Research And Application Of Fluorescence Biosensor Based On Signal Amplification Technology

With the development of biochemical analysis,the simple,rapid,and sensitive detection of biomolecules has great significance in medical diagnosis,food analysis,and other fields.However,the development of new signal amplification technology has become an important research direction for detection of target molecule that can’t be detected because of its low concentration.In this study,a series of novel biosensing assay systems were developed based on metal-enhanced fluorescence effect（MEF）of nanoparticles,rolling circle amplification（RCA）,hybridization chain reaction（HCR）,exonuclease amplification,and aggregation-induced emission（AIE）.Moreover,the proposed strategise exhibited an excellent specificity and sensitivity for the detection of bioactive small molecule,target DNA and metal ions.The thesis included the following chapters:（1）A fluorescent aptasensor was developed for highly specific and sensitive detection of adenosine triphosphate based on silver nanoparticle enhanced fluorescence and fluorescence resonance energy transfer（FRET）.Fluorophore-functionalized aptamers and quencher-carrying strands were immobilized on nanoparticles to form a AgNP-enhanced FRET sensor.The resulting sensor showed lower background fluorescence intensity in the duplex state due to the FRET effect between fluorophores and quenchers.In the presence of ATP,the quencher-carrying strands of the duplex were displaced leading to the disruption of the FRET effect.As a result,the fluorescent intensity was increased by the fluorophore-aptamer near the surface of AgNP.This method had good selectivity and had been successfully applied to the analysis of ATP in human serum samples.The method has potential application value in biological detection with wide application range,strong selectivity and convenient operation.（2）A fluorescent platform was designed for ultrasensitive DNA detection based on rolling circle amplification and hybridization chain reaction.Firstly,target DNA and padlock DNA were hybridized.Circular DNA was formed by using T4 ligase.The rolling circle amplification reaction was occurred with the help of Phi29 DNA polymerase,and amount of repeat single-stranded DNA was produced for initiating the hybridization chain reaction.This method had good selectivity and high sensitivity for the detection of the target DNA.（3）A novel method was developed for fluorescence detection of Hg²⁺based on nuclease cycle signal amplification technology and aggregation-induced emission.Thymine-thymine（T-T）mismatches in the toehold domains could serve as specific recognition elements for Hg²⁺binding.The formed T-Hg²⁺-T base pairs could initiate toehold-mediated strand displacement reaction.The duplex was digestion into two segments of single stranded DNA by by exonuclease III（Exo III）.One part was used for circulation to amplify the signal,while the other part was used for forming a triple helix with Substrate DNA labeled TPE-N₃（tetraphenylethylene derivative）.Once the triple helix structure formed,the fluorescence intensity was increased due to the partially restricted rotation of the phenylene rings.