Quantum dot loaded liposomes as fluorescent labels for immunoassay.

Liposomes loaded with water-soluble and water-insoluble quantum dots (QD) were for the first time applied as labels in different heterogeneous immunoassays for the determination of food contaminants, using mycotoxin zearalenone (ZEN) as a model. A great deal of work was devoted to the optimal choice of phospholipids for the liposomes preparation and to the factors which are important for the stability and size of obtained liposomes. Thin-film hydration and reverse-phase evaporation techniques were evaluated in terms of stability of the obtained liposomes and their efficiency for QD loading. Conjugation of liposomes with proteins and the influence of cross-linkers to the nonspecific interaction of the obtained liposomes with the surface of microtiter plates and cartridges were investigated and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester was found as the optimal cross-linker. The limits of detection (LOD) for ZEN of fluorescence-labeled immunosorbent assays were 0.6 µg kg(-1), 0.08 µg kg(-1), and 0.02 µg kg(-1), using QD, liposomes loaded with water-soluble QD, and water-insoluble QD, respectively. Similarly, the developed qualitative on-site tests using the different QD labels and taking into account the EU maximum residues level for ZEN in unprocessed cereals showed cutoff levels of 100, 50, and 20 µg kg(-1).

Liposomes loaded with quantum dots for ultrasensitive on-site determination of aflatoxin M1 in milk products.

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin-protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 µg kg(-1). For qualitative on-site tests, the cutoff was set at 0.05µg kg(-1), taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5% (2.6% and 3.3%, respectively).

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 IC(50) 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 µg 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.

Lanthanide-to-quantum dot Förster resonance energy transfer (FRET): Application for immunoassay.

Förster resonance energy transfer (FRET) between lanthanide ion complexes (L) acting as donors and luminescent semiconductor quantum dots (QD) acting as acceptors is discussed in the terms of advantages and disadvantages for its application in immunoassay. L-QD-FRET is potentially a powerful tool that can be used to detect and confirm formation of immunocomplexes, but until now it had very limited practical analytical application. Therefore, the main aim of this review is to analyze all possibilities, advantages, and disadvantages of L-QD-FRET in immunoassay applications. Considering L and QD respectively applied as donor and acceptor, the most advantageous properties for analytical purposes are large decay time of L complexes and the high absorption of QD. L complexes' extremely long decay times make it possible to directly detect FRET through enhancement of QDs decay time as a result of energy transfer. Very high QD absorption predetermines extremely large Förster radii (ca. 10nm), which means that FRET can be utilized for proteins and protein complexes, such as antigen-antibody systems.

Sensitive immunochemical approaches for quantitative (FPIA) and qualitative (lateral flow tests) determination of gentamicin in milk.

Three kinds of immunoassays for the determination of gentamicin in milk samples were developed and validated. First, a fast and easily-performed fluorescence polarization immunoassay was used for characterization of the employed polyclonal antibody. The calculated Kaff were (1.9±0.4)×10(9)М(-1) and (6.0±0.2)×10(6)М(-1) for the high- and low-affinity fractions respectively. The assay was characterized with a good sensitivity, the limit of detection being 5µgkg(-1). Two different kinds of detection labels, i.e. colloidal gold (CG) and quantum dots (QDs), were evaluated for use in lateral-flow format with respect to rapid visual on-site testing. The cut-off levels for both qualitative formats were selected based on the maximum level for gentamicin in milk established by the European Commission, 100µgkg(-1), resulting in a 10µgkg(-1) cut-off considering sample dilution. The intra-laboratory validation was performed with sterilized milk samples artificially spiked with gentamicin at concentrations less than, equal to, and greater than the cut-off level. It was shown that milk products could be analyzed without any sample preparation, except for dilution with the buffer solution. The rates of false-positive and false-negative results were below 5% for both labels. The different developed immunoassays were tested towards gentamicin determination in artificially-spiked and naturally contaminated milk samples.

Rapid screening of aflatoxin B1 in beer by fluorescence polarization immunoassay.

This manuscript describes the development of a sensitive, fast and easily-performed fluorescence polarization immunoassay (FPIA) for the mycotoxin aflatoxin B1 (AFB1) in various beer samples, both lager and dark. The highest sensitivity was determined for six poly- and monoclonal antibodies selective towards aflatoxins. The sample pretreatment design was emphasized since beer samples are characterized by extremely diverse matrices. Herein, the choice of sorbent for effective removal of matrix interferences prior to analysis was crucial. The samples were diluted with a borate buffer solution containing 1% PEG 6000 and passed through the clean-up column packed with NH2-derivated silica. This sample pretreatment technique was perfectly suitable for the FPIA of lager beer samples, but for dark beer and ale it did not suffice. An artificial matrix was constructed to plot a calibration curve and quantify the results of the latter samples. The developed immunoassay was characterized by a limit of detection of 1 ng mL(-1). Apparent recovery values of 89-114% for lager and 80-125% for dark beer were established. The FPIA data for AFB1 was characterized by elevated linear regression coefficients, 0.9953 for spiked lager and 0.9895 for dark beer samples respectively.

Preparation and characterization of stable phospholipid-silica nanostructures loaded with quantum dots.

The structural dependence of silica-liposome hybrids on silanization conditions was investigated. Silica coatings protect liposomes against aggregation, degradation, and leakage, which are important for their application in bioimaging. Liposomes loaded with quantum dots were synthesized and attempts to obtain uniformly sized, silica-coated nanocapsules were made.

Silica-coated liposomes loaded with quantum dots as labels for multiplex fluorescent immunoassay.

This manuscript describes synthesis and followed application of silica-coated liposomes loaded with quantum dots as a perspective label for immunoaasay. The hollow spherical structure of liposomes makes them an attractive package material for encapsulation of multiple water-insoluble quantum dots and amplifying the analytical signal. Silica coverage ensures the stability of the loaded liposomes against fusion and internal leakage during storage, transporting, application and also provides groups for bioconugation. For the first time these nanostructures were employed for the sensitive multiplex immunochemical determination of two analytes. As a model system mycotoxins zearalenone and aflatoxin B1 were detected in cereals. For simplification of multiassay results' evaluation the silanized liposomed loaded with QDs of different colors were used. The IC50 values for the simultaneous determination of zearalenone and aflatoxin B1 were 16.2 and 18 µg kg(-1) for zearalenone and 2.2 and 2.6 µg kg(-1) for aflatoxin B1 in wheat and maize, respectively. As confirmatory method, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used.

Novel multiplex fluorescent immunoassays based on quantum dot nanolabels for mycotoxins determination.

The aim of this manuscript was the development of easy-to-operate quantum dots (QDs)-based immunochemical techniques for simultaneous screening of several mycotoxins in cereals. Two different approaches for multiplex fluorescent immunosorbent assay (FLISA) were employed. In the first approach a multiwell plate in which the different wells express a different mycotoxin (deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1) was considered as a multiplex because each sample was pretreated once and then will be distributed over a series of wells within the same plate (single-analyte multiplex, SAM). The entire assay allows the simultaneous determination of all compounds. For the double-analyte multiplex (DAM) two different specific antibodies were co-immobilized in one single well. Zearalenone and aflatoxin B1 were simultaneously determined, provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum, by scanning the assay outcome at two different wavelengths. The limits of detection (LOD) for the simultaneous determination of deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1 by SAM FLISA were 3.2, 0.6, 0.2, 10 and 0.4 µg kg(-1), respectively, while for the DAM FLISA they were 1.8 and 1 µg kg(-1) for zearalenone and aflatoxin B1, respectively. SAM FLISA principle was also presented in a qualitative on-site format and tested for on-site multiplex determination of four mycotoxins in cereals. The achieved cut-off values of 500, 100, 2 and 100 µg kg(-1) for deoxynivalenol, zearalenone, aflatoxin B1 and T2-toxin respectively. For simplification of multiassay results' evaluation the conjugates with QDs of different colors were used.

Immunochemical approach for zearalenone-4-glucoside determination.

Zearalenone-4-ß-D-glucopyranoside (zearalenone-4-glucoside) detection techniques, based on a combination of acidic or enzymatic hydrolysis of the masked mycotoxin to the parent form (i.e. zearalenone), and immunochemical determination of zearalenone-4-glucoside as a difference between the zearalenone concentration after and before cleavage of the glycosidic bond were developed. The limit of detection for zearalenone-4-glucoside, achieved for the enzyme linked immunosorbent assay, was 3 µg kg(-1); the cut-off level for the sum of zearalenone and zearalenone-4-glucoside determination by a qualitative gel-based immunoassay was 50 µg kg(-1). Trifluoromethanesulfonic acid was checked for acidic hydrolysis and resulted in approximately 70% of glycosidic bond cleavage in optimal conditions. Seven different glycoside hydrolases were tested during the design of the enzymatic hydrolysis technique. Enzymatic hydrolysis combined with enzyme linked immunosorbent assay and gel-based immunoassay determinations was applied for the determination of zearalenone-4-glucoside or the sum of zearalenone and zearalenone-4-glucoside in cereal samples. The chosen enzyme (glucosidase from Aspergillus niger) allowed to cleave 102% of zearalenone-4-glucoside in standard solutions and 85% in cereal samples. Liquid chromatography coupled to tandem mass spectrometry was used as confirmatory method. As a result, good correlations between immunochemical techniques and the chromatographic data were obtained. The developed technique is suitable for simultaneous immunochemical determination of zearalenone and its masked form, zearalenone-4-glucoside.

Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine.

A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2 microg L(-1). The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods.

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 microg L(-1) according to EU legislation was achieved with both formats. A more significant colour difference between blank and spiked samples was observed for the gel-based assay making this superior to the membrane-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 samples.