High-sensitivity label-free electrochemical genosensors for carbon nanotube plasmon-assisted detection of somatic mutations in nucleic acids from formalin-fixed paraffin-embedded tissues

Abstract

Currently, molecular genetic testing of somatic point mutations in the genome of cancer tumors for targeted therapy requires high-performance tools for studies of spatial gene expression and spatial genome profiling in formalin-fixed paraffin-embedded (FFPE) tissue samples. We offer new high-performance label-free electrochemical impedimetric deoxyribonucleic-acid (DNA) nanosensors. The sensors based on a platform of crystalline carbon nanotube plasmonic assemblies are fabricated by the Langmuir–Blodgett deposition technique. The carbon-nanotube assemblies are suspended on nanoporous supports. Incorporation of fast Fourier transforms in the impedance spectroscopy led to the introduction of a new information parameter, the integral value of capacitance changes. The allele-sensitive assay based on plasmon-assisted effects in the assemblies of electrically active conjugates between DNA and few-walled carbon nanotubes has been used for discrimination of allele single-nucleotide polymorphisms (SNPs) of Kirsten Rat Sarcoma viral oncogene homologue (KRAS gene). Using 19- and 20-mer single-stranded (ss) probe oligonucleotides and 35- and 47-mer toehold probes, we explored the probe length and the target location. The genotyping technology allows discrimination of single-nucleotide variations in the target 35-base oligonucleotide at the attomolar concentration level. Human genomic DNAs were isolated from FFPE colorectal cancer tumor tissue samples. The KRAS-gene exon 2, codon 12, c.35G$>$A mutation has been successfully discriminated in the genomic DNAs with a significance level, P, of 0.001–0.02. The assay has a sensitivity with 0.33-ng/ml limit of detection (LOD) for native genomic DNA.