Biomedical subjects

Hans-Martin Striebel

Publications and source records attributed to Hans-Martin Striebel.

4 recordsLinked to original sources

PubMed2004

Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection.

Detecting protein-protein interactions other than those of antibody-antigen pairs still represents a demanding and tedious task. In the present work, a novel method as an alternative to current molecular biology-based detection procedures is established. It solely relies on the change of fluorescence decay times of the protein's intrinsic fluorophores tryptophan and tyrosine due to protein-protein interaction. Unlike previously utilized related methods, no labelling of the binding partners is required. This opens the possibility to detect proteins and their natural interactions without perturbation due to chemical alteration. The technique uses immobilization of one of the protein partners onto solid supports, which allows performance of protein binding studies in the microarray format. Fluorescence lifetime experiments of proteins in their different binding states have been applied to protease/protease-substrate pairs, as well as to the tubulin/kinesin system. Different binding behavior of proteins in solution towards protein partners immobilized on protein microarrays was detected with regard to binding specificity and protein amount. This label-free method for analyzing protein microarrays offers broad applicability ranging from principal investigations of protein interactions to applications in molecular biology and medicine.

Fluorescence↗

PubMed2004

Detection of multiple human herpes viruses by DNA microarray technology.

BACKGROUND: The detailed characterization of virus DNA is a challenge, and the genotyping that has been achieved to date has only been possible because researchers have sent a great deal of time and effort to do so. Instead of the simultaneous detection of hundreds of viruses on a single high-density DNA-chip at very high costs per chip, we present here an alternative approach using a well-designed and tailored microarray which can establish whether or not a handful of viral genes are present in a clinical sample. METHODS: In this study we applied a new concept of microarray-based, optimized and robust biochemistry for molecular diagnostics of the herpesviruses. For comparison, all samples were genotyped using standard procedures. RESULTS: The biochemical procedure of a knowledge-based, low-density microarray was established based on the molecular diagnostics of human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The study attempted to optimize parameters of microarray design, surface chemistry, oligonucleotide probe spotting, sample labeling and DNA hybridization to the developed DNA microarray. The results of 12 900 hybridization reactions on about 150 configured herpes virus microarrays showed that the established microarray-based typing procedure was reproducible, virus-specific and sufficiently sensitive with a lower limit of 100 viral copies per mL sample. CONCLUSIONS: The developed method utilizes low-fluorescence background coverslips, epoxy surface chemistry, standardized oligonucleotide probe spotting, PCR-labeling with Cy3 of isolated DNA, array hybridization, and detecting of specific spot fluorescence by an automatic microarray reader. We expect the configured microarray approach to be the method for high-throughput associated studies on human herpes viruses.

Carbocyanines↗

PubMed2004

Enhancing sensitivity of human herpes virus diagnosis with DNA microarrays using dendrimers.

DNA microarray technology has become a promising new tool for the detection and identification of viral pathogens in human plasma and cell cultures. For exploration of this technology, we have developed DNA microarrays that encode capture oligonucleotide probes for different human herpes viruses: herpes simplex virus (HSV) HSV-1, HSV-2, varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and HHV-6. The on-chip hybridization is accomplished with the PCR amplicons of the respective human herpes virus types. In this original article, we attached multiple Cy3-fluorophores to the branched 5' ends of the labeling oligonucleotide primers. For the first time, we experimentally demonstrated that the self-designed, knowledge-based, and focused microarrays specifically hybridized to fluorophore-labeled pathogenic DNAs using dendrimer technology. The fluorescence signal enhancement via the dendrimers was up to 30 times compared with the quenched single Cy3-fluorophore-labeled HSV-1 DNA. The on-chip signal-amplifying effect depended upon the number of branches and the concentration of fluorophore-labeled pathogenic DNAs. Treblers were superior to doublers, as trebler-labeled nucleic acids had fluorescence-signal-enhancing effects over a broad range of labeled DNA concentrations exemplified for the quenched single Cy3-fluorophore-labeled HSV-1 and non-quenched single Cy3-fluorophore-labeled CMV DNAs.

Blood↗

PubMed2003

Virus diagnostics on microarrays.

Whereas the majority of microarray applications still deal with expression analysis for gathering information about levels of gene products at certain cell states, other approaches simply ask the question whether particular genes, which are usually indicative for particular microorganisms and pathogens, are present in a sample or not. Investigations that are more detailed try to evaluate the presence of particular subtypes of a given pathogen. The combination of microarray technology and virus diagnostics promises to generate an ideal platform for fast, sensitive, specific, and parallelized virus diagnostics. Performing virus diagnostics on microarrays, however, requires other basic techniques to be optimized. This is necessary in order to obtain unambiguous and reproducible results, which are compatible with the needs for clinical routine. Parameters that have to be considered include supports, coupling chemistry, chemical oligonucleotide synthesis, signal enhancement strategies, and optimal coordination of PCR reactions, hybridizations, and signal detection, as well as interpretation strategies. Finally, considerations should be given to economic aspects, one chip-one patient strategies and low integrated arrays as a custom-tailored way to fast and accurate diagnostic tools.

Animals↗