The Design Of Ratiometric Fluorescent System And Its Application In The Detection Of Avian Influenza Virus DNA

Avian influenza is an acute respiratory infectious disease caused by the avian influenza virus.Since it was first discovered in 1878,avian influenza has forced tens of thousands of people health worldwide,seriously threatening people’s health and posing a massive threat to the world economy and public health.The occurrence of avian influenza is related to the nucleic acid of the avian influenza virus.Therefore,the detection of avian influenza virus DNA is of great significance.Fluorescent analysis methods have good application prospects due to their high sensitivity,low cost,and easy operation.However,most fluorescence analysis methods are based on the intensity change of a single signal,which are easily affected by external factors,such as instrument and environment,especially in a complex matrix.Ratiometric fluorescence analysis methods demand two sets of output signals,making the quantitative detection results more reliable.In view of this,the nucleic acid sequence of H1N1 DNA was used as the target to construct two different ratiometric fluorescent sensing systems in this thesis to achieve sensitive,accurate and rapid detection.The specific research content is as follows:（1）DNA functionalized red fluorescent quantum dots（DNA-QDs）and DNA-conjugated green fluorescent carboxyl fluorescein（FAM-DNA）were used as fluorescent groups,and the DNA functionalized gold nanoparticles（DNA-AuNPs）were used as a carrier to construct a ratiometric fluorescent sensor system.In this system,DNA-AuNPs and DNA-QDs were first assembled by complementary base pairing.Based on the strand displacement reaction and the blocking effect of the target DNA on the sticky end by introducing FAM-DNA and H1N1 DNA,respectively,a new type of AuNPs-DNA-QDs ratiometric fluorescence sensor system was constructed and successfully applied to detect H1N1 DNA via changing the ratio of the fluorescence signals of two different colors.Under the optimized experimental conditions,the method could detect H1N1 DNA within the concentration range of 2.5 n M-20 n M,and the detection limit was as low as 0.52 n M without amplification.In addition,the method was successfully applied to the spiked experiment in 1%of human serum,and the spiked recovery rate was between 96.67%and 102.43%.（2）Using the red fluorescence generated from the interaction of the nucleic acid molecule"light switch"[Ru（phen）₂（dppz-idzo）]²⁺with the dual-strand H1N1 DNA as the response signal,two-dimensional phenyl-doped carbon nitride nanosheets（PDCN）was used as the reference signal,and a ratiometric fluorescent sensor system was constructed to detect H1N1 DNA by changing the ratio of the fluorescence signals of two different colors.Under the optimized experimental conditions,this method could detect H1N1 DNA in the concentration range of 1 n M-30 n M within 30 min,and the detection limit was 0.27 n M.Compared with the previous system,this system had a wider detection range,higher sensitivity and shorter reaction time.In addition,the method was successfully applied to the spiked experiment in 1%of human serum,with the spiked recovery rate between 96.80%and 103.67%.Finally,this method could also be combined with smartphones to detect H1N1 DNA in the UV light box visually.Compared with the monochrome system without PDCN,the ratiometric fluorescent sensor system showed more obvious colors change and had good application prospects in point-of-care detection.