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Applied Surface Science 253 (2007) 3127–3132

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

Received 31 March 2006; accepted 2 July 2006

Available online 5 September 2006

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.

# 2006 Elsevier B.V. All rights reserved.

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 litho-

graphy 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.

* Corresponding author. Tel.: +91 22 2576 7767.

E-mail address: mukherji@iitb.ac.in (S. Mukherji).

0169-4332/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2006.07.017

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-(2-

aminoethyl) 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 photo-

lithography techniques. The mask used for photolithography

M. Joshi et al. / Applied Surface Science 253 (2007) 3127–31323128

Fig. 1. Prototype of mask used for photolithography. Each window in the mask

is of (2 mm � 2 mm) size.

Fig. 2. Chemical bond structure of SU-8 surface: (a) before sulphochromic

solution treatment and (b) after sulphochromic solution treatment.

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 SU-

8 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 com-

pletely 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,

H2SO4 , Hþ þHSO4� (1)

The chemical reaction associated with the hydrolysis of

surface epoxy group of SU-8 is given by Eq. (2)

(2)

Surface adsorbed water was removed by heating the

samples at 110 8C for 2 h under vacuum. Two percent

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

identify the grafted antibody layer, FITC tagged goat anti-

human HIgG (0.5 ml/ml in PBS) was incubated at room

M. Joshi et al. / Applied Surface Science 253 (2007) 3127–3132 3129

Fig. 3. Grazing angle FTIR of SU-8 surface: (a) before silanization and (b) after silanization showing additional R-NH2 group at 1617 cm�1.

temperature for 1 h. This was also followed by three PBS

rinses.

3. Results

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

AFM was used to study the SU-8 surface morphology.

Fluorescence microscopy was used to identify the grafted

antibody layer.

3.1. Fourier transform infrared spectroscopy

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

M. Joshi et al. / Applied Surface Science 253 (2007) 3127–31323130

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 cm�1 [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 cm�1. 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.

Fig. 4. AFM pictures of SU-8 surface: (a) before silanization, (

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

b) after silanization and (c) after antibody immobilization.

M. Joshi et al. / Applied Surface Science 253 (2007) 3127–3132 3131

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.

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.

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

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/bio-

reactor 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.

M. Joshi et al. / Applied Surface Science 253 (2007) 3127–31323132

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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