Multiplex immunoassay for persistent organic pollutants in tilapia: comparison of imaging- and flow cytometry-based platforms using spectrally encoded paramagnetic microspheres.

Recent developments in spectrally encoded microspheres (SEMs)-based technologies provide high multiplexing possibilities. Most SEMs-based assays require a flow cytometer with sophisticated fluidics and optics. A new imaging super-paramagnetic SEMs-based alternative platform transports SEMs with considerably less fluid volume into a measuring chamber. Once there SEMs are held in a monolayer by a magnet. Light-emitting diodes (LEDs) are focused on the chamber to illuminate the SEMs - instead of lasers and they are imaged by a charge-coupled device (CCD) detector, offering a more compact sized, transportable and affordable system. The feasibility of utilising this system to develop a 3-plex SEMs-based imaging immunoassay (IMIA) for the screening of persistent organic pollutants (POPs) was studied. Moreover the performance characteristics of 3-plex IMIA were critically compared with the conventional 3-plex flow cytometric immunoassay (FCIA). Both SEM technologies have potential for the multiplex analysis of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs) in buffer and fish extract with insignificant differences in assay sensitivities. Furthermore, we developed a faster and simpler, modified QuEChERS-like generic POPs extraction from tilapia fillet using sodium hydrogen carbonate as one of the salt additives and dispersive solid-phase extraction (dSPE) as a clean-up. Finally, a preliminary in-house validation using 40 different blank and spiked tilapia fillet samples was performed in both systems and the results obtained were critically compared. The lower-cost imaging SEMs-based system performed similarly to the original flow cytometer and, in combination with the new quicker QuEChERS-like extraction, it has high potential for future rapid screening of POPs in several other sample matrices such as other fish species, vegetable refined oils and environmental samples.

Multiplex screening of persistent organic pollutants in fish using spectrally encoded microspheres.

Persistent organic pollutants (POPs) are environmental and food-related contaminants of global public health concern and known to be carcinogenic and endocrine disruptors. Their monitoring is essential, and an easy-to-use, rapid, and affordable multianalyte screening method with simplified sample preparation can be a valuable tool prior to instrumental analysis. For this purpose, a flow cytometric immunoassay (FCIA), based on a spectrally encoded microbeads technology, was developed for the multiplex detection of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (BDEs) in buffer and fish extracts. The sensitivities of the assays in the three-plex FCIA format were similar to the individual FCIAs for the marker compounds benzo[a]pyrene (BaP), 3,3',4,4'-tetrachlorobiphenyl (PCB77), and 2,2',4,4'-tetrabromodiphenyl ether (BDE47) in buffer with IC(50) values of 0.4, 20, and 2 µg L(-1), respectively. Apart from the three markers, we could detect at least 14 other POPs. Extracts of fish with different fat content, prepared with a simplified extraction and cleanup procedure, had an insignificant influence on the overall three-plex FCIA performance, with the exception of some impact on the PAHs detection. The performance of the three-plex FCIA, in combination with the simple extraction procedure, is adequate for regulatory control in accordance with the required limits.

Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food.

Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC(50) of 0.3 microg L(-1) using a monoclonal antibody (Mab22F12) against BaP, similar to the IC(50) of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction procedure which are under development.

Biosensor discovery of thyroxine transport disrupting chemicals.

Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds.