Colorimetric Sensor for Label Free Detection of Porcine PCR Product (ID: 18)

This report described the use of $40±5 nm$ in diameter citrate‐coated gold nanoparticles (GNPs) as colorimetric sensor to visually detect the presence of a 17‐base swine specific conserved sequence and nucleotide mismatch in the mixed PCR products of pig, deer and shad cytochrome b genes. The size of these PCR amplicons was 109 base‐pair and was amplified with a pair of common primers. Colloidal GNPs changed color from pinkish‐ red to purple‐gray in 2 mM PBS buffer by losing its characteristic surface plasmon resonance peak at 530 nm and gaining new features between 620 and 800 nm in the absorption spectrum indicating strong aggregation. The particles were stabilized against salt induced aggregation, retained spectral features and characteristic color upon adsorption of single‐stranded DNA. The PCR products without any additional processing were hybridized with a 17‐nucleotide swine probe prior to exposure to GNPs. At a critical annealing temperature (55° C) that differentiated between the match and mismatch pairing, the probe was hybridized with the pig PCR product and dehybridized from the deer’s and shad’s. The interaction of dehybridized probe to GNPs prevented them from salt‐induced aggregation, retaining their characteristic red color. The assay did not need any surface modification chemistry or labeling steps. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The assay obviated the need of complex RFLP, sequencing or blotting to differentiate the same size PCR products. We find the application of the assay for species assignment in food analysis, mismatch detection in genetic screening and homology study among closely related species.