The surface of ZnO nanocrystal was functionalized with the amino groups using 3-aminopropyltriethoxysilane APTES and tetraethyl orthosilicate TEOS [25].. Surface modification of ZnO nano
Trang 1N A N O E X P R E S S Open Access
Effect of substrate (ZnO) morphology on enzyme immobilization and its catalytic activity
Yan Zhang1, Haixia Wu1*, Xuelei Huang2, Jingyan Zhang2and Shouwu Guo1*
Abstract
In this study, zinc oxide (ZnO) nanocrystals with different morphologies were synthesized and used as substrates for enzyme immobilization The effects of morphology of ZnO nanocrystals on enzyme immobilization and their catalytic activities were investigated The ZnO nanocrystals were prepared through a hydrothermal procedure using tetramethylammonium hydroxide as a mineralizing agent The control on the morphology of ZnO nanocrystals was achieved by varying the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system The surface of as-prepared ZnO nanoparticles was functionalized with amino groups using
3-aminopropyltriethoxysilane and tetraethyl orthosilicate, and the amino groups on the surface were identified and calculated by FT-IR and the Kaiser assay Horseradish peroxidase was immobilized on as-modified ZnO
nanostructures with glutaraldehyde as a crosslinker The results showed that three-dimensional nanomultipod is more appropriate for the immobilization of enzyme used further in catalytic reaction
Introduction
The composition, size, and surface characteristics of
sub-strates have been considered as the important factors that
affect the physical/chemical properties of the immobilized
enzymes [1-3] However, for conventional bulk solid
sub-strates, such as glass slides [4], polymer monoliths [5,6],
and silica beads [3], the control on their morphologies and
functionalizing their surfaces are usually laborious
Nanos-caled materials, such as metal [7] and metal oxide
nano-spheres [8], carbon nanotubes [9,10], graphene oxide
nanosheets [11], have also been utilized as the substrates
for enzyme immobilization It has been demonstrated that
the size of nanostructured materials might play significant
roles to regulate the catalytic activity of the immobilized
enzymes [3] The preparation of nanostructured materials
with controlled chemical composition, size, morphology,
and the surface functionalization have witnessed great
pro-gressive achievements during the last two decades [12,13]
However, the systematic study of the effects of the
mor-phology of the nanoscale substrates on the enzyme
immo-bilization remains to be expanded ZnO nanocrystals have
unique physical/chemical properties and pronounced bio-compatibility, which are beneficial for many practical applications In addition, a variety of nanostructures of ZnO, such as nanospheres [14], nanowires [15], nanorods [16], nanonails [17], nanotubes [18], nanotetrapods [19], nanotablets [20], and nanoflowers [21], have been pre-pared successfully Therefore, ZnO nanocrystals are ideal materials to study the effect of the morphology of the sub-strate on the catalytic efficiency of the immobilized enzymes Horseradish peroxidase (HRP) was used as a model enzyme because it has been wildly studied and used
in many fields, such as organic syntheses [22], phenol removal [23], biosensor, and drug delivery [24]
In this study, we report the effects of the morphology
of the ZnO nanocrystals on the enzyme immobilization ZnO nanocrystals with different morphologies, including nanosphere, nanodisk, and nanomultipod, were fabri-cated simply through a hydrothermal procedure The surface of ZnO nanocrystal was functionalized with the amino groups using 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) [25] Glutar-aldehyde was used as a crosslinker to immobilize the HRP enzyme molecules on the surface of as-modified ZnO nanocrystals Then the enzyme loading and catalytic activity were evaluated
* Correspondence: haixiawu@sjtu.edu.cn; swguo@sjtu.edu.cn
1
National Key Laboratory of Micro/Nano Fabrication Technology, Key
Laboratory for Thin Film and Microfabrication of the Ministry of Education,
Research Institute of Micro/Nano Science and Technology, Shanghai Jiao
Tong University, Shanghai 200240, PR China
Full list of author information is available at the end of the article
© 2011 Zhang et al; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,
Trang 2Results and discussion
ZnO nanocrystals with different morphology
Figure 1 shows the SEM images of ZnO nanocrystals
with different morphologies The nanospheres, with
dia-meter of approximately 30 nm, were obtained when pure
methanol was used as the hydrothermal reaction solvent
as shown in Figure 1a When the ratio of methanol to
water was adjusted to 1:1, ZnO nanodiscs, approximately
85 nm in width and 25 nm in thickness, were acquired as
shown in Figure 1b Figure 1c shows the ZnO
nanomulti-pods composed of several rods of 100 nm in diameter
and 200 nm in length when the methanol/water ratio
reaches 1:9 This illustrates that the ratio of methanol/
water is certainly a crucial factor to control on the
mor-phology of ZnO nanocrystals Although the
morpholo-gies of aforementioned ZnO nanocrystals are different,
the XRD patterns are all well consistent with the
stan-dard wurtzite ZnO structure as shown in Figure 2 Thus,
the effect of crystal form on the surface modification and
enzyme immobilization can be neglected
Surface modification of ZnO nanocrystals
In order to immobilize enzyme, the surface of ZnO
nano-crystals were functionalized with amino groups using
APTES/TEOS Figure 3a shows a typical image of coated
ZnO nanodiscs, where a thin film with uniform thickness
of 2 nm formed on the surface Comparing the
Zeta-potentials of the bare ZnO and the as-modified ZnO
nanocrystals (Figure 3b), it can be deduced that the
sur-face electrostatic state of ZnO nanocrystals was changed
The surface groups of the modified ZnO were further
characterized by FT-IR spectra Through comparing the
FT-IR spectra before and after modification in Figure 3c,
except for a few peaks at 3430, 1630, and 433 cm-1
corre-sponding to water (moisture) and ZnO nanocrystal, the
peaks at 2936.3 and 2872 cm-1of the C-H stretching
vibration [26], and the peaks at 1330 and 1560 cm-1of the stretching vibration of C-N and bending vibration of N-H can be found In addition, the strong absorption peaks at 3428.6 and 1633.5 cm-1, assigned to N-H bend-ing vibrations, are overlapped with the bendbend-ing vibration
of the absorbed H2O [27] These results confirmed the presence of amino groups on the ZnO nanocrystals surface
The amount of amino groups and the thickness of the coating layer can be controlled by adjusting the ratio of TEOS to APTES When the ratio of TEOS to APTES was 1:1, a coating layer of approximately 2 nm can be generated on the surface of ZnO nanocrystal, but, at the same time, lots of isolated SiO2 nanocrystals were formed, as shown in Figure 4a When the ratio of TEOS
to APTES was 1:4, a layer with uniform thickness of about 2 nm was formed as shown in Figure 4b When the ratio of TEOS to APTES was decreased to 1:10, no fully covered coating layer can be generated, as shown
in Figure 4c According to the standard curve of glycine obtained by Kaiser Assay, the amount of amino groups
on the surface of ZnO nanocrystals was deduced When the ratios of TEOS to APTES were 1:1, 1:4, and 1:10 used for the surface modification, the amounts of amino groups on the surface of ZnO nanodisks were 0.03, 0.07, and 0.02 mmol/g, respectively These results show that the ratio of TEOS to APTES used for the surface modi-fication determines the uniformity of the coating layer
as well as the amount of amino groups
The aforementioned procedure was also performed on the surface modifications of ZnO nanospheres and nano-multipods with TEOS to APTES ratio of 1:4 Figure 5 depicts the TEM images of the nanosphere and nanomul-tipod after the modification Similar to the nanodisks, there are thin coating layers formed both on nanospheres and nanomultipods The amounts of amino groups on
Figure 1 SEM images of ZnO nanocryatals (a) Nanospheres, (b) nanodisks, and (c) nanomultipods prepared using mixtures of methanol and water with different volume ratios of 10:0, 1:1 and 1:9 as solvents, respectively.
Trang 3the surfaces of ZnO nanospheres and nanomultipods
modified with TEOS to APTES ratio of 1:4 were also
measured, which are 0.127 and 0.044 mmol/g,
respec-tively The specific surface areas of ZnO nanospheres,
nanodisks, and nanomultipods are 33.59, 11.99, and
11.85 m2/g, respectively, which were measured using
Brunauer-Emmett-Teller (BET) method Thus,
consider-ing the surface areas of different morphologies, the
sur-face densities of amino groups on ZnO nanospheres,
nanodisks, and nanomultipods were calculated, which
are determined to be 3.78, 5.94, and 3.71 μmol/m2
, respectively
The effects of morphologies of ZnO nanocrystals on HRP
immobilization and their activity
The two aldehyde groups (-COH) of glutaraldehyde can
bond separately to the amino groups of HRP and
as-modi-fied ZnO [28], and thus, glutaraldehyde was used as a
crosslinker to immobilize HRP molecules on the modified
ZnO nanocrystal surfaces As shown in Figure 6, the
high-est loadings of HRP on the ZnO nanospheres, nanodisks
and nanomultipods were 0.094, 0.275, 0.240 mg/m2,
respectively From Figure 6, we find that the
immobiliza-tion of HRP on ZnO nanomultipods can reach the highest
loading at the lowest ratio of glutaraldehyde to amino
groups The maximum loading of HRP on ZnO
nanomul-tipods was higher than that on the nanospheres, but, as
high as that on the nanodisks, even if the surface density
of amino groups on ZnO nanomultipods was relatively
lower than that of the other two
The catalytic activity of the HRP immobilized on
dif-ferent ZnO nanocrystals was assayed through phenol
oxidation reaction Soluble HRP was also characterized
as a control Their kinetic parameters were obtained from the Lineweaver-Burk equation, and the data are summarized in Table 1 1/Km, which express the affinity
of phenol compounds to HRP, and the Kmof the HRP immobilized on ZnO nanospheres, nanodisks and nano-multipods were tagged as K (s), K (d), and K (m),
Figure 2 XRD patterns of ZnO nanocryatals (a) Nanospheres, (b)
nanodisks, and (c) nanomultipods.
Figure 3 The surface functionality of ZnO nanodisks before and after modification (a) The TEM images of ZnO modified by TEOS:APTES (1:4 in volume) (b) Zeta-potential curves, and (c) FT-IR spectra of ZnO nanodisks before and after modification.
Trang 4respectively Table 1 shows that Km(s) was unexpectedly
higher than that of the free HRP, while Km(d) and Km
(m) are close to that of free HRP suggesting that the
substrate affinity to the HRP was not affected by the
immobilization on these two ZnO materials Thus, we
can assume the morphology of the ZnO nanocrystals
must be an important fact that affects the affinity of
phenol compounds to HRP
The catalytic property of the immobilized HRP was
further characterized by the catalytic efficiency (Kcat/Km)
The catalytic efficiency of immobilized HRP was much
lower than that of free HRP, which coincide with the
report of the literature [29] As shown in Table 1, the
cata-lytic efficiency values of immobilized HRP on the
nanospheres, nanodisks and nanomultipods were 0.78, 1.09, 1.28 mM-1s-1, respectively Through comparing the
Kcat/Km value of the HRP immobilized on three ZnO nanocrystals with different morphologies, the nanomulti-pod ZnO nanocrystals are apparently favorable for enzyme immobilization
Those all may due to the three dimensional structural feature of nanomultipods, which could affect the enzyme interaction with the immobilized substrate and conforma-tion of the enzyme, and result in increasing the enzyme loading on the solid substrate and catalytic efficiency Because the nanomultipods have several pods, and the spaces among the pods are limited, the glutaraldehyde is difficult to self-polymerize, and the amino groups on the
Figure 4 TEM images of amino group modified ZnO nanodisks (a-c) TEM images of modified ZnO nanodisks with different TEOS:APTES ratios of 1:1, 1:4, and 1:10.
Figure 5 TEM images of amino group-functionalized ZnO nanocrystals (a) Nanospheres and (b) nanomultipods using TEOS and APTES with the ratio of 1:4 in volume.
Trang 5multipods are more efficiency to immobilize HRP While,
nanospheres and nanodisks have opened spaces,
glutaral-dehyde tends to polymerize Thus, HRP loadings on
nano-spheres and nanodisks need more glutaraldehyde to reach
the maximum loadings Due to the opened structure and
lower surface density of amino groups, the loading of HRP
on nanospheres is lowest Thus, HRP loadings on
nano-spheres and nanodisks need more glutaraldehyde to reach
the maximum loadings Compared with nanodisks and
nanospheres, the HRP loading on ZnO nanomultipods
reached the highest when its glutaraldehyde to amino
groups ratio was lower, which indicates that every HRP
molecule needs less glutaraldehyde to immobilize HRP on
the surface of ZnO nanocrystal and less conformation
happened Therefore, the 3D structure of nanomultipods
and the lowest ratio of Nglutaraldehyde/N-NH2may result in
the stabilization of HRP immobilized on nanomultipods,
which indicates that the 3D nanomultipod is more
appro-priate for the immobilization of enzyme and its catalytic
efficiency
Conclusions
ZnO nanocrystals with different morphologies, including
nanosphere, nanodisk, and nanomultipod, were prepared
via hydrothermal reactions using the mixtures of
metha-nol/water with different volume ratios The surfaces of
ZnO nanocrystals were modified with amino groups using TEOS and APTES to study the effect of the mor-phology of the materials to the enzyme immobilization The surface density of the amino groups and the thick-ness of coating were controlled by tuning the ratio of TEOS to APTES It was demonstrated when the ratio of TEOS to APTES was 1:4, a layer of uniform thickness of approximately 2 nm can be generated on surface of the ZnO nanocrystals
HRP molecules were immobilized on the modified ZnO nanocrystal surfaces using glutaraldehyde as a crosslinker
It was illustrated that the enzyme loading on the ZnO nanostructure was in the order of nanospheres < nano-multipod < nanodisks, while the nanonano-multipod reached the highest loading when its glutaraldehyde to amino groups ratio was lower than the other two, causing less conformation change of HRP on the ZnO surface, leading
to a higher catalytic efficiency In brief, the 3D nanomulti-pod is more appropriate for the immobilization of enzyme and for being used in catalytic reaction than the other two, which has great implications for the many ongoing studies
of enzyme immobilization and applications of the immobi-lized enzymes
Methods
Materials
Zn(Ac)2·2H2O, (CH3)4NOH (25%), Glutaraldehyde (25%), phenol (99%), 4-aminoantipyrine (4-AAP), methanol, and
H2O2(30%) were purchased from Sinopharm Chemical Reagent Company, Shanghai, China APTES (≥98.0%) was bought from Sigma-Aldrich, USA TEOS was obtained from Linfeng Chemical Reagent Company, Shanghai, China HRP was purchased from Majorbio Biotech Com-pany, USA All reagents were used as-received
Synthesis of ZnO nanocrystals
The ZnO nanocrystals were prepared through a thermal procedure using tetramethylammonium hydro-xide as a mineralizing agent The control on the morphology of ZnO nanocrystals was achieved by vary-ing the ratio of CH3OH to H2O, which were used as solvents in the hydrothermal reaction system In a typi-cal experiment, Zn(Ac)2·2H2O (5 mmol) was dissolved
in 15 mL of the mixture of methanol and water with different volume ratios in a flask under vigorous stirring Then, 15 mL of (CH3)4NOH was dropped into the flask
Figure 6 The enzyme loadings on different morphologies of
ZnO nanocrystals The loadings of HRP with different ratios of
glutaraldehyde and amine groups on the surface of the modified
ZnO nanocrystals.
Table 1 The loadings and kinetic properties of the HRP immobilized on ZnO nanocrystals
sample Enzyme loading (mg/m 2 ) K m (mM) K cat / K m (mM -1 s -1 )
Trang 6as mineralizing agent The as-obtained turbid
suspen-sion was transferred into a Teflon inner reactor of
stain-less-steel autoclave The autoclave was heated at 200°C
for 24 h for the hydrothermal reaction The reaction
solution was then cooled down to room temperature
and the solid product was separated from the reaction
mixture by centrifugation (4000 rpm), and was washed
with ethanol and water alternately, each for three times
Characterization of the ZnO nanocrystals
The size, morphology, BET surface area, and crystallinity
of the ZnO nanocrystals before and after the surface
modification were characterized using scanning electron
microscope (Zeiss ultra 55, Germany), transmission
elec-tron microscopy (TEM) (JEM-2100, Japan), accelerated
surface area, porosimetry system (Micromeritics ASAP
2010 M+C, USA), and X-ray powder diffraction (XRD)
(BRUKER-AXS, Germany) The surface functionalities of
the ZnO nanocrystals after the modification were
stu-died using FT-IR with a Nicolet 5700 Fourier transform
infrared spectrometer (Thermo Electron, USA), and
Zeta potentials obtained on Nicomp 380/ZLS (America)
Surface functionalization of ZnO nanocrystals
A typical surface functionalization process was as
follows
In general, 100 mg of ZnO nanocrystals was suspended
into 20 mL of ethanol (pH = 10.8) under sonication
Then, 15μL of TEOS and 60 μL of APTES were added
into the ethanol solution, and the mixture was stirred for
5 h The solid product was filtered and washed with
etha-nol, and then dried at room temperature The amounts of
amino groups on the surface of ZnO nanocrystals were
measured by Kaiser Assay At first, the standard curve of
glycine obtained by Kaiser Assay Glycine were mixed
with 2 mL of acetate buffer (0.6 M, pH = 4.5) and 2 mL
of 10 mg/mL ninhydrin ethanol solution The mixture
was treated at 90°C for 20 min After centrifugation at
10,000 rpm for 3 min, the amino groups’ concentration
in the supernatant was monitored spectrophotometrically
565 nm, and then, 10 mg of modified ZnO was utilized
to monitor spectrophotometrically as glycine Then, the
amount of amino groups was calculated based on the
standard curve of glycine
100 mg modified ZnO was suspended into 20 mL pH
7.0 0.1 M phosphate buffers, and then 750 μL of 25%
glutaraldehyde solution was added to the mixture The
mixture was incubated at room temperature, 200 rpm
for 2 h After filtration and washing with the phosphate
buffer, the samples were utilized to immobilize HRP
HRP immobilization
For HRP immobilization, 100 mg of functionalized ZnO
nanocrystals was added into 1 mL, 0.1 M, and pH 7.0 of
potassium phosphate buffer containing 100 μg of HRP The mixture was incubated at 4°C for 2 h with 240 rpm shaking, and then centrifuged at 10,000 rpm for 3 min The supernatant was collected The sediments were cen-trifuged and rinsed alternately three times with 0.1 M,
pH 7.0 phosphate buffer solution to remove non-specifi-cally adsorbed enzymes The solid was stored at 4°C for further measurements The supernatants were employed
to determine the enzyme loading on modified ZnO
Characterization of the immobilized HRP
The enzyme loading is obtained by subtracting the amount of the left HRP in the supernatant from the total HRP added Free HRP and the immobilized HRP activity was assayed by colorimetric method using 4-AAP as pre-viously described [2] In brief, the immobilized enzyme was added into 1 mL of 0.1 M, pH 7.0, phosphate buffer which contained 60 mM of phenol, 14.38 mM of 4-AAP, and 1.21 mM of hydrogen peroxide, and then reacted at 30°C for 3 min The initial catalytic reaction rates of the enzyme in the supernatant and the immobilized enzyme were determined by measuring the UV absorbance of the reaction mixture at 510 nm The double reciprocal plots
of the rates and substrate concentrations were plotted to obtain Kmand Kcat according to the Lineweaver-Burk equation
Abbreviations 4-AAP: 4-aminoantipyrine; APTES: aminopropyltriethoxysilane; BET: Brunauer-Emmett-Teller; HRP: horseradish peroxidase; TEM: transmission electron microscopy; TEOS: tetraethyl orthosilicate; XRD: X-ray powder diffraction; ZnO: zinc oxide.
Acknowledgements This study was financially supported by the National “973” Program (Nos 2007CB936000 and 2010CB933900), the NSFC (No 20774029 and
No 20906055) of China, the State key laboratory of bioreactor engineering (No 2060204), and China postdoctoral science foundation
(No 20100470131).
Author details 1
National Key Laboratory of Micro/Nano Fabrication Technology, Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, PR China 2 State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, 200237, PR China
Authors ’ contributions
YZ conducted the experiments and performed data analysis XH helped in designing the immobilized enzyme test JZ participated in the design and helped in compiling the data of immobilization enzyme interpretation HW helped during the most of operation and data interpretation of analytic equipments used SG conceived basic idea of this technique and supported the organization of this article.
Competing interests The authors declare that they have no competing interests.
Received: 24 March 2011 Accepted: 13 July 2011 Published: 13 July 2011
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doi:10.1186/1556-276X-6-450 Cite this article as: Zhang et al.: Effect of substrate (ZnO) morphology
on enzyme immobilization and its catalytic activity Nanoscale Research Letters 2011 6:450.
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