Sensing and Bio-Sensing Research 42 (2023) 100595

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Sensing and Bio-Sensing Research

journal homepage: www.elsevier.com/locate/sbsr

Magnetite chitosan hydrogel nanozyme with intrinsic peroxidase activity

for smartphone-assisted colorimetric sensing of thiabendazole
Abera Merga Ariti a, Seada Abdo Geleto a, Beamlak Teshome Gutema b,
Efrata Getachew Mekonnen b, Yitayal Admassu Workie a, c, Ebrahim M. Abda b, d, **,
Menbere Leul Mekonnen a, c, *
a
Industrial Chemistry Department, Addis Ababa Science and Technology University, Addis Ababa, P.O. Box 1647, Ethiopia
b
Biotechnology Department, Addis Ababa Science, and Technology University, Addis Ababa, P.O.Box 1647, Ethiopia
c
Nanotechnology Center of Excellence, Addis Ababa Science and Technology University, Addis Ababa, P.O.Box 1647, Ethiopia
d
Bioprocess and Biotechnology Center of Excellence, Addis Ababa Science and Technology University, Addis Ababa, P.O. Box 1647, Ethiopia

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

Keywords: Although nanozymes proved a promising utility in sensing applications, they often lack a rational approach in
Chitosan hydrogel their design. Herein, magnetite chitosan hydrogel (MCH) is reported as a rational peroxidase nanozyme for the
Fe3O4 nanoparticles smartphone-assisted colorimetric detection of thiabendazole (TBZ). Chitosan due to its polycationic nature,
Peroxidase nanozyme
renders a microenvironment similar to amino acids in HRP enzyme. Characterizations of the nanozyme using
Colorimetric sensors
Thiabendazole, smartphone sensors.
SEM, XRD, and XPS confirmed the distribution of Fe3O4NPs on the chitosan matrix. The peroxidase activity was
demonstrated using TMB and H2O2 as substrates which resulted in a characteristic absorption at 652 nm. MCH
nanozyme showed a 24% higher peroxidase activity in acidic pH than the pristine Fe3O4 confirming the role of
chitosan in boosting the electron transfer. Kinetics result suggested the catalytic reaction followed a Michalis-
Menten model with Km and Vmax of 0.45 mM and 15 μM/min for TMB and 2.8 mM and 4.18 μM/min for
H2O2 respectively. These values are competitive with natural HRP enzymes reported before. Further, MCH
nanozyme showed improved thermal and temporal stability as well as reusability retaining 80% of its activity
after the 4th cycle. TBZ showed concentration-dependent inhibition on the peroxidase activity. The degree of
inhibition exhibited a linear relationship with the concentration of TBZ from 0.1 to 100 μM (R2 = 0.998)
enabling the detection of TBZ down to 0.73 and 1.84 μM in a spectrometer and smartphone-based readouts
respectively. The results show the potential of the prepared nanozyme as a point-of-need sensor for food safety
monitoring.

1. Introduction
Enzymes are biocatalysts that have important utility in catalysts of
various biotransformation and industrial processes. Due to their high
catalytic activities, selectivity, and substrate specificity, natural en­
zymes are attractive in biomedical[1,2], industrial processes [3], envi­
ronmental monitoring[4,5], and food safety applications[6]. In
addition, compared to other biorecognition elements, enzymes offer
wider substrate choice and simpler design making sensor development
easier[7]. Despite this, natural enzymes have intrinsic limitations hin­
dering their applications in extreme operating conditions such as in high
temperatures and salinity[8]. In addition, their lower operational
stability, high cost of purification, and difficulty in reusing make their
routine applications, especially in resource-limited areas strenuous. In
this regard, enzyme memetic materials often called nanozymes could
help to overcome the intrinsic limitations of natural enzymes while
benefiting from their salient features.
Nanozymes are nanomaterial-based artificial enzymes with intrinsic
catalytic activity[9]. Nanozymes have become a promising bio­
nanotechnological tool as they are advantageous over natural enzymes
in terms of adjustable catalytic activity, low cost, ease of mass produc­
tion, and high stability[10,11]. Furthermore, developments in nano­
technology have made the synthesis and modification of nanomaterials
easier for achieving improved catalytic efficiency[12]. After the study

* Corresponding author at: Industrial Chemistry Department, Addis Ababa Science and Technology University, Addis Ababa, P.O. Box 1647, Ethiopia.
** Corresponding author at: Biotechnology Department, Addis Ababa Science and Technology University.
E-mail addresses: ebrahim.mama@aastu.edu.et (E.M. Abda), menbere.leul@aastu.edu.et (M.L. Mekonnen).

https://doi.org/10.1016/j.sbsr.2023.100595
Received 22 July 2023; Received in revised form 10 October 2023; Accepted 17 October 2023
Available online 18 October 2023
2214-1804/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
A.M. Ariti et al.
on the peroxidase activity of Fe3O4 by Gao and his colleagues,[13]
various nanomaterials are reported to have enzyme memetic properties.
Among them, peroxidase memetic nanozymes have wider applications,
particularly in colorimetric sensors[14,15] antimicrobials[16,17], etc.
Despite a large number of reports, peroxidase nanozymes often lack
rational design in resembling the nanomaterial both to the microenvi­
ronment and catalytic center of the natural enzymes. This limits nano­
zymes from enjoying the intrinsic enzyme activity with higher catalytic
efficiency[18,19]. An aspect of enhancing the performance of nano­
zymes often involves the functionalization of the nanoparticles with
different matrices. For instance, histidine-functionalized Fe3O4 showed
improved peroxidase activity over its pristine form[20]. Recently, the
Liu group also demonstrated enhanced peroxidase activity of liposome-
modified Fe3O4 even breaking the pH limit reported for peroxidase
nanozymes[21]. However, the use of biomolecules for routine applica­
tions such as point-of-need sensors would not be viable both practically
and economically. Hence a rational design of peroxidase nanozymes
based on cheaper and sustainable scaffolds could be more attractive for
sensor development.
In this study, Chitosan-Fe3O4 hydrogel (MCH) is reported as a
rational design of peroxidase nanozyme. Chitosan (Poly-N-acetylglu­
cosamine), is chosen due to its hydroxyl and amino groups which could
endow the nanozyme similar microenvironment as the amino acids in
the horseradish peroxidase (HRP) enzyme. Chitosan is a biodegradable
and abundant biopolymer [22] which are important factors for sus­
tainable industrial-scale production. MCH was synthesized by co-
precipitation of Fe2+/3+ on chitosan matrix forming magnetite hydro­
gel. After extensive characterization and optimization, the peroxidase
activity is tested using TMB and H2O2 as substrates. Further, the pre­
pared MCH nanozyme is applied for smartphone-assisted colorimetric
sensing of thiabendazole based on its inhibition effect on the nanozyme
activity.
Thiabendazole (TBZ) is a pesticide used in the post-harvest process
for preventing the spoilage of fruits.[23,24] However, overexposure to
TBZ causes simple adverse reactions to carcinogenic effects in humans
[25,26]. The minimum residue limit of TBZ set by the European Union
for different fruit types ranges from 0.05 to 10 mg/Kg, making reliable
measurement critical [27]. The most frequently used analytical methods
such as ELISA[28], HPLC- UV–Vis[29], and SERS[25,30] are reported
for the detection of TBZ. However, these methods often require expertise
skill, sophisticated instrumentation, and lengthy sample preparation
steps which make point-of-need rapid analytical decision-making diffi­
cult. In this regard, nanozyme-based colorimetric sensors offer simpler,
rapid yet reliable detection platforms for rapid analytical decision-
making. The prepared MCH nanozyme featuring the catalytic activity
of the HRP enzyme represents a rational nanozyme design with robust
performance for a smartphone-based colorimetric detection of TBZ.
Such analytical schemes are more relevant for resource-limited settings
as they allow easy and low-cost point-of-need inspection tools in the
food supply chain.
2. Materials and methods
2.1. Materials
All chemicals and reagents used for this research were of analytical
grade and used without further purification. Chitosan low molecular
weight (C6H11NO4)n with 85% degree of deacetylation was purchased
from Central Drug House (PLtd. Delhi (India). Ferric chloride (FeCl3,
98%) and ferrous sulfate heptahydrate (FeSO4.7H2O, 98.5%) were
purchased from Loba Chemie Pvt. Ltd., Colaba Mumbai (India). Acetic
acid (CH3COOH, 99%), sodium acetate (CH3COONa, 99%), and
hydrogen peroxide (H2O2, 30%) were purchased from Alpha Chemika,
India. Thiabendazole standard (C10H7N3S, 99%) was purchased from
Sigma Aldrich, USA. 3,3,5,5-Tetramethylbenzidine (C16H2ON2, 99%)
was obtained from Acros Organics. Sodium hydroxide (NaOH 99.5%) is
2
Sensing and Bio-Sensing Research 42 (2023) 100595
purchased from Blulux Laboratories, India. Ultrapure water (18.2 MΩ
cm) by a Milli-Q system was used for all solution preparation. Fruit
samples such as Apples and Bananas were purchased from a local mar­
ket. Acetate buffer was used for all experimental measurements.
2.2. Synthesis of nanozyme
The magnetic chitosan hydrogel (MCH) was synthesized by co-
precipitation of Fe2+/3+ on the chitosan matrix as reported elsewhere
with a slight modification.[31] First, 0.5 g chitosan was dissolved in 50
mL acetic acid solution (1%, v/v) to obtain 2%, (w/w) chitosan solution.
Next, a total of 5 mL of magnetite precursor containing 3.5 mmol
FeCl3⋅6H2O and 1.75 mmol FeSO4.7H2O was added into the chitosan
solution and stirred for 2 h to obtain a homogeneous solution. The
mixture was soaked in 100 mL of NaOH solution (0.625 mol/L) for 4 h.
Finally, the resulting product was washed with deionized water and
stored at 4 ◦ C in a refrigerator until being used.
2.3. Characterization of nanozyme
All absorption measurements for optimization, quantitative and ki­
netic analysis were done using a UV − Vis spectrometer (JASCO 770).
Scanning electron microscopy (FESEM, JSM-6500F JEOL) was used to
confirm the surface morphology of the synthesized magnetite chitosan
hydrogel. An X-ray diffraction spectrophotometer (XRD-7000, DRA­
WELL) was used to evaluate the crystallographic nature and phase
composition of the nanocomposite. The functional groups present on the
nanozyme were investigated using an FT-IR spectrophotometer (Nicolet
Evolution-300). The surface composition and chemical states were
studied by X-ray photoelectron spectrometer VG ESCA Scientific Theta
Probe with monochromatic Al Kἀ (1486.6 eV) as an X-ray source.
2.4. Peroxidase activity of magnetic chitosan hydrogel
The preliminary peroxidase-like activity of the prepared material
(MCH) was examined using TMB and H2O2 substrates in acetate buffer
(pH 4). In a typical assay, 200 μL of TMB (6 mM) and H2O2 (10 mM)
were mixed with 1.75 mg MCH in 3 mL acetate buffer. The reaction
mixture was incubated at different times and the absorbance of the
resulting blue-colored product from the oxidation of the chromogenic
substrate was scanned from 200 to 800 nm. The effect of substrate
concentration, time, mass of nanozymes, and pH were optimized using
one factor at a time approach. Control reactions for the peroxidase ac­
tivity test were also performed in parallel. The thermal stability, of the
nanozyme was also studied by conducting the catalytic reaction at
different temperatures in a water bath system. The temporal stability of
the MCH nanozyme was also investigated by measuring the peroxidase
activity of the nanozyme dispersed in a buffer at different times. Recy­
clablity of the nanozyme was also studied by conducting the peroxidase
activity of the nanozyme which was used in previous cycles after
washing in a buffer.
The steady-state kinetics of the prepared MCH nanozymes was
investigated by varying the concentration of one of the substrates at a
time. The resulting absorbance from the oxidation of TMB was measured
by a UV–visible spectroscope at a fixed wavelength (652 nm) at a 1 min
interval. The enzyme kinetic parameters were then calculated from the
Lineweaver-Burk plot (eq. 1) which is the double-reciprocal plot of the
Michalis-Menten equation. (Eq. 2)
1
=
km 1
+
1
(1)
[v] Vmax [S] Vmax
Vmax
V= + [s] (2)
km
Where V is the initial velocity, Vmax is the maximum velocity of the
reaction, S is the initial concentration of the substrate and Km is the
A.M. Ariti et al. Sensing and Bio-Sensing Research 42 (2023) 100595

Michaelis constant indicating the affinity of the enzyme to the substrate. described above. The percent recovery was then calculated according to
Eq. 3. All data in this study were analyzed using Origin Lab software
(2022)
2.5. Colorimetric detection of TBZ
Cspiked − Cunspiked
%R = x 100 (3)
A colorimetric detection assay was developed as follows. 200 μL of Cadded
TBZ calibration standards, were first incubated with 3 mL of 0.58 mg/L
Where Cspiked and Cunspiked are the concentrations found in TBZ
of nanozyme followed by the addition of 200 μL of the 6 mM of sub­
spiked and unspiked fruit matrices and Cadded is the actual concentration
strates. The absorbance of the resulting blue-colored solution was then
of TBZ added to the fruit samples.
measured at 652 nm. A calibration curve showing the relationship be­
tween the concentration of TBZ and Abs (Abs vs C) was plotted from
3. Result and discussion
which the analytical figures of merits were calculated. The selectivity of
the sensor was evaluated by running an analysis of the mixture of
3.1. Synthesis and characterization of nanozyme
different interferents. Further, a model sensor platform for point-need
application in food safety was prepared. Typically, the hydrogel con­
The MCH nanozyme was successfully prepared by co-precipitation of
taining well-plates with a mobile phone-based color reading application
Fe2+/Fe3+ salts on the acidic solution of chitosan. Fig. 1a shows the SEM
(Spotxel® Reader 1.1) was designed, tested, and optimized as a proof-of-
micrograph of the prepared MCH nanozyme. As seen in the images, the
concept for the utility of the developed sensor as a point-of-need sensing
magnetic nanoparticles are dispersed on the chitosan flakes. The EDX
platform.
spectrum in Fig. S1 shows the presence of Fe, N, C, and O which is
The applicability of the sensor for real sample analysis was examined
consistent with the expected elemental composition of magnetite
by analysis of TBZ spiked fruit matrix. Typically, Bananas and Mango
nanoparticles and chitosan. Out of these elements, Fe accounts for
collected from a local market were juiced and an acetonitrile extract of
3.75% by weight indicating a rational design of nanozymes in which the
the juice was spiked with two concentration labels of standard TBZ. 200
metal centers need to be lower in composition similar to natural
μL of the spiked samples were then analyzed with a similar protocol

Fig. 1. (a) SEM micrograph; (b) XRD pattern of MCH nanozyme; (c) FTIR spectrum of MCH nanozyme and chitosan (CH).

3
A.M. Ariti et al.
enzymes.
The crystallinity and phase composition of MCH nanozyme was
studied by powered XRD (Fig. 1b). The peak centered at 2θ 20.2◦ rep­
resents diffraction from chitosan flakes and is consistent with the pris­
tine chitosan.[32,33] This is also consistent with commercial chitosan
(Fig. S2). While the peak at 2θ 30.17◦ , 35.62◦ , 43.16◦ , 53.49◦ , 57.11◦ ,
62.71◦ , and 74.26◦ can be assigned to diffractions from (220), (311),
(400), (422), (511), (440), and (533) planes of magnetite phase (Fe3O4)
respectively. The peaks are consistent with the JCPDF No. 65–3107 and
reveal the formation of a pure Fe3O4 phase. The crystallite size calcu­
lated by the Debye-Sheerer formula was found to be 12.29 nm.
FT-IR analysis was performed to investigate the functional groups in
the prepared nanozyme and compare them with the pure chitosan. As
seen in Fig. 1c the broad bands at 3460 cm− 1 correspond to the –OH
stretching vibration overlapped with the NH2 stretching mode. The
small shoulder peak at 1652 cm− 1, corresponds to C– – O stretching from
residual amide which could be due to incomplete deacetylation in the
manufacturing process of chitosan. While the peak at 1599 cm− 1 cor­
responds to the N–H bending. This peak disappeared in the composite
(MCH) indicating the association of the amine group with iron. The
medium broad peak at 1432 cm− 1 represents C–N stretching. In addi­
tion, the peak at 1076 cm− 1 represents the C–O stretching of glycosidic
linkage in chitosan. The bands are consistent with previous reports on
similar chitosan-based materials[34–36].
The nanozyme surface chemical states were further investigated by
XPS. As seen in the wide scan XPS spectrum of MCH (Fig. 2a), Fe, O, C,
and N were identified. The high-resolution spectrum of Fe 2p (Fig. 2b)
depicts two peaks at BE 723.02 and 709.53 eV which are characteristics
Fig. 2. (a) Wide scan XPS spectrum of MCH; high-resolution XPS spectrum of (b) Fe2p (c); O1s (d) N1s.
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Sensing and Bio-Sensing Research 42 (2023) 100595
of the Fe 2p½ and Fe 2p3/2 of Fe3O4. A narrow scan spectrum of O1s
(Fig. 2c) shows two peaks at BE ca 531.1 and 528.5 eV which corre­
sponds to Fe coordinated and organic oxygen[37,38]. Deconvolution of
N1s high-resolution spectrum (Fig. 2d) results in a peak at 398.6 eV and
397.7 eV which corresponds to Fe coordinated N and a free amino
group. From this, it can be learned that the iron in MCH is associated
with N and O atoms. This is consistent with the natural HRP enzyme
where the heme Fe exists in coordination with the amino acids[38,39].
3.2. Peroxidase activity of MCH nanozyme
The initial peroxidase activity test on MCH nanozyme was conducted
using H2O2 and TMB substrates in acetate buffer (pH 4). As shown in
Fig. 3a and the inset photos, the reaction between TMB and H2O2
resulted in a blue color with characteristics of absorption at 652 nm.
However, the blue color was not observed when H2O2 and TMB were
mixed without MCH under similar conditions. Further, the addition of
TMB into MCH in the absence of H2O2 did not yield the expected blue
color. The results illustrate the peroxidase mimetic activity of MCH in
catalyzing the TMB-H2O2 reaction. The blue color formed is attributed to
the charge transfer complex formed due to the catalytic oxidation of
TMB [13,40]. The reaction between TMB and H2O2 reached equilibrium
in 30 min indicating a rapid catalytic reaction (Fig. S3). Interestingly,
chitosan owes peroxidase activity (Fig. 3b) which was improved by 42%
with the addition of Fe3O4. This significant improvement could be
ascribed to the presence of polycationic chitosan which facilitates the
electron transfer similar to the amino acids microenvironment in the
natural HRP[41,42].
A.M. Ariti et al.
Fig. 3. (a) Peroxidase memetic activity of MCH; (b) comparison of peroxidase activity; effect of (c) pH; (d) mass of the nanozyme on the peroxidase activity.
As shown in Fig. 3c, the MCH nanozymes works best in acidic con­
dition with optimum activity at pH 3.6. The MCH nanozyme retains 50%
of its optimum activity between pH 2 and 5. Further, whether the source
of peroxidase activity is from the leached iron in an acidic solution or the
intact MCH structure was also investigated. As seen in Fig. S4, the
leached solution obtained after washing the nanozyme in the acid buffer
doesn't have significant peroxidase activity. This indicates any peroxi­
dase activity comes from the intact MCH structure.
The mass of nanozyme required for catalyzing the chromogenic re­
action was optimized by varying the amount of nanozyme at fixed
substrate concentrations. As seen in Fig. 3d, 1.75 mg of MCH was op­
timum to effectively catalyze 6 mM of substrates in a 3 mL reaction
volume. The specific activity was then calculated from the slope of the
plot of unit activity (b) vs mass of nanozyme The unit activity was
calculated using eq. 4 from the initial rate measurement. Accordingly,
the specific activity was calculated to be 3.050 × 10− 2 U/mg. (Fig. S5)
V ΔA
bnanozyme= × (4)
εl Δt
where b - the unit activity of the nanozyme (U = μmol/min), V – volume,
ℇ- absorptivity coefficient, l-path length, A- absorbance, and t- time.
3.3. Enzyme kinetics
Steady-state kinetics of the nanozyme was performed at different
concentration ranges of TMB and H2O2. The results (Fig. 4a-b) show the
catalytic reaction follows a typical Michaelis-Menten model. Km and
Vmax determined from the Linewaver-Burk plot (Fig. 4c-d) were 0.45 mM
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Sensing and Bio-Sensing Research 42 (2023) 100595
and 15 μM/min for TMB and 2.8 mM and 4.18 μM/min for H2O2
respectively. As shown in Table 1 the Km values obtained are competi­
tive with natural HRP enzyme and lower than Ag-MoS2 which was
previously reported. This could be ascribed to enhanced catalytic re­
actions in MCH emerging from multiple iron centers dispersed in the
chitosan matrix. However, the lower Km in Fe3O4 NPs could be due to the
high surface area arising from their small size (30 nm). Despite this, the
use of such pristine nanomaterials could lead to aggregation which re­
duces their catalytic activity for practical applications. Hence the use of
chitosan in our nanozyme could help not only in creating an amino acid
microenvironment but also prevent the potential aggregation of Fe3O4
NMs.
3.4. Stability and recyclability of MCH nanozyme
Stability is an important factor in which nanozymes rival natural
enzymes. Hence the thermal stability of the MCH nanozyme was studied
by conducting the peroxidase activity assay at different operational
temperatures. As seen in Fig. 5a, the peroxidase memetic activity
increased with temperature up to 65 ◦ C and gradually declined after­
ward losing only 2% of its room temperature activity at 95 ◦ C. Further,
the temporal stability of the MCH nanozyme was examined by
measuring the peroxidase activity of MCH dispersed in a buffer at
different time intervals. As shown in Fig. 5b, the nanozyme retains
86.67% of its peroxidase memetic activity after one month. Hence both
stability studies suggest a robust nanozyme activity.
Recyclability is also another important factor required for the prac­
tical application of nanozymes. The peroxidase memetic activity of the
recovered MCH nanozyme was studied for four successive cycles. The
A.M. Ariti et al. Sensing and Bio-Sensing Research 42 (2023) 100595

Fig. 4. Michaelis-Menten curve for (a) TMB; (b) H2O2 substrates; Lineweaver-Burk plot of for (c) TMB; (d) H2O2.

A colorimetric detection assay was then carried out using the inhi­
Table 1 bition of the peroxidase activity of MCH by TBZ. Accordingly, different
Comparison of Kinetic parameters.
concentrations of TBZ were incubated with the MCH-TMB-H2O2
Nanozyme Substrate Km (mM) V max(μM/min) Ref mixture. Fig. 6c depicts a gradual decline in absorption at 652 nm with
TMB 0.1 344 increasing TBZ concentration. This suggests that more catalytic centers
Fe3O4 NPs [43]
H2O2 154 978 could be occupied with increased concentration of TBZ preventing
TMB 0.44 1.38 oxidation of TMB hence the blue color fades gradually. Fig. 6d demon­
HRP [43]
H2O2 3.7 –
TMB 0.7 6.37
strates a typical calibration curve relating the decrease in absorption
Ag-MoS2 [44] intensity (ΔA) with the TBZ concentration (C). As seen in the figure, the
H2O2 2.29 5.25
TMB 0.45 15 calibration curve exhibited a linear relationship between ΔA vs C from
MCH (Chitosan-Fe3O4) This study
H2O2 2.8 4.18 0.1 to 100 μM (R2 = 0.9987). The limit of detection and quantification
was calculated using the ICH guideline[46] as, LOD = 3SE/m and LOQ =
10SE/m, where SE and m are the standard error of the y-intercept and
recovered nanozyme was washed with distilled water before each cycle
slope, respectively. Consequently, LOD and LOQ were found to be 0.73
of the assay. As illustrated in Fig. 5c, the nanozyme maintained its
80.74% of peroxidase activity after the fourth cycle suggesting excellent
μM and 2.43 μM respectively. The results demonstrate acceptable
analytical figures of merits for TBZ detection as per the residue limit set
performance.
by the European Union [27].

3.5. Colorimetric detection of TBZ 3.6. Smartphone-assisted colorimetric detection of TBZ

The colorimetric detection of TBZ both in a spectrometer and
smartphone application is illustrated in Fig. 6a. First, the effect of TBZ
on the peroxidase activity of MCH was studied by adding TBZ (10 μM)
into the nanozyme-TMB-H2O2 mixture. As seen in Fig. 6b, TBZ sup­
pressed the intensity of blue color which was accompanied by a decrease
in absorption intensity at 652 nm. This confirms the inhibition effect of
TBZ on the peroxidase activity of MCH which could be ascribed to
occupation of active sites by TBZ.[44,45] Further, the presence of any
oxidoreductase activity on TBZ was checked by adding 10 μM TBZ to the
MCH–TMB in the absence of H2O2. As seen in Fig. 6b and inset photo a,
the assay didn't bring the expected blue color. This indicates the
mechanism involved by TBZ is purely inhibition of the peroxidase ac­
tivity of MCH. The result is in agreement with previous reports with a
different nanozyme.[45].
Due to the significance of onsite pesticide detection, portable
analytical tools are highly sought after in food safety monitoring. Hence
in this study, we attempted a proof-of-concept smartphone-assisted
detection of TBZ using the Spotxel®1.1 color reading application
installed on a Samsung Galaxy A30S smartphone. As shown in the digital
image in Fig. 5e, the smartphone readout RGB color intensity resulting
from the inhibition assay is directly related to TBZ concentrations
(0.1–100 μM, R2 = 0.997) with LOD 1.84 μM. Where ΔI is the difference
between color intensity before and after the addition of TBZ. When the
concentration of TBZ increases, the absorbance at 652 nm decreases
because of increased inhibition of the catalytic reaction which is
responsible for the absorbance. Hence the one with the smaller TBZ
concentration has a lower ΔI because it has a relatively higher absor­
bance which is closer to the control (no TBZ) making ΔI, smaller and vice

6
A.M. Ariti et al.
Fig. 5. (a) Thermal stability; (b) temporal stability; (c) recyclability of MCH nanozyme (C represents the cycle number).
versa. The result demonstrates the potential of MCH nanozyme for point-
of-need pesticide screening in the food supply chain.
3.7. Detection of TBZ fruit matrix
The application of the developed sensor in real sample analysis was
studied by the detection of TBZ spiked fruit matrix in a similar assay
protocol as above. The concentration of TBZ in each spiked sample was
then determined using the regression equation of the calibration curve.
The recovery of the method was then calculated by using Eq. 3. As seen
in Table 2 the % recovery spans from 87.4 to 112.7%, which is accept­
able to the standard analytical method validation ranges (80–120%).
Thus, the developed sensor demonstrated excellent accuracy for TBZ
detection in fruit samples.
3.8. Interference test
The performance of the method in the presence of other interferents
was studied. Accordingly, 100 μM of potential interferents were mixed
with 10 μM TBZ-MCH-TMB-H2O2 mixture in a similar assay condition.
As shown in Fig. 7, all of the interferents have an inhibitory effect on the
peroxidase activity of MCH, however, none of them have brought more
inhibition as that of TBZ illustrating the selectivity of the colorimetric
detection. This could be due to the structure of TBZ with two rings taking
more adsorption space compared with other metal ions and small mol­
ecules such as ascorbic and citric acid. So the result indicates that the
developed nanozyme-based colorimetric sensor can effectively detect
TBZ in the presence of these interferents which are potentially available
in the fruit matrix.
3.9. Comparison of sensor performance
Table 3 shows a summary of recent nanozyme-based colorimetric
detection reports on different pesticides based on nanozymes. To the
best of our knowledge, this is the second nanozyme-based colorimetric
detection of TBZ. Although a comparison of different pesticides couldn't
be accurate, this table could highlight the LOD labels of different
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Sensing and Bio-Sensing Research 42 (2023) 100595
nanozymes for pesticide detection. Hence, the MCH nanozyme owes
comparable even lower LOD than previously reported common nano­
zymes. However, the LOD is slightly lower than that reported for Ag-
MoS2. Although Ag-MoS2 has a lower detection limit, its composition is
different from the natural HRP. Hence the rational design used in this
study outweighs the small difference in LOD because rationally designed
nanozymes could be more important than functional memetic-only
nanozymes.
4. Conclusion
In summary, magnetic chitosan hydrogel (MCH) is demonstrated as a
rational peroxidase mimic for the smartphone-assisted colorimetric
detection of TBZ, which is a common antifungal agent in the postharvest
process of fruits. The MCH nanozyme represents a rational and simpler
colorimetric sensor for point-of-need applications. The peroxidase ac­
tivity of the MCH was studied and optimized using TMB and H2O2
substrates resulting in a 24% higher activity than the pristine Fe3O4 NPs
in acidic conditions. This improved activity could be due to two reasons:
(1) the polycationic nature of chitosan which enhances electron transfer
similar to the amino acid microenvironment in HRP enzyme; and (2), the
chitosan matrix could prevent the aggregation of Fe3O4 NPs which in
turn improves their catalytic activity. Overall multifunctional group
biopolymers such as chitosan are attractive materials for developing
rational nanozymes with enhanced activity. With a typical Michaelis-
Menten-based kinetic model, the MCH showed strong substrate speci­
ficity which is competitive with the reported Km value for HRP. The
concentration-dependent inhibition effect of TBZ on the peroxidase ac­
tivity of MCH was successfully used for a smartphone-assisted colori­
metric detection of TBZ, demonstrating The smartphone-generated color
intensity is directly related to the degree of inhibition resulting in a
lower detection limit acceptable as per the EU residue limit for TBZ. The
results of the study showed the utility of MCH nanozyme for point-of-
need applications in food safety monitoring.
A.M. Ariti et al. Sensing and Bio-Sensing Research 42 (2023) 100595

Fig. 6. (a) Scheme for colorimetric TBZ detection; (b) inhibition effect of TBZ on MCH peroxidase activity (the inset phots a = MCH + TMB+ TBZ; b = MCH + TMB+
H2O2 c = TBZ+ MCH + TMB+ H2O2); (c) absorption spectra of TMB-H2O2-MCH in the presence of different concentration labels of TBZ; (d) calibration curve for the
TBZ detection (ΔA vs C), ΔA is the difference of Abs before and after TBZ addition; (e) calibration for TBZ using a smartphone application; ΔI: ΔA is the difference in
color intensity before and after TBZ addition (the photo above the graph represents the inhibition assay result which was later digitized by the color reader app).

Software; Beamlack T. Gutema: investigation, Material preparation,

Table 2
Software; Efrata G. Mekonnen: investigation, Material preparation,
Recovery results of TBZ from real fruit sample matrices (n = 3).
Software; Yitayal A. Workie: Supervision; Editing, Ebrahim M.Abda:
Sample TBZ added (M) TBZ found (M) Recovery (%) RSD (%) Supervision, Editing, Conceptualization; Bing-Joe Hwang: Investigation,
1 × 10− 5
9.64 × 10–6 96.4 4.00 Resources, Menbere L. Mekonenen: Funding acquisition, Conceptual­
Apple 7
1 × 10− 1.087 × 10–7 108.7 4.71 ization, Resources, Methodology, Writing-Review & Editing.
5
1 × 10− 8.74 × 10–6 87.4 6.05
Banana 7
1 × 10− 1.127 × 10–7 112.7 5.20

Declaration of Competing Interest

Authorship contribution
The authors declare no known competing financial or other interests
Abera M.Ariti: Investigation, Material preparation, Writing original nor personal relationships that could have appeared to influence the
draft, Software, Seada A. Geleto: investigation, Material preparation, work reported in this paper.

8
A.M. Ariti et al. Sensing and Bio-Sensing Research 42 (2023) 100595

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