BeschreibungOne of the main challenges for further development of biochip technology is the strength of signals produced by probe-target interaction, especially for low probe and target concentrations. This is expected to become an increasingly important issue as chip technology drifts to smaller high-density chips. The signal intensity mainly depends on the functionality of the chip surface, the immobilization capacity and the density of attachment and accessibility for the interacting molecules. Reactive chip surfaces which fulfill these demands have been developed for DNA and protein immobilization: Vinylbenzylthiocyanate-co-polymers were prepared and coated onto the chip by using a quadruple film applicator. To bind biomolecules the functional polymers were either activated by UV-light to form a reactive isothiocyanate group or reduced to a thiol-group, which was used for a disulfide exchange reaction. Well-defined highly branched graft polymers have been prepared by "living" free radical polymerization. The polymerization reaction was started by using initiator-transfer-terminator (iniferters) agents, such as benzoinmethylether or sodium N, N-diethyldithiocarbamate trihydrate. Surface characterization in terms of branch architecture, hydrophilicity/ hydrophobicity and density of reactive groups was performed by fluorescence imaging and contact angle measurements. Photografted and non grafted regions were visualized by a fluorescence microscope on staining certain chain groups with appropriate fluorescent dyes. The developed surface chemistries were evaluated by model oligonucleotide-, DNA-, and protein assays and compared with commercially available reactive glass slides. Short DNA sequences (up to 70 bases) and partial complementary DNA of up to 5000 nucleotide bases were arrayed onto the chip and hybridized. The model protein assay was based on a classical antibody-antigen reaction and an ELISA test. Important parameters of evaluation were immobilization capacity, signal-to-noise ratio, spot morphology, and reproducibility of results. Raw data of fluorescence emission and images of the arrays, delivered by a non-confocal fluorescence imager, were the basis for filtering, normalisation and statistical analysis. For statistical analysis usually means, medians or log transformed signal intensity ratios are computed. Background information, coefficients of variation, standard deviations and spot morphologies allowed us to filter out spots, which were worth further investigation . Quality control procedures from literature were applied to our data set. Furthermore, we worked out our own statistical procedure which allowed us to compare results from slides of different experiments and charges as will be presented at the conference.
|22 Apr. 2003
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