ABSTRACT
An innovative label-free DNA genosensing assay based on a direct hybridization followed by DPASV in the presence of [Fe(CN)6]4-/3- was developed for recognizing the H. influenza genome in human plasma samples. To attain this objective, Zn-based MOF was synthesized and combined with carboxymethyl cellulose (CMC), which were immobilized on the surface of Au electrode and AuNPs were immobilized on the Zn-based MOF/CMC/Au-modified electrode surface. The genosensing bio-assay provides high specificity, sensitivity, and good performance for the determination of L-fuculokinase gene from the Haemophilus influenza genome. Various characterization techniques were applied including Fe-SEM, EDS, FT-IR, and XRD for investigation of morphological features and particle size. Under optimal conditions LOD and LOQ were 1.48 fM and 3.23 fM, respectively. Moreover, a wide linear range was obtained ranging from 0.1 pM-10 nM for t-DNA. The recoveries and RSDs were 98.4-103% and 2.2-3.2, respectively. The fabricated biosensing assay presented high selective ability of one, two, and three-base mismatched sequences. In addition, negative control of the genosensing assay for investigation of the selectivity was performed by the t-DNAs of Salmonella typhimurium and Shigella flexneri bacteria. Likewise, reproducibility and repeatability of the related bio-assay were investigated. It is to be noted that the organized genosensing bio-assay can be straightforwardly reused and regenerated to assess the hybridization process.
Subject(s)
Biosensing Techniques/methods , DNA, Bacterial/blood , Electrochemical Techniques/methods , Haemophilus influenzae/chemistry , Metal-Organic Frameworks/chemistry , DNA, Bacterial/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Gold/chemistry , Haemophilus influenzae/enzymology , Humans , Limit of Detection , Metal Nanoparticles/chemistry , Nucleic Acid Hybridization , Phosphotransferases (Alcohol Group Acceptor)/genetics , Reproducibility of Results , Zinc/chemistryABSTRACT
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⯵M to 1 ZM. Moreover, engineered geno-assay selectively differentiates the complementary sequence from target sequences with one, double and three base mismatch sequences.
