DNA microarrays for visual detection of human pathogenic microorganisms based on tyramine signal amplification coupled with gold label silver stain

摘要

The utility of DNA microarrays is severely limited by their restricted sensitivity. Tyramine signal amplification (TSA) coupled with gold label silver stain (GLSS) was introduced in DNA microarrays for visual detection of human pathogenic microorganisms. First, a TSA system was introduced to the microarrays after the microarrays were prepared and hybridized with biotinylated targets. This procedure leads to large amounts of biotinconjugated tyramine depositing at the site of enzyme reaction under HRP catalysis. Second, streptavidin- nanogold was introduced and accumulated by specific binding of biotin and streptavidin. Finally, silver staining was performed. The images of the spots were scanned with a visible light scanner and quantified with ArrayVision 7.0 software. Detection conditions were systematically optimized. Then the sensitivity among TSA coupled with GLSS, GLSS, and TSA coupled with Cy3 was compared. The optimized conditions were: streptavidin-HRP (1 mgmL~(-1)) dilution 1:1500, biotin-tyramine dilution 1:200 (+0.5% H_2O_2), streptavidin-nanogold dilution 1:100 (all diluted in 1×PBS +1% BSA) and silver stain time of 10 min. The sensitivity of TSA coupled with GLSS was 100-fold higher than that of GLSS, and was identical with that of TSA coupled with Cy3. Meanwhile, the specificity of the microarrays were not affected. This implied that TSA coupled with GLSS was a sensitive visual detection method and would be an ideal alternative to fluorescence-based detection for DNA microarrays.