| Surface-enhanced Raman scattering(SERS)is a powerful spectral analysis method that combines traditional Raman scattering with emerging micro-nano science and technology.It can detect fingerprint spectral information of analyte molecules with extremely high sensitivity.SERS has made breakthrough progress in various research fields in recent years.However,the application of SERS in various industries is still limited by the problems of poor uniformity,poor stability,insufficient sensitivity of the SERS active substrate,and the inability to achieve higher economic benefits in scientific research and practical applications.For example,the most widely used solid-state SERS substrates prepared by the metal nanoparticle film formation method.That is,metal nanoparticles are added dropwise to the surface of substrates such as silicon wafers,glass sheets,or aluminum sheets,and dried to form a film on the integrated SERS substrates.The overall structure size of such an integrated substrate is much larger than the size of the detection unit,and the preparation process is often susceptible to environmental factors,resulting in"coffee ring effect"and the like,resulting in generally poor substrate uniformity and stability;Moreover,such a traditional preparation method has a small batch size and a single functional structure,which severely limits the potential efficacy of SERS substrates and large-area high-throughput applications.Therefore,it is necessary to develop new SERS substrate preparation strategies.With the advancement of micro-nano technology,in order to improve the basic performance of SERS substrates,researchers introduced arrayed micro-nano structures into SERS substrate preparation.For example,the SERS substrate is prepared by directly forming the arrayed structure on the macro substrate through photolithography,template assisting,and the like.Although these substrates can significantly improve the uniformity and stability of the SERS signal,they rely on high-precision instruments.At the same time,the size of the array unit is much smaller than the detection unit,so that the arrayed structure does not match the actual detection,which also limits its application value.With the innovation of interface assembly technology,SERS substrates with excellent performance can still be obtained through a simple self-assembly process.For example,the nano-structure unit derived from the metal nano-particle sol is self-assembled on the surface of a large-scale substrate by chemical or physical methods,and the micro-features of the nano material and the macro-structure of the substrate surface are synthesized to prepare a highly active and practical SERS substrate.Optimizing the self-assembly process can effectively improve the SERS performance of the substrate such as uniformity and stability;and regulating the surface structure of the substrate can develop potential applications for the substrate.Meanwhile,soft photolithography based on polydimethylsiloxane(PDMS)has attracted widespread attention in surface structure regulation and applications due to its flexibility,transparency,and biocompatibility.Therefore,this thesis proposes an arrayed SERS substrate preparation strategy based on nanoparticle immersion and adsorption self-assembly.This arrayed SERS substrate realizes the size coupling of array micropores and laser spots.It can be used in conjunction with a motorized stage to highlight the surface microporous structure and to achieve arrayed qualitative and quantitative detection of actual biological samples such as urease concentration in human saliva.First,this paper combines the requirements of SERS active substrate,self-assembly,and the characteristics of metal nanoparticle sols to prepare silver nanostars(Ag NSs),which are highly branched multi-tip star-shaped nanoparticles.By analyzing the ultraviolet-visible absorption spectrum of the Ag NSs nanoparticle solution,it shows the broadband local surface plasmon resonance effect from blue light to near-infrared light,which proves that it is expected to become a SERS substrate active material working under wide-wavelength laser excitation;and based on the solution state,the stability of Ag NSs solution prepared with weak surfactant was investigated.The FDTD method was used to simulate the local electromagnetic field of star-shaped nanoparticles.It was found that the electromagnetic field was significantly enhanced and the number of local plasmon resonance locations increased.It was proved that Ag NSs will produce more and stronger hot spot.By detecting the Raman signal of 4-mercaptobenzoic acid(4-MBA),the significant SERS enhancement effect of Ag NSs was confirmed,and the enhancement factor(EF)was as high as 1.11*10~6.Then,combining the characteristics of practical applications,self-assembly,and PDMS substrates,PDMS substrates with regular hexagonal microwell arrays with different sizes were designed and prepared.The size coupling of microwells with different sizes and laser spots was explored during Raman detection.Based on the microscopic image,gray analysis,coating rate calculation and SERS test results,four main experimental factors affecting the preparation of the self-assembled arrayed microwells SERS substrate were analyzed.The optimal experimental conditions were determined as 200?m microwells size,Ag NSs solvent with 80%ethanol,Ag NSs concentration of 1.0 mg/m L,and 4 h soaking time.Under these conditions,the SERS substrate obtained by immersion and adsorption self-assembly achieves the best adsorption efficiency and adsorption uniformity,and the obtained SERS spectrum reaches the maximum signal intensity and the minimum relative standard deviation(RSD).The results of SERS analysis showed that the RSD of the integrated intensity of the4-MBA SERS spectrum measured at the characteristic peaks of 1076 cm-1 and 1585 cm-1on a single substrate were 7.52%and 4.38%,respectively;The RSD of SERS spectra at 1076cm-1 and 1585 cm-1 measured on different substrates were 4.42%and 4.49%,respectively.The integrated intensity at 1076 cm-1 and 1585 cm-1 measured by the same substrate after about 2 weeks also decreased by only about 17%.Compared with several traditional SERS substrates,the excellent uniformity,reproducibility and stability of the array microwells SERS substrate are confirmed again.Finally,this paper proposes to use the arrayed microwells SERS substrate as the detection platform,and based on the catalytic reaction of urease and urea,the portable peak Raman spectrometer is used to detect the characteristic peak integrated intensity of urea at 1003 cm-1.When the urea concentration in the reaction solution is constant,the urease concentration is detected,and the correlation between the urease concentration and the change in the integrated intensity of the characteristic peak of urea SERS is established to realize the quantitative detection of urease.As a result,a standard linear correlation was obtained in the range of 0.5-4?g/m L,and the correlation coefficient R~2 was 0.995.In addition,the urease concentration in actual saliva samples of different humans can be measured with a recovery rate of more than 95%.Such an automated detection platform highlights the advantages of the arrayed microwells SERS substrate.It integrates a portable Raman spectrometer and a motorized stage,which not only achieves stable and accurate determination of urease concentration in saliva,but also simplifies the test process,saving time and effort.This article proposes an arrayed microwells SERS substrate preparation strategy based on immersion adsorption self-assembly.By exploring the experimental factors affecting the self-assembly effect,the optimized experimental conditions are used to achieve high uniformity,reproducibility and stability of SERS substrate preparation.And actively explore its practical application in the detection of human saliva samples.Such an arrayed substrate design strategy based on the size coupling of microwell and laser spot makes the substrate accurate in actual SERS detection,high signal strength,good uniformity and stability,etc.And it is expected to become a potential detection tool in the field of biological sample automation and high-throughput detection. |
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