Construction Of Biosensor Based On High-efficient Raman Substrate And Nucleic Acid Amplification Strategy

Surface-enhanced Raman scattering（SERS）technique has been widely applied in material chemistry,biomedicine,molecular imaging,archaeology and many other fields with the advantages of high sensitivity,resistance to photobleaching,long-term monitoring and quick nondestructive detection.In addition,the SERS-biosensor shows wide application prospects in disease biomarker analysis.The substrate with excellent Raman performance can significantly improve the biosensor sensitivity for quantitative detection of trace biological samples.Moreover,the effective signal amplification strategies were significant for improving the analysis performance of the sensor such as hybridization chain reaction（HCR）,strand displacement amplification（SDA）and catalytic hairpin assembly（CHA）.Therefore,three kinds of biosensors were constructed with a variety of efficient SERS substrate and nucleic acid amplification strategy for high-selective detection of uracil DNA glycolase.The main works are as follows:1.SiO₂@Ni@C and Au nanocages as SERS substrate with structure switching strategy for sensitive uracil DNA glycolase detectionSurface enhanced Raman scattering（SERS）were widely used for the detection of proteins and other biological molecules with the advantage of high sensitivity and rapid nondestructive.However,Raman signals of these molecules were weak,so direct detection would lead to low sensitivity.Compared with the gold nanospheres,there were more SERS hot spots at the edge and vertex of the inner cavity of the gold nanocages（Au NCs）,which could significantly enhance the Raman signal.Therefore,in this work,we employed DNA structure switching strategy for SERS biosensor combining Au NCs as SERS platform to analyze trace amounts of uracil DNA glycolase（UDG）.First,we utilized NH₂-functionalized SiO₂@Ni@C nanospheres to immobilize hairpin DNA（R）.Next,Raman label toluidine blue（TB）and probe DNA（S₁）were attached to Au NCs through Au-SH bond,forming TB-Au NCs-S₁ complex.In the presence of target UDG,they could specifically recognize and hydrolyze the uracil bases,generating apyrimidinic（AP）sites.In this way,the structure switching process were initiated and hairpin DNA（R）were reconfigured to hairpin DNA（R′）.The generated R′could grab S₁ to form complementary DNA double chain,leading TB-Au NCs-S₁ coupled with SiO₂@Ni@C-NH₂.With the 633 nm laser excitation,we could obtain a strong Raman signal.By this simple protocol,the SERS assay could achieve high selective detection of UDG in a concentration range from 5×10^-5 U m L^-1 to1 U m L^-1 without other enzyme assisted.Therefore,the findings indicated that this SERS-based assay may open new potential in protein detection.2.Ag/TiO2 network SERS substrate contributed by charge transfer and electromagnetic effect for uracil DNA glycolase detectionThe common noble metal such as gold and silver were used as Raman enhancement substrates.The single electromagnetic enhancement effect would limit the improvement of the Raman sensors sensitivity.Semiconductor have good biocompatibility.In addition,these materials can significantly improve the Raman signal through chemical enhancement effect.Metal/semiconductor composites can enhance the Raman signal of signal molecules through the synergistic effect of electromagnetic enhancement and chemical enhancement,thus improving the sensitivity of immunsensor.In this work,a network SERS substrate was constructed with Ag nanocubes（Ag NCs）and TiO₂ nanospheres linked by mutual amplification DNA probe for uracil DNA glycolase（UDG）detection.Impressively,the introduction of TiO₂semiconductor could greatly enhance the Raman intensity.The superior performance of this net substrate contributed to the the synergistic effect of surface plasmon resonance（SPR）of Ag NCs and charge transfer（CT）of semiconductor TiO₂.Moreover,the Raman reporter methylene blue（MB）was away from the Ag substrate through hairpin DNA structure switching,which could decrease the background signal.Based on this assay,we successfully constructed the SERS biosensor for UDG detection with a low detection limit of 1.2×10^-5 U m L^-1 and the concentration range was 7.5×10^-5 U m L^-1 to0.1 U m L^-1.Furthermore,this proposed assay offered a reliable strategy to design new SERS substrate combining metal and semiconductor with final applications in analysis of biomolecule and disease diagnosis.3.High-efficient Raman enhanced substrate Fe₃O₄@P-4VP@Au NSs combined with DNA cascade recycling amplification strategy for construction of uracil DNA glycolase biosensorNucleic acid amplification technology which could significantly improve the sensitivity of Raman sensors would cause nucleic waste.In addition,the low efficiency of single cycle strategy made it difficult to achieve trace biomolecule detection.In this work,Fe₃O₄@P-4VP@Au NSs was ultilized in a SERS biosensor with use of cascaded recycling amplifications strategy for detection of UDG.Compared with gold nanosphere,Fe₃O₄@P-4VP@Au NSs substrate could greatly enhance the Raman intensity which attributed to the strong electric fields comes from narrow nanogaps between Au nanoparticles.The experimental operation would be simplified through magnetic separation.In this process,the substrate material could be enriched to achieve better Raman enhancement.Moreover,a large number of trigger DNA could be obtained for the introduction of the cascaded recycling amplifications strategy.Moreover,this strategy could avoid the waste of nucleic chain.By this simple protocol,the SERS assay could achieve sensitive UDG detection in a concentration range from2.5×10^-5 U m L^-1 to 10 U m L^-1 with a detection limit down to 6.9×10^-5 U m L^-1.Therefore,the findings indicated that this SERS-based assay may open new potential in clinical applications.