Real-Time Monitoring And Quantitative Analysis Of Mechanically Induced Directional Differentiation Of Neural Stem Cells By Semiconductor Raman

Neural stem cells have been recognized for their therapeutic potential in neurodegenerative diseases due to their pluripotent properties,which enable them to replace damaged cells and reconstruct neural networks.However,the clinical applications of stem cell therapy are hampered by certain risks,including uncontrolled proliferation and differentiation of transplanted cells,as well as the possibility of tumorigenesis.To address these issues,there is an urgent need to develop safe and controllable methods to induce neural stem cells into specific functional cells.Current biochemical methods used to regulate neural stem cell behavior are limited by their cumbersome operation,complex processes,and potential risks of contamination,making them difficult to translate to clinical practice.In contrast,mechanical regulation of neural stem cells not only avoids these risks but also leverages the cells’mechanical memory to prolong the effects.In addition,although various analytical methods have been utilized to monitor the process of stem cell differentiation,these conventional techniques often exhibit strong destructive and invasive characteristics.Furthermore,these methods are constrained by certain temporal and spatial limitations,making it difficult to achieve long-term and sensitive monitoring of large-scale stem cell differentiation.Conversely,Raman spectroscopy possesses sensitive responses to molecular structures,symmetries,electronic environments,and bond relationships,providing molecular fingerprint information with high resolution and clear bands.In addition,the low invasiveness and fast and convenient operational modes of Raman spectroscopy have led to its widespread application in the characterization of stem cell differentiation.Here,we developed two platforms that can regulate the mechanical microenvironment and evaluate differentiation outcomes at the multicellular and single-cell levels,respectively.In Chapter 2,we first constructed a chiral MXene-based platform for the regulation and monitoring of neural stem cells.The movement of chiral Ti₃C₂ MXene nanoparticles under circularly polarized light can adjust the mechanical microenvironment of neural stem cells,reshape the cell skeleton,and shorten the differentiation period to 14 days.Meanwhile,Ti₃C₂ MXene exhibits a strong localized surface plasmon resonance（LSPR）effect,with an enhancement factor of up to 2.31*10⁸ when adsorbing methylene blue.Based on the distance sensitivity of the LSPR effect and charge transfer effect,we designed a Turn-OFF Raman detection platform using hairpin aptamers,achieving highly sensitive and selective detection of functional neural marker dopamine（DA）with a detection limit as low as 2.1 pg/m L.Combined with Raman imaging technology,in situ monitoring of the secretion of dopamine by neurons was achieved.To avoid the uncertainty caused by cell-cell interactions and heterogeneity in regulating cell fate in neural stem cell clusters,a microfluidic chip capable of single-cell capture and manipulation was designed in our work in Chapter 3.Additionally,a cell factory chip with multi-level micro-nano-angstrom topography was constructed by combining chemical synthesis methods.Under the joint influence of multiple forces,we successfully achieved precise control of the mechanical microenvironment at the pN level,while shortening the differentiation period and increasing the proportion of neural stem cells differentiating into GABAergic neurons.Furthermore,the CuCo₂O₄semiconductor nanosites can also serve as SERS substrates,significantly enhancing the Raman signal of methylene blue molecules（EF=1.97*10⁷）.Afterwards,by utilizing the affinity difference between the adaptor and target molecules and complementary chains,a Turn-OFF type Raman probe was designed,which realized a rapid,sensitive,and specific response to the concentration of functional neuron markers.The detection limit can reach 0.32 n M,and it can be integrated as a cell quality assessment module with microfluidic cell factories to achieve non-destructive,batch,and rapid evaluation of cell differentiation results.