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IEEE INDIA ANNUAL CONFERENCE 2004. INDICON 2004
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Anhydrous Silanization and Antibody
Immobilization on Hotwire CVD Deposited
Silicon Oxynitride Films
Manoj Joshi, Sunil Singh, BibhuSwain, Samadhan Patil,
Rajiv Dusane, Ramgopal Rao, Soumyo Mukheji
Absrruct--Hotwire C M (HWCVD)deposited silicon rich nitride
films were treated with 0 2 plasma using RF plesma setup. The
thickngs o f this oxpitride film was measured using
spectroscopic ellipsometry. The film was treated with 1343aminoethyl) aminopropyl]-trlmethoxysilene (AEAPS) followed
by Immobilizatioa of Human immunoglobulin (HIgG) on it.
Surface morphology at various stages of experimentation was
studied using AFM. Antibody Lmmobllized surface is further
bvestigated using fluomcence mImscopy.
Keywords-
Financial support from the Govemment of India under the
National Programme on Smart Materials is gratefully
acknowledged.
HWCVD, AEAPS, silanization,HIgG
I. INTRODUCTION
,.
However the first technique suffers from the low density of
antibody immobilization due to lack of enough Si02 sites
available for forming silanol bonds and second require.few
hours of chemical oxidization.
Silicon nitride (Si,N,) is widely used as an insulating
thin film as well as a passivation layer in semiconductor
industry. Its Young's modulus is higher than silicon and its
intrinsic stress can be controlled by the specifics of the
deposition process. To minimize the HF etch rate and reduce
residual stress, nitride may be deposited with an excess of
silicon (silicon rich nitride) thereby creating an effective
masking material against alkaline etch solutions. These
properties attract its use in the microfabrication of various
structures of Micro Electro Mechanical Systems (MEMS).Its
hydrophobic nature may be effectively used to overcome
problems like stiction. Further, silicon rich nitride films may
act as an effective barrier against the mobile ion diffusion,
(e.g. sodium and potassium ions found in biological
environments) which increases its use in Bio-MEMS [I].
The electrical and mechanical properties of silicon nitride can
be altered as per requirements of the application by choosing
suitable deposition process parameters. Silicon n i ~ d ecan
also be used to detect biomolecules by suitably modifying its
surface. One such example has been demonstrated in pHISFET used as glucose sensor, by treating silicon nitride
surface at the gate of ISFET with 0
2 plasma (21. Silanization
ahd antibody immobilization may be possible on the native
oxide layer formed on silicon nitride surface [3] or by
oxidizing siticon nitride surface chemically [4].
This paper demonstrates antibody immobilization on
hotwire CVD (HWCVD)deposited silicon rich silicon nitride
that can also be used as a structural material for Bio-MEMS.
The surface of the silicon nitride is treated with 02 plasma in
a RF plasma setup to get a thin layer of oxynitride film which
does not alter its mechanical-properties. The oxynitride film
is subjected to silanization followed by antibody
immobilization.
The surface morphology of silicon nitride at the various
stages of experimentation is studied with Atomic Force
Microscopy (AFM).The. thickness -of oxynitride film is
measured using spectroscopic ellipsometry. The antibody
immobilization is examined using fluorescence microscopy.
11. MF;I'HODOLOGY
A. Deposition of silicon nitride in Hotwire CVD
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Manoj Joshi.
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School of Biosciences and Bioengineering. Indian institute of Technology
Borrbay.
Sunil Singh,Bibhu Swain, Rajiv Dusane,
Department of Metallurgical Engineering and Materials Science, h&an
Institute of Technolow Bombay.
Samadhan F'atil, Ramgopa! b o ,
Department of Elecmcal Engineering, Indian Institute of Technology
Bombay,Mumba,India.
Soumyo Mukherji
Correspondingauthor- Tel. +91 22 2576-7767
Email- mukherji @cc.iitb.ac.in
gas inlet
fig. I Schematic cross section of hotwire depition chamber used for the
deposition of silicon rich nitride.
0-7803-6909-3/04/$20.00
022004 IEEE
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MDLAN INSTITUTE OF TECHNOLOGY, KHARAGPUR 721302, DECEMBER 20-22,2004
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Hotwire CVD is hown for low temperature deposition
and high growth rate of the films [5]. O& HWCVD setup
consists of deposition chamber (Fig.1) connected:to an ultra
high vacuum system (UHV). Silicon wafer (P-type,Clll>)
was cleaned with standard RCA cleaning and loaded on the
substrate heater.
The process parameters of HWCVD deposited silicon nitride
are as shown in Table 1.
Tablel. ptoccss pamneters for HWCVD deposited Silicon NiMdc
I Base nressure
1 IO* Torr
S a 4 flow rate
1sccm
NH3 flow rate
20sccm
Substrate Temerature
300'C
I Time of deposition
1 20min.
I
COSilanization and Antibody Immobilization
[3-(2-aminoethyl)
aminopropyll-bimethoxysilane
(AEAPS)was obtained fiom Sigma Aidrich USA and HIgGI
FITC tagged goat antihuman IgG from Bangalore Genei,
I
The etch rate of the silicon rich nitride obtained film was
found very low in BHF.
B. Oxygen plasma treatment to silicon nitride
..
India. Silicon nitride samples treated with 0;plasma (silicon
oxynitride) were subjected to sulphochromic sotution ( I d
DI water with 500ug KzCrz07added With 20ml H2S04) for
10 minutes followed by DI .water rinse. This removes any
native carbon impurities and creates OH groups on Si02
surface by opening siloxane bonds (Silanol sites). Surface
adsorbed water was removed by heating the sample at ISO'C
for two hours under vacuum. 1% AEAPS solution in ethanol
was prepared in argon ambient. To maintain orientation of
NH2 group of AEAPS on the surface away from oxynitride
sufface, the pH of silane solution was made 5.2 by adding
acetic acid followed by dipping the sample in it for 5
minutes. The excess amount of silane on the oxynitride
surface was removed by rinsing in ethanol followed by
condensation at 1IO'C in argon ambient.
.
1% aqueous solution of the homo-bifunctional agent,
glutaraldehyde, was used as a linker. Silanized oxynitride
surface was dipped in it for 30 minutes followed by
incubation of HIgG (0.5 mV ml in PBS) for 1 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/d solution of BSA in PBS, followed by rinse in PBS
thrice, To identify the grafted antibody layer, FITC tagged
goat anti-human HIgG (0.5 mV ml in PBS) was incubated for
1 hour, rinsed in PBS and stored at 4*C [7j.
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Fig. 2 Schematic cross section of RF plasma system
HWCVD deposited Si,N, film on silicon substrates was
used for O2plasma treatments in a RF plasma system at 13.56
MHz. The process parameters for the RF 02 plasma treatment
are as shown in Table 2.
Tablc2. Process parameters for 0 2 p~asmatreatment
Time for O2treatment
O2flow rate
The growth of oxynitride m a y be layer-by-layer
oxidation process in which the oxygen atoms replace
nitrogen atom to form an oxide layer at the oxide nitride
interface. This has been explained as a four-step process, (i)
oxygen injection at the oxide-plasma interface (ii) transport
of the oxygen species through the growing oxide; (iii)
transformation (oxidation reaction) at the oxide-nitride
interface; (iv) transport of nitrogen species back to the oxideplasma interface [ 6 ] .
I IOsccm
Fig. 2 shows the schematic cross section of RF plasma
system used for this purpose. All the plasma treatments were
carried out with highly enriched oxygen gas.
111. RESULTS
A. Atomic Force Microscopy (AFM)
The AFM system used was Digital Instrument
Nanoscope 111. The high aspect ratio SiSN4super tips probes
integrated on Si3N4cantilever were used in contact imaging
mode. Silicon nitride surface before and after 02 plasma
treatment was observed in contact mode AFM.Fig.3a shows
the surface morphology of HWCVD deposited silicon nitride
and Fig.3b shows the same surface after 02 plasma treatment.
It was observed that the surface morphology of silicon nitride
changes with the 0
2 plasma treatment. The R M S surface
roughness of silicon nitride was 1.08 nm and grain height
was 0.06 nm. For oxynitride surface, the R M S surface
roughness found was 3.8 nm and grain height was 11.6 nm.
This shows that during transformation of silicon
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IEEE INDlA ANNUAL CONFERENCE 2004. MDlCON 2004
oxynitride surface AFM was done in tapping mode. In
contact mode AFM,the contact force excreted by scanning
tip on loosely adsorbed protein molecules affect the
resolution of AFM imaging adversely [8]. The high aspect
ratio silicon super tip probes were used in tapping imaging
mode. Tapping Mode AFM operates by scanning a tip
attached to the end of an oscillating cantilever across the
sample surface. The cantilever is oscillated at or slightly
below its resonance frequency (-300 &) with amplitude
ranging typically from 20nm to l o b The tip lightly taps
on the sample surface during scanning, contacting the surface
at the bottom of its swing. R M S surface roughness of
silanized oxynitride surface was 5.21 nm and grain height
was 25.06 nm The RMS surface roughness of antibody
immobilized silicon oxynitride surface was 3,96 nm and
grain height was 15.67 nm
The R M S roughness and grain height of antibody
immobilized surface closely resembles with silicon
oxynitride surface indicating that the antibody
immobilization is quite dense and uniform on silicon
oxynibide surface.
E. Fluorescence Microscopy
Silicon nitride surfaces with.,and without 02 plasma
treatment subjected to silanization and antibody
itlrmobi~ization.To identify the .grafted antibody layer, FITC
tagged goat anti HIgG was incubated on it and observed
under fluorescent microscope. A- Zeiss Axioskop-2 MAT
microscope with fluorescence excitation wavelength of 450490 nm and emission sensitiviv above 520 run w a s used for
the studies. The sampIes were observed using simple optical
microscopy (Fig.4a and FigAc) for preliminary identification
'
of surface features. Following.this, fluorescence micrographs
of the sample surfaces at the same spots were obtained
'
(Fig.4b and Fig.4d,).
As observed from these micrographs, weak fluorescence
was detectable fiom the nitride surface wi+out plasma
treatment at the areas corresponding to incubation of FITC
tagged antibodies (Fig.4b). , a s may be due SilaniZation and
antibody immobilization on.'native oxides of silicon nitride or
the adsorption of antibodies oninitride surface. Brighter
fluorescence was observed from-similar areas of the nitride
surface treated with O2plasma (Fig4d).
C Spectroscopic Ellipsomei?y
The thiclmess of the oxynitride film on the silicon nitride
surface was investigated using spectroscopic ellipsometry
using a SENTECH Instruments-GmbH, model SE 800
ellipsometer. The position of the arm was located at 70'
'
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Fig. 3 AFM images of (a) silicon nitride surfnce (b) silicon oxyni@de
surface (c) silanized oxpitride surface (d) Antibody lmmpbilized silicon
axpitride surface
nitride in to oxynitride there is drastic'change in sirface
morphology.
. .
' Tapping mode AFM is preferred for tbe AkM of soft
surfaces. Hence for silanized and antibody immobilized'
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INDIAN INSTITUTE OF TECHNOLOGY, KHARAGPUR 721302, DECEMBER 20-22,2004
applications in micro-bio-systems. Oxygen plasma treatments
at elevated temperatures may fiuther increase the density of
Si02 on silicon nitride surface. Antibody immobilization
using silanization on such surface may give higher density of
immobilization.
ANKNOWLEDGEMENT
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The authors thank Professors R. La1 and R. Pinto from
Electrical Engineering Department, IIT 3ombay, for their
heIpful discussion experimental support. Authors also thank
Professor S.Major for his support in using AFM.
REFERENCES
MaIuf, “An Introduction to Microelectromechanical Systems Engineering,” Artech House,
Basten, London.
[l] Nrtdim
b.
[2]
Li-Te Yin, Jug-Chun Chou et. al., “Characteristics of
Silicon Nitride after 0 2 Plasma Surface Treatment for
pH-ISFET Applications,” IEEE Transactions on
Biomedical Engineering, vol. 48,no.3, March 2001.
[3] R. A. Williams & H. W. Blanch, “Covalent
immobilization of protein monolayers for biosensor
application,” Biosensors & BioeIectronics 9 (1994)159167.
C
[43 Sang-Ho Lee a, Chang-So0 Lee b, DongiSik Shin,
“Microprotein patterning using a lift-off process with
fluorocarbon tluIl film,” Sensors and Actuators, B 99,
(2004) 623-632.
B. Patil, A h A. K d h a r , R.O.Dusane,
‘Transparent silicon nitride alloy fiIms by hot wire CVD
technique” presented at the seminar on semiconductor
physics and devices central University, . Hyderabad;
.
March 57,1999.
[5] Samadhan
d
Fig. 4 Micrographs of antibody in&bhzod (a) silicon nitride surfacc undcr
normal optical microscope @) silicon nitride surface unda fluoresecnt
microscope (c) silicon oxjmitride surface under normal optical microscope
(d) silicon nitride surfacc under fluorescent microscope.
witb respect to the stage, and wavelength used was 350 to
’
BOO m. Elipsometry measurements were made on several
different spots on silicon nitride and silicon oxynitride
surfaces. Thickness of HWCVD deposited silicon nitride film
was 200 nm (RI-1.985)and for oxynitride film it was 61.56
nm (RI-1.765).
(61 Octmvien Buiu, Gray PKennedy et.al, “Structural
Analysis of Silicon Dioxide and Silicon Oxynitride
Films Produced using an Oxygen Plasma,’! IEEE
Transactions on Plasma Science, VOL.26, N0.6,
Dec.1998
Immunosensor for
(71 Bhagwati Prasad, “Capacitive
Fibronectin,” ‘Ph.D. thesis’ submitted to school of
Bioscience and Bioengineering,.IIT Bombay, 2000.
IV CONCLUSION
In order to obtain the antibody immobilization on silicon
rich nitride, O2plasma treatment can be used. In this case O2
plasma trea’meit was carried out at room temperature.
Above results demonstrate QZ plasma treatment on silicon
rich nitride is able to generate oxynitride film of reasonable
thichess. Subsequently .this oxfitride film can be used to
immobilize antibodies via the process of silanization. Since
silicon is widely used material in silicon derived
microsystems such immobilization has great potential for
(81 Hong Xing You, Chirstophes R.Lowe, “AFM Studies of
Protein Adsorption,,” Journal of colloid and interface
science, ~01.182,pp 586-601, 1996.
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