Insights into the "free state" enzyme reaction kinetics in nanoconfinement

Article abstract of DOI:10.1039/c3lc41319e

The investigation of enzyme reaction kinetics in nanoconfined spaces mimicking the conditions in living systems is of great significance. Here, a nanofluidics chip integrated with an electrochemical detector has been designed for studying "free state" enzyme reaction kinetics in nanoconfinement. The nanofluidics chip is fabricated using the UV-ablation technique developed in our group. The enzyme and substrate solutions are simultaneously supplied from two single streams into a nanochannel through a Y-shaped junction. The laminar flow forms in the front of the nanochannel, then the two liquids fully mix at their downstream where a homogeneous enzyme reaction occurs. The "free state" enzyme reaction kinetics in nanoconfinement can thus be investigated in this laminar flow based nanofluidics device. For demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of beta-d-glucose. The reaction product hydrogen peroxide (H ₂O₂) can be electrochemically detected by a microelectrode aligning to the end of nanochannel. The steady-state electrochemical current responding to various glucose concentrations is used to evaluate the activity of the "free state" GOx under nanoconfinement conditions. The effect of liquid flow rate, enzyme concentration, and nanoconfinement on reaction kinetics has been studied in detail. Results show that the "free state" GOx activity increases significantly compared to the immobilized enzyme and bath system, and the GOx reaction rate in the nanochannel is two-fold faster than that in bulk solution, demonstrating the importance of "free state" and spatial confinement for the enzyme reaction kinetics. The present approach provides an effective method for exploiting the "free state" enzyme activity in nanospatial confinement.

Full text of DOI:10.1039/c3lc41319e

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Insights into the ‘‘free state’’ enzyme reaction kinetics
in nanoconfinement3
Cite this: Lab Chip, 2013, 13, 1546
Chen Wang,ab De-Kai Ye,a Yun-Yi Wang,a Tao Lu*b and Xing-Hua Xia*a
The investigation of enzyme reaction kinetics in nanoconfined spaces mimicking the conditions in living
systems is of great significance. Here, a nanofluidics chip integrated with an electrochemical detector has
been designed for studying ‘‘free state’’ enzyme reaction kinetics in nanoconfinement. The nanofluidics
chip is fabricated using the UV-ablation technique developed in our group. The enzyme and substrate
solutions are simultaneously supplied from two single streams into a nanochannel through a Y-shaped
junction. The laminar flow forms in the front of the nanochannel, then the two liquids fully mix at their
downstream where a homogeneous enzyme reaction occurs. The ‘‘free state’’ enzyme reaction kinetics in
nanoconfinement can thus be investigated in this laminar flow based nanofluidics device. For
demonstration, glucose oxidase (GOx) is chosen as the model enzyme, which catalyzes the oxidation of
beta-D-glucose. The reaction product hydrogen peroxide (H2O2) can be electrochemically detected by a
microelectrode aligning to the end of nanochannel. The steady-state electrochemical current responding
to various glucose concentrations is used to evaluate the activity of the ‘‘free state’’ GOx under
nanoconfinement conditions. The effect of liquid flow rate, enzyme concentration, and nanoconfinement
on reaction kinetics has been studied in detail. Results show that the ‘‘free state’’ GOx activity increases
significantly compared to the immobilized enzyme and bath system, and the GOx reaction rate in the
nanochannel is two-fold faster than that in bulk solution, demonstrating the importance of ‘‘free state’’
and spatial confinement for the enzyme reaction kinetics. The present approach provides an effective
method for exploiting the ‘‘free state’’ enzyme activity in nanospatial confinement.
Received 29th November 2012,
Accepted 14th January 2013
DOI: 10.1039/c3lc41319e
www.rsc.org/loc
reaction system, enzymes are usually immobilized on nanos-
tructured materials4–10 or walls of microchannels11–14 and
Introduction
single/array nanochannels.15,16 When substrate solutions are
brought intocontact with the immobilized enzymes, hetero-
geneous enzyme catalytic reactions take place. For nanostruc-
tures, layered materials4–6 and mesoporous materials7–10 are
often used to immobilize enzymes, mimicking confined
environments in organisms. The size of the nanocavities in
layered nanostructures is usually comparable to biomolecules,
and thus allows the biomolecules to be tightly incorporated
into the interlayer space of the layered structures. The
alternative matrix for nanomaterials/biomolecules hybrid
construction, mesoporous materials, can concentrate enzymes
in the pores, resulting in fast reaction rates and considerably
reduced reaction times.10 However, for nanostructured mate-
rials immobilized with enzymes, mass diffusion into the
nanocavity is unfavorable, thus only a part of the biomolecules
(those in contact with the liquids) can play their function
efficiently. Therefore, it is difficult to investigate the influence
of liquid flow rate on the biomolecular behaviours, and the
biochemical reaction kinetics cannot be evaluated in situ. For
microchannels11–14 and single/array nanochannels,15,16 the
enzymes can be steadily immobilized onto the channel walls,
allowing the enzyme reactions to occur in continuous flow
Enzymes are key proteins in living systems, catalyzing almost
all chemical reactions in living beings and taking part in
various biological reactions within biological cells.
Understanding of the enzymatic reaction kinetics in confined
nanospace would help us unravel the chemical processes
occurring in life activities. Up to now, most enzyme activity
assays are performed in diluted solutions, which contain only
one or a few different types of biomolecules in vitro, which is
very different from the crowded environmental in vivo
conditions. Thus, researchers have begun moving beyond the
conventional bath bulk methods to space-confined systems,
which can well mimic the confined environments in living
systems.1–3
Enzyme assays in space confinement include heteroge-
neous and homogeneous reaction systems. In a heterogeneous
aState Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry
and Chemical Engineering, Nanjing University, Nanjing 210093, China.
E-mail: xhxia@nju.edu.cn; Fax: (+86) 25 83686106
bSchool of Basic Sciences, China Pharmaceutical University, Nanjing 211198, China.
E-mail: lutao@cpu.edu.cn; Fax: (+86) 25 86185179
Electronic supplementary information (ESI) available: See DOI: 10.1039/
3
c3lc41319e
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mode. In this method, the enzymes can be used circularly, and
the immobilized biomolecules show a higher stability due to
the confined nanospace.
and used to study the reaction kinetics of immobilized glucose
oxidase (GOx).15 The results demonstrated the significant
nanoconfinement effect on immobilized enzyme reaction
kinetics. In addition, fluorescence labelling of biomolecules
and simultaneous product purificaiton30 were also demon-
strated on the fabricated micro/nanofluidics device.
In brief, the immobilization method for enzyme assays
presents many advantages including ease of enzyme recycling,
higher stability due to the immobilized state, and enhanced
enzyme concentrations due to the sample enrichment in
nanostructures. However, a reduced enzymatic activity is
usually observed because of possible changes in the enzyme
conformation and steric hindrance of the enzyme catalytic
sites. Furthermore, there are difficulties for inactivated
enzyme replacement. In comparison, ‘‘free state’’ enzymes in
a homogeneous system in micro/nanochannels can directly
address the challenges faced in the heterogeneous reaction
systems, which allows for more active catalysis sites of the
enzyme to be fully developed. Thus, it is expected that
homogeneous enzyme reaction systems may provide a better
strategy to study enzyme reaction kinetics.
Up to now, many studies on homogeneous enzyme activity
in microfluidic chips have been reported. In these cases,
utilization of miniaturized enzyme reactors can reduce sample
consumption and mimic confinement spaces in living
systems.17–26 The first example of homogeneous biocatalysis
on a microscale was described by Bao et al.17 Thereafter,
microfluidics based enzyme microreactors have been widely
used in analytical chemistry or biochemistry. Recently,
Yamashita et al.26 studied nonimmobilized enzyme reaction
kinetics in a microchannel at laminar flow. Their results
showed that a ‘‘free state’’ enzyme in a microchannel could
retain highly catalytic activity and fast reaction rate as
compared to the immobilized enzymes. In another report,27
an air-pressure-based control system was developed to study
In this work, a nanofluidics ‘‘free state’’ enzyme nanor-
eactor with a tunable size of nanochannels has been fabricated
and the homogeneous enzyme reaction kinetics in nanocon-
finement is investigated using GOx as a model system. The
nanofluidics device was fabricated using the same UV-ablation
technique developed in our laboratory.29 The fluid flow in the
nanochannel is predicted as being laminar in nature as the
Reynolds number Re is far below 2000 in the nanochannel.26,27
However, the two fluids will be mixed immediately when they
enter into the final nanochannel via a Y-shaped junction due
to the nanoconfinement effect. The layout of the Y-shaped
nanofluidics chip and the principle for the enzyme reaction
kinetics study are illustrated in Fig. 1. First, solutions of the
enzyme (GOx) and its substrate (glucose) are simultaneously
and continuously supplied from two single streams into a
nanochannel through a Y-shaped junction. When these two
solutions meet in the nanochannel, the laminar flow forms at
the beginning of the junction, then the mixing process takes
place through transverse diffusion of the adjacent flows in the
nanochannel. Simultaneously, a homogeneous enzyme reac-
tion occurs, generating the product hydrogen peroxide (H2O2)
which is detected by a microelectrode aligning to the end of
the nanochannel (the position of the microelectrode is shown
in Fig. S1, ESI ). Therefore, the ‘‘free state’’ enzyme reaction
3
kinetics in nanoconfinement can be investigated using the
steady state electrochemical response to H2O2 on this
nanofluidics device integrated with an electrochemical detec-
tor. The effect of liquid flow rate, enzyme concentration, and
nanoconfinement on the kinetic parameters of the homo-
geneous enzyme reaction has been studied. The present work
provides an effective method for performing the ‘‘free state’’
enzyme activity assay in nanospatial confinement, unravelling
the chemical processes occurring in living systems.
homogeneous enzyme reaction kinetics in
a Y-shaped
extended nanospace channel. Results indicated that the
enzyme reaction rate in the extended nanospace was increased
by a factor of about two compared with the bulk and
microchannel systems. We used a micro/nanofluidic chip to
concentrate the enzyme and then studied the kinetics of the
homogeneous enzyme reaction at extremely high concentra-
tions.28 It was found that the time for enzyme preconcentra-
tion (e.g., enzyme concentration) had a significant effect on the
enzymatic reaction kinetics and the corresponding linear
response range of the substrate. At the proper enrichment
time of the enzyme, the enzyme reaction is kinetically
controlled; and the resulting Km is very small, indicating a
higher enzyme activity. These reports inspired us to further
explore the ‘‘free state’’ enzyme activity in confined space (e.g.,
nanometer scale channels), investigating in detail the effect of
existing state and space confinement on the enzyme reaction
kinetics.
Effective and precise fabrication of nanoscaled structures is
the prerequisite for studying the nanoconfinement effect of
enzymatic reactions. Recently, we developed a UV-ablation
method for fabrication of nanochannels with nanometer
precision.29 Using this approach a series of nanofluidics
devices with different dimensions were successfully fabricated
Fig. 1 Schematic layout of the nanofluidics chip. Green and pink colors denote
enzyme and substrate, respectively; yellow denotes the reaction product. The
product of the enzymatic reaction, hydrogen peroxide, can be electrochemically
determined as indicated by the rise of the current (right panel of the figure)
when the substrate, glucose, is introduced. The working electrode is aligned to
the end of the nanochannel with a distance of 20 mm.
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ephthalate) (PET) photomask (Fig. 2B).29 After that, the PC
plate was washed with water followed by N2 gas for drying. The
nanochannel with a Y-shaped nanochannel network formed
on the UV-exposed region (Fig. 2C). Finally, a PDMS cover with
two reservoirs (e, s) was sealed to the PC plate reversibly, and a
complete nanofluidics chip was constructed (Fig. 2D). The UV
light came from a 30 W low-pressure mercury lamp with a
power density of 5 mW cm22. The plate-to-lamp distance was
controlled as 2 cm. The PET photomask was commercially
available with the designed pattern blank since PET film
blocks beams in the wavelength range of 200–285 nm.31 In the
present work, the depth of the channel was controlled as about
80 nm for a 90 min UV irradiation according to the etching
rate reported previously,29 and the width was 200 mm. The total
length o–r was 8 mm and e (or s)–o was 4 mm. The point ‘‘o’’
denotes the junction of the ‘‘Y-shape’’ nanochip, at which two
single streams converge into a single nanochannel; r is the end
of the nanochannel; e and s are the two reservoirs for enzyme
and substrate respectively. To prevent nonspecific binding of
GOx onto the PDMS surface, we introduced bovine serum
albumin (1% solution in 10 mM PBS (pH7.0), Sigma, St. Louis,
MO) into the nanochannel and then incubated for several
hours for each new nanochip. This process results in
passivation of the nanochannel wall with BSA. After that, the
nanochannels were flushed with buffer solution until the
current of the power supply levelled off.
Experimental
Materials and reagents
Glucose oxidase was obtained from Sigma and used as
received. Glucose was from Nanjing Chemical Reagent
Company. Glucose stock solutions were allowed to mutarotate
overnight
at
room
temperature
before
use.
Polydimethylsiloxane (PDMS) precursor and curing agent
(Sylgard 184) was from Dow Corning, Midland, MI, USA.
Polycarbonate was from Suzhou Plastic Co. (Suzhou, China).
Poly(ethyleneterephthalate) (PET) was from DIKA Official
Limited Company, Suzhou, China. Bovine serum albumin
(BSA) was obtained from Nanjing Chemical Reagent Company.
Phosphate buffer (PBS, pH 7.0, 10 mM) solution was used as
the buffer system. All solutions were kept in a freezer to
prevent deterioration and were filtered with a 0.22 mm syringe
filter to remove particulate prior to use. All aqueous solutions
were prepared from deionized water (18 MV?cm21, PURELAB
Classic, PALL, USA).
Fabrication of the nanofluidics device
The nanofluidics chip was composed of an upper PDMS cover
with two reservoirs and a polycarbonate (PC) substrate with
nanochannels. Fig. 2(A–D) illustrates the fabrication of
nanofluidics chips using a previously described method.15,29,30
In brief, a PC plate was first cleaned with isopropanol and
water, dried under a warm air stream (Fig. 2A), then exposed to
254 nm UV light in the air with the use of a poly(ethyleneter-
Characterization
A scanning electron microscope (SEM, Sirion, FEI Company,
Holland) and an inverted fluorescence microscope (Nikon, Ti-
U, Japan) equipped with a digital CCD camera were used to
characterize the morphology of the nanochannels fabricated.
Electrochemical measurements in nanofluidics
A home-made plexiglass holder combined with a 3-D micro-
manipulator was used to fix the nanofluidics chip and house
the detector and reservoirs as reported previously.32,33 Proper
sealing of the solution reservoir at the end of the nanochannel
was achieved with the help of silicone grease and double-sided
adhesive tape.
A
three-dimensional micromanipulator
(Shanghai Lianyi Instrument Factory of Optical Fiber and
Laser, Shanghai, China) was integrated on the plexiglass
holder to precisely position the working electrode. Alignment
of the working electrode (platinum ultramicroelectrode, 10 mm
in diameter) to the nanochannel end was performed under a
microscope (Jiangnan Optical Instrument Factory, Nanjing,
China), as shown in Fig. S1, ESI . A saturated Ag/AgCl
3
Fig. 2 Schematic layout of the nanofluidics chip fabrication process. (A) UV light
irradiated PC under a PET photomask; (B) pattern transfer of the photomask to
the PC plate surface. The gray color denotes the PET photomask, and the purple
arrows denote the UV light; (C) formation of the nanochannel on the PC surface;
(D) formation of the final nanofluidics chip through sealing the PDMS with two
reservoirs (e for enzyme and s for substrate) on to the PC with nanochannels
reversibly. Total length o–r = 8 mm, s (or e)–o = 4 mm. Symbols: o is the junction
of ‘‘Y-shape’’ nanochip, at which two single streams converge into a single
nanochannel; r defines the end of the nanochannel; e and s are the two
reservoirs for enzyme and substrate respectively. The single nanochannel was
achieved by exposing a PC substrate to UV irradiation at a lamp-to-plate
distance of 2.0 cm. (E, F) Scanning electron microscope (SEM) images of the
nanochannel formed after 120 min UV irradiation. (E) Depth of the channel
(about 110 nm), and (F) width of the nanochannel (about 200 mm).
reference electrode, a platinum counter electrode, and a
ground Pt electrode for CE were also placed in the reservoir
along with the working electrode. Amperometric detection was
carried out in
a three-electrode configuration on an
Electrochemical Workstation CHI 650 (Shanghai Chenhua
Instrument Company, Shanghai, China).
‘‘Free state’’ GOx reaction kinetics detection in bulk solution
The same three-electrode configuration on an Electrochemical
Workstation CHI 650 (the platinum ultramicroelectrode work-
ing electrode, a saturated Ag/AgCl reference electrode, and a
platinum counter electrode) was used for the electrochemical
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detection of the ‘‘free state’’ GOx reaction product H2O2, and
for the investigation of the enzyme reaction kinetics in
solution.
glucose into the enzyme stream was calculated as 20 s (the enzyme
molecule is much larger than glucose, therefore the diffusion of
GOx into the glucose stream is relatively slow and can be ignored).
In the present work, the time for liquids flow through the single
nanochannel behind junction is calculated as 40 s assuming the
liquid flow rate is 200 mm s21. Therefore, the complete mixing of
samples can be achieved through adjusting the liquid flow rate ,
400 mm s21 (flow time > 20 s) in the nanofluidics chip. For
demonstration, a fluorescence small molecule probe (fluorescein
isothiocyanate, FITC) and PBS buffer solutions were simulta-
neously driven through the ‘‘Y-shaped’’ nanofluidics chip. The
flow behaviour in the nanochannel behind the junction was
investigated using an inverted fluorescence microscope. The photo
Safety considerations
The high-voltage power supply should be handled with
extreme care to avoid any electric shock.
Results and discussion
Nanofluidics device fabrication
Upon irradiation of a PC film under a PET photomask (200 mm
wide), continuous decomposition of the PC occurs, forming a
nanochannel on the surface of the PC surface. By controlling
the UV irradiation time for 90 min with a lamp-to-plate
distance of 2.0 cm, a nanochannel with an 80 nm depth was
fabricated according to our previously reported etching rate of
images (shown in Fig. S2, ESI ) show that complete mixing was
3
achieved within 1 mm, which is consistent with the analysis
described here.
Influence of detection potential
0.015 nm s .
21 15,29,30 Because of the difficulty in characterizing
When enzyme (GOx) and substrate (glucose) solutions are
driven electrokinetically from two separated channels to a
single channel through a ‘‘Y-shaped’’ junction, the enzyme
and glucose solutions mix each other through transverse
diffusion at the entering point of the Y-junction and thus a
homogeneous enzyme reaction occurs, generating hydrogen
peroxide, as represented in eqn (1). The activity of GOx can be
estimated by measuring the concentration of hydrogen
peroxide. Since hydrogen peroxide is electroactive, it can be
electrochemically detected (eqn (2)) on a platinum ultrami-
croelectrode aligning to the end of the nanochannel.
an 80 nm-deep nanochannel on a broad PC surface, a
nanochannel with 120 min UV ablation (about 110 nm in
depth) was chosen for obtaining SEM images. Fig. 2E and F
show the depth (about 110 nm) and width (about 200 mm) of
the channel, respectively.
It is worth mentioning that channel collapse could occur
due to the larger aspect ratio of width to depth of the
nanochannel. In the present work, reversible bonding of PC
and PDMS was used to avoid channel collapse. However, it was
still found that there is no electrical current passing through
the nanochannel when the UV-ablation time is less than 20
min, indicating a completely blocked nanochannel caused by
PDMS collapsing. Only with a UV irradiation time longer than
25 min, an electrical current flowing through starts appearing,
indicating the existence of a nanochannel. Therefore, the real
nanochannel depth used in our experiment should be smaller
than 80 nm with 90 min UV irradiation.
GOx
b-D-glucosezO2 ꢀ? D-gluconic acidzH2O2
(1)
(2)
z0:8 V vs Ag=AgCl
H2O2 {{{{{{{{{{{? O2z2Hzz2e-
To achieve the optimum detection potential, a hydrody-
namic voltammogram of a platinum ultramicroelectrode from
a 10 mM PBS (pH 7.0) buffer solution containing 1 mM
hydrogen peroxide was collected with a liquid flow rate of 200
mm s21 in the nanochannel. The detection potential ranged
from 0.50 to 1.0 V vs. Ag/AgCl. Fig. 3 shows that the response
current increases with the detection potential from 0.50 to 0.80
V with a nearly linear dependence and then levels off gradually
at higher potentials. Since higher anodic detection potentials
will result in higher background current in electrophoresis–
electrochemical detection, 0.80 V (vs. Ag/AgCl) was chosen for
the detection of hydrogen peroxide in the subsequent
experiments, which is in accordance with our previous
reports.15,16,28
Fluids flow and mixing in a Y-shaped nanofluidics chip
The Reynolds number (Re) is a dimensionless quantity
describing the behaviour of fluids in channels, which is
defined as Re = vlr/g, where v is the fluid velocity, l is a
characteristic channel dimension, r is the fluid density and g
is the fluid viscosity. For Re > 2000, turbulent flow occurs. On
the contrast, laminar flow remains at Re , 2000. For the
nanofluidics chip used in this work, the Reynolds number is
significantly lower than 1 using either the width or the depth
of the channel as the l value (v = 200 mm s21, r = 1.0 g cm23, g =
1 cP). Therefore, when the two fluids flow through the
Y-shaped nanofluidics chip, a laminar flow state remains near
the junction. Once the two streams of fluids meet in the
Y-shaped nanochannel, transverse diffusion between the two
fluids starts, and these two fluids will gradually mix
Determination of the enzyme reaction product, H2O2
thoroughly within a short time (or a short distance). The
The steady-state current response for the ‘‘free-state’’ GOx to
0.50 mM glucose solution with an enzyme concentration of
6.50 mM (10 mg ml21) and flow rate of 200 mm s21 is shown in
Fig. 4. Results show that after the addition of enzyme and
glucose samples, the oxidation current starts to increase at
about 60 s and then increases rapidly. After 120 s, it reaches a
pffiffiffiffiffiffiffiffi
distance of transverse diffusion, x, is given by x~ 2Dt, where
D is the diffusion constant, its typical value being about 1029 m2
s21 for small molecules; t is the fluid flow time in the
nanochannel. For the nanochannel used (x = 200 mm), the time
for completed transverse diffusion of the small molecule of
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Fig. 5 Influence of the liquid flow rate on the steady-state current response of
the reaction product. Glucose solutions at four different concentrations (5.00,
10.00, 15.00, 25.00 mM) were used.
Fig. 3 Hydrodynamic voltammogram of a Pt ultramicroelectrode (10 mm in
diameter) in a solution of 10 mM PBS (pH 7.0) containing 1 mM hydrogen
peroxide. The liquid flow rate is controlled as 200 mm s21 by a high-voltage
power supply.
S1, respectively. Glucose solutions at four different concentra-
tions (5.00, 10.00, 15.00, 25.00 mM) and an enzyme solution at
a fixed concentration (6.50 mM) were electrokinetically driven
through the nanofluidics chip under different flow rates. The
electrochemical responses to four glucose concentrations
(5.00, 10.00, 15.00, 25.00 mM) at varied flow rates are shown
in Fig. 5. It can be seen that the current response first
increases with the flow rate in a nearly linear relationship,
then levels off at high flow rate. This phenomenon can be
explained by the counterbalance of two factors, i.e., the
difference in reaction time and mass transport velocity. First,
as the liquid flow rate increases, the amount of reaction
product reaching the electrode surface increases with the flux,
resulting in an enhanced current response. However, as
described above, when the liquid flow rate exceeds 400 mm
steady-state value. Five parallel measurements with a 0.50 mM
glucose solution through the nanofluidics chip under the
same conditions show good reproducibility (the relative
standard deviation, RSD = 4.53%, n = 5, Fig. S3, ESI ). These
3
results demonstrate that it is quite reasonable to take
advantage of the steady-state current profiles to unravel the
‘‘free state’’ enzyme reaction kinetics in a laminar flow
confined in a nanochannel.
Effect of liquid flow rate
The liquid flow rate affects the current response of the
reaction product and thus the enzyme reaction kinetics
parameters.16,28 To investigate the influence of the liquid flow
rate on the steady-state current response of the reaction
product, four different liquid flow rates (100, 200, 330, 400 mm
s
21, the samples cannot be mixed thoroughly since the flow
s
21) were used. The flow rate is controlled by a laboratory-
time t is too short for completing the transverse diffusion of
samples, leading to a reduced amount of reaction product.
Therefore, a decreased current response trend would appear as
the flow rate exceeds 400 mm s21. These two factors function
simultaneously, resulting in the obvious differences in the
electrochemical response to the same glucose solution at
different flow rates. In the following experiment, a liquid flow
rate of 200 mm s21 is chosen for investigation of ‘‘free state’’
GOx reaction kinetics in nanoconfinement, which can render
samples fully mixed (40 s flow time) whilst maintaining a
relatively large current response.
made high-voltage power supply (0–5000 V) through platinum
electrodes placed in the reservoirs. For a detailed description
of the liquid flow rate measurement and corresponding
parameters, refer to the supporting information and Table
Study of ‘‘free state’’ GOx reaction kinetics confined in the
nanochannel
To investigate the ‘‘free state’’ GOx reaction kinetics confined
in the nanochannel, solutions at different concentrations of
GOx and glucose were electrokinetically driven through the ‘‘Y-
shaped’’ nanofluidics chip. The steady current response to the
reaction product, hydrogen peroxide, was used to monitor the
GOx reaction kinetics. The results are shown in Fig. 6. The
Lineweaver–Burke plots are shown in Fig. S4, ESI . It can be
3
Fig. 4 Steady-state current response of the enzyme reaction product, hydrogen
peroxide, on a Pt ultramicroelectrode at 0.80 V to a 0.50 mM glucose solution in
10 mM PBS (pH 7.0) with an enzyme concentration of 6.50 mM and a flow rate
clearly seen that for all concentrations of GOx, the current
response to glucose first increases with the glucose concentra-
tion, then levels off gradually at high glucose concentrations.
of 200 mm s21
.
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‘‘free state’’ GOx reaction in the present nanofluidics chip will
be of great significance in clinical diagnosis.
For investigation of the enzyme reaction kinetics, the
famous Michaelis–Menten equation was used. The Michaelis
constant (Km) of the homogeneous enzyme reaction was
determined using the Lineweaver–Burke form of the
Michaelis–Menten equation, which can be easily constructed
based on the Lineweaver–Burke plots (Fig. S4, ESI ). For
3
electrochemical detection, the Michaelis–Menten equation can
be modified as eqn (3), where, iss is the steady-state current of
the enzymatic reaction product, C the substrate concentration,
imax the maximum current measured under the saturated
substrate conditions, and Km the Michaelis constant.16,28,34
The catalytic constant of the enzyme reaction (kcat),
characterizing the enzyme reaction kinetics in a particular
reactor, can be calculated based on eqn (4),35,36 where Vmax is
the maximum reaction rate measured under saturated
substrate conditions, and [E] the enzyme concentration. The
Vmax values are determined from the imax values according to a
standard H2O2–current calibration,37 which can be easily
achieved by driving a series of different concentrations of
H2O2 solutions through the nanofluidics chip for electroche-
mical measurement. Given the reaction time 40 s for liquid
flow rate of 200 mm s21 in the nanochannel, the maximum
reaction rate is thus given by vmax = [H2O2]/t (for detailed
experimental conditions, refer to supporting information and
Fig. 6 Steady-state current responses to hydrogen peroxide produced by the
enzyme reaction as a function of glucose concentration for four different
enzyme concentrations (0.65, 2.60, 6.50, 13.00 mM). The liquid flow rate is
controlled as 200 mm s21 by a high-voltage power supply.
This trend shows the typical kinetic properties of enzyme
reactions, and is similar to our previous reports.15,16,28 To
investigate the effect of enzyme concentration on the reaction
kinetics, GOx solutions at four different concentrations (0.65,
2.60, 6.50, 13.00 mM) were used while keeping the same liquid
flow rate of 200 mm s21. The results in Fig. 6 indicate that the
enzyme concentration has a significant effect on the steady
current response of the reaction product. First, for the same
concentration of glucose, the current response increases with
the enzyme concentration due to the increase of the reaction
product. This increasing trend becomes more obvious as the
glucose concentration increases, indicating a higher enzyme
concentration is favorable for achieving complete reaction
when large amounts of substrate exist. Second, the enzyme
concentration also influences the linear response range to
glucose. As indicated in Table 1, the linear range first increases
with the enzyme concentration, increasing from 0.65 to 6.50
mM, then diminishes as the enzyme concentration increases
further. The first enlarged linear range is due to the increased
amount of product caused by the increase in enzyme
concentration. However, with the enzyme concentration
further increasing, the electrochemical current response
increases considerably, which can lead to a reduced linear
range to glucose.28 Therefore, an optimum linear range (0–
10.00 mM) of substrate is achieved at an enzyme concentration
of 6.50 mM. Since the normal human blood glucose concentra-
tion ranges from 4.00 to 6.00 mM and diabetes refers to 8.00
mM or higher, the extended linear range achieved from the
Fig. S5, ESI ).
3
1
1
Km
~
z
(3)
iss
imax
imaxC
vmax = kcat[E]
(4)
The effect of the enzyme concentration on the Michaelis
constant, the maximum reaction rate and the catalytic
constant are listed in Table 1. The resulting Km value of GOx
remains nearly constant (3.62–4.04 mM) with an average value
of 3.88 mM (RSD = 4.74%), indicating a reaction-kinetics-
controlled process in the nanofluidics chip at a 200 mm s21
flow rate. The maximum current and maximum reaction rate
increases with the enzyme concentration due to the higher
amount of reaction product being generated at high enzyme
concentrations. However, the resulting values of kcat maintain
similar values with an average of 22.91 s21(RSD = 4.27%),
which indicates the same enzyme reaction rate in the same
nanoreactor providing samples are fully mixed.
Table 1 Effect of enzyme concentration on enzyme reaction kinetics and linear response range of the substrate
[Enzyme] (mM)
Km (mM)
imax (nA)
vmax (mM s21
)
kcat (s21
)
Linear range (mM)
Nanofluidics
0.65
2.60
6.50
13.00
3.97
3.62
4.04
3.88
3.88
4.74%
15.09
0.11
0.43
0.97
2.10
14.82
61.83
140.30
305.32
22.81
23.78
21.58
23.49
22.91
4.27%
9.50
0.10–5.00
0.10–5.00
0.10–10.00
0.10–8.00
Average value
RSD
Bulk
13.00
17.61
123.53
0.10–5.00
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Lab on a Chip
mechanism that stabilizes protein folds in the cell. Our
previous study also showed that confinement in a nanochan-
nel larger than the size of the enzyme molecule can increase
enzyme activity to some extent.15 In the nanoscale space
provided herein, the enzyme molecules can be stabilized by
folding forces, eliminating some expanded configurations of
the unfolded chain, which is more favorable to the equili-
brium shifting from the unfolded state to the native state, and
thus a higher enzyme activity may be achieved. Secondly, the
laminar flow confined in the nanochannel can provide an
ideal interface for a homogeneous enzyme reaction due to the
accelerated biomolecule diffusion and mixing in the nanoscale
channel, therefore, allowing a faster enzyme reaction, and
accordingly a larger kcat value.
Fig. 7 The Lineweaver–Burke plot for substrate concentrations ranging from
0.05 to 15.00 mM for free GOx in bulk solution, resulting in a Km value of 15.09
mM. The inset shows the steady-state current response to hydrogen peroxide
produced by the enzyme reaction as a function of glucose concentration. The
concentration of free glucose oxidase is 13.00 mM.
The present results for ‘‘free state’’ enzymes are also
compared to the ones for immobilized GOx reported pre-
viously.15 In ref. 15, the values of Km ranged from 10.06 to 4.02
mM for four different dimensions of nanofluidic chips,
demonstrating the existence of a nanoconfinement effect.
For a nanofluidics chip of the same dimensions fabricated
with a 90 min UV light ablation, the Km for the immobilized
GOx is 6.96 mM,15 which is larger than the value (3.88 mM) for
the ‘‘free state’’ GOx in the laminar flow confined nanochan-
nel. A small Km indicates a more efficient complex formation
between enzyme and substrate, and also a higher conversion
efficiency of substrate to product. The higher catalytic activity
of ‘‘free state’’ GOx is due to the fully developed active catalysis
sites in the homogeneous system, which could be decreased by
an altered enzyme conformation and steric hindrance in
enzyme-immobilized systems. In addition, for the immobi-
lized state of the enzyme, the covalent binding of the enzyme
to the channel wall could reduce the activity of the enzyme as
well.40
Effect of nanoconfinement on ‘‘free state’’ GOx activity
To investigate the nanoconfinement effect on ‘‘free state’’ GOx
activity, the free enzyme kinetics of GOx in bulk solution was
investigated under the same reaction conditions (e.g., a
reaction-kinetics-controlled process). The results are shown
in Fig. 7 and Table 1 (the detailed experiment data are shown
in Fig. S6, ESI ). The K value of 15.09 mM and maximum
3
m
current of 17.61 nA in solution are determined according to
Fig. 7. The reaction rate Vmax and the catalytic constant (kcat
are calculated as 123.53 mM s21 and 9.50 s21 using the same
H O –current calibration (Fig. S7, ESI ) method as described
)
3
2
2
above in nanofluidics. The resulting kcat value in bath solution
is also in accordance with the reported value.37
From the results, it is clear that the average Km value of ‘‘free
state’’ GOx in nanofluidics (3.88 mM) is nearly three times
smaller than in the bulk system (15.09 mM), and the reaction
rate kcat value increases by a factor of more than twofold when
changing from bulk to nanfluidics (an average value of 22.91
s21 in nanofluidics). The smaller value of Km indicates a
higher enzyme activity. Therefore, GOx activity increases in
nanospace confinement. This behavior might be due to two
reasons. First, the nanochannel provides an ideal confined
environment for accommodation of the enzyme, which
mimics the real conditions in living systems, which are very
different from the diluted bulk solutions in most reports on
enzyme activity assays. The crowded medium efficiently
restricts the available conformational space which protein
chains can occupy. The folding stabilities of proteins could be
increased in confined spaces. Accordingly, proteins have
higher stabilities inside the pores within chromatography
columns, the interiors of ribosomes, or the steric occlusion
inside cells.1,38 It has been reported that even when a protein
approaches a single planar wall, the equilibrium between the
native and unfolded states can be affected, leading to more
proteins in the native state.1 In another report,39 it has been
proven that the confinement of a protein in nanocages could
increase its thermodynamic stability in a way similar to the
Conclusions
In summary, a nanofluidics device integrated with an electro-
chemical detector has been used to study ‘‘free state’’ GOx
reaction kinetics in nanoconfined environments. In this device,
enzyme and substrate can be efficiently mixed in a nanochannel
through laminar flow due to the shortened diffusion time in
nanochannels, and thus an accelerated reaction rate can be
expected. Results show that the ‘‘free state’’ GOx activity in
nanospatial confinement increases significantly compared to
the immobilized and solution systems as indicated by the
remarkable decrease of the Michaelis–Menten constant Km, and
the reaction rate increases by a factor of about two compared
with the rates in bulk solution, demonstrating the importance
of ‘‘free state’’ and spatial confinement on the enzyme activity,
which would help us understand the fundamentals of enzyme
reactions confined in nanospaces.
Acknowledgements
This work was supported by the Grants from the National 973
Basic Research Program (2012CB933800), the National Natural
1552 | Lab Chip, 2013, 13, 1546–1553
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