Research On Novel Optical Biosensor Based On Functional Nucleic Acid And Isothermal Signal Amplification Technology

In recent years,with the progress of science and technology,biosensor has become one of the important development directions of modern biotechnology.Among them,optical biosensor is widely used in biology,chemistry,food,medicine,environment,military and other fields because of its advantages such as high sensitivity,good selectivity,simple operation,fast analysis speed and low cost.The discovery of functional nucleic acid probe breaks through the traditional understanding that nucleic acid is only a carrier of genetic information storage and transport.As a nucleic acid molecule with special functions such as high affinity and high specificity,it has great advantages in the field of analytical chemistry.Isothermal signal amplification technology is widely used in the detection of various analytes because it can achieve fast and efficient signal amplification at constant temperature.The combination of functional nucleic acid probe and isothermal signal amplification technology provides a new design idea and platform for the construction of biosensor system.In this thesis,three simple,fast,ultrasensitive and highly specific optical biosensors were constructed by using the specificity of functional nucleic acid probes（aptamers,molecular beacons,DNAzyme）,,combined with enzyme-assisted amplification,enzyme-free chain replacement and catalytic hairpin self-assembly.Firstly,a simple and robust fluorescence sensing strategy has been developed for the detection of pathogenic bacteria by the combination of the dual functionality of phi29 DNA polymerase with isothermal circular strand displacement polymerization（ICSDP）.The strategy relies on target-triggered formation of a mature primer that initiates the cyclic strand displacement polymerization reaction with the aid of dual functional phi29,thus,amplified detection of the target can be achieved.To our knowledge,this work is the first report where dual functional phi29-assisted ICSDP has been employed for fluorescence sensing of pathogenic bacteria.It is worth noting that a hairpin pre-primer is introduced that can be trimmed into a mature primer for initiating ICSDP via the 3’→5’proofreading exonuclease activity of phi29,which contributes to the ultrahigh specificity of the strategy owing to the elimination of the unwished nonspecific extension.On the basis of the present amplification strategy,our biosensor exhibits excellent specificity and sensitivity toward S.typhimurium with an excellent detection limit as low as 1.5 cfu mL^-1.In addition,the strategy offers the advantages of a simplified operation,shortened analysis time,and highly sensitive detection of pathogens with only a one-step reaction.Furthermore,by redesigning the corresponding binding molecules,the proposed strategy can be easily extended for the detection of a wide spectrum of analytes.Hence,the dual functional phi29-assisted ICSDP strategy indeed creates a robust and convenient fluorescence sensing platform for the identification of pathogenic bacteria and related food safety analysis.Secondly,a novel fluorescence sensing strategy for ultrasensitive and highly specific detection of adenosine triphosphate（ATP）has been developed by the combination of the proximity ligation assay with bidirectional enzymatic repairing amplification（BERA）.The strategy relies on proximity binding-triggered the release of palindromic tail that initiates bidirectional cyclic enzymatic repairing amplification reaction with the aid of polymerase and two DNA repairing enzymes,uracil-DNA glycosylase（UDG）and endonuclease IV（Endo IV）.A fluorescence-quenched hairpin probe with a palindromic tail at the 3′end（MB）is skillfully designed that functions as not only the recognition element,primer,and polymerization template for BERA but also the indicator for fluorescence signal output.On the basis of the amplification strategy,this biosensor displays excellent sensitivity and selectivity for ATP detection with an outstanding detection limit of 0.81 pM.Through simultaneously enhancing the target response signal value and reducing nonspecific background,this work deducted the background effect,and showed high sensitivity and reproducibility.Moreover,our biosensor also shows promising potential in real sample analysis.Therefore,the proximity-enabled BERA strategy indeed creates a simple and valuable fluorescence sensing platform for ATP identification and related disease diagnosis and biomedical research.Thirdly,a label-free and enzyme-free biosensing platform for ultrasensitive detection of S.Typhimurium has been constructed by the ingenious coupling of three-way G-quadruplex DNAzyme（GQH DNAzyme）with 12 polycytosine-templated silver nanoclusters（dC12-AgNCs）.The detection mechanism is based on the concept,that is,cysteine（Cys）can sensitively and rapidly reacts with dC12-AgNCs via the formation of Ag-S bond and enhances the fluorescence of dC12-AgNCs.It is worth mentioning that this is the first time that three-way GQH DNAzyme has been used for S.Typhimurium detection.On the basis of the present amplification strategy,our biosensor displays excellent analytical performance toward S.typhimurium with an outstanding detection limit of 6.3 cfu m L^-1.What’s more,the method is simple in operation,short in reaction time,and does not require biomolecule labeling,and establishes a new sensing platform for S.Typhimurium analysis.Meanwhile,by replacing the corresponding aptamer for target recognition,our proposed strategy can further extend for detection of various analytes in related food safety analysis and clinical diagnosis.