Research On Biosensing Methods Based On Isothermal Nucleic Acid Amplification

Developing highly sensitive trace analyte detection methods has important research significance in the fields of food safety detection,disease diagnosis,and environmental monitoring.Biosensors have the advantages of low cost,high sensitivity,simple,fast,and strong specificity,which have broad application prospects in the field of trace analysis.However,the traditional biosensing method which generates a signal output unit from a target object has been unable to meet the detection requirements of trace analytes.In contrast,the isothermal nucleic acid signal amplification method has the advantages of simple operation,strong specificity,flexible sequence design and strong signal amplification ability,which provides a powerful means for the highly sensitive detection of trace analytes in complex environmental systems.In this thesis,three kinds of biosensing methods with high sensitivity,strong specificity,and simple operation were constructed based on the highly specific recognition ability of functional nucleic acid and the efficient amplification ability of the signal amplification technology of constant temperature nucleic acid.The efficient detection of target analytes in fish,serum,and milk samples was successfully realized.The research content of this thesis mainly includes three parts:（1）A Hemin/G quadruplex mediated aptasensor was constructed for highly sensitive ATP detection and fish freshness assessment.The specific binding between ATP and two aptamer subunits was used to pull the two divided G-quadruplex subunits closer,thus producing Hemin/G quadruplex DNAzyme with simulated horseradish peroxidase catalytic activity on the electrode surface.The DNAzyme was used to catalyze electrochemical reduction of H₂O₂ to generate cathode current to achieve quantitative detection of ATP.The results show that the concentration of ATP has a good linear relationship with the cathode current in the range of 0.1～2μM,and the detection limit is 0.04μM.Finally,this method was applied to detect the ATP content in carp samples at different storage temperatures,and the assessment of carp freshness was achieved by establishing its correlation with TVC content.（2）An autocatalytic rolling circle amplification coupled catalytic hairpin self-assembly（ARCA-CHA）system was constructed for the detection of avian influenza virus H5N1 DNA.In this study,H5N1 DNA was used to open the recognition module H₃ and release the initiator chain T,which in turn activates the ARCA-CHA reaction.Using alternating cyclic activation between RCA and CHA to generate a significantly enhanced fluorescence resonance energy transfer（FRET）signal achieved high sensitivity and specificity of H5N1 DNA.The results showed that there was a good linear relationship between the concentration of H5N1 DNA and FRET signal values in the range of 0.1 n M to 10 n M,and the detection limit was 90 p M.Finally,the efficient detection of H5N1 DNA in fetal bovine serum（FBS）was successfully achieved using the established method.（3）A self-driven hybridization chain reaction（SHCR）system was developed for the detection of Kanamycin.In this study,Kanamycin was specifically bound to its aptamer to release the initiating chain I,which then activated the SHCR reaction.The cyclic self-activation of the SHCR reaction generated exponentially amplified FRET signals to achieve high sensitivity and specificity detection of Kanamycin.The experimental results show that the Kanamycin concentration in the range of 50 p M to 10 n M has a good linear relationship with the FRET signal value,and the detection limit is 40 p M.Compared with the conventional HCR system,the sensitivity is improved by about 5800 times.Finally,the efficient detection of Kanamycin in milk powder,milk,and honey samples was successfully achieved using the established method.