| With the advancement of medical technology,people are making great progress in conquering cancer.Numerous medical studies have shown that if cancer can be detected and diagnosed early,cancer can be treated promptly,which will effectively reduce cancer mortality.Tumor markers are the product of tumorigenesis and proliferation processes.The content of tumor markers in human tissues and body fluids has become an important indicator for clinical diagnosis of cancer.Tumor markers are mainly classified into protein,hormones and nucleic acids.Nucleic acid tumor markers are closely related to the uncontrolled expression of genes and the occurrence and development of tumors.More and more attention has been paid to its detection with high sensitivity,high throughput and high specificity.In general,traditional nucleic acid detection methods,such as fluorescence quantitative polynucleotide chain reaction?RT-qPCR?,are difficult to meet tumors due to extremely low levels of tumor nucleic acid markers in human serum and easy degradation of nucleic acids after serum sampling.The need for rapid screening and early diagnosis.However,the detection method based on Surface-enhanced Raman scattering?SERS?technology has the advantages of high sensitivity,non-destructiveness and wide detection range.It is of great significance to apply it to the qualitative and quantitative detection of tumor nucleic acids.In this thesis,several novel microarray SERS substrates were designed and fabricated.Two detection methods based on SERS technology for microRNA?microRNA?were proposed.High specificity and sensitivity of miRNA-106a and miRNA-21 related to pancreatic cancer and non-small cell lung cancer were achieved respectively.The main contents of the paper are as follows:1.Preparation of silver-coated coronal silica column array substrate and detection of tumor marker miRNA-106a.Using polystyrene?PS?spheres as templates,large area coronal silicon column arrays were fabricated by metal assisted etching and wet chemical etching.Ag/Si CPA substrates were obtained by in situ growth of silver nanoparticles.The experimental results show that the prepared substrate has excellent surface enhanced Raman scattering?SERS?characteristics.At the same time,the prepared rhodamine molecule?R6G?labeled DNA hairpin probe was linked to the substrate,after complementary hybridization with miRNA-106a,SERS signal detection was conducted to obtain the corresponding dose-response curve.The results showed that the detection of miRNA-106a based on the SERS characteristics of the substrate?Ag/Si CPA?had advantages of good specificity and high sensitivity,with the detection range of 1 fmol·l-1-100 pmol·l-1 and the detection limit of0.917 fmol·l-1.2.Detection of tumor marker miRNA-21 based on sandwich SERS structure and enzyme splicing techniqueFirstly,a layer of hexagonal densely packed PS sphere arrays was self-assembled on silicon wafers,and then a layer of silver nanoparticles is sputtered on the surface of PS spheres by magnetron sputtering technology to form a silver-coated hexagonal densely packed PS sphere array substrate?Ag/PS HCA?.Simultaneously,sea urchin Au nanoparticles were synthesized by chemical reduction method,and link it to 4-Mercaptobenzoic acid?4-MBA?to prepare Au@4-MBA probe.Then thiol-modified and amino-modified DNA 21 were linked to Ag/PS HCA substrate and Au@4-MBA probes respectively to construct Ag/PS HCA-DNA21-Au@4-MBA sandwich structure.The next,it was hybridized with the tumor marker microRNA-21,then double-stranded specific shear enzymes?DSN?were added,and finally SERS signal was detected.The experimental results show that the detection of miRNA-21 is not only highly sensitive but also greatly specific,and the detection limit is 0.853 fmol·L-1. |
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