The SERS Method Based On Specific Reaction Detects Biomarkers

Surface-enhanced Raman scattering?SERS?has become a mature vibrational spectroscopic technique during the last decades and the number of applications in the chemical,material,and in particular life sciences is rapidly increasing.SERS phenomenon was commonly observed from some nanometal substrate such as Ag,Au,Cu colloids that absorbed special molecules,and some nanoparticle assemblies such as dimers,tetrahedrons,satellites,chains helices,and nanocrystals,which caused raman scattering signal to greatly enhance compared with normal raman signal.?SERS?,as a molecular fingerprint spectrum,is one of the promising technologies as it exhibits a unique blend of advantages,such as ultrasensitive,non-invasive probing,compatibility with aqueous solution,minimal sample preparation,and label-free monitoring for specific analyte in complex matrices.Giving all of these features,SERS has been widely used in various areas,such as food safety,environment,bio-diagnosis,medicine,chemistry,etc.This work was based on specific reactions for SERS detection of three biomarkers.The work is as follows:1.Abnormal glutathione?GSH?levels are related to several diseases including cancer.In this paper,surface-enhanced Raman spectroscopy?SERS?based on a2-?pyridyldithio?ethylamine?PDEA?modified AgNPs@Si wafer is proposed for intracellular glutathione?GSH?detection.PDEA plays multifunctional roles in the method,including the participation in the thiol-disulfide exchange reaction and the contribution with a great SERS signal reporter.With the addition of GSH,the disulfide bond of PDEA will be broken,releasing the pyridine ring in PDEA and resulting in a signal-off SERS response.The developed specific reaction-based SERS assay can detect GSH as low as 2.5×10^-7 M.Where after,we employed this method to evaluate the cellular GSH levels,finding the levels in cancer cells are higher than that in normal cells.The PDEA modified AgNPs@Si-based SERS protocol demonstrates good selectivity and high sensitivity as well as robustness,which is suitable to evaluate cellular GSH levels.2.Based on the protein glycosylation reaction in combination with the SERS tag,a SERS biosensor that can be developed to determine N-acetylglucosamine glycosyltransferase?OGT?activity and screen their inhibitor.We designed a polypeptide containing a glycoproteinase recognition site.It is modified with biotin at one end and cysteine at the other end.The biotin links to the magnetic beads by the interaction between biotin and avidin.Cysteine can capture gold nanoparticles loaded with SERS probes,non-radioactive N-acetylglucosamine?O-GlcNAc?can be used as a sugar donor.The peptides enzymatic hydrolysised by glycosylase can escape the subsequent excision of proteinase K.The complete polypeptide chain captures the gold nanoparticles to the surface of the magnetic beads through cysteine,resulting in a strong SERS signal.When the activity of glycosylase is low,there are few polypeptides that undergo glycosylation and more polypeptides are cleaved by protein K,resulting in inefficient capture of gold nanoparticles and the overall SERS signal of the magnetic beads is low.This strategy is used to establish a correlation between glycosylase activity and SERS intensity to achieve quantitative determination of glycosylase.It does not require complex labeling procedures,combined with the effect of magnetically enhanced SERS detection,can achieve specific sensitivity detection of glycosylase,and is of great significance for the study of protein glycosylation and the el ucidation of OGT biological effects.There are huge application potentials in drug discovery and clinical diagnosis.In addition,the method can be used as a universal sensing platform to determine other glycosyltransferases by designing a suitable polypeptide substrate.3.The SERS method was developed based on signal amplification strategy to detect DNA methylase DNMT1 activity.We designed a DNA strand S1 with methylation sites and poly A at one end.By the action of poly A and nanoparticles,S1was attached to the AgNPs@Si substrate surface.The Au NP-DNA complex was prepared by attaching a thiol-containing DNA strand S2 and S3 to the surface of the prepared gold nanoparticle by a gold-sulfur bond.The S2 chain is longer and can be complementary to the S1 terminal base.However,the S3 chain is shorter which acts as a steric hindrance to prevent some of the S2 linkage on the gold nanoparticles from being too dense.The SERS"hot spot"is formed by the S1 base complementary pairing with the Au NP-DNA complex to achieve signal amplification.Methylene blue?MB?can be attached to the surface of the substrate by channel bonding and electrostatic adsorption with S3 to produce SERS activity.The double-stranded methylation was achieved by the action of DNA methyltransferase 1?DNMT1?on the S1 with a methylation site.The restriction endonuclease BssHII could not recognize the site of cleavage and the SERS signal remained.When DNMT1 activity is weak,S1 with methylation site is recognized and cleaved by restriction endonuclease BssHII,and gold nanoparticles leave the surface of AgNPs@Si substrate so the SERS signal of MB is weakened.This method is of great significance for the study of DNA methylation and shows the potential for clinical application.