# Fibronectin Adsorption to Nanopatterned Silicon Surfaces

Authors: I. Salakhutdinov; P. VandeVord; O. Palyvoda; H. Matthew; G. Tatagiri; H. Handa; G. Mao; G. W. Auner; G. Newaz
Journal: Journal of Nanomaterials
 (2008)

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## Abstract

The possibility of using surface topography for guidance of different
biological molecules and cells is a relevant topic that can be applied to a wide
research activity. This study investigated the adsorption of fibronectin to a
diffraction grated silicon surface. The rectangular grating profile featured a
controlled surface with 350 nm period and a corrugation depth of
90 nm. Results demonstrated that the controlled surface had a
significantly positive effect on the fibronectin binding. Thus, nanoscale
surface topography can enhance fibronectin binding.


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## Body

## 1. Introduction

The possibility
of using surface topography for guidance of different biological molecules and
cells is a relevant topic that can be applied to a wide research activity [1].
One important subject is the attachment of various plasma and extracellular
proteins to biomaterial surfaces [2]. Fibronectin (FN) is a well defined
extracellular protein consisting of two dimer subunits, each is about 250
kilodaltons (kD) and an elongated shape with dimensions 45nm×9nm×6nm
[3]. FN plays a key role in cell
adhesion and mediating cell response, thus it will be used as a model for
protein adherence in our study. Although FN adsorption on different materials
has been investigated very actively, it has not been identified how FN adherence is altered in response to nano- and
micropattern roughness. We used diffraction grating on silicon substrate as
controllable roughness to investigate alterations in FN adsorption. Diffraction grating fabrication
technologies are well developed, thus it is possible to fabricate diffraction
gratings with a wide range of grating periods, corrugation depth, and grating
profile.Many engineering applications have focused on
biomimetic sensors based on waveguide technology. Considering new advances in
microelectromechanical systems/nanoelectromechanical systems (MEMS/NEMS)
fabrication, soft lithography, and the development of smart adhesives,
integration of complementary metal-oxide-semiconductor (CMOS)
and MEMS/NEMS should be further explored to provide the infrastructure for
integration of the whole silicon-based sensory system especially in controlling
host-biomaterial interactions. Any
attempt to make a sophisticated, functional surface for biointeractions must
take into account the highly developed ability of biological systems to
recognize specially designed features on the molecular scale [4]. The
materials used in BioMEMS/BioNEMS devices must exhibit desirable micro-/nanoscale
tribological and mechanical properties [5]. From the cellular perspective, the
interactions of cells with each other and extracellular materials (proteins,
matrices, solid surfaces, etc.) are of vital importance to proper cell
functioning. These interactions have major effects on the proliferation,
differentiation, migration, and organization of cells [6, 7]. When designing
novel biomaterials properties, one must understand that when an implant surface
comes into contact with physiological solutions, proteins adsorb immediately on
material surface. This adsorption is
known to cause conformational changes in the native protein structure with the
possibility of subsequently promoting or inhibiting nearby cells to interact
with material, thus leading to implant integration or rejection [4, 6, 8].
Recent studies on protein/material surface interactions have increased the
knowledge base on this topic and this relationship appears to be mediated by a
class of high molecular weight glycoproteins that are involved both in these
interactions and in the actual structure of extracellular matrices. Some of the
most intensively studied glycoproteins are FN, laminin, Willebrand protein,
thrombospondin, and vitronectin [9–11]. The general structural outline of FN consists
of a dimer of two subunits, each is about 250 kilodaltons (kD) [12]. Each
subunit is folded into an elongated and flexible arm 60 nm long, and the two
subunits are joined by disulfide bonds very near their C-termini. Within each
subunit, there is a series of tightly folded globular domains; each specialized
for binding to other molecules such as collagen,
glycosaminoglycans, transglutaminase, or to cellular membrane receptors [13, 14]. Since it is known that cells
may never see the native biomaterial, the configuration of the absorbed proteins is of utmost importance in
cell activation and response. By optimally designing a surface for a specific
protein conformational change, we must take into account how the protein 3D
topography and chemical structure will affect its absorption onto the material
surface. To further investigate
the phenomenon of protein adsorption and the effect of nanoscale modulation of
the surface, we chose to examine how nanoscale modulation affects FN binding to
silicon surfaces.


## 2. Grating Characterization

As mentioned, the mechanisms of protein absorption to patterned structures are not
clear yet. We chose diffraction grating technology for two reasons. Firstly,
there are several results with regards to the role of periodic structures
positively affecting the attachment of biological objects, with emphasis on
cells [15, 16]. Secondly, diffraction gratings are one of
themost widely used optical instruments that are
very well investigated both theoretically and practically. Diffraction grating
technologies are recognized to permit for a defined grating period, corrugation
profile, and corrugation depth.There are three main practical diffraction
grating profiles: sinusoidal, trapezoidal, and rectangular. Figure1 presents a
simplified model for optimal grating profile and hypothesized protein
attachment based on assumptions described in [17].Attachment
model for different diffraction grating profiles.
(a)(b)(c)The next parameter to determine is corrugation depth. We expect that optimal corrugation depth will
correspond to the maximum of diffraction efficiency. Figure 2 presents results of calculations of
diffraction efficiency verses the corrugation depth made by modified C-method [18].Figure 2
Diffraction efficiency at
1st order dependence versus the corrugation depth.In total, we decided to use
nanopatterned surfaces with a period about 350 nm (175 nm plateaus and 175 nm
valleys), a corrugation depth about 90 nm and rectangular grating profile to
explore protein adsorption onto silicon surfaces.


## 3. Technology and Experiment

### 3.1. Grating Fabrication

P(boron)-type silicon wafers from Silicon Quest International, (Santa
Clara, Calif, USA), with〈1‐0‐0〉 orientation with
thickness equal to 510–540μm; material resistivity was 4–20 Ω-cm, were utilized
for this study. Diffraction gratings
were fabricated by optical holography. As a laser source, we used Coherent
INNOVA 300C FReD Ar laser with frequency doubling. The diffraction grating was
fabricated on the silicon substrates by holography with UV5 photoresist as a
mask material. The mask structures were etched by RIE DryTek systemat the following conditions: C2F6—40 sccm; O2—8 sccm; RF power—120 W; pressure—223 mTorr.
These conditions resulted in diffraction gratings with rectangular
profile having size of 3mm×5mm within the total 10mm×10mm silicon substrate.


### 3.2. Atomic Force Microcope (AFM) and Scanning Electron Microscope (SEM) Measurements

In order to assess surface
topography of the grated surfaces, AFM (Nanoscope III, Digital Instruments/VEECO) was used. All the AFM images were
obtained using an E scanner with maximum scan area14.2×14.2μm2.
Height, deflection, and friction images were obtained in contact mode in
ambient air with silicon nitride tips (NP, VEECO). The scan rate used was 0.8–1 Hz. Integral and proportional gains were approximately 2.0 and 3.0,
respectively. Figure 3 presents an example of AFM measurements for the
fabricated gratings. It is very important that our samples have highly
homogeneous nanopatterned structure. Five different areas of the diffraction
grating were analyzed to check the periodicity and the depth of the grating.
Nanoscope software was used to analyze the images. Using sectional analysis,
the periodicity of the grating was found to be 355 nm ± 0.08 nm and the height
was found to be 87 nm ± 3 nm (Height information might not be very accurate as
may be the tip is not reaching the bottom most point of the grating).AFM
image of fabricated diffraction grating used as controllable rough surface and
the sectional height profile along the line.
(a)(b)In order to verify AFM measurements, we made
SEM image of the fabricated grating. These measurements confirmed the high
uniformity of the fabricated gratings; this is an important factor for our
biomedical research.In
summary, the grating was found to be highly uniform in periodicity and height
at various places. A 2D image of a two-dimensional grating is shown in Figure5.
The homogeneity was demonstrated to be 0.02% for the grating period and 2.0%
for the grating corrugation depth.


### 3.3. Protein Adsorption Assay

Prior to protein absorption,
all silicon samples were  cleaned via the RCA cleaning procedure. After which,
human FN
(Sigma, St. Louis, Mo, USA) was reconstituted to a final concentration of 10μg/ml in phosphate buffered saline (PBS).
Protein was adsorbed onto experimental silicon surfaces by immersion in the
prepared FN solution
for 2 hours at room temperature with gentle rotation. Additional samples were
also immersed in PBS solution without protein to be used as control surfaces.
After incubation, the solutions were removed and the samples were carefully
washed 3 times with PBS to eliminate any unbound protein. Care was taken in
order to prevent the drying of the protein-coated surfaces before further analysis.


### 3.4. Immunodetection of Adsorbed Proteins

Patterned and control
surfaces were removed from PBS and incubated with 2% bovine serum albumin (BSA;
Sigma, St. Louis, Minn, USA) solution for 2 hours in room temperature in order
to block later nonspecific antibodies binding. Immunostaining procedure was
performed with FN
chicken antihuman antibodies (Invitrogen, Chicago, Ill, USA) diluted1∶1000.
Following a PBS wash, samples were then incubated for 2 hours with an Alexa
Fluor 488 goat antichicken IgG (H+L) (Invitrogen, Chicago Ill, USA) diluted
1∶200. Both primary and secondary antibodies were individually diluted in PBS
with 1% BSA. Between each step of the immunostaining procedure, samples were
repeatedly washed with PBS. For each assay, an additional control was prepared
consisting of a protein-coated sample submitted to the same described procedure
but instead of incubating with the primary antibody, PBS was used. Thus, the protein-coated
samples were exposed to the secondary antibody only as a control. Subsequently,
the silicon wafers were mounted on glass slides using ProLong Gold Antifade
reagent (Invitrogen, Chicago Ill, USA). Immunostaining results were observed
and recorded using fluorescent microscope (Nikon Eclipse TE2000-U). Ten digital
images of each sample (n=3) were captured and analyzed for average pixel
intensity on both the patterned and nonpatterned areas using Image J Software.
The results demonstrated a significantly higher (P=01) level of
fluorescence on the patterned area as compared to the nonpatterned surface (Figure 4). These
results strongly indicate a higher level of FN protein attachment occurring on
the patterned surface as compared to the nonpatterned surface.Figure 4
SEM
image of the fabricated grating.(a)
Fluorescence micrograph of the border between the patterned surface (right) and
the nonpatterned surface (left) (200x). (b) Graph depicts the average pixel
density from the fluorescence microscopy analysis.
(a)(b)Figure 6
2D diffraction grating
with grating periodΛ=351.2 nm.


### 3.5. Further Development

The mechanisms of why there was an increase
in protein attachment on the patterned surfaces are not clear yet. Thus it will
be interesting to investigate variations in nanopatterned structures (size and
shapes) which could be effective for alterations in protein attachment. After
fabrication of 1D diffraction grating by deep UV lithography, we also
fabricated 2D gratings with the same periodΛ=351.2 nm in both coordinates (Figure 5).To fabricate such structures, we
simply exposed the surface twice. Prior to the second exposure, we rotated structure
on 90°. We expect that 2D gratings will give another prospective structure for
examining changes in protein adsorption. It has been shown that 2D periodical structure
is a good candidate for using of tuning localized plasmons for the surface-enhanced
Raman scattering [19]. Future studies
will compare protein adhesion of 1D and 2D nanopatterned surfaces.


## 3.1. Grating Fabrication

P(boron)-type silicon wafers from Silicon Quest International, (Santa
Clara, Calif, USA), with〈1‐0‐0〉 orientation with
thickness equal to 510–540μm; material resistivity was 4–20 Ω-cm, were utilized
for this study. Diffraction gratings
were fabricated by optical holography. As a laser source, we used Coherent
INNOVA 300C FReD Ar laser with frequency doubling. The diffraction grating was
fabricated on the silicon substrates by holography with UV5 photoresist as a
mask material. The mask structures were etched by RIE DryTek systemat the following conditions: C2F6—40 sccm; O2—8 sccm; RF power—120 W; pressure—223 mTorr.
These conditions resulted in diffraction gratings with rectangular
profile having size of 3mm×5mm within the total 10mm×10mm silicon substrate.


## 3.2. Atomic Force Microcope (AFM) and Scanning Electron Microscope (SEM) Measurements

In order to assess surface
topography of the grated surfaces, AFM (Nanoscope III, Digital Instruments/VEECO) was used. All the AFM images were
obtained using an E scanner with maximum scan area14.2×14.2μm2.
Height, deflection, and friction images were obtained in contact mode in
ambient air with silicon nitride tips (NP, VEECO). The scan rate used was 0.8–1 Hz. Integral and proportional gains were approximately 2.0 and 3.0,
respectively. Figure 3 presents an example of AFM measurements for the
fabricated gratings. It is very important that our samples have highly
homogeneous nanopatterned structure. Five different areas of the diffraction
grating were analyzed to check the periodicity and the depth of the grating.
Nanoscope software was used to analyze the images. Using sectional analysis,
the periodicity of the grating was found to be 355 nm ± 0.08 nm and the height
was found to be 87 nm ± 3 nm (Height information might not be very accurate as
may be the tip is not reaching the bottom most point of the grating).AFM
image of fabricated diffraction grating used as controllable rough surface and
the sectional height profile along the line.
(a)(b)In order to verify AFM measurements, we made
SEM image of the fabricated grating. These measurements confirmed the high
uniformity of the fabricated gratings; this is an important factor for our
biomedical research.In
summary, the grating was found to be highly uniform in periodicity and height
at various places. A 2D image of a two-dimensional grating is shown in Figure5.
The homogeneity was demonstrated to be 0.02% for the grating period and 2.0%
for the grating corrugation depth.


## 3.3. Protein Adsorption Assay

Prior to protein absorption,
all silicon samples were  cleaned via the RCA cleaning procedure. After which,
human FN
(Sigma, St. Louis, Mo, USA) was reconstituted to a final concentration of 10μg/ml in phosphate buffered saline (PBS).
Protein was adsorbed onto experimental silicon surfaces by immersion in the
prepared FN solution
for 2 hours at room temperature with gentle rotation. Additional samples were
also immersed in PBS solution without protein to be used as control surfaces.
After incubation, the solutions were removed and the samples were carefully
washed 3 times with PBS to eliminate any unbound protein. Care was taken in
order to prevent the drying of the protein-coated surfaces before further analysis.


## 3.4. Immunodetection of Adsorbed Proteins

Patterned and control
surfaces were removed from PBS and incubated with 2% bovine serum albumin (BSA;
Sigma, St. Louis, Minn, USA) solution for 2 hours in room temperature in order
to block later nonspecific antibodies binding. Immunostaining procedure was
performed with FN
chicken antihuman antibodies (Invitrogen, Chicago, Ill, USA) diluted1∶1000.
Following a PBS wash, samples were then incubated for 2 hours with an Alexa
Fluor 488 goat antichicken IgG (H+L) (Invitrogen, Chicago Ill, USA) diluted
1∶200. Both primary and secondary antibodies were individually diluted in PBS
with 1% BSA. Between each step of the immunostaining procedure, samples were
repeatedly washed with PBS. For each assay, an additional control was prepared
consisting of a protein-coated sample submitted to the same described procedure
but instead of incubating with the primary antibody, PBS was used. Thus, the protein-coated
samples were exposed to the secondary antibody only as a control. Subsequently,
the silicon wafers were mounted on glass slides using ProLong Gold Antifade
reagent (Invitrogen, Chicago Ill, USA). Immunostaining results were observed
and recorded using fluorescent microscope (Nikon Eclipse TE2000-U). Ten digital
images of each sample (n=3) were captured and analyzed for average pixel
intensity on both the patterned and nonpatterned areas using Image J Software.
The results demonstrated a significantly higher (P=01) level of
fluorescence on the patterned area as compared to the nonpatterned surface (Figure 4). These
results strongly indicate a higher level of FN protein attachment occurring on
the patterned surface as compared to the nonpatterned surface.Figure 4
SEM
image of the fabricated grating.(a)
Fluorescence micrograph of the border between the patterned surface (right) and
the nonpatterned surface (left) (200x). (b) Graph depicts the average pixel
density from the fluorescence microscopy analysis.
(a)(b)Figure 6
2D diffraction grating
with grating periodΛ=351.2 nm.


## 3.5. Further Development

The mechanisms of why there was an increase
in protein attachment on the patterned surfaces are not clear yet. Thus it will
be interesting to investigate variations in nanopatterned structures (size and
shapes) which could be effective for alterations in protein attachment. After
fabrication of 1D diffraction grating by deep UV lithography, we also
fabricated 2D gratings with the same periodΛ=351.2 nm in both coordinates (Figure 5).To fabricate such structures, we
simply exposed the surface twice. Prior to the second exposure, we rotated structure
on 90°. We expect that 2D gratings will give another prospective structure for
examining changes in protein adsorption. It has been shown that 2D periodical structure
is a good candidate for using of tuning localized plasmons for the surface-enhanced
Raman scattering [19]. Future studies
will compare protein adhesion of 1D and 2D nanopatterned surfaces.


## 4. Discussion

FN is a representative of a cell adhesion
protein that is present in both plasma and the extracellular matrix. Altering
the attachment of this protein suggests that our nanopatterned structures may
lead to changes in the acceptance level of biomaterials by the host. Rechendorff
et al. proposed that protein shape effects its interaction with biomaterial
surface [20]. They created random nanosize rough surface by evaporation of
tantalum films, with surface roughness in the range between 2.0 and 32.9 nm.
They determined that fibrinogen, due to of its elongated shape, is much more
sensitive to the surface roughness as compared to bovine serum albumin, a
protein which has a nearly globular shape.


## 5. Conclusions

We proposed a simple model for protein attachment regarding grating corrugation
profile. From our model, we examined diffraction gratings with rectangular
grating profile.We
found that diffraction grating could serve as a controlled rough surface for FN.
Our results strongly indicate a higher level of FN attachment occurring on the
patterned surface. Thus the nanopatterned surface has a significant positive
effect on the binding of FN.Such
a positive result for FN, which plays a key role in cell adhesion and mediating
cell response, proves that cell attachment could be improved on investigated
nanopatterned structures.



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*Source: 543170-2008-03-24.xml*