The Application Of Isothermal Nucleic Acid And Label-free Fluorescent Signal Amplification Strategy In The Field Of The Gene Detection

The detection of the specific nucleotide or gene sequence has important application value in clinical diagnosis,gene mutation,and biological defense.In recent years,researchers have been working to develop more sensitive and simpler biosensor technology for the detection of specific nucleic acids.The fluorescence biosensor has become one of the important research directions because of the characteristics of simplicity,good selectivity and fast response.However,most fluorescence biosensor technologies require labelling and nanomaterial to achieve signal amplification.Therefore,three types of the fluorescence biosensors were designed and developed with label and nanomaterial-free technology for signal amplification,and the feasibility,experimental parameters,and analytical performance of the biosensor were investigated in details in the research,which provided valuable references for developing the fluorescence biosensors with high sensitivity,simplicity,and cost-effectiveness.The main results were as follows:1.A fluorescence biosensor for ultrasensitive and simple detection of the mecA gene was proposed by using an exonuclease III(Exo III)-assisted cascade signal amplification strategy.In the system,the hairpin probe(HP)acted as the target recognition element and the signal reporter.Without the mecA gene,the HP cannot be digested by Exo III,as the G-rich sequences are blocked in the stem of the HP.In the presence of the mecA gene,the hybridization of the mecA gene with the 3’ end-extruding HP probe triggers the digestion reaction of Exo III,liberating the mecA gene and the mecA gene analogue.Both the released mecA gene and the mecA gene analogue can hybridize with other HP and activate another round of the cleavage reaction.Consequently,the released free G-quadruplex is“lit up” by N-methylmesoporphyrin IX(NMM),displaying a dramatically enhanced fluorescence intensity.This sensing platform showed a high sensitivity towards the mecA gene with a detection limit as low as 2.4 fM without any labelling,immobilization,or washing steps.The designed sensing system also exhibits excellent selectivity for the mecA gene in the presence of other interfering DNA sequences.Furthermore,the presented biosensor is robust and has been successfully applied for the detection of the mec A gene in a real food sample milk with satisfactory results.2.A fluorescence biosensor for simple detection of the HIV-1 gene was proposed by using the toehold-mediated strand displacement reactions(TMSDRs)-assisted cascade signal amplification strategy.The hybridization of the HIV-1 gene with the LS of the SP changes its stability and leads to the exposure of a new toehold.The FS binds to the new toehold and liberates a G-quadruplex and the HIV-1 gene through the TMSDRs.Therefore,the HIV-1 gene can be cyclically reuse to liberate many G-quadruplex,which can interact with NMM to generate dramatically increased fluorescence signal for the target detection.This sensing platform showed a high sensitivity towards the HIV-1 gene with a detection limit as low as 1.9 pM without any labelling,immobilization,or washing steps.The designed sensing system also exhibits an excellent selectivity for the HIV-1 gene compared with other interference DNA sequences.Furthermore,the presented biosensor is robust and has been successfully applied for the detection of the HIV-1 gene in a real biological sample with satisfactory results,suggesting that this method is promising for simple and early clinical diagnosis of the HIV infection3.A fluorescence biosensor was proposed for simple detection of the Kras gene by using the three-way DNA junction-driven catalyzed hairpin assembly(CHA)strategy.In this system,a three-way DNA junction probe(JP)and two hairpin probes(H1 and H2)were designed.The S1 and the S2 can activate the CHA when they are linked together.The S1 and S2 can co-recognize the target Kras gene to form a JP,which make the toehold and branch-migration regions.The hybridization of the JP with the terminal toehold region of the H1 through toehold strand displacement reaction(TSDR)leads to the exposure of a new toehold.Upon binding to the new toehold of the H1,the H2 displace the hybridized JP and expose the G-quarduplex through the second TSDR to active the DNA active.Thus,the JP can be cyclically reuse to liberate many G-quadruplex,which can interact with NMM to generate dramatically increased fluorescence signal for the target detection.This sensing platform showed a high sensitivity towards the Kras wild type with a detection limit as low as 2.7 fM without any labelling,immobilization,or washing steps.The designed sensing system also exhibits an excellent selectivity for the Kras wild type compared with other interference DNA sequences.Furthermore,the presented biosensor is robust and has been successfully applied for the detection of the Kras wild type in a real biological sample with satisfactory results,suggesting that this method is promising for simple and early clinical diagnosis of the genetic diseases.