Study On Construction Of High-sensitivity Biosensing Method Based On Functional Nucleic Acid Acids

Functional nucleic acids include aptamers,DNases,ribozymes,ribosome switches,and the like.As a functional molecule,functional nucleic acid is often combined with colorimetric,fluorescence,electrochemical and other analytical methods to construct biosensors.In this paper,functional nucleic acids are combined with fluorescence analysis methods to achieve sensitive and specific detection of SARS-Co V-2 virus markers,pathogens,and disease markers.The main work of this paper is as follows:(1)Based on the strategy of combining toehold activation reaction and fluorescence resonance energy transfer,a method for simultaneous detection of SARS-Co V-2 N gene fragment and S protein was established.This method uses a connector to connect the S protein aptamer complementary strand and N gene to form a joint toehold,and based on the joint toehold,a hairpin structure probe h DNA is designed.The hairpin structure probe is modified with carboxyfluorescein JOE and poly A,and poly A can be adsorbed on the nanometer Gold surfaces,based on the FRET effect,have fluorescence quenching.When the complementary strand of the S protein aptamer and the N gene exist at the same time,the hairpin structure probe is opened,the fluorophore is far away from the gold nanoparticles,and the fluorescence is recovered,thereby realizing the simultaneous detection of the S protein and the N gene.(2)In this study,a double-hairpin structure probe capable of discriminating single nucleotide variation(SNV)was designed,which could discriminate between perfect match(MT)and single base mismatch(WT).Taking advantage of the high-efficiency transcription of T7 RNA polymerase and the characteristics of double-hairpin structure probes to distinguish single nucleotide variations,the T7 promoter sequence was introduced into the double-hairpin DNA structure to achieve an exponential increase in sensitivity.Multicomponent DNase was then used to eliminate the interference caused by the single base mismatch sequence.Finally,the double-hairpin structure probe,T7 RNA polymerase and multi-component DNase were combined to establish a biosensing platform for the detection of single nucleotide variation(SNV)with high specificity.The results showed that the biotransducer could rapidly detect covid-19 mutant pseudovirus samples and methicillin-resistant Staphylococcus aureus,and the detection limit of covid-19 mutant pseudovirus samples was 0.96 copy/μL,which provided a high level of detection for pathogens.a very good platform.(3)The binding region and unzipping region of T7 promoter were split into T7-a and T7-b,and the effects of T7 promoter binding region and unzipping region and the combination of modified T7 promoter on transcription efficiency were discussed.The results showed that the binding region of the T7 promoter was the key to affecting the transcription efficiency,and the two parts of the split T7 promoter were hybridized with the template strand in an end-to-end manner to achieve the best transcription effect.Then,the T7 RNA polymerase transcription template strand is encoded as the complementary sequence of malachite green aptamer and the complementary sequence of Broccoli aptamer,respectively.Based on the transformation of T7promoter and the competition of target protein,aptamer and complementary strand,enterotoxin exists.B and Staphylococcus aureus,the RNA aptamers that bind to the fluorescent dyes malachite green and DFHBI-1T were transcribed,respectively,producing red and green fluorescence.A method for the detection of enterotoxin B and Staphylococcus aureus was established.This method The linear range for the detection of enterotoxin B was 0.8～90 pg/m L,and the detection limit was 0.1086 pg/m L;the linear range for the detection of Staphylococcus aureus was 6×10~2～6×10~4 cfu/m L.On this basis,the template chain was redesigned,and the detection of liver cancer markers alpha-fetoprotein(AFP)and human serum albumin(HSA)was realized by the same method.The detection limit of alpha-fetoprotein was 1.290 pg/m L;The limit of detection for human serum albumin was 45.90 pg/m L.The method can simultaneously detect multi-component samples,and has good application prospects in the detection of pathogens and disease markers.