A novel nucleic acid based bio-assay toward recognition of Haemophilus influenza using bioconjugation and DNA hybridization method

Abstract

Haemophilus influenza (H. influenza) is a gram negative coccobacillus pathogenic microorganism. H. influenza produces beta-lactamases, and it is also able to modify its penicillin-binding proteins, so it has gained resistance to the penicillin family of antibiotics. In this work, a novel sensitive approach was established for the monitoring of H. influenza using DNA based bio-assay. For the first time, specific sequence of thiolated probe of Haemophilus influenza (SH-5′-AAT TTT CCA ACT TTT TCA CCT GCA T-3′) was immobilized on the surface of gold (Au) electrode. Square wave voltammetry (SWV) was carried out in toluidine blue (TB) solution for DNA hybridization and targeting of cDNA sequence of Haemophilus influenza. Field scanning electron microscope (FE-SEM) was applied to investigation of the electrode morphology and estimate of particle size. In the optimal conditions, the planned strategy could detect target DNA (5′-ATG CAG GTG AAA AAG TTG GAA AAT T-3′) down to 1 ZM with a linear range from 1 μM to 1 ZM. Moreover, engineered geno-assay selectively differentiates the complementary sequence from target sequences with one, double and three base mismatch sequences.