The effect of pore size in an ultrasensitive DNA sandwich-hybridization assay for the Escherichia coli O157:H7 gene based on the use of a nanoporous alumina membrane

Abstract

The authors describe a rapid method for the detection of the Escherichia coli O157:H7 (E. coli O157:H7) bacterial gene. The DNA sandwich-hybridization impedimetric assay is based on the use of a nanoporous alumina membrane in combination with gold/silver core/shell nanoparticles (Ag@AuNPs) that act as tags for impedance signal amplification. The probe oligonucleotides were immobilized on the walls of the nanopores. This is followed by hybridization, first with target (analyte), then with reporter oligonucleotides labeled with Ag@AuNP tags. The impedimetric signal results from target oligo hybridization with probe oligos and co-hybridization with labeled reporter oligos, which increases the blocking degree of the nanopores. The assays were tested with membranes in nanopore sizes of 20 nm, 50 nm and 100 nm. The assay performs best in case of 100 nm nanopores where the limit of detection is as low as 11 pM, with a linear detection range that extends from 50 pM to 200 nM. This indicates its potential for rapid and ultrasensitive gene detection. [Figure not available: see fulltext.]