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 μg/L and 10 μg/kg, 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.     

Multiplex flow-through immunoassay formats for screening of mycotoxins in a variety of food matrices

Two multi-analyte flow-through immunoassay formats for rapid detection of mycotoxins in a variety of food matrices (peanut cake, maize, and cassava flour) were developed and evaluated. The selected food matrices are typical staple foods and export products for most low-income communities around the world. The assay formats included gel-based and membrane-based flow-through assays and were based on the principle of indirect enzyme-linked immunosorbent assay. Using the same immunoreagents, the performance characteristics of both assays were compared. To the best of our knowledge, this is the first report on such a comparison. The gel-based format was developed to screen for ochratoxin A, fumonisin B₁, deoxynivalenol, and zearalenone detection at cut-off values of 3, 1,250, 1,000, and 200 μg kg⁻¹, respectively, while the membrane-based format can be used to screen ochratoxin A, aflatoxin B_1, deoxynivalenol, and zearalenone at the following cut-offs: 3, 5, 700, and 175 μg kg⁻¹, respectively. The applicability of these assay formats was demonstrated by evaluating the performance characteristics of both tests through performing multiple experiments on different days. Both assays were further evaluated by analyzing naturally contaminated samples in the laboratory and also in the field under tropical conditions (Cameroon, West Africa). The false-negative rate with both formats was less than 5%, which is in good agreement with Commission Decision 2002/657/EC regarding the performance of analytical methods intended for screening purposes.     

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α?=?0.05) affect the recoveries of ergot alkaloids.

Anodic‐Stripping Voltammetric Immunoassay for Ultrasensitive Detection of Low‐Abundance Proteins Using Quantum Dot Aggregated Hollow Microspheres

A new anodic‐stripping voltammetric immunoassay protocol for detection of IgG1, as a model protein, was designed by using CdS quantum dot (QD) layer‐by‐layer assembled hollow microspheres (QDHMS) as molecular tags. Initially, monoclonal anti‐human IgG1 specific antibodies were anchored on amorphous magnetic beads preferably selective to capture F_ab of IgG1 analyte from the sample. For detection, monoclonal anti‐human IgG1 (F_c‐specific) antibodies were covalently coupled to the synthesized QDHMS. In a sandwich‐type immunoassay format, subsequent anodic‐stripping voltammetric detection of cadmium released under acidic conditions from the coupled QDs was conducted at an in situ prepared mercury film electrode. The immunoassay combines highly efficient magnetic separation with signal amplification by the multilayered QD labels. The dynamic concentration range spanned from 1.0 fg mL^?1 to 1.0 μg mL^?1 of IgG1 with a detection limit of 0.1 fg mL^?1. The electrochemical immunoassay showed good reproducibility, selectivity, and stability. The analysis of clinical serum specimens revealed good accordance with the results obtained by an enzyme‐linked immunosorbent assay method. The new immunoassay is promising for enzyme‐free, and cost‐effective analysis of low‐abundance biomarkers.     

Calculation of phase diagrams of ternary M-Ga-Sb (M = In,Al) systems by convex hull approach

The ternary semiconductor phase diagrams of M-Ga-Sb (M = In, Al) are calculated by means of the convex hull approach. The software package TernAPI is tested as an effective tool for these purposes. The new facilities and advantages of this package are proven. It enables one to construct ternary phase diagrams based on thermodynamic properties of all phases existing in systems. The changes in phase region shapes and phase transformations with temperature can be studied by means of the TernAPI package. Selection of reliable thermodynamic models of solutions is of crucial importance for correct calculations of phase equilibria. It is shown that an adequate phase diagram can be constructed when a polynomial model of liquid solutions in the Al-Ga-Sb system is used.     

Elastic contact between nominally plane surfaces in the presence of roughness and adhesion

The solution of the problem of interaction with regard to the forces of adhesive (molecular) attraction of two nominally plane half-spaces one of which is elastic and the surface of the other has a regular relief is presented. The surface mutual approach dependence on the applied nominal pressure and the effective specific work of adhesion are analyzed for various parameters of adhesive interaction and micro-geometry of the surfaces.     

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).

Multi-analyte homogenous immunoassay based on quenching of quantum dots by functionalized graphene

We propose a homogenous multi-analyte immunoassay based on the quenching of quantum dot (QD) fluorescence by means of graphene. Two QDs with emission maxima at 636 and 607 nm were bound to antibodies selective for mouse or chicken immunoglobulins, respectively, and graphene functionalized with carboxylic moieties was employed to covalently link the respective antigen. The antibody-antigen interaction led graphene close enough to QDs to quench the QD fluorescence by resonance energy transfer. The addition of free antigens that competed with those linked to graphene acted as a “turn-on” effect on QD fluorescence. Fluorescence emitted by the two QDs could be recorded simultaneously since the QDs emitted light at different wavelengths while being excited at the same wavelength and proved to be linearly correlated with free antigen concentration. The developed assay allows measuring both antigens over 2–3 orders of magnitude and showed estimated limits of detection in the nanomolar range. This approach is thus a promising universal strategy to develop homogenous immunoassays for diverse antigens (cells, proteins, low-molecular-mass analytes) in a multi-analyte configuration.