OWLS APPLICATION NOTES NO-007

LABEL-FREE IMMUNOSENSOR FOR OCHRATOXIN - A

using OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY (OWLS) detection
Abstract
Ochratoxins are toxic metabolites produced by Penicllium verrucosum and several
Aspergillus species, 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 development of suitable validated and official
anal ytical m ethods and rapi d screeni ng tests for cost-effecti ve food control on a l arge
sc al e. OW LS offered a hi ghl y sens iti v e label -free method to develop imm obili zed antigen-
BSA conjugate based competitive immunosensor.

Application of OWLS sensors Standard inhibition curve

of the immobilized antigen conjugate based competitive
as competitive immunosensor for the detection of aflatoxin immunosensor
Ochratoxin A

Surface Chemistry of OWLS sensors

Amino functionalisation of waveguide surface by 3-
ami nopropyl tri ethoxysil ane

Imm obi lis ati on of Oc hratoxin-BSA c onjugate
(10µg/ml ) on the OW LS s ens or s urfac e b y
gl utaral deh yde (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
immobilised 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
proportional to the Ochratoxin A content in the
samples.
The sensitive detection range of the competitive
d e t e c t i o n m e t h o d w a 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 O c h r a t o xi n A .
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.

Com pari ng the res ults from OW LS m easurem ent to

that obtained by ELISA method, the calculated
regression coefficient (R2) for Ochratoxin A was
0.97. It can be stated that the competitive
imm unosensor based on O W LS detection coul d be
suitable for the quick determination of Ochratoxin
level in grain samples.

References
1. Vörös, J. J. Ramsden, G. Csucs, I. Szendrõ, S.M. De
Paul, M. Textor, N. D. Spencer (2002): Optical Grating
Measuri ng c ycl es of di fferent dil uti ons of m onoclonal anti - Coupler Biosensors. Biomaterials 23 3699-3710.
ochratoxin A antibody (1 - 0.01µg/ml, 2 - 0.1µg/ml, 3 - 2. Adányi N ., Levkovets I.A., Rodri guez-Gi l S., Ronal d
0.25µg/ml, 4 - 0.5µg/ml, 5 - 1.0µg/ml, 6 – 2.5µg/ml, 7 – A., Váradi M. Szendrı I. (2006): Development of
5.0µg/ml, 8 - 10µg/ml, 9 –25µg/ml, 10 – 1.0µg/ml) 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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