Fast Deoxynivalenol Determination in Cereals Using a White Light Reflectance Spectroscopy Immunosensor

Deoxynivalenol (DON) is a mycotoxin produced by certain Fusarium species and found in a high percentage of wheat and maize grains cultured worldwide. Although not so toxic as other mycotoxins, it exhibits both chronic and acute toxicity, and therefore methods for its fast and accurate on-site determination are highly desirable. In the current work, we employ an optical immunosensor based on White Light Reflectance Spectroscopy (WLRS) for the fast and sensitive immunochemical label-free determination of DON in wheat and maize samples. The assay is completed in 12 min and has a quantification limit of 2.5 ng/mL in buffer corresponding to 125 μg/kg in whole grain which is lower than the maximum allowable concentrations set by the regulatory authorities for grains intended for human consumption. Several extraction protocols have been compared, and the highest recovery (>90%) was achieved employing distilled water. In addition, identical calibration curves were received in buffer and wheat/maize extraction matrix providing the ability to analyze the grain samples using calibrators in buffer. Recoveries of DON from spiked wheat and maize grain samples ranged from 92.0(±4.0) to 105(±4.0)%. The analytical performance of the WLRS immunosensor, combined with the short analysis time and instrument portability, supports its potential for on-site determinations.

Identification, partial characterization, and use of grey mullet (Mugil cephalus) vitellogenins for the development of ELISA and biosensor immunoassays

Vitellogenin (Vtg) has proven to be a sensitive and simple biomarker in determining sex, sexual maturity, and xenoestrogenic effects in fish. Thus, our investigation has been focused on identification, partial characterization, and quantification of grey mullet (Mugil cephalus) Vtg through the use of a variety of biochemical and immunological analytical techniques. Mullet is considered both a promising aquaculture candidate and an important species for improving sediment quality in polyculture systems. In the first part of this work, grey mullet Vtg was purified from plasma of 17β-estradiol (E2)-induced male fish by a one-step chromatographic protocol, and partially characterized. Specific polyclonal antibodies were then raised against the mullet Vtg, and both an indirect ELISA and an optical immunosensor were set up and validated to quantify plasma Vtg. The indirect ELISA and the optical immunosensor assay developed showed linear measuring in the range 56.8–1047.1 ng mL−1 and 70–739 ng mL−1 Vtg concentrations in standard solutions, respectively. The results obtained suggest that the indirect ELISA allows Vtg detection over a wide dynamic range, thus resulting more suitable for rapid and sensitive sample screening. Therefore, we suggest that the direct immunosensor is a promising tool which needs more investigation to improve the sensitivity.

Covalent immobilization of rabbit-antiaflatoxin-antibodies onto the poly-acrylamideacrylonitrile as well as hybrid material UREASIL and developing an optical immunosensor

The aim of this work is to describe a covalent immobilization of antibodies onto the poly- acrylamide-acrylonitrile or hybrid material UREASIL and creation of optical immunosensor for determination of aflatoxin Bl. For this purpose, mouse-anti-aflatoxin B1 antibodies with oxidized carbohydrate moieties were covalently immobilized on the membranes of polyacrylamide- polyacrylonitrile copolymer, as well as the hybrid material UREASIL. To determine the affinity> binding of the immobilized antibody with afatoxin Bl was used "sandwich" method. Associated with the immobilized antibody sought ingredients interact with a surplus of secondary’ signal antibodies. The described method has been developed as a model system, which can easily be applied for the determination of aflatoxins in samples of different origin. To the best of our knowledge, this is the first study to show that in the establishment of biosensor was used hybrid material UREASIL.

Ανίχνευση C-αντιδρώσας πρωτεΐνης και D-διμερών σε μικροσυστοιχίες με φασματοσκοπία ανάκλασης λευκού φωτός

Cardiovascular diseases are one of the main death causes in the modern world. Forthis reason, the determination of the concentration in the serum of particular proteins,called cardiac markers, is vital both for determining the extent of the damage after anischemic incident as well as for predicting a cardiac disease. The aim of this workwas to develop a method to simultaneously detect two markers that can help inpredicting a heart attack, namely C-reactive protein (CRP) and D-Dimers usingantibodies microarrays in combination with an optical immunosensor based on whitelight reflectance spectroscopy and allows the detection of biomolecular reactions inreal time without the use of labels. In particular, detection is based on monitoringchanges in interference spectrum created upon reflection of white light on a siliconsurface covered with a suitable film of transparent material on which the analytespecificantibodies are immobilized. At first, suitable antibodies were selected throughthe development of non-competitive enzyme immunoassays for the two analytes inmicrotitration wells. After that, the immunochemical assays were transferred to theoptical sensor, wherein the thickness and composition of the transparent filmdeposited on the silicon surface as well as the method for the chemical modificationof the surface and immobilization of antibodies were optimized. Subsequently, all theparameters of immunochemical determinations with the sensor were optimizedincluding antibody concentrations, immunoreaction time, flow rate, etc. By applying the optimal conditions, calibration curves for both markers were obtained and therepeatability and reliability of the measurements performed with the developedimmunosensor were evaluated. Finally, discrete zones of the two specific antibodiesagainst CRP and D-Dimers were created by spotting them onto the same surfaceand the surfaces were used for the simultaneous detection of the two analytes inserum samples. The values determined for both analytes were in good agreementwith those determined for the same samples with reference methods, demonstratingthe reliability of the determinations performed with the developed immunosensor andits potential for application in the simultaneous determination of two markers inclinical samples.

Magnetically-actuated, bead-enhanced silicon photonic immunosensor

Magnetic actuation has been introduced to an optical immunosensor technology resulting in improvements in both rapidity and limit of detection for an assay quantitating low concentrations of a representative protein biomarker.