Development of immunosensor based on OWLS technique for determining Aflatoxin B1 and Ochratoxin A.

Mycotoxins are toxic secondary metabolites produced by a number of different fungi, and can be present in a wide range of food and feed commodities including cereal grains, oil seeds, dried fruits, apple juice, wine and meat products from animals fed contaminated meal. Many mycotoxins are highly resistant, and survive food processing, and therefore enter the food chain and provide a threat to human health. The optical waveguide lightmode spectroscopy (OWLS) technique has been applied to the detection of Aflatoxin and Ochratoxin in both competitive and in direct immunoassays. After immobilizing the antibody or antigen conjugate for the direct or indirect measurement, respectively, the sensor chip was used in flow-injection analyser (FIA) system. When using non-competitive method, sensor responses were obtained first only at analyte concentrations of 5-10 ng ml(-1). In both cases, the responses were very unstable. For competitive sensor investigation with the sensitized chip first the optimal dilution rate of monoclonal antibodies was determined, for the measurement of Ochratoxin A and Aflatoxin B1 the monoclonal antibody stock solution was diluted to 1 microg ml(-1) and to a 1:400 dilution, respectively. During the competitive measurement standard solutions were mixed with monoclonal antibodies at the appropriate concentration, the mixture was incubated for 1 min and injected into the OWLS system. The sensitive detection range of the competitive detection method was between 0.5 and 10 ng ml(-1) in both cases. After the establishment of the indirect method, barley and wheat flour samples were measured, and the results were in good correlation by those measured by enzyme linked immuno-sorbent assay (ELISA). Regression coefficient between the two methods for Ochratoxin and Aflatoxin was determined as 0.96 and 0.89, respectively.

Modification of the surface of integrated optical wave-guide sensors for immunosensor applications.

Methods have been developed which enable attachment amino and epoxy groups to the surface of integrated optical wave-guide sensors for immunosensor applications. The SiO2-TiO2 surfaces were modified by use of the trifunctional silane reagents gamma-aminopropyltriethoxysilane (APTS) and gamma-glycidoxypropyltrimethoxysilane (GOPS) in organic and/or in inorganic phases. Silanization methods were optimized taking into consideration the concentration of silane reagent used and the temperature and time of reaction. To evaluate the layers formed, immobilization experiments were undertaken on the modified surfaces using the bovine serum albumin (BSA)-anti-BSA IgG antibody model molecule pair. The regenerability of the sensitized surfaces was also studied.

Determination of the ratio of D- and L-amino acids in brewing by an immobilised amino acid oxidase enzyme reactor coupled to amperometric detection.

To determine the quantity of free amino acids, the D- and L-forms separately, is an important task in modern nutritional studies. The aim of our present work was to develop rapid, routine methods for fast determination of the different forms of free amino acids. We utilized two enzymes (L-amino acid oxidase, D-amino acid oxidase) with broad specificity. In our home-made reactors, the enzymes were immobilized in a thin-layer Plexi-cell on natural protein membrane. The enzyme-cell was built into a FIA system and the hydrogen peroxide generated during the enzymatic reaction was determined by an amperometric detector. The electrode potential was fixed at +100 mV. The parameters for the biochemical and electrochemical reactions were optimized in each case. The optimal pH value for measuring L- and D-amino acids was found ca. 8.8 and 9.5, respectively. The LAO reactor could be used for more than 900 measurements, while the DAO reactor for about 1000 measurements. The working concentration range was between 0.1-3 and 0.2-3 mM, respectively. The same standard solution (L- and D-Methionine, 1 mM) was injected 25 times sequentially and the standard deviations were 2 and 2.7%, respectively. After determining the optimal parameters, the specificity of the immobilized enzyme preparations towards different amino acids and in samples from different stages of brewing was investigated.

Application of biosensor for monitoring galactose content.

The quality and quantity of different sugars play a very important role in studying the carbohydrate metabolism of yeast. During the bioprocesses there is a need to follow the concentrations of these sugars. Authors have reported on the development of biosensors for determination of glucose and maltose previously. The aim of this research was to construct a sensor for determining galactose in fermentation broths to prepare the basis for an online monitoring system. Using a modified thin-layer enzyme cell connected to an electrochemical detector cell, a biosensor has been developed for this purpose. Galactose was oxidized with immobilized galactose oxidase enzyme (EC 1.1.3.9) and the hydrogen peroxide generated during the enzyme reaction was determined with an amperometric detector. The parameters for the biochemical and electrochemical reactions were optimized. The pH optimum of 6.6 was found when using phosphate buffer. The buffer solution completed by micro elements (Mg2+, Se2+) gave more stable signs. The activities for raffinose, lactose, glycerol and dihydroxyacetone were 68, 16, 6 and 430%, respectively. With the thin-layer cell more than 900 samples were measured in 6 weeks. Samples obtained from different fermentations were measured with the newly developed galactose sensor and the results were compared with the standard UV method. The correlation coefficient was 0.991. The results showed that the application of the new biosensor was successful.

Studying the bienzyme reaction with amperometric detection for measuring maltose.

If a fermentation process is followed by a change in the concentration of maltose, information on the level of the maltose is important. A new type of sensor was developed for measuring the content of maltose in fermentation broth. The base of the sensor is a thin-layer reactor, in which a protein membrane was chosen for the immobilization of enzymes. To measure maltose, we investigated the influence of enzymes such as alpha-glucosidase (EC 3.2.1.20) and amyloglucosidase (EC 3.2.1.3) on the effectiveness of conversion to glucose. Because amyloglucosidase proved to be more effective, a mixture of amyloglucosidase and glucose oxidase was applied for the determination of maltose. To develop the best measuring technique, the consequences of changes in different parameters, such as the optimal ratio of enzymes, role of pH value and that of the flow rate, were studied. An amperometric measuring cell with Pt-Ag/AgCl-Pt electrodes was used at +600 mV operating potential. The results indicate that the maltose content in different types of fermentation broth can be determined by the new measuring cell in the range of 0.2-4 mM maltose. The cell was successfully tested in the fermentation of brewer's yeast.