Synthesis of copper ion incorporated horseradish peroxidase-based hybrid nanoflowers for enhanced catalytic activity and stability

Article abstract of DOI:10.1039/c5dt01250c

In this study, we report the preparation, catalytic activity and stability of a hybrid nanoflower (hNF) formed from horseradish peroxidase (HRP) enzyme and copper ions (Cu²⁺). We studied the morphology of hNFs as a function of the concentrations of copper (Cu²⁺) ions, chloride ions (Cl^-) and HRP enzyme, the pH of the buffer solution (phosphate buffered saline), and the temperature of the reaction. The effects of morphology on the catalytic activity and stability of hNFs were evaluated by oxidation of guaiacol (2-methoxyphenol) to colored 3,3-dimethoxy-4,4-diphenoquinone in the presence of hydrogen peroxide (H₂O₂). The enhanced activity of hNFs synthesized (from 0.02 mg mL^-1 HRP in 10 mM PBS (pH 7.4) at +4°C) was 17595 U mg^-1, which was ～300% higher than free HRP in PBS, where it achieved an activity of 5952 U mg^-1. In terms of stability, these hNFs stored in PBS buffer at +4°C and room temperature (RT = 20°C) lost 4% and 20%, respectively, of their initial catalytic activities within 30 days. Finally, we demonstrated that these hNFs can be utilized as sensors for the detection of dopamine.

Full text of DOI:10.1039/c5dt01250c

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Synthesis of copper ion incorporated horseradish
peroxidase-based hybrid nanoflowers for
enhanced catalytic activity and stability†
Cite this: DOI: 10.1039/c5dt01250c
a
b
b,c
a
Burcu Somturk, Mehmet Hancer, Ismail Ocsoy* and Nalan Özdemir*
In this study, we report the preparation, catalytic activity and stability of a hybrid nanoflower (hNF) formed
2
+
from horseradish peroxidase (HRP) enzyme and copper ions (Cu ). We studied the morphology of hNFs
2
+
−
as a function of the concentrations of copper (Cu ) ions, chloride ions (Cl ) and HRP enzyme, the pH of
the buffer solution (phosphate buffered saline), and the temperature of the reaction. The effects of mor-
phology on the catalytic activity and stability of hNFs were evaluated by oxidation of guaiacol (2-methoxy-
2 2
phenol) to colored 3,3-dimethoxy-4,4-diphenoquinone in the presence of hydrogen peroxide (H O ).
The enhanced activity of hNFs synthesized (from 0.02 mg mL− HRP in 10 mM PBS (pH 7.4) at +4 °C) was
17 595 U mg− , which was ∼300% higher than free HRP in PBS, where it achieved an activity of 5952 U
mg−1. In terms of stability, these hNFs stored in PBS buffer at +4 °C and room temperature (RT = 20 °C)
lost 4% and 20%, respectively, of their initial catalytic activities within 30 days. Finally, we demonstrated
that these hNFs can be utilized as sensors for the detection of dopamine.
1
1
Received 31st March 2015,
Accepted 21st April 2015
DOI: 10.1039/c5dt01250c
www.rsc.org/dalton
procedures have limited application of the chemical modifi-
cation method.
In contrast to chemical modification, immobilization of
1
. Introduction
6
–8
As natural biocatalysts, enzymes have received considerable
attention owing to their unique properties, including high enzymes in/on solid supports has offered a simple, alternative,
catalytic activity, stability, selectivity, low toxicity and water generally requiring one-step reaction. In general, immobilized
solubility. Accordingly, they have found widespread use in enzymes show improved stability, making them efficient, re-
various scientific and technical fields, including chemistry, usable and economical. Over the past two decades, enzymes
biochemistry, medicine, pharmaceutical science and industry have been immobilized in/on various supports, such as meso-
1
–5
(textile, food, etc.).
However, the instability of the free porous silica, inorganic or organic nanomaterials, polymer
enzymes in aqueous solution strictly limits their applications. strands and sol–gel materials through covalent, non-covalent,
9
–13
To address this issue, two common methods, chemical modifi- entrapment and cross-linking interactions.
Nanomaterials
cation and immobilization, have been used to enhance the with their high surface-to-volume ratios serve as platforms
enzyme catalytic activity and stability. Although chemically capable of loading significantly more enzyme. Immobilization
modified enzymes have exhibited high stability, they have of enzymes generally enhances the stability, presumably due to
not shown a remarkable increase in catalytic activity. Further- reduced mobility and attachment of more enzymes per volume
more, the time-consuming and labor-intensive synthetic unit of the support compared to the free enzyme dissolved in
1
4–25
solution.
However, increased catalytic activity is generally
limited due to mass transfer limitations between the enzyme
and the substrate and conformational changes in the enzyme.
Therefore, there is still a high demand for immobilized
enzymes with enhanced catalytic activity and stability.
Recently, Zare and co-workers reported an elegant approach
for the synthesis of immobilized enzymes in the form of nano-
a
26–34
Department of Chemistry, Faculty of Science, Erciyes University, Kayseri,
8039 Turkey. E-mail: ozdemirn@erciyes.edu.tr
3
b
Nanotechnology Research Center (ERNAM), Erciyes University, Kayseri,
3
8039 Turkey. E-mail: ismailocsoy@erciyes.edu.tr
c
Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University,
Kayseri, 38039 Turkey
Electronic supplementary information (ESI) available: SEM images of Cl− and
†
flowers with highly enhanced catalytic activity and stabi-
HRP enzyme concentration dependent hNFs and the SEM image of hNFs after
activity measurements; photos of guaiacol in PBS buffer, the mixture of hNFs
and guaiacol before activity measurements and the mixture of hNFs and guaia-
col stored at RT for 2 months; activities of hNFs synthesized at different pH
values. See DOI: 10.1039/c5dt01250c
35,36
lity.
Zare and co-workers also reported synthesis of a
membrane containing laccase nanoflowers for detection of
3
7
phenol. In subsequent studies, Zeng and co-workers utilized
the same synthetic route to produce CaHPO
4
-α-amylase-based
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3
8
nanoflowers
and Yang and co-workers used enzyme-in- First, hNFs and free HRP were dissolved in 1 mL of PBS in two
organic nanoflowers as sensors for detection of hydrogen per- separate reaction tubes and then 1 mL of 22.5 mM hydrogen
3
9
oxide and phenol. Despite these reports, the underlying peroxide (H
2
O
2
) and 1 mL of 45 mM guaiacol were added to
and
principles of nanoflower formation and increased catalytic each reaction tube. The final concentrations of H
2 2
O
activity and stability still remain elusive, and further studies guaiacol in the tubes were determined as 7.5 mM and 15 mM,
are needed to explore effects of reaction parameters on the respectively. The change in color was visually observed with
morphology of nanoflowers and to elucidate the mechanism of naked eyes and the changes in absorbance values were also
enhancement of enzyme activity and stability. Herein, we monitored for 3 min at 470 nm at 25 °C using a UV-Vis
present a study on the formation, catalytic activity and stability spectrophotometer.
of hybrid nanoflowers (hNFs) containing horseradish peroxi-
dase (HRP) enzyme and copper ions (Cu ). The main goals
2
+
2.4 Dopamine detection test
were to evaluate the effect of reaction parameters on formation Various concentrations of dopamine (DA) (10, 20, 40 and 80 µg
−
1
of hNFs and to explain the enhancements in the activity and mL ) were dissolved in a 1 mM HCl solution. Standard reac-
stability at different storage temperatures. The NFs exhibited tion mixtures containing 1 mL of 22.5 mM H O and different
2
2
dramatically enhanced activity due to these potential reasons: volumes of DA (30 µL through 240 µL) were freshly prepared
1) the high surface area of hNFs, which alleviates mass-trans- and then the same concentrations of 100 μL hNFs and free
fer limitations, (2) cooperative effects due to nanoscale- HRP were separately added to these mixtures. The final
(
entrapped HRP, and (3) favorable HRP conformation in hNFs.
volume of the mixtures was adjusted to 3 mL by the addition
of PBS (0.1 M, pH 6.8). The change in color was visually
observed and the changes in absorbance values were moni-
tored for 50 min at 297 nm at 25 °C using a UV-Vis
spectrophotometer.
2
. Experimental
2
.1 Chemicals and materials
Copper(II) sulfate pentahydrate (CuSO
bovine serum (BSA) (lyophilized powder) and peroxidase from
4
2
·5H O), albumin from
3
. Results and discussion
horseradish (HRP) (lyophilized powder, beige, ∼150 U mg−
1
,
Enzyme Commission (EC) Number 1.11.1.7), guaiacol and Morphology evolution with various reaction parameters and
dopamine hydrochloride were obtained from Sigma-Aldrich. the mechanism underlying enzyme activity and stability in
Salts (NaCl, KCl, Na HPO , KH PO , CaCl ·2H O, MgCl ·6H O) terms of hNF morphology, storage temperature and storage
2
4
2
4
2
2
2
2
were also purchased from Sigma-Aldrich. Ultrapure water (18.2 time were systematically studied. It is well known that reaction
MΩ; Millipore Co., USA) was used in all experiments.
parameters, including precursor concentrations, reaction
temperature and reaction time, can influence the morphology
of all types of nanomaterials and their physical and chemical
2
+
2
.2 Synthesis of HRP–Cu hybrid nanoflowers
The synthesis of hNFs was accomplished using a modified properties. In addition, the precursor concentration ratio has a
3
5
method. First, a 120 mM CuSO
4
stock solution in ultrapure significant impact on the formation of hybrid nanomaterials
4
1–44
water was freshly prepared. Then, 60 µL of CuSO stock solu- by controlling the number of nucleation centers.
In this
4
tion was added to 9 mL of 10 mM PBS solution (pH 7.4), con- report, the hNF formation mechanism is described in terms of
taining 0.02 mg mL− HRP. This final mixture was vigorously several reaction parameters.
1
agitated, and was then left undisturbed for incubation for
We chose HRP for this study, owing to its wide catalytic
7
2 hours at two different temperatures, +4 °C and room temp- activity, high sensitivity and substrate specificity. It has been
erature (RT = 20 °C). After incubation, the growth process of widely used in many applications, including removal of
hNFs was complete and the mixture was centrifuged to obtain phenols from polluted water, organic synthesis, biosensor
4
5–48
a blue-colored precipitate (indication of hNF formation). The design, and clinic and micro-analytical studies.
collected hNF powder was washed at least 3 times with water
3
.1 Preparation and characterization of hybrid nanoflowers
and centrifuged at 12 000 rpm for 20 min in order to remove
unreacted components. Finally, the hNF precipitates were In this synthesis strategy described above, Cu ions were
2
+
dried under vacuum at room temperature.
rationally and successfully combined with HRP to form flower-
like hybrid structures called “hybrid nanoflowers”. The pro-
posed mechanism of hNF formation is illustrated in Fig. 1.
2
.3 Enzyme activity measurement
In all activity and stability measurement experiments, the The hNFs contain three major components: phosphate ions
−
1
2+
2+
hNFs (prepared from 0.02 mg mL HRP and 0.8 mM Cu ) from the PBS solution, Cu ions and HRP enzyme. The syn-
and an identical concentration of free HRP were used. The thesis of hNFs involves three major successive steps: nuclea-
activities of hNFs were determined by colorimetric and spec- tion, growth process and formation. To generate uniform
troscopic methods using guaiacol as a chromogenic sub- hNFs, it is first necessary to understand the nucleation step, in
4
0
2+
strate. In activity measurements with hNFs and free HRP, which Cu ions react with phosphate to create primary copper
a standard protocol (pH 6.8, 0.1 M KH PO , 25 °C) was applied. phosphate complexes. The amine groups in the backbone of
2
4
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2+
Fig. 1 Possible formation mechanism of HRP–Cu hNF divided into three parts: (I) nucleation, (II) growth and (III) completion of the entire hNF for-
mation. Blue dots represent the copper phosphate complex.
HRP preferentially coordinate to copper phosphate to initiate slowly than that of hNFs at RT. In addition, we assume that
the nucleation of nanocrystals. In the growth process, the HRP molecules bind to copper phosphate nanocrystals more
primary nanocrystals were continuously fed with HRP to tightly at +4 °C than at RT. In a further study, the effects of
2
+
produce large petals on the nanocrystals. The key feature of HRP and Cu concentrations on the formation of the hNFs
the growth process is the action of HRP as “glue” to form large were determined. When the concentration of HRP was
assembled branches of HRP (connected to each other), resem- decreased from 0.1 mg mL− to 0.02 mg mL , the hNFs were
1
−1
bling flower petals, on copper phosphate nanocrystals. In the produced more uniformly and monodisperse (Fig. S1A and
2
+
formation, hierarchically and kinetically grown nanocrystals S1B in the ESI†). Furthermore, the concentration of Cu was
reached the saturation level to complete the growth process. As also highly important for initiation of the nanocrystals and the
a consequence, the hNFs were efficiently produced at the end growth process. From our results, we determined that the
2
+
of the anisotropic growth process.
optimal Cu concentration was around 0.8 mM to properly
2
+
The hNFs were characterized using SEM, EDX analysis, form the hNFs. We demonstrated that the Cu ion is an indis-
Bradford assay, UV-Vis spectrometry and XRD. The effect of pensable component and a driving force to form primary
reaction temperatures on the morphology of the hNFs was nanocrystals. For instance, no blue-color precipitates were
2
+
2+
demonstrated with SEM images (Fig. 2). The hNFs synthesized observed without Cu and when the concentration of Cu
2
+
in PBS (pH 7.4) at +4 °C had spherical shapes with diameters was below 80 µM in PBS because the Cu concentrations were
of ∼5.5 µm size (Fig. 2A) while the hNFs synthesized at RT not high enough to reach the point of supersaturation needed
gave blooming structures with ∼6.5 µm in size. We speculate to initiate nucleation and subsequent growth of copper phos-
2
+
that the growth process of the hNFs at +4 °C occurred more phate nanocrystals. In addition, the Cu concentration was
Fig. 2 SEM images of hNFs synthesized at different incubation temperatures (A) +4 °C. Inset: the SEM image of a hNF of (A) with 25.00k× magnifi-
cation, (B) RT. Inset: the SEM image of a hNF of (B) with 25.00k× magnification.
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2
+
also a key for determining the number of Cu binding sites, that HRP molecules were predominately protonated and posi-
2
+
which generate separate petals and are fundamental for the tively charged at pH values below the pI, the presence of Cu
formation of the hNFs. In a typical hNF synthesis, HRP and cations and positively charged HRP molecules resulted in very
2
+
2+
Cu were accordingly mixed in a 10 mM PBS solution contain- strong repulsions between Cu ions and HRP and also among
ing ∼140 mM Cl to form the hNFs. When the Cl− concen- HRP molecules, preventing incorporation of Cu and HRP.
tration was increased 50 fold to ∼7 M, the blue-colored However, formation of hNFs was achieved at pH 6 through pH
precipitates were not observed. We attribute this to the for- 9, as shown in the SEM images presented in Fig. 3.
−
2+
mation of soluble Cu(II) chloride complexes, which prevented
Spherical hNFs were obtained at pH 6 and 8 (Fig. 3A and
the growth of nanocrystals. However, even though blue precipi- B), whereas the shapes were splayed at pH 9 (Fig. 3C) due to
tates were observed at the bottom of the reaction tubes in the repulsion among negatively charged HRP molecules. It is also
−
absence of Cl , no hNFs were formed due to the incomplete noteworthy to mention that the hNFs were not able to form at
growth process, as shown in SEM images in Fig. S1C and S1D pH 10 and above because of strong negative repulsions, which
in the ESI.†
expel petals of HRP rather than forcing them to bind to each
For further morphology evolution study, the NFs were syn- other. Zeta potential measurements were used to determine
thesized at various PBS pH values (pH 6, 8 and 9). The HRP the charge density and change of charge density on the sur-
4
4
isoelectric point (pI) is 5.5, where free HRP is expected to be faces of hNFs synthesized at different pH values. The hNFs
neutral, while above and below this value the protein is at pH 6 were negatively charged with −3.89 mV zeta potential
expected to be negative and positive, respectively. At pH 3, 4 due to the slight deprotonation of HRP. When increasing pH
and 5, no blue colored precipitates were observed. Considering values, the hNFs became more negative. The zeta potentials of
Fig. 3 Formation of the hNFs at different buffer pH values. (A) pH 6, (B) pH 8, and (C) pH 9.
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Fig. 4 (A) EDX analysis of the hNFs, (B) XRD patterns of the hNFs (black line). The peak position of the Cu
3
(PO
4
)
2
·3H
2
O (red line, JCPDS 00-022-
0
548) located for comparison.
hNFs at pH 7.4, 8, and 9 were −9.35 mV, −14.30 mV and +4 °C was 17 595 U mg− , which was ∼300% higher than free
1
−
1
−
19.71 mV, respectively. The elemental composition of the HRP in PBS, where it achieved an activity of 5952 U mg .
hNF powders was analyzed by EDX and peak positions of However, an activity of 9587 U mg− was obtained by the use of
1
Cu (PO ) in the hNFs were analyzed by XRD. Fig. 4A clearly the hNFs synthesized at RT but stored at +4 °C (data not
3
4 2
shows that the weight and atomic percentages of Cu in the shown here). In terms of stability, the hNFs stored at +4 °C
hNFs were about 9.3% and 2.8%, respectively. Fig. 4B also and RT lost only 4% and 20%, respectively, of their initial
reveals that most of the diffraction peaks of Cu (PO ) in the activities within 30 days (Fig. 5C). For comparison, Fig. 5C also
3
4 2
hNFs were consistent with those of JCPDS card (00-022-0548). demonstrates that the same concentration of free HRP stored
However, we realized that some diffraction peaks may belong at +4 °C and RT lost 65% and 92%, respectively, of its initial
to KCuCl and KCuCl crystals.
activity within 30 days. These findings indicate that (1) HRP in
the hNFs is much more stable than free HRP in solution; and
2
3
3
.2 Enzymatic activity and stability measurement of hybrid
(
2) the storage temperature affects the activity and stability of
nanoflowers
both hNFs and free HRP. To corroborate the high stability and
The catalytic activities and stabilities were determined using reusability of these hNFs (synthesized at +4 °C but stored at
guaiacol as a model substrate for hNFs synthesized under the RT), the nanoflowers used for activity measurements after 30
following experimental conditions: (1) at +4 °C and pH 7.4; (2) day storage at RT were stored at RT for two more months.
RT and pH 7.4; (3) +4 °C and pH 6, 8, and 9. In general, HRP, Although these hNFs lost their flower-like shapes and
an oxidoreductase, efficiently catalyzes reactions between the appeared as pressed disc shaped structures (Fig. S2A in the
target compound (H-atom donor) and hydrogen peroxide, ESI†), they exhibited increased activity compared to HRP in
H
2
O
2
(H-atom acceptor). In addition, HRP also catalyzes the solution but their activity was reduced (∼31%) compared to
oxidation of several organic and inorganic substrates in the fresh hNFs (data not shown). The color change of guaiacol by
4
7–50
presence of H O .
In this study, the addition of hNFs to a the oxidation reaction catalyzed by these pressed disc shaped
mixture of guaiacol and H
resulted in efficient and rapid structures was also observed visually (Fig. S2B(iii) in the ESI†).
oxidation of guaiacol to produce the orange colored quinone The mixture of guaiacol and H in PBS is initially colorless
2
2
2 2
O
O
2 2
type structure, 3,3-dimethoxy-4,4-diphenoquinone (Fig. 5A). before addition of hNFs (Fig. S2B(i) in the ESI†). However,
This process allowed us to monitor the activity both visually upon addition of fresh hNFs, the color of the mixture changed
with the naked eye and spectrophotometrically with the to intense dark red (Fig. S2B(ii) in the ESI†).
increase in the absorbance of the product at 470 nm.
To gain further insight into the impact of reaction tempera-
First, hNFs with 98% encapsulation yield (synthesized at ture on catalysis and stability, both formation and storage of
4 °C, pH 7.4) were dissolved in 1 mL of PBS (pH 6.8) and the hNFs were carried out at RT (pH 7.4). Fig. 5D shows that these
+
solution was mixed with guaiacol (45 mM, 1 mL) and H O
hNFs lost 29.9% of their initial activity within 30 days, whereas
2
2
(
22.5 mM, 1 mL) in a reaction tube. The resulting mixture was the hNFs synthesized at +4 °C showed a lower activity loss
divided into two parts, which were stored at two different (20%), as mentioned above. In addition, the oxidation of
temperatures (+4 °C and RT) to determine the influence of guaiacol to colored 3,3-dimethoxy-4,4-diphenoquinone was
storage temperatures (+4 °C and RT) on the catalytic activity spectrophotometrically determined at 470 nm, which allows
and stability of these particular hNFs. Fig. 5B demonstrates us to quantitatively analyze the activity performance of hNFs
that the enhanced activity of hNFs synthesized and stored at and free HRP and to monitor their kinetics. The overall results
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Fig. 5 Activity and stability of nanoflowers and free HRP enzyme towards guaiacol. (A) Formation of the orange quinone type structure “3,3-
dimethoxy-4,4-diphenoquinone” from HRP nanoflower catalyzed reaction of guaiacol with H . (B) Enhanced activity of nanoflowers synthesized
2
O
2
at +4 °C and free HRP measurements. (C) Stability of nanoflowers synthesized at +4 °C measured at +4 °C (blue line) and RT (green line) and free
HRP enzyme measured at +4 °C (purple line) and RT (red line.) (D) Stability of nanoflowers synthesized at +4 °C (green line) and RT (blue line) and
free HRP enzyme (dark red line) measured at RT.
show that the formation temperatures of hNFs play a role in initial activity, whereas those prepared at pH 8 and pH 6 lost
the activity and stability of hNFs. We suggest that the hNFs 16.8% and 21.5% of their initial activities, respectively, within
should be synthesized and stored at +4 °C to obtain the best 30 days. Among hNFs prepared at different pH values, the
activity and stability. The hNFs appeared more compact when hNFs prepared at pH 7.4 showed the best catalytic activity and
prepared at +4 °C and HRP assembled with the favorable con- stability with 4% activity loss within 30 days (previously shown
formation in hNFs and both effects may result in decreased in Fig. 5C) as shown in Fig. S3 in the ESI† for comparison.
mass transfer limitations and induce an enhancement in Their high activity is attributed to the morphology of hNFs
activity and stability.
with a high surface-to-volume ratio and a suitable HRP
Although we proved that hNFs synthesized under particular conformation.
experimental conditions (at +4 °C and pH 7.4) exhibited dra-
3.3 Visual and spectrometric detection of dopamine
matically improved activity and stability, a further study was
carried out to demonstrate the activity performance of the Dopamine (DA), one of the important catecholamine neuro-
hNFs, which were prepared at different pH values. As pre- transmitters, is vital to human life. Controlling its level in the
sented in the SEM images above, the hNFs produced at mammalian central nervous system provides proper signal
different pH values had different shapes and sizes. We believe transport in nerve cells. In particular, low levels of DA are
that these morphological differences substantially affect activi- associated with several serious neurological diseases, such as
4
4,51
ties and stabilities of hNFs. Accordingly, hNFs were syn- schizophrenia, Huntington’s disease and Parkinsonism.
thesized at various pH values at +4 °C (pH 6, 8 and 9) and Inspired by the excellent catalytic activity of hNFs, we used
stored at +4 °C. The hNFs prepared at pH 9 lost 11% of their them as sensors to detect DA by oxidation. We selected the
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activities and stability of a series of hNFs synthesized under
different conditions were evaluated, and the results showed
that the hNFs exhibited dramatically enhanced activity and
stability compared to those of free HRP. We determined that
the hNFs obtained from 0.02 mg mL− HRP in a PBS solution
1
(
at +4 °C and pH 7.4) exhibited the best activity and stability
within 30 days of storage at +4 °C using guaiacol as a model
substrate in the presence of H O . Furthermore, the hNFs were
2
2
also successfully applied as sensors for visual and spectro-
metric detection of the oxidation of dopamine in the presence
of H O to produce an orange colored quinone-type product.
2
2
Author contribution
B.S. performed experiments. I.O. and N.Ö. conceived the origi-
Fig. 6 Kinetics of oxidation of dopamine by HRP nanoflowers and free nal idea and I.O. and N.Ö. supervised the project. I.O., N.Ö.
HRP enzyme. Inset: color changes in dopamine with respective concen- and M.H. designed the project. All authors contributed to the
trations (10, 20, 40, 80 µg mL− ).
1
manuscript writing.
Conflict of interest
hNFs synthesized at +4 °C in 10 mM PBS (pH 7.4) because of
their excellent catalytic activity for the oxidization of DA in the
presence of H to an orange colored quinone-type product.
The authors declare no financial interest.
2 2
O
The oxidation of DA was visually and spectrometrically
detected as shown in Fig. 6. The hNFs catalyzed highly rapid
and efficient DA oxidation compared to free HRP. In the stan-
dard oxidation procedure, a specified amount of the hNFs was
mixed with a series of DA concentrations (10, 20, 40, 80 µg
Acknowledgements
We thank Menekşe Sarihan at the Erciyes University Nano-
technology Research Center for assistance with SEM and EDX
measurements. We also appreciate Fatma Kilic Dokan at the
Erciyes University Technology Research and Implementation
Center for assistance with XRD measurements. This work was
supported by a grant from the Erciyes University Scientific
Research Office (FBG-2014-5376)2014-2016. We appreciate the
comments on the manuscript by Dr Basri Gulbakan as well as
the suggestions of Colonel Savas Karatepe.
−
1
mL ) in 10 mM PBS buffer (pH 6.8) and absorbance changes
were monitored by UV-Vis spectrometry at 485 nm, as well as
by visual observation. The color intensified as the concen-
tration of DA was increased. In addition, as the concentration
−1
−1
of DA was raised from 10 µg mL to 80 µg mL , the hNF
activity also increased. As a result, the oxidative activities of
the hNFs were much higher than that of free HRP at all DA
concentrations. It is known that KM (Michaelis constant) is an
important characteristic feature for an enzyme and it shows
the affinity of the enzyme to the substrate and the robustness
of the enzyme–substrate complex. For this reason, the KM References
values of free HRP and hNFs were determined as 0.059 and
1
J. Kim, J. W. Grate and P. Wang, Trends Biotechnol., 2008,
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R. Mesing, Immobilized Enzymes for Industrial Reactors,
Academic Press, 1995.
0
.031 mM, respectively via a plot of 1/V versus 1/[S] by the
method of Lineweaver and Burk towards dopamine. The lowest
of the hNFs dictates that they have enough specificity and
efficiency to react with dopamine compared to free HRP.
2
2
K
M
5
3
4
5
4
. Conclusion
In conclusion, we have investigated the effects of reaction para-
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2
+
hNFs composed of Cu ions and HRP enzyme. We experi-
2
+
mentally explained the impacts of the concentrations of Cu
−
ions, Cl ions and HRP enzyme, the pH values of PBS, and the
reaction temperature on the formation of hNFs. The catalytic
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