Study Of Novel Au/Ag Alloy Nanoparticle-based SERS Nanoprobes For Biomedical Analysis

Surface-enhanced Raman scattering spectroscopy?SERS?not only provides molecular vibration information,but also has been widely used in biomedical imaging,biosensing and disease diagnosis because of its narrow scattering peaks for multiplex analysis.Especially in recent years,the development of colloidal plasma metal nanomaterials has attracted more and more attention in the application of SERS technology in the analysis and detection of complex biological systems.However,existing SERS detection still face many challenges in biomedical imaging and biosensing.First,conventional SERS substrates are generally made of gold or silver nanomaterials,while the SERS enhancement effect of the gold nanomaterials is not ideal.Although the silver nanomaterials have better SERS enhancement,they have poor chemical stability and high biological toxicity.Secondly,the Raman reporters commonly used in biological system detection have multiple Raman peaks in the fingerprint region(<1800 cm^-1).These Raman peaks easily overlap with the Raman peak of endogenous substances,which will seriously affect the analysis and detection.In addition,existing SERS detection methods are generally more concerned with the detection of a single target,and few studies have devoted to the development of a convenient and reliable SERS sensing array for simultaneous analysis and detection of various disease markers.In this thesis,SERS technology was used as the main analytical method.Firstly,AuAg alloy nanoparticles with excellent SERS enhancement properties were synthesized,and then combined with cell silent region Raman reporter and sensor array strategy.A series of new SERS sensing methods for biomedical imaging and disease marker detection were constructed.The details are as follows:?1?Alkyne-DNA-functionalizedAu/Agalloynanoparticles?Alkyne-DNA-AuAgNPs?for ratiometric SERS imaging assay of endonuclease activity in live cells.Au/Ag allloy nanoparticles?AuAgNPs?were synthesized as SERS substrates,which not only have chemical stability and biocompatibility similar to gold,but also have plasma-enhancing properties similar to silver.Functionalization of AuAgNPs by using 4-thiophenylacetylene?MPAE?and single-stranded DNA?ssDNA?labeled 3-??4-phenylethynyl?benzylthio?propionic acid?PEB?.Alkyne as Raman reporter avoiding the interference of complex components on the SERS signal.In the presence of endonucleases,ssDNA is cleaved,PEB molecules are released from the surface of the particles,the SERS signal(2215 cm^-1)of the alkyne group in PEB is decreased,and the SERS signal(1983 cm^-1)of the alkyne group in MPAE remains unchanged.Highly sensitive,non-interfering analysis of endonucleases in vitro and in vivo was achieved based on ratio peak intensity I₁₉₈₃/I₂₂₁₅.?2?Porous SiO₂-coated Au/Ag alloy nanoparticles?AuAg@p-SiO₂NPs?for the alkyne-mediated ratiometric SERS imaging analysis of hydrogen peroxide?H₂O₂?in live cells.An ultra-thin porous SiO₂ shell-coated Au/Ag alloy nanoparticle?AuAg@p-SiO₂NPs?was prepared.This novel SERS substrate has the excellent properties of AuAgNPs,the ultra-thin porous SiO₂ shell avoiding particle agglomeration while eliminating the use of traditional organic capping ligands,enabled the particles with clean surface and highly available hot spots.The AuAg@p-SiO₂NPs functionalized with 4-mercaptophenylboronic acid?MPBA?and4-mercaptophenylacetylene?MPAE?,and then incubated with dopamine?DA?as bridging molecules to incorporate 3-??4-phenylethynyl?benzylthio?propanoic acid?PEB?to the surface of AuAgNPs.In the presence of H₂O₂,the borate is converted to phenol,PEB derivatives is released from the surface of AuAgNPs,the SERS signal at2214 cm^-1 is significantly reduced,and SERS signal at 1986 cm^-1 remains unchanged.The quantitative analysis of H₂O₂ and interference-free Raman imaging of exogenous and endogenous H₂O₂ in living cells were achieved based on the ratio peak intensity I1986/I2214.?3?Nitrile-functionalized Au/Ag alloy nanoprobe and DNA hydrogel-based SERS sensor array for miRNAs-marked cancer screening.The target miRNA-responsive DNA hydrogel was used as a switch for the SERS sensing detection channel.In the presence of the target miRNA,the activity of the MNAzyme is restored,the substrate linkage chain is broken in the DNA hydrogel,the hydrogel is disintegrated.Polypeptide-biotin?Bio-PP?and 4-mercaptobenzonitrile?MPBN?functionalized AuAgNPs will pass through the damaged DNA hydrogel and combined with Bio-PP/SA?polypeptide-biotin/streptavidin system?,the SERS sensor produces strong Raman signal for miRNA quantification.Moreover,by simply adjusting the recognition sequence of DNA hydrogel in the SERS sensing array detection channel,simultaneous detection of 9 miRNAs can be achieved,and it has broad application prospects in miRNA-marked cancer screening.?4?Nitrile-functionalized Au/Ag alloy nanoprobe and target catalyzed DNA hairpin self-assembly based SERS sensing array for simultaneous detection of multiple miRNAs.The SERS sensing wells were modified with the hairpin 1corresponding to the target miRNA.AuAgNPs were modified by the hairpin 2corresponding to miRNA and the Raman reporter molecule 4-mercaptobenzonitrile?MPBN?,formed SERS tags.In the presence of the target miRNA,the catalytic hairpin self-assembly reaction?CHA?is initiated,and the hairpin 1/hairpin 2 hybrid duplex is formed.The miRNA is released to continue to initiate the CHA reaction,and the signal is amplified cyclically.The SERS tags were coupled to the SERS sensing well,strong SERS signals were detected and enabled highly sensitive quantification of the target miRNA.In addition,this method is combined with SERS arrays and successfully applied to the detection of multiple miRNAs.The highly sensitive determination of endogenous miRNAs in RNA extracts of MCF-7 cells,HepG2 cells and L02 cells is of great significance for the clinical diagnosis of miRNA-related diseases.?5?DNA hydrogel and portable glucometer readout based biosensing array for miRNA identification and quantitative detection analysis.The amylase encapsulated in the DNA hydrogel is separated from the amylose in the external solution.After the target miRNA is added,the MNAzyme activity is restored,and the substrate cleavage reaction in the DNA hydrogel is cyclically,resulting in the disintegration of the hydrogel.The amylase is released into the solution to catalyze the hydrolysis of amylose,producing a large amount of glucose and enhancing the signal of portable glucometer?PGM?.A portable detection of the miRNA via the PGM signal is implemented.In addition,through the simple and rational design of the target recognition sequence,the DNA hydrogel sensing platform has been successfully applied to the detection of various miRNAs,achieving the detection of endogenous miRNAs in cellular RNA extracts from HeLa cells,HepG2 cells,MCF-7 cells and L02cells.