Novel Biosensing Methods Based On Nucleic Acid Probe And Nanomaterial

Biosensor holds great potential in practical analysis and scientific research because it has advantages such as high sensitivity, high selectivity, low cost, and the ability to be used in complex system. Nucleic acid probes and nanomaterials have been widely used in the field of biosensors and exhibit great achievements. Therefore, they can provide more sensor design strategies and platforms. This doctoral thesis is based on advantages of nucleic acid probes and nanomaterials in biosensing and biochemical analysis applications, combining with the sophisticated signal amplification methods, focusing on the research hotspots, establishing several biosensing and biochemical analysis methods for sensitive detection of small molecules and histone acetylase activity, lable-free detection of biological molecules, cell surface proteins in situ detection and imaging, and mRNA quantitative analysis. The developed methods are simple, high sensitive, specific, low cost, and can also be applied in complex system. The detailed contents are described as follows.Split aptamer represents one of the major mechanisms in developing sensors toward varying analytes. Current split aptamer assays have been limited by their inferior sensitivity, while novel split aptamer-based strategies with efficient signal amplification has not been reported. The challenge for combining signal amplification approaches with split aptamer lies in the lacking of downstream biochemical reactions that are specific to the assembly of split aptamer fragments. In chapter 2, we developed a novel, highly sensitive split aptamer mediated endonuclease amplification (SAMEA) strategy for the construction of aptameric sensors. We realize that the assembly of split aptamer fragments in response to its target actually brings two tail sequences in close proximity, forming a three-way junction structure that is essential for proximity-dependent hybridization. This realization motivates us to investigate the hypothesis of using the three-way junction structure as the substrate of DNA endonuclease IV (Endo IV). This investigation reveals a new finding that Endo IV efficiently cleaves the substrate in a three-way junction structure with substantial signal amplification. Based on this new finding, a new split aptamer-based signal amplification strategy is then developed via a combination of split aptamer with proximity-dependent hybridization mediated Endo IV amplification. To our knowledge, this is the first time that split aptamer has been coupled to a signal amplification method. Therefore, it may provide a new paradigm for the design of ultrasensitive aptameric sensors, because incorporation of efficient signal amplification in split aptamer can substantially enhance the detection sensitivity. To demonstrate the concept, we choose two small molecules, adenosine and cocaine, as the model targets in this study. The results revealed that the developed strategy offers ～105-fold sensitivity improvement as compared to previous amplification-free split aptamer assays.Histone deacetylases (HDACs) is one of the most importan protein post-translational modification enzymes. Identifying and understanding HDACs are critical in the study of cell biology and disease treatment and prevention. However, it is still a challenge to develop facile, cost-effective, and versatile HDACs quantitative activity assays. In chapter 3, a novel fluorescent nanosensor has been developed for detecting the concentration of histone deacetylase (HDACs) based on graphene oxide (GO)-peptide nanoassembly. The formation of GO-peptide nanoassembly is nonemissive due to the strong adsorbption of fluorophore-labeled peptide to the surface of GO via aromatic and hydrophobic residues. In the presence of HDAC1, the deacetylation of lysine generates the sustrate for rLys-c, which is an endoproteinase and can specifically cleave lysine residues at the carboxyl-terminal. Upon the cleavage of rLys-c, the peptide was cut into two fragments, releasing high fluorescence signals. The developed nanobiosensor was shown to be highly sensitive and selective for quantification of HDAC 1 with a wide linear detection range (from 100 pM to 100 nM) and a detection limit of 70 pM. In virtue of these advantages, we can know that this strategy provide a simple, rapid but cost-effective platform for HDACs detection.In chapter 4, We developed a novel label-free biosensor for biomolecules detection based on the thioflavin T (ThT)-induced conformational change of guanine-rich oligonucleotide and self-assembled aptamer/GO nanosheets nanoassembly. This biosensor consists of two regions:one is the aptamer itself for recognition of the target biomolecules of interest; the other is a G-rich oligonucleotide sequence capable to fold into a G-quadruplex structure when induced by ThT. With the aptamer sequence absorbed on the GO surface, we obtain a self-assembled aptamer/GO architecture as the label-free sensor. The presence of the target would specifically bind to the target, which induces the release of the aptamer sequence away from the GO surface. Then, ThT can bind to the G-rich oligonucleotide region of the probe to form G-quadruplex conformation with a substantially enhanced fluorescence signal. To demonstrate the developed biosensor, we apply the aptamer/GO-based biosensor to detecting human a-thrombin and adenosine monophosphate (AMP). The result revealed that the proposed biosensor provides a label-free, sensitive, selective and rapid method for sensing different analytes.Detection of specific biomarkers in cell membranes is critical for cell biology and disease theranostics. In chapter 5, we develop a versatile terminal protection assay strategy for wash-free quantification and imaging of cell surface proteins using small molecule-linked DNA with programmable signal sequences. DNA probes are designed to link toa small molecule ligand at 3’end for specific recognition of the target cell surface protein and a programmablesignal sequence at 5’terminal for delivering detectable responses. Binding of the small molecule ligand to target protein enables protection of the DNA probes from exonuclease I mediated degradation, leaving the surface-binding probes intact while the non-binding probes degraded. This strategy thus allows wash-free detection of the cell surface protein via the selectively protected signal sequence. By programming the signal sequences asperoxidase-like DNAzyme, quantitative polymerase chain reaction (qPCR) targeting DNA and Ag nanoclusters (AgNCs) forming DNA template based on our new finding that theexonuclease I is able to quench the fluorescence of AgNCs, we can develop this strategy into a versatile platform for colorimetric detection, qPCR quantification and fluorescence imaging of the cell surface protein. This platform is demonstrated using a folate-linked DNA probe for folate receptor detection on tumor eel surface. The results revealed that this strategy enables highly selective and sensitive detection of the tumor cells as well as quantification and localization of the membrane protein on the cells, implying its potential in membrane protein based biomedical and clinical applications.Quantification of the expression levels of a particular mRNA is crucially important for identification of the subtypes of disease and clinical diagnosis. In chapter 6, we developed a strategy based on hybridization chain reaction induced fluorescence enhancement of silver clusters for mRNA detection. Different from traditional HCR, four hairpin probes were designed here. While hairpin probes HI has been extended with G-rich sequence at its 3’end, and hairpin probes H3 has been extended 28 nt segment at its 5’end for hybridization with a part of P5 probe which contains DNA template for silver clusters preparation. The presence of mRNA triggers orderly catalyzed hybridization among hairpin probes H1, H2, H3 and H4. As a result, long chain DNA polymer structures were generated. The hybridization with presynthesis Ag NCs/P5 brings the G-rich overhang close to Ag NCs, which greatly enhances the fluorescent intensity of AgNCs. This assay is capable of effectively detecting target mRNA. Compared with previously reported mRNA detection methods, this method does not need any chemical modifications. In adition, the method has other advantages like high sensitivity, and can be used for the determination of mRNA in complex cellular extracts.