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 Standard inhibition curve

as competitive immunosensor for the detection of of the immobilized antigen conjugate based competitive
A f l a t o xi n B 1 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%).
The sensitive detection range of the competitive
Competitive assay format 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 .
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 Sample measurement
injected into the O W LS system. During
E xt r a c t i o n p r o c e d u r e
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
W eight 1 g of sample and add 10 mL of
for binding of the antibody to the toxin conjugate acetoni tril e/water (6:4; v/v) m i xture. (C ompl ete
immobilized on the sensor surface. Upon incubation, cereal grains should be grinded)
only antibodies remaining in free form in the sample
Stirred for 5 min
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
Decante and filtrate using a UF mem brane with
sensor surface. Thus, the amount of antibodies 1 00 . 00 0 N MW L.
bound to the surface of the chip was inversely (5 min centrifugation at 5000 rpm)

Dilute the filtrate at 100 fold dilution with 100 fold
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 dil uti on of acetoni tril e/water mi xture i n TR IS buffer
samples.

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
10000x
Mass (arbitrary unit)

50
1000x
40 Com pari ng the res ults from OW LS m easurem ent to
100x that obtained by ELISA method, the regression
30 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 .
200x
20 It can be stated that the competitive immunosensor
400x based on OW LS detection could be suitable for the
10
400x 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
0 samples.
0 2 4 6
-10
References
Time (min)
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
Selection of optimal antibody dilution for competitive
2. Adányi N ., Levkovets I.A., Rodri guez-Gi l S., Ronal d
measurement of Aflatoxin B1
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.
3. www.owls-sensors.com

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