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OWLS APPLICATION NOTES NO-011

TEST MEASUREMENT OF LABEL-FREE


IMMUNOSENSOR USING BSA - ANTI BSA MODEL
MOLECULE PAIR
Using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection

I n t h e c a s e o f g l a s s t y p e m e t a l o xi d e s u r f a c e s s u c h
Coverage (ng/cm2)
as SiO2-TiO2, mainly hydroxyl groups are present, 600
which offer relatively few possibilities for covalent 5
anti-BSA IgG r
immobilization of biomolecules. To widen the circle 500
r
4 r
3
of covalent coupling methods, the surface of the r 1 r 2 r
waveguide has to be modified by silanization using 400
BSA
reactive silane reagents for introducing functional
groups of all sorts (e.g. aliphatic amine, sulfhydryl, 300
aromatic amine and epoxy) onto the surface of
200 A - distilled water 1 - 10µg/ml IgG
inorganic materials. Amino groups are formed by γ- B - 2.5% glutaraldehyde 2 - 25µg/ml IgG
aminopropyltriethoxysilane (APTS). D C - TRIS buffer, pH=7.4 3 - 50µg/ml IgG
100 D - 200 µg/ml BSA (TRIS) 4 - 100µg/ml IgG
B
C r - regeneration solution, 0.1 M HCl 5 - 200µg/ml IgG
0
0 20 40 60 80 100 120
A
Time (min)

Experiment performed on amino surface

r - regeneration solution, 0.1 M HCl


Coverage (arbitrary unit) S - 50µg/ml IgG standard
350
r r r r r
300

250

200

150
Evaluation of layers formed 100
S S S S S

To test the layers formed, immobilization 50


e xp e r i m e n t s we r e u n d e r t a k e n o n t h e m o d i f i e d
0
surfaces using the bovine serum albumin (BSA) -
150 200 250 300 350 400
anti-BSA antibody model molecule pair.
Time (min)

Sensor responses obtained for 50µg/ml anti-BSA


antibody standards

T h e a c t i v a t i o n o f t h e a m i n o s i l a n i z e d s e n s o r s wa s
p e r f o r m e d b y i n j e c t i n g g l u t a r a l d e h yd e ( 2 . 5 % i n
d i s t i l l e d w a t e r ) . T h a n t h e s u r f a c e w a s w a s h e d wi t h
distilled water and tris buffer (42 mM, pH 7.4) for a
few minutes and BSA (10 g/ml) in TRIS buffer was
i m m o b i l i z e d o n t h e s u r f a c e . I t wa s f o l l o we d b y
washing with buffer and injecting 0.1 M HCl to
remove molecules bound slightly to the surface.
After this step the chip was ready to measure IgG
m o l e c u l e s . M e a s u r e m e n t s we r e c a r r i e d o u t b y
injecting the IgG standard solutions. Antibodies
b o u n d t o t h e a n t i g e n w e r e w a s h e d o f f wi t h 0 . 1 M H C l
after each cycle. The system proved to be stable,
responses did not decrease significantly during the
measurement. Calibration curve for anti-BSA antibody standards

References
1. Vörös, J. J. Ramsden, G. Csucs, I. Szendrõ, S.M. De
Paul, M. Textor, N. D. Spencer (2002): Optical Grating
Coupler Biosensors. Biomaterials 23 3699-3710

2. www.owls-sensors.com

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Web: http://www.owls-sensors.com/; E-mail: info@owls-sensors.com Copyright @ Microvacuum, 2006