Surface-enhanced Raman Scattering Based-detection Methods For Amino Acids And Peptides

As an ultrasensitive and selective analytical technique,surface-enhanced Raman scattering（SERS）has attracted more and more attentions from the field of life sciences and been widely used in areas such as spectroelectrochemistry,catalysis,biomolecules detection and materials.We proposed several detection methods for amino acids and peptides on the basis of SERS advantages including rapidity,ultrasensitivity,in situ and nondestructive detection.It can be divided into two parts.On one hand,combining traditional chemical derivatization with SERS technique,target molecules-amino acids and peptides,were derived and quantified indirectly by measuring SERS signals of derivative reactants or products with large Raman cross section.The approach was applied to ultrasensitive determination in practical samples successfully.On the other hand,we prepared a dual-fuctional SERS active substrate-Ag@Ti O2 for rapid enrichment and SERS detection of phosphoamino acids.The method will provide a new way for the exploration of protein phosphorylation.The major contributions of this work are as follows:（1）Histidine（His）and tyrosine（Tyr）determination SERRS assay based on azo coupling with p-aminothiophenol（PATP）A simple and highly sensitive surface-enhanced resonance Raman scattering（SERRS）-based approach coupled with azo coupling reaction has been put forward for quantitative analysis of His and Tyr.We chose PATP which was reduced into diazonium ions by HNO2 under acidic condition.The obtained diazonium ions which act as electrophilic reagent react with the target molecule-His and Tyr under alkaline condition,producing azo compounds with the structure of-N=N-.The SERRS signals were obtained by mixing the azo solution with prepared SERS active substrate-silver nanoparticles（Ag NPs）in a certain ratio.The quantitative analysis for His and Tyr was achieved through the correlation relationship between the intensity of characteristic peaks and log concentration of amino acids.The whole experiment process is simple without any separation or concentration steps and fast because the azo reaction can be completed in 1 min.Further,we can differentiate His and Tyr from their mixture due to their structural difference.The proposed method has been applied to the quantitative detection of His and Tyr in real biological samples.（2）SERRS assay based on azo coupling with sulfanilic acid for ultrasensitive detection of His,Tyr and thyrotropin-releasing hormone（TRH）Sulfanilic acid was chosen as azo component for His and Tyr in order to solve the problem about interference of blank sample.The results indicate that the interference has been successfully removed at the cost of sensitivity in a certain degree.We applied this SERRS-based method for TRH determination.Ultrahigh sensitivity of this approach originates from two factors: changing TRH to an azo compound and the SERRS effect at 532 nm excitation wavelength.The lowest detectable concentration of TRH was found to be as low as 1 pg m L-1,which is 10-fold lower than the lowest normal reference value in human serum reported in previous literature.In comparison with conventional TRH identification and quantification methodologies,radioimmunoassay（RIA）and subsequent various hyphenated techniques,the main advantages of this study include（1）simplicity and rapidity: the whole experiment process can be prepared within 2 minutes;（2）ultrasensitivity and selectivity: the detection limit is the lowest until now.The SERRS spectrum exhibits unique and abundant vibrational information specific to the TRH-derived azo dye,ensuring the selectivity for TRH analysis;（3）practicability and reliability: the SERRS-based assay coupled with the azo reaction was successfully applied to the detection of TRH in human serum and the results we obtained coincided with the ELISA results basically.The approach described here provides a rapid,simple and ultrasensitive platform for TRH quantitative analysis in practical applications.（3）SERS-based approach combined with ninhydrin derivatization for total amino acids quantificationwe propose a simple and sensitive SERS method for the determination of total amino acids without any separation steps.The procedure described here is based on the ninhydrin derivatization reaction with amino acids,followed by SERS measurements of the producing mixtures loaded on a self-assembled Ag NPs film.A good linear correlation between excess ninhydrin SERS signals and the log values of the total amino acids concentrations is obtained;the detection limit of the method is 4.3 ×10-9 M.The derivatization reaction is reliable and the whole experimental procedure is very simple.The sensitivity of the proposed protocol allows quantitative analysis of total amino acids at picomoles levels without any separation procedures.On the basis of the conventional ninhydrin reaction,we put forward a simple SERS method for determining the total amino acids concentrations with high sensitivity,which is a promising way for routine detection.（4）Enrichment and detection of phosphoamino acids by Ag@Ti O₂According to the selective and reversible affinity of phosphate groups to semiconductor nanomaterial — Ti O2,we prepared a dual-functional SERS active substrate-Ag@Ti O2 for enrichment and SERS characterization of phosphoamino acids by controlling Ti O2 thickness.We chose O-phosphate-L-serine（OPLS）and O-phosphate-L-tyrosine（OPLT）as targets for enrichment and determination with prepared Ag@Ti O2 and got their fingerprint information in different concentrations.Furthermore,we are attempting to apply the substrate for the phosphorylated protein detection,which would provide new ideas and means for the study on protein phosphorylation.