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-
9638
ꢀ 2008 Wiley-VCH Verlag GmbH & Co.KGaA, Weinheim
Chem. Eur. J. 2008, 14, 9633 – 9640