A new route to the considerable enhancement of glucose oxidase (GOx) activity: The simple assembly of a complex from CdTe quantum dots and GOx, and its glucose sensing

Article abstract of DOI:10.1002/chem.200800681

A new complex consisting of CdTe quantum dots (QDs) and glucose oxidase (GOx) has been facilely assembled to achieve considerably enhanced enzymatic activity and a wide active temperature range of GOx; these characteristics are attributed to the conformational changes of GOx during assembly. The obtained complex can be simultaneously used as a nanosensor for the detection of glucose with high sensitivity. A mechanism is put forward based on the fluorescence quenching of CdTe QDs, which is caused by the hydrogen peroxide (H ₂O₂) that is produced from the GOx-catalyzed oxidation of glucose. When H₂O₂ gets to the surface of the CdTe QDs, the electrontransfer reaction happens immediately and H₂O₂ is reduced to O₂, which lies in electron hole traps on CdTe QDs and can be used as a good acceptor, thus forming the nonfluorescent CdTe QDs anion. The produced O₂ can further participate in the catalyzed reaction of GOx, forming a cyclic electrontransfer mechanism of glucose oxidation, which is favorable for the whole reaction system. The value of the Michaelis-Menton constant of GOx is estimated to be 0.45 mML^-1, which shows the considerably enhanced enzymatic activity measured by far. In addition, the GOx enzyme conjugated on the CdTe QDs possesses better thermal stability at 20-80°C and keeps the maximum activity in the wide range of 40-50°C. Moreover, the simply assembled complex as a nanosensor can sensitively determine glucose in the wide concentration range from micro- to millimolar with the detection limit of 0.10 μM, which could be used for the direct detection of low levels of glucose in biological systems. Therefore, the established method could provide an approach for the assembly of CdTe QDs with other redox enzymes, to realize enhanced enzymatic activity, and to further the design of novel nanosensors applied in biological systems in the future.

Full text of DOI:10.1002/chem.200800681

FULL PAPER
DOI: 10.1002/chem.200800681
A New Route to the Considerable Enhancement of Glucose Oxidase
GOx) Activity: The Simple Assembly of a Complex from CdTe Quantum
Dots and GOx, and Its Glucose Sensing
(
[
a]
Lihua Cao, Jian Ye, Lili Tong, and Bo Tang*
Abstract: A new complex consisting of
CdTe quantum dots (QDs) and glucose
oxidase (GOx) has been facilely assem-
bled to achieve considerably enhanced
enzymatic activity and a wide active
temperature range of GOx; these char-
acteristics are attributed to the confor-
mational changes of GOx during as-
sembly.The obtained complex can be
simultaneously used as a nanosensor
for the detection of glucose with high
sensitivity.A mechanism is put forward
based on the fluorescence quenching of
CdTe QDs, which is caused by the hy-
drogen peroxide (H O ) that is pro-
in electron hole traps on CdTe QDs
and can be used as a good acceptor,
thus forming the nonfluorescent CdTe
QDs anion.The produced O 2 can fur-
ther participate in the catalyzed reac-
tion of GOx, forming a cyclic electron-
transfer mechanism of glucose oxida-
tion, which is favorable for the whole
reaction system.The value of the Mi-
chaelis–Menton constant of GOx is es-
stability at 20–808C and keeps the
maximum activity in the wide range of
40–508C.Moreover, the simply assem-
bled complex as a nanosensor can sen-
sitively determine glucose in the wide
concentration range from micro- to
millimolar with the detection limit of
0.10 mm, which could be used for the
direct detection of low levels of glucose
in biological systems.Therefore, the es-
tablished method could provide an ap-
proach for the assembly of CdTe QDs
with other redox enzymes, to realize
enhanced enzymatic activity, and to
further the design of novel nanosensors
applied in biological systems in the
future.
À1
timated to be 0.45 mmL , which shows
the considerably enhanced enzymatic
activity measured by far.In addition,
the GOx enzyme conjugated on the
CdTe QDs possesses better thermal
2
2
duced from the GOx-catalyzed oxida-
tion of glucose.When H O gets to the
2
2
Keywords: enzymes
·
glucose
surface of the CdTe QDs, the electron-
transfer reaction happens immediately
and H O is reduced to O , which lies
oxidase · nanomaterials · quantum
dots · sensors
2
2
2
Introduction
logical properties of biomolecules conjugated on nanomate-
rials may be regulated by the way of assembly.In particular,
enzymes, as important biomacromolecules, play a prominent
role in biological reaction systems.Thus, the research on the
improvement of enzymatic properties including activity, sta-
bility, and active temperature range has attracted increasing
In recent years, the assembly of nanomaterials and biomo-
lecular complexes as nanosensors for biological analyses and
applications has become a hot research field with the devel-
opment of inorganic–biological hybrids such as nanostruc-
[1–9]
[1,2]
ture-conjugated DNA, proteins, and enzymes.
The bio-
attention.
Nanomaterials, with their interesting properties such as
large surface-to-volume ratio, high catalytic efficiency, and
high surface reaction activity have been favorably adopted
as potential materials to play a catalytic role in enzyme-
[
a] Dr.L.Cao, J.Ye, L.Tong, Prof.B.Tang
College of Chemistry, Chemical Engineering and Materials Science
Engineering Research Center of Pesticide and Medicine Intermediate
Clean Production, Ministry of Education
Key Laboratory of Molecular and Nano Probes
Ministry of Education
Shandong Normal University, Jinan 250014 (China)
Fax : (+86)531-8618-0017
E-mail: tangb@sdnu.edu.cn
[10–12]
based biosensing systems.
The increased surface area of
nanoparticles can provide a better matrix for the immobili-
zation of enzymes, and then enables larger amounts of en-
zymes to immobilize on the particles.The multipoint combi-
nation of enzyme molecules to nanoparticle surfaces can im-
prove the enzyme–substrate interaction effectively by avoid-
ing the potential aggregation of free enzymes, which would
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200800681.
Chem. Eur. J. 2008, 14, 9633 – 9640
ꢀ 2008 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
9633

[
13–15]
lead to the enhancement of enzymatic activity.
In addi-
detection of lower levels of glucose in biological systems.
Therefore, the established method could provide an ap-
proach for the assembly of CdTe QDs with other redox en-
zymes, to realize enhanced enzymatic activity, and to further
the design of novel nanosensors applied in biological sys-
tems in the future.
tion, the enzyme attached on nanomaterial surfaces can
reduce protein unfolding and turbulence, and thus ensure
[
16]
the enhanced stability of enzymes in solution. So, much
effort has been made to develop different materials to be
used as assembled matrices for enzymes, including magnet-
ite nanoparticles, gold nanoparticles (AuNPs), titania sol–gel
membranes, carbon nanotubes, and other oxides such as
[17–21]
ZrO2.
Of these, the materials widely used for investiga-
Results and Discussion
tion of the activity and stability of enzymes are AuNPs and
magnetite nanoparticles.At present, AuNPs have been iden-
tified as a suitable biocompatible material and have been
used to improve the enzymatic activity favorably in enzyme-
Design of the CdTe QDs–GOx complex: Our strategy for
designing the nanocomplex is based on the facile covalent
conjugation between CdTe QDs and GOx.The obtained
CdTe QDs–GOx can be used as a nanosensor for simultane-
ous assay of GOx enzymatic activity, thermal stability, and
glucose sensing.A mechanism is put forward based on the
fluorescence quenching of CdTe QDs, which is caused by
the hydrogen peroxide (H O ) that is produced from the
[22]
based analytical systems. The studies of different enzymes
assembled onto magnetite nanoparticle surfaces have also
demonstrated the potential of enhancing the enzymatic ac-
[23,24]
tivity.
However, the investigation of the enhanced activ-
ity, stability, and active temperature range of enzymes is still
limited to an initial stage.
2
2
GOx-catalyzed oxidation of glucose.When H O gets to the
2
2
Glucose, an important bioactive substance, plays a promi-
nent role in the natural growth of cells.Therefore, it is nec-
essary to regulate the glucose levels in biological systems.
surface of the CdTe QDs, the electron-transfer reaction hap-
pens immediately and H O is reduced to O , which lies in
electron hole traps on CdTe QDs and can be used as a good
acceptor, thus forming the nonfluorescent CdTe QDs anion.
2
2
2
[25]
At present, glucose oxidase (GOx) has been widely em-
ployed in the determination of glucose based on the
enzyme-catalyzed oxidation mechanism using optical and
The produced O can further participate in the catalyzed re-
2
action of GOx, forming a cyclic electron-transfer mechanism
of glucose oxidation, which is favorable for the whole reac-
tion system, as is shown in Scheme 1.
[26–28]
electrical methods.
However, the direct sensing of glu-
cose with high sensitivity accompanied by the considerably
enhanced activity of GOx has
not yet been realized.Quantum
dots (QDs), owing to their
good optical characteristics,
high catalytic effects, and high
electron-transfer
efficiency,
have been used as a preferable
material in enzyme-based bio-
logical analyses and applica-
[29,30]
tions.
have been reported to monitor
biocatalytic transformations
For example, QDs
Scheme 1.The structure of the new assembled CdTe QDs–GOx complex and a schematic illustration of its glu-
cose sensing principles.
and to probe the activities of
polymerase, telomerase, and ty-
[31,32]
rosinase.
Herein, we have
made a new assembly of CdTe QDs and the GOx complex,
and the obtained CdTe QDs–GOx can be used as a nano-
sensor for simultaneous assay of GOx enzymatic activity,
thermal stability, and glucose sensing.Our results indicate
that the lower value of the Michaelis–Menton constant of
Characterization of the CdTe QDs–GOx complex: In our
study, the CdTe QDs–GOx complex was assembled by using
coupling reagents, followed by purification with an ultrafil-
tration membrane, and was characterized by excitation and
fluorescence spectra, and TEM and confocal fluorescence
images, which are shown in Figures 1, 2, and 3.
À1
GOx is estimated to be 0.45 mmL , which shows the con-
siderably enhanced enzymatic activity measured by far.In
addition, the GOx enzyme conjugated on the CdTe QDs
possesses better thermal stability at 20–808C and keeps the
maximum activity in the wide range of 40–508C.The confor-
mation of GOx has been demonstrated to change greatly
during the assembly process.Moreover, the simply assem-
bled nanosensor can sensitively determine glucose over a
wide concentration range from micro- to millimolar with a
detection limit of 0.10 mm, which could be used for the direct
From the experimental results in Figure 1, the maximum
excitation peak of the CdTe QDs redshifts from l=336 to
340 nm and the maximum fluorescence emission peak red-
shifts from 520 to 525 nm after its conjugation with GOx,
which is attributed to the increase of the final size of the
CdTe QDs–GOx complex after surface modification of GOx
with the larger molecular weight. In addition, as can be
seen from the TEM (Figure 2) and confocal fluorescence
images (Figure 3) of CdTe QDs before and after conjugation
[9]
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Enhancement of Glucose Oxidase Activity
FULL PAPER
plex was confirmed by circular dichroism (CD) spectra.As
can be seen from Figure 4, the CD spectra of GOx and the
CdTe QDs–GOx complex are similar to each other, indicat-
ing that GOx retains its biological activity after conjugation.
In addition, the CD spectral peak of CdTe QDs–GOx be-
comes weaker because of removing excess GOx in the con-
jugation.And it also proves that ultrafiltration is a practical
way of purifying the CdTe QDs–GOx complex.The data
from the CD spectra reveal that the tertiary structure of
[33]
GOx remains mostly intact.
Confirmation of the mechanism based on the fluorescence
quenching of CdTe QDs: It is well known that d-glucose
can be oxidized quickly to gluconic acid and H O in the
2
2
Figure 1.The excitation spectra and fluorescence spectra observed from
CdTe QDs (a and b, respectively) and the CdTe QDs–GOx complex (c
and d, respectively).Solutions were prepared in PBS buffer (10 m m,
pH 7.4).
[26–28]
presence of GOx.
To illustrate the mechanism of the
fluorescence quenching of the CdTe QDs–GOx nanosensor
after the introduction of glucose, catalase, which is a scav-
enger of H O , was used in the reactive system.Figure 5
2
2
shows that the addition of cata-
lase to the nanosensor solution
before glucose sensing blocks
the decrease in the fluorescence
and does not produce any influ-
ence on the fluorescence of the
nanosensor.In addition, control
experiments indicate that d-glu-
cose by itself does not affect
the fluorescence of the CdTe
QDs.These results demonstrate
that H O generated by bioca-
2
2
talysis of GOx indeed results in
fluorescence quenching.The
precise mechanism that stimu-
lates the decrease in the fluo-
rescence of CdTe QDs–GOx is
based on the electron-transfer
reaction that occurs on the sur-
Figure 2.TEM images of a) CdTe QDs, b) the CdTe QDs–GOx complex, and c) after addition of glucose to
b).
(
face of CdTe QDs.When H O2
2
gets to the surface of the CdTe
QDs, it is reduced to O imme-
2
diately, which lies in electron
hole traps on the CdTe QDs
and can be used as a good ac-
ceptor, thus forming the non-
Figure 3.Confocal fluorescence images of a) CdTe QDs, b) the CdTe QDs–GOx complex, and c) after addition
of glucose to (b).
fluorescent
anion.
CdTe
The produced O2
QDs
[
34–37]
can further participate in the
catalyzed reaction of GOx,
with GOx, the average diameter of the CdTe QDs increases
clearly, showing that the GOx has been well conjugated on
the CdTe QDs.Therefore, the conjugation between carboxyl
group-coated CdTe QDs and GOx, using 1-ethyl-3-(3-dime-
thylaminopropyl)carbodiimide (EDC) and N-hydroxysucci-
nimide (NHS) as coupling reagents, is feasible.
forming a cyclic mechanism of glucose oxidation, which is
favorable for the whole reaction system.The catalytic kinet-
ic assay on glucose sensing revealed that the fluorescence
decreases with reaction time, which also suggests a cyclic
mechanism (Figure 6).Furthermore, the TEM and confocal
fluorescence images of CdTe QDs–GOx (Figures 2 and 3)
indicate that the morphology of the nanosensor is not de-
stroyed and remains intact after the addition of glucose,
confirming that the fluorescence quenching is not caused by
Biological activity of the CdTe QDs–GOx complex: The
biological activity of the assembled CdTe QDs–GOx com-
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9635

B.Tang et al.
CdTe QDs and GOx during the catalyzed oxidation of glu-
cose, which leads to the fluorescence quenching of CdTe
QDs.And, thus, CdTe QDs can sensitively respond to H O ,
2
2
[38]
in accord with the recently reported CdSe/ZnS QDs.
The enzymatic activity assay on GOx: The enzymatic activi-
ty of GOx assembled on the CdTe QDs was investigated by
monitoring the decrease in fluorescence intensity during oxi-
dation of glucose.Because GOx can catalyze the oxidation
of glucose to produce H O , it is expected that for a given
2
2
enzymatic activity, the rate of H O production would in-
2
2
crease as the glucose concentration increases.So the extent
of fluorescence quenching embodies the changes of GOx en-
zymatic activity.In addition, as the biocatalytic reaction pro-
ceeds, the luminescence quenching of CdTe QDs–GOx is
enhanced, in accord with the theory of the catalytic kinetic
study and the proposed cyclic mechanism.In the experi-
ment, we carried out the assays upon interaction of glucose
with the sensing system for a fixed reaction time of ten mi-
nutes (Figure 6).As can be seen from Figure 7a, the relative
fluorescence intensity increases with the increasing glucose
concentration up to 6.0 mm, after which it reaches satura-
tion.According to the theory of kinetics of enzyme-cata-
Figure 4.CD spectra of a) free GOx and b) GOx conjugated on the CdTe
QDs.Samples were dissolved in PBS (10 m m, pH 7.4). The initial concen-
À1
tration of the GOx solution was 1.00 mgmL
.
lyzed reactions, the Michaelis–Menton parameter (K ) can
m
Figure 5.The fluorescence spectra of a) the CdTe QDs–GOx nanosensor,
b) after addition of glucose to (a), and c) the catalase introduced before
glucose sensing.
Figure 6.The change of fluorescence intensity with the sensing reaction
time.
surface oxidation of CdTe QDs, but is an electron-transfer
mechanism.Therefore, the glucose sensing mechanism using
the new assembled CdTe QDs–GOx nanosensor is based on
formation of the cyclic electron-transfer mechanism between
Figure 7.a) The relative fluorescence intensity changes after addition of
different concentrations of glucose to the sensing solution.b) Line-
weaver–Burke plots of the GOx enzyme–glucose reaction.
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Enhancement of Glucose Oxidase Activity
FULL PAPER
be determined by analysis of enzyme–substrate reactions.In
our present assay system, the parameter was estimated by
[39]
using the Lineweaver–Burke plot (Figure 7b).The value
À1
of Km was calculated to be 0.45 mmL , indicating the con-
siderably enhanced activity of GOx for glucose after its con-
À1
jugation with CdTe QDs relative to the value of 5.85 mmL
À1
for free GOx and 3.74 mmL for GOx immobilized on the
[22]
gold nanoparticles that had the lowest value reported.
The secondary and tertiary structures of the enzyme are
known to play an important role in the enzymatic activi-
[
40,41]
ty,
so the conformational changes of GOx were studied
by CD and fluorescence spectral experiments.As can be
seen from Figure 4, the CD spectrum of free GOx has char-
acteristic peaks at l=210 and 220 nm, which are retained in
the native conformation.Whereas, GOx conjugated on the
CdTe QDs shows a CD minimum peak at l=208 nm, dem-
onstrating that the precise enzymatic conformation, includ-
ing a helix, b sheet, b turn, and random coil, changes greatly
compared with the free GOx.The conformational changes
can also be illustrated by fluorescence measurements on
GOx, which are shown in Figure 8.The fluorescent amino
Figure 9.The changes of fluorescence intensity in glucose sensing at the
temperature of 20–808C.
the maximum activity at 40–508C and a sharp activity reduc-
tion occurs when the temperature is above 508C.Although
the activity of GOx decreases at higher temperature, the rel-
ative activity of GOx still remains at 808C.Our results show
that the conjugated GOx enzyme exhibits a wider active
temperature range than the free GOx enzyme, the thermal
stability of which has been reported frequently in the litera-
[22,43–45]
ture.
The reason behind the thermal stability enhance-
ment of GOx may lie in the large surface areas on CdTe
QDs, which reduce enzyme unfolding and turbulence, lead-
ing to the conformational changes of GOx, and thus ensure
[16]
the enhanced stability of enzymes in solution.
Glucose sensing based on the CdTe QDs–GOx complex:
On the basis of the considerably enhanced GOx enzymatic
activity, we performed the sensitive determination of glucose
by using the assembled CdTe QDs–GOx nanosensor.The
experimental conditions including the pH of the solution
and the amounts of the sensing solution were optimized (see
the Supporting Information).Under optimal conditions, as
can be seen from Figure 10, the fluorescence intensity grad-
Figure 8.The excitation spectrum of a) GOx, and the fluorescence spec-
tra of b) GOx and c) GOx conjugated on the CdTe QDs.
acid residues in proteins are a useful indicator of the local
[42]
and overall conformation. And the maximum emission of
proteins is highly dependent on the environment around it.
In our study, the native GOx has a maximum fluorescence
peak at l=343 nm when excited at the maximum excitation
wavelength of 293 nm.A redshift to 350 nm is observed in
GOx after its conjugation with CdTe QDs, which indicates
that the conformation of GOx changes in the process of as-
sembly.Therefore, the considerably enhanced activity of
GOx by the new assembly of GOx and CdTe QDs is due to
the favorable changes of its conformation.
Thermal stability assay on the GOx enzyme: The thermal
stability of covalently conjugated GOx was investigated in
the range of 20–808C, as can be shown in Figure 9.Our re-
sults indicate that the conjugated GOx remains active over
a wide range of temperature.It is observed that GOx has
Figure 10.The changes in fluorescence intensity of the CdTe QDs–GOx
nanosensor at different concentrations of glucose for a fixed reaction
time of 10 min under optimal experimental conditions.
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9637

B.Tang et al.
ually decreases as the concentration of glucose increases.In
addition, the decrease of fluorescence intensity is directly
proportional to the concentration of glucose in the range of
perature range.The assembly of GOx to CdTe QDs is facile,
and the assembled nanosensor has good stability, which en-
sures its potential for bioanalytical applications.Moreover,
the simply assembled nanosensor can sensitively determine
glucose over a wide concentration range from micro- to mil-
limolar with the detection limit of 0.10 mm, which could be
used for the direct detection of lower levels of glucose in
complicated biological systems.These results indicate that
CdTe QDs with good luminescence, high catalytic effects,
and electron-transfer efficiency may become a promising
nanomaterial for the enhancement of enzymatic activity.In
addition, the new method of assembly provide an approach
for the assembly of CdTe QDs with other redox enzymes, to
realize enhanced enzymatic activity, and to further the
design of novel nanosensors applied in biological systems in
the future.
5.0 mm to 1.0 mm (Figure 11).Statistical analysis gave us a
Experimental Section
Figure 11.Linear plot of relative fluorescence intensity as a function of
glucose concentration.
À1
Materials: Glucose oxidase (GOx, 200 Umg ), catalase (1340 units per
mg solid, from bovine liver), thioglycolic acid (TGA), 1-ethyl-3-(3-dime-
thylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS)
were obtained from Sigma (Aldrich). d-Glucose was purchased from
Amresco Corporation.Tellurium powder (99%) and sodium hydrogen
boride (99%) were obtained from China Medicine Group Shanghai
Chemical Reagent Corporation.All chemicals were used without further
purification.Ultrapure water used in the experiment was purified with a
Milli-Q (electric resistivity 18.2 MWcm ) water purification system.A
00K Nanosep filter (Pall Corporation, USA) and Microcon YM-30-
0000 NMWL (Millipore, USA) were used as the ultrapurification instru-
value for the detection limit as low as 0.10 mm towards the
concentration of glucose, thus the sensitivity of this method
is a clear improvement on other GOx-based glucose detec-
[26–28]
tion methods.
Therefore, our new assembled CdTe
À1
QDs–GOx nanosensor can be applied to the ratiometric de-
tection of glucose with high sensitivity and simplicity.
1
3
mentation.
Physical instrumentation and methods: Excitation and fluorescence spec-
tra were carried out with an Edinburgh FLS920 spectrofluorimeter (Ed-
inburgh Instruments, Scotland) equipped with a xenon lamp and a quartz
cuvette (1.0 cm optical path). Spectrometer slits were set to 2.0 nm. TEM
images were performed on a Hitachi Model H-800 instrument (Japan).
CD spectra were obtained on a circular dichroism spectrometer (J-810,
JASCO, Japan); a JASCO cell of path length 0.10 cm was used. Confocal
fluorescence microscopy images were obtained on a LSM510 confocal
laser-scanning microscope (Carl Zeiss).Centrifugation was carried out on
a Sigma 3K 15 centrifuge.
Conclusion
In summary, we have assembled a new CdTe QDs–GOx
complex, and thus achieved considerably enhanced enzy-
matic activity and widened the active temperature range of
GOx.The obtained complex can be used as a nanosensor
for simultaneous assay on GOx enzymatic activity, thermal
stability, and glucose analysis.A mechanism is put forward
based on the fluorescence quenching of CdTe QDs, which is
Preparation and purification of water-soluble CdTe QDs: CdTe QDs
2
+
were prepared by using the reaction between Cd and a NaHTe solution
in the presence of thioglycolic acid (TGA) as a stabilizer according to
caused by the H O that is produced from the GOx-cata-
2
2
[
46]
lyzed oxidation of glucose.When H O reaches the surface
the literature. To remove excess thioglycolic acid, the as-prepared QDs
were precipitated with an equivalent amount of 2-propanol, and then re-
dispersed in ultrapure water and precipitated with 2-propanol twice.The
pellet of purified QDs was dried overnight at room temperature under
vacuum, and the final product in the powder form could be redissolved
in ultrapure water (100 mL).The aggregated nanoparticles that appeared
during the process of redissolving were removed by ultrafiltration using a
2
2
of the CdTe QDs, the electron-transfer reaction occurs im-
mediately and H O is reduced to O , which lies in electron
hole traps on CdTe QDs and can be used as a good accept-
or, thus forming the nonfluorescent CdTe QDs anion.The
2
2
2
produced O can further participate in the catalyzed reac-
2
1
00K Nanosep filter under centrifugation (12000 rpm, 5 min).The upper
tion of GOx, forming a cyclic electron-transfer mechanism
on glucose oxidation, which is favorable for the whole reac-
tion system.The lower value of the Michaelis–Menton con-
phase was discarded.The obtained homogeneous QDs were in the lower
phase and used as the stock solution.
Assembly of the CdTe QDs–GOx complex: Glucose oxidase (GOx) was
dissolved in phosphate-buffered saline solution (PBS, 10 mm, pH 7.4) to
À1
stant is estimated to be 0.45 mmL , which shows the consid-
À1
erable enhanced enzymatic activity measured by far.In ad-
dition, the GOx enzyme conjugated on the CdTe QDs ob-
tains better thermal stability at 20–808C than free GOx and
keeps the maximum activity in the wide range of 40–508C.
The conformational changes of GOx are attributed to the
above-enhanced enzymatic activity and the wide active tem-
obtain a solution (1.0 mgmL ) that was stored at 48C.The conjugation
proceeds by NHS and EDC forming active esters to conjugate the car-
boxyl groups of QDs to the primary amine groups of GOx.Briefly, EDC
(
0.50 mg) and NHS (0.25 mg) were added to the CdTe QDs stock solu-
tion (1.00 mL) to activate the QDs in PBS (10 mm, pH 7.4), and it was
then incubated for 30 min at RT with continuous gentle mixing.Next, the
activated QDs and GOx solution (100 mL) were incubated at room tem-
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Chem. Eur. J. 2008, 14, 9633 – 9640

Enhancement of Glucose Oxidase Activity
FULL PAPER
perature for another 2 h with continuous gentle mixing, and then stored
at 48C overnight.The GOx-conjugated QDs were separated from the so-
lution by removing free GOx as well as other small molecules through ul-
trafiltration under centrifugation (12000 rpm, 5 min) with a YM-30K ul-
trafilter.The final concentrated CdTe QDs–GOx complex was diluted to
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