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An automated flow-through multi-mycotoxin immunoassay using the stand-alone Munich Chip Reader 3 platform and reusable biochips was developed and evaluated. This technology combines a unique microarray, prepared by covalent immobilization of target analytes or derivatives on diamino-poly(ethylene glycol) functionalized glass slides, with a dedicated chemiluminescence readout by a CCD camera. In a first stage, we aimed for the parallel detection of aflatoxins, ochratoxin A, deoxynivalenol, and fumonisins in cereal samples in a competitive indirect immunoassay format. The method combines sample extraction with methanol/water (80:20, v/v), extract filtration and dilution, and immunodetection using horseradish peroxidase-labeled anti-mouse IgG antibodies. The total analysis time, including extraction, extract dilution, measurement, and surface regeneration, was 19 min. The prepared microarray chip was reusable for at least 50 times. Oat extract revealed itself as a representative sample matrix for preparation of mycotoxin standards and determination of different types of cereals such as oat, wheat, rye, and maize polenta at relevant concentrations according to the European Commission regulation. The recovery rates of fortified samples in different matrices, with 55–80 and 58–79 %, were lower for the better water-soluble fumonisin B1 and deoxynivalenol and with 127–132 and 82–120 % higher for the more unpolar aflatoxins and ochratoxin A, respectively. Finally, the results of wheat samples which were naturally contaminated with deoxynivalenol were critically compared in an interlaboratory comparison with data obtained from microtiter plate ELISA, aokinmycontrol® method, and liquid chromatography–mass spectrometry and found to be in good agreement.

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V(L)) and heavy (V(H)) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V(L) and V(H) chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V(L) and V(H) domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V(L) and V(H) combinations. On the basis of our data, it was shown that the V(H) plays the major role for specific binding of B[a]P, whilst the V(L) can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V(L) in providing cross-reactivity.

The toxic nature of polycyclic aromatic hydrocarbons (PAHs), in particular benzo[a]pyrene (B[a]P), neccessitates the monitoring of PAH contamination levels in food and the environment. Here we introduce an indirect immunoassay format using electro-switchable biosurfaces (ESB) for the detection of B[a]P in water. The association of anti-B[a]P antibodies to microelectrodes is analyzed in real-time by measuring changes in the oscillation dynamics of DNA nanolever probes, which are driven to switch their orientations by high-frequency electrical actuation. From the association kinetics, the active concentration of anti-B[a]P, and hence the B[a]P contamination of the sample, can be determined with picomolar sensitivity. The detection limit of the assay improves with measurement time because increasingly accurate analyses of the binding kinetics become possible. It is demonstrated that an exceedance of the permissible 10 ng/L (40 pM) limit for B[a]P is detectable in an unprecedented short assay time (1 h), using a simple three-step workflow involving minimal sample preparation. The reproducibility was satisfying with standard deviations below 5%. Further, the utility of the assay for practical applications is exemplified by analyzing a river water sample.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, which can cause cancer in humans. The maximum tolerable limit of benzo[a]pyrene (B[a]P) in drinking water was set to 10 ng/L by the European Commission (Council Directive 98/83/EC), because of its highly carcinogenic and mutagenic effect on humans. In the present investigation, mice were immunized with B[a]P-bovine serum albumin conjugates and 110 generated hybridoma cell lines screened by different techniques to identify clones that produce anti-B[a]P antibodies. Subsequently, a new automated flow-through biochip noncompetitive direct chemiluminescence immunoassay (CLEIA) was compared with conventional indirect and direct enzyme-linked immunosorbent assays (ELISAs). It was demonstrated that the microchip-based screening method compared to ELISA was fast and very sensitive with use of only nanoliter volumes of supernatant. Forty clones could be evaluated in less than 5 min. Six high affinity monoclonal antibodies with different cross-reactivities (CR) for individual PAHs were identified by the chip-based assay and indirect microtiter plate ELISA. In comparison, the direct ELISA in the microtiter plate failed to identify three of these clones. The four antibodies with the highest affinity had half maximum inhibitory concentrations (IC 50 values) between 0.31 and 0.92 μg/L for B[a]P. Affinity constants of these four antibodies were determined by surface plasmon resonance using a water soluble B[a]P-peptide. The observed CR pattern of the four monoclonal antibodies for 16 tested PAHs was quite different. Only one specific antibody for B[a]P was observed, while others were more suitable for class-specific PAH determination.

Ochratoxin A (OTA) can contaminate foodstuffs in the ppb to ppm range and once formed, it is difficult to remove. Because of its toxicity and potential risks to human health, the need exists for rapid, efficient detection methods that comply with legal maximum residual limits. In this work we have synthesized an OTA conjugate functionalized with a water-soluble peptide for covalent immobilization on a glass biochip by means of contact spotting. The chip was used for OTA determination with an indirect competitive immunoassay format with flow-through reagent addition and chemiluminescence detection, carried out with the stand-alone automated Munich Chip Reader 3 (MCR 3) platform. A buffer model and real green coffee extracts were used for this purpose. At the present, covalent conjugate immobilization allowed for at least 20 assay-regeneration cycles of the biochip surface. The total analysis time for a single sample, including measurement and surface regeneration, was 12 min and the LOQ of OTA in green coffee extract was 0.3 μg L−1 which corresponds to 7 μg kg−1.

Pathogen detection is important for health and safety reasons. Several outbreaks all over the world have shown the need for rapid, qualitative, quantitative, and, particularly, multianalyte detection systems. Hence, a multichannel flow-through chemiluminescence microarray chip for parallel detection of pathogenic bacteria was developed. The disposable chip made of acrylonitrile-butadiene-styrene (ABS) copolymer was devised as a support for a multiplexed sandwich immunoassay. Calibration and measurement was possible in one experiment, because the developed chip contains six parallel flow-through microchannels. Polyclonal antibodies against the pathogenic bacteria Escherichia coli O157:H7, Salmonella typhimurium, and Legionella pneumophila were immobilized on the chip by microcontact printing in order to use them as specific receptors. Detection of the captured bacteria was carried out by use of specific detection antibodies labelled with biotin and horseradish peroxidase (HRP)-streptavidine conjugates. The enzyme HRP generates chemiluminescence after adding luminol and hydrogen peroxide. This signal was observed by use of a sensitive CCD camera. The limits of detection are 1.8 x 10(4) cells mL(-1) for E. coli O157:H7, 7.9 x 10(4) cells mL(-1) for L. pneumophila, and 2.0 x 10(7) cells mL(-1) for S. typhimurium. The overall assay time for measurement and calibration is 18 min, enabling very fast analysis.