Biomedical subjects

Claus B V Christensen

Publications and source records attributed to Claus B V Christensen.

6 recordsLinked to original sources

PubMed2006

Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films.

Allele-specific hybridization to a DNA microarray can be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose mutations in the human beta-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding melting point temperature ( approximately 20 degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments conducted using a target DNA specific for the TC tag of the immobilized probes showed that the spotting and hybridization procedure had a variance of 20%, indicating that signal differences as low as twofold could be detected between perfect match and mismatch. Together, our results show that the use of microarrays of TC-tagged probes that have been immobilized on agarose films grafted onto glass is a robust and inexpensive genotyping method.

Alleles↗

PubMed2005

Diagnostic and analytical applications of protein microarrays.

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein-protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.

Anti-Bacterial Agents↗

PubMed2004

Development of a multiplex microarray microsystem.

A hybrid multiplex microarray microsystem has been developed that consists of 32 individually addressable array reaction chambers, supporting the use of multichannel pipettes for addition of up to 8 samples simultaneously. Discrimination between Campylobacter jejuni and Campylobacter coli bacteria was observed in DNA samples containing Campylobacter spp., with the same specificity and sensitivity as when compared to a full-size microarray. The spinloaded multiplex microarray microsystem described provides a novel and convenient test format for simultaneous low-density microarray analysis and is universally adaptable to other DNA, protein or small molecule microarray based applications.

Biocompatible Materials↗

PubMed2004

Quantitative microarray pesticide analysis.

To replace a pesticide immunoassay based on microtiter plates, we have developed a quantitative, competitive microarray immunoassay, which permits rapid and highly sensitive quantification of the dichlobenil degradation product 2,6-dichlorobenzamide (BAM), and the prominently used herbicide atrazine. The pesticide analysis is based on the competitive binding of fluorescence conjugated monoclonal antibodies (mAb) to their respective analytes. Lowest detection limits were calculated to 1 ng/l (5 pM) for BAM and 3 ng/l (10 pM) for atrazine. Corresponding IC(50) values were, 10 ng/l (50 pM) for BAM and 34 ng/l (160 pM) for atrazine, respectively. In comparison to the existing microtiter plate immunoassay, the microarray was found to be up to 20-fold more sensitive. Compared to the gas chromatography with mass spectroscopy (GCMS) analysis performed on more than 1000-fold concentrated samples, the microarray-based immunoassay was even 10-fold more sensitive using non-concentrated samples. Measuring both analytes simultaneously did not affect assay sensitivity compared to single analyte quantification. Besides a gain in sensitivity and the possibility of multiplex quantification, assay times and assay complexity were reduced drastically with the microarray platform compared to the microtiter plate immunoassay and GCMS, suggesting that the microarray based immunoassay is a viable method for measuring picomolar amounts of analytes, e.g. clinically relevant analytes.

Antibodies, Monoclonal↗

PubMed2004

Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate.

An agarose film has been proposed as an efficient substrate for producing microarrays. The original film preparation procedure was simplified significantly by grafting the agarose layer directly onto unmodified microscope glass slides instead of aminated glass slides, and the blocking procedure was replaced with a wash in 0.1x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) without decreasing the performance of the produced microarrays. Characterization of the grafted agarose film using atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the agarose film had a 10-fold increase in surface roughness compared to glass and that the interior of the agarose film was porous, with pore sizes between 100-500 nm. A comparison of hybridization on aldehyde-activated agarose-coated microarray slides and commercial amino-reactive microarray slides showed that aldehyde-activated agarose-coated slides had the highest signal-to-noise ratio of 850, suggesting that the aldehyde-activated agarose microarray slides are suitable in applications where analytes have a wide concentration range. By immobilizing the DNA probes using ultraviolet (UV) light, the signal-to-noise ratio was further increased to 3000 on the agarose microarray slides. The specificity of the UV cross-linked DNA probes was demonstrated using 21 and 25 bp long capture probes, enabling discrimination of target molecules differing in only one base.

Adsorption↗

PubMed2003

Detection of analyte binding to microarrays using gold nanoparticle labels and a desktop scanner.

Microarray hybridization or antibody binding can be detected by many techniques, however, only a few are suitable for widespread use since many of these detection techniques rely on bulky and expensive instruments. Here, we describe the usefulness of a simple and inexpensive detection method based on gold nanoparticle labeled antibodies visualized by a commercial, office desktop flatbed scanner. Scanning electron microscopy studies showed that the signal from the flatbed scanner was proportional to the surface density of the bound antibody-gold conjugates, and that the flatbed scanner could detect six attomoles of antibody-gold conjugates. This detection system was used in a competitive immunoassay to measure the concentration of the pesticide metabolite 2,6-dichlorobenzamide (BAM) in water samples. The results showed that the gold labeled antibodies functioned comparably with a fluorescent based immunoassay for detecting BAM in water. A qualitative immunoassay based on gold-labeled antibodies could determine if a water sample contained BAM above and below 60-70 ng L(-1), which is below the maximum allowed BAM concentration for drinking water (100 ng L(-1)) according to European Union legislation.

Antibodies, Monoclonal↗