Biomedical subjects

Markus Ehrat

Publications and source records attributed to Markus Ehrat.

4 recordsLinked to original sources

PubMed2005

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.

Bacteremia↗

PubMed2005

Tuned graft copolymers as controlled coatings for DNA microarrays.

DNA microarrays have become a powerful tool for expression profiling and other genomics applications. A critical factor for their sensitivity is the interfacial coating between the chip substrate and the bound DNA. Such a coating has to embrace the divergent requirements of tightly binding the capture probe DNA during the spotting process and of minimizing the nonspecific binding of target DNA during the hybridization assay. To fulfill these conditions, most coatings require a passivation step. Here we demonstrate how the chain density of a graft copolymer with a polycationic backbone, poly(l-lysine)-graft-poly(ethylene glycol), can be tuned such that the binding capacity during capture probe deposition is maximized while the nonspecific binding during hybridization assays is kept to a minimum, thus alleviating the requirement for a separate passivation procedure. Evidence for the superior performance of such coatings in terms of signal-to-noise ratio and spot quality is presented using an evanescent field-based fluorescent sensing technique (the ZeptoREADER). The surface architecture is further characterized using optical waveguide lightmode spectroscopy and time-of-flight secondary ion mass spectrometry. Finally, in a model assay, we demonstrate that expression changes can be detected from 1 microg of total mRNA sample material with a limit of detectable differential expression of +/-1.5.

DNA↗

PubMed2002

Immunoaffinity screening with capillary electrochromatography.

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.

Antibodies↗

PubMed2002

Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

Protein Array Analysis↗