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www.elsevier.com/locate/apsusc
Applied Surface Science 253 (2007) 8119–8124
Printing technologies for fabrication of bioactive and regular
microarrays of streptavidin
C.Z. Dinu a, V. Dinca b,*, J. Howard a, D.B. Chrisey c,d
a Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germanyb Institute of Electronic Structure and Laser, Foundation for Research and Technology, Hellas, Heraklion, Greece
c US Naval Research Laboratory, USAd Rensselaer Polytechnic Institute, Troy, NY, USA
Available online 6 March 2007
Abstract
In this study, we report and compare two methods for fabricating patterns of streptavidin protein using soft litography microprinting technique
(mCP) and laser-based method termed ‘matrix assisted pulsed laser evaporation direct write’ (MAPLE DW). The mCP approach is a parallel
deposition technique capable of X depositions per stamper. The technique is limited in more sophisticated multicomponent deposition by the size
of patterns that can be produced and the features obtained during the transfer process. The computer-aided design/computer-aided manufacturing
(CAD/CAM) ability of MAPLE DWovercomes the limitations of the mCP approach. (i) We establish the science and engineering principles behind
the effective transfer of microarrays and (ii) we explore issues regarding the direct immobilization, morphology and function of the deposited
protein at the interface with an aqueous environment and in the precision of controlled ligand-receptor reactions. In summary, our objective was to
develop simple, robust microfabrication techniques for the construction of model 2D and 3D bioscaffolds to be used in fundamental bioengineering
studies.
# 2007 Published by Elsevier B.V.
Keywords: Bioengineering studies; Ligand-receptor; Microfabrication
1. Introduction
Proteins are building blocks of living organisms; they play
numerous roles in vivo such as sensing the environment,
processing the information, acting as molecular recognition and
catalytic units [1,2]. Integrating their functionality in engi-
neered environments and in relation with other biological
ligands can produce sensing devices and 3D biomolecular
interfaces.
Heterogenous functionalization of solid surfaces by micro
and nanopatterning provides powerful strategies to generate
active bio-interfaces. Different technologies can be applied:
layer by layer [3,4], molecular imprinting [5–7], Langmuir–
Blodgett technique [8–10], plasma-spray [11,12], microcontact
printing [13,14], laser-particle guidance [15–17], liquid or
droplet microdispensing approaches [17]. Most of the listed
* Corresponding author.
E-mail address: [email protected] (V. Dinca).
0169-4332/$ – see front matter # 2007 Published by Elsevier B.V.
doi:10.1016/j.apsusc.2007.02.199
immobilization techniques even if they give reproducible
results, they lack in proving that the native conformation of the
deposited biomaterial and hence its functionality is preserved.
Also, many times they require stepwise processes, high-quality
starting materials, modified surface coatings, expensive organic
precursors, solvents and surfactants [18]. Therefore, the success
of any transfer printing technology would depend on the
reliable control of the dynamic process of delivery of the
‘‘ink = donor’’, the quality of the ‘‘ink’’ and the acceptance and
quality of the ‘‘paper = acceptor’’.
Microcontact printing (mCP) – referred to as soft litography
[19] – has emerged as a fast stamping technique with capability
to process large surfaces in one run. In mCP an elastomer
polydimethylsiloxane (PDMS) is used to form a stamp to be
coated with an ‘‘ink’’ containing the biological molecules to be
transferred [20]. The main function of this technology is to
establish the direct contact with the substrate, in a precise
manner and a given printing time by reducing as much as
possible any mechanical forces that might affect the viability of
the transferred material [21,22]. With mCP different biological
Fig. 1. Schematic diagram of microcontact printing technique.
C.Z. Dinu et al. / Applied Surface Science 253 (2007) 8119–81248120
materials have been printed: alkanthiols [19], proteins [13],
DNA [23] or cells [24]. However, it was shown that mCP
printing could lead to ill-defined patterns, to aggregations and
loss of activity of the printed material [19]. Therefore,
alternative methods have to be exploited. Ideally, any
alternative methods should bring a defined quantity of
biomolecules in contact with a surface and as such induce
adsorption with high spatial control and minimum loss of
functionality.
Recent research has focused on the direct writing of
biological material by laser transfer technology matrix assisted
pulsed laser evaporation direct write (MAPLE DW) [25–27].
Generally, MAPLE DW uses a laser pulse to transfer material
from a ‘‘donor’’ to an ‘‘acceptor’’ surface. The material is
transferred in micron sized voxel patterns with high precision,
high resolution and in a computer-aided design/computer-aided
manufacturing (CAD/CAM) manner [27,28]. In order to
preserve the functionality and to prevent high evaporation
rates or possible cross-contamination a ‘‘buffer’’ is used as
embedding medium [27].
In this paper, we used mCP and MAPLE DW to manufacture
patterns of streptavidin on glass surfaces. We perform a
comparative study by addressing issues related to homogenous
protein pattern formation, interface adhesion reactions and
printing; in some cases, we also study protein activity after
transfer by evaluating its functionality through a ligand-
recognition reaction. Our goal was to develop a method able to
produce mesoscopic patterns of organic molecules with similar
morphological integrity while with reduced size, in a controlled
and reliable manner and at predetermined sites.
2. Materials and methods
2.1. Cleaning the acceptor surfaces
The acceptor surfaces were cover slips glass substrates
(Corning, US, 22 mm � 22 mm) previously cleaned in a
sequential manner. Briefly, the cleaning consisted in: sonication
in 10% soap solution for 5 min, rinsing with double distillated
water, sonication in 70% ethanol solution for 5 min, rinsing
with double distillated water (all liquid reagents were from
Sigma, Germany). The surfaces were dried right before use to
avoid any contamination.
2.2. Microcontact printing
PDMS stamps were fabricated by casting and curing Sylgard
184 (Dow Corning, Germany) elastomeric polymer against
photoresist micropatterned silicone masters (TU Dresden
Facility, Germany). Different stamp configurations were used
for these experiments: stripes or squares geometries. The
PDMS stamps were first cured for 4 h at 65 8C and then exposed
to inking streptavidin protein solution (fluorescein labeled and
unlabelled; 100–200 mg/ml) (Pierce, US) for various amounts
of time (1, 3 and 5 min, respectively). Excess solution was
removed with a stream of nitrogen gas. Subsequently, the inked
stamp was brought in direct contact with the cleaned glass by
applying a low mechanical force (Fig. 1). Once the stamp was
removed, the glass was analyzed in epi-fluorescence or used to
form a perfusion chamber.
2.3. Perfusion chamber experiments
In order to determine whether the deposited patterns are
functional, we performed a fluorescence assay in a perfusion
chamber. The perfusion chamber was built up from a cleaned
glass (Corning, US, 24 mm � 50 mm) and the patterned cover
glass separated by a double side tape spacer [29]. The chamber
was precoated with a casein containing solution (0.8 mg/ml,
Sigma, Germany) for 5 min (to avoid any subsequent unspecific
binding) and subsequently with a solution containing biotin
rhodamine labeled (1 mg/ml, Pierce, US) for 5 min, in dark, at
room temperature. After the incubation, any unbound biotin
C.Z. Dinu et al. / Applied Surface Science 253 (2007) 8119–8124 8121
was washed away (PBS, Sigma, Germany) and the sample was
analyzed with the epifluorescence microscope.
2.4. Microscope evaluation
The perfusion chamber was mounted on an inverted
fluorescence microscope Axiovert 200 M (Zeiss, Jena, Ger-
many) equipped with a high magnification (100�, N.A. = 1.4)
oil immersion objective with the appropriate blue and red
(FITC and TRITC) filters. Images were acquired using a 16-bit
high resolution cooled digital frame transfer CCD (Micromax,
512 BIT Visitron, Germany) and captured on a computer
equipped with Metamorph imaging software (Visitron,
Germany).
2.5. MAPLE DW-general set-up
MAPLE DW used a ArF excimer laser (193 nm laser
wavelength, 30 ns; 1 Hz pulse width) (Lambda Physik 305i, US)
to transfer biomolecules from a donor-coated surface, the ribbon,
to an acceptor surface, the glass. The laser fluence was varied
between 25 and 35 mJ/cm2. The deposition was done in ambient
conditions (air and room temperature). In order to decrease the
laser beam a circular aperture (100 mm) was used (Fig. 2).
The ribbon was an UV transparent quartz disk coated with a
solution at neutral pH. The solution contained streptavidin
(1 mg/ml, Pierce, US) embedded in a non-toxic, plasma-
consistency methylcellulose medium (1%, Sigma, Germany).
Using a pipette, 50 ml of the neutral solution was spread onto
the ribbon and spun in four steps as it follows: 20 s at 1200 rpm,
10 s at 100 rpm, 10 s at 300 rpm and last step, 20 s at 3000 rpm.
Homogenous layers with a thickness of about 100 mm were
obtained.
The acceptor surfaces were cover slips glass substrates
(Corning, US, 22 mm � 22 mm) cleaned as previously
Fig. 2. The MAPLE DW set-up. With the laser irradiation and through an evapora
surface. An aperture was used to control the diameter of the deposited patterns.
described. The ribbon was separated from the acceptor by
100–200 mm space. The gap between the ribbon and substrate
was controlled with a z-stage translation.
2.6. Microscope evaluation
An inverted fluorescence microscope Olympus IX71 (US)
equipped with high magnification objectives (10�, 20�, 50�)
was used to analyze the MAPLE deposited micropatterns.
Images were acquired in single reflection, using a V-shaped
optical path with apochromatic relay lenses.
3. Results and discussions
3.1. Soft lithography (mCP)
Using mCP patterns of proteins were printed on a glass
substrate. For this, a PDMS stamp was used to deliver labeled
streptavidin (Fig. 3a–c). In some cases, unlabelled streptavidin
was printed and served as template for further ligand-
recognition reaction (Fig. 3d). Briefly, fluorescein labeled
biotin was incubated with the printed streptavidin in a perfusion
chamber. During the incubation process, the streptavidin target
specifically bound biotin capture molecules from the aqueous
solution. Any unbound molecules were washed away. The
fluorescence was quantified with an inverted epifluorescence
microscope.
Microcontact printing was an excellent way of creating
active streptavidin patterns on a solid glass substrate. The
mechanism of transfer was due to the fact that binding between
the cleaned glass surface and the proteins was stronger than the
adhesion force between the proteins and the PDMS stamp.
However, the transfer was not only localized to the stamp
patterns: there were also molecules transferred between the
patterns (in line scans images quantifying the fluorescence
tion process, proteins were transferred from the donor surface to the acceptor
Fig. 3. Deposition of streptavidin molecules by soft lithography. Herein scale bar was 10 mm. (a) Fluorescence micrographs of fluorescein labeled streptavidin
printed on a glass surface in a square like geometry. The pattern size was about 5 mm. (b) Fluorescence micrographs of fluorescein labeled streptavidin on a glass
surface in a square like geometry. The pattern size was about 10 mm. (c) Fluorescence micrographs of fluorescein labeled streptavidin on a glass surface in a stripes
like geometry. The transferred stripes were about 2 mm. (d) Fluorescence micrographs of rhodamine labeled biotin bound specifically to streptavidin previously
printed on the substrate. The streptavidin was transferred in a stripe like geometry (feature size of about 2 mm) and served as template for further ligand: labeled biotin.
C.Z. Dinu et al. / Applied Surface Science 253 (2007) 8119–81248122
intensity on and in between the patterns: images not shown).
This was presumably due to the fact that not only the inking
molecules were transferred but also parts of the PDMS stamp.
With the reduction in pattern size, defects in the transfer
appeared: the patterns were not well defined, nor homogenous.
More important, they were not anymore continuous (i.e. no
free-standing structures). This was mainly due to the instability
of the stamp features to mechanical stresses applied at the
transfer [19].
Finally, we tested the functionality of the immobilized
streptavidin. For this, site-specific attachment of biotin to the
streptavidin bioscaffold gave the patterns the property of
changing their optical characteristics as they switch between
two different states-free/bound. The observed fluorescence
signal allowed us to conclude that the deposited patterns were
viable.
3.2. Matrix assisted pulsed laser evaporation direct write
(MAPLE DW)
Using MAPLE DW patterns of streptavidin were deposited
on a glass substrate. The fraction of the molecules expelled
from the ribbon was directly correlated to the laser fluence
(Fig. 4). To determine if any reduction in pattern size was
possible, an aperture with fixed opening was used (Fig. 5). We
observed how rate of energy deposition affected desorption of
biomolecules from the ribbon. When less energy was used, a
smaller degree of overheating was reached at the end of the
laser pulse and thus a less violent evaporation occurred. For
high fluences (F1 and F2, respectively), the patterns were not
homogenous being formed from splashed particles. With
decreasing the laser fluence, regular mesoscopic patterns were
formed and no observable splashing occurred.
The mechanical or adhesive properties and thus the adhesion
force in vapor is always less than in vacuum due to the capillary
condensation of the water around the surface contact sites [30].
As such, one specific goal was to control the adhesion of the
streptavidin patterns to the glass at room temperature
conditions. It is known that adhesion between protein and
glass surfaces varies as a function of ionic strength (IS), pH,
loading force, isoelectric point (IP) and the residence time
[31,32]. As such, the adhesion force between the protein and the
surface increases with residence time and decreases with IS and
pH. Changes in pH can alter the conformation of the protein
therefore preserving the IP is mostly desired when preserving
the protein activity [33]. This was achieved by using an
Fig. 4. Optical micrographs of streptavidin protein patterns deposited on a glass substrate by MAPLE DW. During the transfer process, the optical fluence was varied
from 35 to 25 mJ/cm2 with F1 (in a) > F2 (in b) > F3 (in c) > F4 (in d). The changes with the fluence were revealed.
C.Z. Dinu et al. / Applied Surface Science 253 (2007) 8119–8124 8123
embedding media that preserved the IP [34] by acting as an
energy absorber [25,35] and causing a limitation of the
diffusion and an apparent stabilization of the protein [36].
Restricting the laser beam by using variable apertures
proved that if aimed, reduction in the pattern size was possible.
Precisely, we show that by using a circular aperture one can
Fig. 5. Micrographs of streptavidin patterns obtained using MAPLE DWand an
aperture that limited the laser beam.
obtain patterns with the same morphological integrity, well-
defined, not ill-masked as in the case of mCP and with a reduced
size (up to 10 mm). This is presumably due to improving the
beam quality of the excimer laser [37]. The preceding spatial
filtering and the aperture removed as much of the non-coherent
beam as it could. This was critical for focusing and for beam
uniformity and combined with the final aperture leaded to small
transferred patterns.
When compared with mCP, in MAPLE we clearly show a
reduction in size complementary to well defined and with the
same morphological characteristics patterns. Also, we show
that there is no transfer outside the pattern given by the fact that
the media presumably favorized the high absorption to the
substrate and cancelled the external splashing. In comparison
with mCP, MAPLE DW has also an improved resolution and
C.Z. Dinu et al. / Applied Surface Science 253 (2007) 8119–81248124
moreover the system is envisaged as an automated CAD/CAM
device, where micropatterns with desired location can be
produced. Key advantages of the system are the automated
interface, the rapid process time, the ease of operation and the
ability to dynamically interrupt the process any time after
achieving the requested patterns.
4. Conclusions
We have presented and characterized two microfabrication
techniques to print biological molecules on biomaterial
surfaces: the first is based on soft lithography and the second
one is based on laser printing. Regardless of the technique used,
the possibility of generating bioscaffolds with well-defined
geometry, morphology and at the micron scale enables complex
bioengineering studies.
Acknowledgements
The Office of Naval Research Global (ONR) financially
supported the travel for the MAPLE research (contract: STEP
4046). Chrisey gratefully acknowledges the Office of Naval
Research which supported the research.
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