Graphene nanomaterial based fluorescence resonance energy transfer (FRET) Biosensor for biomolecule and cell detection

Pao Yue-kong Library Electronic Theses Database

Graphene nanomaterial based fluorescence resonance energy transfer (FRET) Biosensor for biomolecule and cell detection


Author: Shi, Jingyu
Title: Graphene nanomaterial based fluorescence resonance energy transfer (FRET) Biosensor for biomolecule and cell detection
Degree: M.Phil.
Year: 2015
Subject: Biosensors
Nanostructured materials.
Hong Kong Polytechnic University -- Dissertations
Department: Interdisciplinary Division of Biomedical Engineering
Pages: xx, 144 pages : illustrations (some color)
Language: English
InnoPac Record:
Abstract: Nowadays biodetection is popular in many fields, such as food safety, medical diagnostics, drug discovery and environment detection. Conventional biodetection methods including immunological assay (ELISA) and nucleic acid based assays (PCR) are primarily hampered by time-consuming and labor-intensive procedures with relatively low sensitivity. Therefore, the development of rapid, direct and sensitive biodetection methods is of great importance. Fluorescence resonance energy transfer (FRET) is a direct and sensitive method, which can detect biological phenomena in nanoscale. However, traditional based FRET is mainly based on organic fluorophores which have short fluorescence life time and low stability. The research in this dissertation is focused on developing graphene nanomaterial based fluorescence resonance energy transfer (FRET) biosensors for rapid and ultrasensitive detection of various biological species, including staphylococcus aureus (S. aureus) mecA gene sequence, bacterial protein toxin and circulating tumor cells in breast cancer (MCF-7).The whole study includes three parts. The first part of this thesis is focused on development of a FRET biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) for rapid and sensitive detection of specific mecA gene sequence of staphylococcus aureus. This FRET biosensor platform is realized by immobilization of capture probes on GQDs and conjugation of reporter probes on AuNPs. Target oligonucleotides then co-hybridize with capture probes and reporter probes to form a sandwich structure which brings GQDs and AuNPs to close proximity to trigger FRET effect. The fluorescence signals before and after addition of targets are measured and the fluorescence quenching efficiency could reach around 87%. The limit of detection (LOD) of this FRET biosensor was around 1 nM for S. aureus gene detection. Experiments with both single-base mismatched oligonucleotides and double-base mismatched oligonucleotides demonstrated the specificity of this FRET biosensor.
The second part of this study describes a graphene oxide (GO) based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin serotype A light chain (BoNT-LcA) enzymatic activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) is optimally designed and synthesized with the centralized recognition/cleavage sites. BoNT-LcA can specifically cleave SNAP-25-GFP substrate immobilized on GO surface, releasing the fragment connected with GFP into the solution. By monitoring fluorescence signal recovery, the target BoNT-LcA protease activity is detected sensitively and selectively with the linear detection range from 1 fg/mL to 1 pg/mL. The LOD for BoNT-LcA is around 1 fg/ml. Moreover, stability and reliability of this GO-peptide based FRET biosensor is also investigated. In the third part of this dissertation, a fluorescence labeled epithelial cell adhesion molecule (EpCAM) aptamer/GO nanocomplex based FRET biosensor is developed for sensitive detection of breast cancer circulating tumor cells (CTCs).The FRET sensing platform is constructed by absorption of FAM-EpCAM aptamer (donor) on GO (acceptor) surface via π-π stacking interaction. Target MCF-7 cancer cells can be detected through affinity interaction between FAM-EpCAM aptamer and EpCAM protein that expressed on the surface membrane of MCF-7 cells, resulting in the release of FAM-EpCAM aptamer from GO surface. By monitoring fluorescence signal recovery, the target MCF-7 cancer cells can be detected sensitively and selectively.

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