. Specific calculation process was
shown in the Supporting Information online.
3
Food Chemistry 396 (2022) 133654
2.6. Analysis of volatile substances in samples under 4℃ storage
environment
2.6.1. Solid-phase microextraction (SPME) conditions
The 6 g sample was placed in a 15 ml headspace vial and then
equilibrated at 60 ◦ C for 15 min. A SPME fiber module (50/30 μm DVB/
CAR/PDMS, 1 cm, gray; ANPEL Laboratory Technologies (Shanghai)
Inc.) was used to extract volatiles from meat samples at 60 ◦ C for 40 min.
2.6.2. Gas chromatography–mass spectrometer (GC–MS) conditions
The Trace Ultra ITQ1100 GC-MS system (Thermo Scientific, Wal­
tham, MA) was used to analyze volatile compounds. The carrier gas is
helium, and the carrier gas flow rate is 1.6 ml/min without splitting. The
temperature of the injection port is 250 ◦ C. Heating program: the initial
temperature is 40 ◦ C, keep it for 2 min, then increase to 100 ◦ C at a rate
of 5 ◦ C/min, and finally rise to 230 ◦ C at a rate of 6 ◦ C/min, and keep it
for 5 min.
2.7. Determination of total viable count and H2S-producing bacteria
count in meat samples during storage
According to previous research (Zhai et al., 2019), The study used
the plate streak method to calculate the total viable count (TVC) and the
number of H2S-producing bacteria (H2S-PBC) in meat samples. Meat
samples (5 g) were mixed with 25 ml of PBS buffer solution, and the
mixture was homogenized to extract the suspension. The appropriate
concentration of the dilution (10 μl) was poured onto the surface of the
medium, and the TVC was measured after culturing at 37 ◦ C for 2 days.
And the diluted solution was poured on the surface of Trisaccharide iron
agar medium and incubated at 20 ◦ C for 3 days to identify H2S-PBC.
Bacterial counts are expressed in colony forming units (CFU) per gram of
meat sample, and then converted to a logarithmic 10-based value, which
is log (CFU/g).
X. Huang et al.
Fig. 3. (a) Fluorescent spectra of CuNCs/CNQDs nanocomposites at concentrations of sodium sulfide from 0 to 3 μM; (b) CIE chromaticity diagram of the (x, y)color
coordinates of the CuNCs/CNQDs nanocomposites varying with sulfide; (c) The linear relationship between the concentration of sulphides from 0 to 3 μm and the
intensity of the fluorescence ratio (I645/I440); (d) The fluorescence intensity ratio(I645/I440) of different interfering substances reacting with CuNCs/CNQDs
nanocomposites;
2.8. Application of paper-based H2S sensor in monitoring meat spoilage
Pork and Chicken breast were purchased from local supermarkets
and kept below 4 ◦ C. 25 g of fresh chicken breast and pork were put into
disposable plastic packaging boxes. The paper-based sensor was placed
on the bottom of the box and were stored at 4 ◦ C.
3. Results and discussions
3.1. Structural analysis of CuNCs/CNQDs
DPA is used as a reducing agent for synthesizing copper clusters. The
process is that SH is combined with copper, and the pH of the entire
reaction environment is 2.66. Under this solvent condition, the COOH
group is deprotonized to COO–, while the NH2 group is protonized to
NH+ 3 . CuNCs and CNQDs have the opposite charge, so they are adsorbed
together under physical conditions through electrostatic interaction.
This nanocomposite exhibits two emission peaks at the excitation
wavelength of 365 nm (Fig. 2(a)).
As can be seen from the (TEM) images, nitrogen-doped carbon
quantum dots are bound together with aggregated copper clusters.
(Fig. 2(b)) In the enlarged image, it can be clearly seen that the lattice
distance of CuNCs/CNQDs is 0.535 and that of CNQDS is 0.224. Fig. 2(c)
shows the potential changes before and after the mixing of CuNCs and
CNQDs. In addition, Fig. S1 shows CNQDs and CuNCs/CNQDs in a
dispersed state.
Fig. 2(d) shows an X-ray photoelectron spectroscopy (XPS) spectrum
4
Food Chemistry 396 (2022) 133654
of the CuNCs/CNQDs complex, which includes the combined energy of
elements C, N, O, Cu, S, etc. Fig. 2(e) shows the energy level of the Cu
element, with no Cu2p3/2 satellite peak near the 942 eV, thus proving
that there is no existence of Cu(II). There are 933.0 eV and 952.7 eV
peaks in the spectrum, corresponding to Cu2p1/2 and Cu2p3/2 respec­
tively (Jia, Yang, Li, Li, & Wang, 2014). Considering that the combi­
nation of 2p3/2 between Cu (0) and Cu (I) can be relatively close, we
believe that the valence of copper elements in CuNCs/CNQDs is between
0 and + 1 (Chen et al., 2021). From Fig. 2(f), we can further analyze the
surface structure of CuNCs/CNQDs by the Fourier transform-infrared
(FT-IR) spectrum. The sealing agent on the surface of CuNCs contains
the carboxylate (COO–) and the amino (NH2) group, which is confirmed
by the characteristic peaks of the COO–(1494 cm− 1) stretch mode and
the feature peaks of the NH2 (3410 cm− 1) stretch mode (Jia et al., 2014).
And the CuNCs/CNQDs surface contains the characteristic peaks of
CNQDs, which include the feature peaks of the stretch vibration modes
of C–– O (1610 cm− 1) and C–N (1406 cm− 1) (Zhou, Jin, Zhao, Chu, &
Peng, 2021). The feature peaks of these spectra clearly indicate the
composition of CuNCs/CNQDs.
3.2. Analysis of CuNCs/CNQDs characteristics
At 365 nm excitation wavelengths, CuNCs/CNQDs nanocomposites
have twin emission peaks at 440 nm and 645 nm. During the assembly of
CuNCs and CNQDs, the fluorescence intensity of CNQDs is reduced. This
phenomenon can be interpreted as fluorescent resonance energy transfer
(Li et al., 2020). And in the solvent environment with pH of 2.66, the
X. Huang et al. Food Chemistry 396 (2022) 133654

Table 1 different sulphides, the emission peaks at 440 nm remain almost con­
Comparison of previously reported methods and this work. stant, while the emission peaks at 645 nm decrease gradually, indicating
No. Probe Strategy LOD Analyte Ref. that the fluorescence intensity of copper clusters is quenched by the
Used influence of sulfur ions. As shown in Fig. S2, the peak corresponding to
1 Resorufin Fluorescence 17.3 H2S (Sun et al., Cu 2p3/2 shifted to higher binding energy, accompanied by the
probe μM 2022) appearance of a satellite peak at 942.08 eV, confirming that the Cu2+
2 CDs Fluorescence 200 nM H2S (Liu & Han, was present after the reaction. Also, when the sulphide concentration
2016) reaches 1 mM, the fluorescence of CuNCs almost completely disappears
3 Eu3+/ Fluorescence 145 nM H2S (Zhou et al.,
Cu2+@ZnPDA 2021)
and the fluorescent color of the solution gradually changes from red to
4 C8-ISPBI Colorimetry 120 nM H2S (Zheng et al., blue, which is consistent with the CIE graph (Fig. 3(b)). In the range of
2021) 0 to 3 μmolL(exp)− 1, I645/I440 is proportional to the concentration of
5 Au/Ag Colorimetry 167ppt S2− (Huang et al., sulphides, the correlation coefficient is 0.9923 (Fig. 3(c)), and the
nanorods 2014)
detection limit is calculated at 63.7 nmolL(exp)− 1 based on a sign.
6 PPy/CV conductivity 500 S2− (Yavarinasab
impedance ppb et al., 2021) al-to-noise ratio of 3:1 (As shown in S7-S8). This shows that the
7 MnO2 CTL 280 nM H2S (Chen et al., detection sensitivity at low sulfur concentration is high, which has
2015) certain detection advantages compared with the previously reported
8 P-dots Fluorescence 450 nM S2− (Wang et al., methods (Table 1).
2017)
9 AuNCs/CDs Fluorescence 350 nM S2− (Nejad et al.,
In order to evaluate the anti-jamming of CuNCs/CNQDs nano­
2020) composites to other interfering substance, In the selectivity test in the
10 CuNCs/CNQDs Fluorescence 4.52ppt H2S This work presence of various interfering substances, the concentration of the
67.2 S2− interfering substances is consistent with the concentration of sulfur ions.
nM
As shown in Fig. 3(d), sulfur ions have a large change in the fluorescence
ratio of the complex and the reaction to other interfering substance is not
electrostatic interaction between the two particles is maximized by the obvious. Therefore, we believe that CuNCs/CNQDs are highly selective
deprotonation and protonization of the cluster surface group. These in detecting sulfur ions.
effects greatly enhance the composition and arrangement of molecules
and reduce the energy loss through the non-radiant rotational relaxation
3.4. Construction of a ratio-based fluorescent paper-based sensor
channel, thus significantly improving the emission efficiency (Jia et al.,
2014).
Inspired by the reaction results of CuNCs/CNQDs solution and sul­
fide ions, we further tried to construct a paper-based sensor to detect
3.3. Analysis of selectivity and sensitivity of CuNCs/CNQDs probe for hydrogen sulfide gas. During the deterioration of chicken meat, various
detecting sulfide volatile gases other than H2S will be produced (Fig. S3). Among them,
we selected some representative volatile organic compounds to test the
Firstly, we studied that the fluorescence changes of the combination selectivity of the sensor, including acetone, hexane, cyclohexane, chlo­
of sulphides with CuNCs/CNQDs nanocomposites at different concen­ roform, trichloroethylene, ethylene, isopropanol, methanol, ethanol,
trations. As shown in Fig. 3(a), the CuNCs/CNQDs nanocomposite has toluene, and trimethylamine. It can be seen from the figure (Fig. 4(a))
two emission peaks at 440 nm and 645 nm. With the addition of that the probe has a certain selectivity to H2S.The CuNCs/CNQDs

Fig. 4. (a) Photographs of paper-based sensors exposed to different volatile organic compounds (VOCs) under 365 nm light for 48 h; (b) photograph of the gas sensor
device; (c) At different concentrations of hydrogen sulfide gas, the (R-R0)/B value of the paper-based sensor varies over time; (d) In the range of 0 to 45.2 ppt
concentration of hydrogen sulfide gas, the linear relationship between hydrogen sulfide gas concentration and (R-R0)/B value; (e) In the range of 0 to 45.2 ppt
concentration of hydrogen sulfide gas, the linear relationship between hydrogen sulfide gas concentration and ΔE.

5
X. Huang et al.
Fig. 5. The color evolution of cuNCs/CNQDs paper-based sensors for moni­
toring the deterioration of fresh chicken breasts and pork at 4 ℃.
methanol solution (50 mg/ml) of 10 μl is dripped on the filter paper (d =
1 cm, d is the side length of the filter paper) and then dried with ni­
trogen. The paper base is placed in a custom-made ackli containers for a
transparent and sealed H2S gas generator (Fig. 4(b)). In order to
generate gaseous H2S, different concentrations of sodium sulfide solu­
tion are added to the sample pool, and excess dilute sulfuric acid is
injected into the sample pool by Micro syringe. The concentration of
hydrogen sulfide is controlled by the content of sodium sulfide
(Table S1), and the paper color is monitored by a quad-UV meter and
recorded on a smartphone. Therefore, we calculated the content of
hydrogen sulfide gas in the atmosphere according to Henry’s law (Eq.
(1)).
6
Food Chemistry 396 (2022) 133654
n(H2 S, g) • R • T n0 (Na2 S) − n(H2 S, g)
P= = KB • 2 O)•ρ(H2 S)
(1)
V(H2 S, g) n0 (Na2 S) − n(H2 S, g) + v(HM(H O)
2
The paper-based color gradually changes from red to blue over time
(Fig. S4), and the captured image (R-R0)/B value stabilizes after 48 h
(Fig. 4(c)). As shown in Fig. 4(d), in the range of hydrogen sulfide
concentrations of 0 to 45.2 ppt, the paper-based image (R-R0)/B values
collected showed a linear correlation with the gas concentration (y =
2.7032x + 0.0289, R2 = 0.9981), with a detection limit of 4.35 ppt (As
shown in S9-S10). In addition, the reflected paper base is measured by a
chromic aberration meter. We found that the paper-based color differ­
ence (ΔE) also has a linear relationship with hydrogen sulfide gas (y. =
13.5522x + 17.979, R2 = 0.988) (Fig. 4(e)).
Therefore, CuNCs/CNQDs paper-based sensors can not only improve
the portability and practicality of The CuNCs/CNQDs probe, but also
enable low-concentration hydrogen sulfide fluorescence quantitative
detection and color-comparison semi-quantitative detection. Therefore,
CuNCs/CNQDs paper-based sensors can not only improve the portability
and practicality of The CuNCs/CNQDs probe, but also enable low-
concentration hydrogen sulfide fluorescence quantitative detection
and color-comparison semi-quantitative detection.
3.5. Application of paper-based hydrogen sulfide sensor in meat spoilage
monitoring
Under the irradiation of a 365 nm UV lamp, the paper H2S sensor
shows a visible color change as the chicken breast and pork are stored at
4 ◦ C (Fig. 5). At the same time, in the control experiment, the paper-
based sensor did not show a significant color change during storage.
When using paper-based sensors to monitor chicken breast and pork, the
red in the paper-based sensor gradually fades to blue after 3 to 4 days.
The spoilage of chicken breast and pork was evaluated by TVC and H2S-
PBC. From Fig. 6(a), it can be seen that the initial TVC of chicken breast
is 4.68 log(CFU/g), which gradually increases to 8.92 log(CFU/g) after
8 days of storage at 4 ◦ C. At the same time, H2S-PBC increased from 3.67
log(CFU/g) to 8.87 log(CFU/g). This indicates that most of the spoilage
bacteria in chicken breast have the ability to produce H2S.
Similarly, TVC and H2S-PBC in pork increased from 3.67 to 9.82 log
(CFU/g), and from 3.47 to 9.74 log(CFU/g). A polynomial is used to
determine the relationship between the number of microorganisms and
the paper-based sensor (R-R0)/B value. For chicken breast meat samples,
the quadratic equations between (R-R0)/B value and TVC (Fig. 6(a)) and
(R-R0)/B value and H2S-PBC (Fig. 6(b)) were established, where R2
values were respectively 0.9757 and 0.9753. For pork samples, a similar
quadratic fit was also established between (R-R0)/B value and TVC
(Fig. 6(c)) and between (R-R0)/B value and H2S-PBC (Fig. 6(d)) Equa­
tion, R2 is 0.9447 and 0.9748, respectively. According to European
Community legislation, 5 × 106 CFU/g, or ~ 6.7 log(CFU/g), is the
maximum acceptable amount of raw meat. Therefore, in this study,
when the TVC of the chicken breast sample is 6.6 log(CFU/g), the (R-
R0)/B value of the paper-based sensor is 0.0202 (Fig. 6(a)), indicating
that if the (R-R0)/B value of the paper-based sensor is (R-R0)/B value is
higher than 0.0202, the chicken breast cannot be eaten. When the TVC
content in the pork sample is about 6.6 log (CFU/g), the (R-R0)/B value
of the paper-based sensor is 0.0256. It shows that if the (R-R0)/B value of
the paper-based sensor is higher than 0.0256, The pork cannot be eaten.
4. Conclusion
In summary, this study developed a simple fluorescence detection
method for sulfur ions and a visual fluorescence detection method for
hydrogen sulfide gas. First, we prepared a ratio fluorescent probe by
electrostatic assembly, and the probe reacted with sulfide to generate
CuS. The fluorescence of the CuNCs/CNQDs nanocomposites changed
from red to blue as the sulfide concentration increased. The minimum
X. Huang et al.
Fig. 6. The (R-R0)/B value of the paper sensor with the changes of TVC for (a) chicken breast and (c) pork, and the (R-R0)/B value of the paper sensor with the
changes of H2S-PBC for (b) chicken breast and (d) pork, during storage at 4 ◦ C.
limit of sulfur ion detection by this method is 62.7 nM. Secondly, in
order to overcome the limitations of probe detection in solution, CuNCs/
CNQDs were loaded on paper to achieve visual fluorescence quantitative
detection of hydrogen sulfide gas and semi-quantitative detection of
chromatic aberration. The ratio fluorescent paper-based sensor can
detect hydrogen sulfide gas with a minimum limit of 4.35 ppt after being
loaded by the probe. Then the CuNCs/CNQDs was applied to detect H2S
generated from chilled chicken breast and pork during storage. It is
showed good selectivity towards H2S against other volatile gases
generated from chicken breast and pork during storage. The CuNCs/
CNQDs presented visible color changes when it was used as H2S sensor
to monitor chilled meat spoilage. This work opens up a way for the
accurate and quantitative detection of hydrogen sulfide gas, and pro­
vides a reference for the on-line nondestructive monitoring of meat
freshness.
CRediT authorship contribution statement
Huang Xiaowei: Conceptualization, Methodology, Writing – orig­
inal draft. Sun Wei: Methodology. Li Zhihua: Supervision, Funding
acquisition, Writing – review & editing. Shi Jiyong: Validation. Zhang
Ning: Validation, Investigation. Zhang Yang: Data curation. Zhai
Xiaodong: Resources. Hu Xuetao: Data curation. Zou Xiaobo: Super­
vision, Funding acquisition, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The National Natural Science Foundation of China (31801631,
7
Food Chemistry 396 (2022) 133654
1601360061), Jiangsu Natural Science Foundation for Excellent Young
Scholars (BK20200103), China Postdoctoral Science Foundation
(2020M683372), Jiangsu Association for Science and Technology youth
Talent Promotion Project, The Earmarked Fund for China Agriculture
Research System, Young talent Development Program of Jiangsu Uni­
versity. Science and Technology Innovation Agricultural Project of
Shanghai Science and Technology Commission(19391902100).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.133654.
References:
Ahn, Y. J., Han, S. H., & Lee, G.-J. (2021). Rapid and simple colorimetric detection of
hydrogen sulfide using an etching-resistant effect on silver nanoprisms. Microchimica
Acta, 188(4), 129. https://doi.org/10.1007/s00604-021-04783-4
Borch, E., & Agerhem, H. (1992). Chemical, microbial and sensory changes during the
anaerobic cold storage of beef inoculated with a homofermentative Lactobacillus sp.
or a Leuconostoc sp. International Journal of Food Microbiology, 15(1), 99–108.
https://doi.org/10.1016/0168-1605(92)90139-T
Chen, C., Zhao, D., Lu, L., Yang, F., & Yang, X. (2015). A simple and rapid colorimetric
sensor for sulfide anion detection based on redox reaction of ABTS with Au (III).
Sensors Actuators B: Chemistry, 220, 1247–1253. https://doi.org/10.1016/j.
snb.2015.07.044
Chen, Y., Lim, J.-Y., Wu, X. H., Heo, J. S., Fengying, Z., Yuan, J. F., … Ren, W. X. (2021).
Development of H2S and HClO dual-responsive fluorescent probe for cancer
recognition. Dyes and Pigments, 195, 109666. https://doi.org/10.1016/j.
dyepig.2021.109666
El-Shamy, A. G. (2021). New nano-composite based on carbon dots (CDots) decorated
magnesium oxide (MgO) nano-particles (CDots@MgO) sensor for high H2S gas
sensitivity performance. Sensors Actuators B: Chemistry, 329, 129154. https://doi.
org/10.1016/j.snb.2020.129154
Ghosh, S., & Biswas, S. (2021). Ultrafast and nanomolar level detection of H2S in aqueous
medium using a functionalized UiO-66 metal-organic framework based fluorescent
chemosensor. Dalton Transactions, 50(33), 11631–11639. https://doi.org/10.1039/
d1dt01456k
Hu, X., Cao, H., Dong, W., & Tang, J. (2021). Ratiometric fluorescent sensing of ethanol
based on copper nanoclusters with tunable dual emission. Talanta, 233, 122480.
https://doi.org/10.1016/j.talanta.2021.122480
X. Huang et al.
Huang, H., Li, Q., Wang, J., Li, Z., Yu, X.-F., & Chu, P. K. (2014). Sensitive and Robust
Colorimetric Sensing of Sulfide Anion by Plasmonic Nanosensors Based on Quick
Crystal Growth. Plasmonics, 9(1), 11–16. https://doi.org/10.1007/s11468-013-
9592-0
Jia, X., Yang, X., Li, J., Li, D., & Wang, E. (2014). Stable Cu nanoclusters: From an
aggregation-induced emission mechanism to biosensing and catalytic applications.
Chemical Communications (Cambridge, England), 50(2), 237–239. https://doi.org/
10.1039/c3cc47771a
Kaushik, R., Ghosh, A., Singh, A., & Jose, D. A. (2018). Colorimetric sensor for the
detection of H2S and its application in molecular half-subtractor. Analytica Chimica
Acta, 1040, 177–186. https://doi.org/10.1016/j.aca.2018.08.028
Kim, T. J., Lee, Y. J., Ahn, Y. J., & Lee, G.-J. (2019). Characterization of H2S releasing
properties of various H2S donors utilizing microplate cover-based colorimetric assay.
Analytical Biochemistry, 574, 57–65. https://doi.org/10.1016/j.ab.2019.03.021
Li, H. T., Zhao, K., Ma, Y., Jia, Y. Y., Liu, Y. F., Gao, S., … Zhang, X. X. (2019).
Quantitative detection of trace H2S based on ultraviolet differential optical
absorption spectroscopy. IOP Conference Series: Earth and Environmental Science, 354
(1), 012067. https://doi.org/10.1088/1755-1315/354/1/012067
Li, W., Hu, X., Li, Q., Shi, Y., Zhai, X., Xu, Y., … Kang, S. (2020). Copper nanoclusters @
nitrogen-doped carbon quantum dots-based ratiometric fluorescence probe for lead
(II) ions detection in porphyra. Food Chemistry, 320, 126623. https://doi.org/
10.1016/j.foodchem.2020.126623
Li, X.-C.-Q., Yang, T., Wang, J., & Huang, C.-Z. (2021). CdTe Quantum Dots-Electrospun
Nanofibers Assembly for Visual and Portable Detection of Cu2+. Chinese Journal of
Analytical Chemistry, 49(2), 207–215. https://doi.org/10.1016/S1872-2040(20)
60079-2
Li, Z., Guo, S., & Lu, C. (2015). A highly selective fluorescent probe for sulfide ions based
on aggregation of Cu nanocluster induced emission enhancement. Analyst, 140(8),
2719–2725. https://doi.org/10.1039/c5an00017c
Lin, D., Yan, M., Lin, S., & Chen, S. (2014). Increasing prevalence of hydrogen sulfide
negative Salmonella in retail meats. Food Microbiology, 43, 1–4. https://doi.org/
10.1016/j.fm.2014.04.010
Ling, Y., Wu, J. J., Gao, Z. F., Li, N. B., & Luo, H. Q. (2015). Enhanced Emission of
Polyethyleneimine-Coated Copper Nanoclusters and Their Solvent Effect. The
Journal of Physical Chemistry C, 119(48), 27173–27177. https://doi.org/10.1021/
acs.jpcc.5b09488
Liu, B., & Han, S. (2016). Determination of trace hydrogen sulfide by using the
permanganate induced chemiluminescence of carbon dots. Microchimica Acta, 183
(11), 3087–3092. https://doi.org/10.1007/s00604-016-1957-6
Liu, J., Chen, X., Zhang, Y., Gao, G., Zhang, X., & Hou, S. (2018). A novel xanthene-based
colorimetric and fluorescence probe for detection of H2S in living cells. Journal of
Luminescence, 204, 480–484. https://doi.org/10.1016/j.jlumin.2018.08.053
Ma, J. Y., Chen, P. C., & Chang, H. T. (2014). Detection of hydrogen sulfide through
photoluminescence quenching of penicillamine-copper nanocluster aggregates.
Nanotechnology, 25(19), 195502. https://doi.org/10.1088/0957-4484/25/19/
195502
Mourão, J., Rebelo, A., Ribeiro, S., Peixe, L., Novais, C., & Antunes, P. (2020). Atypical
Non-H2S-Producing Monophasic Salmonella Typhimurium ST3478 Strains from
Chicken Meat at Processing Stage Are Adapted to Diverse Stresses. Pathogens, 9(9).
https://doi.org/10.3390/pathogens9090701
Food Chemistry 396 (2022) 133654
Nejad, M. A. F., Bigdeli, A., & Hormozi-Nezhad, M. R. (2020). Wide color-varying
visualization of sulfide with a dual emissive ratiometric fluorescence assay using
carbon dots and gold nanoclusters. Microchemical Journal, 157, 104960. https://doi.
org/10.1016/j.microc.2020.104960
Paul, N., Sarkar, R., Sarkar, R., Barui, A., & Sarkar, S. (2020). Detection of hydrogen
sulfide using BODIPY based colorimetric and fluorescent on-off chemosensor.
Journal of Chemical Sciences, 132(1), 21. https://doi.org/10.1007/s12039-019-1724-
4
Shu wang, C., Xiao wei, Y., Zhen zhen, W., Tong tong, S., & Shuli, S. (2019). Inversion
algorithm for detection of H2S gas concentration by differential absorption
spectroscopy. Proceedings of SPIE, 11189.
Sinju, K. R., Bhangare, B., Pathak, A., Patil, S. J., Ramgir, N. S., Debnath, A. K., &
Aswal, D. K. (2022). ZnO nanowires based e-nose for the detection of H2S and NO2
toxic gases. Materials Science in Semiconductor Processing, 137, 106235. https://doi.
org/10.1016/j.mssp.2021.106235
Sun, Y., Tang, X., Zhang, K., Liu, K., Li, Z., & Zhao, L. (2022). Hydrogen sulfide detection
and zebrafish imaging by a designed sensitive and selective fluorescent probe based
on resorufin. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy,
264, 120265. https://doi.org/10.1016/j.saa.2021.120265
Tian, X., Cao, Y., Chen, J.-J., Liu, K., Wang, G.-S., & Gao, X.-M. (2019). Hydrogen
sulphide detection using near-infrared diode laser and compact dense-pattern
multipass cell. Chinese Physics B, 28(6), 063301. https://doi.org/10.1088/1674-
1056/28/6/063301
Wang, C., Sun, J., Mei, H., & Gao, F. (2017). Organic semiconductor polymer nanodots as
a new kind of off-on fluorescent probe for sulfide. Microchimica Acta, 184(2),
445–451. https://doi.org/10.1007/s00604-016-2027-9
Wen, Z. Q., Song, S., Hu, T., Wang, C., Qu, F., Wang, P., & Yang, M. (2019). A dual
emission nanocomposite prepared from copper nanoclusters and carbon dots as a
ratiometric fluorescent probe for sulfide and gaseous H2S. Mikrochimica Acta, 186(4),
258. https://doi.org/10.1007/s00604-019-3295-y
Yavarinasab, A., Janfaza, S., Tahmooressi, H., Ghazi, M., Tasnim, N., & Hoorfar, M.
(2021). A selective polypyrrole-based sub-ppm impedimetric sensor for the detection
of dissolved hydrogen sulfide and ammonia in a mixture. Journal of Hazardous
Materials, 416, 125892. https://doi.org/10.1016/j.jhazmat.2021.125892
Zhai, X., Li, Z., Shi, J., Huang, X., Sun, Z., Zhang, D., … Wang, S. (2019). A colorimetric
hydrogen sulfide sensor based on gellan gum-silver nanoparticles bionanocomposite
for monitoring of meat spoilage in intelligent packaging. Food Chemistry, 290,
135–143. https://doi.org/10.1016/j.foodchem.2019.03.138
Zhang, Y., Wang, Y., Liu, Y., Dong, X., Xia, H., Zhang, Z., & Li, J. (2019). Optical H2S and
SO2 sensor based on chemical conversion and partition differential optical
absorption spectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy, 210, 120–125. https://doi.org/10.1016/j.saa.2018.11.035
Zheng, J., Noh, H. L., Chun, H. W., Oh, B. M., Lee, J., Choi, S.-K., … Kim, J. H. (2021).
Highly sensitive, selective, and rapid response colorimetric chemosensor for naked
eye detection of hydrogen sulfide gas under versatile conditions: Solution, thin-film,
and wearable fabric. Sensors Actuators B: Chemistry, 341, 130013. https://doi.org/
10.1016/j.snb.2021.130013
Zhou, Q., Jin, B., Zhao, P., Chu, S., & Peng, R. (2021). rGO/CNQDs/ZIF-67 composite
aerogel for efficient extraction of uranium in wastewater. Chemical Engineering
Journal, 419, 129622. https://doi.org/10.1016/j.cej.2021.129622