polymers

Article
Dual-Mode Ce-MOF Nanozymes for Rapid and Selective
Detection of Hydrogen Sulfide in Aquatic Products
Qi Cheng 1,† , Xiaoyu Du 1,† , Zuyao Fu 1 , Zhaoyang Ding 1,2,3, * and Jing Xie 1,3, *

1 College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;
[email protected] (Q.C.); [email protected] (X.D.); [email protected] (Z.F.)
2 Marine Biomedical Science and Technology Innovation Platform of Lin-Gang Special Area,
Shanghai 201306, China
3 Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China
* Correspondence: [email protected] (Z.D.); [email protected] (J.X.);
Tel.: +86-21-6190-0369 (Z.D.); +86-21-6190-0351 (J.X.)
† These authors contributed equally to this work.

Abstract: Increasing concern over the safety of consumable products, particularly aquatic prod-
ucts, due to freshness issues, has become a pressing issue. Therefore, ensuring the quality and
safety of aquatic products is paramount. To address this, a dual-mode colorimetric–fluorescence
sensor utilizing Ce-MOF as a mimic peroxidase to detect H2 S was developed. Ce-MOF was pre-
pared by a conventional solvothermal synthesis method. Ce-MOF catalyzed the oxidation of
3,3’,5,5’-tetramethylbenzidine (TMB) by hydrogen peroxide (H2 O2 ) to produce blue oxidized TMB
(oxTMB). When dissolved, hydrogen sulfide (H2 S) was present in the solution, and it inhibited the
catalytic effect of Ce-MOF and caused the color of the solution to fade from blue to colorless. This
change provided an intuitive indication for the detection of H2 S. Through steady-state dynamic
analysis, the working mechanism of this sensor was elucidated. The sensor exhibited pronounced
color changes from blue to colorless, accompanied by a shift in fluorescence from none to light
blue. Additionally, UV–vis absorption demonstrated a linear correlation with the H2 S concentration,
ranging from 200 to 2300 µM, with high sensitivity (limit of detection, LOD = 0.262 µM). Fluorescence
intensity also showed a linear correlation, ranging from 16 to 320 µM, with high selectivity and
sensitivity (LOD = 0.156 µM). These results underscore the sensor’s effectiveness in detecting H2 S.
Citation: Cheng, Q.; Du, X.; Fu, Z.;
Furthermore, the sensor enhanced the accuracy of H2 S detection and fulfilled the requirements for
Ding, Z.; Xie, J. Dual-Mode Ce-MOF
Nanozymes for Rapid and Selective
assessing food freshness and safety.
Detection of Hydrogen Sulfide in
Aquatic Products. Polymers 2024, 16, Keywords: H2 S; nanozyme; metal–organic frameworks; dual-mode sensor; food freshness
1747. https://doi.org/10.3390/
polym16121747

Academic Editor: Chen-I Yang

1. Introduction
Received: 2 June 2024 Hydrogen sulfide (H2 S) is a colorless, corrosive, flammable and toxic gas under stan-
Revised: 18 June 2024 dard conditions, characterized by a distinct rotten egg odor at low concentrations. Although
Accepted: 18 June 2024
it has traditionally been recognized as a key indicator of food spoilage, recent studies have
Published: 20 June 2024
revealed its significant role as a small redox-active molecule in cell signaling pathways [1].
However, H2 S gas generated during food manufacturing processes can adversely affect
food quality. For instance, H2 S produced by yeast during winemaking can compromise
Copyright: © 2024 by the authors.
the taste and flavor of wine [2]. Numerous studies have shown that long-term exposure to
Licensee MDPI, Basel, Switzerland. low doses of H2 S is associated with headaches, vertigo, respiratory disorders and other
This article is an open access article related symptoms, and it may cause respiratory diseases. When ingested through food,
distributed under the terms and H2 S penetrates cell membranes, disrupts the respiratory chain and interferes with cellular
conditions of the Creative Commons respiration. Concurrently, H2 S, recognized as a physiologically active gas, participates
Attribution (CC BY) license (https:// in various physiological processes within the body, including the facilitation of vascular
creativecommons.org/licenses/by/ smooth muscle relaxation and the modulation of nerve transmission [3]. Excess H2 S con-
4.0/). centrations can lead to respiratory system paralysis, impairing normal cognitive functions

Polymers 2024, 16, 1747. https://doi.org/10.3390/polym16121747 https://www.mdpi.com/journal/polymers

Polymers 2024, 16, 1747 2 of 18

and behaviors. Abnormal H2 S levels are closely associated with symptoms of various
diseases, including acute infectious diseases, food poisoning and severe conditions like
Down syndrome, diabetes and Alzheimer’s disease [4], sometimes resulting in sudden
death. Given the health risks associated with H2 S exposure, detecting its presence has
become a matter of significant concern.
H2 S is now recognized as a third gas transmitter alongside nitric oxide (NO) and car-
bon monoxide (CO) [5]. The detection of gases produced during food manufacturing helps
standardize processes and quality control, and it also helps with continuously monitoring
their status and preservation and tracing their origin. Establishing a fluorescence-based
detection method for the quantitative and visual assessment of H2 S in aquatic products
holds significant practical implications for ensuring food safety and human health.
Traditional analytical techniques include gas chromatography (GC), electrochemical
analysis, metal-induced sulfide precipitation [6], plasma atomic emission [7], surface-
enhanced Raman scattering [8] and airfield effect transistors [9]. However, these methods
necessitate skilled operators and employ complex, costly and stationary equipment [10]. In
contrast, the fluorescence method offers higher sensitivity and convenience. Moreover, col-
orimetric sensors also provide a user-friendly and easy-to-deploy solution for routine food
monitoring, as they are easily understood by consumers and do not require sophisticated
instrumentation. In addition, their portability and cost-effectiveness make them ideal for
widespread adoption in food safety protocols. In summary, the combination of colorimetric
and fluorescence methods can achieve multiple advantages, such as portability, sensitivity
and a low cost [11].
Colorimetry has garnered significant attention in bioanalysis and environmental
science due to its affordability, simplicity and visual monitoring capabilities. To facilitate
the translation of detection signals into perceptible color alterations, a variety of compounds
and materials have been engineered and synthesized as colorimetric indicators. In the
realm of H2 S sensing, certain colorimetric approaches have been developed, leveraging
the catalytic properties of metal–organic frameworks (MOFs) or similar catalysts that
oxidize specific substrates in response to H2 S. The reaction products then interact with
color-developing agents, leading to a visible color change that is correlated with H2 S
concentration. This strategy allows for a direct and intuitive assessment of H2 S levels,
which is valuable for environmental and medical monitoring applications.
However, natural peroxidases exhibit high catalytic activity but are susceptible to
environmental conditions such as pH and temperature, which can lead to decreased activity
or complete inactivation [12]. Consequently, natural peroxidases are impractical for use
outside laboratory settings. To enhance the applicability of colorimetric detection, nanoma-
terials have emerged as promising alternatives to natural peroxidases. Nanomaterial-based
enzymatic mimics, or nanozymes, offer several advantages, including simple and cost-
effective synthesis, high structural and chemical stability, ease of storage and compatibility
with a wide range of operating temperatures and pH values, rendering them more suitable
for field-based determinations.
Enzymes, as a class of biological macromolecular catalysts, play pivotal roles in organ-
ismal biotransformation and industrial processes. Their notable advantages, such as high
efficiency, exceptional selectivity and environmental friendliness, render them indispens-
able tools across various sectors of modern industry. However, the inherent limitations of
natural enzymes—including sensitivity to environmental factors, poor operational stability,
high costs and short lifespans—have restricted their broad application, particularly in
challenging conditions. Consequently, substantial efforts have been devoted over the past
few decades to surmounting these constraints. Prominent strategies encompass directed
evolution, protein and enzyme engineering, enzyme immobilization, artificial enzyme
design and medium regulation. With dynamic advancements in modern nanomaterials
science, supramolecular chemistry and protein engineering, artificial enzyme engineering
has emerged as a promising avenue to address these challenges. Artificial enzyme nanoma-
terials, exemplified by nanozymes, are gaining traction as effective alternatives to natural
Polymers 2024, 16, 1747 3 of 18

enzymes due to their controllable catalytic activity, adaptable design, heightened stability
and favorable biocompatibility. As a result, they offer compelling prospects for diverse
industrial applications [13].
Nanozymes, defined as nanomaterials with natural enzyme-like properties, have
gained recognition as promising artificial enzymes in recent years. These nanozymes
effectively merge the benefits of traditional chemical catalysts and biocatalysts, exhibiting
high and tunable catalytic efficiency, exceptional stability, recyclability, ease of manufac-
ture and cost-effectiveness. Consequently, both the fundamental research and practical
applications of nanozymes are flourishing, demonstrating significant potential in areas
such as biosensing, environmental resource protection, food safety and antimicrobial and
antioxidant applications [14]. Metal–organic frameworks (MOFs), characterized by their
porous and crystalline structure, consist of a periodic network formed by the self-assembly
of organic units (typically organic ligands such as carboxylic acids and pyridines) and
metal ions or clusters (commonly transition metals like Cu2+ , Zn2+ and Fe2+ ). Due to their
abundant active sites, customizable properties and robust stability, MOFs are considered
promising candidates for nanozyme applications [15–18].
Additionally, MOFs feature large specific surface areas, adjustable pore sizes and
shapes, easy modification and numerous unsaturated metal sites, which make them highly
suitable for applications in gas adsorption separation, catalysis, sensing, biomedicine and
more [19].
In recent years, a variety of MOF-based nanozymes from the oxidoreductase (such as
peroxidase, oxidase, catalase and superoxide dismutase) or hydrolase (such as carbonic
anhydrase, phosphatase, protease and nuclease) families have been developed and have
achieved significant success in analytical chemistry, environmental management, disease
diagnosis and treatment. The outstanding catalytic activity of these nanozymes has also
led to their widespread use in constructing colorimetric biosensors, transforming them
into powerful and effective tools for real-time detection. Innovative colorimetric sensing
strategies have been developed by integrating nanozymes with colorimetric sensors [20].
Furthermore, various nanomaterials, including carbon-based nanomaterials, metal nanopar-
ticles, metal oxide nanoparticles and quantum dots, have been employed for real-time,
miniaturized and highly selective H2 S detection [21]. MOF-based techniques for detecting
H2 S through fluorescence, catalytic and electrochemical properties have been extensively
explored, contributing to advancements in the field [22].
In this study, a dual-mode colorimetric–fluorescence sensor based on a Ce-MOF
nanozyme was developed. The fluorescence resonance energy transfer (FRET) between
Ce-MOF and TMB resulted in the generation of bright blue fluorescence in the solution.
Furthermore, as the concentration of Na2 S increased, oxTMB in the solution was reduced
back to TMB, resulting in a decrease in oxTMB and a corresponding increase in fluorescence
emission intensity within the detection system. This indicated an enhanced FRET effect.
For H2 S detection (Scheme 1), visual fluorescence detection of H2 S was achieved. Ce-MOF
was synthesized using solvothermal synthesis, demonstrating an excellent dual-mode
colorimetric fluorescence response. Therefore, the FRET-based Ce-MOF provided a highly
selective and sensitive fluorescence detection strategy for H2 S analysis. The prepared
Ce-MOF exhibited exceptional stability and selectivity under the given detection conditions
while maintaining uniform particle size distribution of the nanozyme. Based on this
approach, our study successfully detected H2 S levels in aquatic products and confirmed its
significant potential for application in food freshness assessments.
Polymers 2024, 16, 1747
x FOR PEER REVIEW 4 4of
of 18

Scheme 1. Schematic representation of the preparation of Ce-MOF and the colorimetric–fluorescent

Scheme 1. Schematic representation of the preparation of Ce-MOF and the colorimetric–fluorescent
sensing of H2S.
sensing of H2 S.
2.
2. Materials
Materials and
and Methods
Methods
2.1. Materials
2-hydroxy-terephthalic acid (H22BDC–OH), TMB, ammonium cerium nitrate, 3,3′,5,5′- 3,3′ ,5,5′ -
tetramethylbenzidine,
tetramethylbenzidine, N,N-dimethylformamide(DMF),
N,N-dimethylformamide(DMF), acetic acetic acid,
acid, anhydrous
anhydrous ethanol,
ethanol,
sodium acetate,
acetate, hydrogen
hydrogenperoxide,
peroxide,sodium
sodiumsulfide
sulfideand
andother
othersubstances
substancesused forfor
used selec-
se-
tivity and
lectivity anti-interference
and anti-interferencetesting,
testing,such
suchasasmetal
metalion
ionsolutions,
solutions,werewere purchased
purchased from
Aladdin (Shanghai,
(Shanghai,China)
China)and
and used
used as as received.
received. Fresh
Fresh shrimp
shrimp and salmon
and salmon were were pur-
purchased
from Lotus
chased fromSupermarket (Shanghai,
Lotus Supermarket China).China).
(Shanghai, The buffer solution
The buffer of acetic
solution acid/sodium
of acetic acid/so-
acetateacetate
dium (0.2 M)(0.2
wasM)obtained by mixing
was obtained 82 mL of
by mixing 82 acetic
mL ofacid (0.2acid
acetic M) (0.2
and M)
18 mL
andof18sodium
mL of
acetate (0.2
sodium M). (0.2 M).
acetate

2.2. Apparatus
2.2. Apparatus
2.2.1. Morphology Characterization
2.2.1. Morphology Characterization
The morphologies of Ce-MOF were investigated using a field emission scanning elec-
The morphologies of Ce-MOF were investigated using a field emission scanning elec-
tron microscope (Tescan Mira 3 XH, Tescan, Brno, Czechia) for SEM and SEM–energy
tron microscope (Tescan Mira 3 XH, Tescan, Brno, Czechia) for SEM and SEM–energy
dispersive spectroscopy (EDS) analyses. Additionally, high-resolution images were cap-
dispersive spectroscopy (EDS) analyses. Additionally, high-resolution images were cap-
tured with a transmission electron microscope (Talos F200X G2, Thermo Fisher, Waltam,
tured with a transmission electron microscope (Talos F200X G2, Thermo Fisher, Waltam,
MA, USA) for TEM analysis. The particle size distribution of Ce-MOF was measured using
MA, USA) for TEM analysis. The particle size distribution of Ce-MOF was measured us-
a dynamic light scattering (DLS) system (Malvern Zetasizer Nano ZS, Malvern Instruments
ing a dynamic light scattering (DLS) system (Malvern Zetasizer Nano ZS, Malvern Instru-
Ltd., Westborough, MA, USA). The system was stabilized at 25 ◦ C with deionized water
ments Ltd., Westborough, MA, USA). The system was stabilized at 25 °C with deionized
(0.01 mg·mL–1 ) serving as the dispersion medium in a four-sided quartz cuvette. Approxi-
water (0.01 mg·mL –1) serving as the dispersion medium in a four-sided quartz cuvette.
mately 2 mg of the powder sample was used for each test. For SEM and EDS analyses, a
Approximately
scanning voltage2of mg 15 of
kVthewaspowder sample
employed. was were
Samples used prepared
for each test. For SEM
by applying andtoEDS
them the
analyses, a scanning voltage of 15 kV was employed. Samples were prepared
surface of a conductive adhesive using cotton swabs, coating with gold and then examining by applying
them
in theto the surface
electron of a conductive
microscope chamber. adhesive
For TEM,using cottonvoltage
a scanning swabs, of
coating
200 kV with
wasgold and
utilized.
then examining
Samples in the electron
were dispersed microscope
in ethanol chamber. For
and homogenized via TEM, a scanningSubsequently,
ultrasonication. voltage of 200a
kV was utilized. Samples were dispersed in ethanol and homogenized via
droplet of the suspension was placed on a copper mesh carrier and observed in the TEM ultrasonication.
Subsequently, a dropletproduced
once dry. Free radicals of the suspension
by memetic was placed onCe-MOF
peroxidase a copper mesh
were carrier
tested and ob-
by electron
served
paramagnetic resonance (EPR) (Bruker, A300, Mannheim, Germany). The fluorescencewere
in the TEM once dry. Free radicals produced by memetic peroxidase Ce-MOF life-
tested
time wasby determined
electron paramagnetic
with HORIBA’sresonance
highly (EPR) (Bruker,
sensitive A300,
integrated Mannheim,spectrometer
fluorescence Germany).
The fluorescence
(FluoroMax-4, lifetimeKyoto,
HORIBA, was determined
Japan). with HORIBA’s highly sensitive integrated flu-
orescence spectrometer (FluoroMax-4, HORIBA, Kyoto, Japan).
Polymers 2024, 16, 1747 5 of 18

2.2.2. XPS Analysis

X-ray photoelectron spectroscopy (XPS) measurements were conducted using the
EscaLab 250Xi XPS system (Thermo Fisher Scientific, Horsham, UK). This analysis offered
detailed insights into the chemical states and elemental composition of the sample. For the
measurements, approximately 10 mg of the sample was used. Key parameters included an
operating voltage of 12.5 kV, a filament current of 16 mA and the use of Al Kα radiation as
the excitation source. The measurements were performed with a pass energy of 30 eV and
step size increments of 0.1 eV.

2.2.3. FT-IR Analysis

The synthesized MOFs were characterized using Fourier transform infrared spectroscopy
(FT-IR) with a Nexus 410 FT-IR spectrometer (Thermo Nicolet, Waltham, MA, USA). Spectra
were recorded across a scanning range from 400 to 4000 cm−1 for samples weighing
approximately 10 mg. Analysis of the FT-IR spectra enabled the identification of specific
functional groups within the MOF materials by analyzing peak positions along the IR curve.

2.2.4. XRD Analysis

X-ray diffraction (XRD) patterns were acquired using an Ultima IV multipurpose X-ray
diffractometer (manufactured in Tokyo, Japan). Powder X-ray diffraction (XRD) patterns
were measured on a Bruker Focus D8 diffractometer operating at 40 kV using Cu-Kα X-ray
radiation (λ = 1.54056 nm). The instrumental parameters for the XRD analysis comprised
a scanning range from 5 to 80 degrees, a scanning speed of 12 degrees per minute and
a sample quantity of approximately 50 mg. The obtained data were then analyzed and
processed using Jade 4.6.0 software.

2.2.5. Brunauer–Emmett–Teller (BET) Analysis

Nitrogen adsorption–desorption isotherms were measured by the BET technique.
Low-pressure gas adsorption studies were performed on the 3Flex Analyzer (Micromeritics
Instrument, Norcross, GA, USA), a fully automated microporous gas analyzer, with relative
pressures up to 1 atmosphere. The low temperature was controlled using a liquid nitrogen
bath at 77 K. The apparent surface area was determined by applying BET and Langmuir
models from nitrogen adsorption isotherms collected at 77 K.

2.2.6. Optical Detection

UV–vis absorption spectra were recorded using a U-3900 UV–vis spectrophotometer
(Hitachi, Tokyo, Japan), and fluorescence spectra were captured with an F-7000 fluores-
cence spectrophotometer (Hitachi, Japan). Additionally, short-wave ultraviolet detec-
tion of the samples at 365 nm was performed using a handheld UV lamp (Spectroline,
Melville, NY, USA).

2.3. Synthesis of Ce-MOF

Ce-MOF was synthesized using the classical solvothermal method [23]. First, 164.5 mg of
ammonium cerium nitrate was dissolved in 15 mL of DMF, and 109.3 mg of 2-hydroxyterephthalic
acid was dissolved in 45 mL of DMF. Both solutions were sonicated until fully dissolved.
The solution containing 2-hydroxyterephthalic acid was then transferred to a 100 mL round-
bottomed flask and immersed in silicone oil at 120 ◦ C. A magnetic stirrer was used to
maintain stirring at 200 rpm. When the solution in the flask reached approximately 120 ◦ C,
600 µL of glacial acetic acid was added, followed by the slow addition of 15 mL of the
ammonium cerium nitrate solution at a rate of 1 drop every 3 s. After the complete addition
of the ammonium cerium nitrate solution, the opening of the flask was gently covered
with tinfoil, and stirring was continued at 200 rpm for 6 min. Subsequently, the magnetic
stirrer was turned off, and the temperature was maintained at 120 ◦ C for 3 h to allow for
the synthesis and growth of Ce-MOF. Afterward, the heating was turned off, the flask
was removed from the silicone oil, and the solution was cooled to room temperature. The
Polymers 2024, 16, 1747 6 of 18

solution was then evenly divided into four 50 mL centrifuge tubes, and 15 mL of anhydrous
ethanol was added to each tube. After balancing, the tubes were centrifuged at 12,000 rpm
at 4 ◦ C for 20 min. Centrifugation was accomplished, the supernatant was removed, and
20 mL of anhydrous ethanol was added to each tube. This washing step was repeated three
times to ensure the removal of any impurities. Once the three washes were completed, the
washed precipitate was collected and placed in an oven at 75 ◦ C for 24 h until completely
dried, yielding the final product.

2.4. The Mimetic Peroxidase-like Activity of Ce-MOF Nanozyme

TMB was used as the color developing substrate. Ce-MOF catalyzes TMB to ox-TMB
only in the presence of Ce-MOF, TMB and H2 O2 , and the solution was obviously blue,
which indicated that it had the activity of simulating peroxidase. The UV–vis absorption
peak was obtained at 652 nm, and the ionic system of the solution changed. In order to
better understand the properties of Ce-MOF intermediates during enzyme conversion in
the presence of hydrogen peroxide and TMB, 1.25 mL of Ce-MOF (0.05 mg·mL–1 ), 8 mL of
buffer solution and 0.75 mL of H2 O2 (100 mM) were prepared for EPR.
To further analyze its catalytic activity, the same concentration of Ce-MOF was used
in acetic acid/sodium acetate buffer solution (pH = 4, 0.2 M). Various concentrations of
TMB (1.5–43.26 mM) and H2 O2 (0.01–2 mM) were tested as the kinetic parameters of the
substrates. The absorbance of the solution at 652 nm was rapidly recorded following the
final addition of each varying concentration of substrate. The maximum reaction rate (Vmax )
and the Michaelis constant (Km ) were then calculated using the relevant Equation (1).
  
1 1 Km 1
= + (1)
V [S] Vmax Vmax

Here, V is the enzymatic reaction efficiency (the initial rate of the catalytic reac-
tion at different substrate concentrations). [S] is the concentration of substrate TMB or
hydrogen peroxide.

2.5. Optimization of Experimental Conditions

Amounts of 150 µL of H2 O2 (100 mM), 250 µL of Ce-MOF (0.05 mg·mL−1 ) and 150 µL
of TMB (10 mM) were mixed in a 2 mL centrifuge tube, and 1450 µL of the buffer solution,
acetic acid/sodium acetate (pH = 4), was added. The mixture reacted at room temperature
(25 ◦ C). During the reaction, Ce-MOF catalyzed TMB to ox-TMB, and the solution was
obviously blue. Finally, the UV–vis absorption peak was collected at 652 nm. Similar
methods were used to perform optimization experiments with a single variable, and
multiple 2 mL centrifugal tubes were prepared. The range of the H2 O2 concentration
optimization test was 1–400 mM, and the range of the TMB concentration optimization test
was 1–15 mM. The pH optimization test range of the buffer solution of acetic acid/sodium
acetate was 2.4–8.2, and the reaction time of the optimized system was 5–50 min.

2.6. Selectivity and Anti-Interference Experiment

In order to study the selectivity and anti-interference of the detection system to H2 S, the
same concentration of selected interfering substances was tested under the same detection
conditions. Glutamic acid (Glu), glutathione (GSH), cysteine (Cys), anions (NO2 − , S2 O4 2− ,
SO3 2− , NO3 − , Br− , SO4 2− , CO3 2− ) and cations (Na+ , Ca2+ , K+ , Cu2+ , Mg2+ , NH4 + ) were
included. The UV–vis absorbance (the corresponding concentration in the detection system
was 192 µM) and fluorescence intensity (the corresponding concentration in the detection
system was 320 µM) were measured by adding sodium sulfide and an equal amount of
interfering substances, respectively. After the solution completely reacted for 30 min, the
solution was placed under the U-3900 ultraviolet spectrophotometer at 652 nm to test
its absorbance. All fluorescence spectra were measured using an excitation wavelength
of 325 nm. The anti-interference test was conducted to measure the UV–vis absorbance
and fluorescence intensity in the same way by adding more than the same amounts of
Polymers 2024, 16, 1747 7 of 18

interfering substances in the test system without adding sodium sulfide. After the test was
completed, sodium sulfide of equal concentration was added to each tube, and the UV–vis
absorbance and fluorescence intensity were determined again [24,25].

2.7. Colorimetric–Fluorescence Sensing Analysis

The catalytic activity of the Ce-MOF nanozyme was investigated by using TMB, a
typical oxidase substrate, in a buffer solution of acetic acid/sodium acetate (pH = 4).
Additionally, 50 mg of sodium sulfide powder was weighed and dissolved in 10 mL of the
buffer solution to prepare a 5 mg·mL−1 sodium sulfide solution, which was sonicated for
future use. A series of 2 mL centrifuge tubes was prepared, each with a total volume of
2 mL of solution. Amounts of 150 µL of H2 O2 (100 mM), 250 µL of Ce-MOF (0.05 mg·mL−1 )
and 150 µL of TMB (10 mM) were added to this mixture, along with varying volumes of
acetic acid/sodium acetate buffer (pH = 4) to ensure proper mixing. The final concentration
of Ce-MOF in the detection system was 6.25 µg·mL−1 . The tubes were then incubated at
25 ◦ C for 30 min. Once a noticeable color change was observed, one tube was set aside as a
control, and varying volumes of the 5 mg·mL−1 sodium sulfide solution, serving as a source
of H2 S, were added to the remaining tubes. The color changes in the solution were visually
observed under sunlight and ultraviolet light. The UV–vis absorbance of the solutions was
measured at a specific wavelength of 652 nm using a spectrophotometer. Additionally,
fluorescence emission spectra were recorded with a fluorescence spectrophotometer, setting
the excitation wavelength to 325 nm.

2.8. Detection of H2 S in the Real Samples

The enzyme-labeled strip of 8 tubes was prepared, and 300 µL of buffer solution acetic
acid/sodium acetate was added to each tube. The fresh salmon was cut into 25 pieces of
similar size and stored in a refrigerator at −20 ◦ C. On days 0–9, four pieces of salmon fillet
and solution-containing enzyme-labeled strip were randomly selected every day, sealed in
white foam dishes using transparent plastic wrap and kept in a refrigerator at 4 ◦ C. The
H2 S released by the fish body was absorbed by the buffer solution. After 9 days, the test
system solution (no buffer solution added) was configured and reacted for 30 min. The
solution in the enzyme-labeled strip was dropped into the mixed solution and reacted
for 2 min, and colorimetric pictures were taken in a camera obscura. Then, the mixed
solution was absorbed into the eight tubes, and an ultraviolet lamp was irradiated to
observe the fluorescence change. Shrimp was purchased and frozen at −20 ◦ C. For days
0–5, 5 shrimp and enzyme-labeled strips containing the solution were randomly selected
every day, sealed in a white foam plate and kept in a refrigerator at 4 ◦ C. The H2 S released
by the shrimp was absorbed by the buffer solution, and the shrimp was stored for 5 days.
Colorimetric and fluorescence changes were observed by the same test method as that used
for the salmon.

3. Results and Discussion

3.1. Characterization
Ce-MOF was prepared by the reaction of ammonium cerium nitrate and ligand 2-
hydroxyterephthalic acid at 120 ◦ C with DMF as a solvent. The successful preparation of
Ce-MOF was confirmed by various characterization techniques [26]. SEM (Figure 1a), TEM
and DLS showed the spheroidal morphology characteristic of Ce-MOF, with a size range
of about 452.4 nm (Figure 1c). Higher resolution images by TEM showed uniform-sized
nanoparticles of Ce-MOF (Figure 1b), which was further verified by dynamic light scattering
results. X-ray energy dispersive spectroscopy (EDS) showed that Ce-MOF contained Ce,
C and O elements, and all elements were evenly dispersed in the Ce-MOF nanozyme
structure (Figure 1d).
OR PEER REVIEW 8 of 18

Polymerscontained
Ce, C and O elements, and all elements were evenly dispersed in the Ce-MOF 8 of 18
2024, 16, 1747
nanozyme structure (Figure 1d).

Figure 1. MorphologyFigure
of Ce-MOF: (a) SEM
1. Morphology image; (b)
of Ce-MOF: TEMimage;
(a) SEM image;(b) (c)
TEM DLS analysis;
image; (c) DLS(d) EDS (d)
analysis; images.
EDS images.

The XRD pattern of Ce-MOF ◦

The XRD pattern of Ce-MOF displayed three displayed
diffraction three
peaksdiffraction
centered peaks centered
at 8.9°, at 8.9 ,
25.6°
25.6◦ and 40.7◦ (Figure 2a), which aligned well with the spectra synthesized by the
and 40.7° (Figure 2a), which aligned well with the spectra synthesized by the predecessors
predecessors [27], and the successful synthesis of Ce-MOF was confirmed. In the FTIR
[27], and the successful synthesis
spectrum (Figureof2b),
Ce-MOF
extensivewas confirmed.
asymmetric In thevibrations
stretching FTIR spectrum
at 3324 cm(Figure
−1 indicated
2b), extensive asymmetric stretching vibrations at 3324 cm −1 indicated abundant hydroxyl−1
abundant hydroxyl groups (O-H) on the surface of Ce-MOF. The peak at 1415 cm cor-
groups (O-H) on theresponded
surface of to the symmetric
Ce-MOF. The stretching
peak atof1415
the carboxylic acid group (O=C–O),
cm−1 corresponded and asym-
to the sym-
metric stretching vibrations were observed at 1571 cm−1 . Additionally, absorption at
metric stretching of the carboxylic acid group (O=C–O), and asymmetric stretching vibra-
1495 cm−1 suggested the presence of asymmetric C=C tensile vibrations in the benzene
tions were observed at[28].
ring 1571 cmresults
These
−1 . Additionally, absorption
verify the successful at 1495
coordination cm−1 suggested
of ammonium the with
cerium nitrate
presence of asymmetric C=C tensile vibrations
2-hydroxyterephthalic in the
acid [29]. The benzene
absolute surfacering [28].
value forThese
Ce-MOF results ver-m2 /g.
was 16.353
ify the successful coordination of ammonium
According to Figure ceriumisotherm
2c, the adsorption nitrateofwithCe-MOF2-hydroxyterephthalic
exhibited an inflection point
within
acid [29]. The absolute the low-pressure
surface value forregion.
Ce-MOF The was
adsorption
16.353 and
mdesorption
2/g. According curve did not overlap
to Figure 2c, in the
high-pressure region, and there was a hysteresis loop. According to the classification of the
the adsorption isotherm of Ce-MOF exhibited an inflection point within the low-pressure
adsorption isotherms, characteristics of type II and type IV were seen, indicating that they
region. The adsorption and desorption
are mesoporous materials curve
[30].did not overlap
According to Figurein 2d,
thethehigh-pressure
size diagram of region,
the aperture
and there was a hysteresis loop.size
had an average According
of 2.52 nm. toThisthe
canclassification
prove that Ce-MOF of the adsorptionmaterial.
is a mesoporous iso-
XPS spectra were analyzed to elucidate the surface
therms, characteristics of type II and type IV were seen, indicating that they are mesopo-composition of Ce-MOF, particu-
larly focusing on the oxidation states of cerium (Ce). The comprehensive XPS spectrum
rous materials [30]. According to Figure 2d, the size diagram of the aperture had an aver-
revealed Ce 3d, O 1s and C 1s signals, consistent with the elemental mapping (Figure 2e).
age size of 2.52 nm. High-resolution
This can proveXPS that Ce-MOF
spectra of Ce is
3daindicated
mesoporous material.of Ce4+ and Ce3+ on the
the coexistence
surface of Ce-MOF (Figure 2h). Herein, 886.28 and 900.48 eV belong to the valance state of
3+ , and 881.58 and 904.48 eV denote the 4+ state for Ce [31]. Moreover, the high-resolution
C 1s spectrum displayed peaks at 286.18, 284.78 and 288.58 eV, corresponding to C–O,
C–C and O–C=O bonds (Figure 2f), and the O 1s spectrum showed peaks at 532.48 and
531.48 eV for O–C=O and Ce–OH, confirming the chemical environment of the surface
(Figure 2g) [22].
 region. The adsorption and desorption curve did not overlap in the high-pressure region,
and there was a hysteresis loop. According to the classification of the adsorption iso-
therms, characteristics of type II and type IV were seen, indicating that they are mesopo-
Polymersrous materials
2024, 16, 1747 [30]. According to Figure 2d, the size diagram of the aperture had an aver- 9 of 18
age size of 2.52 nm. This can prove that Ce-MOF is a mesoporous material.

Figure 2. Characterization of Ce-MOF: (a) XRD spectra; (b) FT-IR spectra; (c) Nitrogen adsorption–
desorption isotherm; (d) BJH pore diameter; (e) XPS spectra; (f) C 1s spectra; (g) O 1s spectra; (h) Ce
3d spectra.

3.2. Peroxidase Mimetic Activity Analysis of Ce-MOF

The peroxidase-like activity of Ce-MOF was evaluated using TMB as the substrate.
Experimental parameters, including pH, TMB concentration, H2 O2 concentration and
reaction time, were optimized to maximize peroxidase activity. The optimal conditions
were determined to be a pH of 4, a TMB concentration of 10 mM and an H2 O2 concentration
of 100 mM. After a reaction time of 30 min, the color change in the solution was readily
observable to the naked eye. Thus, the optimal reaction duration was established at 30 min
under balancing detection efficacy and visibility.
A steady-state kinetic analysis was conducted to assess the peroxidase mimetic activity
of Ce-MOF. As the TMB concentration increased from 1.5 mM to 43.26 mM and as the
H2 O2 concentration rose from 0.01 mM to 2 mM, the reaction rate of Ce-MOF peroxidase
steadily increased. This response exhibited typical Michaelis–Menten kinetics, indicating
that substrate catalysis adhered to the characteristics of this model. The values of Km
and Vmax were determined through fitting the Lineweaver–Burk plot (Figure 3). The
Km value for Ce-MOF with H2 O2 was 0.07773 mM, which reflects the affinity between
the enzyme and its substrate. A lower Km value indicates a stronger affinity between
Ce-MOF and its substrate compared to natural horseradish peroxidase (HRP) and other
reported nanozymes. Ce-MOF exhibited comparable or superior affinity and catalytic
efficiency for H2 O2 , suggesting enhanced catalytic activity, making it suitable for use in
subsequent colorimetric assays. At the same time, the maximum reaction rates of the
Ce-MOF substrates, H2 O2 and TMB, were 15.16 × 10−6 Ms−1 and 6.69 × 10−6 Ms−1 ,
respectively, which were also higher than the maximum reaction rates of some nanozymes
toward H2 O2 substrates (Table 1), indicating that Ce-MOF has excellent and higher catalytic
activity. It also exhibited strong peroxidase-mimicking activity [32].

Table 1. Comparison of kinetic parameters of H2 O2 .

Nanozymes Km (mM) V max (10−6 Ms−1 ) Ref.

Fe3 O4 NPs 154 5.7333 [33]
MOF-808 1.06 0.0139 [34]
Au-Ni/g-C3 N4 0.16 0.0234 [35]
 Polymers 2024, 16, 1747 10 of 18

Table 1. Cont.

Nanozymes Km (mM) V max (10−6 Ms−1 ) Ref.

PtCu 0.37 0.0114 [36]
Ag-MoS2 2.29 0.0875 [37]
Polymers 2024, 16, x FOR PEER REVIEW HRP 3.7 0.0870 [38] 10 of 18
Ce-MOF 0.07773 15.16 This work

Figure 3. Steady-state kinetic analysis: (a,b) Kinetic study for the substrate of H2O2 of Ce-MOF based
Figure 3. Steady-state kinetic analysis: (a,b) Kinetic study for the substrate of H2 O2 of Ce-MOF based
on the Michaelis–Menten equation; (c,d) Kinetic study for the substrate of TMB of Ce-MOF based
on the Michaelis–Menten equation; (c,d) Kinetic study for the substrate of TMB of Ce-MOF based on
on the Michaelis–Menten equation.
the Michaelis–Menten equation.

Table 1. Comparison
3.3. Colorimetric of kinetic parameters
Characteristic Analysis ofofCe-MOF
H2O2.

Nanozymes The feasibility analysis K

ofmthe dual-mode colorimetric–fluorescence
(mM) Vmax (10−6 Ms−1) sensor was carried
Ref.
out according to a previous method [39]. As shown in Figure 4a, TMB alone (purple line)
Fe3O4NPs 154 5.7333 [33]
and TMB combined with H2 O2 (red line) exhibited no significant UV–vis absorption peak
MOF-808 1.06 0.0139 [34]
at 652 nm. However, upon the addition of Ce-MOF (blue line), the UV–vis absorption
Au-Ni/g-C3N4 0.16 0.0234
spectrum displayed a pronounced peak at 652 nm. This peak was notably diminished [35]
PtCu when the mixture was treated0.37 with a 5 mg·mL−1 sodium0.0114 [36]
sulfide solution, indicating a
Ag-MoS2 2.29 0.0875 [37]
significant reduction in the concentration of Ce-MOF following the absorption of H2 S. This
HRP observation could also suggest3.7 that the catalytic activity of the Ce-MOF nanozyme
0.0870 [38] was
Ce-MOF substantially decreased after0.07773
the addition of H2 S, and it implies
15.16 that the presence of H2 S
This work
might inhibit the Ce-MOF nanozyme’s catalysis in the TMB oxidation reaction [32].
3.3. Colorimetric Characteristic Analysis of Ce-MOF
The feasibility analysis of the dual-mode colorimetric–fluorescence sensor was car-
ried out according to a previous method [39]. As shown in Figure 4a, TMB alone (purple
line) and TMB combined with H2O2 (red line) exhibited no significant UV–vis absorption
peak at 652 nm. However, upon the addition of Ce-MOF (blue line), the UV–vis absorption
spectrum displayed a pronounced peak at 652 nm. This peak was notably diminished
when the mixture was treated with a 5 mg·mL−1 sodium sulfide solution, indicating a sig-
Polymers 2024, 16, x FOR PEER REVIEW 11 of 18
Polymers 2024, 16, 1747 11 of 18

Figure 4. Colorimetric
Figure 4. Colorimetriccharacteristic analysis:(a)
characteristic analysis: (a)Feasibility
Feasibilityof of colorimetric
colorimetric detection
detection of dual-mode
of dual-mode
sensor; (b) EPR spectrum of DMPO-
sensor; (b) EPR spectrum of DMPO- OH. OH.

In In ordertotoidentify
order identify thetheactive
activefree radicals
free produced
radicals duringduring
produced Ce-MOF catalysis,catalysis,
Ce-MOF the EPR the
technique was used to directly detect reactive oxygen species (ROS)
EPR technique was used to directly detect reactive oxygen species (ROS) using 5,5-dime- using 5,5-dimethyl-1-
pyrroline N-oxide
thyl-1-pyrroline (DMPO)
N-oxide as a trapping
(DMPO) probe. Itprobe.
as a trapping could be observed
It could that ·OH (hydroxyl
be observed that ·OH (hy-
radical) radiated in the Ce-MOF solution for 20 min in a four-wire spectrum of 1:2:2:1,
droxyl radical) radiated in the Ce-MOF solution for 20 min in a four-wire spectrum of
which further confirms that the hydroxyl radical was the main intermediate, and ·OH
1:2:2:1, which further
was produced in theconfirms
system, from thatwhich
the hydroxyl
it can be radical
inferredwas
that the
·OHmainwas theintermediate,
main ROS and
·OH was produced in the system, from which it can be inferred
in the Ce-MOF system (Figure 4b) [40]. It was verified that the catalytic pathway that ·OH wasofthethemain
ROS in theinvolved
solution Ce-MOFgenerating
system (Figure 4b) [40].
a hydroxyl It was
radical, verified
oxidizing TMB that the catalytic
to oxTMB pathway of
and changing
thethe
solution
solutioninvolved generating
from colorless to bluea [41].
hydroxyl radical, oxidizing TMB to oxTMB and chang-
ing the Ten 2 mLfrom
solution centrifuge tubestowere
colorless blueprepared,
[41]. and 150 µL of H2 O2 (100 mM), 250 µL
ofTenCe-MOF
2 mL(0.05 mg·mL−tubes
centrifuge
1 ) and 150 µL of TMB (10 mM) were added, respectively, and
were prepared, and 150 µL of H2O2 (100 mM), 250 µL of
mixed (0.05
Ce-MOF with mg·mL
different−1)volumes
and 150of µLthe
of buffer
TMB (10 solution
mM) wereof acetic acid/sodium
added, acetate
respectively, andfor
mixed
30 min, making sure that the solution was blue after the full reaction. After the reaction,
with different volumes of the buffer solution of acetic acid/sodium acetate for 30 min,
0.0, 6.0, 9.8, 19.7, 32.9, 41.3, 46.0, 55.2, 60.5 and 74.0 µL sodium sulfide solutions were
making
addedsure that
to ten 2 mLthecentrifuge
solution was tubes,blue
andafter the volume
the total full reaction.
of the After
mixedthe reaction,
solution 0.0, 6.0,
in each
9.8,centrifuge
19.7, 32.9,tube
41.3,was
46.0, 55.2,
fixed to 60.5
be 2 and
mL. The74.0concentration
µL sodium sulfide of H2 S solutions
in the system werewasadded
0.000,to ten
2 mL0.192, 0.314, 0.631, 1.054, 1.323, 1.474, 1.768, 1.938 and 2.371 mM, respectively. After 2 min, tube
centrifuge tubes, and the total volume of the mixed solution in each centrifuge
wasthe fixed to be 2of
absorption mL.
theThe concentration
series of concentration of H 2S in the
gradient systematwas
solutions 6520.000,
nm was 0.192, 0.314, 0.631,
determined,
and1.323,
1.054, the UV–vis
1.474,absorption
1.768, 1.938 spectrum was obtained.
and 2.371 As the concentration
mM, respectively. After 2 min, of sodium sulfide
the absorption of
increased, the characteristic peak of oxTMB gradually decreased,
the series of concentration gradient solutions at 652 nm was determined, and the UV–vis and it could be seen
that the UV–vis
absorption spectrum absorption of the sensor
was obtained. As thehad a good correlation
concentration with the
of sodium concentration
sulfide increased, the
of Na2 S (0–2300 µM) (Figure 5a). By fitting this value to the Na2 S concentration, the
characteristic peak of oxTMB gradually decreased, and it could be seen that the UV–vis
fitted equation was Y = −0.3558X + 1.1913 (R2 = 0.9942) with a limit of detection (LOD) of
absorption of the sensor had a good correlation
0.262 µM (Figure 5a inset). The limit of detection was calculated with the concentration
by Equation (2), of Na 2S (0–2300
where σ
µM) (Figure 5a). By fitting this value to the Na 2S concentration, the fitted equation was Y
is the standard deviation of the blank and k is the slope of the linear fitting equation.
= −0.3558X + 1.1913 (R2 = 0.9942) with a limit of detection (LOD) of 0.262 µM (Figure 5a
inset). The limit of detection was calculated 3σ
LOD =by Equation (2), where σ is the standard
(2) de-
k fitting equation.
viation of the blank and k is the slope of the linear
The sensor colorimetric response had a detection
3σ range of 200–2300 µM. As the con-
LOD
centration of H2 S in the solution increased, the=color transitioned from blue to colorless, (2)
k
demonstrating the inhibitory effect of H2 S on the catalytic activity of Ce-MOF. This effect
could be attributed to several mechanisms, including strong coordination between H2 S and
the active sites of Ce-MOF, a reduction in the formation of intermediate hydroxyl radicals
and a decrease in the levels of peroxide or oxTMB.
16, x FORPolymers
PEER REVIEW
2024, 16, 1747 12 of 18
12 of 18

Figure 5. Dual-mode colorimetric–fluorescence

Figure sensor respondssensor
5. Dual-mode colorimetric–fluorescence to different
respondsH2Stoconcentrations: (a)
different H2 S concentrations:
Linear relationship(a)between UV–vis absorption
Linear relationship and different
between UV–vis absorptionHand
2S concentrations; (b) Fluorescence
different H2 S concentrations; (b) Fluorescence
spectrum of different H2S concentrations
spectrum of different H2 Sunder excitation
concentrations of 325
under nm; (c)of
excitation Linear relationship
325 nm; between between
(c) Linear relationship
fluorescence spectrum and different H 2 S concentrations; (d) Fluorescence lifetime of Ce-MOF.
fluorescence spectrum and different H2 S concentrations; (d) Fluorescence lifetime of Ce-MOF.

3.4. Analysis ofresponse

The sensor colorimetric Fluorescence
hadProperties of Ce-MOF
a detection range of 200–2300 µM. As the con-
The solution
centration of H2S in the excitation increased,
wavelengththeof Ce-MOF was determined
color transitioned by blue
from the UV–vis absorption curve.
to colorless,
demonstrating the inhibitory effect of H2S on the catalytic activity of Ce-MOF. This the
It can be seen from Figure 4a that, when only Ce-MOF existed in solution, wavelength
effect
corresponding to the UV–vis absorption peak appeared at 325 ± 5 nm. Therefore, 325 nm
could be attributed to several mechanisms, including strong coordination between H2S
was set as the fluorescence emission of the excitation wavelength determination test system.
and the active sites of Ce-MOF, a reduction in the formation of intermediate hydroxyl
The peak fluorescence emission occurred at 410 ± 5 nm. Eleven 2 mL centrifuge tubes were
radicals and a decrease
prepared inand
themixed
levelswith
of peroxide
150 µL oforHoxTMB.
O (100 mM), 250 µL of Ce-MOF (0.05 mg·mL−1 ),
2 2
150 µL of TMB (10 mM) and different volumes of the buffer solution acetic acid/sodium
3.4. Analysis of Fluorescence
acetate for 30Properties of Ce-MOF
min, making sure that the solution was blue after the full reaction. After
the reaction, 0.0, 0.5,
The excitation wavelength of Ce-MOF 1.0, 1.5, 2.0,was
3.0, 4.0, 5.0, 6.0, 8.0by
determined and
the10.0 µL sodium
UV–vis sulfide solutions
absorption
that had turned were added to eleven 2 mL centrifugal
curve. It can be seen from Figure 4a that, when only Ce-MOF existed in solution, the wave- tubes, and the total volume of
the mixed solution in each centrifugal tube was also fixed to 2 mL. The concentration
length corresponding to the UV–vis absorption peak appeared at 325 ± 5 nm. Therefore,
of H2 S in the system was 0.0, 16.0, 32.0, 48.0, 64.0, 96.1, 128.1, 160.2, 192.2, 256.3 and
325 nm was set as theµM,
320.0 fluorescence
respectively.emission
After theof the excitation
reaction for 2 min,wavelength determination
the fluorescence intensity of this series
test system. The of peak fluorescence emission occurred at 410 ± 5 nm. Eleven
concentration gradient solutions was determined at an excitation wavelength 2 mL centri-of 325 nm,
fuge tubes were and prepared and mixed with 150 µL of H
the fluorescence spectrum, as shown in Figure 5b, was obtained. Ce-MOF
2 O 2 (100 mM), 250 µL of
(0.05 mg·mL ), 150 µL
−1 In of TMBto(10
order mM) investigate
further and different thevolumes of the
fluorescence buffer solution
response of Ce-MOF acetic
to H2 S, the
acid/sodium acetateemission
for 30 spectra
min, of Ce-MOF
making suretreated
that with different concentrations
the solution was blue afteroftheH2 Sfull
(0–320
reac-µM) in the
tion. After the reaction, 0.0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0 and 10.0 µL sodium sulfide
solutions that had turned were added to eleven 2 mL centrifugal tubes, and the total vol-
ume of the mixed solution in each centrifugal tube was also fixed to 2 mL. The concentra-
tion of H2S in the system was 0.0, 16.0, 32.0, 48.0, 64.0, 96.1, 128.1, 160.2, 192.2, 256.3 and
320.0 µM, respectively. After the reaction for 2 min, the fluorescence intensity of this series
Polymers 2024, 16, 1747 13 of 18

buffer solution of acetic acid/sodium acetate were recorded. As shown in Figure 5b, the
fluorescence intensity of Ce-MOF at 410 nm gradually increased with the increase in the
concentration of Na2 S in the H2 S source. A rate-based fluorescence method was used to
evaluate the detection ability of Ce-MOF. As shown in Figure 5c, the fluorescence intensity
of the sensor I410 had a good correlation with the concentration of Na2 S (0–320 µM). The
fitting equation is as follows: Y = 1.2768X + 1360.1600 (R2 = 0.9665). More importantly,
under the irradiation of 365 nm ultraviolet light, the color of the detection liquid showed a
significant change from no obvious fluorescence to obvious bright blue fluorescence, which
could realize the visual detection of the sensor. The Ce-MOF sensor had a detection range
of 0–320 µM for H2 S. With a limit of detection of 0.156 µM, the detection of H2 S by Ce-MOF
was more sensitive than previously reported. The colorimetric detection method was
directly visible to the naked eye in sunlight and was simple and portable, so we fabricated
a colorimetric sensing method. According to Table 2, it can be seen that the colorimetric
limit of detection of our sensor was lower than that of other colorimetric sensors but still
higher than that of most fluorescence sensors. Therefore, we also established a more precise
fluorescence detection method, and a dual-mode colorimetric–fluorescent sensor was made
in our work. By comparing the LOD values of the sensors studied by previous researchers,
it can be found that our Ce-MOF material had a low LOD among a variety of sensors using
colorimetric, fluorescent or dual-mode colorimetric–fluorescence methods, indicating its
high sensitivity.
It can be seen from the ultraviolet curve and fluorescence spectrum that the fluo-
rescence emission wavelength of the sensor ranged from 350 nm–500 nm, and the blue
line (TMB + H2 O2 + Ce-MOF) and green line (TMB + H2 O2 + Ce-MOF + Na2 S) in the
UV–vis absorption spectrum peaked at around 350 nm–450 nm. Fluorescence lifetimes
were used to explore the mechanisms underlying the establishment of the dual-mode
Ce-MOF nanozyme sensor. The overlap of the fluorescence emission spectrum of Ce-MOF
(energy donor) and the UV–vis absorption spectrum of oxTMB (energy acceptor) shown in
Figure 4a provide the conditions of FRET. As shown in Figure 5d, the fluorescence lifetime
of Ce-MOF was 24 ns, which changed to 16 ns after oxTMB was added. The change in
the fluorescence lifetime indicated that the enhanced fluorescence between Ce-MOF and
oxTMB was based on the shortened fluorescence lifetime of Ce-MOF as an energy donor
by FRET [42]. In addition, with the increase in the concentration of Na2 S, the oxTMB in
the solution was reduced to TMB, the oxTMB decreased, and the fluorescence emission
intensity of the detection system at 410 nm gradually increased, indicating that the FRET
effect was enhanced [43,44].
Table 2. Comparison of other sensors for detecting H2 S.

Sensors Types LOD Ref.

CR-DNP Fluorescence 0.4 µM [45]
Pb(btc)-1 Colorimetric 110 µM [46]
P1 Fluorescence 0.66 µM [47]
Probe L Fluorescence 0.372 µM [5]
Microplate
cover-based Colorimetric 1.48 µM [48]
colorimetric assay
Eu(tdl)2 abp Fluorescence 0.64 µM [49]
GG-AgNPs Colorimetric 0.81 µM [50]
Colorimetric 0.262 µM
Ce-MOF This work
Fluorescence 0.156 µM
Polymers 2024, 16, x FOR PEER REVIEW 14 of 18
Polymers 2024, 16, 1747 14 of 18

The evaluation
3.5. Selectivity of selectivity and anti-interference performance are key factors in the
and Anti-Interference
analysis
The of accuracy.ofTo
evaluation evaluateand
selectivity the anti-interference
specific perception of H2S by are
performance Ce-MOF, Glu, in
key factors GSH
the and
Cys, various potential interfering substances such as anions (NO
analysis of accuracy. To evaluate the specific perception of H2 S by Ce-MOF, Glu, GSH2 −, S2O42−, SO32−, NO3−, Br−,

SO , COvarious
and42−Cys, 32−) andpotential
cations (Na +, Ca2+, K+, Cu2+, Mg2+, NH4+) were added
interfering substances such as anions (NO2 − , Sinto 2the
− detection
2 O4 , SO3 ,
2−
system−
NO3 , Br −
in equal 2 −
, SO4 amounts 2 −
, CO3 )under the same
and cations + 2+
(Na concentration +
, Ca , K , Cu test2+ 2+
conditions.
, Mg 4+
, NH ) were All UV–vis
added ab-
sorbance and fluorescence
into the detection system in spectra were recorded.
equal amounts under the same concentration test conditions.
As canabsorbance
All UV–vis be clearly seen in Figure 6a,b,
and fluorescence the presence
spectra of these interfering substances did
were recorded.
As can be clearly seen in Figure 6a,b, the
not cause a significant change in the color of the reaction presence of these interfering
solution. substances
In contrast, the peak
did not cause
UV–vis a significant
absorption change in the
was significantly color of
reduced, andthethe
reaction solution.
fluorescence In contrast,
intensity was the
signifi-
peak UV–vis absorption was significantly reduced, and the fluorescence
cantly enhanced after the introduction of H2S. In the selective test, it is worth noting that intensity was
significantly
sulfite, enhanced
thiosulfate andafter the introduction
cysteine, which are of veryH2 S. In the to
similar selective
H2S, hadtest,little
it is worth noting
effect on the Ce-
that sulfite, thiosulfate and cysteine, which are very similar to H2 S, had little effect on the
MOF nanozyme, and their UV–vis absorbance ratio and fluorescence intensity ratio were
Ce-MOF nanozyme, and their UV–vis absorbance ratio and fluorescence intensity ratio
not obvious compared with H2S. These results show that the Ce-MOF-nanozyme-based
were not obvious compared with H2 S. These results show that the Ce-MOF-nanozyme-
H2S detection method had excellent selectivity and anti-interference capability, and the
based H2 S detection method had excellent selectivity and anti-interference capability, and
established
the established method
method hadhad
significant
significantselectivity
selectivityandandcould
couldspecifically
specifically distinguish
distinguish andand de-
tect H2H
detect S despite
S despitethe presence
the presenceofofpotential
potentialinterfering
interfering compounds.
compounds.
2

Figure 6. Selectivity and anti-interference of Ce-MOF: (a) UV–vis absorption spectrum from the
Figure 6. Selectivity
responses of Ce-MOFand anti-interference
in the presence of H2ofS Ce-MOF: (a) UV–vis
or coexistence absorption
with other species;spectrum from the re-
(b) Fluorescence
sponses of Ce-MOF in the presence of H2S or coexistence with other species; (b) Fluorescence inten-
intensity from the responses of Ce-MOF in the presence of H2 S or coexistence with other species.
sity from the responses of Ce-MOF in the presence of H2S or coexistence with other species.
3.6. Detection of H2 S e in Real Samples
3.6. Detection
Monitoringof H
H22SSeconcentrations
in Real Samplesis used as a fast, sensitive, non-destructive and stable
method to evaluate
Monitoring H2Sthe freshness of aquatic
concentrations is used asproducts. Fresh salmon
a fast, sensitive, and shrimpand
non-destructive were
stable
selected to
method as evaluate
the real detected objects,
the freshness and the products.
of aquatic colorimetric andsalmon
Fresh fluorescence changes
and shrimp in se-
were
Ce-MOF
lected as sensors
the realwere monitored
detected objects, 4 ◦ Cthe
at and for colorimetric
9 days of salmon deterioration changes
and fluorescence and 5 days
in Ce-
of shrimp deterioration (Figure 7a). The salmon stored at 4 ◦ C were selected on days
MOF sensors were monitored at 4 °C for 9 days of salmon deterioration and 5 days of
0, 4 and 9, and the shrimp samples were picked on days 0, 2 and 4 (Figure 7b). The
shrimp deterioration (Figure 7a). The salmon stored at 4 °C were selected on days 0, 4 and
buffer solution of acetic acid/sodium acetate on different days reacted with the mixed
9 , and the shrimp samples were picked on days 0, 2 and 4 (Figure 7b). The buffer solution
solution of the test system for 2 min, and the color changes in the solution were observed
of acetic acid/sodium acetate on different days reacted with the mixed solution of the test
under daylight and ultraviolet light, respectively. It was observed that the colorimetry of
system for gradually
the sensor 2 min, andchanged
the colorfrom
changes
blue in
to the solution
colorless, andwere observed under
the fluorescence daylight
of the sensor and
ultraviolet light, respectively. It was observed that the colorimetry of the
gradually increased, which further confirmed that the sensor had a significant response sensor gradually
changed from blue to colorless, and the fluorescence of the sensor
to different H2 S concentrations generated by decomposition during the degradation of gradually increased,
which
aquaticfurther
productsconfirmed
[51]. that the sensor had a significant response to different H2S con-
centrations generated by decomposition during the degradation of aquatic products [51].
Polymers 2024, 16, x FOR PEER REVIEW 15 of 18
Polymers 2024, 16, 1747 15 of 18

Figure 7. Application of Ce-MOF in real samples: (a) H2 S detection in shrimp; (b) H2 S detection
Figure 7. Application of Ce-MOF in real samples: (a) H2S detection in shrimp; (b) H2S detection in
in salmon.
salmon.
4. Conclusions
4. Conclusions
In summary, this work proposed and fabricated a novel nanozyme dual-mode sensor
and established a colorimetric–fluorescence method for the real-time visual determination
In summary, this work proposed and fabricated a novel nanozyme dual-mode sensor
of H2 S. This was achieved by utilizing Ce-MOF as a peroxidase mimetic enzyme and a dual-
and established a colorimetric–fluorescence method for the real-time visual determination
mode probe. The Ce-MOF nanozyme exhibited a rapid response (<5 min), high selectivity
ofand
H2exceptional
S. This wassensitivity
achieved(LOD by utilizing
= 0.262 µMCe-MOF as a peroxidase
in colorimetric method; mimetic enzyme
LOD = 0.156 µM and a
dual-mode probe.
in fluorescence The Ce-MOF
method). The sensornanozyme
comprised exhibited
Ce-MOF,a3,3’,5,5’-tetramethylbenzidine
rapid response (<5 min), high se-
lectivity
(TMB) and andHexceptional
O
2 2 . Ce-MOF sensitivity
acted as (LOD
a = 0.262
catalyst for µM in
hydrogencolorimetric method; LOD = 0.156
peroxide decomposition,
·
µM in fluorescence method). The sensor comprised Ce-MOF, 3,3’,5,5’-tetramethylbenzi-
generating OH that oxidized TMB into oxTMB. Additionally, energy transferred from
Ce-MOF
dine (TMB)to and
TMBHthrough FRET. As
2O2. Ce-MOF theas
acted concentration
a catalyst for of hydrogen
H2 S increased in the decomposition,
peroxide solution,
the FRET effect strengthened, leading to enhanced fluorescence signals
generating ·OH that oxidized TMB into oxTMB. Additionally, energy transferred from Ce- with gradient
intensity. Furthermore, this sensor was successfully applied to detect H2 S contents in
MOF to TMB through FRET. As the concentration of H2S increased in the solution, the
salmon and shrimp samples, which allowed us to accurately monitor their freshness levels,
FRET effect strengthened, leading to enhanced fluorescence signals with gradient inten-
ranging from fresh to medium fresh to spoiled states. In summary, the improved nanozyme
sity. Furthermore,
dual-mode sensor notthisonly
sensor wasmore
enabled successfully appliedoftoHdetect
precise detection H2S contents in salmon
2 S but also demonstrated
and shrimp
excellent samples, which
visualization allowed
performance when us analyzing
to accurately monitorsuch
real samples theirasfreshness levels, rang-
food products.
ing from fresh to medium fresh to spoiled states. In summary, the improved nanozyme
Author Contributions:
dual-mode sensor not Q.C. data enabled
only curation and writing—original
more draft preparation.
precise detection of H2S butX.D.also
investigation,
demonstrated
methodology and formal analysis. Z.F. validation and visualization; Z.D. and J.X. writing—review
excellent visualization performance when analyzing real samples such as food products.
and editing. All authors contributed to the study conception and design. All authors have read and
agreed to the published version of the manuscript.
Author Contributions: Q.C. data curation and writing—original draft preparation. X.D. investiga-
Funding:
tion, This work
methodology andwas supported
formal by the
analysis. Z.F.National Keyand
validation R&Dvisualization;
Program of China (Grant
Z.D. and J.X.No.
writing—
2023YFD2401405)
review andAll
and editing. Shanghai Local
authors College Capacity
contributed to theBuilding Foundation (Grant
study conception No. 23010502400).
and design. All authors have
read and agreed
Institutional to the
Review published
Board version
Statement: of the manuscript.
Not applicable.
Funding: This work
Data Availability was supported
Statement: by theinNational
All data analyzed this study Key R&D Program
are included of China
in the published (Grant No.
article.
2023YFD2401405) and Shanghai Local College Capacity Building Foundation (Grant No.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
23010502400).
personal relationships that could have appeared to influence the work reported in this paper.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: All data analyzed in this study are included in the published article.
Conflicts of 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.

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