Download Abstract: Epitope-Enhanced Assay Sensitivity For Imaging

Epitope-Enhanced Assay Sensitivity For Imaging Nanoplasmonics Biosensing
A. Frangolho*1,2, P. Pellacani 1, A. Valsesia1, W. Haasnoot 2, C.S. Nielsen 3, G. Marchesini 1,4
1PLASMORE SRL, Italy;2RIKILT-Institute of Food Safety,The Netherlands; 3Schafer-n, Denmark;
4 Biosypher, United kingdom
Imaging nanoplasmonics (iNP) allows the study of biomolecular interaction in highly multiplexed manner and in
real time. iNP is very sensitive to local changes of refractive index caused by the absorption of a molecule in the
proximity of a metallic nanoplasmonic surface. In order to specifically detect the absorbed molecules different assay
formats can be used. In a direct assay an antibody is immobilized on the surface and the binding of the antigen can
be measured. However, the success of this format is dependent on the molecule’s molecular weight. On the other
hand, an inhibition assay can be used with the advantage of signal enhancement due to the antibody mass.
iNP features a high intensity-fast decaying evanescent wave compared with traditional SPR and the total sensed
volume around each nanostructure is below 100 nm. For assay towards proteins, the main bottleneck of the
inhibition assay format for iNP technology is that the immobilization of a large molecule may decrease the sensed
volume that can be occupied by the antibody. In the current study we propose a solution by immobilizing only the
linear active epitope of less than 20 aminoacids in length instead of the full protein. This allows a higher local
binding site density , therefore higher number of antibodies can be bound and sensed increasing the specific signal
and stability of the chip to regenerations.
Within the IMPRESS project various allergens are been mapped for their epitopes to produce a high density
microarray for detection in food matrices using an iNP device, the Impressor (Fig.1). We evaluated the feasibility of
using the epitope of bovine kappa-casein to build an inhibition assay and compared it with standard assay in two
different technologies, iNP and SPR. Bovine kappa-casein is a protein used to detect cow milk in goat milk, a
common food fraud and adulteration. One epitope-containing-peptide was first identified and immobilized on a
nanochip and a biacore CM5 chip used for benchmarking and the maximum signal and inhibition of the binding of
the antibody by free kappa-casein was measured. Preliminary results show approximately an order of magnitude
sensitivity enhancement in both SPR and iNP technologies when the epitope is used directly compared to the
immobilization of the full protein. Measurements of chip stability show the remarkable capabilities of this new
technology and approach to immunoassay design.
Fig. 1 – Impressor