Nanochemistry and biotechnology

Influence of cross-linking reagents on the operation parameters of bioselective elements of SPR immunosensors based on intermediate layer of recombinant Staphylococcal protein A 
Bakhmachuk A.O.1,2, Gorbatiuk O.B.1,3, Dons’koi B.V.4, Rachkov A.E.1, Soldatkin A.P.1,2

1 Institute of Molecular Biology and Genetics, NAS of Ukraine. Akademika Zabolotnoho Str., 150, Kyiv-03680, Ukraine. 
E-mail: a.bakhmachuk@gmail.com
2 Institute of High Technologies, Taras Shevchenko National University of Kyiv. Pr. Akademika Hlushkova, 2, korp.5, Kyiv-03022, Ukraine.
3 State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine. Velyka Vasyl’kivska Str., 57/3, Kyiv-03150, Ukraine.
4 Institute of pediatrics, obstetrics and gynecology AMS of Ukraine. Maiborody Str., 8, Kyiv-04050, Ukraine. 

During immunosensor creation for antigen detection, after immobilization of antibodies their antigen-binding activity is usually much lower than the activity of the same antibodies in solution. The main reasons for this are believed to be random orientation of antibodies on the sensor surface and steric hindrance caused by the influence of the surface of a solid substrate. The use of an intermediate layer containing immunoglobulin-binding proteins (e.g. Staphylococcal protein A (SPA)), that selectively bind Fc fragment of antibody leaving Fab fragment available for antigen detection, is a promising approach. In order to ensure the efficient immobilization of SPA via a strong interaction of thiol group with the gold sensor surface while preserving its immunoglobulin-binding activity, one cysteine residue has been introduced into a non-essential part of recombinant SPA. 
One of serious drawbacks of this approach is impossibility of the multiple usage of such bioselective elements, because during regeneration the affinity bonds between the immunoglobulin-binding proteins and antibodies are destroyed as well as the bonds between antibodies and antigens. However, the use of bifunctional reagents, that covalently bind proteins, can successfully solve this problem. 
This work is aimed to compare the operation parameters (e.g. sensitivity, specificity, reproducibility of sensor signal) of bioselective elements of surface plasmon resonance immunosensor based on intermediate layer of recombinant SPA after its covalent binding to antibodies using two well-known for such purposes cross-linking reagents: glutaraldehyde and bissulfosuccinimidyl suberate (BS3).