Novel amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotubes-screen printed electrodes for the determination of trace levels of non-amplified DNA in genetically modified (GM) soy

Abstract

A novel amperometric genosensor based on PNA probes covalently bound on the surface of Single Walled Carbon Nanotubes – Screen Printed Electrodes (SWCNT-SPEs) was developed and validated in samples of non-amplified genomic DNA extracted from genetically modified (GM)-Soy. The sandwich assay is based on a first recognition of a 20-mer portion of the target DNA by a complementary PNA Capture Probe (CP) and a second hybridization with a PNA Signalling Probe (SP), with a complementary sequence to a different portion of the target DNA. The SP was labelled with biotin to measure current signal by means of a final incubation of an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). The electrochemical detection was carried out using hydroquinone diphosphate (HQDP) as enzymatic substrate. The genoassay provided a linear range from 250 pM to 2.5 nM, LOD of 64 pM and LOQ of 215 pM Excellent selectivity towards one base mismatch (1-MM) or scrambled (SCR) sequences was obtained. A simple protocol for extraction and analysis of non-amplified soybean genomic DNA without sample treatment was developed and validated. Our study provides insight into how the outstanding recognition efficiency of PNAs can be combined with the unique properties of CNTs in terms of signal response enhancement for direct detection of genomic DNA samples at the level of interest without previous amplification.