Biomedical subjects

Rudolf Rigler

Publications and source records attributed to Rudolf Rigler.

6 recordsLinked to original sources

PubMed2003

Gene expression analysis using single molecule detection.

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.

Actins↗

PubMed2002

Parallel flow measurements in microstructures by use of a multifocal 4 x 1 diffractive optical fan-out element.

We have developed a multifocal optical fluorescence correlation spectroscopy system for parallel flow analyses. Multifocal excitation was made possible through a 4 x 1 diffractive optical fan-out element, which produces uniform intensity in all four foci. Autocorrelation flow analyses inside a 20 microm x 20 microm square microchannel, with the 4 x 1 fan-out foci perpendicular to the flow direction, made it possible to monitor different flows in all four foci simultaneously. We were able to perform cross-correlation flow analyses by turning the microstructure, thereby having all four foci parallel to the direction of flow. Transport effects of the diffusion as a function of flow and distance could then also be studied.

Fluorescent Dyes↗

PubMed2002

Parallel fluorescence detection of single biomolecules in microarrays by a diffractive-optical-designed 2 x 2 fan-out element.

We have developed a multifocal diffractive-optical fluorescence correlation spectroscopy system for parallel excitation and detection of single tetramethylrhodamine biomolecules in microarrays. Multifocal excitation was made possible through the use of a 2 x 2 fan-out diffractive-optical element with uniform intensity in all foci. Characterization of the 2 x 2 fan-out diffractive-optical element shows formation of almost perfect Gaussian foci of submicrometer lateral diameter, as analyzed by thermal motion of tetramethylrhodamine dye molecules in solution. Results of parallel excitation and detection in a high-density microarray of circular wells show single-biomolecule sensitivity in all four foci simultaneously.

Lasers↗

PubMed2002

Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy.

Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.

Amino Acid Sequence↗

PubMed2002

Brownian motion of single molecules in electric fields.

The behavior of negatively charged single rhodamine-labeled molecules excited to fluorescence in confocal volume elements has been investigated in oscillating voltage gradients between 1 and 15 kV/cm. The effective charge of rhodamin labeled deoxy-uridine triphosphate (Rh-dUTP) is dependent on the field strength applied and likely is due to differences in the screening by counter ions. It can be shown that the attractive forces generated by an oscillating dipole can increase the concentration of Rh-dUTP by a factor 2. Likewise a substantial increase of concentration can be obtained for Rh-labeled DNA molecules when linear voltage gradients are applied. The importance for diagnostic analysis at the single molecule level is discussed.

DNA↗

PubMed2002

(13)C-(1)H NMR relaxation and fluorescence anisotropy decay study of tyrosine dynamics in motilin.

Tyrosine ring dynamics of the gastrointestinal hormone motilin was studied using two independent physical methods: fluorescence polarization anisotropy decay and NMR relaxation. Motilin, a 22-residue peptide, was selectively (13)C labeled in the ring epsilon-carbons of the single tyrosine residue. To eliminate effects of differences in peptide concentration, the same motilin sample was used in both experiments. NMR relaxation rates of the tyrosine ring C(epsilon)-H(epsilon) vectors, measured at four magnetic field strengths (9.4, 11.7, 14.1, and 18.8 Tesla) were used to map the spectral density function. When the data were analyzed using dynamic models with the same number of components, the dynamic parameters from NMR and fluorescence are in excellent agreement. However, the estimated rotational correlation times depend on the choice of dynamic model. The correlation times estimated from the two-component model-free approach and the three-component models were significantly different (1.7 ns and 2.2 ns, respectively). Various earlier studies of protein dynamics by NMR and fluorescence were compared. The rotational correlation times estimated by NMR for samples with high protein concentration were on average 18% longer for folded monomeric proteins than the corresponding times estimated by fluorescence polarization anisotropy decay, after correction for differences in viscosity due to temperature and D(2)O/H(2)O ratio.

Animals↗