OWLS APPLICATION NOTES NO-006

LABEL-FREE IMMUNOSENSOR FOR Aflatoxin B
using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection

Abstract
Aflatoxins are toxic metabolites produced by Aspergillus species (mainl y A. Flavus, A. parasiticus, and A. nomius) and can be present in a wide range of food and feed commodities. Because of the persistence of Aflatoxins in the food chain, exposure to the compound is a potential human health hazard. This has prompted adoption of regulatory limits in several countries which, in turn, implies the devel opm ent of sui table vali dated and offi ci al anal yti cal methods and rapi d screeni ng tests for cost-effec ti ve food control on a large sc al e. OW LS offered a hi ghl y s ensitiv e label -free m ethod to develop immobilized antigen- BSA conjugate based competitive immunosensor.

Application of OWLS sensors
as competitive immunosensor for the detection of A f l a t o xi n B 1

Standard inhibition curve
of the immobilized antigen conjugate based competitive aflatoxin immunosensor

Surface Chemistry of OWLS sensors
Amino functionalisation of waveguide surface by 3-aminopropyltriethoxysilane Immobilisation of A f l a t o xi n - B S A conjugate (10µg/m l) on the OW LS sensor surface by glutaraldehyde (2,5%).

Competitive assay format
S t a n d a r d s a n d s a m p l e s w e r e m i xe d wi t h a n t i b o d y , i n c u b a t e d f o r a c e r t a i n p e r i o d a n d t h e m i xt u r e w a s injected into the O W LS system. During d e t e r m i n a t i o n , t o xi n p r e s e n t i n t h e s a m p l e c o m p e t e s for binding of the antibody to the toxin conjugate immobilized on the sensor surface. Upon incubation, only antibodies remaining in free form in the sample m i xt u r e b i n d t o t h e a n t i g e n s i m m o b i l i z e d o n t h e sensor surface. Thus, the amount of antibodies bound to the surface of the chip was inversely p r o p o r t i o n a l t o t h e A f l a t o xi n B 1 c o n t e n t i n t h e samples.

The sensitive detection range of the competitive d e t e c t i o n m e t h o d wa s b e t w e e n 0 . 5 - 1 0 n g m l - 1 wh e n m e a s u r i n g A f l a t o xi n B 1 .

Sample measurement
E xt r a c t i o n p r o c e d u r e
W eight 1 g of sample and add 10 mL of acetoni tril e/water (6:4; v/v) m i xture. (C ompl ete cereal grains should be grinded) Stirred for 5 min Decante and filtrate using a UF mem brane with 1 00 . 00 0 N MW L. (5 min centrifugation at 5000 rpm) Dilute the filtrate at 100 fold dilution with 100 fold dil uti on of acetoni tril e/water mi xture i n TR IS buffer

Optimization of antiserum dilution
The antibody concentration employed is one of the parameters of key importance, because increasing t o xi n c o n c e n t r a t i o n s i n t h e s a m p l e r e s u l t i n l a r g e r decreases in the assay signal.

70 60 Mass (arbitrary unit) 50 1000x 40 100x 30 200x 20 10 0 -10 0 2 Time (min) 4 6 400x 400x 10000x

Com pari ng the res ults from OW LS m easurem ent to that obtained by ELISA method, the regression c o e f f i c i e n t ( R 2 ) f o r A f l a t o x i n wa s c a l c u l a t e d 0 . 9 7 . It can be stated that the competitive immunosensor based on OW LS detection could be suitable for the q u i c k d e t e r m i n a t i o n o f A f l a t o xi n l e v e l i n g r a i n samples.

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 36993710 Adányi N ., Levkovets I.A., Rodri guez-Gi l S., Ronal d A., Váradi M. Szendrı I. (2006): Development of immunos ens or bas ed on OW LS t echni que f or determining Aflatoxin B1 and Ochratoxin A • Biosensors and Bioelectronics,22(6) 797-802. www.owls-sensors.com

Selection of optimal antibody dilution for competitive measurement of Aflatoxin B1

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MicroVacuum Ltd. H-1147, Kerékgyártó u. 10., Budapest, Hungary
Web: http://www.owls-sensors.com/; E-mail: info@owls-sensors.com

Phone: + 36 1 252 1991; +36 1 467 0108

Fax: +36 1 221 7996

Copyright @ Microvacuum, 2006

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