Catalytic evaluation of biocompatible chitosan-stabilized gold nanoparticles on oxidation of morin

Article abstract of DOI:10.1016/j.carbpol.2021.117699

Herein, we present a study on the catalytic evaluation of biocompatible chitosan-stabilized gold nanoparticles (CH-AuNPs) on the oxidation of morin as a model reaction. Biocompatible CH-AuNPs have been characterized through several analytical methods such as TEM, UV–vis, DLS and zeta potential analyses. CH-AuNPs have a small size (10 ± 0.4 nm) with a narrow size distribution and high positive surface charge (+40.1 mV). CH-AuNPs has been demonstrated to be highly active nanocatalysts for the oxidation of morin with the assistance of H₂O₂ as an oxidant compared with control experiments. The oxidation reaction follows a pseudo-first-order reaction. The kinetic studies show that apparent rate constant (k_app) is positively correlated with the concentrations of CH-AuNPs and H₂O₂, while it is negatively correlated with morin concentration. Furthermore, the reusability tests have been performed and the results demonstrate the long-term stability and reusability of CH-AuNPs without any loss of catalytic activity. Cytotoxicity studies exhibit that CH-AuNPs have low toxicity and they are biocompatible with HeLa and MCF-7 cells.

Full text of DOI:10.1016/j.carbpol.2021.117699

Carbohydrate Polymers 258 (2021) 117699
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Catalytic evaluation of biocompatible chitosan-stabilized gold
nanoparticles on oxidation of morin
,
,b,
Onur Buluta b, M. Deniz Yilmaza
*
a Department of Bioengineering, Faculty of Engineering and Architecture, Konya Food and Agriculture University, 42080 Konya, Turkey
b Research and Development Center for Diagnostic Kits (KIT-ARGEM), Konya Food and Agriculture University, 42080 Konya, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chitosan
Herein, we present a study on the catalytic evaluation of biocompatible chitosan-stabilized gold nanoparticles
(CH-AuNPs) on the oxidation of morin as a model reaction. Biocompatible CH-AuNPs have been characterized
through several analytical methods such as TEM, UV–vis, DLS and zeta potential analyses. CH-AuNPs have a
small size (10 ± 0.4 nm) with a narrow size distribution and high positive surface charge (+40.1 mV). CH-AuNPs
has been demonstrated to be highly active nanocatalysts for the oxidation of morin with the assistance of H2O2 as
an oxidant compared with control experiments. The oxidation reaction follows a pseudo-first-order reaction. The
kinetic studies show that apparent rate constant (kapp) is positively correlated with the concentrations of CH-
AuNPs and H2O2, while it is negatively correlated with morin concentration. Furthermore, the reusability
tests have been performed and the results demonstrate the long-term stability and reusability of CH-AuNPs
without any loss of catalytic activity. Cytotoxicity studies exhibit that CH-AuNPs have low toxicity and they
are biocompatible with HeLa and MCF-7 cells.
Gold nanoparticles
Morin
Oxidation
Catalysis
1. Introduction
such as easy synthesis, high catalytic activity, small size (a few
nanometer-scale), and distinguished optical characteristics (Corma &
Catalytic oxidation is one of the major techniques to degrade toxic
and hazardous organic compounds, which are extensively used by in-
dustrial processes, in wastewater (Chahbane et al., 2007; Liotta, 2010;
Ribeiro, Silva, Figueiredo, Faria, & Gomes, 2016; Segal, Suib, & Foland,
1997; Shahidi, Roy, & Azzouz, 2015; Wang, Weinstein, White, & Stahl,
2018). Catalytic oxidation is also employed to remove unwanted stains
on textile fabrics and residual lignin in wood pulp (Dannacher, 2006;
Hage & Lienke, 2006; Wieprecht, Hazenkamp, Rohwer, Schlingloff, &
Xia, 2007; Yurdakul, Oktem, & Yilmaz, 2019). In recent years, transition
metal nanoparticles as nanocatalysts in catalytic oxidation processes
have received a great deal of research interest in both scientific and
industrial studies because they offer various fantastic features such as
high surface area to volume ratio, high catalytic efficiency, easy removal
from the solution, good dispersion of the active sites, and easy diffusion
of the reactants to nanoparticle surface (Aiken, Lin, & Finke, 1996;
Astruc, 2007; Li et al., 2015; Polzer, Wunder, Lu, & Ballauff, 2012; Xaba
& Meijboom, 2017; Xing, Zhou, Ma, & Wu, 2011; Zhang et al., 2013).
Among various transition metal nanoparticles, gold nanoparticles
(AuNPs) as oxidation catalysts are special due to their unique properties
Garcia, 2008; Ilunga & Meijboom, 2016a, 2017; Ndolomingo & Meij-
boom, 2016; Scire & Liotta, 2012; Thompson, 2007). However, bare
gold nanoparticles are unstable and easily aggregated in solutions due to
their high surface energies. When AuNPs are agglomerated, their cata-
lytic activity is considerably decreased. To increase the stability and to
prevent the aggregation of AuNPs in solution, stabilizing agents
including organic ligands, polymers, and dendrimers have been exten-
sively used (Saha, Agasti, Kim, Li, & Rotello, 2012). The studies on
catalytic oxidation using dendrimer encapsulated AuNPs have been re-
ported first time by Meijboom and coworkers (Nemanashi & Meijboom,
2015). In their studies, poly(amidoamine) PAMAM stabilized AuNPs
were synthesized and their catalytic activities were investigated by
using morin dye as a model compound in the presence of hydrogen
peroxide (H2O2).
In recent years, carbohydrate polymers such as chitosan, cellulose,
hyaluronic acid, and alginate have been frequently used as stabilizing
agents for AuNPs due to their environmentally-friendly nature, abun-
dance in nature, easy availability and affordable prices (Chen et al.,
2015; Dey, Sherly, Rekha, & Sreenivasan, 2016; Esumi, Takei, &
* Corresponding author at: Department of Bioengineering, Faculty of Engineering and Architecture, Konya Food and Agriculture University, 42080 Konya, Turkey.
E-mail address: deniz.yilmaz@gidatarim.edu.tr (M.D. Yilmaz).
https://doi.org/10.1016/j.carbpol.2021.117699
Received 13 December 2020; Received in revised form 21 January 2021; Accepted 22 January 2021
Available online 27 January 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.

O. Bulut and M.D. Yilmaz
Carbohydrate Polymers 258 (2021) 117699
Yoshimura, 2003; Huang & Yang, 2004; Hussain, Iqbal, & Mazhar,
2009; Lee et al., 2012; Leiva et al., 2015; Pandey, Goswami, & Nanda,
2013; Pradeepa et al., 2016). Among them, the use of chitosan, a
biocompatible and biodegradable natural polysaccharide obtained by
the partial deacetylation of chitin (Donmez, Oktem, & Yilmaz, 2018;
Perez-Obando et al., 2019), for the synthesis of AuNPs as a biocompat-
ible reducing and capping agent has been studied by many researchers
for various purposes such as drug delivery, antimicrobial and other
biomedical applications (Chen, Zhao, & Wang, 2020). In general, these
nanomaterials are considered to be chemically stable, non-toxic and
biocompatible. In this regard, chitosan-based AuNPs in the range of
10ꢀ 20 nm exhibited bactericidal effects against antibiotic resistant
bacteria without any significant toxic effects on human cells (Regiel--
Futyra et al., 2015). A chitosan-AuNPs based material was reported as a
drug delivery system which enables efficient loading and targeting of
chemotherapeutics to human breast cancer cells (Fathy, Mohamed,
Elbialy, & Elshemey, 2018). Although a number of studies were reported
on the synthesis, characterization and biomedical applications of
chitosan-AuNPs hybrid materials, there is no report on the catalytic
activity of chitosan-stabilized AuNPs (CH-AuNPs) as biocompatible
nanocatalysts in catalytic oxidation processes.
removed from the water bath and cooled immediately to room tem-
perature. The prepared CH-AuNPs were centrifuged at 15,000 rpm for
1 h and washed three times with 1 % acetic acid to remove excess re-
actants. The residue was redispersed in 1 % acetic acid and stored at 4 ◦C
for further use.
3.2.2. Catalytic oxidation of morin in solution
The oxidation of morin was carried out in a glass beaker with a
magnetic stirrer at room temperature (25 ◦C) in 10 mM carbonate buffer
at pH 10. The stock solutions of morin (3.2 mM), H2O2 (30 % in water),
and CH-AuNPs (10 nM in 1% acetic acid) were used respectively. The
reactions were initiated by mixing of appropriate amounts of reagents in
a UV cuvette. At given time intervals, samples were taken and analyzed
by UV–vis spectrophotometer. Apparent rate constants (kapp) were
calculated using Eq. (1).
C
A
ꢀ ln = ꢀ ln =kₐₚₚt
C₀ A₀
(1)
where C0 and C are the concentrations at 0 min and at a given time, and
A0 and A are the absorbance values at 390 nm at 0 min and at given
time, respectively.
Herein, we report, for the first time, the use of CH-AuNPs as
biocompatible and stable catalysts for the oxidative degradation of
morin as a model compound combined with H2O2 as the oxidant. The
catalytic process is monitored via UV–vis spectroscopy by recording the
changes in maximum absorbance peak of morin at 390 nm. The full
kinetic analysis, biocompatibiliy, and reusability of CH-AuNPs are also
evaluated. The possible mechanism for the degradation of morin was
also discussed.
The analysis of the kinetic data was performed in terms of the
Langmuir-Hinshelwood model using Eq. (2) (Wunder, Polzer, Lu, Mei, &
Ballauff, 2010; Wunder, Lu, Albrecht, & Ballauff, 2011).
nꢀ 1
m
k. S. Kmnorin . [morin] . (KH . [H2O2])
2O2
kapp
=
(2)
n
m
(1 + (Kmorin . [morin]) + (KH . [H2O2] ) )2
2O2
where k is the molar rate constant per unit square of the catalyst, and
morin and KH2O2 are the adsorption constants of the two reactants. The
K
parameters n and m are the Freundlich exponents for morin and H2O2,
rerspectively. According to this model, H2O2 reacts on the surface of the
nanocatalysts yielding reactive oxygen species (ROS). Subsequently,
morin adsorbs on the empty sites of the catalysts and undergoes a sur-
face reaction. The degradation products of morin desorbes from the
surface and the nanocatalysts continue to be used into the next catalytic
cycle.
2. Hypotheses
The surface functionalization with biocompatible chitosan may
improve the catalytic efficiency of gold nanoparticles and can be a
potent strategy for decreasing cytotoxicity of nanocatalysts.
3. Experimental section
3.2.3. Cell viability assay
3.1. Materials
Human cervical carcinoma (HeLa) and human breast cancer (MCF-7)
cell lines were obtained from American Type Culture Collection. Both
cell lines were maintained in Dulbecco’s Modified Eagle Medium
(DMEM) supplemented with 10 % heat-inactivated fetal bovine serum
Unless otherwise noted, all chemicals and solvents are analytical
grade and used without further purification. Commercially available
chitosan (high molecular weight around 310ꢀ 375 kDa, > 75 % deace-
tylated), morin hydrate, HAuCl4.3H2O, acetic acid (glacial), hydrogen
peroxide (30 % in H2O), Na2CO3, NaHCO3, HCl, NaOH, t-BuOH, NaN3,
and p-benzoquinone were all purchased from Sigma-Aldrich and used as
received. Purified water (resistivity > 18 MΩ cm) from the Arium® Mini
Water Purification System was used in all experiments.
(FBS), penicillin (100 IU/mL) and streptomycin (100
μg/mL) in a hu-
midified incubator with 5% CO2 at 37 ◦C.
The effects of CH-AuNPs on cell viability was determined by a
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Mosmann, 1983). Briefly, HeLa and MCF-7 cells were
seeded in 96-well cell culture plates with a density of 104 cell/well and
incubated for 48 h. After adhesion of cells, the medium was discarded,
and cells were washed with phosphate buffered saline (PBS). Each well
was treated with fresh medium containing CH-AuNPs at different con-
centrations (ranging from 0.05 nM to 6.80 nM), and cells were incu-
Transmission electron microscopy (TEM) examination was
completed on a Jeol 2100 F HRTEM electron microscope equipped with
an Orius SC1000CCD camera. For TEM measurements, the samples were
prepared by dispersing the powder samples in ethanol, after which they
were dispersed and dried on carbon film on a Cu grid. DLS and zeta
potential measurements were carried out by Malvern Zetasizer Nano
ZS90 analyzer. UV–vis absorbance spectra were recorded on Agilent
Cary 60 UV–vis spectrophotometer.
bated for 24 h. Then, each well was treated with 10
(5 mg/mL in PBS). After 4 h of incubation with MTT, 100
μ
L solution
μ
L of sodium
dodecyl sulfate (SDS) solution (1:10, w/v) was added to each well and
incubated overnight for the solubilization of MTT into purple formazan
crystals. Sunsequently, the absorbance of each well was measured at
570 nm using a microplate reader. Results obtained from control cells
were accepted as 100 % cell viability, and the relative viability of treated
cells was calculated accordingly.
3.2. Methods
3.2.1. Synthesis of CH-AuNPs
CH-AuNPs were synthesized as follows (Zhao et al., 2019): Briefly,
0.125 g of chitosan was dissolved in 95 mL of 1% acetic acid in water.
The solution was heated to 90 ◦C. To this solution, 5 mL of 10 mM
HAuCl4. 3H2O was added with continuous stirring and the color of the
solution turned to yellow. The mixture was stirred at 90 ◦C for 1 h and
its color changed to wine red. Then, the reddish colored solution was
2

O. Bulut and M.D. Yilmaz
Carbohydrate Polymers 258 (2021) 117699
4. Results and discussion
increases the hydrodynamic particle size and vice versa. In addition, the
zeta potential value of CH-AuNPs in 10 mM carbonate buffer at pH 10
was found to be ꢀ 6.05 mV (Fig. S3), indicating the deprotonation of
amino groups of chitosan and confirming the aggregation behavior of
CH-AuNPs in basic solution due to the reduced electrostatic
stabilization.
4.1. Characterization of nanocatalyst
Before the catalytic evaluation, as-prepared CH-AuNPs were char-
acterized. The morphology and size of CH-AuNPs were investigated by
transmission electron micsopcopy (TEM). The TEM images showed non-
aggregated and spherical nanoparticles (Fig. 1A) with the average mean
particle size is around 10 ± 0.4 nm (Fig. 1B). The UV–vis spectrum of
the as-synthesized CH-AuNPs is also shown in the inset of Fig. 1A. A
strong band at 520 nm has been attributed to a surface plasmon reso-
nance (SPR) peak, indicating the formation of CH-AuNPs. To determine
the surface charge and hydrodynamic size of CH-AuNPs, zeta potential
and dynamic light scattering (DLS) measurements were performed in 1%
acetic acid solution. The zeta potential value of CH-AuNPs was found to
be +40.1 mV (Fig. 1C). This positive charge has been ascribed to the
protonated amine groups of chitosan, confirming that the surface of
catalysts was covered with chitosan biopolymers. The hydrodynamic
size of CH-AuNPs was measured by DLS method. As shown in Fig. 1D,
the hydrodynamic size of catalysts (78 nm) is greater than that of the
size estimated by TEM (10 nm). The reason for different sizes is that
TEM measures the solid AuNPs core by ignoring the chitosan coating
and the solvent layer, whereas DLS measures the solid inorganic core
along with chitosan layer on nanoparticles and the hydration layer
(Yilmaz, 2016; Yurdakul et al., 2019). Furthermore, positively charged
chitosan polysaccharides are able to form aggregates in acidic medium
(Collado-Gonzalez et al., 2015). The intensity distribution analysis as
shown in Fig. 1D detects such aggregates which are present in the so-
lution and these aggregates protect individual AuNPs from agglomera-
tion. Further DLS studies were performed to better understand the
structure of CH-AuNPs in acidic and basic solutions. In Figs. S1 and S2, it
is outlined a comparison between the size distributions corresponding to
CH-AuNPs in acidic and basic solutions. Particle sizes are much smaller
in acidic medium (Fig. S1) than that of basic solution (Fig. S2). This
result can be attributed to the electrostatic stabilization of CH-AuNPs by
the protonated amine groups of chitosan in acidic solution. The distri-
butions by number, volume and intensity indicate that an increase in pH
4.2. Catalytic activity of nanocatalyst
The catalytic oxidation of morin, commonly used polyphenolic dye
in the oxidation processes, in the presence of H2O2 as the oxidant was
chosen to study the catalytic activity of CH-AuNPs (Bingwa, Bewana,
Haumann, & Meijboom, 2017). The catalytic process was monitored
using UV–vis spectrophotometer at room temperature. As shown in
Fig. 2A, the maximum absorbance peak of morin (0.1 mM in 10 mM
carbonate buffer, pH = 10) at 390 nm gradually decreased within
30 min and the color of the solution turned from intense yellow to
colorless as a result of the oxidation of morin. It is important to note here
that SPR peak of CH-AuNPs at 520 nm remained unchanged during the
time of the reaction, confirming the stability of CH-AuNPs in the
oxidation solution. Fig. 2B outlines the kinetics of the oxidation of morin
in the presence and absence of H2O2 and CH-AuNPs. The results indicate
that uncatalyzed reactions result in low oxidation rates during the
course of the reaction, while the reaction rate dramatically increased in
the presence of CH-AuNPs. Once the logaritmic data of absorbance
changes is plotted against the reaction time, a linear curve was obtained,
indicating pseudo-first-order reaction kinetics relating to morin con-
centration by using a large excess of H2O2 (Fig. 2C) (Nemanashi &
Meijboom, 2015). The kapp values were calculated by using Eq. 1 and
found to be (1.21 ± 0.07) × 10ꢀ 3 s-1 in the presence of both CH-AuNPs
and H2O2, (5.04 ± 0.05) × 10-5 s-1 in the presence of H2O2 without
CH-AuNPs and (1.12 ± 0.04) × 10-5 s-1 in the presence of catalyst
without H2O2.
4.3. Effect of nanocatalyst concentrations
The reaction kinetics as a function of catalyst concentration were
Fig. 1. (A) TEM image of as-synthesized CH-AuNPs with UV–vis spectrum and the color in solution (inset) and (B) corresponding particle size distribution histogram.
(C) Zeta potential and (D) hydrodynamic size distribution histogram of CH-AuNPs in 1 % acetic acid.
3

O. Bulut and M.D. Yilmaz
Carbohydrate Polymers 258 (2021) 117699
Fig. 2. (A) The UV–vis spectra of catalytic oxidation of morin (0.1 mM) by H2O2 (490 mM) and CH-AuNPs (2.5 nM) in 10 mM carbonate buffer at pH 10 and 298 K.
(B) Kinetic spectral changes and (C) the corresponding first-order plots for the catalytic oxidation of morin as a function of time with and without catalyst.
studied by varying the concentration of CH-AuNPs while morin and
H2O2 concentrations kept constant. Fig. 3A shows the first order plots
over time using different concentrations of CH-AuNPs and a linear
relation was observed at each concentration. It is clear that the con-
centration of morin decreases with increasing CH-AuNPs concentration.
The apparent rate constants (kapp) were calculated from the Eq. 1 and
they were plotted against the concentration of CH-AuNPs (Fig. 3B). A
linear relation is observed, indicating that the active sites increased with
increasing catalyst concentration which boosted the formation of active
radical species to enhance the catalytic activity. This linear relationship
can be related to the increase of the surface area required for the cata-
lytic reaction (Ilunga & Meijboom, 2016b).
morin conentration and keeping H2O2 concentration constant. Another
set of experiment was performed by a constant morin concentration
while varying H2O2 concentration. The plots of logarithmic data versus
time (Fig. 4A and B) and the apparent rate constants as function of the
concentrations of either morin (Fig. 4C) or H2O2 (Fig. 4D) are depicted
in Fig. 4. It is found that the increase of morin concentration leads to the
decrease in the kapp values, while the increase of H2O2 concentration
results in the increase of kapp
.
This observation is attributed that both reactants compete for the
empty site on CH-AuNPs and proposed mechanism of the oxidation of
morin using CH-AuNPs in the presence of H2O2 is outlined in Fig. 5
(Ncube, Hlabathe, & Meijboom, 2015; Ndolomingo & Meijboom, 2016).
The catalytic reaction on CH-AuNPs surface follows the
Langmuir-Hinshelwood model (Nemanashi & Meijboom, 2015; Xiao
et al., 2019; Yu et al., 2019). According to this model, both morin and
H2O2 were adsorbed onto the CH-AuNPs surface with a fast and
reversible fashion. CH-AuNPs catalyzed H2O2 to produce superoxide
radical, then superoxide reacted with adsorbed morin. Degradation
products, 2,4-dihydroxybenzoic acid and 2,4,6-trihydroxybenzoic acid
(Ilunga & Meijboom, 2016a), removed from the surface generating the
new available active sites for next catalytic cycle.
4.4. Effect of H2O2 and morin concentrations
To investigate the reaction mechanism in more detail, the kinetic
studies were carried out by determining kapp values while varying the
4.5. Analysis of reactive oxygen species
To verify the existince of superoxide radical in the reaction envi-
ronment, the effects of radical scavengers on the degradation of morin
were investigated. Sodium azide (NaN3), t-butanol and p-benzoquinone
•
were chosen as singlet oxygen (1O2), superoxide (O2¡) and hydroxyl
•
( OH) radical scavengers, respectively (Zhang, Nie, Hu, & Hu, 2011).
The degradation rate of morin was obviously decreased in the presence
of p-benzoquinone (Fig. 6). Although there was no significant effect of
NaN3 on reaction rate, t-butanol had a slight influence on the oxidation
•
of morin. These results indicated that superoxide (O2¡) was the major
•
ROS generated in the reaction environment, whereas hydroxyl ( OH)
radical was the minor.
4.6. Reusability and stability of nanocatalyst
The reusability and stability are crucial properties for any hetero-
geneous catalyst for practical applications. The reusability tests of CH-
AuNPs were evaluated by repeated additions of morin to a solution of
the reaction. It is found that CH-AuNPs have not been lost their catalytic
Fig. 3. (A) The first-order plots of different concentrations of CH-AuNPs and
(B) the influence of catalyst concentration on the apparent rate constants (●
0.25 nM, ◼ 0.5 nM, ◆ 1.0 nM, ▴1.5 nM, × 2.0 nM).
4

O. Bulut and M.D. Yilmaz
Carbohydrate Polymers 258 (2021) 117699
Fig. 4. (A) The first order kinetic plots of different concentrations of morin (● 32
μ
M, ◼ 56
μ
M, ◆ 80
μ
M, ▴ 128
μ
M) at constant concentrations of H2O2 (490 mM)
and CH-AuNPs (2.5 nM) and (B) different concentrations of H2O2 (● 25 mM, ◼ 50 mM, ▴ 100 mM, ◆ 150 mM) at constant concentration of morin (0.1 mM) and CH-
AuNPs (2.5 nM) at 298 K. (C) Influence of morin concentration and (D) H2O2 concentration on apparent rate constants, kapp
.
Fig. 5. Proposed mechanism and reaction scheme of the catalytic oxidation of morin by H2O2 on the surface of CH-AuNPs according to Langmuir-Hinshelwood
model for a bimolecular surface reaction.
activity even after five times of use (Fig. 7A). The kapp values of repeated
catalytic cycles were calculated by using Eq. (1) and found to be
1.61 × 10ꢀ 3 s-1 for 1st run, 1.30 × 10ꢀ 3 s-1 for 2nd run, 1.32 × 10ꢀ 3 s-1
for 3rd run, 1.38 × 10ꢀ 3 s-1 for 4th run, and 1.35 × 10ꢀ 3 s-1 for 5th run,
respectively, proving the reusability of CH-AuNPs. As can be seen in
TEM micrograph of recycled CH-AuNPs after 5th run (Fig. 7B), the
morphology and the structure of the catalyst remained unchanged.
Furthermore, the average mean particle size of recycled CH-AuNPs is
around 10 ± 0.6 nm (Fig. 7C) and the SPR band is located at 520 nm
(Fig. 7D). These results demonstrate that the structure of CH-AuNPs is
stable during the consecutive catalytic reactions. Therefore, CH-AuNPs
have unique long-term stability and can be reused without noticable
loss of catalytic activity for the oxidation of morin.
Fig. 6. Kinetics of the oxidation of morin (0.1 mM) in the presence of CH-
AuNPs (2.5 nM) and H2O2 (490 mM) in 10 mM carbonate buffer at pH 10 at
298 K with 100 mM NaN3 (▴), 100 mM t-BuOH (◼), 100 mM p-benzoqui-
none (●).
5

O. Bulut and M.D. Yilmaz
Carbohydrate Polymers 258 (2021) 117699
Fig. 8. The viability of HeLa and MCF-7 cell lines after 24 h of incubation with
various concentrations of CH-AuNPs determined by MTT assay.
control experiments. The kinetic studies showed that apparent rate
constant (Kapp) was related to the concentrations of CH-AuNPs, morin
and H2O2, and the oxidation reaction followed a pseudo-first-order re-
action. Furthermore, the reusability tests were performed and the results
demonstrated the long-term stability and reusability of CH-AuNPs
without any loss of catalytic activity. Cytotoxicity studies demon-
strated that CH-AuNPs have low toxicity and they are biocompatible
with HeLa and MCF-7 cells. We believe that present research will pave
the way for the development of environmentally friendly, biocompat-
ible, and highly stable nanocatalysts stabilized by biocompatible mate-
rials with fascinating and challenging oxidation properties. To this end,
further studies are ongoing in our research laboratory.
Fig. 7. (A) Time dependence of the reaction as a function of the repeated ad-
ditions of morin (0.1 mM) in the presence of H2O2 (490 mM) using CH-AuNPs
(2.5 nM) in 10 mM carbonate buffer at pH 10. (B) TEM micrograph of recycled
CH-AuNPs after 5th run. (C) Corresponding particle size histogram and (D)
UV–vis spectrum.
4.7. Studies on cell viability and cytotoxicity
The MTT assay was performed to evaluate the effect of CH-AuNPs on
the viability of two human cell lines, namely; HeLa and MCF-7. This
assay is based on reduction of a yellow solution of tetrazolium salt to a
purple colored formazan compound by mitochondrial dehydrogenases
present in metabolically active and viable cells. Therefore, the amount
of formazan is proportional to the amount of viable cells. As depicted in
Fig. 8, the effects of CH-AuNPs on the viability of HeLa and MCF-7 cells
are represented. The tested concentrations of CH-AuNPs ranged from
0.05 nM to 6.80 nM in both cell lines, which yielded a similar
concentration-dependent trend on the cell viability after 24 h of treat-
ment. In the range from 0.05 nM up to 0.85 nM, the cytotoxic effects of
CH-AuNPs were not statistically significant on both cell lines (p < 0.05).
However, increasing in the range from 1.70 nM to 6.80 nM, CH-AuNPs
start to exhibit gradual cytotoxic effects in which the viability of HeLa
and MCF-7 cells reduced to 75.65 ± 4.67 % and 83.82 ± 2.18 % at
1.70 nM, respectively. At the highest concentration of 6.8 nM, the
viability of HeLa and MCF-7 cells reduced to 63.32 ± 3.44 % and
71.42 ± 6.36 %, respectively. These results showed that a wide range of
concentrations of CH-AuNPs, even the concentrations used in this study,
did not exhibit any significant cytotoxic effect on both HeLa and MCF-7
cell lines.
CRediT authorship contribution statement
Onur Bulut: Investigation. M. Deniz Yilmaz: Investigation, Writing
- review & editing.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
We would like to thank Konya Food and Agriculture University
(KFAU) for financial support of this research. We extend our gratitude to
KFAU Research and Development Center for Diagnostic Kits (KIT-
ARGEM) for the use of the facilities.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2021.117699.
5. Conclusions
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