<br/>DNA detection technologies play an important role in diagnostic applications in the areas of public health and biomedicine. Zinc finger proteins (ZFPs) are the most common DNA-binding domains and multiple ZF domains can be assembled to bind to any DNA sequence of interest. In this work, six finger ZFPs were engineered to bind to 18 base pairs of DNA within the stx2 gene that encodes for E. coli O157 shiga toxin and the tetM gene (tetracycline resistance gene) with high specificity. Here, we designed a novel sensing technology to detect ARGs in bacteria using a graphene oxide-based biosensor utilizing ZFPs. Our approach relies on the on and off effect of fluorescence signal in the absence and presence of target ARGs, respectively. Two-dimensional graphene oxide (GO) sheet possesses unique electronic, thermal, and mechanical properties. In this study, we take advantage of the quenching ability of GO to create a novel method for detecting the specific double-stranded DNA sequences within ARGs. Quantum dot-labeled ZFPs can be adsorbed onto GO via stacking interactions of aromatic and hydrophobic residues in conjunction with hydrogen bonding interaction between hydroxyl or carboxyl groups of GO and hydroxyl or amine groups of the protein. The fluorescence signal of QD-labeled ZFPs is quenched due to fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and GO when they are in close proximity. In the presence of target DNA, the bound protein-DNA complex is dissociated from the GO surface due to the conformational changes, thereby restoring the fluorescence signal. Here, we optimized GO concentrations used in the assay and QD-labeling on ZFPs and determined the sensitivity of our system. This study could bring new capabilities to molecular diagnostics and broaden the spectrum of GO applications in public health and clinical diagnostics.

Novel application of 2D-nanosheet and engineered DNA-binding proteins for specific gene detection

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.