Kapillar-Immuno-Assay mit elektrochemischer Detektion

Alkylphenolethoxylate werden als Bestandteile von Waschmitteln und als Weichmacher eingesetzt und gelangen so in großer Zahl ins Abwasser. Alkylphenole, als relativ hydrophobe Abbauprodukte, gelten aufgrund ihres hohen östrogenen Potential als Umwelthormone (endocrine disruptors). Deshalb werden verschiedene klassische und biochemische Analysenmethoden für die Substanzklasse entwickelt. Das hohe Probenaufkommen verlangt nach schnellen Nachweisverfahren, die mit Hilfe von automatisierbaren Fließ-Systemen realisiert werden kann. In dieser Arbeit präsentieren wir einen elektrochemischen Fließ-Immunoassay (EFIA), der eine Kombination eines empfindlichen Biosensors mit einem Kapillar-Immuno-Reaktor darstellt. Im vorgestellten Ansatz werden Antikörper gegen die Zielsubstanzen Nonyl-,Oktylphenol, und Nonylphenolethoxylat in Mikro-Kapillaren immobilisiert, in denen Kompetition stattfindet. Dazu wird die Probenlösung mit Tracer versetzt und gemeinsam in den Kapillaren inkubiert. Nach einem anschließenden Spülschritt wird die gebundene Menge markierte Analyt quantifiziert, indem das aminophenylierte Substrat des Tracers injiziert wird. Nach zeitlich kontrollierter Hydrolyse wird das gebildete p-Aminophenol am Biosensor vorbei geführt und dabei vermessen. Als Markierungssubstanz für den Tracer wird ß-D-Galactosidase verwendet, welche Amino- und Nitrophenylgalactopyranosid hydrolysiert und so Amino- bzw. Nitrophenol freisetzt. Die Tracer werden durch chemische Kopplung der zur Immunisierung verwendeten Haptene mit dem Enzym (ßGal) dargestellt

Modular microfluidic system as a model of cystic fibrosis airways

A modular microfluidic airways model system that can simulate the changes in oxygen tension in different compartments of the cystic fibrosis (CF) airways was designed, developed, and tested. The fully reconfigurable system composed of modules with different functionalities: multichannel peristaltic pumps, bubble traps, gas exchange chip, and cell culture chambers. We have successfully applied this system for studying the antibiotic therapy of Pseudomonas aeruginosa, the bacteria mainly responsible for morbidity and mortality in cystic fibrosis, in different oxygen environments. Furthermore, we have mimicked the bacterial reinoculation of the aerobic compartments (lower respiratory tract) from the anaerobic compartments (cystic fibrosis sinuses) following an antibiotic treatment. This effect is hypothesised as the one on the main reasons for recurrent lung infections in cystic fibrosis patients

Micro immuno supported liquid membrane (mu-ISLM) extraction

A mu-ISLM (micro Immuno Supported Liquid Membrane) extraction system coupled in a sequential injection analysis (SIA) configuration, capable of simultaneous sample cleanup and enrichment was developed. The capacity of the extraction channels - donor and acceptor, respectively - were reduced from originally 10 to 1.65 mu L. The acceptor channel surface was covered with a gold layer enabling immobilisation of antibodies via an alkanthiol self-assembled monolayer. The mu-ISLM-SIA system resulted in very high extraction efficiency and enrichment factor (Extraction efficiency (E): 136%; Enrichment factor (E-e): 544) and a subsequent low detection limit for the model analyte simazine of 0.1 ng/L

Flow immunochemical bio-recognition detection for the determination of interleukin-10 in cell samples

On- and off-line heterogeneous non-competitive flow immunoassays for the determination of Interleukin-10 are described. The sample containing IL-10 is mixed, either on-line in a reaction coil or off-line in a test tube, with fluorescent labelled anti-IL-10 antibodies to form an antibody-antigen complex. The labelled unbound antibodies are trapped on an immobilized IL-10 column whereas the IL-10-antibody complexes are eluted and detected downstream by a fluorescence detector. The optimization of the systems was performed with respect to choice of affinity support, flow rate, carrier buffer additives, pH and antibody-antigen association. Both bio recognition assays were tested with a spiked cell medium and the IL-10 detection limits in this matrix was found to be 8 fmol using the off-line incubation mode and 40 fmol using the on-line incubation mode. The sample through-put was 26 and 40 samples per hour in the on-line and off-line incubation modes, respectively. IL-10 identification in the sample fractions was achieved using MALDI-TOF MS

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 mum depth and 25 pm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-mug 1(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 mug 1-1, and IC50 values of 0.096 and 0.130 mug 1(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCI buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124%, at atrazine fortification levels of 0.5-3 and 80-240 mug 1(-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved

Fluorescence polarisation for immunoreagent characterisation

Antibodies were characterised using fluorescence polarisation, a homogeneous assay technique in which all reagents are in solution. Kinetic studies on the association and dissociation of the immunocomplex were performed. A competitive assay was used and the sensitivities, operational linearities, as well as the specificities of the immunoassays were experimentally determined for various antibody preparations with specificity for triazines. Detection limits for atrazine in water samples were determined to be within the range of 0.08-0.4 ng ml-1 using a 5-min incubation time and a 0.5-ml sample volume