Tyramide signal amplification as universal detection method on protein coated microbeads

Abstract

Quantitative analysis of a target molecule in a microbead-based fluorescent assay requires a specific labeling procedure. For nucleic acid analysis the hybridization with florescent labeled oligonucleotides is the most common method. However, disadvantages are the necessity for direct labeling of p robes and the sensitivity to detect low amounts of target molecules. In this study we established an alternative detection method for biomolecules on microbeads, the tyramide signal amplification (TSA). Hereby, biomolecules are detected by enzymatically activated and fluorophore-conjugated tyramides that bind to specific protein residues. This method has proven to be a versatile and robust enzyme amplification technique for sensitive immunohistochemical detection. Now, we present the feasibility of the TSA procedure to detect hybridized biotinylated oligonucleotide probes bound to protein coated microbead surfaces TSA was performed using fluorescent, size-encoded and streptavidin coated microbeads that were loaded with dual-biotinylated DNA capture probes, prepared from polymerase chain reaction. Beside streptavidin alone for surface coating of those microbeads, we applied different quantities of streptavidin in combination with bovine serum albumin, immunoglobulin G or Protein G/A, to check for positive effects on the resulting signal intensities through specific binding of tyramide molecules. For this method, streptavidin turned out as appropriate protein for the surface binding, without the need for further molecules. In comparison to a standard detection with common streptavidin-fluorophore-conjugates TSA showed its advantage in the detection of low probe amounts down to a concentration of 3.3·10-4 ng/μL.