Nanoparticle sensor for label free detection of swine DNA in mixed biologicalsamples

Abstract

We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specificconserved sequence and nucleotide mismatch in PCR-amplified and non-amplifiedmitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color frompinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected bythe dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance ofnew features in the 620–800 nm regions in their absorption spectra. The particles werestabilized against salt-induced aggregation upon the adsorption of single-stranded DNA.The PCR products, without any additional processing, were hybridized with a17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 °C) that differentiated matched and mismatched base pairing, the probe was hybridized topig PCR product and dehybridized from the deer product. The dehybridizedprobe stuck to GNPs to prevent them from salt-induced aggregation and retainedtheir characteristic red color. Hybridization of a 27-nucleotide probe to swinemitochondrial DNA identified them in pork–venison, pork–shad and venison–shadbinary admixtures, eliminating the need of PCR amplification. Thus the assaywas applied to authenticate species both in PCR-amplified and non-amplifiedheterogeneous biological samples. The results were determined visually and validated byabsorption spectroscopy. The entire assay (hybridization plus visual detection) wasperformed in less than 10 min. The LOD (for genomic DNA) of the assay was6 µg ml − 1 swine DNA in mixed meat samples. We believe the assay can be applied for speciesassignment in food analysis, mismatch detection in genetic screening and homology studiesbetween closely related species.