| Lung cancer is one of the most common primary malignant tumors,and the incidence and mortality rate of lung cancer are the highest among cancers in China,and the five-year survival rate is only 10%-20%.Early diagnosis and screening can significantly improve the survival rate of lung cancer,but there are still many limitations in the current means of early lung cancer screening,and the detection of tumor biomarkers has opened up new ideas and methods for early lung cancer diagnosis.Circular RNA(circRNA)is a novel biomarker,as a non-coding RNA that can act as a ce RNA(competing endogenous RNA)and regulate gene expression in mammals,showing great potential in cancer diagnosis and targeted therapy.The research of circRNA is still in its infancy,and the main research direction is focused on exploring the expression and functional mechanisms of circRNA.Traditional detection methods such as northern blotting and RT-PCR(reverse transcription-polymerase chain reaction)are too complicated and expensive to meet the needs of detection and research.Therefore,it is crucial to develop a method for highly sensitive quantitative analysis of tumor biomarker circRNA.Raman spectroscopy and surface-enhanced Raman spectroscopy(SERS)have attracted the attention of a wide range of researchers due to their advantages of high sensitivity,high specificity,fast and nondestructive detection.SERS spectroscopy can even realize single-molecule detection under special conditions.In recent years,they have been widely used in field of life science,showing great potential for application in cancer diagnosis,progression detection and recurrence prediction.In this thesis,we used laser tweezer Raman spectroscopy as a means to investigate the detection of circRNA associated with lung cancer,explored the effect of circRNA on lung cancer cells,and developed a new method for highly sensitive quantitative detection of circRNA in cells and blood based on SERS technology.The main studies are as follows.1.Laser tweezer Raman spectroscopy(LTRS)was combined with PCA-LDA(principal component analysis-linear discriminant analysis)algorithm to develop a study on the function of circRNA in lung cancer cells and intracellular cells.Firstly,the high quality Raman spectra of three live cells BEAS-2B,H1650 and HCC827 were collected using laser Raman spectroscopy system,and the biochemical differences between them were analyzed,and the high sensitivity and high specificity of discriminative distinction between the three was achieved by combining with PCA-LDA analysis.Then,lung cancer cells were transfected with small interfering RNA(si RNA),and the changes in Raman peaks attributed to proteins in the cells before and after transfection were found using spectral changes,demonstrating that the target circ SATB2 has a relevant function in regulating protein expression.The results suggest that this method can be a potential tool for studying circRNA cell function.2.A dual-labeled ratio-calibrated SERS probe was designed for quantitative detection of intracellular circRNAs.The probe uses two Raman signal molecules,the reporter molecule ROX located on the DNA signal chain to reflect the concentration of circRNA,and the internal standard molecule 4MBN embedded inside the nanoparticle,which can calibrate the acquired Raman spectral signal in real time and improve the reliability of spectral detection.A detection limit of 0.043 p M can be achieved using a signal-ratio calibrated SERS probe.In situ intracellular imaging can be achieved using the Raman signals of ROX and 4MBN,and the intensity of the labeled molecular SERS spectra reflects the concentration of the target circRNA inside the cell.The results obtained by this method are consistent with the conventional method q RT-PCR,which is expected to become an effective detection and analysis tool for single-cell circRNA research.3.A SERS sensing platform based on a "sandwich" structure was prepared for the highly sensitive quantitative detection of tumor marker circRNAs.The platform consists of two parts: a two-dimensional SERS substrate formed by Ag nanoparticles self-assembled on a silicon wafer,and a SERS probe with a core-shell nanoparticle structure,which are connected by a circular amplification process initiated by the target circRNA.The quantification of target circRNA concentration using the ratio of Raman reporter molecules to the second-order peak intensity of the in silico wafer makes this SERS sensing platform with good sensitivity,specificity and reliability,and excellent performance in serum,providing a convenient and efficient potential means for in vitro detection of circRNA.Combined with the current situation of circRNA clinical needs at home and abroad,closely following the international research hotspots of cancer detection,this thesis has developed a series of methods for circRNA research and detection based on laser tweezer Raman spectroscopy and surface-enhanced Raman spectroscopy,which is expected to provide a reliable sensitive research method for circRNA detection. |
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