Bioimprinting for multiplex luminescent detection of deoxynivalenol and zearalenone.

A sensitive tool for simultaneous quantitative determination of two analytes in a single spot with the use of a bioimprinted protein is presented for the first time. BSA is chosen as a scaffold for generation of binding sites specific towards two compounds. A multiplex immunosorbent assay for screening of two cereal-born mycotoxins, deoxynivalenol and zearalenone, in wheat and maize is realized with the use of fluorescent silica coated quantum dots as labels. Water-soluble fluorescent nanostructures consist of core/shell Cd-QDs enrobed in silica shells to ensure their solubility. The mycotoxins are simultaneously detected by scanning the assay outcome at two different wavelengths, since two QD@SiO2 labelled conjugates fluorescent in different parts of the spectrum. The assay is combined with a rapid extraction technique. The limits of detection for the simultaneous determination were 100 and 700 µg kg-1 in both matrices for zearalenone and deoxynivalenol, respectively. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to confirm the obtained results.

Imprinted proteins as a receptor for detection of zearalenone.

A strategy to design an immunoassay based on imprinted proteins to detect a foodborne toxin zearalenone (ZEN) has been proposed. Proteins were used as scaffolds for generation of binding cavities with a high specificity against ZEN. Different proteins and different bioimprinting approaches were tested. The imprinted proteins primarily prepared in a tube and then immobilized on a microwell plate, and directly prepared in the microwell plate were compared for an application in immunosorbent detection of ZEN in naturally contaminated wheat and maize samples. The assay was combined with a rapid extraction technique. Specific interactions between the imprinted proteins and the target in a µg kg-1 linear range was achieved. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using the developed immunoassay for the analysis of ZEN with a sensitivity matching the maximum permitted level for ZEN in unprocessed cereals established by the European Commission (100 µg kg-1).

Capacitive sensor for detection of benzo(a)pyrene in water.

An affinity sensor based on capacitive transduction was developed to detect benzo(a)pyrene (BaP) in river water. Two types of recognition elements, the synthetic receptor analogues molecularly imprinted polymers (MIPs) and natural monoclonal antibody (mAb) were tested for this type of biosensor. Different polymerization strategies to obtain MIPs were compared. One approach comprised a preliminary batch synthesis of beads that were subsequently coupled covalently to an electrode surface. Another approach consisted of the in-situ synthesis of MIPs directly onto the electrode surface using electropolymerization. The latter approach provided the best results. To choose the optimal recognition element mAb and MIP-modified electrodes different sets were evaluated with regards to their sensitivity, selectivity, linear range and re-usability. The mAb-modified electrodes were considerably more sensitive toward BaP (ng L-1 range vs µg L-1 range for the MIP-modified one), and showed a broader linear working range than the MIP-modified electrodes. The latter revealed more suitable for group-selective determination of PAHs. The developed capacitive sensor was tested for the detection of BaP in naturally-contaminated water samples collected in different places of Ghent, Belgium. The results obtained with the sensor were coherent and reproducible, and were in a good agreement with the confirmation technique, HPLC-FlD.

Affinity sensor based on immobilized molecular imprinted synthetic recognition elements.

An affinity sensor based on capacitive transduction was developed to detect a model compound, metergoline, in a continuous flow system. This system simulates the monitoring of low-molecular weight organic compounds in natural flowing waters, i.e. rivers and streams. During operation in such scenarios, control of the experimental parameters is not possible, which poses a true analytical challenge. A two-step approach was used to produce a sensor for metergoline. Submicron spherical molecularly imprinted polymers, used as recognition elements, were obtained through emulsion polymerization and subsequently coupled to the sensor surface by electropolymerization. This way, a robust and reusable sensor was obtained that regenerated spontaneously under the natural conditions in a river. Small organic compounds could be analyzed in water without manipulating the binding or regeneration conditions, thereby offering a viable tool for on-site application.

Polymer-coated fluorescent CdSe-based quantum dots for application in immunoassay.

The paper describes all stages of synthesis and characterization of biocompatible CdSe-based core/shell quantum dots (QDs) and their application as fluorescent label for immunoassay. Special attention was focused on development of maleic anhydride-based amphiphilic polymers for QDs solubilization in aqueous media. In this work two PEG-amines were tried for polymer modification: monoamine Jeffamine M 1000 used previously in some researches and diamine Jeffamine ED-2003 applied for the first time for QDs solubilization. The use of different Jeffamines allows us to obtain QDs with carboxyl or amine functional groups available for conjugation. The influence of polymer composition on optical properties of the nanocrystals and their stability in aqueous solutions as well as on their conjugation with biomolecules was studied. QDs with different coatings were used as biolabels in quantitative fluorescence microtiter plate immunoassay and qualitative on-site column test. It was found that quantum dots covered with amphiphilic polymer prepared from poly(maleic anhydride-alt-1-octadecene) and Jeffamine ED-2003 retained up to 90% of their initial brightness, easily conjugated with protein and showed low non-specific adsorption. In optimized conditions the obtained QDs were successfully used for determination of mycotoxin deoxynivalenol in wheat and maize samples by fluorescence microtiter plate immunoassay with an IC50 of 220 µg kg(-1) and by on-site column test with cut-off of 500 µg kg(-1).

Development of suspension polymerized molecularly imprinted beads with metergoline as template and application in a solid-phase extraction procedure toward ergot alkaloids.

The first successfully developed molecularly imprinted polymer toward six ergot alkaloids and their respective epimers is described. A new imprinting molecule, metergoline, was used as template analogue in the production of suspension polymerized beads. These spherical particles functioned as selective sorbent in a solid-phase extraction column. The application of this column in the cleanup of barley samples prior to liquid chromatography coupled with tandem mass spectrometry allowed simple and cost-efficient sample preparation. The performance of the imprinted polymer and a non-imprinted control polymer was evaluated. This includes determination of the recovery values and the matrix effect of each of the 12 tested ergot alkaloids as well as a cross-reactivity study with 25 common mycotoxins. The binding isotherms were obtained for metergoline, thus allowing comparison with other (imprinted) sorbents. A comparison between bulk and suspension polymerization is provided to determine the appropriate production technique.

Interplay between hydrogen-bond formation and multicenter pi-electron delocalization: intramolecular hydrogen bonds.

The specific case of intramolecular hydrogen bonds assisted by pi-electron delocalization is thoroughly investigated using multicenter delocalization analysis. The effect of the pi-electron delocalization on the intramolecular hydrogen-bond strength is determined by means of the relative molecular energies of "open" and "closed" structures, calculated at the B3LYP/6-311++G(d,p) level of theory. These relative energies are compared to variations in the multicenter electron delocalization indices and covalent hydrogen-bond indices, which are shown to correlate very well with the relative strength of the intramolecular hydrogen bonds studied. The multicenter electron delocalization indices and covalent bond indices have been computed using the quantum theory of atoms in molecules approach. The hydrogen bonds are formed with oxygen, nitrogen, or sulfur as acceptor atom, which are also the atoms considered to be bonded to the donor hydrogen. Malonaldehyde is taken as reference; the substitution of oxygen by other atoms at the acceptor and donor positions and the effect of the aromaticity have been studied. The results shown here match perfectly with the qualitative expectations derived from the resonance models. In addition, they provide a quantitative picture of the role played by the pi-electron delocalization on the relative strength of intramolecular hydrogen bonds.

Interplay between hydrogen bond formation and multicenter pi-electron delocalization: intermolecular hydrogen bonds.

The interplay between aromatic electron delocalization and intermolecular hydrogen bonding is thoroughly investigated using multicenter delocalization analysis. The effect on the hydrogen bond strength of aromatic electron delocalization within the acceptor and donor molecules is determined by means of the interaction energies between monomers, calculated at the B3LYP/6-311++G(d,p) level of theory. This magnitude is compared to variations of multicenter electron delocalization indices and covalent hydrogen bond indices, which are shown to correlate perfectly with the relative values of the interaction energies for the different complexes studied. The multicenter electron delocalization indices and covalent bond indices have been computed using the quantum theory of atoms in molecules approach. All the hydrogen bonds are formed with oxygen as the acceptor atom; however, the atom bonded to the donor hydrogen has been either oxygen or nitrogen. The water-water complex is taken as reference, where the donor and acceptor molecular environments are modified by substituting the hydrogens and the hydroxyl group by phenol, furan, and pyrrole aromatic rings. The results here shown match perfectly with the qualitative expectations derived from the resonance model.