| Author: | Oudeng, Gerile |
| Title: | Two-dimensional molybdenum disulfide based nanoprobes for nucleic acid detection |
| Advisors: | Yang, Mo (BME) |
| Degree: | Ph.D. |
| Year: | 2020 |
| Subject: | Hong Kong Polytechnic University -- Dissertations Biosensors Nucleic acids |
| Department: | Department of Biomedical Engineering |
| Pages: | xx, 136 pages : color illustrations |
| Language: | English |
| Abstract: | In the past decade, nanomedicine has provided new avenues for diagnosis and treatment through the application of nanomaterials and nanotechnology. Many types of nanomaterials have been used in a variety of biomedical applications such as biosensing, bioimaging and therapy. Two-dimensional nanomaterials are widely used in different biomedical fields due to their biocompatibility and unprecedented electronic and optical properties. In particular, MoS₂ nanosheets has attracted more and more biomedical problems due to their excellent optical absorption, large surface area, strong adsorption capacity to biomolecules and low cytotoxicity. It has been developed for a variety of applications such as biomolecular detection, gene delivery, cell imaging and therapy. However, when we reviewed the application of MoS₂ nanosheets in biosensing, we found that most biosensors are based on liquid phase detection. Conventionally, liquid-based bioassays require the extraction of biological samples and subsequent detection in tubes. Recent biosensing strategies based on the liquid phase provide many experiences for extracellular detection, but it also has some drawbacks. First, in this type of detection, samples are not in original physiological environment and many biological factors are eliminated. Based on the biocompatibility and optical properties of MoS₂ , we developed a biofunctionalized MoS₂ nanosheets for intracellular detection of miRNA. On the other hand, liquid based detection in tubes usually requires large number of samples and rarely involves high-throughput and multicomplex assay. In order to make up for this deficiency, we explored the possibility to combine the MoS₂ in a solid based droplet microarray. To explore the intracellular sensing application of MoS₂, we developed a biofunctionalized the MoS₂ nanosheets probe for in-situ miRNA expression detection at single-cell level. The strategy was based on the folic acid-polyethylene glycol (FA-PEG) functionalized MoS₂ nanosheets with adsorbed dye-labeled single-stranded DNA (ssDNA) probes. Once the nanoprobes are internalized intocancer cells, the hybridization between the probes and target miRNA results in the detachment dye-labeled probes from MoS₂ nanosheets surface, leading to the green fluorescence recovery. In this probe, MoS₂ nanosheets offer advantages of high fluorescence quenching efficiency and extremely low toxicity. The FA conjugation could protect the probes and improve cancer cell transfection efficiency. The ability of this nanoprobe for endogenous miRNA detection in living single cancer cells is demonstrated for two types of cancer cells with different miRNA-21 expression (MCF-7 and Hela cells). With the cancer targeting and PEGylated probe protection, the detection period (1-4 h) was shorter than many in vitro tests (10 h). This functionalized MoS₂ nanosheets based nanoprobes could provide a sensitive and real-time detection of intracellular miRNA detection platform. The second part of the work presents the 2D MoS₂ nanosheets modified dendritic droplet microarray (DMA) for rapid and sensitive detection of two different human immunodeficiency virus (HIV) genes. DMA slides were made by hydrophilic and hydrophobic patterning of dendritic substrates. The abundant dendritic ends of the hydrophilic spots are functionalized with positive amino groups, which electrical adsorb the MoS₂ nanosheets. The sensing process is then achieved via the fluorescence resonance energy transfer (FRET) between MoS₂ and fluorescent dye-labeled HIV probes. The dye-labeled HIV probe is adsorbed onto MoS₂ by van der Waals force, then in the presence of the target HIV gene, the target sequences hybridized with the single strand probe sequences to form the double-stranded deoxyribonucleic acid (dsDNAs). In dsDNAs, the bases of genes were hided into the gene skeleton, thus the adsorption of MoS₂ are significantly weakened. In addition, dsDNAs have stronger negative surface charge compare with that of ssDNAs, which enhanced the electrostatic repulsion of dsDNAs and the negative charged MoS₂. These two factors made the dsDNAs detached from the MoS₂, the fluorescence signals can be gradually recovered. The detection of HIV-1 and HIV-2 genes are realized by monitoring the fluorescence signal change of the probes from "turn-off" to "turn-on". By modifying the DMA with MoS₂ nanolayer, we have developed a low-cost (<150 nL) biosensor that can detect individual HIV genes from 10 nM to 50 nM and can also implemented the multicomplex targets detection in a chessboard like mode. This strategy combined the parallelization, partitioning and miniaturization of DMA with the biocompatible 2D MoS₂, opening the way for applying the 2D nanomaterials in fast and simple sensing devices. |
| Rights: | All rights reserved |
| Access: | open access |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| 991022378658103411.pdf | For All Users | 8.05 MB | Adobe PDF | View/Open |
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