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Biomedical subjects

W Fritzsche

Publications and source records attributed to W Fritzsche.

At least 19 recordsLinked to original sources
PubMed2005

Chip-based electrical detection of DNA.

A variety of methods have been developed for the detection of the binding of the complementary strand of DNA to a gene chip using electrical rather than the established optical signal techniques. Chip-based DNA sensors offer sensitivity, specificity, parallelisation and miniaturisation for the detection of selected DNA sequences or mutated genes associated with human diseases. Problems associated with the established fluorescence-based optical detection technique include the high equipment costs and the need to use sophisticated numerical algorithms to interpret the data. These problems generally limit its use to research laboratories and make it hard to adapt this detection scheme for on-site or point-of-care use. An electrical readout might be a solution to these problems. A review of a number of different approaches to achieve an electrical readout for a DNA chip is presented. The review covers various methods that are based on the use of metal nanoparticles as labels and also electrochemical methods that use polymer-modified electrodes, DNA-specific redox reporters, and DNA-mediated charge transport techniques.

DNA↗
PubMed2001

DNA monolayer on gold substrates characterized by nanoparticle labeling and scanning force microscopy.

Monolayers of single-stranded DNA on gold substrates were studied by scanning force microscopy. Complementary DNA probes labeled by gold nanoparticles were applied for contrast enhancement. Substrate regions modified with DNA could be visualized in a highly specific manner. The influence of the solution concentration on the surface density of adsorbed nanoparticles could be visualized. Because individual label particles can be easily detected, this labeling technique opens the way for characterization of DNA monolayers with a lateral resolution in the nanometer range.

Base Sequence↗
PubMed2001

DNA-gold conjugates for the detection of specific molecular interactions.

DNA chips are an emerging technology for parallel detection of DNA molecules, with applications ranging from medicine to environmental monitoring. The typical set-up includes fluorescence labeling for detection of binding events on the chip surface. Here another labeling technique based on gold nanoparticles is presented. These labels are much more stable, and their optical signal is less influenced by the environment. The specificity of gold-labeled DNA probes and the ease of detection using optical reflection or transmission is demonstrated. In conclusion, gold-labeling is a promising candidate for more robust and reliable DNA-chip detection.

Affinity Labels↗
PubMed2000

Chip-based optical detection of DNA hybridization by means of nanobead labeling.

A new scheme for the detection of molecular interactions based on optical readout of nanoparticle labels has been developed. Capture DNA probes were arrayed on a glass chip and incubated with nanoparticle-labeled target DNA probes, containing a complementary sequence. Binding events were monitored by optical means, using reflected and transmitted light for the detection of surface-bound nanoparticles. Control experiments exclude significant influence of nonspecific binding on the observed contrast. Scanning force microscopy revealed the distribution of nanoparticles on the chip surface.

Base Sequence↗
PubMed2000

DNA probes on chip surfaces studied by scanning force microscopy using specific binding of colloidal gold.

Single-stranded DNA was covalently bound on chip surfaces using two different silanization procedures. The resulting surfaces were characterized by fluorescence and scanning force microscopy using sequence-complementary DNA molecules with labels. Colloidal gold (30 nm) was used as the topographic label. Scanning force microscopy revealed the individual labels on the surface and their distribution. Steps of silane layers or DNA-modified surfaces prepared using an elastomeric mask provided internal controls for comparison of modified with unmodified surfaces.

Base Sequence↗
PubMed1999

Salt-dependent chromosome viscoelasticity characterized by scanning force microscopy-based volume measurements.

Metaphase chromosomes prepared according to the standard spreading procedure exhibit viscoelastical behavior after rehydration. The salt-dependency of this elasticity was investigated using contact mode scanning force microscopy (SFM). Therefore, chromosomes were imaged in solutions of different ionic strength (0.3 x PBS and water). The elasticity was probed by stepwise increase of the loading force of the scanning tip, resulting in a set of images. The images were used for the determination of the height and the apparent volume of each chromosome, and these values were the base for a characterization of the viscoelastical response of the chromosomes under different salt conditions. Lower ionic strength resulted in a greater response of the chromosome structures to applied loading forces.

Chromosomes↗
PubMed1997

Mapping elasticity of rehydrated metaphase chromosomes by scanning force microscopy.

Scanning force microscopy was used for mapping the viscoelastic properties of metaphase chromosomes. These properties were probed by scanning with various imaging forces and subsequent calculation of the difference image. The procedure allows a mapping of the viscoelastic behavior expressed as force-dependent indentation of the local surface feature and results in an image with material contrast. The approach is demonstrated on rehydrated metaphase chromosomes, which were spread and air-dried before rehydration in aqueous buffer. The rehydration resulted in a swelling of the chromosome structure and was accompanied by drastic changes in the viscoelastic properties. For comparisons, force-distance curves on metaphase chromosomes were accumulated; these curves were also used for the calculation of the stiffness curve. The demonstrated approach of mapping viscoelasticity by differential scanning force microscopy allows the detection of domains with varying mechanical properties in biomolecules such as chromosomes.

Chromosomes↗
PubMed1997

Scanning Force Microscopy Study of Methanol-Induced Changes in the Distribution of Silver Particles in Colloidal Metal Films

Colloidal silver particles were attached to a solid surface for use as a substrate for surface-enhanced Raman scattering and surface enhanced fluorescence spectroscopies. The enhancement of Raman scattering is dramatically increased after exposure of the substrates to methanol solution. Scanning force microscopy was used to visualize the substrate before and after the methanol treatment, and a statistical evaluation of the particle distribution revealed some differences. The nearest-neighbor distance was decreased after the exposure to methanol. This effect suggests that an aggregation of the particles occurred. This aggregation is probably based on increased attractive interactions and lateral mobility of the particles in methanol.

Journal Article↗
PubMed1997

Chicken erythrocyte nucleosomes have a defined orientation along the linker DNA--a scanning force microscopy study.

The orientation of nucleosomes was investigated using scanning force microscopy (SFM) of hypotonically spread chicken chromatin. A virtual cross section parallel to the substrate at half maximum height of the nucleosomal structure revealed an elliptical shape. The orientation of the major axis of this ellipse was investigated in reference to the direction of the axis of the nucleosomal chain. An alignment of the nucleosomes along the nucleosomal chain was observed, with more than 50% of the nucleosomes aligned with the long axis of the chain within < or = 30 degrees deviation. The alignment distribution peaked at 10-20 degrees. The application of SFM-based image processing for the structural investigation of a protein-DNA complex demonstrates the potential for this approach in structural molecular biology.

Algorithms↗
PubMed1997

Application of atomic force microscopy to visualization of DNA, chromatin, and chromosomes.

The scanning force microscope (SFM, also called the atomic force microscope, AFM) provides a new and powerful method for visualization and manipulation of biological samples. Its high precision and sensitivity allow the investigator to interrogate samples at very high spatial resolution and simultaneously accumulate a variety of data types, including topography, viscoelasticity, chemical properties, and local friction. We provide here a brief review of the literature describing the current state of the art in the application of SFM to the study of DNA, chromatin and chromosomes, and some examples from this laboratory. Suggestions for future directions of this technology are also presented.

Animals↗
PubMed1996

Scanning force microscopy reveals ellipsoid shape of chicken erythrocyte nucleosomes.

Scanning force microscopy was used to investigate the conformation of hypotonic spread chicken erythrocyte nucleosomes. Nucleosomal chains were prepared in low-salt conditions and fixed before centrifugation onto glass coverslips and air drying. The images of single nucleosomes were isolated by image processing, and the height and geometry of the resulting three-dimensional structures were investigated. An average nucleosome height of 4.2 +/- 1.1 nm was determined. A virtual cross section at half-maximum height of the nucleosome structure was used for a characterization of the nucleosome geometry. The shape of this cross section was best described by an ellipse with an aspect ratio (major/minor axis) of approximately 1.30.

Animals↗
PubMed1996

Volume determination of human metaphase chromosomes by scanning force microscopy.

The scanning force microscopy (SFM) yields the topography of the investigated surface. A procedure was developed which starts from this three-dimensional information to estimate the volume of a biological specimen. The volume of spread human metaphase chromosomes was determined in air and rehydrated in aqueous buffer. A difference of the determined volume of a air-dried metaphase chromosome set was found compared to values from electron microscopic investigations, and could be correlated with differences in the hydration state of the chromosomes. SFM-based relative volumes of air-dried chromosomes resembles literature data regarding volume range and distribution. Possible application of SFM-based relative volume measurements for chromosome classification purposes is discussed.

Algorithms↗
PubMed1995

Scanning force microscopy of microtubules and polymorphic tubulin assemblies in air and in liquid.

We have investigated microtubules (MTs) and polymorphic assemblies, formed in vitro from isolated microtubule protein, by scanning force microscopy (SFM) in air and in liquid. Immobilization of MTs was achieved by placing a drop of the assembly solution on a polylysine-coated coverslip. After washing with taxol and air drying, the characteristic microtubular fibrous morphology appeared in the SFM. The MTs formed a network similar to that obtained by transmission electron microscopy (TEM). A height of approximately 9.5 nm for dried MTs was computed from the surface topography. Glutaraldehyde fixation of the MTs yielded higher structures (approximately 14 nm), which swelled to approximately 20 nm after rehydration, a value close to the MT diameter of approximately 25 nm determined from TEM images of ultrathin sections. The protofilament pattern of the MTs and surface attached MT-associated proteins were not apparent from SFM, although the height along the long axis of the MTs appeared slightly modulated. In addition to MTs, various polymorphic tubulin assemblies including ribbons, hoops and double-walled MTs were visualized by SFM.

Air↗
PubMed1995

Scanning force microscopy of chromatin.

Scanning force microscopy (SFM) is a new method to obtain the topography of surfaces with nanometer-resolution. The ability to image under liquids makes the technique attractive for biological applications, especially for the determination of the ultrastructure of biomolecules under native conditions. One growing field of interest is the investigation of chromatin and chromatin-related structures. Different levels of chromatin condensation were the subject of several previous SFM investigations, from the nucleosomal chain, to the 30-nm fiber, ending with the metaphase chromosome. The SFM yielded new information on such fundamental problems as the core spacing of the nucleosomal chain, the internal structure of the 30-nm fiber and the banding mechanism of metaphase chromosomes. Other investigations dealt with the SFM characterization of polytene chromosomes. This paper reviews the state-of-the-art in SFM chromatin research and discusses future developments in this field.

Animals↗
PubMed1994

Probing chromatin with the scanning force microscope.

With the scanning force microscope (SFM), one can image the topography of biological material adsorbed at air-solid or liquid-solid interfaces with up to nanometer resolution. In principle, fixation, contrast enhancement, and labeling are not required. We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin ultrastructures in the SFM. A beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying. The beads-on-a-string morphology of the basic nucleosomal assembly was well delineated. The nucleosomes appeared as round protrusions with an apparent height of 4-6 nm. The histogram of center-to-center distances between adjacent nucleosome cores along the filament axis had a peak at approximately 30 nm. Reversible changes in the three-dimensional structure were observed upon exposure of air-dried samples of metaphase chromosomes to solutions of different ionic strengths.

Animals↗