Preparation Of Noble Metal/Semiconductor SERS Substrate And Its Bioassay Application

Tumor markers can be divided into six categories,namely embryonic antigens,sugar chain antigens,hormones,enzymes and isozymes,proteins and oncogene products.Highly sensitive trace detection of tumor markers contributes to the clinical diagnosis and therapeutic observation of cancer becuase tumor markers is closely related to the occurrence and development of tumors.Compared with the commonly used biological detection methods,the tumor marker detection method based on surface-enhanced Raman scattering(SERS)has good characteristics,containing high detection sensitivity,good biocompatibility,and specificity,to have wide detection ranged and attracted more and more attention.For example,about the SERS-based tumor marker immunoassay method,the"sandwich" structure(immune substrate-test substance-immunoprobe)has gradually gained practical application as a main detection scheme.However,in the case of oncogene product tumor markers,miRNA detection faces many challenges due to factors such as low miRNA concentration in serum of early cancer patients and easy degradation of miRNA after serum sampling.Therefore,with reference to SERS-based tumor marker immunoassay methods,especially the design and preparation of SERS substrates with high enhancement properties,good signal reproducibility and stability,it is of great significance for the detection of oncogene product tumor marker miRNAs.In our work,we have carried out research on the preparation technology of noble metal/semi conductor nanostructure SERS substrate to apply in the biological detection of miRNA,which achieved good results.The main contents are summarized as follows:1.Preparation of Ag/Ti02 nanowires(NWs)SERS substrate and its bioassay for miRNA-21.Firstly,the titanium sheet is hydrothermally reacted with sodium hydroxide,and the reaction product is hydrolyzed to obtain a Ti02 NWs structure.Then,Ag/Ti02 NWs SERS substrate was prepared by depositing Ag on the surface of Ti02 NWs structure by magnetron sputtering.The SERS spectra of the Raman-labeled molecule 4-mercaptobenzoic acid(4MBA)show that the Ag/Ti02 NWs SERS substrate not only has high SERS enhancement properties also good uniformity.Furthermore,Ag/TiO2 NWs SERS substrate was fabricated by immobilizing the 3’-NH2-ssDNA on the surface of Ag/TiO2NWs substrate.Au@Ag core-shell NPs by linking with 4MBA and 5’-NH2-ssDNA is used to prepare Au@Ag@4MBA@5’-NH2-ssDNA probe.We combined Au@Ag@4MBA@5’-NH2-ssDNA probes and SERS substrate with miRNA-21 to form a"sandwich" structure and realized biodetection of genetic tumor markers,which the limit of detection was as low as 0.75 fmol·L-1.It can be seen that the prepared Ag/Ti02 NWs SERS substrate have great SERS enhancement due to the electromagnetic enhancement effect of Ag nanoparticles and the chemical enhancement caused by charge transfer between 4MBA,Ag and TiO2,and applied to the highly sensitive detection of miRNA-21.2.Preparation of Ag/ZnO nanopencils SERS substrate and its bioassay for miRNA-106a.Using a zinc sheet as the source material,ZnO of nano-pencil structure substrate was prepared in situ on a zinc sheet by a two-step hydrothermal method.Then by the magnetron sputtering technique,the Ag NPs was spurted on the surface of the prepared ZnO nanopencils substrate to obtain Ag/ZnO nanopencils substrate of high sensitivity and uniformity.And the bioassay scheme based on SERS for miRNA-106a was proposed.Furthermore,Ag/ZnO SERS substrate was fabricated by immobilizing the 3’-NH2-ssDNA on the surface of Ag/ZnO nanopencils substrate.And we combined Au@Ag@4MBA@5’-NH2-ssDNA probes and SERS substrate with miRNA-106a to form a sandwich" structure and realized biodetection of miRNA-106a overexpression in renal cell carcinoma,which the limit of detection was as low as 57.5 fmol·L-1.