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 as SiO2-TiO2, mainly hydroxyl groups are present, which offer relatively few possibilities for covalent immobilization of biomolecules. To widen the circle of covalent coupling methods, the surface of the waveguide has to be modified by silanization using reactive silane reagents for introducing functional groups of all sorts (e.g. aliphatic amine, sulfhydryl, aromatic amine and epoxy) onto the surface of inorganic materials. Amino groups are formed by γaminopropyltriethoxysilane (APTS).

600 500

Coverage (ng/cm2)
anti-BSA IgG r 1 r 2 r 3 r 4 r 5 r

400
BSA

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A D B C A - distilled water B - 2.5% glutaraldehyde C - TRIS buffer, pH=7.4 D - 200 µg/ml BSA (TRIS) r - regeneration solution, 0.1 M HCl 1 - 10µg/ml IgG 2 - 25µg/ml IgG 3 - 50µg/ml IgG 4 - 100µg/ml IgG 5 - 200µg/ml IgG

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60 Time (min)

80

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120

Experiment performed on amino surface

350 300 250 200 150

Coverage (arbitrary unit)
r r

r - regeneration solution, 0.1 M HCl S - 50µg/ml IgG standard r r r

Evaluation of layers formed
To test the layers formed, immobilization 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 surfaces using the bovine serum albumin (BSA) anti-BSA antibody model molecule pair.

S

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S

S

S

S

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300 Time (min)

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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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