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Project ID: QLK4-CT-2002-02323
Finanziato nell'ambito di: FP5-LIFE QUALITY
Paese: Austria

Hydrophobic biochip surfaces

Fabrication, protocol and hybridization conditions of hydrophobic chip surfaces made of epoxy resin (so-called ARChip Epoxy) and PST-co-VBT (so-called ARChip UV) were optimized for use in an oligonucleotide chip for EDC detection.

The lowest oligonucleotide concentration leading to a significant hybridization signal was 2 µM. Fluorescence signals resulting from probes of concentrations higher than 20 µM did not further increase signal intensity. Both, SH- and NH2-modified oligonucleotides led to a significant hybridization signal, whereas almost no signal occurred, when unmodified oligonucleotides were employed.

The hybridization fluorescence of SH-and NH2-modified Alf1b50 strongly depends on the pH of the print buffer. Whereas both modifications are readily immobilized at pH 7 generating the same strong signal, NH2-modified oligonucleotides show an improved, optimum binding at pH 8 resulting in a 20-fold increased signal.

When adding a 33dT spacer to a 17-bases oligonucleotide, the hybridization efficiency was increased by 50%. The signal-to-noise ratio (S/N) increased by 2 to 3 times, when exposed to UV-light after spotting.

Efficient blocking of reactive surface groups after arraying is critical for a reduced fluorescence background. The epoxy chip blocked with ethanolamine tend to provide a more homogeneous background, which is clearly expressed by the coefficient of variance at 10, 1 and 0.1 µM guide dot concentration (with ethanolamine (ETA)): 6.3%, 7.8%, 34.3%; without ETA: 30.5%, 32.8%, 45.0%).

The effect of high guide dot concentration on neighbouring spots is most obvious with 10 µM Cy5-labelled oligonucleotide: compared with 1 and 0.1 µM Cy5-fluorescent oligonucleotide the background is double. The increased local background is a result of the background contamination of hybridized probes around the bright guide dots.

The choice of print buffer is critical for spot homogeneity and signal strength and thus has direct impact on data analysis and data reliability. 20 different buffers and buffer additives were tested and optimized in oligonucleotide arrays. A series of ARChips Epoxy was produced and distributed among the partners for EDC testing.

ARChip UV was optimized with respect to chip fabrication, photoactivation (pre- and post-activation, no activation) and print buffer & additives. The immobilization capacity was effected by the UV energy density used for photoactivation of the vinylbenzylthiocyanate group and was estimated from the signal difference of fluorescent, amino-modified Alf1b after spotting and after hybridization.

The decrease in fluorescence signal was taken as a measure for the unbound Alf1b washed off during the washing and blocking steps and was inversely proportional to the immobilization capacity. Increasing the energy density of the 254 nm UV light from 60 to 450 mJ cm-2 resulted in an increase in immobilization capacity from 20 to 80%. Thus, an energy density of 450 mJ cm-2 was chosen for the activation step in all experiments.

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Claudia PREININGER, (Scientist)
Tel.: +435-05503527
Fax: +435-05503666