Amperometric determination of hazelnut traces by means of Express PCR coupled to magnetic beads assembled on disposable DNA sensing scaffolds

Abstract

A disposable amperometric sensor using magnetic microcarriers has been designed and implemented to be used in combination with the so called Express PCR to detect the presence of hazelnut traces in foodstuffs through the detection of Cor a 9 allergen coding sequence. The developed procedure involves the use of streptavidin-modified magnetic microbeads (Strep-MBs), specific biotinylated capture and detector probes which hybridize with a specific region of the gene encoding the protein Cor a 9, and appropriate primers for PCR amplification. A 50-mer synthetic target DNA or unmodified 100-bp PCR products were selective captured via sandwich hybridization with capture probe modified MBs and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a commercial streptavidin–peroxidase (Strep-HRP) conjugate and the final modified MBs were magnetically captured onto a screen-printed carbon electrode to perform amperometric detection using the H2O2/HQ system. A LOD of 0.72 pM was obtained for the synthetic target and the applicability studies demonstrated the possibility to detect the denatured PCR amplified samples obtained using only 20 pg of genomic DNA extracted from hazelnut. RSD values obtained, below 10% in all cases, confirmed the good reliability of extraction, amplification and quantification protocols involved in the developed methodology. The strict specificity of the designed primers and selected probes for hazelnut was demonstrated by performing PCR amplification of genomic DNA extracted from different hazelnut varieties and other species of similar families (pistachio, cashew, walnut and tangerine) and analyzing the resultant amplicons by the developed electrochemical sensor. The reliable and sensitive results achieved indicate that Express PCR in conjunction with an electrochemical DNA sensor, used for the first time in this work, provides a suitable sensitive, specific, and cost-effective method for routine food allergens determinations, particularly useful for resource-limited settings.