| Breast cancer is the most fatal disease threatening women’s health.Detection of circulating tumor cells(CTC)in breast cancer can provide an effective approach for the early screening and diagnosis,monitoring metastasis and recurrence,tracking treatment effect and evaluation prognosis.However,CTCs are rare in blood and present significant heterogeneity among individual cells.Traditional population-based measurements often mask features of a small number of key subsets in CTC.Therefore,analyzing tumor cells at single-cell resolution is of great significance for accurate diagnosis of cancer.The excellent performance of surface enhanced Raman spectroscopy(SERS)in many aspects such as sensitivity,non-invasiveness,multiplexing etc.,has provided a new approach for CTC detection of breast cancer.In the thesis,we aim at developing a single cell detection technique for early diagnosis of breast cancer.Combined with functional nanoparticles and microfluidic platforms,a series of SERS technique-based methods are established for phenotype,migration and intracellular environment detection of CTCs,which may pave the way for the early diagnosis and imaging of breast cancer.The main innovative results of the thesis are summarized as follows:(1)A new SERS-based method for phenotype identification of CTCs with high sensitivity is proposed.Firstly,we designed a fluidic microarray with narrow gaps to capture CTCs in blood,and studied the effect of gap size on cell capture rate;secondly,we constructed a core-shell SERS nanoprobe with DNA aptamer as a recognition molecule to specifically recognize three kinds of biomarkers on breast cancer cell membrane;combined with multivariable analysis algorithm,SERS spectra from individual cells were collected and analyzed,the phenotypic information was profiled,and a classification model of the cell subtypes was constructed.Combined with the microstructure of the microfluidic chip,we are not only able to effectively separate CTC from blood,but also realize the phenotypic identification and analysis of breast cancer cells on a scale of single cells,which provides a new method for accurate diagnosis of breast cancer.(2)A novel SERS-microfluidic platform for simultaneous analysis of biological phenotype and chemotaxis on breast cancer single cells was proposed.We designed a microfluidic chip that integrates two functions: SERS-based phenotype profiling and mobility analysis.The migration channels on the chip were developed to observe the cell response to chemokine gradients,which is able to assess the invasiveness of cancer cells.Using this platform,the variation of protein phenotype and chemotaxis of SKBR3 cells prior to and after epithelial-mesenchymal transition were observed successfully.Our results demonstrate that the platform provides an effective means for identifying invasive subpopulations in CTCs,and can simultaneously obtain static(multiple biomarker expressions)and dynamic information(chemotaxis)of a single cell,which enhances the diagnostic ability of the platform and provides a new method for accurate diagnosis of metastatic cancer.(3)A highly stable SERS nanosensor is proposed for intracellular p H detection of breast cancer cells.The sensor is comprised of gold nanostars as plasmonic substrate and p H-sensitive Raman molecules as the sensing unit.Polyarginine,a biocompatible polymeric peptide was externally coated on the sensor,which provides an excellent affinity towards cells.The experimental results show that the nanosensor presents good stability in a complex biological environment and high cell loading ability,being quite suitable for p H detection in intracellular environments.Subsequently,the nanoparticle endocytosis process and p H changes in breast cancer cells were observed via 2D and 3D Raman imaging method,which is of great significance to understand the interaction between nanoparticles and individual cells and the inner homeostasis of the cancer cells. |
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