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A certain group of mycotoxins, the ergot alkaloids, has caused countless deaths throughout human history. They are found in rye and other cereals and ingesting contaminated foods can cause serious health problems. To identify contaminated food exceeding the legal limits for ergot alkaloids, a portable and cost-effective test system is of great interest to the food industry. Rapid analysis can be achieved by screening for a marker compound, for which we chose ergometrine. We developed a magnetic bead-based immunoassay for ergometrine with amperometric detection in a flow injection system using a handheld potentiostat and a smartphone. With this assay a limit of detection of 3 nM (1 μg/L) was achieved. In spiked rye flour, ergometrine levels from 25 to 250 μg/kg could be quantified. All results could be verified by optical detection. The developed assay offers great promise to meet the demand for on-site ergometrine detection in the food industry.
Toxic secondary metabolites produced by fungi (aka mycotoxins) are often identified as contaminants in cereals or other food and pose a severe health risk, since they can be carcinogenic, neurotoxic, mutagenic or harmful in other respect. The highly nephrotoxic ochratoxin A (OTA) is one of the most important and widespread mycotoxins found in grains such as rye, wheat or oats. Thus, the fast and cost-effective detection of OTA is of great importance, to ensure that the toxin concentration is below the legal limits. Currently, the mycotoxin detection in mill operations is typically performed by means of the antibody-based ELISA method, which requires extensive laboratory equipment and specialized personnel. It was the aim of this study to develop an ELISA-inspired sensor method enabling the on-site quantification of OTA. Based on a classical competitive ELISA, an electrochemical detection system was developed. Anti-OTA-antibodies were captured on magnetic beads on which the competitive affinity reaction between OTA and an OTA- horseradish peroxidase (HRP) tracer was performed. Here the OTA concentration is inversely proportional to the amount of the bound OTA-HRP tracer, which was electrochemically quantified. For this purpose, oxidized tetramethylbenzidine (TMB), which is enzymatically produced by the reduction of H2O2 by the OTA-HRP tracer, is quantified by amperometry with screen-printed electrodes in a flow system. With this method a detection limit of 250 ng L-1 could be achieved which meets the current requirements for the detection of OTA in food and feed.
Superparamagnetic hybrid polystyrene-core silica-shell beads have emerged as promising alternatives to traditional in flow cytometry-based competitive antibody assays [1]. These materials consist of a polystyrene core and a silica shell, in which magnetic nanoparticles are embedded, facilitating the handling and retention in tests. The outer silica surface allows for easy modification through silane chemistry, allowing the attachment of antibodies, or other molecules of interest. Ochratoxin A (OTA), a mycotoxin that can be found in grain products, coffee, cacao, or grapes, was chosen as the main target analyte to detect [2]. In this study, previously in house produced anti-OTA antibodies [3] were attached to the surface of the particles and the whole system was used as detection entity. In a first approach, the system was used for the development of a competitive cytometry assay using an OTA-fluorescein (OTA-F) adduct as competitor and marker. In this assay the fluorescence emitted by the OTA-F competitor on the surface of the particle was detected at a wavelength of 518 nm using a 533/30.H filter and was correlated to the forward scatter (FSC) to distinguish it from the excess of competitor still in solution. Under optimised conditions, the final assay showed a limit of detection of 0.03 nM. In a second approach, a simplified ready-to-inject fluidic system was built based on a laser (488 nm) and a photomultiplier detector to measure the signal of competitor still in solution. The competition step was carried out in a vial and the whole mixture was injected into the fluidic system. To avoid signal scattering, the particles were separated in-line using a magnet and only the OTA-F competitor still in solution was detected, reaching a limit of detection of 1.2 nM. With the aim to reduce user manipulation, the final assay is still under development for in-line incubation during the competitive step.
Das Ziel der Arbeit war die Entwicklung einer magnetischen Anreicherungsmethode für eine Bead-basierte Substanzbibliothek zur Identifizierung von Proteinbindern. Im Ramen des Forschungsbelegs wurde bereits eine Anreicherungsmethode für ein Modellsystem einer Peptidbibliothek entwickelt. Das Modellsystem bestand dabei aus zwei verschiedenen Peptiden. Als Positivkontrolle wurde das FLAG-Peptid gewählt, welches selektiv gegen den Anti-FLAG-Antikörper bindet, und ein weiteres Peptid wurde als Negativkontrolle gewählt. Anhand dieses Modellsystems konnte die zuvor entwickelte Anreicherungsmethode von 20.000 auf 1.000.000 Beads vergrößert werden. Hierbei wurde ein Anreicherungsfaktor von 818 und eine gute Wiederfindungsrate von 82% erreicht.
Increasing contamination of environmental waters with pharmaceuticals represents an emerging threat for the drinking water quality and safety. In this regard, fast and reliable analytical methods are required to allow quick countermeasures in case of contamination. Here, we report the development of a magnetic bead-based immunoassay (MBBA) for the fast and cost-effective determination of the analgesic diclofenac (DCF) in water samples, based on diclofenac-coupled magnetic beads and a robust monoclonal anti-DCF antibody. A novel synthetic strategy for preparation of the beads resulted in an assay that enabled for the determination of diclofenac with a significantly lower limit of detection (400 ng/L) than the respective enzyme-linked immunosorbent assay (ELISA). With shorter incubation times and only one manual washing step required, the assay demands for remarkably shorter time to result (< 45 min) and less equipment than ELISA. Evaluation of assay precision and accuracy with a series of spiked water samples yielded results with low to moderate intra- and inter-assay variations and in good agreement with LC–MS/MS reference analysis. The assay principle can be transferred to other, e.g., microfluidic, formats, as well as applied to other analytes and may replace ELISA as the standard immunochemical method.