Detection Of Low Abundance Protein Base On Rolling Circle Amplification

Objective:Protein is one of the most important macromolecular compound in thehuman body. Sensitive protein detection is critically important in basicdiscovery research and clinical practice, because a few molecules of proteinare sufficient to affect the biological functions of cells and triggerpathophysiological processes. However, many protein molecules are invery low concentration in the body and so signal amplification is one of themost critical issues in low-abundancy protein analysis. This study combinedwith immune response and rolling circle amplification to set up a highthroughput test of low abundance protein, and use the vascular endothelialgrowth factor(VEGF) as a model protein to verify the practicability of theproposed strategy.Method:Combination of high sensitivity and specificity of rolling circleamplification（RCA）method with Antigen-antibody immune response. Thisstudy propose a new cascade signal amplification strategy for the low-abundance protein detection. According to the streptavidin as a bridge, the multiplex anchoring of biotinylated single-strand oligonucleotide willbound to the biotinylated immunocomplex. The RCA was initiated toproduce long single-strand DNA, which contained hundreds oftandem-repeat sequences for linear periodic hybridization of a largenumber of biotinylated probes and the periodic assembly of SA-HRP torealize signal amplification.Result:In the optimized experimental conditions, the RCA strategy show alimit of detection of about10fM. Compare to the conventional ELISA,this method show a higher sensitivity and could meet low abundanceprotein detection.ConclusionRolling circle amplification strategy has a very strong signalamplification ability, minimal non-specific adsorption and low backgroundsignal. It could provide technical support for a variety of low-abundanceprotein detection and would become a powerful tool for new proteinmarkers of disease screening and clinical diagnostics.