Novel Biosensing Methods Based On Dnazyme Fluorescent Probes And Nanomaterials

With the rapid development of science research,the importance of biosensing technology in many fields has become increasingly prominent;it has been widely used in biomedicine,medical diagnosis,environmental monitoring,food safety and other fields.In recent years,various new materials with unique macroscopic quantum tunneling effect,large surface area,small size effect,good biocompatibility and orientation ability have been discovered and provided a powerful impetu for the researches and developments of biosensing technology by introduced these new materials into biological research.In this dissertation,we constructed three simple and rapid detection biosensors using deoxyribozyme and nanomaterials,which have achieved the analysis of microRNA（miRNA）,adenosine triphosphate（ATP）and cysteine（Cys）.The main contents are as following:（1）In chapter 2,we report a target-assisted self-cleavage DNAzyme probes for signal amplified imaging of miRNA-222 and miRNA-223 in living cells.The analytical platform included a dual-labeled（FAM and Dabcyl）8-17 DNAzyme strand（DNAzyme 1 probe）for miRNA-222 detection,and another dual-labeled（TAMRA and BHQ2）8-17 DNAzyme strand（DNAzyme 3 probe）for miRNA-223 detection.The DNAzyme probe comprised three essential structural components:a substrate segment containing a cleavage site,a target-binding sequence which was complementary with the target miRNA,and a Mg²⁺-dependent 8-17 DNAzyme segment.In the absence of target miRNAs,the catalytic activity of DNAzymes was inhibited and the fluorescence of FAM and TAMRA were efficiently quenched by Dabcyl and BHQ2,respectively.While in the presence of target miRNAs,target miRNAs could hybridize with the target-binding sequence of DNAzyme probes,facilitating the formation of an internal hairpin structure to yield an“active”DNAzyme,which then cleaved the substrate segment in the presence of Mg²⁺.The cleavage making the dyes apart from the quenchers.As a result,the fluorescence of FAM and TAMRA were recovered.Meanwhile,the target miRNAs were bind to another DNAzyme probes to drive another catalytic cycle.The fluorescence intensity of DNAzyme 1 and DNAzyme 3probes increased linearly with the miRNA-222 and miRNA-223 concentration in the range of2-50 nM and 2-25 nM,respectively,and the detection limits were determined to be 0.68 nM and 0.45 nM for miRNA-222 and miRNA-223,respectively.（2）In chapter 3,based on the ability of graphitic carbon nitride（g-C₃N₄）nanomaterials to adsorb ssDNA and the ssDNA to enhance the peroxidase activity of g-C₃N₄ nanosheets,we have designed a new kind of label-free visual sensing platform for ATP detection.In the presence of ATP aptamer ssDNA,the peroxidase activity of g-C₃N₄ nanosheets was strong,TMB was oxidized,and the absorbance of the solution was strong.ATP has stronger affinity towards ATP aptamer ssDNA,which led to the ssDNA separated from the surface of the g-C₃N₄ nanosheet,therefore,the peroxidase activity of the g-C₃N₄ nanosheet was reduced,and the absorbance of the solution was weaker,thereby achieving the purpose of specifically detecting ATP.The absorbance of the solution decreased linearly with the ATP concentration in the range of 0-0.35 mM,and the detection limit was 0.02 mM.（3）In chapter 4,based on the fluorescence quenching effect of mercury ions(Hg²⁺)and the fluorescence properties of carbon dots（CDs）,we constructed a simple and label-free“turn-off-on”fluorescence sensing platform for the analytical detection of Cys in solution.We used ATP as a raw material by hydrothermal method to synthesize a kind of N/P co-doped CDs,which capable of generating strong fluorescence under 320 nm excitation.In the presence of Hg²⁺,the fluorescence of CDs was efficiently quenched by Hg²⁺due to the occurrence of charge transfer process when Hg²⁺was close to the surface of the CDs.When in the presence of Cys and Hg²⁺,the Hg-S bond was formed by the strong affinity between Cys and Hg²⁺,which led to the leave of Hg²⁺from the surface of CDs,and the fluorescence restore of CDs,thereby achieving the purpose of detecting Cys.The fluorescence intensity of CDs increased linearly with the Cys concentration in the range of 0-400μM,and the detection limit was 340 nM.