A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of TEM-1 β- lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogenspecific DNA sequences present in E. coli O157:H7 on the lab-on-a chip. ZFPs customdesigned to detect shiga toxin in E. coli O157:H7 were immobilized on the cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic. Pathogen-specific double-stranded DNA was directly detected by engineered ZFPs immobilized on the COC chip, providing a detection limit of 10 fmole of target DNA in colorimetric assay. Therefore, in this study, we demonstrated a great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a chip technology for detection of pathogens.
Antibiotic resistance is a serious, and rapidly growing global threat. Here, we designed a novel screening method to detect antibiotic resistance genes (ARGs) in bacteria using a graphene oxide-based biosensor utilizing engineered ZFPs. Two-dimensional
graphene oxide (GO) sheet possesses unique electronic, thermal, and mechanical properties. The quenching ability of GO can create novel methods for detection of biomolecules. Our approach utilizes quenching of fluorescence signal by GO in the absence of target ARGs, but restoring the signal in the presence of target ARGs. Quantum dot (QD)- labeled ZFP can bind to 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. Due to fluorescence resonance energy transfer (FRET) between QD and GO when they are in close proximity, fluorescence signal of QD-labeled ZFP is expected to be quenched. In the presence of target DNA, the bound DNA-protein complex is released from GO, restoring the fluorescence signal.
| Identifer | oai:union.ndltd.org:WKU/oai:digitalcommons.wku.edu:theses-4084 |
| Date | 01 October 2018 |
| Creators | Ha, Dat Thinh |
| Publisher | TopSCHOLAR® |
| Source Sets | Western Kentucky University Theses |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses & Specialist Projects |
Page generated in 0.0021 seconds