Nanobiosensor for the detection and quantification of specific DNA sequences in degraded biological samples

Abstract

A 27-nucleotide AluI fragment of swine cytochrome b (cytb) gene was integrated to a 3-nm diameter citrate- tannate coated gold nanoparticle to fabricate a species specific nanobiosensor. The biosensor was applied to authenticate pork adulteration in autoclaved pork-beef mixtures. The sensor was found to be sensitive enough to detect 0.5 pork in raw and 2.5-h autoclaved mixed samples in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic targets from moderate to extreme target concentrations and a sigmoidal relationship was found. The kinetic curve was used to develop a convenient method for quantifying and counting target DNA copy number. The biosensor probe was hybridized with a target DNA that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed meat products or extensively degraded samples where PCR-based identification technique might not work due to the degradation of comparatively longer DNA. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences in degraded samples to address a range of biological problems such as food analysis, biodiagnostics, environmental monitoring, genetic screening and forensic investigations.