Application of 2D‐nanosheet Graphene Oxide and Engineered DNA‐binding Proteins for Specific Gene Detection

Abstract

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 desired DNA sequences of interest. In this work, six finger ZFPs were engineered to bind to 18 base pairs of DNA within the stx2gene that encodes for E. coli O157 shiga toxin and the tetM gene (tetracycline resistance gene) with high specificity. Here, we further optimized a novel sensing technology to detect antibiotic resistance genes (ARGs) in bacteria using a graphene oxide-based biosensor utilizing the engineered ZFPs. 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 (ds) DNA sequences within ARGs. Quantum dot (QD)-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 QDs and GO when they are in close proximity. In the presence of target DNA, the bound protein-DNA complex will be dissociated from the GO surface due to the conformational change, thereby restoring the fluorescence signal. Here, we were able to improve the sensitivity of our system, providing a limit of detection as low as 100 pM. This study could bring new capabilities to molecular diagnostics and broaden the spectrum of GO applications in public health and clinical diagnostics.