Gel electrophoresis separation and origins of light emission in fluorophores prepared from citric acid and ethylenediamine

We investigated light emission of hydrothermally treated citric acid and ethylenediamine (EDA) with various precursor ratios using gel-electrophoresis. We show that this relatively simple approach can deliver significant insights into the origins of photoluminescence. We found that products of the synthesis consist of both positively and negatively charged species and exhibit large dispersion in electrophoretic mobility (i.e. charge-to-size ratio). We observed that despite the large dispersion of the reaction products the blue light emission is confined to discrete bands clearly identifiable in the gel. We demonstrate clear evidence that this emission originates from the negatively charged light molecular fraction with the highest mobility which shows no excitation-dependent light emission. This molecular fluorophore exhibits spectral characteristics similar to previously reported 1,2,3,5-tetrahydro-5-oxo-imidazo[1,2-a]pyridine-7-carboxylic acid (IPCA). Secondary gel electrophoresis run performed on the bands extracted from the first run indicates that no further separation takes place. On the basis of our experimental results, we conclude that relatively stable binding exists between IPCA and EDA-derived product. Thus, the products of the reaction contain IPCA both in molecular form and in complexes with EDA-derived products. We conclude that excitation-dependent emission is related to the fluorophore binding to the positively charged EDA-derived products with a positive charge

Silanized liposomes loaded with luminescent quantum dots as label for mycotoxin detection

Silanized liposomes loaded with luminescent quantum dots was developed as a perspective label for immunoaasay. Silica coverage ensures the stability of the liposomes against fusion and internal leakage and also simplifies bioconugation

Determination of Ochratoxin A in colored food products: sample preparation and an immunoassay test method

A method is proposed for the purification of highly colored food products (red wine, red pepper) for the immunochemical test determination of Ochratoxin A (OTA) with visual detection. The method is based on passing an analyzed sample (wine diluted with a solution of polyethylene glycol and sodium hydrocarbonate or water-ethanol extract of pepper diluted with a solution of sodium hydrocarbonate) though an adsorbent layer. Criteria for selecting the adsorbent are considered, and silica gels with aminopropyl and trimethylaminepropyl groups are used as the optimal ones. A test system for the determination of OTA combines the indicated purification method with the immunoaffinity preconcentration and immunoenzyme detection. The developed approach has allowed the test determination of OTA in red wine and red pepper at levels of 2 mu g/L and 10 mu g/kg, respectively

Luminescent quantum dots as labels for multiparametric immunoassay

Use of quantum dots as highly sensitive labels in immunochemical assay for simultaneous screening of multiple analytes is described

New Immunochemically-based Field Test for Monitoring Benzo[a]pyrene in Aqueous Samples

Polycyclic aromatic hydrocarbons (PAHs) represent health and environmental concerns. The present study was designed to develop a highly sensitive and reliable immunochemically-based non-in stru mental field assay to monitor the presence of benzo[a]pyrene (BAP) in aqueous samples at the ppt-level using visible test evaluation. The corresponding gel-based immunoassay was set up to combine preconcentration and detection of the target analyte using anti-PAH antibodies and horseradish-BAP tracer conjugate in one single cartridge. Water sample preparation, such as extraction, centrifugation, or filtering was found to be unnecessary. No interference by higher water-soluble PAHs at 4000-fold excess compared to BAP was observed. The assay was configured as a qualitative test (positive/negative) at the cut-off level of 5 ng L-1, however, this level can be adapted to the required analyte concentration by rather simple adjustment of the anti-PAH antibody concentration of the test zone. Test validation was performed with real samples such as surface water, melted snow, and tap water using high performance liquid chromatography with fluorescent detection (HPLC-FLD) with solid phase extraction for validation

Quantum dot based rapid tests for zearalenone detection

Three different kinds of immunosorbent assays with luminescence detection were developed for the determination of zearalenone (ZEN), a secondary toxic metabolite of Fusarium fungi. CdSe/ZnS core/shell quantum dots (QDs) were used as a label in quantitative micro-well plate immunoassays (fluorescent-labeled immunosorbent assay, FLISA) and in qualitative column test methods. As carriers for QD-based column tests, sepharose gel (for covalent binding of antibody) and polyethylene frits (for physical absorption of antibody) were used and compared. The application of QDs as a label resulted in a fourfold decrease in the IC50 value with FLISA (0.1 ng mL(-1)) with a detection limit of 0.03 ng mL(-1) when compared with the traditional immunosorbent assay which makes use of horseradish peroxidase as the enzyme label. The cutoff levels for both qualitative column test methods were selected based on the maximum level for ZEN in unprocessed cereals established by the European Commission (100 mu g kg(-1)) as 5 ng mL(-1) taking into account extraction and dilution. The different developed immumoassays were tested for ZEN determination in raw wheat samples. As a confirmatory method, liquid chromatography coupled to tandem mass spectrometry was used. The obtained results allow using FLISA and both qualitative column test methods for the analysis of analytes with very low established maximum limits, even in very complicated food matrices, owing to the high dilution of the sample extract

Non-instrumental immunochemical tests for rapid ochratoxin A detection in red wine

Gel-based and membrane-based flow-through immunoassay formats were investigated for rapid ochratoxin A (OTA) detection in red wine. The flow-through set-Up consisted of an antibody containing gel or membrane placed at the bottom of a standard solid-phase extraction column (i.e the flow-through column), combined with a clean-up column. Different clean-up methods were studied for red wine clarification and purification The optimal method consisted of passing wine, diluted with an aqueous Solution containing 1% polyethylene glycol (PEG 6000) and 5% sodium hydrogencarbonate, through strong anion exchange (SAX) silica. An immunoassay for OTA detection in red wine was optimized and a cut-off level at 2 mu g L-1 according to EU legislation was achieved with both formals A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the inembiane-based assay The proposed rapid gel-based test was compared with a standard immunoaffinity column - high-performance liquid chromatography - fluorescent detection (IAC-HPLC-FLD) method and a good correlation of the results was obtained for naturally contaminated wine sample