Study On The Construction And Bioimaging Analysis Of Ratiometric SERS Nanoprobes In Cellular Silent Region

Surface-enhanced Raman scattering(SERS)is a powerful and sensitive analytical technique,which has been widely used in chemistry,materials science,analytical science,surface science,biomedicine and other fields due to its advantages of low autofluorescence,resistance to photobleaching,lack of phototoxicity,narrow emission peak,multiplex detection,etc.Especially with the continuous improvement of instruments and technologies,SERS has attracted more and more attention in the research of complex biological systems.However,SERS also faces some urgent problems to be solved in practical application.For example,due to the random distribution of hot spots on the SERS substrate and the fact that only analytes located in the hot spots could contribute to the overall SERS signal.Therefore,it is still a great challenge for quantitative SERS detection.In addition,the Raman shift of Raman reporters in SERS nanotags or nanoprobes is mostly located in the fingerprint region(<1800 cm^-1),which is easy to overlap with the Raman peaks of endogenous species and difficult to distinguish,thus affecting the accuracy of the experimental results.In order to solve these problems,SERS technology is used as the analytical method in this dissertation,and utilizing Raman reporter or SERS substrate of Raman signal located in the cellular silent region(1800-2800 cm^-1)to fabricate different SERS nanoprobes for ratiometric SERS imaging analysis in cell.The specific research contents are as follows:(1)A ratiometric SERS nanoprobe(SWCNT/Ag/AuNPs/MPP)was developed for imaging hypoxic living cells or tissues.The hypoxia SERS nanoprobe was prepared by assembling azo-alkynes derivatives(4-((4-mercaptophenyl)diazenyl)phenyl 3-((4-(phenylethynyl)benzyl)thio)propanoate,MPP) on single-walled carbon nanotubes(SWCNTs)surface-functionalized with Ag/Aualloy nanoparticles(SWCNT/Ag/AuNPs).Under a normoxia condition,the two Raman scattering signal of MPP(2207 cm^-1)and SWCNTs(2578 cm^-1)will be shown and can be distinguished in the Raman spectra.Under a hypoxic condition,MPP preassembled on the surface of the SWCNT/Ag/AuNPs are reduced stepwise by various reductases and eventually removed from the surface of the SWCNT/Ag/AuNPs,resulting in the loss of characteristic alkyne Raman bands at 2207 cm^-1.But the band at 2578 cm^-1,attributed to the 2D-band of SWCNTs(internal standard),is not changed.Thus allowing the detection of different levels of hypoxia based on the ratiometric peak intensity(I₂₅₇₈/I₂₂₀₇)in vitro and in vivo.(2)Ag/Aualloy nanoparticles-coated single-walled carbon nanotubes-based for imaging analysis of intracellular deoxyribonuclease I(DNase I)activity was developed.SERS nanoprobe was prepared by wrapping hairpin DNA(HPDNA)on single-walled carbon nanotubes(SWCNTs)surface-functionalized with Ag/Aualloy nanoparticles by?-?stacking and intercalating methylene blue(MB)molecule into the double helix of HPDNA as the signal element.Intracellular DNase I was activated when the cells were treated with SERS nanoprobe and apoptotic agent of 2-phenethyl isothiocyanate(PEITC)in turn.The double helix of HPDNA immobilized on the surface of SWCNTs can be hydrolyzed into DNA fragments by DNase I,MB molecules release from HPDNA and eventually remove from the surface of the SWCN/Ag/AuNPs.As a result,the strong SERS signal of MB molecules at 1393 cm^-1decreases or disappears,whereas the band at 2575 cm^-1,attributed to the 2D-band of SWCNTs(internal standard),remains almost unchanged,enabling the determination of DNase I in living cells based on the ratiometric peak intensity(I₂₅₇₅/I₁₃₉₃).(3)Alkyne/ruthenium(?)complex(PBTD/Ru(?))and AuAgalloy nanoparticles(AuAgNPs)-based ratiometric SERS nanoprobe(PBTD/Ru(?)-AuAg@p-SiO₂)for in vitro and ex vivo tracking of carbon monoxide(CO)was developed.Based on the strong Raman enhancement property of AuAgNPs and good hydrophilicity and biocompatibility of silicon dioxide(SiO₂)material,porous SiO₂coated AuAgNPs(AuAa@P-SiO₂)were firstly synthesized,and then PBTD/Ru(?)was modified on the surface of AuAgNPs to achieve the construction of ratiometric SERS nanoprobe for CO detection.In the presence of CO,the displacement of the alkyne ligand by CO results in a decrease of the alkyne vibrations at 2206 cm^-1and an increase of the metal carbonyl complex signals at 2100 cm^-1.Both of these signals are located in the cellular silent region,where natural cellular components do not generate a Raman signal.The effective ratiometric detection of CO in real-time and interference-free SERS imaging of CO was achieved based on the ratiometric peak intensity(I2100/I2206).(4)4-ethynyl-phenol(EP)-based SERS nanotag(AgAu-EP@Au)for cell imaging analysis has been developed.The synthesis approach of AgAu-EP@Aunanotags was based on the utilization of silver nanoparticles(AgNPs)as seed for Audeposition by combination of galvanic replacement reaction between Agand Auprecursor and Auprecursor reduction using L-ascorbic acid(L-AA)in the presence of polyvinypyrrolidone(PVP)as a stabilizer.This SERS nanotag consists of a hollow AgAualloy core,Raman reporter EP with a specific Raman signal in the cellular silent region and the protective shell of Aushell.The investigation of the AgAu-EP@Aunanotag showed that the SERS nanotags had good signal reproducibility,structural stability and excellent anti-interference ability in complex environment.After the modification of 4-mercaptobenzenitrile(MBN)on the surface of AgAu-EP@Aunanotag,a novel ratiometric SERS nanoprobe(AgAu-EP@Au/MBN)was synthesized and applied to cell imaging analysis.The successful preparation and application of this ratiometric SERS nanoprobe indicated that the SERS nanotags could be applied to the detection of different analytes after simple modification,which further proved its good versatility.