Fluorometric determination of the p53 cancer gene using strand displacement amplification on gold nanoparticles.

Abstract

A fluorometric assay is described for the tumor suppressor gene p53. The method is based on strand displacement amplification on gold nanoparticles (GNPs). A FAM-labeled hairpin probe (HPP) is used that can hybridize to the GNP-confined linker strand, and the green fluorescence of the FAM label is quenched by the GNPs. In the presence of the p53 gene, it will hybridize with the HPP. This leads to fluorescence recovery. The primer then hybridizes with the opened HPP and induces the polymerization/displacement reactions. As a result, the hybridized p53 gene is released and, in turn, hybridizes with another HPP on the surface of the GNPs. This triggers the next round of hybridization/enzymatic polymerization/displacement reactions. This results in efficient strand displacement amplification and generates a substantially amplified signal. The method is referred to as GNP-HPP because it involves the use of GNPs and a HPP. The method allows the target DNA (p53) to be quantified down to 1.6 pM concentrations with a linear response in the 5 pM to 1nM concentration range. In addition, mutant p53 genes can be easily distinguished from the wild-type gene. The method is highly sensitive, selective, and has a low background signal. Graphical abstract Schematic presentation of a chain hybridization signal amplification system (GNP-HPP) based on the use of gold nanoparticles (GNP) as a quenching source for the tumor suppressor gene p53 detection. The hairpin probe (HPP) having a 5'-end modified fluorophore was used as a signalling probe.