| Raman spectroscopy is a noninvasive strategy and molecular recognition technology,which can provide the intrinsic molecular fingerprint information of analytes.However,the Raman scattering cross section of most analytes are very small(10-30?10-25 cm2),and this inherent defect directly leads to the insensitivity of the Raman signal.Surface-enhanced Raman Scattering Spectroscopy(SERS)technology can effectively enhance the Raman signal of molecular adsorbed on the substrate surface,and the enhancement effect can be magnified by 1014?1015 times.So that the sensitivity can down to"single molecule"recognition level.Therefore,SERS technology has been widely used in materials science,environmental monitoring,food safety and even show outstanding advantages in life samples(such as DNA,proteins,viruses,bacteria,living organisms,etc).It has become a crucial tool for single molecule and single cell analysis.The development of SERS mainly revolves around how to design SERS substrates with superior performance to improve the detection reproducibility and sensitivity.Conventional SERS substrates more or less show disadvantages such as poor uniformity,poor reproducibility and disordered arrangement.Therefore,most conventional SERS substrates cannot generate repeatable and stable signals.In addition,SERS is a surface phenomenon and shows near-field effect,only when the analyte molecule is close to the surface of the substrate,it shows good enhancement effect.When the analyte molecule locates far from the substrate surface,the SERS signal become weak or even disappears.This is very unfavorable for the detection of biological macromolecules.This thesis aims to overcome the limitations(signal instability and near-field effects)of conventional SERS substrate.It is a meaningful and fundamental issue to explore.Based on these challenges,this paper designed and fabricated various two-dimensional ordered nanoarrays as SERS substrates through different methods,then using these substrates as sensors to detect different biomarkers.We obtained good SERS detection results.The specific research contents and results are as follows:1.This chapter designed and fabricated a periodical gold nanohole array(NHA)as SERS substate to detect DNA methylation.This substrate can generate controllable"hot spots"and improve the detection reproducibility.In addition,the NHA shows tunable surface plasmon resonance(SPR)property,we can optimize the SPR peak to the region where we are interested through adjusting the structural parameters.The intensity changes of the ring breathing vibrational band of cytosine(785 cm-1)is regarded as a marker of Raman detection of DNA methylation.We used finite-difference time-domain(FDTD)simulation to tune the SPR peak of substrate to 785cm-1 to match well with the vibrational bands of the analyte,which dramatically enhances the detection sensitivity.The designed PGNA was fabricated via electron beam lithography(EBL)technique and its SERS activity was evaluated by Raman reporters.We further used this method for DNA methylation detection,and related characterization showed that the detection limit as low as 1%.2.The dynamic balance between DNA methylation and demethylation is closely related to the biological development and diseases.Therefore,the development of highly sensitive DNA epigenetic modification(including 5-methylcytosine,5-hydroxymethylcytosine,5-formylcytosine and 5-carboxylcytosine)methods is particularly important.This chapter chose the NHA substrate with the highest SERS enhancement factor to detect different epigenetic modifications.We can identify the four different epigenetic modification based on the difference of the characteristic peaks of the Raman spectra.This method was then used for monitoring the dynamic DNA epigenetic alterations in the TET protein-mediated oxidation process.It proved that the reaction is a three-step continuous reaction,and the rate constants of each step are 0.6,0.25 and 0.15 min-1.3.Tuning the SPR of nanomaterials to the near infrared(NIR)region and thus achieving wavelength-matching between plasmon and NIR excitation is one elementary way to avoid native autofluorescence from the biological samples as well as generate SERS enhancement.Herein,we engineered a gold triangle-shaped nanoholes array(NHA)with more intense and wavelength-matched SPR at 785 nm as the SERS-active substrate.The array was fabricated through master-assisted replication method.Then,we used this NHA substrate as sensing platform for the ultrasensitive detection of MTase activity combine with hybridization chain reaction(HCR).Our method achieved a broad linear with low detection limit.It has a good clinical application prospect.4.Conventional SERS substrates show near-field effect.Driven by this challenge,this chapter reported a novel SERS substrate.We sandwiched a 20-nm NHA in a symmetrical dielectric environment with close refractive index and formed a kind of"dielectric layer+metal film+dielectric layer"symmetrical structure.The electromagnetic(EM)field on both sides of the metal film may couple with each other,then enhanced the EM intensity and extended the EM field strength,giving the substrate long-range surface enhanced Raman effect.The long-range effect of the LR-SERS substrate was experimentally demonstrated using Raman reporter-modified straight DNA as rulers.The EF at 22 nm above the LR-SERS substrate surface remains 4.1×105.Using this advantage,the LR-SERS substrate was used as a sensing platform to detect mi RNA coupling of HCR signal amplification strategy.The developed sensor achieved a broad detection linear range with a low detection limit.The SERS effect has been greatly improved.5.A large-area densely arranged gold hexagonal nanoplate film was formed through Langmuir-Blodgett(LB)method,and then sandwiched between a symmetrical dielectric environment with close refractive index as a long-range SERS substrate.The long-range performance of this substrate was theoretically predicted through FDTD simulation,and then confirmed experimentally.We used this substrate to detect protein S,a characteristic marker of new coronavirus disease.The detection method showed high accuracy and sensitivity,and the detection limit was as low as 1?10-10 g/m L.This method can effectively distinguish healthy human saliva and infected human saliva containing protein S through principal component analysis.This detection method provides a new idea for the clinical detection of new coronavirus infection. |
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