IEEE/EMBS International Summer School on Medical Devices and Biosensors (ISSS-MD)

Characterization of Anhydrous Silanization and Antibody Immobilization on

Silicon dioxide Surface
Manoj Joshil, M.Goyal', R.Pinto2, S. Mukherjil*
'School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India.
2 Department of Electrical Engineering, Indian Institute of Technology Bombay, Mumbai, India.

Abstract- Formation of uniform and cluster free silane The thicker silane layer on the sensor surface alters the
monolayer is one of the fundamental prerequisite for affinity mechanical properties of the biosensor. It also induces
cantflever based biosensors. We report anhydrous silanization stresses on the sensor surface which may leads to difficulties
protocol for uniform silane monolayer on silicon dioxide (SiO2) s n the s ensor suracewicayi edo difficulTi
surface using [3-(2-aminoethyl) aminopropyll-trimethoxysiane
(APTES) and characterized by AFM, spectroscopic anticipate these problems silane monolayer with controlled
ellipsometry and FTIR. Silanization coverage is controled by thickness on biosensor is essential.
availability of surface water. The roughness of the resulting In the conventional protocol of aqueous phase
silanized surface following our protocol is in the range of SiO2 silanization, the aminosilanes undergo hydrolysis and
surface roughness. Silanized SiO2surface is used to immobilize polymerization in the bulk phase before depositing and
human immunoglobulin (HlgG) on it. FITC tagged goat forming bonds with the silicon dioxide surface [1]. However,
antihuman IgG is allowed to react with HIgG. The immobilized this results in the formation of polysilane networks prior to
surface is further characterized using Fluorescent spectroscopy deposition. Thus polymerization of multifunctional silane
and Fluorescent Microscope. Characterization results obtained molecules is observed both parallel and perpendicular to the
from anhydrous silanization protocol are compared with the solfces leadtoforatoofiane me cule te
conventional aqueous silanization protocol. surface. This leads to formation of silane molecule clusters
and non-uniform silane layer thickness on sensor surface.
Keywords - affinity cantilever, silanization, APTES, FITC In anhydrous silanization, trace water molecules on the
support surface are utilized to hydrolyze the silane
I. INTRODUCTION molecules to form siloxane bonds. The degree of
silanization depends on the concentration of water
Chemical modification of SiO2 surface by molecules present on the surface when the aminosilane is
organofunctional silane is a well-known technique for applied. Reduced silane coverage can be obtained by heating
biosensor applications. Most of the approaches however the surface at high temperature (to remove adsorbed water
produce non-uniform silane thickness and clustering of molecules) prior to application of the aminosilane [2].
silane molecules on the sensor surface which reduce the The main goal of this paper is to demonstrate the
sensitivity of affinity cantilever. The sensitivity of the qualitative improvement of silanized surface in anhydrous
affinity cantilever biosensor depends on maximum silanization, which is a prerequisite for improving the
deflection of cantilever due to surface force generated by sensitivity of affinity based cantilever biosensors. The
immobilized antibody on its surface as shown in Fig.l. surface morphology of the silanized surface is studied with
Maximally reproducible surface force can be achieved only AFM and thickness measurement using spectroscopic
if antibodies immobilized on cantilever are in uniform single ellipsometer. The orientation of silane molecule is
layer. investigated using FTIR. It is shown that anhydrous
Cantilever Antiboies Surface Force silanization technique produce dense and uniform antibody
Xs )!y .3N .
<9ZY a <; t-> immobilization. Characterization results obtained from
anhydrous silanization protocol are compared with the
conventional aqueous silanization protocol.

SIlIcon II. METHODOLOGY

Silicon wafer of resistivity 4-6 Q.cm and orientation

Fig. I Cantilever surface with uniform antibody immobilization to develop
surface force <100> was selected and cleaned by RCA cleaning followed
*Co---------- author- ;9122 - -----by
*Corresponding author- Tel. +91 22 2576-7767 dry thermal 0oxidation
tikeso m PE at 11 00°C
a forbandfo
1 hour to get oxide
im
Email- mukherji(icc.iitb.ac.in thickness of 100 m. APTES was obtamed from Sigma
-----__ -- --Aldrich USA and HIgG/ FITC tagged goat antihuman IgG
Financial support from the Government of India under the National from Bangalore Genei. To generate silanol sites on oxidized
Programme on Smart Materials is gratefully acknowledged. silicon, samples were dipped in sulphochromic solution

0-7803-8612-4/04/$20.00 © 2004 IEEE 7

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(Iml DI water with 500ug K2Cr2O7 added with 20ml H2SO4)
for 10 minutes followed by DI water rinse. This removes
any native carbon impurities and creates OH groups on SiO2
surface by opening siloxane bonds. Surface adsorbed water
was removed by heating the sample at 2000C (1400C in
aqueous protocol) for two hours under vacuum.
1% APTES solution in ethanol (95:5 % ethanol water
mixtures in aqueous protocol) was prepared in argon
ambient [3]. To maintain orientation of NH2 group of
APTES on the surface away from SiO2 surface, the pH of
silane solution was made 3.2 by adding acetic acid followed
by dipping SiO2 surface in it for 5 minutes. (In aqueous
protocol APTES is allowed to hydrolyze for 5 minutes
followed by dipping SiO2 surface in it for 2 minutes).The
excess amount of silane on the SiO2 surface is removed by
rinsing in ethanol followed by condensation at 110°C in
argon ambient. The silanization steps mentioned above are
carried in the silanization set up fabricated at
Microelectronics Laboratory, IIT Bombay as shown in Fig.2.
Fig. 2 Silanization set up fabricated at Microelectronics Laboratory, IIT
Bombay
1% aqueous solution of the homo-bifunctional agent,
glutaraldehyde, was used as a linker. Silanized SiO2 surface
was dipped in it for 30 minutes followed by incubation of
HIgG of different concentrations for I hour. The unsaturated
aldehyde sites and non-specific adsorption sites on the
antibody immobilized surface were blocked by dipping the
samples for 1 hour at room temperature in 2mg/ml solution
of BSA in 0.05M PBS, followed by rinse in 0.05 M PBS
thrice. To identify the grafted antibody layer, FITC tagged
goat anti-human HIgG (lml/ ml in PBS) was incubated for 1
hour, rinsed in PBS and stored at 4°C.
M RESULTS
The silanization on the SiO2 surface, using aqueous and
anhydrous protocol was characterized using various tools.
AFM was used to study the surface morphology of the
silane layer produced in both protocols. The thickness of the
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silane layer at different silane concentrations was measured
with spectroscopic ellipsometer and the orientation of silane
molecule on the SiO2 surface investigated with FTIR. The
antibody immobilization on silanized SiO2 surface was
studied with fluorescence spectroscopy and fluorescent
microscope.
A Atomic Force Microscopy
The AFM system used was DI Nanoscope AFM. The
high aspect ratio Si3N4 super tips probes integrated on Si3N4
cantilever were used. All the images presented here are
obtained using contact imaging mode. We observed that
silanization of SiO2 surface by anhydrous protocol produces
smoother and cluster free silane monolayer as compared to
aqueous silanization protocol. Size of the cluster on the SiO2
surface due to aqueous silanization shown in Fig. 3 (a) was
in the range of 1 50nm to 250nm.
E 0+~ ~ ~ ~ ~ ~ ~ ~
Fig. 3 (a) Silanized SiO2 surface using aqueous silanization showing
clusters of silane molecules on the surface
The SiO2 surface modified with anhydrous silanization
was found uniform and cluster free as shown in Fig.3
(b).The RMS surface roughness of silanized SiO2 surface in
aqueous silanization was 1.4 nm where as in anhydrous
silanization it was 0.5 nm, which is in the range of RMS
roughness of the SiO2 surface
40
y.s
Fig. 3 (b) Silanized SiO2 surface using anhydrous silanization showing
uniform and cluster free surface
 In order to obtain high resolution images of the protein
molecules using AFM, molecules should be firmly attached
to solid support to resist the contact force excreted by
scanning tip in the contact imaging mode [4]. Even in the
covalent immobilization there are some loosely adsorb
proteins which contaminate the scanning tip, giving rise to
an increase of interaction between the tip and protein,
thereby affect adversely the important factor limiting high
resolution AFM imaging of protein molecule structure.
Atmost care was taken at every step of immobilization to
minimize non specific protein bindings and removal of
loosely coupled antibodies.
Fig. 4 (a) Antibody immobilized surface using aqueous silanization
1o.
n | | - - 1 i sdiane
D.S z
'.'.
Fig.4 (b) Antibody immobilized surface using anbydrous silanization
The RMS surface roughness of the antibody
immobilized surface using aqueous silanization shown in
Fig.4 (a) was found 3.5 nm where as for antibody
immobilized surface with anhydrous silanization shown in
Fig.4 (b) was 0.56 nm. This shows that anhydrous
silanization produces better quality surface for the
miniaturized biosensors.
B Elipsometry
The silane layer thickness on the sensor surface is one
of the important parameter in simulation and
characterization of affinity cantilever biosensor. This is
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investigated using spectroscopic ellipsometry.The
Elipsometry instrument used was SENTECH Instruments
GmbH, model SE 800. The position of the arm was located
at 700 with respect to the stage, and wavelength used was
350 to 800 nm. The refractive index of silane film used was
1.446. We prepared silanized SiO2 surface of different silane
concentration for both aqueous and anhydrous protocols.
The Elipsometry measurements were made on an area of
lcm2 and on several different spots on each sample surface
of SiO2.
By varying the silane concentration the change in silane
layer thickness is as shown in Fig. 5. It was found that the
silane layer thickness due to anhydrous silanization is lower
than aqueous silanization. We may get further more
controlled silane layer thickness using gas phase silanization
[5]
The appropriate selection of the silane concentration for
miniaturized biosensors can be decided by finding out the
surface roughness of silanized SiO2 using AFM [6].
. 76 . __Aqueous
ai 5, . . / - ~~~~~silanization
__ silanization
0-
0 1 2 3
percentage
Fig. 5 Silane layer thickness for various silane concentration detemiined by
Elipsometry
C FTIR Studies
For getting the efficient immobilization of antibodies, it
is essential that amino group (NH2) of silane on the surface
must be oriented away from the surface. This was
investigated by using infrared spectroscopy. We used
Nicolet Magna-IR spectrometer-550 in the grazing angle
mode. The instrument gives maximum sensitivity at the
angle of incidence 860. But to adjust the maximum gain of
FTIR instrument, it needs large samples size on which silane
layer may not have uniform thickness and gives errors in
results. Hence we selected angle of incidence 800.The
polarized Infrared light was used to scan the silanized SiO2
surface.
The silane concentration on the SiO2 surface used for
FTIR study was 2%. To get the maximum sensitivity from
the FTIR instrument, we used a quarter piece of the 2 inch
wafer. The care was taken to get uniform silane layer on
quarter pieces. The wave number associated with the free
NH2 group is in the range of 3300 cmn to 3500 cm-' [7]. We In Fig. 7 antibody immobilized with aqueous silanization
found the peaks of free NH2 group of APTES on the sio2 shows relatively higher fluorescent intensity due to
surface at 3361 cmn' as shown in Fig. 6. attachment of antibody on silane clusters on SiO2 the surface.
E Fluorescent Microscopy

Aqueous s.lanizatlon Fluorescence microscopy was used to compare the

quality of antibody immobilized surface using both, aqueous
and anhydrous silanization protocols. We used ZEISS
Axioskope-2 MAT fluorescent microscope. Silanized SiO2
nhydrous silanization
X . A- surfaces prepared with 1% silane solution in aqueous and
anhydrous silanization protocol were immobilized using
HIgG (0.5mg/ml in PBS). To identify the grafted antibody
3600 3361 3000 layer, a drop of FITC tagged goat antihuman IgG (mImlml in
Wave number (cm-1) PBS) is incubated on it.
Fig. 8 (a) and (b) shows the fluorescence images on
Fig. 6 FTIR of aqueous and anhydrous silanized SiO2 surface. The peaks SiO2 surface treated with aqueous and anhydrous
associated with free NH2 group on the silanized surface at 3361cm71. silanization protocols. We observed green fluorescence on
the area under the curve due to drop of FITC tagged goat
The amplitude of Nwa2 group peaks associated with antihuman IgG. We also observed dense and uniformn
aqueous silanization was found more than anhydrous fluorescence on the sample treated with anhydrous protocol
silanization compared with the sample treated with aqueous silanization.

D Fluorescence Spectroscopy
The amount of antibody immobilization on silanized
Si02 surface due to both aqueous and anhydrous protocol
was investigated using fluorescence spectroscopy. We used
Perkin-Elmer LS55 Luminescence spectrometer (PMT-
R928). To find out the excitation wavelength of FITC ,
tagged goat antihuman HIgG, UV spectroscopy was done. PBS) wu
The peak absorption was at 294 nm wavelength. Four Si02
samples of each 1cm2 area were silanized at 1% silane
solution by both aqueous and anhydrous silanization. On
these samples, HIgG of different concentration (0.2mg/ml to Fig. 8 (a) Fluorescence image of antibody immobilized SiO2 surface treated
0.8mg/ml in PBS) followed by FITC tagged goat antihuman with aqueous silanization protocol. Area under the curve due to drop of
HIgG (Iml/ml in PBS) wasimmobilized. FITC tagged goat antihuman IgG shows the fluorescence emission
The peak intensity of emission was observed at higher
wavelength of 440 n 2m.

Fluorescent Intensity at 440 nm

'1O000
d800
j600 -Silanization
400 __ aAqueous
S i ~~~~~~~~~~~Slianization
~200j - _
g0
U. 00.10.50 1
~~~~~~~~~~~Fig.
8 (b) Fluorescence image of antibody immobilized SiO2 surface treated
Anltbody concentration with anhydrous silanization protocol. Area under the curve due to drop of
(mg/ml) FITC tagged goat antihuman IgG shows the fluorescence emission
Fig. 7 Change in fluorescent intensity with antibody concentration. The
excitation wavelength used was 294 mn and peak emission observed at 440
nm

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 IV. CONCLUSION [6] J. N. Volle, G. Chambon, A. Sayah, "Enhanced
sensitivity detection of protein immobilization by
In order to achieve the controlled silane layer fluorescent interference on oxidized silicon,"
thickness using anhydrous silanization on the biosensor Biosensors and Bioelectronics, vol 19, pp 457-464,
surface, we need to control the surface adsorbed water. This 2003
can be done by controlling the temperature of dehydration
process prior to silanization. The temperature varying from [7] Koji Nakanishi, "Infrared Absorption Spectroscopy,"
1400C to 2000C can give variable silane coverage on the Holden-Day, Inc., San Francisco, 1962.
SiO2 surface. It is reported in the literature that the loss of
hydroxyl group on the surface occurs at temperatures greater
than 200°C. Another alternative for liquid phase silanization
can be gas phase silanization. The main challenge associated
with gas phase silanization is to maintain the orientation of
amino group.
We achieved cluster free and uniform silane monolayer
using anhydrous silanization. The RMS surface roughness
of the silane layer obtained from anhydrous silanization
protocol was 0.5 nm which is in the range of SiO2 surface
roughness. The minimum thickness of the silane layer
achieved using anhydrous silanization protocol was
approximately 4 nm. The layer of antibodies immobilized on
sio2 surface using anhydrous silanization protocol,
observed under fluorescent microscope, was found dense
and uniform as compared to aqueous silanization protocol.

ACKNOWLEDGMENT

The authors thank Professors R. Lal and R. Rao from

Electrical Engineering Departnent, IIT Bombay, for their
helpful discussion on the fabrication of silanization setup
and experimentation.

REFERENCES
[1] Plueddemann E. P, "Silane Coupling Agents," 2nd
Ed, Plenum Press, New Yark, 1991.
[2] Wayne Yoshida, Robert P.Castro, "Multilayer
Alkoxysilane Silylation of oxide surface," Langmuir
vol. 17, pp 5882-5888, 2001.
[3] Dr. Bhagwati Prasad, "Capacitive Immunosensor for
Fibronectin," 'Ph.D. thesis' submitted to school of
Bioscience and Bioengineering, IIT Bombay, 2000.
[4] Hong Xing You, Chirstopher R.Lowe, "AFM Studies of
Protein Adsorption," Journal of colloid and interface
science, vol.182, pp 586-601, 1996.
[5] Ulf Jonsson, Goran Olofsson, "Chemical Vapour
Deposition of Silane," Thin Films, vol.124, pp1l7-123
1985

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