BeschreibungBiochip technology has been revolutionizing most fields of molecular biology. One 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. In order to achieve higher signals and lower detection limits we aim at a) the optimization of chip process parameters, b) the use of gold particles as labels in surface enhanced fluorescence detection, and c) increased surface reflectivity by addition of SiO2 and TiO2 layers. Surface chemistries, such as porous poly(styrene-co-4-vinylbenzylthiocyanate) (PST-co-VBT)1, 2 and hydrogel-doped sol-gel which provide high immobilization capacity and appropriate density for protein attachment have been developed: PST-co-VBT was illuminated with 254 nm UV light to achieve the desired photoisomerization SCN-NCS and subsequent binding of proteins. Providing binding sites for both thiols and amines, PST-co-VBT was used for simultaneous immobilization of Au-labelled oligonucleotides and proteins. The length of the oligonucleotides, carrying the gold particles, was tuned to the distance at which resonance between the metal and the fluorescent target-protein occurred, resulting in signal enhancement. Silica-co-polymers of hydrogels were generated for protein immobilization to combine the mechanical stability of silanes with the biocompatibility of hydrogels. Enhancement of the reflectivity by incorporation of high index refractive TiO2-layers between two low-refractive polymer-layers or between the glass-substrate and the polymer-layer was achieved by hydrothermal synthesis of nanostructured TiO2-particles or by Sol-Gel technology3. An increase of the reflection of light with desired wavelength was expected by coating a TiO2-layer, which was characterised by a tuneable refractive index and a defined thickness, on the chip- substrate (glass) or by coating high refractive index TiO2- layers between low refractive index layers (SiO2 or polymer). These interference coatings were characterized by the refractive index of the layers, the film thickness and the stack design. Process parameters that were optimized were print buffer, humidity control during arraying, slide agitation, drop size, probe and target concentration, etc. Important parameters of evaluation were immobilization capacity, signal-to-noise ratio and spot morphology. Quantitative quality control procedures were applied to compare the results from slides of different immobilization chemistry and detection technique, experiments and charges.
|4 Apr. 2004 → 7 Apr. 2004
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