Construction Of High-performance SERS Active Substrates And Their Application In Biosensing

Surface enhanced Raman scattering（SERS）has become one of the most promising trace analysis techniques in the field of biomedical and pharmaceutical identification due to its unique fingerprint characteristics,non-destructive property and high specificity.However,there are still several problems to be solved in the practical portable detection,such as low sensitivity,poor reproducibility and low sampling efficiency.In this paper,the functional SERS substrates with high sensitivity and good repeatability were constructed by preparing high-performance nanoparticles,selecting specific functional substrates,designing signal enhancement strategies,and realized trace analysis and accurate identification of biomedical samples.The main research contents of this paper are as follows:（1）In order to further improve the sensitivity and reproducibility of SERS immunoassay,a SERS-based coronavirus disease 2019（COVID-19）biosensor was developed for the ultrasensitive detection of severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）in untreated saliva in combination with the specific immunoreactivity of antibody antigen.The SERS-immune substrate was fabricated by a novel oil/water/oil（O/W/O）three-phase liquid-liquid interfaces self-assembly method,forming two layers of dense and uniform gold nanospheres（Au NSs）films to ensure the reproducibility of SERS immunoassay.The detection was performed by an immunoreaction between the SARS-Co V-2 spike antibody modified SERS-immune substrate,spike antigen protein and Raman reporter-labeled immuno-silver nanoparticles（Ag NPs）.The electromagnetic field coupling between the Au NSs in the immune substrate and the immune Ag NPs could further improve the sensitivity of the biosensor.This SERS-based biosensor was able to detect the SARS-Co V-2 spike protein at concentrations of 0.77 fg m L^-1in phosphate-buffered saline and 6.07 fg m L^-1in untreated saliva.The designed SERS-based biosensor exhibited excellent specificity and sensitivity for SARS-Co V-2 virus without any sample pretreatment,providing a potential method and choice for the early diagnosis of COVID-19.（2）In order to solve the problems of difficult sampling and easy contamination in complex environments,a capillary-based SERS substrate with high sampling efficiency and good reproducibility was designed and prepared for trace detection and identification of fentanyl.Through encapsulating gold trisoctahedra（Au TOH）in capillary tube for the first time,the SERS substrate was constructed by combining the superior SERS properties of Au TOH and the advantages of capillaries in SERS signal amplification,facile sample extraction,and portable trace analysis.The effects of the size and density of Au TOH on SERS performance were investigated by experiments and simulations,which showed that the maximum SERS enhancement was obtained for Au TOH with size of 75 nm when particle density reached 74.54countsμm^-2.The proposed SERS substrate possesses good reproducibility with the relative standard deviation（RSD）of less than 5%.Based on this advantage,the substrate was employed as a sensor for trace detection of fentanyl spiked in aqueous solution and serum samples at different concentrations with limit of detection（LOD）as low as 1.86 ng m L^-1and 40.63 ng m L^-1,respectively.In addition,we also validated the feasibility of the designed sensor for accurate identification of trace fentanyl adulterated in heroin at mass concentration down to 0.1%（10 ng in 10μg total）,which facilitates portable tracking analysis.（3）In order to further enhance the application of field portable analysis,a wearable and highly sensitive glove-based SERS substrate was constructed for the field identification of tramadol and midazolam and the precise identification of methamphetamine powder.The glove-based SERS substrate was constructed by integrating the flexible adhesive tape substrate assembled with Au TOH and wearable gloves,and combining the uniform and dense polystyrene microsphere opal photonic crystals to further enhance the Raman signal.Compared with the conventional press-peel transfer method,the direct homogeneous assembly of Au TOH onto 3 M adhesive tape by the interface self-assembly method effectively improves the signal reproducibility.The effect of the number of Au TOH assembly layers on SERS performance was investigated to obtain the optimal detection sensitivity.The prepared adhesive tape-based substrate has good flexibility,and different degrees of bending and stretching do not significantly affect the repeatability of the glove-based SERS substrate,with RSDs less than 6%after bending and stretching.The glove-based SERS substrate was employed as a sensor,incorporating its advantages in efficient sample extraction and portable analysis to achieve trace detection of tramadol and midazolam in serum with LODs as low as69.19 ng m L^-1and 35.03 ng m L^-1,respectively.In addition,the prepared biosensor can accurately identify methamphetamine powder samples in complex binary mixtures.This work provides feasible scheme and validation basis for the design and preparation of easy-to-use wearable SERS sensors.In summary,this paper have developed three functional SERS substrates with high sensitivity and reproducibility to address the practical problems existing in field trace analysis of biopharmaceuticals by selecting specific substrates and designing signal enhancement strategies,realizing the portable detection with high specificity and reproducibility.The development of these novel SERS biosensors provides new ideas and perspectives for the research of biological portable assays.