| Nucleic acid molecules play a role of storing and transmitting genetic information in the synthesis of protein,called "the code of life".Many diseases are strongly associated with abnormal expression of nucleic acid,and the relevant nucleic acid has been expressed even before obvious symptoms are present.Highly sensitive detection of nucleic acid markers can not only diagnose and treat diseases early,but also,in some cases,prevent the spread of diseases and improve public safety.Conventional nucleic acid detection technology still has some shortcomings,such as strict requirements on equipment,complex operation and high cost,so it is urgent to develop a more portable and low-cost nucleic acid detection platform.As a point-of care testing(POCT)platform,the lateral flow test strip(LFTS)can quickly get test results on-site with portable analysis equipment and matching reagents.With the advantages of rapid analysis,simple operation and low cost,it has great potential in the real time diagnosis of clinical diseases.However,due to the low expression of nucleic acid markers at the early stage of disease,it is urgent to develop the nucleic acid lateral flow test strip(NALFTS)with high sensitivity.The main methods to improve the sensitivity of NALFTS include signal label amplification strategy,nucleic acid amplification technology and nucleic acid signal amplification technology.Fluorescent nanoparticles(FNs)are synthesized by loading or wrapping large amounts of fluorescent dyes,quantum dots(QDs)and other fluorescent materials with high fluorescence intensity and stability.As signal labels,compared with colloidal gold nanoparticles(AuNPs)or QDs,it can effectively improve the sensitivity of NALFTS.In addition,compared with protease-dependent nucleic acid amplification techniques,enzyme-free nucleic acid signal amplification techniques don’t rely on environmentally sensitive and expensive proteases,greatly reducing the cost,simplifying the operation and improving the accuracy and reproducibility.Based on this,in this paper,combining enzyme-free nucleic acid signal amplification and signal label amplification strategy,NALFTS was constructed for highly sensitive detection of H1N1 influenza virus and miRNA-21.The main research contents are as follows:1.Entropy-driven amplification strategy-assisted lateral flow assay biosensor for ultrasensitive and convenient detection of nucleic acids.In this work,a fluorescent NALFTS based on enzyme-free isothermal entropydriven amplification strategy was firstly designed for rapid and sensitive detection of disease nucleic acid markers.Firstly,the substrate strand was composed of the report probe FNs-DNA,a biotin-modified linker strand and a by product strand.When the target nucleic acid is present,the entropy-driven amplification reaction is initiated and a large number of FNs-DNA are released.Then,FNs-DNA flowed along the NC membrane by capillarity and hybridized with the capture probe of test line,resulting in the accumulation of FNs and producing obvious fluorescent bands for the detection of target DNA.The detection limits for target DNA and H1N1-RNA were 1.43 pM and 2.02 pM,respectively.Entropy-driven amplification is driven by the entropy increase of the system,and the base pairs remain unchanged during the amplification process,thus greatly reducing the interference of complex environments.In addition,the use of double-stranded substrate probes,instead of hairpin probes,not only allows flexible sequence design to develop a common nucleic acid testing platform,but also increases the stability of the probes.Therefore,this biosensor shows great application potential in the real-time detection of nucleic acid at the early stage of diseases.2.Lateral flow test strip combined with enzyme-free cascade amplification strategy for convenient and sensitive detection of miRNA.In this work,based on enzyme-free cascade amplification technology,a fluorescent NALFTS was developed for the ultrasensitive detection of miRNA-21.The enzyme-free cascade amplification reaction consisted of HCR and DNAzyme in tandem,in which DNAzyme was initially enclosed in H2.When miRNA-21 is present,it triggers the cascade amplification reaction,and then releasing large amounts of DNAzyme to cut the substrate strand.The severed substrate strand could not be hybridized with the report probe FNs-DNA and then captured by the capture DNA,resulting in no fluorescent signal on the test line.However,when the target was not present,the substrate strand was hybridized with report probe FNs-DNA and capture DNA to form a sandwich structure on test line,where bright fluorescent signal appears.The target concentration can be detected by measuring the fluorescence signal intensity of the test line,and the detection limit is 0.79 pM.Based on the single step nucleic acid signal amplification technique,this work combines the multi-step cascade amplification technique with NALFTS to further improve the sensitivity and accuracy,and shows great potential in the real time diagnosis of diseases nucleic acid3.Dual-mode lateral flow test strip based on signal label amplification strategy for rapid and highly sensitive detection of miRNA.In this work,a double-mode NALFTS based on signal label amplification strategy was constructed for the highly sensitive detection of miRNA-21.FNs-DNA and AuNPs-DNA formed a reticular structure by complementary hybridization of report DNA 1 and report DNA 2,used as dual-mode amplified report probes.When miRNA21 was present,the dual-mode amplified report probes were hybridized with miRNA21 and captured by capture probes fixed on the test line.Finally,a large number of AuNPs and FNs were collected on the test line and simultaneously presented visual red bands and strong fluorescent bands.By measuring the fluorescence signal intensity,this method can detect a minimum concentration of 1.0 pM miRNA-21.By combining the FNs and AuNPs labels,not only realizing the effect of signal amplification,but also performing the capability of dual-mode detection,which further improves the sensitivity and application ability of NALFTS. |
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