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.

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.

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.

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 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).