ADDIS ABABA SCIENCE AND TECHNOLOGY UNIVERSITY

MAGNETIC CHITOSAN HYDROGEL NANOZYME

WITH INTRINSIC PEROXIDASE ACTIVITY FOR

COLORIMETRIC SENSING OF THIABENDAZOLE

M.Sc. Thesis Research Proposal

BY
ABERA MERGA ARITI

Advisor: MENBERE LEUL (Ph.D.)

Coadvisor: ASSELEFECH SORSA (Ph.D.)

DEPARTMENT OF INDUSTRIAL CHEMISTRY

COLLEGE OF APPLIED SCIENCES

DECEMBER, 2022
ADDIS ABABA, ETHIOPIA
Approval Page

Title: Magnetic Chitosan Hydrogel Nanozymes with Intrinsic Peroxidase Activity for
Colorimetric Sensing of Thiabendazole.

Student Name: Abera Merga

Signature, Date: ----------------------------------------------------------

Approved by the examining committee members:

Name Academic Rank Signature Date

Advisor: Menbere Leul Ph.D. ------------------------------------------------------------------

Co-Advisor: Asselefech Sorsa Ph.D. ------------------------------------------------------------------

Examiner: -------------------------------------------------------------------------------------------------------------------------------------------------

Examiner: ---------------------------------------------------------------------------------------------------------------------------------------------------

Chairperson: --------------------------------------------------------------------------------------------------------------------------------------------------

Name Signature Date

DGC Chairperson: -------------------------------------------------------------------------------------------------------------------------------------

Dean/ Associate ------------------------------------------------------------------------------------------------------------------------------------

Dean for Graduate

Program:

Table of Contents
Acknowledgment............................................................................................................................iv

i
List of abbreviations and Acronyms................................................................................................v
List of Figure and Scheme..............................................................................................................vi
List of Tables.................................................................................................................................vii
Executive Summary.....................................................................................................................viii
Introduction......................................................................................................................................1
1.1 Statement of the Problem.......................................................................................................3
1.2 Significance of the Study.......................................................................................................4
1.3 Scope of the Study.................................................................................................................5
1.4 Objectives of the Study..........................................................................................................5
1.4.1 General objective.............................................................................................................5
1.4.2 Specific objectives...........................................................................................................5
2. Literature Review........................................................................................................................6
2.1 Nanozymes.............................................................................................................................6
2.2 Enzymatic Activity of Nanozymes........................................................................................6
2.3 Peroxidase Sensor...................................................................................................................7
2.4 Chitosan.................................................................................................................................8
2.4.1 Properties of chitosan......................................................................................................8
2.4.2 General applications of chitosan.....................................................................................9
2.5 Colorimetric Sensor based on Peroxidase Nanozymes..........................................................9
2.6 Peroxidase based Nanozymes for Food Safety Monitoring.................................................10
2.7 TBZ as a Food Concern.......................................................................................................12
2.8 Current Detection Methods for TBZ....................................................................................12
3. Research Methodology..............................................................................................................14
3.1 Chemicals and Apparatus.....................................................................................................14
3.2 Experimental Methods.........................................................................................................15
3.3 Synthesis of Magnetic Chitosan Hydrogel Nanocomposite.................................................15
3.4 Characterization of Magnetic Chitosan Hydrogel Nanocomposite.....................................16
3.5 Enzymatic Activity of Magnetic Chitosan Hydrogel Nanocomposite.................................16
3.6 Enzyme Kinetics of Magnetic Chitosan Hydrogel Nanocomposite....................................16
3.7 Colorimetric Detection of TBZ............................................................................................17
3.8 Detection of TBZ in Real Fruit Matrix................................................................................17

ii
 3.9 Development of Point-of-need Sensor Platform..................................................................18
3.10 Data Analysis.....................................................................................................................18
4. Work Plan..................................................................................................................................19
5. Budget Breakdown....................................................................................................................20
References......................................................................................................................................21

iii
Acknowledgment

I want to express my gratitude to my advisors, Menbere Leul (Ph.D.) and Asselefech Sorsa
(Ph.D.), for their direction, counseling, and suggestions. Additionally, I want to thank Yitayal
Admassu (Ph.D.) for his help and advice. Moreover, I would like to thank the Industrial
Chemistry Department, the College of Applied Sciences, and Addis Ababa Science and
Technology University for arranging for me to write my research proposal during my vacation.

iv
List of abbreviations and Acronyms
BSA Bovine serum albumin

CQD Carbon quantum dots

CC-PdNPs Carboxylated chitosan–coated palladium nanoparticles

DDA Degree of deacetylation

DMSO Dimethyl sulfoxide

ELISA Enzyme Linked Immunosorbent Assay

EPA Environmental Protection Agency

GC-MS Gas chromatography-mass spectrometry

HPLC High-performance liquid chromatography

HRP Horse radish peroxidase

LOD Limit of Detection

LOQ Limit of Quantification

MCH Magnetic chitosan hydrogel

MNPs Magnetic nanoparticles

O-GQD Oxygenated graphene quantum dots

RNA Ribonucleic acid

ROS Reactive oxygen species

SEM Scanning electron microscope

SERS Surface-Enhanced Raman Spectroscopy

TMB Tetramethylbenzidine sulfate

XPS X-ray photoelectron spectroscopy

XRD X-ray powder diffraction

v
List of Figure and Scheme
Figure 1 Chemical structure of thiabendazole.................................................................................2
Scheme 1 The steps (work packages) followed in the methodology

vi
List of Tables
Table 1 Peroxidase based-nanozymes for the detection of pesticide............................................11
Table 2. Lists of chemical and apparatus required to perform the research..................................14
Table 3. The estimated time table required to finalize this research.............................................19
Table 4. The budget required for accomplishing the research.......................................................20

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Executive Summary
Nowadays, food safety has gained increasing attention as a major global public health threat.
Due to the extensive use of pesticides in food production, which exposes people to adverse health
effects in a number of ways, including through the consumption of tainted food. Thus, an
effective detection method of pesticides is critical for ensuring food safety throughout the food
supply chain. However, due to the complexity of food matrices and the trace level of food
contaminants, developing reliable and efficient detection methods of pesticides has been a
difficult task. Thiabendazole (TBZ), a benzimidazole derivative, is a widely used pesticide that
protects citrus, apples, and pears from mold, rot, and blight during storage and transportation.
Exposure to thiabendazole can cause nausea, vomiting, headache, weakness, drowsiness, and
loss of appetite. At high doses, it can cause thyroid hormone imbalances and liver damage.
Conventional detection methods for TBZ requires longer sample pretreatment steps, and
expertise skills which makes rapid decision making difficult. Therefore, nanomaterials with
enzyme mimetic activity, often called nanozymes are promising alternative materials for rapid
colorimetric detection of TBZ and other pesticides. Nanozymes have high stability, tunability,
and low cost as compared to natural enzymes-based detection schemes. Herein, magnetic
chitosan hydrogel (MCH) nanocomposite will be synthesized via a facile in-situ method.
Chitosan will be dissolved in acetic acid solution and its protonated form will undergo gelation
with iron ions (Fe3+/Fe2+). After that MCH will be obtained through alkali treatment. The
synthesized nanocomposites will be characterized with SEM, XRD, XPS, FT-IR, and UV-Vis
spectrophotometers. The peroxidase mimicking activity of synthesized nanomaterial will be
investigated using 3,3′,5,5′-tetramethylbenzidine (TMB)/H 2O2) substrates. All parameters
affecting the enzyme activity such as pH, time, and amount of nanozyme will be optimized. The
colorimetric detection of TBZ will be based on its enzyme inhibition effect which could be then
correlated with the concentration of TBZ detection in a real fruit sample (apple). The percent of
recovery, LOD, LOQ, and linearity will be calculated from the regression of the calibration
curve obtained. Finally, a mobile phone-based sensor platform for point need application in food
safety will be prepared

Keywords: Food safety; Magnetic hydrogel; Nanozymes; Thiabendazole

viii
Introduction

Enzymes, as powerful biocatalysts, are mainly composed of proteins while a few are catalytic
RNA molecules[1]. Whereas traditional chemical or industrial catalysts are frequently used in
harsh conditions such as high temperature, high pressure, organic solvents, and extreme pH
environments [2, 3], enzymes are primarily used to catalyze the conversion of biomolecules, and
these reactions are typically carried out under relatively mild conditions [4]. Natural enzymes
have been widely used in industrial, medical, and biological fields due to their high catalytic
activities and substrate specificity [5]. Although promising, they frequently have inherent flaws
such as high preparation and purification costs, low operational stability, catalytic activity
sensitivity to environmental conditions, and difficulties in recycling and reusing [6]. These limit
their further applications in food processing, biosensing, environmental protection, biomedicine,
and so on. To overcome these limitations, scientists have explored alternative artificial enzymes
mimicking the natural enzymes as affordable and more stable substitute called (“nanozymes”).

Nanozymes are nano-materials (with nanoscale sizes 1−100 nm) based artificial enzymes with
intrinsic catalytic activity [7]. Depending on their catalytic activity, nanozymes are classified as
peroxidase, oxidoreductase, superoxide dismutase, catalase, and hydrolase. In 2007, Gao and his
colleagues reported Fe3O4 nanozymes for the first time as having intrinsic peroxidase mimicking
activity [8]. Inspired by this discovery, researchers conducted intensive studies and reported
different kind of nanozymes based on metals (Au, Ag, Pt, Pd, Ir), metal-oxides (CeO 2, Co3O4,
V2O5, V2O3) carbon, single-walled carbon nanotubes (SWNTs), carbon dots (C dots), fullerene,
graphene-oxide (GO)), and other nanomaterials based, (such as Prussian blue [Fe(III)Fe(II)
(CN)6]−, metal organic frameworks (MOFs), and metal chalcogenides (CuS, MnSe, MoS 2, FeSe))
[9].

Nanozymes have become a promising bio-nanotechnological product as they are advantageous

over natural enzymes in terms of high and adjustable catalytic activity, low cost, ease of mass
production, high stability, and durability [10-12]. Compared with natural enzyme, they also have
similar or even better catalytic efficiency following the same enzyme kinetics. Their activity,
selectivity, and specificity of nanozymes are due to their special properties like size, morphology
and large surface area [13]. Thereby, based on the merits, a great many nanozymes-based

1
biosensors has been successfully developed and widely applied in biomedicine, environment,
and agricultural fields [14].

Nanozymes have contributed in agricultural practices, intentional adulteration in processed

foods, and food supply chain contamination, all of which endanger consumer health [15]. Due to
the prevalent use of pesticides from agriculture to the food supply chain, their residues are the
most common sources of the food safety problem [16]. Thiabendazole (TBZ), a benzimidazole
derivative (4-(1H-1,3-benzodiazol-2-yl)-1,3-thiazole), is one of the widely used pesticides and
fungicide, prevent fruit such as citrus, apples and pears from mold, rot, and blight, thus keeping
their freshness before waxing step during storage and transport [17] (Figure 1). Overexposure to
TBZ is reported to cause nausea, vomiting, headache, weakness, drowsiness, and lack of
appetite. It is hepatotoxic and could be even carcinogenic at higher doses [16].

Figure 1 Chemical structure of thiabendazole.

Monitoring the quantity of these pesticide residues throughout the food supply chain is essential
from the perspective of defending consumer health. Though the traditional chromatographic-
spectroscopic methods for the determination of TBZ pesticides provide trustworthy analytical
methods, they necessitate, extensive sample preparation and specialized knowledge, which
makes quick decision-making at the sample point challenging [18]. Therefore, the searching of
simple, affordable and trustworthy analytical methods with point-of-need is in demand. The
creation of nanozymes-based colorimetric sensors is crucial for providing simple, quick,
affordable, and appropriate point-of-need food safety monitoring tools. As a new development,
nanozymes-based colorimetric sensors show some excellent advantages over traditional
techniques, including greater selectivity and sensitivity, more precise target recognition, faster
detection times, and better signal readout [19].

Since their discovery as the first reported peroxidase mimetic, ferromagnetic nanoparticles have
received the most attention. However, the van der Waals attractive forces of Fe 3O4 nanoparticles

2
make them easily aggregate into clusters, limiting their catalytic activities [20]. However, current
peroxidase or other nanozyme selection often lacks rational design in resembling the
nanomaterial both to the microenvironment and catalytic center of the natural enzymes. This
limits nanozymes from having intrinsic enzyme activity and reduces their catalytic efficiency. In
this regard, the use of Chitosan, a natural polymer with both amino and hydroxyl functional
groups, as a scaffold for the metal center could help in copying the amino acid microenvironment
in the natural enzymes.

Hence in this study, magnetic-chitosan hydrogel will be synthesized, characterized and probed
for its peroxidase memetic nanozyme activity for colorimetric detection of TBZ. The hydroxyl
and amino groups in chitosan gives the nanozyme a reductive microenvironment for the metal
similar to the natural amino acids in peroxidase enzyme. This improves electron transfer and
offers the prepared magnetic hydrogel an intrinsic peroxidase activity. The synthesized hydrogel
will be characterized using state of the art analytical tools. This material's peroxidase mimicking
activity will be investigated using 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2O2 as substrates.
UV-Vis spectrophotometers will be used to monitor the resulting colored solution. All
parameters influencing enzyme activity, such as pH, time, and nanozyme amount, will be
optimized. The colorimetric detection of TBZ will be based on its ability to inhibit enzymes,
which will then be correlated with its concentration. This method demonstrates nanozyme-based
detection that is simple, quick, and low-cost. As a result, we will develop and report a new,
simple, rapid, low-cost, and unique MCH hydrogel-based peroxidase mimicking nanozyme for
colorimetric pesticide detection.

3
1.1 Statement of the Problem
These days, food safety has become one of the most concerned topics, since some pesticide
residues on vegetables and fruits, especially endogenous pesticides, which are not easy to be
removed, would be the great risk to human health [21, 22]. Thiabendazole is a pesticide largely
used in vegetables and fruits treatment to prevent mold, blight, and other diseases along the food
supply chain. However, people could be exposed to thiabendazole as they consume such fruits
and this causes various health problem. High doses of these pesticides are still likely to cause
thyroid hormone imbalances and liver damage. Because of the toxicity it posed on the human the
ability to accurately and rapidly detect trace amounts of pesticides has become more and more
important for the health and environment protection.

Different conventional analytic techniques have been reported for the detection of TBZ. Though
these methods show high sensitivity, accuracy, and reliability for food contaminant detection,
they are complicated, laborious, and time consuming, particularly depending on the expensive
instruments with well-trained personnel. Thus, they are difficult to meet the needs of the fast and
on-site screening of massive samples and apply in some situations like in some developing
countries and poor areas without detection instruments and specialists.

Attentively, Enzyme based methods are a better alternative than the conventional method to
monitor pesticide residues in food and environmental samples, due to their high catalytic activity
and specificity features. However, natural enzymes have intrinsic limitations and are expensive
to be used especially in resource-limited areas. Hence it’s very important to find a substitute for
natural enzymes called (“nanozymes’’). Nanozyme which combine enzyme-like properties with
nanoscale features are a promising alternative to replace and overcome these limitations of
natural enzymes.

Nanozymes with peroxidase-like activity have been used as a catalyst to decompose H 2O2 into
OH radicals which oxidizes TMB to give blue color. This colorimetric detection can be used to
monitor TBZ as it is inhibited in the presence of TBZ. As a result, a method for monitoring TBZ
in production process of vegetables and fruits matrices that is simple, rapid, low- cost, and
suitable point-of-need food safety monitoring tool in the food supply chain is needed. Therefore,
the proposed research will synthesize a novel MCH hydrogel nanocomposite and develop
colorimetric detection method for TBZ in fruits with peroxidase activity.

4
1.2 Significance of the Study
The searching of simple, rapid and low-cost nanozyme-based colorimetric sensor and point-of-
need sensor platform for detection of thiabendazole pesticide residue in the food supply chain is
necessary. This could help rapid decision making at the point of sample possible and serve as a
better alternative than the conventional chromatographic/spectroscopic methods which often
require lengthy and costly analytical procedures for pesticide determination. Currently,
peroxidase or other nanozyme selection often lacks rational design in resembling both the
microenvironment and the catalytic Centre of natural enzymes. This limits nanozymes from
exhibiting intrinsic enzyme activity and lowers their catalytic efficiency.

Hence the study will address limitations in constructions of nanozymes which often lacks both
functional and structural resemblance to their corresponding natural enzymes. It also contribute
in providing a simpler, rapid detection tool for food safety monitoring. Moreover, it will add up
to the nanobiotechnological efforts in finding effective nanozymes with intrinsic enzyme activity

1.3 Scope of the Study

The scope of this research is developing simple, rapid, and low-cost nanozymes-based
(nanozymes) colorimetric sensing for one type of pesticide, namely thiabendazole. The
thiabendazole is applicable for controlling pests in fruits but its overexposure causes health
problems. In this work, only apples will be assessed for pesticides using the developed
technique.

1.4 Objectives of the Study

1.4.1 General objective
 To develop MCH hydrogel nanozyme-based colorimetric sensor for TBZ detection.

1.4.2 Specific objectives

 To synthesize MCH hydrogel nanocomposite.
 To characterize the prepared nanocomposite using UV-Visible spectrophotometer, FT-IR,
XPS, XRD and SEM.
 To investigate the catalytic activity and enzyme kinetics of the prepared nanozymes.
 To develop a colorimetric assay for the detection of TBZ.
 To determine the analytical figures of merits.

5
  To develop a model cell phone-based colorimetric sensing platform applicable for point-
of-need food safety monitoring of TBZ.

2. Literature Review
2.1 Nanozymes
The recent convergence of nanotechnology has sparked a surge of study towards the
development of functional nanomaterials with capabilities similar to those seen in enzymes. The
term ‘Nanomaterial’ refers to materials which have a size ranging from one to hundred
nanometer. The science of nanotechnology has gained the focus of scientist due to its attractive
properties. Nanozyme is a general term assigned to nanomaterials with intrinsic enzyme like
characteristics [23]. The word was first coined by Scrimin, Pasquato and their coworkers in 2004
to describe thiol monolayer protected gold clusters exhibiting excellent ribonuclease-like activity
Nanozymes were initially classified as enzyme-nanomaterial hybrid and Nanomaterials with
intrinsic catalytic property. However, the hybrid system was used to improve the stability and
durability. The catalytic activity was only due to the natural enzyme modified on the
nanomaterial whereas, nanozymes catalytic property is intrinsic and comes from the
nanomaterial itself [12]. The first report of nanoparticle with intrinsic peroxidase like property of
Fe3O4 by Gao et al., 2007. The nanoparticle enzyme like activity was studied well as they were
able to interpret the enzyme science through elaboration of the typical Michaelis−Menten
kinetics with lower Km (high affinity for the substrate, H 2O2) and higher catalytic activity (K cat)
compared to the natural enzyme Horse-radish peroxidase (HRP).

According to Yan et al., [24], a typical nanozyme exhibit various features such as nanomaterial
made material with specific structure; follows the same enzymatic kinetics and catalytic
mechanism as natural enzyme; typical nanoscale factors such as size, morphology and surface
affect its activity and nanozymes can be used as enzyme substitutes. The various kind of specific

6
nanostructure and the change in the nanoscale factors grants the nanozyme to have distinct
physicochemical properties. Such tunability of nanozymes is what made them a possible
alternative for the replacement of natural enzyme. Nanozymes' multifunctionality allows them to
be used in a variety of applications, including biosensing, immunoassays, cancer detection and
treatment, and environmental management.

2.2 Enzymatic Activity of Nanozymes

Nanozymes exhibit intrinsic enzymatic activity mainly as a result of typical nanoscale factors
such as size, morphology and surface. There exists a significant physicochemical property due to
the nano-scale size of the materials. As a result of many suspended bonds and under coordination
the atoms on the surface of nanoparticles are unstable having excess energy. The instability of
these atoms arose from the dangling and unsaturated bonds on the surface because of the small
size leading the particles to have high surface atomic number, atomic energy and area [9]. There
are different types of nanozymes based on their enzymatic activities: peroxidase, oxidase,
superoxide dismutase, catalase, and hydrolase mimicking activities [23].

2.3 Peroxidase Sensor

The mechanism of peroxidase mimicking activity is based upon the act of the nanozyme on
peroxide to produce transient reactive intermediates which will interact quickly with another
substrate [25]. Different nanozymes have been synthesized to mimic the peroxidase-like activity
of horseradish peroxidase, glutathione peroxidase and halo peroxidase.

Horse radish peroxidase (HRP) mimicking nanozymes have been reported by Jiang et al., [26] to
describe the peroxidase-like activity of chitosan stabilized silver nanoparticles to decompose
H2O2 and produce free ·OH radicals for detection purposes. Zu et al., [27] reported
functionalized nano-MoS2 nanoflowers for antibacterial activity in the presence of H 2O2. CuO
nanoparticles have been reported to have the HRP-mimicking activity to generate free ·OH
radicals for the degradation of phenol. BSA-templated MnO 2 nanoparticles are used as
peroxidase mimicking nanozyme for immunoassays purpose in the presence of H 2O2 using the
typical HRP substrates ophenylenediamine (OPD) and TMB [28]. Huang et al., [29]. reported
application of GO–Se nanocomposites with excellent glutathione peroxidase (GPx)-like
properties as an antioxidant Natalio et al., [30] showed that vanadium pentoxide nanowires act
like naturally occurring vanadium haloperoxidases to prevent marine biofouling by which the

7
nanozyme catalyzed the oxidation of bromide ions to hypobromous acid (HOBr) in the presence
of bromide ions and hydrogen peroxide.

Han et al., [31] developed Au-Nps based paper chip with POD like activity for a simple, rapid,
selective, and onsite detection of Hg ions. In this work, Au-Hg amalgam is formed which
promote the catalytic activity of the nanozyme against TMB and H 2O2 for colorimetric detection
in environmental samples. Chitosan functionalized MoSe 2 nanosheet with POD and OD like
property was synthesized for the colorimetric detection of Hg ions in the presence of TMB [32].
The chitosan was used to capture Hg2+ ions on the surface of the nanosheet to be reduced into
Hg0 with a limit of detection of 3.5 nM. Generally, peroxidase mimic activity mechanism
contains mostly of the formation of reactive oxygen species (ROS) and the electron transfer
process [33].

2.4 Chitosan
Chitosan is a linear cationic polysaccharide mainly found on the exoskeleton of crustacean and
cell wall of mushrooms composed of D-glucosamine and Nacety-D-glucosamine. Unique
physico-chemical properties of chitosan bestows them to form excellent films, gels, suspensions
and act as a wall material in encapsulation with a gamut of applications in the fields of
biotechnology, food technology, nanotechnology, medicine, agriculture and textiles. Amine
group and hydroxyl groups of chitosan can act as an excellent site for functionalization of bio-
recognition elements and other components in the development of biosensors. Biocompatibility
with cell surfaces and proteins makes them the favorite biomaterial for the fabrication of
diagnostic devices and sensors [34]. Furthuremore nanomaterials and nanocomposites containing
chitosan have been reported to have peroxidase like catalytic activity for biosensor applications.

2.4.1 Properties of chitosan

The properties of chitosan depend on degree of deacetylation (DDA) and molar mass; these
parameters influence the physical, chemical, and also biological properties. Crystallinity is also
an important parameter because it controls many properties such as swelling in water. Chitosan
is generally a semicrystalline material, it crystallizes in the orthorhombic system and two types
of products are available: chitosan I with a low DDA is more disordered than chitosan II with a
high DDA [35]. The presence of the amine functions, the charge, and the properties of chitosan
vary with the pH. At low pH (3-4), the amines protonate and become positively charged. It is the

8
only polycation from natural origin. Whereas at high pH, chitosan cannot attract hydrogen, so it
does not gain positive charges and will remain insoluble [36].

Chitosan shows better adhesion properties to negatively charged surfaces. Its cationic character
in an acid medium allows it to react with negatively charged biological compounds. Besides, the
nature of the glycosidic bonds also gives chitosan an excellent film-forming property. Chitosan
presents several biologic properties, it is non-toxic, biocompatible, bioactive, and biodegradable
polymer [35].

2.4.2 General applications of chitosan

Chitosan has many applications in different fields because of its various properties listed above
especially its polycationic character which is unique among natural polymers. It finds
applications in economically promising sectors such as the food, cosmetics , pharmaceutical,
agricultural industries, and in the development of sensors and biosensors [37]. It can be used as
an encapsulation material for drugs since it allows the controlled release of the drug or any other
substance [38]. Chitosan is also largely used for water treatment due to its chelating property
which allows it to eliminate heavy metals, even in very small quantities [39]. Moreover, in the
field of cosmetics, the film-forming and cationic properties of chitosan are exploited in many
hair and skincare creams and lotions [40]. Chitosan is also capable of triggering defense
mechanisms in plants against infections and parasitic attacks [41].

2.5 Colorimetric Sensor based on Peroxidase Nanozymes

Nanozymes have been widely used to construct colorimetric sensors due to their advantages of
cost-effective, high stability, good biocompatibility, and ease of modification. The emergence of
nanozymes greatly enhances the detection sensitivity and stability of the colorimetric sensing
platform. Recent significant research has focused on designing various sensors based on
nanozymes with peroxidase-like activity for colorimetric analysis. However, with the deepening
of research, nanozymes with peroxidase-like activity also expose some problems, such as weak
affinity and low catalytic activity. In view of the above issues, existing investigations have
shown that the catalytic properties of nanozymes can be improved by adding surface
modification and changing the structure of nanomaterials [42].

9
Before 2007, the colorimetric assays based on these substrates called peroxidase based
colorimetric sensors because of using a peroxidase enzyme for the measurements and since 2007,
with introducing Fe3O4 magnetic nanoparticles that exhibited excellent peroxidase - like activity,
a wide variety of peroxidase mimic colorimetric sensors developed, including noble metals,
metal oxides, metal sulfides/metal selenides, carbon and metal-organic frameworks (MOF), etc
for the determination of different analytes. For instance, Shao-Bin He et al., [43] report a highly
sensitive colorimetric sensor for detection of iodine ions using carboxylated chitosan–coated
palladium nanozyme. In this study, a new type of Pd nanozyme was prepared by a facile one-pot
approach by using carboxylated chitosan as the stabilizer. Owing to the synergistic effect of
carboxylated chitosan stabilized Pd nanoparticles (CC-PdNPs) can effectively catalyze the H 2O2-
mediated oxidation of TMB accompanied by a blue color change (oxidized TMB), indicating the
peroxidaselike activity of CC-PdNPs Xufeng Zhu et al., [44] reported Cationic
chitosan@Ruthenium dioxide hybrid nanozymes for photothermal therapy enhancing ROS-
mediated eradicating multidrug resistant bacterial infection. Herein, RuO 2 nanosheets are
modified with positively charged quaternary ammonium-chitosan (QCS) to improve
biocompatibility, and enhance the interaction between RuO 2 nanozymes and bacterial
membranes.

2.6 Peroxidase based Nanozymes for Food Safety Monitoring

Food is intricate, and food quality and safety are indispensable elements. With the rising public
demands for safe and nutritious food products, a range of innovative, interdisciplinary, and
multidimensional concepts have emerged in the last few decades. Moreover, food safety
monitoring is essential for consumer protection as well as for the food industry thus, screening of
food contaminants and quantification of food constituents is becoming vital for food industries
and consumer [45]. Nanozymes, apart from their therapeutic efficacies, are gaining increasing
interest in safety and quality monitoring in the agri-food sector. These enzyme-mimetic
nanomaterials are proven to detect and monitor food safety in real-time with high selectivity,
stability, reliability, and recyclability. Nanozymes sourced from various nanomaterials like
carbon, metal, and their oxides, metal–organic frameworks, and others offer several advantages
over natural enzymes in food-based applications [11]. Apart from real-time monitoring, these are
also known for their robustness to harsh food processing environments, ease of production, and

10
smooth and facile surface modifications. Nanozymes undergo single and multiple enzyme
pathways during food analysis applications, facilitating the on-demand tailorable activity [46].

Contaminants, if present above the permissible limits in foods, can lead to serious health
concerns. Genetically modified foods and high-risk foods like milk, meat, and several other
commodities should be monitored consistently for quality and safety [47]. The maximum
residual limit in case of pesticides and antibiotics, and permissible levels of microbes and toxins
are to strictly comply to maintain healthy trade practices. From those food contaminants,
pesticides is the critical one that have been widely applied in the agricultural field to reduce the
losses in agricultural production caused through pests and insects [48].

About 1.8 billion of the population engaged in agriculture use pesticides to protect food and
commercial products. It was also estimated that around 25 million agricultural workers
experience accidental pesticide poisonings each year [49]. Carbamates, organochlorines,
fumigants, etc., are different classes of pesticides that interfere with the human body’s normal
functioning, resulting in malfunctioning of the kidneys, nervous system, and respiratory system.
Hence, the rapid and real-time monitoring of these contaminants is of paramount importance. A
study using gold nanorods exhibited superior peroxidase-like activity than HRP and detected
malathion to a LOD of 1.78 μg/mL [50] (Table 1). On the other hand, Singh and co-workers [51]
combined Pd with Au nanozyme to detect malathion, which exhibited high peroxidase-like
activity from pH 2 to 6 with a LOD of 60 ng/mL, confirming the advantage of Pd’s addition to
Au.

Table 1 Peroxidase based-nanozymes for the detection of pesticide.

Pesticides Nanozyme Mechanism/ Transductio Dete Linear LOD Ref.

used Assay n ction range
methodology time
Atrazine Fe3O4- POD mimics Colorimetric 40 2 - 20 2.98 [52]
TiO2/rGO min μg/L μg/L
Triazophos AuNPs Bio-barcode 2.5 × 10-2 1.96*10-
amplification - 40.0 2 ng/mL [53]
competitive - - ng/mL
immunoassay

11
Parathion NiONPs Nanozyme Electrochemi 0.1–30 0.024
based cal μM μM [54]
electrochemical -
sensing

Malathion Au POD mimics Colorimetric 1.78

nanorods μg/mL
Au/Pd 60
ng/mL

Methyl GeO2 POD mimics Colorimetric 50 0.1–50 14 fM [55]

paraoxon min pM

Chlorpyrifo Ag3PO4 OXD mimics Colorimetric 3 min - 9.97 ppm [56]

s NPs
2.7 TBZ as a Food Concern
Thiabendazole (2-(4 -thiazolyl) benzimidazole) is one of the most commonly used pesticide,
which is also used as a fungicide to control postharvest decay caused by fungi in various fruits
and vegetables. The persistence of thiabendazole residues in agricultural products destined to
human consumption is important problem. Several studies have been performed to demonstrate
residues in edible parts of raw fruits. To protect consumers from risks related to thiabendazole in
food, European Union legislation has established maximum residue limits (MRLs) for raw fruit
and vegetables. For fruits like oranges, apples, pears and bananas, the MRL is 5 mg kg −1. Due to
the absence of legal threshold values for processed products such as juices, they have been
normally considered as the MRLs stated for raw fruits [57].

TBZ has a host of adverse effects including nephrotoxicity, hepatotoxicity, carcinogenicity, and
teratogenicity and is thus classified by U.S. Environmental Protection Agency (EPA) as likely to
be carcinogenic at doses high enough to cause disturbance of the thyroid hormone balance.
Because of the toxicity it posed on the human and environments, there is increasing interest to
develop rapid screening methods for TBZ in various food samples at different concentration
levels [58].

2.8 Current Detection Methods for TBZ

Several works have been done for the detection of TBZ in food samples, traditional techniques
such as gas chromatography (GC), liquid chromatography - mass spectrometry (LC-MS), high-

12
performance liquid chromatography (HPLC) and combined HPLC with fluorescence detection
and with ultraviolet detection are used [59]. Xu and his research group [60] developed a
modified quick, easy, cheap, effective, rugged and safe (QuEChERS) method combined with
HPLC for the determination of TBZ in food samples, achieving a LOD of 2.6 ng/mL and the
recoveries in the range of 92.9–103.9% with relative standard deviations (RSD) lower than 6.5%.

However, in these methods, complicated sample preparation, lengthy detection procedure,

requirement of operational skills and expensive equipment or reagents limit their further
applications for rapid and in situ detection for TBZ. Therefore, in recent years, nondestructive
techniques such as spectroscopy and surface-enhanced Raman spectroscopy (SERS) have been
proposed to overcome the above disadvantages, in particular, SERS has demonstrated its
excellent sensitivity in the detection of a wide range of pesticides. As the time for collecting the
spectra is usually a few seconds, it can provide rapid evaluation. The main challenge toward the
analytical application of SERS has been the lack of substrates and uniform signal enhancement
suitable for large-scale production [61].

Electrochemistry provides powerful analytical techniques for sensors, with the advantages of
instrumental simplicity, low cost, miniaturization, work on-site, and the ability to measure
pollutants in complex matrices with minimal sample preparation. For example, Mola. et al., [18]
develop sensitive electrochemical detection of thiabendazole in fruits using Ag- MoS 2 electrode
and they investigated high sensitivity, fast response, high selectivity, lower cost, and
convenience for point-of-need application by using Ag nanoparticles (NPs) modified MoS 2 (Ag
MoS2) applied to the surface of glassy carbon (GC) to produce a robust electrochemical sensor
for the detection of thiabendazole. The developed sensor exhibited a linear range between 1– 10
μM with LOD down to 0.1 μM. Analysis of TBZ in mango and banana matrices gave a recovery
of 91.6–100.4% indicating the suitability of the sensor for food safety monitor.

13
3. Research Methodology
3.1 Chemicals and Apparatus
Table 2. Lists of chemical and apparatus required to perform the research

Chemicals and reagents Equipment Glassware

Acetic acid (99.5%) Aluminum foil Centrifuge Measuring cylinder

Sodium hydroxide (99.8%) Spatula Sonicator Erlenmeyer flask
Ferric chloride Balance X-ray Volumetric flask
Ferrous sulfate Crucible photoelectron Test tubes
deionized water Magnetic stirrer (XPS) Beakers
Thiabendazole standard Micropipette spectroscope. Vacuum filter
(C10H7N3S, ≥99%) Filter paper Scanning Electron Thermometer
Hydrogen peroxide (30%) (Wattman No.1) Microscope (SEM)
Dimethyl sulfoxide (99.9%) Hotplate X-Ray Diffraction
Acetate buffer Oven drier (XRD)
Chitosan (deacetylation, 80– Freeze drier Fourier transforms
95%), Juicer machine infrared
Acetone spectroscope
Acetonitrile (FTIR)
UV-visible
spectroscope
PH meter

14
3.2 Experimental Methods
This research project is divided into three work packages (Scheme 1).

Nanozymes synthesis
Wor
k
plan Nanozyme characterization
1

Wor Enzymatic activity test

k Enzyme kinetics study
plan
2
Hydrogel based colorimetric sensor development for
Wor pesticide detection
k Development of point-of-need sensor platform
plan
3

Scheme 1. The steps (work packages) followed in the methodology

WP1-Synthesis and Characterization

3.3 Synthesis of Magnetic Chitosan Hydrogel Nanocomposite

The magnetic chitosan hydrogel nanocomposite (MCH) will be synthesized via in situ as
reported in literature [62]. Briefly, 2 g chitosan will be dissolved in 50 mL acetic acid solution
(1%, v/v) and chitosan solution (2%, w/v) will be obtained. A total of 5 mL of magnetite
precursor containing 3.5 mmol FeCl3·6H2O and 1.75 mmol FeSO4.7H2O will be added into the
chitosan solution and stirred for 2 h to obtain a homogeneous solution. The mixture will be
soaked in 100 mL of NaOH solution (0.625 mol/L) for 4 h, and OH − will be diffused into the
mixture. The OH− ions will induce deprotonation of chitosan and crystallization of magnetite,
and MCH will be obtained. Finally, deionized water will be used to rinse the MCH to bring
neutral and will be stored at 4℃ in a refrigerator until being used.

15
3.4 Characterization of Magnetic Chitosan Hydrogel Nanocomposite
The morphology of the synthesized MCH hydrogel nanocomposite will be examined using
scanning electron microscope (SEM). The functional groups of the synthesized nanocomposite
will be evaluated by FT-IR spectrophotometer. The optical property of the prepared nanomaterial
will be studied by a UV-visible spectrophotometer. The mechanism, formation, and interaction
(binding energy) of prepared material will be clarified by X-ray photoelectron (XPS)
spectrophotometer. The crystalline structure and phase composition will be evaluated by X-ray
diffraction (XRD) technique.

WP2-Enzymatic Activity Test

3.5 Enzymatic Activity of Magnetic Chitosan Hydrogel Nanocomposite

First, the peroxidase-like activity of the prepared nanocomposite material will be examined using
TMB/ H2O2 substrates in acetate buffer. In a typical enzyme activity assay, 200 µL of TMB and
H2O2 each 6 mM will be mixed with 60µg MCH in 2 mL acetate buffer. The reaction mixture
will be incubated at different times and the absorbance of the resulting blue-colored product from
the oxidation of the chromogenic substrate will be scanned from 200-800 nm. The effect of
substrate concentration (0.5, 2, 4, 6, and 8mM), time (10, 20, 30, 40, and 50 sec) the mass of
nanozymes (20 µg, 40µg, 60µg, 80µg and 100µg), and pH (1, 3, 5, 7, and 9) will optimized by
varying each factor at a time.

3.6 Enzyme Kinetics of Magnetic Chitosan Hydrogel Nanocomposite

The steady-state kinetics of the prepared nanozymes (MCH hydrogel nanocomposite) will be
investigated by varying the concentration of TMB at a fixed H 2O2 concentration and vice versa.
The resulting absorbance from oxidation of TMB will be measured by a UV-visible spectroscope
at a fixed wavelength (652 nm) at a 1-min interval. The enzyme kinetic parameters will be then
calculated from the Line-weaver-Burk plot (equation 1) which is the double-reciprocal plot
derived from the Michalis Menten equation.

1 km 1 1
= + (Equation…..1)
[ v ] V max [ S ] V max

16
Where, V is initial velocity, V max is the maximum velocity of the reaction, S is the initial
concentration of the substrate and Km is Michaelis constant indicates the affinity of the enzyme
to the substrate. [63]

WP3-Application

3.7 Colorimetric Detection of TBZ

For detection of the TBZ, 200 µL of various concentration label of TBZ external standards will
be first incubated with 2 mL of nanozymes (60µg /mL) in a test tube for 20 min. After which,
200 µL of the substrates (6 mM each) will be mixed and the absorbance will be then measured
using UV-visible spectroscope at 652 nm. The analytical figures of merits such as LoD, LoQ,
and linearity will be determined from the calibration curve (Abs vs C) plot according to the
International Conference on Harmonization (ICH) guideline [18]. Selectivity of the sensor will
be evaluated by running analysis of mixture of citrate, ascorbic acid and other potential
interferents in the fruit matrix with TBZ both individually and altogether.

3.8 Detection of TBZ in Real Fruit Matrix

Apple fruit sample will be bought from a local market. These fruit matrices will be used to
demonstrate the applications of the developed nanozymes based sensor in real samples. The fruit
sample will be first juiced with a juicer machine. The juice will be then centrifuged with
acetonitrile for 10 min at 6000 rpm, and then the supernatant will be filtered, diluted with in a
buffer solution at the optimum PH and stored at 4 ℃. Standard solutions of TBZ with two
different concentrations will be prepared and spiked to the extract. 200 µL of the spiked extract
will be then incubated with 2 mL of nanozymes after which 200 µL of the substrates (6 mM
each) will be added. The absorbance of this mixture will be then measured by UV-visible
spectroscope and after this a known concentration of TBZ standard will be spiked and the %
recovery will be calculated (Equation 2). The effect of interferents on the colorimetric detection
of TBZ will also studied by mixing TBZ with citric and ascorbic acid and other commonly
found chemicals in fruits (Malic acid, tartaric acid, flavonoids, phenolic acids, coumarins,
limonoids, carotenoids, pectin and others [64]

Cspiked−Cunspiked
%R= x 100 (Equation….2)
Cadded

17
Where Cspiked and Cunspiked are the concentrations found in TBZ spiked and unspiked fruit matrices
and Cadded is the actual concentration of TBZ added to the fruit samples.

3.9 Development of Point-of-need Sensor Platform

A model sensor platform for point need application in food safety will be prepared. Typically,
the hydrogel containing well-plates with mobile phone-based color reader will be designed,
tested and optimized at a proof of concept for the utility of the developed sensor materials as
point-of need sensing platform.

3.10 Data Analysis

The data analysis techniques in this work will be performed using origin lab software and mobile
phone-based application software.

18
 4. Work Plan
All the activity performed in this research are set in program according to following the table.

Table 3. The estimated time table required to finalize this research

Time (Month)

December January February March April May June

Title and
proposal
submission
Material
purchasing
Activity

Synthesis and
characterization

Enzymati
c activity

Detection,
analysis and
data
collection
Thesis
writing
and
Report

19
5. Budget Breakdown
Table 4. The budget required for accomplishing the research

No Item Unit Quantity Unit price Total Price

(Birr)
1 Thiabendazole standard g 250 1000 1000

2 Hydrogen peroxide L 3 500 500

3 Sodium hydroxide g 250 1000 1000

4 3,3,5,5-tetramethylbenzidine (TMB), g 5 1800 1800

5 Peroxidase enzyme (HRP) g 5 2600 2600

6 Chitosan (deacetylation, 80–95%) g 5 2000 2000

8 Iron (II) sulfate heptahydrate g 100 300 300

9 Acetone L 2L 300 600

10 Acetonitrile L 3L 300 900

11 Acetic acid L 3L 200 600

12 Ferric chloride g 250 600 600

14 Fruit sample (apple) kg 5kg 120 600

18 Stationery, binding, transport expense, 12,500 12,500

and characterization cost
Total lump sum 25,000.00

20
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