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Biomedical subjects

F F Bier

Publications and source records attributed to F F Bier.

13 recordsLinked to original sources
PubMed2003

Dielectrophoretic manipulation of DNA.

The characterisation and spatial manipulation of cells by AC electrokinetic methods such as dielectrophoresis and electrorotation is well established. However, applications to submicroscopical objects like viruses and molecules have been rare. Only recently has the number of such studies risen more quickly due to the availability of suitable electrodes and a growing need for single molecule techniques. Of special interest is the spatial control of single DNA molecules for genetic investigations as well as for the building of well defined structures with nanometre resolution. Here a review is given of dielectrophoretic studies dealing with single and double stranded DNA emphasising single molecule aspects.

Journal Article↗
PubMed2001

Feature-size limitations of microarray technology--a critical review.

The appeal of microarray technology is the possibility of large-scale parallel determination of a variety of variables simultaneously. Hence, microarray technologies attract the interest of both the scientific and business worlds alike. High-throughput screening has been the major focus of the utilization of microarray technologies in recent years, and has provided the strong driving force for developments in this field. DNA chip and biochip technologies have been developed as a consequence of worldwide activity in genome research. This review focuses on microarray-based analysis and emphasizes some of its principal constraints, especially detection limits.

Animals↗
PubMed1999

Changing functionality of surfaces by directed self-assembly using oligonucleotides--the Oligo-Tag.

A method is presented to modify surfaces for biotechnological applications. Oligonucleotides have been coupled covalently to a pre-activated surface. Complementary oligonucleotides hybridize to the surface, which are conjugated with functionalities. The oligonucleotides serve as "Oligo-Tags" for these functionalities that now are linked specifically and reversibly. The approach might be used to change DNA-arrays into arrays of arbitrary ligands. We demonstrate the method with an optical wave guide grating coupler as a sensing surface using two different haptens as examples for a variety of functionalities. The haptens were 2,4-dichlorophenoxyacetic acid and atrazin and are recognized by specific antibodies. The surface created was completely regenerable by alkaline washing or temperature increase without any loss of binding capacity. Specificity was demonstrated by competitive binding of antibody in presence and absence of analyte; unspecific binding has not been observed.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1997

Real-time observation of affinity reactions using grating couplers: determination of the detection limit and calculation of kinetic rate constants.

The use of integrated optical grating couplers for the analysis of bioaffinity reactions in order to calculate kinetic rate constants was investigated. The specificity of the sensor surface was determined by adsorptive or covalent attachment of the specific ligands. As an evanescent field sensor, the specific interaction of the corresponding ligand could be observed in real time and without labels. The detection limit in terms of the molecular weight of the analyte was studied by the specific binding of biotinylated proteins of different molecular weights to avidin-loaded sensors. It was shown that grating coupler sensors allowed detection of compounds of at least 2000 daltons using high-affinity receptors, while the direct sensing of low molecular analytes, such as biotin, could not be significantly achieved. Association rate constants were calculated for the interaction of the different biotinylated proteins to avidin-covered sensors from single binding curves. Due to the strong binding between avidin and biotin, the dissociation of the formed complex could not be observed. Kinetic rate constants and equilibrium constants were determined by studying the interaction of human immunoglobulin with the immobilized receptor, protein G. For the four human immunoglobulin subclasses a high affinity to protein G was determined with affinity constants ranging from 3.3 to 8.4 x 10(8) M-1.

Antibody Affinity↗
PubMed1997

Nucleic acid based sensors.

Nucleic acids may be analyte or molecular recognition elements in biosensors. Both aspects merge in the genosensor approach, where detection of special sequences is facilitated by hybridization of a target nucleic acid to a complementary immobilized template. All three roles of nucleic acids in biosensors are discussed and the state of sensor development reviewed. With the invention of evolutionary synthesis strategies applied to nucleic acids new types of biomolecular receptors are accessible. The impact of aptamers and ribozymes on biosensor development is discussed.

Adsorption↗
PubMed1996

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1996

High sensitive competitive immunodetection of 2,4-dichlorophenoxyacetic acid using enzymatic amplification with electrochemical detection.

The amplification cycle consisting of NADH independent oligosaccharide dehydrogenase (ODH) and laccase has been recently reported to be highly sensitive to several catecholamines and p-aminophenol. A competitive immunoassay for 2,4-dichlorophenoxyacetic acid has been developed by combining this amplification cycle with beta-galactosidase as enzyme label resulting in p-aminophenol as product. The combination of enzymatic amplification cycles with a competitive immunoassay yields a highly sensitive measurement of 2,4-dichlorophenoxyacetic acid. Using a monoclonal antibody the linear range of the assay was between 0.02 and 100 ng/l and the c(50) was found at 0.2 ng/l; the detection limit was at 5 pg/l (25 fmol/l) corresponding to 5 amol.

Journal Article↗
PubMed1996

Label-free observation of DNA-hybridisation and endonuclease activity on a wave guide surface using a grating coupler.

Hybridisation of nucleic acid oligomers to an immobilised target has been observed in real time using evanescent field technology. A biotinylated 24-mer with random sequence including the EcoRI recognition site was immobilised via streptavidin onto a grating coupler wave guide surface. Hybridisation of 22-mer, 15-mer and 8-mer was observed. Activity of restriction endonuclease EcoRI was visualised by measurement of the loss of bound DNA after incubation.

Base Sequence↗
PubMed1993

Enhancement of immunoassay sensitivity by molecular modification of competitors.

The potential of weak competitors to enhance the sensitivity of competitive immunoassays is described. Several triazine derivatives have been analyzed for their use as competitors. Their binding properties were determined using an enzyme-linked immunosorbent assay (ELISA) with a monoclonal antibody specific to triazines. Selected derivatives were immobilized onto the surface of a fibre optic sensor and atrazine was determined in a competitive manner using a fluorescein-labelled antibody. Using the weak binding competitor 11-(4-ethylamino-6-methylthio-s-triazine-2-yl)undecanoic acid (TE11S) the detection limit for atrazine could be lowered 100-fold in comparison to 2-aminohexylamino-4-ethylamino-6-isopropylamino-s-triazine (AHA), the previously used competitor.

Antibodies, Monoclonal↗
PubMed1990

Denaturation behaviour of DNA-protein-complexes detected in situ in metaphase chromosomes in suspension by Hoechst 33258 fluorescence.

The denaturation behaviour of DNA-protein complexes in metaphase chromosomes in suspension was analysed in situ by Hoechst 33258 fluorescence. The results indicate that due to the stability of the dye molecule and the product of the molecular extinction coefficient and the quantum yield at different temperatures, Hoechst 33258 is a suitable probe for the detection of double-stranded DNA. Thus, it is possible to monitor the concentration of double-stranded DNA in a suspension by measuring the total fluorescence intensity. The fluorescence denaturation profiles of DNA (calf thymus) were found to be comparable to absorption measurements. The decrease in fluorescence of metaphase chromosomes in suspension with increasing temperature may therefore be used to detect conformational changes of DNA in situ.

Animals↗
PubMed1990

Improved preparative separation of human B and T lymphocytes and determination of relative mobilities of B and T lymphoblast cell lines by free flow electrophoresis.

The improved quantitative separation of human B and T lymphocytes by free flow electrophoresis is presented. Buffer systems with different compositions are tested analytically and partly preparatively for their effect on the separation profile. The best result is achieved with a modified phosphate-buffered saline and allows the enrichment of unstimulated, functionally intact B and T lymphocytes as well as monocytes in three distinct fractions. Furthermore, the electrophoretic mobilities of six established acute lymphoblastic leukemia cell lines are determined with regard to the mobilities of the healthy cells.

B-Lymphocytes↗
PubMed1989

Determination of the electrophoretic mobility of chromosomes by free flow electrophoresis. I. Morphology and stability.

Isolated metaphase chromosomes of several fibroblastoid cell lines (Chinese hamster, Chinese hamster x human hybrid) were subjected to free flow electrophoresis (FFE) to study their electrophoretic mobility (EM). The morphology and stability of the chromosomes were unaffected by FFE as examined by cytogenetic methods and flow cytometry. The chromosomes of the complement all showed similar EM under most of the conditions applied. At neutral pH the EM of the chromosomes had the same sign as free DNA and about 2/3 of its magnitude. The variation of EM with buffer parameters such as ionic strength, valence of counterions, buffer capacity and dielectric constant of the solvent were investigated. Thermal denaturation increased the EM of the chromosomes by 20%. Partial denaturation might offer a possibility to separate or enrich large amounts of chromosomes by FFE.

Animals↗