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 mu g kg(-1). For qualitative on-site tests, the cutoff was set at 0.05 mu g 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)

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

Forster 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 Land 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 Forster radii (ca. 10 nm), which means that FRET can be utilized for proteins and protein complexes, such as antigen-antibody systems

A luminescence immunoassay test method for determining benzo[a]pyrene in natural water

A test method is developed for determining benzo[a]pyrene in natural water, based on the use of a polyethylene filter (frit) with adsorbed specific antibodies, placed within a transparent column. In passing a test solution, the analyte is adsorbed on the frit similarly to the process implemented in immunoaffinity preconcentration. The added conjugate of a labeled analyte takes the remained vacant binding sites of antibodies. Luminescent semiconductor nanoparticles (quantum dots) CdSe/ZnS, used as labels, enable visual determination under irradiation with UV light. The limit of detection for benzo[a]pyrene in water is similar to 0.5 ng/mL

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

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

Simultaneous determination of several mycotoxins by rapid immunofiltration assay

A method is developed for the simultaneous rapid determination of three mycotoxins, zearalenone (ZEN), ochratoxin A (OTA), and fumonisin B1 (Fum) by membrane immunofiltration analysis using a marker enzyme of alkaline phosphatase (AP) and two mycotoxins, deoxynivalenol (DON) and total T-2 toxin (T2) and HT-2 toxin (HT2) with horseradish peroxidase (HRP). The analysis is based on a competitive interaction between the antigene, free and bound to the enzyme, and antibodies immobilized on a membrane. The procedures of membrane fabrication and the conditions of mycotoxin to determination in model mixtures and extracts from wheat, corn, and silage are optimized. The influence of sample preparation on the results of analysis is studied. It is shown that the additives of polymers favor the reduction of the matrix effect in the analysis of complex matrixes using conjugated HRP. The methods developed allow the determination of mycotoxins at a level of the maximal permissible concentrations legislated by EU directives. The corresponding values (mu g/kg) are 50, 2.5, and 500 in wheat; 100, 2.5, and 500 in corn; and 125, 25, and 1250 in silage for the simultaneous quantification of ZEN, OTA, and Fum (AP marker). For the determination of DON and total T2/HT2 with HRP, 1250 (1000) and 100 (500) in wheat and corn(silage). The procedures were validated by the analysis of spiked and naturally contaminated samples. The analysis of 10 samples takes 25 min

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 mu g kg(-1), 0.08 mu g kg(-1), and 0.02 mu g 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 mu g kg(-1)