Surface-Enhanced Raman Scattering Based Detection Methods For Protein Biomarkers

As an ultrasensitive detecting technique,Surface-Enhanced Raman Scattering(SERS)has aroused extensive attention in biological science.Detection of biomarkers and early diagnosis is an important development direction of SERS in the field of life science.This thesis is focusing on developing SERS based detection methods for protein biomarkers.SERS-based methods have advantages of high sensitivity and multiplex fingerprint message,based on which we succssfully established several SERS-based detection methods for protein biomarker based early diagnosis,protein identification and protein-protein interactions and so forth.The major contributions of this thesis are as follows:1.Detection of glycoprotein with variant discriminationα-Fetoprotein(AFP)is an important tumor biomarker,a glycoprotein.The overexpression of its variant protein(AFP-L3)is associated with hepatocellular carcinoma(HCC).Accordingly,several hospitals have begun to employ the ratio of AFP-L3 to the total AFP level(AFP-L3%)as new diagnostic evidence for HCC.In this chapter,we present a novel concept for the early diagnosis of HCC based on the combination of Raman frequency shift and intensity change,and developed surface-enhanced Raman scattering-based immunochips via AFP-L3%.In the first step of the study,the frequency shift of 4-mercaptobenzoic acid(MBA)was applied for the quantitative determination of total AFP based on the AFP and anti-AFP interaction on MBA modified silver chips.5,5-Dithiobis(succinimidyl-2-nitrobenzoate)(DSNB)-modified immunogold was then incorporated with AFP-L3 antibodies for sandwich immunoreaction on the chips.As a result,we found that a typical Raman band intensity of DSNB presented an exponential linear relationship with the concentration of AFP-L3.Thus,the AFP-L3% can be calculated according to the concentrations of AFP-L3 and total AFP.The most important advantage of the proposed method is the combination of AFP-L3% and frequency shifts of SERS,which exhibits excellent reproducibility and high accuracy,and significantly simplifies the conventional detection procedure of AFP-L3%.Application of the proposed method with the serum of patients with HCC demonstrated its great potential in early liver cancer diagnosis.2.Antibody-free detection of protein biomarkers in human serumIn this chapter,a systematical SERS investigation of a novel Raman probe,perylenetetra carboxylic acid(PTCA)on different substrates was performed at first.Furthermore,we found an obvious wavelength dependent and abnormal SERS spectra,which can be ascribed to be different charge transfer between PTCA and substrates.To our surprise,it is found to directly correlate with the protein types,revealing different frequency-shift and intensity change,according to which protein biomarkers even homologous proteins with very similar molecular structures can be discriminated with the aid of hierarchical cluster analysis.Furthermore,the feasibility of the proposed approach has been proved in early liver cancer diagnosis with clinical samples.All the results indicate that PTCA as a universal SERS probe has great potential in rapid,accurate,and direct protein biomarker discrimination in cancer diagnosis.3.Frequency-shift based SERS detection of protein carbonylationIn the previous two chapters,frequency-shift based surface-enhanced Raman spectroscopy has exhibited great potential applications in bioanalytical chemistry and biomedicine in recent years.The basis and the crucial factors determining frequency shifts are,however,still unclear.Herein,in this chapter,we have systematically investigated how solvents,antigens,and antibodies affect the band shifts in SERS-based immunoassays.By applying the charge transfer theory together with the Stark effect and time-dependent density functional theory(TDDFT)calculation,mechanistic insights into the frequency shifts in immunoreactions is proposed and discussed in detail.Accordingly,the experimental condition is further optimized and is successfully applied for the first time to detect carbonylated proteins,promising diagnostic biomarkers for human diseases.This study provides theoretical guidance for designing SERS frequency shift-based immunoassays and paves a new avenue for further applications of the strategy in clinical diagnosis.4.Detection of His-tagged proteinAlthough SERS has exhibited great potential in protein identification and quantification.The poor spectral reproducibility,originating from random protein immobilization on SERS substrates,still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels.In this chapter,spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement.We have accordingly prepared iminodiacetic acid(IDA)-functionalized silver substrates,which are used for capturing His-tagged proteins via nickel-imidazole coordination.The controlled immobilization enables excellent SERS spectral reproducibility.Furthermore,the interactions between two model proteins,Erv1C(C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1)and AFP,and their ligands Cyt c(cytochrome c)and ATRA(alltrans-retinoic acid)are examined,respectively.The results indicate that the IDAfunctionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS.As a proof-of-concept study,the proposed functionalized SERS-active substrates combined with immobilized metalaffinity chromatography will be useful for mechanism studies on protein-ligand interactions,understanding the structural basis of protein functional versatility and drug design.