University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Ursula Bilitewski

Abbrev:
Bilitewski, U.
Other Names:
Address:
Gesellschaft für Biotechnologische Forschung mbH, (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany
Phone:
+49-531-61810
Fax:
+49-531-6181515
Email:
ubi@gbf.de

Citations 8

"Investigations With Respect To Stabilization Of Screen-printed Enzyme Electrodes"
J. Mol. Catal. B 1999 Volume 7, Issue 1-4 Pages 67-76
J. T. Schumacher, I. Münch, T. Richter, I. Rohm and U. Bilitewski

Abstract: Immobilization of enzymes on screen-printed electrode surfaces was performed by entrapment in UV-polymerizable, screen-printable pastes. The use of glucose oxidase, lactate oxidase, xanthine oxidase and horseradish peroxidase gave corresponding sensors in flow injection analysis (FIA) systems. The influence of various additives on different enzymes in the immobilization matrix was investigated. Activation as well as stabilization was achieved in some cases. An FIA system including dialysis-modules and stabilized glucose and lactate electrodes was successfully used to monitor animal cell cultivations.

"Online Monitoring Of Monoclonal Antibody Production With Regenerable Flow Injection Immuno Systems"
J. Biotechnol. 1994 Volume 32, Issue 3 Pages 213-220
Andreas Gebbert, Manuel Alvarez-Icaza, Heinz Peters, V. Jäger, Ursula Bilitewski* and Rolf D. Schmid

Abstract: In this paper two systems for the observation of the production of mouse-IgG during the cultivation of hybridoma cells in a perfusion reactor are presented. The direct immunosystem is based on the detection of changes in capacitance of a dielectric layer (tantalum oxide) on a metal surface (tantalum) when antibodies bind to immobilized anti-antibodies. The sensor consisted of a 25 nm tantalum oxide layer, electrochemically grown onto a laser patternized 1 micron thick tantalum layer. The indirect system is based on an automated fluorimetric sandwich ELISA system with β-galactosidase conjugated secondary antibodies. Two cultivations of mouse hybridoma cells in a 2-1 perfusion reactor were performed. The first cultivation was monitored with the capacitance system, the second cultivation was monitored with the fluorimetric system.
Tantalum Fermentation broth Capacitance Immunoassay Fluorescence Perfusion

"Comparison Of Different Biosensor Systems Suitable For Bioprocess Monitoring"
J. Biotechnol. 1993 Volume 31, Issue 3 Pages 257-266
U. Bilitewski*, W. Drewes, J. Neermann, J. Schrader, R. Surkow, R. D. Schmid and J. Bradley

Abstract: To achieve effective bioprocess monitoring, sensing systems are required which are suitable for an online determination of substrates, inhibitors, nutrients or products. Such devices may utilise biochemical principles, i.e. the specific interaction of biochemical receptors with their surroundings. They can be constructed either as in situ sensors or as flow-through sensors connected to the process via sampling devices. Hence, characteristic features of an in situ glucose electrode are described, e.g. analytical range, sensitivity and stability. The sensor was based on mediated electron transfer from the enzyme glucose oxidase to the graphite electrode, the mediators being tetrathiafulvalene (TTF) or dimethylferrocene (DMF). Additionally, various flow injection analysis (FIA) systems based on oxidases, which were immobilized either on controlled pore glass or in a membrane, were characterized with respect to analytical ranges and sensitivities and applied to glucose, lactate and glutamate determinations in off-line samples taken from an animal cell cultivation.
Glucose Lactate Glutamate Fermentation broth Sensor Electrode Electrode Method comparison Process monitoring Controlled pore glass

"Control Of Microbial Activity By Flow Injection Analysis During High Cell Density Cultivation Of Escherichia Coli"
J. Biotechnol. 1993 Volume 27, Issue 2 Pages 143-157
T. Ding, U. Bilitewski*, R. D. Schmid, D. J. Korz and E. A. Sanders

Abstract: The application of an automated flow injection analysis (FIA) system for online determination of microbial activity, during high cell density cultivations of Escherichia coli is reported. Based on a bioelectrochemical principle, the FIA method used a redox mediator (potassium hexacyanoferrate(III)) to facilitate electron transfer from the microorganisms to an electrochemical detector. Assays were carried out using a new sampling device which provided aseptic operation by use of a valve and chemical sterilisation. No sample dilution or pretreatment was necessary for biomass concentrations up to approximately 40 g l-1. The sample volume was 0.5 mL and the overall analysis time was 5 min. FIA signals were found to correlate well with the oxygen uptake rate (OUR). Changes in metabolic activity due to low substrate levels or high inhibitor concentrations in the cultivation medium became obvious from the FIA signals.
Microbial activity Bacteria Fermentation broth Electrochemical analysis Process monitoring

"Detection Of Methamidophos In Vegetables Using A Photometric Flow Injection System"
Environ. Monit. Assess. 1997 Volume 44, Issue 1-3 Pages 375-382
J. Lui, A. Günther and U. Bilitewski

Abstract: A flow injection analysis (FIA) system using immobilized acetylcholinesterase (AChE) has been used for the detection of methamidophos in vegetable extracts. Methamidophos is one of the most commonly used organophosphate insecticides in South East Asia. AChE was immobilized onto magnetic particles; using a magnetic reactor, the particles could easily be separated from the test sample. Results show that complex matrices such as vegetable extracts have no inhibitory effect on AChE activity in the FIA system. The presence of methamidophos in the extracts caused AChE inhibition. The response could be followed as an inhibition curve and the inhibition constant calculated. Results show that using 85% AChE residual activity as the detection limit methamidophos could be detected in lettuce and cabbage at 12 and 3 mg/kg vegetable material respectively. In a simulated field situation, cabbage leaves were spiked with 20 to 40 mg/kg methamidophos, homogenized and tested in the FIA system. The corresponding methamidophos levels predicted by the experimental results came very close to the known calculated values. Data presented here suggest that it is feasible to use this system to supplement the traditional chromatographic analysis methods for methamidophos analysis.
Methamidophos Insecticide Leaves Vegetable Spectrophotometry Method comparison

"Automated Determination Of Lactulose In Milk Using An Enzyme Reactor And Flow Analysis With Integrated Dialysis"
Anal. Chim. Acta 1996 Volume 324, Issue 1 Pages 37-45
Michael Mayer, Meike Genrich, Wolfgang Künnecke and Ursula Bilitewski*

Abstract: The automated FIA method was based on the enzymatic hydrolysis of lactulose to fructose and galactose followed by the separation of fructose by dialysis and its detection by fructose dehydrogenase (FDH) catalyzed oxidation with ferricyanide as electron acceptor. The manifold allowed a sample stream (0.1 ml/min) to be merged with the reaction buffer stream (0.1 ml/min) containing 185 iu/ml β-galactosidase in phosphate buffer of pH 5. The flow was propelled through a reaction coil (1.3 m x 0.8 mm i.d.) operated at 50°C and the donor channel of the dialysis cell. The acceptor channel (64 mm x 2 mm x 0.5 mm) of the dialysis cell contained stationary 2 mM potassium ferricyanide in phosphate/citrate buffer of pH 5.5. After a dialysis period of 180 s, the acceptor solution was pumped through the FDH enzyme reactor to an amperometric detector where the ferrocyanate was re-oxidized. The amperometric detector was equipped with a screen-printed Pt electrode at +385 mV vs. Pt reference. The method was applied to the analysis of milk samples using a standard-addition calibration procedure. Lactulose concentrations of up to 12.28 mM were measured and these results were confirmed by a photometric method. The sampling frequency for the proposed method was 17 samples/h.
Lactulose Milk Amperometry Electrode Dialysis Immobilized enzyme Method comparison Standard additions calibration Heated reaction

"Electrochemical Detection Of African Swine Fever Virus In Pig Serum With A Competitive Separation Flow Injection Analysis Immunoassay"
Analyst 1997 Volume 122, Issue 2 Pages 155-159
Matthias Stiene and Ursula Bilitewski

Abstract: Pig serum was diluted 50-fold with PBS containing 0.05% Tween 20. A portion (1 ml) of the resulting solution was incubated with 50 ng biotinylated virus protein VP73 (preparation described) and 250 ng horse-radish peroxidase-labelled mAb 18BG3 (Ingenasa) for 25 min. A portion of the reaction mixture was injected into a carrier stream of 0.1 M phosphate buffer of pH 5.5 (buffer A) and passed through a column of biotinylated glass beads coated with streptavidin (preparation described) where the immunological complex formed during the incubation reaction was trapped. The peroxidase activity of the captured labelled antibodies was determined by passing a solution of H2O2 and hydroquinone (each 2 mM) in buffer A through the column and detecting the enzymatic product amperometrically at a vitreous C electrode at -100 mV vs. Ag/AgCl. The calibration graph was linear from 1-80 ng/ml mAb 18BG3 with a concentration of 50% inhibition at 6 ng/ml.
African swine fever virus Serum Pig Immunoassay Amperometry Electrode Column Glass beads Buffer

"Biochemical Analysis With Microfluidic Systems"
Anal. Bioanal. Chem. 2003 Volume 377, Issue 3 Pages 556-569
Ursula Bilitewski

Abstract: Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.