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: valentina.dinca@inflpr.ro (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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