Silanization and antibody immobilization on SU-8
Manoj Joshi a, Richard Pinto b, V. Ramgopal Rao b, Soumyo Mukherji a,*
a
School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India
b
Department of Electrical Engineering, Indian Institute of Technology Bombay, Mumbai, India
Abstract
SU-8, an epoxy based negative photoresist, has emerged as a structural material for microfabricated sensors due to its attractive mechanical
properties like low Young’s modulus and chemical properties like inertness to various chemicals used in microfabrication. It can be used to
fabricate MEMS structures of high aspect ratio. However, the use of SU-8 in BioMEMS application has been limited by the fact that
immobilization of biomolecules on SU-8 surfaces has not been reported. In this study, the epoxy groups on the SU-8 surface were hydrolyzed in the
presence of sulphochromic solution. Following this, the surface was treated with [3-(2-aminoethyl) aminopropyl]-trimethoxysilane (AEAPS). The
silanized SU-8 surface was used to incubate human immunoglobulin (HIgG). The immobilization of HIgG was proved by allowing FITC tagged
goat anti-human IgG to react with HIgG. This process of antibody immobilization was used to immobilize HIgG on microfabricated SU-8
cantilevers.
Keywords: SU-8; AEAPS; Silanization; HIgG; Antibody immobilization
1. Introduction
Miniaturized biosensors are fabricated using microfabrication
techniques. Materials used for fabrication of such sensors are
silicon, silicon dioxide, silicon nitride, gold, etc. Such materials
are achieved using standard microfabrication techniques, such as
oxidation, chemical vapor deposition, physical vapor deposition,
etc. Patterning these materials requires processes like lithography and etching, which further add to complexity, cost and
production time of sensor fabrication.
SU-8 (glycidyl ether of bisphenol A) polymer is a negative
photoresist and has emerged as a structural material for
biosensors. There are different methods for immobilization of
biomolecules on to a polymer surface, e.g. entrapment,
encapsulation, adsorption, covalent binding, etc. Covalent
immobilization is often necessary for binding molecules that do
not adsorb, adsorb very weakly or adsorb with improper
orientation and conformation to polymer surfaces [1–3]. This
may result in better biomolecule activity, reduced non-specific
adsorption and greater stability.
Covalent immobilization can be achieved on the polymer
surface by modifying it to have at least one functional group,
such as CHO, NH2, SH, etc. which can be used to bind
biologically active molecules.
One of the preferred methods of creating amino groups on
the surface of substrates is by treatment with aminosilanes. In
this paper, we describe a process to immobilize antibodies on
SU-8 surfaces using silanization. The C–O bonds (99 kcal/
mol) in the epoxy group on SU-8 surface are cleaved using
sulphochromic solution, resulting grafting of hydroxyl groups
on it. Such a modified SU-8 surface is treated with aminosilane
followed by antibody immobilization on it.
2. Materials and methods
SU-8 2000 was obtained from MicroChem USA, [3-(2aminoethyl) aminopropyl]-trimethoxysilane (AEAPS) was
obtained from Sigma–Aldrich USA and HIgG/FITC tagged
goat anti-human IgG from Bangalore Genei, India. All other
chemicals were obtained from SD FineChem India Ltd.
2.1. Sample preparation
SU-8 was patterned on silicon wafer using standard photolithography techniques. The mask used for photolithography
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Fig. 1. Prototype of mask used for photolithography. Each window in the mask
is of (2 mm 2 mm) size.
had a chequer-board pattern with alternate windows for silicon
and SU-8 under study (Fig. 1). This would subsequently help to
prove the selectivity of the immobilization process towards SU8 over silicon. The parameters used to obtain the SU-8 surface
were: prebake temperature 70 8C (5 min), UV exposure of 6 s,
post-bake temperature 95 8C (5 min). Silicon surfaces completely covered with SU-8 were also prepared for FTIR and
AFM studies. The process parameters for creating the SU-8
film was the same as mentioned earlier. The surface
modification and antibody immobilization processes after
creation of the SU-8 film were identical for both types
(patterned and solid) of samples.
2.2. Silanization and antibody immobilization
Native oxide from the silicon squares on the patterned
samples was removed by dipping the surfaces in 2% HF for
30 s. All samples were subjected to sulphochromic solution
treatment for 10 min followed by DI water rinse. The chemical
bond structure of SU-8, before and after sulphochromic
solution treatment is as shown in Fig. 2. In sulphochromic
solution, K2Cr2O7 is used as a catalyst and H2SO4 in the ionic
state is given by,
H2 SO4 , Hþ þ HSO4 (1)
The chemical reaction associated with the hydrolysis of
surface epoxy group of SU-8 is given by Eq. (2)
Fig. 2. Chemical bond structure of SU-8 surface: (a) before sulphochromic
solution treatment and (b) after sulphochromic solution treatment.
AEAPS solution in ethanol was prepared in argon ambient
[4,5]. To maintain orientation of NH2 group of AEAPS away
from the surface, the pH of the silane solution was optimized
to 3.7 by adding acetic acid. The samples were kept in the
silane solution for 7 min. The excess amount of silane on the
SU-8 surface was removed by rinsing in ethanol. This was
followed by condensation at 110 8C in argon ambient
for 10 min. The silanized samples were dipped in 1%
aqueous solution of glutaraldehyde (homo-bifunctional cross
linker) for 30 min. They were then ready for antibody
immobilization.
The samples were incubated in HIgG (0.5 ml/ml in
phosphate buffer saline) suspension for 1 h. Loosely
adsorbed antibodies were removed by rinsing the samples
in PBS solution three times. The unsaturated aldehyde
sites and non-specific adsorption sites on the antibody
immobilized surfaces were blocked by dipping the samples
in 2 mg/ml solution of BSA in PBS at room temperature for
1 h, followed by rinsing in PBS for three times [6]. To
(2)
Surface adsorbed water was removed by heating the
samples at 110 8C for 2 h under vacuum. Two percent
identify the grafted antibody layer, FITC tagged goat antihuman HIgG (0.5 ml/ml in PBS) was incubated at room
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Fig. 3. Grazing angle FTIR of SU-8 surface: (a) before silanization and (b) after silanization showing additional R-NH2 group at 1617 cm1.
temperature for 1 h. This was also followed by three PBS
rinses.
AFM was used to study the SU-8 surface morphology.
Fluorescence microscopy was used to identify the grafted
antibody layer.
3. Results
3.1. Fourier transform infrared spectroscopy
The samples were studied at various stages of the process
using different characterization tools. The presence of chemical
bonds on the silanized SU-8 surface was demonstrated using
Fourier transform infrared spectroscopy (FTIR). Tapping mode
The chemical bonds on the SU-8 surface before and after
silanization were studied using Fourier transform infrared
spectroscopy. A Nicolet Magna-IR spectrometer-550 in the
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grazing angle mode was used for this purpose. The polarized
infrared light at an angle of 808 was used to scan the SU-8
covered samples. The wave number associated with the R-NH2
group is in the range of 1560–1640 cm1 [7]. The R-NH2 peak
is absent in the grazing angle FTIR of unmodified SU-8 surface
(Fig. 3a). However, grazing angle FTIR of modified SU-8
surface (Fig. 3b) clearly shows the presence of R-NH2 peak at
1617 cm1. This may be taken as evidence of grafting of
aminosilane on SU-8 surface.
3.2. Atomic force microscopy (AFM)
Digital Instrument Nanoscope III was used for atomic force
microscopy. High aspect ratio silicon cantilevers were used to
obtain the AFM images. Since the samples of SU-8 and the
biolayer on top of it are softer than normal metal/semiconductor
compound films, tapping mode AFM was used to investigate
the SU-8 surface at various stages of experimentation [8].
As shown in Fig. 4a and b, surface roughness of SU-8
increases with the silanization process. The RMS roughness of
the SU-8 surface was found 0.446 nm and for silanized SU-8
surface, it was 2.245 nm. However, the RMS surface roughness
of the antibody immobilized surface was reduced to 1.484 nm
(Fig. 4c). This reduction in the surface roughness is may be due
to the clustering of the antibodies on the silanized SU-8
surface.
3.3. Fluorescence microscopy
The antibody immobilization on SU-8 surface before and
after silanization was investigated using a ZEISS Axioskope-2
MAT fluorescence microscope. SU-8 surface with and without
silanization treatment was subjected to antibody (HIgG)
immobilization. To identify the grafted antibody layer, FITC
tagged goat anti-HIgG was incubated on it and observed under
a fluorescence microscope. Fluorescence excitation wavelength
of 450–490 nm and emission sensitivity above 520 nm was
used for these studies. The samples were observed using normal
optical microscope for preliminary identification of surface
features. Following this, fluorescence micrographs of the
sample surfaces at the same spots were obtained. As observed
from micrographs shown in Fig. 5b, weak and random
fluorescence is detectable on the part of the surface
corresponding to SU-8 without surface modification, although
the complete sample surface was incubated with HIgG and the
drop of FITC tagged goat anti-HIgG was administered. This
may be due to the random and scattered adsorption of
antibodies on the SU-8 surface. Hence, it is inferred that
antibody cannot be immobilized without the silanization of
SU-8.
The silanized SU-8 surface patterned on silicon and gold
surface was incubated with HIgG followed by incubating a drop
of FITC tagged goat anti-HIgG, shows much brighter and more
Fig. 4. AFM pictures of SU-8 surface: (a) before silanization, (b) after silanization and (c) after antibody immobilization.
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Fig. 5. Micrograph of unmodified SU-8 surface treated with HIgG followed by
FITC tagged goat anti-HIgG observed under: (a) optical microscope and (b)
fluorescent microscope.
uniform fluorescence (Figs. 6b and 7b) on SU-8 surface.
This also demonstrates that, antibody immobilization is
selective only on SU-8 as against silicon and gold surface.
The few scattered spots of high fluorescence may be due to
agglomeration of antibodies because of uneven topography of
the surface. The figures demonstrate that, the SU-8 surfaces
treated with aminosilane are more amenable to immobilization
of biomolecules.
3.4. Functionalization of micro-cantilevers
The process of silanization and antibody immobilization
described in this paper can be extended to immobilize the
biomolecules on the SU-8 surface of microfabricated
sensors. In order to test the efficacy of the process described
in this study towards that purpose, SU-8 cantilevers were
fabricated using surface and bulk micromachining. The
fabrication details of SU-8 cantilevers are beyond the scope
Fig. 6. Micrograph of silanized SU-8 surface patterned on silicon and treated
with HIgG followed by FITC tagged goat anti-HIgG and observed under: (a)
optical microscope and (b) fluorescent microscope.
of this paper. Such cantilevers were treated with silanization
followed by antibody immobilization. One such example of
antibody immobilization on SU-8 cantilever is demonstrated
in Fig. 8.
4. Discussion
SU-8 has emerged as a structural material in MEMS due to
its low young’s modulus and/or high aspect ratio structures can
be fabricated using this polymer. However, for biosensor/bioreactor applications, it is critical that the surface of SU-8 be
functionalized with bio-active molecules. In this paper, we
described a method for achieving this goal.
However, there are many challenges involved in the
silanization and antibody immobilization on microcantilever
surfaces. For example, SU-8 has good adhesion with silicon
nitride surfaces and poorer adhesion with gold surfaces. Hence,
SU-8 surfaces spin-coated on gold need to be handled very
carefully during the surface functionalization process.
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Fig. 7. Micrograph of silanized SU-8 surface patterned on gold and treated with
HIgG followed by FITC tagged goat anti-HIgG and observed under: (a) optical
microscope and (b) fluorescent microscope.
Fig. 8. Micrograph of SU-8 cantilever treated with silanization followed by
incubation of HIgG and FITC tagged goat anti-HIgG and observed under: (a)
optical microscope and (b) fluorescent microscope.
Acknowledgements
Authors thank Prof. R. Lal and Prof. P. Apte for their helpful
discussions during experimentation. Authors also thank student
and staff members of iSens group (IIT Bombay), especially Dr.
Sheetal Patil, for providing SU-8 microcantilevers for antibody
immobilization.
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