Registration of T-2 mycotoxin with total internal reflection ellipsometry and QCM impedance methods.

A sensitive optical method of total internal reflection ellipsometry (TIRE) in conjunction with immune assay approach was exploited for the registration of T-2 mycotoxin in a wide range of concentrations from 100 microg/ml down to 0.15 ng/ml. Association constants of 1.4x10(6) and 1.9x10(7)mol(-1)s for poly- and monoclonal T-2 antibodies, respectively, were evaluated from TIRE kinetic measurements. According to TIRE data fitting, binding of T-2 molecules to antibodies (at saturation) has resulted in the increase in adsorbed layer thickness of 4-5 nm. The QCM impedance measurements data showed anomalously large mass increase and film softening, most likely, due to the binding of large T-2 aggregates to antibodies.

Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors.

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer. The glycolysated protein of jp51 may be selectively immobilised from the mixture of retroviral proteins (p24 and jp51), if it is necessary to distinguish infected animals from preliminarily immunised ones by means of a vaccine based on p24 protein. It was shown that the use of Staphylococcal protein A, instead of some lectins as intermediate layer for the Ab immobilisation, does not lead to a more sensitive determination of such low-weight toxins as 2,4-dichlorophenoxyacetic acid (2,4-D). The above-mentioned results were obtained with surface plasmon resonance (SPR) technique.

Planar silicon nitride waveguides for biosensing.

The principles of attenuation of the light intensity due to multiple reflections are realised in a planar silicon oxide (SiO(2))silicon nitride (Si(3)N(4)) waveguiding structure for the purpose of developing optical biosensors with improved sensitivity. The analysis of the experimental data shows that the large difference in refractive indices of core and cladding layers gives rise to an increase in sensitivity by a factor of 3 over previously reported structures. Composite polyelectrolyte self-assembled thin films containing cyclo-tetra-chromotropylene as an indicator and enzymes glucose oxidase or urease were employed in the superstrate as a sensing membrane. Individual enzyme reactions as well as their inhibition by pesticides were studied by monitoring the intensity of light output from the planar waveguide. The results were compatible with those obtained by conventional ultraviolet-visible absorption spectroscopy. The instrument detection limit for Imidacloprid pesticide was found to be as low as 10 ppb in concentration.

Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.

The most sensitive optical method of interferometry was exploited for determination of changes in the refractive index following the adsorption of biological molecules onto the solid surface. Instead of having two waveguiding arms (the main and the reference) in traditional Mach-Zhender interferometer, two ortogonal TM and TE modes propagating through the SiO(2)-Si(3)N(4)-SiO(2) waveguide structure were employed in planar polarization interferometer (PPI). Multiperiodic PPI response was, therefore, formed due to the phase shift between TM and TE modes. A matrix simulation procedure was developed in order to investigate the influence of both the refractive index and molecular layer thickness on the PPI response. Nonspecifical binding of fibrinogen to silicon nitride surface was studied as a model object for PPI testing. The results obtained are in good agreement with the known information about fibrinogen adsorption on the different surfaces. An attempt to introduce the concept of 'surface molecular concentration and molecular polariziability' instead of 'molecular layer thickness and refractivity' was undertaken.

Immobilization of biocomponents for immune optical sensors.

Immobilisation of both human immunoglobulin(IgG) and antiimmunoglobulin (anti-IgG) was performed by means of polyelectrolyte self-assembly. This technique was compared with direct immobilisation of the immune components on bare gold and their covalent binding via glutaraldehyde as a bifunctional reagent. Additionally, the immune components were properly oriented during their immobilisation by using a predeposited layer of the protein A. Methods of the surface plasmon resonance (SPR) and planar interferometry were employed for monitoring the immobilisation as well as specific immune reaction. It was shown that in case of the use of polyelectrolyte self-assembly it is possible to achieve the sensitivity of the analysis up to 30 ng/ml for SPR and up to 1 ng/ml for planar interferometer based immune sensors.