Enzymatic Oxidation of Glucose Confined in a Nanodevice
FULL PAPER
be monitored with electrochemical real-time detection. The
PAA membrane provides a convenient nanofluidic platform
for immobilizing active, accessible biocatalysts by using a co-
valent method. Therefore, the influence of mass transport
on nano-enzyme reaction kinetics with a convective flow
through the nanochannel array of the PAA membrane can
be systematically investigated. For the model system of the
oxidation of glucose by dissolved oxygen catalyzed by im-
mobilized glucose oxidase (GOD), the current response of
the electroactive product hydrogen peroxide can be detected
by the Pt-film electrode arranged at the end of the nano-
channels of the PAA membrane. The results show that the
flow rate has a significant effect on the enzyme reaction ki-
netics and conversion efficiency. The enzymatic reaction is
determined by mass transport at lower flux. In this case, the
detection current and apparent enzyme activity is very sensi-
tor (Zhejiang Huada Limited Company, Zhejiang, China) at 1508C with
the two prepunched holes well aligned.
Immobilization of enzyme: Traditional aminosilane and glutaraldehyde
coupling chemistry was used for the enzyme immobilization. The
APTMS-grafted PAA membrane with the fabricated Pt-film electrode
was mounted in a home-made flow apparatus. A solution containing
2.5% glutaraldehyde in phosphate buffer (10 mm, pH 7.0) continuously
flowed through the PAA membrane with a syringe pump (TS2–60, Multi-
ꢀ
1
syringe pump, Lange, China) at a flow rate of 20 mLmin for 1 h. Trace
amounts of free glutaraldehyde possibly present in the nanochannels
were removed by flowing PBS (10 mm) through the PAA membrane for
10 min to avoid cross-linking with GOD. After the above treatments, glu-
taraldehyde was successfully covalently coupled onto the surface amine
groups on the PAA membrane, which was further used to covalently
bind the amine groups of the enzymes.
The covalent immobilization of GOD was carried out by flowing a solu-
ꢀ1
tion of GOD in pH 7 PBS (2.5 mgmL ; catalog no. G6125, EC 232–
ꢀ1
6
01—0, Type II from Aspergillus niger, 21200 Unitsg , purchased from
Sigma–Aldrich) through the above-treated PAA membrane at a flow rate
ꢀ
1
of 2 mLmin for 1 h. Subsequently, PBS (10 mm) was pumped through
the PAA membrane to remove the immobilized enzyme physically. This
flow method provides a more homogeneous enzyme distribution and en-
sures that the enzymes are immobilized in the interior of the nanochan-
nels of the PAA. The prepared nano-enzyme reactor could be used for
enzyme assays. It could be reused with retained enzyme activity by stor-
ing in a phosphate buffer solution at pH 7.0.
tive to the flow rate. However, at
a
faster flux
ꢀ
1
(
>50 mLmin ), the enzymatic reaction kinetics becomes
the rate-determining step and the detection current and ap-
parent enzyme activity remain constant. These results would
help us to understand the fundamentals of enzymatic reac-
tions confined in nanospaces. The present nano-enzyme re-
actor integrated with a real-time detecting system could find
application in various fields of bioanalysis, such as biosen-
sors, drug screening, and chemical synthesis.
Cell assembly and pressure-driven fluid-flow measurements: The experi-
mental setup with the pressure-driven fluid flow is schematically illustrat-
ed in Figure 1A. Prior to the measurements, the fabricated membrane
was immersed in the buffer solution for at least 30 min to ensure com-
plete wetting. The PAA membrane was clamped between two thin poly-
(
dimethylsiloxane) (PDMS) films and placed between two home-made
half cells. The flowing channels (diameter: 2 mm) on both the half cells
were aligned to the exposed holes of the PAA membrane. Importantly,
the Pt-coated side of the PAA membrane was in contact with the detec-
tion cell (right-side cell), whereas the other side of the PAA membrane
was in contact with the fluid inlet cell (left-side cell).
Experimental Section
Surface modification: The porous anodic alumina (PAA) membrane
Whatman) with the nominal diameter of 200 nm and thickness of 60 mm
was cleaned. The cleaned PAA was immersed into a mixture of 3-ami-
nopropyltrimethoxysilane (APTMS) and acetone (10 mL; 9:1) for about
2 h, thus resulting in grafting aminopropyl functional groups onto the
inner-wall surface of the PAA membrane. The excess silane solution was
removed from the PAA nanochannels by rinsing with copious amounts of
acetone followed by washing with deionized water. The sample was dried
under a stream of nitrogen to remove any impurities and fluid. The re-
maining modification steps, which started from the surface-bound
amines, were carried out after fabrication of the Pt-film electrode on one
side of the PAA membrane and subsequently assembling the PAA mem-
brane in the cell.
The detection half-cell contained PBS (1 mL; 10 mm, pH 7.0) and the
fluid inlet half-cell was connected with a syringe pump. The Pt-film work-
ing electrode on the PAA membrane, a Pt-wire counterelectrode, and the
Ag/AgCl reference electrode in the detection cell forms a three-electrode
electrochemical system for electrochemical characterization. After the
immobilization of the enzyme in the nanochannels of the PAA mem-
brane as mentioned above, a constant flux of glucose solution was driven
(
[
40]
1
through the membrane. As a potential for the oxidation of H
plied to the Pt-film working electrode, the enzymatic reaction product
could be electrochemically detected by using a CHI 900 electro-
2 2
O was ap-
2 2
H O
chemical workstation.
Enzyme kinetics assays: Before the enzyme reaction started, the control
experiment was conducted by initially driving the buffer solution through
the nanochannel-array reactor. The enzyme assays of GOD covalently
immobilized in the nanochannels of the PAA membrane were conducted
at various concentrations (0.05–20 mm) and flow rates of glucose. The
Fabrication of the Pt-film working electrode on the PAA membrane:
The PAA-membrane-based enzyme reactor was clamped between two
cells for the flow experiments (Figure 2A). It is important to make sure
that the Pt-film electrode on one side of the PAA membrane is stable in
the whole experimental process. Therefore, the Pt-film working electrode
on the PAA membrane was fabricated as follows: Briefly, a Pt film was
sputtered on one side of the PAA membrane, thereby serving as the
working electrode (or detector). Sputtering to a thickness of 100 nm for
the Pt film was performed with a current of 15 mA in a vacuum chamber
electrochemical signals of the enzymatic reaction product H
Pt-film electrode at 0.7 V versus Ag/AgCl were recorded.
2 2
O on the
ꢀ
4
at a pressure of 5ꢁ10 mbar (Ar plasma). In this case, the sputtered Pt
[
26]
film did not block the nanopores.
For easy disposal, the Pt-coated
PAA membrane was sandwiched between two poly(ethyleneterephtha-
late) (PET) sheets (thickness: 100 mm; DIKA Official Limited Company,
Suzhou, China) with prepunched holes (diameter: 2 mm). The holes de-
fined the area of the membrane exposed to the contacting solutions. Cu
wire (diameter: 0.2 mm) was placed in electrical contact with the Pt-
coated PAA membrane by Ag conductive epoxy. Extra care had to be
taken so that the Ag conductive epoxy was isolated from the solution. Fi-
nally, the membrane assembly was laminated by using a heating lamina-
Acknowledgements
This work was supported by the Grants from the National 973 Basic Re-
search Program(2007CB714501, 2007CB936404), the National Natural
Science Foundation of China(NSFC, no. 20775035, 20828006, 20890020,
20975047), and the National Science Fund for Creative Research Groups-
AHCTUNGTRENNUNG( 20821063).
Chem. Eur. J. 2010, 16, 10186 – 10194
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
10193