Building three-dimensional nanostructures with active enzymes by surface templated layer-by-layer assembly

Article abstract of DOI:10.1039/b517557g

We show that well-defined three-dimensional nanostructures of functional enzymes can be controllably fabricated by layer-by-layer assembly of avidin and biotinylated horseradish peroxidase on micro-contact printing patterned surface templates. The Royal S

Full text of DOI:10.1039/b517557g

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Building three-dimensional nanostructures with active enzymes by
surface templated layer-by-layer assembly{
Sakandar Rauf,a Dejian Zhou,*ab Chris Abell,b David Klenermanb and Dae-Joon Kang*ac
Received (in Cambridge, UK) 13th December 2005, Accepted 2nd February 2006
First published as an Advance Article on the web 9th March 2006
DOI: 10.1039/b517557g
mainly on opposite sites on the HRP molecule, preventing the two
biotins from the same HRP binding to the same avidin.9
We show that well-defined three-dimensional nanostructures of
functional enzymes can be controllably fabricated by layer-by-
layer assembly of avidin and biotinylated horseradish perox-
idase on micro-contact printing patterned surface templates.
Scheme 1 shows the schematic of our approach (the ideal case).
mCP is used to pattern a gold surface with a well-defined SAM
template, with regions promoting the assembly of enzymes within
a resist background. This is achieved by printing micron-sized
SAM stripes of 11-mercaptoundecylhexa(ethylene glycol) alcohol
(EG6OH)7 as the resistive coating, followed by filling the
unstamped gold regions with a SAM of a biotin terminated thiol,
by solution self-assembly.7 Avidin is then specifically assembled
onto the biotinylated SAM regions via two of its biotin-binding
sites. This leaves two further biotin-binding sites for biotin-HRP
binding. This in turn produces a biotin terminated outmost layer
for binding of a second avidin layer. Repeating the LBL assembly
with avidin and biotin-HRP will produce a controlled growth of
the 3D enzyme structures. Since the enzymes are assembled on top
of a bio-compatible protein (avidin) layer, the immobilized
enzymes should maintain their catalytic function.9 It was
anticipated that such 3D enzyme nanostructures (with multiple
layers of active enzymes) should exhibit higher overall catalytic
activities than conventional immobilized enzyme arrays, which
usually contain just a single layer of enzymes.
The controlled positioning of functional biomolecules on surfaces
at the micro- to nano scale is important to the development of
array-based high-throughput screening techniques, novel biosen-
sors, bioelectronics, tissue engineering, and fundamental studies in
cell biology.1 This has been achieved by inkjet printing, micro-
fluidics, micro-contact printing (mCP), e-beam lithography, and
scanning probe based techniques.2 Most of these arrays however,
contained just a single layer of biomolecules.2 This may limit their
applications as highly sensitive sensors, since a multilayer structure
is often required to increase the sensitivity.3 Layer-by-layer (LBL)
assembly4 on patterned surfaces have been shown as an attractive
approach to achieve multilayer microscale structures with poly-
mers,5 nanoparticles,6 and biomolecules.7
We are particularly interested in the assembly of well-defined
three-dimensional (3D) structures with active enzymes, because
they hold important applications in miniaturised assays, sensors,
and devices.8 Despite extensive LBL assembly studies with
biomolecules, however, to our knowledge, multilayer 3D micro-/
nano structures with active enzymes have not been realized.7,9,10
Presumably nonspecific interactions associated with proteins can
lead to nonspecific adsorption on the resist background. To obtain
well-defined multilayer structures, the nonspecific adsorption must
be minimized. Here, we use a self-assembled monolayer (SAM)
terminated with hexa(ethylene glycol) groups, known for its ability
to reduce nonspecific biomolecule adsorption,11 as our resistive
coating. We choose horseradish peroxidase each labelled with on
average 2.3 biotins (biotin-HRP) as our model enzyme and avidin
as a biocompatible linker because each avidin has 4 biotin binding
sites, two on each side of the protein.12 This takes the advantage of
the extremely strong biospecific binding between the avidin and
biotin (dissociation constant, Kd y 10215 M).12 HRP labelled with
2.3 biotins is expected to be sufficient since biotinylation occurs
To confirm that multilayer avidin/biotin-HRP films can be
assembled controllably, we assembled homogenous films on gold-
coated silicon surfaces (y200 nm gold with y10 nm chromium)
and used ellipsometry to monitor the film thickness as a function
of the number of assembly cycles.13 The gold surface was coated
with a SAM of the biotin-terminated thiol by incubating it in a
aNanoscience Centre, University of Cambridge, 11 J J Thomson Avenue,
Cambridge, UK CB3 0FF
bDepartment of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, UK CB2 1EW. E-mail: dz209@cam.ac.uk;
Fax: (+44) 1223-336362; Tel: (+44) 1223-330107
cSungkyunkwan Advanced Institute of Nanotechnology, Institute of
Basic Science, CNNC and Department of Physics, Sungkyunkwan
University, Suwon, 440-746, Korea. E-mail: djkang@skku.edu;
Fax: (+82) 31-290-5947; Tel: (+82) 31-290-5906
{ Electronic supplementary information (ESI) available: Details of the
experimental procedures, materials and methods, enzyme activity and
AFM measurements. See DOI: 10.1039/b517557g
Scheme 1 Schematic of our approach to assemble 3D structures with
active enzymes.
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because the first avidin layer is not densely packed, with a
thickness of y2.3 nm, which is much smaller than a thickness of
y5.5 nm expected from a densely packed avidin layer (the size of
avidin is y4.0 nm 6 5.5 nm 6 5.5 nm).14
Next we assembled the enzymes on the mCP patterned surface
templates under identical assembly conditions as those used in the
ellipsommetry studies. We used atomic force microscopy (AFM,
tapping mode in air) to monitor the topographic evolution of the
enzyme patterns following the LBL assembly process.6,7 Fig. 2A
shows the topographic image of a mCP-patterned SAM template,
with 2 mm SAM stripes of the EG6OH separated by the biotin-
thiol stripes, on a flat thin-gold coated surface. The EG6OH stripes
are y2.4 nm taller than the biotin-thiol stripes. This agrees well
with the thickness difference between the two SAMs (the EG6OH
and biotin-thiol SAMs are found to be y3.4 and y1.0 nm,
respectively).7 After a bilayer of avidin/biotin-HRP is assembled
onto the template, the topographic contrast between the protein
and EG6OH stripes is reversed (Fig. 2B). The protein stripes are
now y1.2 nm higher than the EG6OH stripes, presumably
because the proteins preferentially assembled onto the biotin
stripes. Despite using the EG6OH SAM, one of the most effective
resistive coatings to biomolecules, some non-specific adsorption on
the resist stripes was still observed.16 We found most of the non-
specifically adsorbed proteins could be removed by treating the
surface with a surfactant (0.1% Tween 20 in PBS) for 1.5 h. The
treatment resulted in an increased height contrast between the two
stripes to y3.2 nm (Fig. 2C). Further, we found that rinsing with
the surfactant after each assembly step significantly reduces
nonspecific adsorption on the resist stripes while still maintaining
the specific assembly on the functional stripes. Each sample with a
given number of bilayers was individually prepared, because the
samples need to be thoroughly rinsed with water to remove salt
and dried before the AFM measurements. Such treatment may
partially denature the assembled proteins.
Fig. 1 Plot of the avidin/biotin-HRP film thickness vs the number of the
assembly bilayers.
biotin-disulfide solution.7 The first avidin layer was prepared by
incubating the surface with an avidin solution for 30 min. After
rinsing with PBS, the surface was incubated with a biotin-HRP
solution for 30 min followed by rinsing with PBS. This constituted
an assembly cycle (denoted as (avidin/biotin-HRP)1). The surfaces
were thoroughly washed but not dried between each assembly step.
A specific number of bilayers was made before each measurement.
This precaution was found to be critical to maintain the protein
function by reducing the possibility of protein denaturation due to
drying.
Fig. 1 shows the thickness of the avidin/biotin-HRP film as a
function of the number of assembly cycles. It is clear that the
protein film thickness increases linearly with the number of the
assembly cycles (R 5 0.998), suggesting a regular and well-
controlled LBL build-up of the enzyme film after the first bilayer.
The average thickness of each avidin/biotin-HRP bilayer,
y1.44 nm is, however, significantly smaller than the combined
thickness of closely packed HRP and avidin layers (y8–10 nm,
estimated from the dimensions of the proteins).14 This suggests
that the proteins are not closely packed within the film, and similar
results have been reported by other groups.10 This is not surprising
By using a surfactant rinse in between each assembly step, multi-
layered 3D structures of avidin/enzyme can be readily fabricated.
Fig. 2D shows the topographic image of the patterned surface after
Fig. 2 AFM topographic images showing the evolution of the avidin/biotin-HRP 3D structures. Images sizes, 20 mm 6 20 mm for A, B, and C, and
40 mm 6 40 mm for D. Images A, B, and C are on the left height scale, and image D is on a different scale shown on the right. (A) A mCP-patterned surface,
the (EG)6OH stripes are y2.4 nm taller than the biotin-thiol stripes. (B) After assembly of a bilayer of avidin/biotin-HRP, the protein stripes are y1.2 nm
higher than the EG6OH stripes. (C) The same as B after a treatment with the surfactant, the protein stripes are y3.0 nm higher than the EG6OH stripes.
(D) After assembly of 9 bilayers of avidin/biotin-HRP, the protein stripes are y12 nm higher than the EG6OH background.
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the assembly of 9 avidin/biotin-HRP bilayers. The height of the
enzyme strips is now y12 nm above the resist background. This
corresponds to a total protein layer thickness of y14.4 nm, taking
into account the original thickness difference between the SAMs.
This thickness broadly agrees with that of the ellipsommetry
datum (y15 nm). The topographic image also shows the resist
background regions are reasonably clean, with little nonspecific
adsorption on the EG6OH region, confirming a well-controlled
layer-by-layer build-up of the 3D enzyme structures on the
functional pattern region.
knowledge, this is the first example of multilayer 3D structures
assembled from active enzymes. Miniaturization of the structures
to the nanoscale should further improve the activity of such
enzyme assemblies. Furthermore, operating in 3D would allow
more functions, unavailable from 2Ds, to be realised. For example,
bi-enzyme and multi-enzyme systems can also be introduced into
these 3D structures to achieve higher sensitivity.18 These 3D
enzyme nanostructures may have wide applications in highly
miniaturized biosensors, biocatalysts, tissue engineering, biochips,
and to study biological processes.
An absorption-based assay was used to investigate the activity
of the immobilized homogenous enzyme films prepared rinsing
with PBS only in between each assembly step without the
surfactant. In the presence of H2O2, the HRP catalyses the
turnover of a non-coloured substrate, Amplex red, into a coloured
product, resorufin, that strongly absorbs at 571 nm.15 The assay
was carried out in PBS with 5 mM H2O2 and 25 mM Amplex red.
The changes in absorbance at 571 nm at different time intervals for
the 1, 5 and 9 bilayer samples were investigated (Fig. S1).{ All
three samples are catalytically active, and the overall activity
increased with the number of the enzyme layers. The enzyme
activity in first bilayer is estimated to be y14% that of the free
enzymes in solution.{ This level of catalytic activity is somewhat
better than some covalently immobilised HRPs on flat surfaces,
where a y5% of the free enzyme activity has been reported.17 The
rate of activity increase as a function of enzyme layers is sub-linear,
with the 5- and 9-bilayer films being only y25% and y40% more
active than the first bilayer. The rate of the catalytic activity
increase is slower than those immobilized on submicron beads,
where multilayer enzymes produced up to 5-fold increase in the
total catalytic activity.9 Presumably the beads have a bigger surface
area, and can also diffuse in solution to improve substrate
accessibility. Nonetheless, we have achieved an improved overall
catalytic activity per unit surface area by using the multilayer
enzyme structure.
We acknowledge the financial support of Interdisciplinary
Research Collaboration in Nanotechnology, UK. This work was
also supported by the SRC program (Center for Nanotubes and
Nanosructured Composites) of MOST/KOSEF and, in part, the
Ministry of Science and Technology of Korea through the
Cavendish-KAIST Cooperative Research Program.
Notes and references
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The fact that the 9-bilayer sample is only 40% more active than
the first bilayer suggests that the enzyme activity of the multilayer
film mainly comes from the outmost enzyme layer, with a minor
contribution from the inner layers. Presumably the exposed
outmost enzymes are most directly accessible to the substrates,
those at the inner layers have to rely on substrate diffusing through
the outer protein layers to reach them, and thus have a much lower
substrate accessibility (and hence the apparent activity). The
contribution from the inner enzymes is reflected by the slightly
higher activity of the 9-bilayer film as compared to the 5-bilayer.
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In summary, we have successfully fabricated well-defined
multilayer 3D enzyme structures by using mCP-patterned SAM
templates to guide the layer-by-layer assembly of avidin and
biotin-HRP. This precisely controls the height and the positions of
the enzyme structures assembled on surface. The assembled
enzymes maintain some catalytic activity, with the overall activity
increasing with the increasing number of the enzyme layers. To our
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