Analytical, thermodynamical and kinetic characteristics of photoluminescence immunosensor for the determination of Ochratoxin A.

Ochratoxin A (OTA) is one of the most widespread and dangerous food contaminants. Therefore, rapid, label-free and precise detection of low OTA concentrations requires novel sensing elements with advanced bio-analytical properties. In the present paper we report photoluminescence (PL) based immunosensor for the detection of OTA. During the development of immunosensor photoluminescent ZnO nanorods (ZnO-NRs) were deposited on glass substrate. Then the ZnO-NRs were silanized and covalently modified by Protein-A (Glass/ZnO-NRs/Protein-A). The latest structure was modified by antibodies against OTA (Anti-OTA) in order to form OTA-selective layer (Glass/ZnO-NRs/Protein-A/Anti-OTA). In order to improve immunosensors selectivity the surface of Glass/ZnO-NRs/Protein-A/Anti-OTA was additionally blocked by BSA. Formed Glass/ZnO-NRs/Protein-A/BSA&Anti-OTA structures were integrated within portable fiber optic detection system, what is important for the development of low cost and portable immunosensors. The immunosensor has been tested in a wide range of OTA concentrations from 10-4ng/ml until 20ng/ml. Interaction isotherms were derived from analytical signals of immunosensor. Association constant and Gibbs free energy for the interaction of Glass/ZnO-NRs/Protein-A/Anti-OTA with OTA were calculated, analyzed and compared with some other related results. Sensitivity range and limit of detection were determined as 0.1-1ng/ml and 10-2ng/ml, respectively. Interaction kinetics of ZnO-NRs with OTA was evaluated. Response time of the immunosensor toward OTA was in the range of 500-800s. Some insights related to the mechanism of PL-signal generation are proposed and discussed.

Gold coated porous silicon nanocomposite as a substrate for photoluminescence-based immunosensor suitable for the determination of Aflatoxin B1.

A rapid and low cost photoluminescence (PL) immunosensor for the determination of low concentrations of Aflatoxin B1 (AFB1) has been developed. This immunosensor was based on porous silicon (PSi) covered by thin gold layer (Au) and modified by antibodies against AFB1 (anti-AFB1). PSi layer was formed on silicon substrate, then the surface of PSi was covered by 30nm layer of gold (PSi/Au) using electrochemical and chemical deposition methods and in such ways PSi/Au(El.) and PSi/Au(Chem.) structures were formed, respectively. In order to find PSi/Au the most efficiently suitable for PL-based sensor design, structure several different PSi/Au(El.) and PSi/Au(Chem.) structures were designed while using different conditions for electrochemical or chemical deposition of gold layer. It was shown that during the formation of PSi/Au structure crystalline Au nanoparticles uniformly coated the surface of the PSi pores. PL spectroscopy of PSi/Au nanocomposites was performed at room temperature and it showed a wide emission band centered at 700nm. Protein A was covalently immobilized on the surface of PSi/Au(El.) and PSi/Au(Chem.) forming PSi/Au(El.)/Protein-A and PSi/Au(Chem.)/Protein-A structures, respectively. In the next step PSi/Au(El.)/Protein-A and PSi/Au(Chem.)/Protein-A structures were modified by anti-AFB1 and in such way a structures (PSi/Au(El.)/Protein-A/anti-AFB1 and PSi/Au(Chem.)/Protein-A/anti-AFB1) sensitive towards AFB1 were designed. The PSi/Au(El.)/Protein-A/anti-AFB1- and PSi/Au(Chem.)/Protein-A/anti-AFB1-based immunosensors were tested in a wide range of AFB1 concentrations from 0.001 upon 100ng/ml. Interaction of AFB1 with PSi/Au(El.)/Protein-A/anti-AFB1- and PSi/Au(Chem.)/Protein-A/anti-AFB1-based structures resulted PL quenching. The highest sensitivity towards AFB1 was determined for PSi/Au(El.)/Protein-A/anti-AFB1-based immunosensor and it was in the range of 0.01-10ng/ml. The applicability of PSi/Au-based structures as new substrates suitable for PL-based immunosensors is discussed.

Specific binding of large aggregates of amphiphilic molecules to the respective antibodies.

The Binding of nonylphenol to respective antibodies immobilized on solid substrates was studied with the methods of total internal reflection ellipsometry (TIRE) and QCM (quartz crystal microbalance) impedance spectroscopy. The binding reaction was proved to be highly specific having an association constant of KA=1.6x10(6) mol(-1) L and resulted in an increase in both the adsorbed layer thickness of 23 nm and the added mass of 18.3 microg/cm2 at saturation. The obtained responses of both TIRE and QCM methods are substantially higher than anticipated for the immune binding of single molecules of nonylphenol. The mechanism of binding of large aggregates of nonylphenol was suggested instead. Modeling of the micelle of amphiphilic nonylphenol molecules in aqueous solutions yielded a micelle size of about 38 nm. The mechanism of binding of large molecular aggregates to respective antibodies can be extended to other hydrophobic low-molecular-weight toxins such as T-2 mycotoxin. The formation of large molecular aggregates of nonylphenol and T-2 mycotoxin molecules on the surface was proved by the AFM study.