Papers by Louis Tiefenauer
Iodinated estradiol tracers were synthesized with three different bridges connecting the radiolab... more Iodinated estradiol tracers were synthesized with three different bridges connecting the radiolabelled moiety to the steroid core: Hemisuccinate, carboxymethyloxime and amide. Taking these iodinated tracers in combination with ten antibodies raised against estradiol-6-CMOalbumine, titers and slopes of calibration curves have been compared to the corresponding data using a 3H tracer. The data indicate that the tracer with the amide bridge is recognized similarly to the tritiated estradiol by all antibodies tested, whereas the two other iodinated tracers exhibit substantial bridge binding. The results suggest that the amide tracer structure can generally be used to improve the quality of estradiol antibodies suffering from bridge binding effects.
A biotinyl-6cr-estradiol derivative (Bio-E,) was synthesized and used as the key component in ant... more A biotinyl-6cr-estradiol derivative (Bio-E,) was synthesized and used as the key component in antigen-and antibody-immobilized ELISA techniques, and the relative merits of the two methods were compared. A precise and reproducible antigen-immobilization was achieved in avidin-coated microtiter plates with Bio-E,. This assay, when completed by the incubation with primary antibody and second antibody-peroxidase conjugate, has a very low detection limit (6 pg/ml estradiol) but required a long incubation time with primary antibody to reach equilibrium. At non-equilibrium conditions, using a high antibody concentration, the assay could be very fast and sensitive. In the antibody-immobilized assay, the Bio-E, was added to compete with the estradiol present in the calibrator or sample and visualized with a streptavidin-peroxidase conjugate. The detection limit is higher (34 pg/ml), but the specificity was superior and the incubation time to reach equilibrium shorter as compared to the antigen-immobilized assay. Therefore, the antibody-immobilized assay appeared to be ideal for the classical ELISA technique, whereas the antigen-immobilized method seemed to be best suited for automated assay systems using antibody in excess.
Antisera used in immunological assay systems for small molecular weight substances are routinely ... more Antisera used in immunological assay systems for small molecular weight substances are routinely prepared by coupling the hapten to a carrier protein via a chemical linker. Often this bridge is partly recognized by the antibody, resulting in reduced sensitivity when an identically structured tracer (e.g. iodine-labelled) is used. Historically, the problem was solved by changing the linking structures in the tracer. An alternative way is exemplified by the development of a very sensitive and specific iodinated radioimmunoassay for melatonin, This new approach involves the design of a linkage identical in the tracer and the antigen that is both very short and closely resembling the structure of the analyte itself.
Designed networks of neurons are potentially very useful to investigate neural activities. Using ... more Designed networks of neurons are potentially very useful to investigate neural activities. Using photolithography microgrooves suited in size for single neurons have been produced on glass chips. Two conducting gold lanes ending in each microgroove allow extracelluar stimulation of the neurons and recording of their activity. A cell adhesive surface was created by functionalization of glass with the adhesion peptide RGDC. In addition, in order to optimize the contact of the neuronal cell membrane to the electrode surface axonin-1, a speci®c neural adhesion protein was used. A recombinant form of axonin-1 was produced and immobilized in a correct orientation on protected gold surfaces through a C-terminal cysteine residue. Neurite outgrowth of neurons cultured on chips derivatized with RGDC or axonin-1 were compared. The developed materials and methods represent a ®rst step towards establishing designed functionalized glass surfaces for neurophysiological investigations.
Biosensor research is strongly interdisciplinary as it requires experience in chemistry, biochemi... more Biosensor research is strongly interdisciplinary as it requires experience in chemistry, biochemistry, biology, material science, electronics and engineering. The recent progress in micro-and nanotechnology allows to miniaturize complex systems as well as to address problems at a molecular level. The architecture and even the function of single molecules on a sensor surface have been investigated and can to some extent even be predetermined. At present, microtechnology is well established in the production of micro-fluid transport systems and has a high potential for cell-culturing and monitoring devices in the future. Three different running projects are presented which illustrate the usefulness of micro-and nanotechnology for biosensor research: 1)Investigations on amperometric immunosensor devices, 2) the measurement of binding forces of individual antigen-antibody pairs, and 3) the fabrication of microchannels suitable for neuron-cell growth and recording. Big efforts, however, will be required to integrate the recognition element of a sensor into a device for an intended application
Antibody single-chain Fv fragment (scFv) molecules that are specific for f luorescein have been e... more Antibody single-chain Fv fragment (scFv) molecules that are specific for f luorescein have been engineered with a C-terminal cysteine for a directed immobilization on a f lat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.
Electrochemical immunosensing requires labeling because most antigen and antibody species are ele... more Electrochemical immunosensing requires labeling because most antigen and antibody species are electrochemically inert. The use of microperoxidase as the label is most promising due to its electrocatalytic activity, which is utilized for signal ampli®cation. A method for coupling the microperoxidase MP-11 to antibody molecules has been developed. The electrocatalytic effect of the conjugate when immobilized on gold electrodes has been demonstrated. Using the streptavidin± biotin system the antigen estradiol was immobilized on gold. Recognition of labeled antibody could be detected electrochemically. #
A modular immunosensor device based on silicon technology with electrochemical detection is prese... more A modular immunosensor device based on silicon technology with electrochemical detection is presented. The sensor surface is functionalized with the antigen estradiol using the streptavidin-biotin system. The immobilized antigen is recognized by ferrocene-labeled antibody molecules in a competitive assay format. The ferrocene redox centers can be detected electrochemically. In order to facilitate the electron transfer, a gold layer structured in nanometer dimensions is used as an electrode. The functionslued silicon chip will be integrated into a micromachined fluid transport system. The resulting immunosensor device for estradiol represents a prototype allowing easy adaptation to other biotinylated analytes since the immobilized streptavidin serves as a universal anchor.
One of the most crucial steps for the successful construction of a biosensor is the appropriate a... more One of the most crucial steps for the successful construction of a biosensor is the appropriate and reproducible coupling of the biological part (e.g. enzyme, antibody) to the inorganic moiety of the device (e.g. electrode, microchip). In this paper three methods of immobilization of avidin to a glassy carbon electrode are described. Depending on the type of immobiliza~on, avidin may lose its biological activity as determined by an enzyme immunoassay, using biotinylated reagents. If avidin is covalently bound to the glassy carbon electrode via the bridge molecule 44'-diaminodiphenylamine, the biological activity is retained. About 1,5 pmol of avidin can be bound to the electrode (3 mm in diameter), resulting in a nearly complete monolayer of protein.
Target-specific superparamagnetic contrast agents may allow the localization of specific tissues ... more Target-specific superparamagnetic contrast agents may allow the localization of specific tissues such as tumors by magnetic resonance imaging (MRI). In this report the preparation and in vitro characterization of tumor-specific superparamagnetic particles (SMP) are described. Particles of uniform size (9.6 f 0.8 nm) were prepared from an alkaline solution of ferric and ferrous ions and isolated by differential centrifugation. The resulting nanoparticle suspension is stabilized in buffer using a polypeptide coat to which a monoclonal antibody, specific to carcinoembryonic antigen (CEA), was covalently attached at the hinge region. The resulting anti-CEA SMP have a hydrodynamic radius of less than 50 nm, and specifically bind to CEA in vitro. The visualization of epitopes, present on a cell surface in very low density as expected for tumor antigens or receptors, may be achieved due to the highRz relaxivity of 300 L mmol-' s-l of the contrast agent described here. Furthermore, the polypeptide coat chosen provides an ideal platform for the attachment of biological modifiers needed for the reduction of the antigenicity and blood clearance rate of anti-CEA SMP. * Corresponding author. Tel.: 0041 56 99 21 11. Fax: 0041 e Abstract published in Aduance ACS Abstracts, August 15, 56 98 26 35. 1993.
A sensitive method based on ferrocene-streptavidin (Fc-Stv) conjugates for the simultaneously amp... more A sensitive method based on ferrocene-streptavidin (Fc-Stv) conjugates for the simultaneously amplified electrochemical and surface plasmon optical detection of DNA target hybridization to peptide nucleic acid-modified gold surfaces is reported. The attachment of Fc-Stv to the biotinylated complementary target DNA not only amplified the surface plasmon resonance signal but also enhanced the electrochemical signal due to the many Fc markers per Stv. The ferrocene redox peak current increased with the increase of the target DNA concentration. Consequently, the amount of hybridized target DNA can be estimated by cyclic voltammetry and chronocoulometry. The detection limit of this DNA sensor is 10 pM (2 fmol, with signal to noise > 3). This sensor was also shown to have high selectivity (at the single-base mismatch level) and good reproducibility. (1) (a) Piuuno, P. A. F.; Krull, U. J.; Hodson, R. H. E.; Damha, M. J.; Cohen, H. Anal. Chem. 1995, 67, 2635-2643. (b) Dore, K.; Dubus, S.; Ho, H.-A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M.
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Papers by Louis Tiefenauer