Studies Of Novel Biosensing Technology Based On DNA Nanostructures And Enzyme-free Nucleic Acid Amplification

The research of biomarkers is of great significance for the observation and analysis of epidemiology,clinical diagnosis and therapy,and it is a powerful and dynamic approach to understand related diseases.The rapid development of biosensor technology provides a reliable,rapid,quantitative,low-cost and high-throughput platform for the detection of biomarkers.These biosensor platforms have enabled the identification of biomarkers and signaling molecules related to cell growth,cell death,and cell metabolism.With the development of medicine towards molecular marker-based diagnosis and highly specific treatments for molecular targets,the demand for highly sensitive detection of biomarkers is also increasing.DNA nanotechnology has provided a powerful tool to create molecular assembly structures and dynamic molecular devices with arbitrary control of size as well as shape,and has a wide range of applications in materials science and biomedical research.Enzyme-free nucleic acid amplification provides an important platform for detecting very low abundance nucleic acids,which can achieve either target amplification or signal amplification.Based on DNA nanostructure and enzyme-free nucleic acid amplification technology,this doctoral dissertation took the unique advantage of DNA nanotechnology to develop a series of novel nano-biosensor platforms for highly sensitive diagnosis of biomarkers.By means of signal amplification and target amplification,high sensitivity and high specificity of target recognition are achieved and applied to in situ imaging of related biomarkers in living cells.The detailed content of this dissertation was described as follows:Biothiols play a vital role in the physiological matrix by participating in the process of reversible oxidation-reduction reaction and important cellular functions such as detoxification and metabolism.Therefore,a facile,sensitive,and selective detection of biothiols in biological samples is highly required.In Chapter 2,we proposed a novel fluorescent nanosensor based on the hybridization chain reaction,which recruited graphene oxide to selectively fluorescence quenching of double-stranded DNA and hairpin DNA and used T-Hg²⁺-T coordination to control hybridization.The proposed strategy provided a simple but highly selective biosensing platform for the detection of biothiols.In the sensing system,the target,biothiols,induced the formation of long duplex chains through disrupting the thymine-Hg（II）-thymine coordination chemistry and initiating a hybridization chain reaction to amplified fluorescence signal.The HCR guaranteed high sensitivity of the proposed method.GO was employed as an excellent fluorescence quencher to reduce the background signal and further help strengthen the detection sensitivity.GO also functioned as the signal controller by selectively adsorbing hairpin DNA and releasing long double-stranded DNA products.Telomerase plays a crucial role in the proliferation of immortalized cells,and most cancers have been shown to have highly expressed telomerase activity.Therefore,the highly sensitive detection of intracellular telomerase activity has great prospects for the early diagnosis and treatment of tumors.In Chapter 3,based on a hybridization chain reaction chain reaction and a novel DNA nanostructure,we achieved highly sensitive detection of intracellular telomerase activity.DNA nanostructures were constructed by streptavidin,which could be used as delivery carriers to efficiently deliver nucleic acid probes into cells.In the presence of telomerase target,the primer probe could be extended to produce telomere repeats on the end of sequences,which could be used as the initial strands to trigger hybridization chain reaction.At the same time,the fluorescent signal was amplified that made telomerase could be detected in vivo.In addition,due to the characteristics of DNA nanostructures,hybridization chain reaction in DNA nanostructures could be triggered in four directions to generate non-linear hybridization chain reaction products.Through the amplification of non-linear hybridization chain reaction and the efficient delivery of DNA nanostructures,the sensitivity of telomerase detection was improved,that enabled sensitive imaging of telomerase in a single cell.The programmability of DNA molecules makes them suitable for making dynamic structures capable of performing sophisticated tasks.DNA nanowalker have proven to be excellent dynamic nanodevices for implementing a variety of special biological functionally at the molecular scales.In Chapter 4,based on Catalyzed Hairpin Assembly and dynamic DNA nanostructures,we herein demonstrated a rationally designed spatially localized bipedal DNA nanowalker built on proximity-based intramolecular Catalyzed Hairpin Assembly（PICHA）that moved along three-dimensional track on gold nanoparticles surface.Interestingly,through spatially arranging DNA hairpins of a CHA circuit on Y-shaped monomer,it is to be noted that the nanowalker could strongly increase the local concentration and showed high-speed kinetics.Moreover,the bipedal nanowalker could achieve self-propelled running in situ without addition of exogenous fuel.With high speed and robust self-propelled walking ability of nanowalker,such progressively moving device shown great potential for programmable circuit and computing patterns.The ability to monitor the expression level and location of specific endogenous mRNA in living cells in real-time will offers great opportunities in molecular biology and disease research.In Chapter 5,on the basic of spatially localized three-dimensional nanowalker,we further transduce the mRNA response signal into the states of bipedal walker to achieve self-propelling DNA nanowalker.The signal output from DNA nanowalker enables ultrasensitive imaging of mRNA in living cells.In proposed strategy,the blocker DNA strand could block the catalytic active sites on the ends of the bipedal walker,which made the legs of bipedal walker close to each other.Due to the proximity effect,the melt temperature between Blocker strands and walker strand could substantially increase,and the background noise of system could be reduced.In present of target mRNA,the bipedal walker could be liberated from Blocker stands,triggering the intramolecular CHA.The CHA reaction is no longer affected by the diffusion limitation in the cytoplasm,and there is no need to add exogenous fuel to drive the DNA nanomachine.Spatially localized three-dimensional nanowalker was applied in sophisticated systems and enabled spatiotemporal sensitive imaging of targets in biological systems.