ALuminogen With Aggregation-Induced Emission Characteristics For Cell Chromatin Density Analysis

Chromatin density refers to the degree of looseness or tightness of chromatin in different cell cycles or in different cell states.During the biological processes of cell rest and activation,cell division and proliferation,cell hypertrophy and cell apoptosis,chromatin density changes dynamically.Currently,the commonly used methods of chromatin density analysis mainly include high-resolution microscope imaging and high-throughput chromosome conformation capture（Hi-C）.High-resolution microscope imaging can achieve the resolution as small as 0.1～0.2 nm,and can provide clear imaging of the cell ultrastructure.However,its sample preparation process is complex,time-consuming,and the cost of detection is very high.Hi-C technology can provide a large amount of chromatin interaction data and can be used to reconstruct the three-dimensional spatial structure of chromatin.However,due to its high cost,long detection cycle and the difficulty in data analysis,it is not suitable for routine detection in general laboratories.Fluorescence is a commonly used analytical tool in the field of life science.As the core of fluorescence imaging technology,fluorescent materials need to have the characteristics of good biocompatibility,high sensitivity and simplicity.However,for many conventional organic fluorescent probes,when they are in the aggregate state or when the concentrations are relatively high,the fluorescence intensity will be greatly reduced or even completely quenched,that is,aggregation-caused quenching（ACQ）.This defect greatly affects the fluorescence intensity and stability,and makes it difficult to complete long-term dynamic observation and signal tracking.In 2001,professor Benzhong Tang’s team from Hong Kong University of Science and Technology discovered an optical phenomenon that is completely opposite to the ACQ phenomenon and named it as aggregation-induced emission（AIE）and proposed that restriction of intramolecular motions（RIM）was the main luminescence mechanism of AIE fluorescent molecules.AIE fluorescent molecules do not emit in the single molecule dispersive state,but emit strong fluorescence in the aggregate or solid state.This feature avoids the limitation of high concentration in which fluorescence is easily quenched,endows excellent light stability and high fluorescence intensity with AIE fluorescent molecules,significantly improves the detection sensitivity,achieves long-term fluorescence imaging and broadens the application of fluorescent materials in the biomedical fields.In the first chapter of this thesis,we designed and synthesized a novel fluorescent probe named as MASPB which exhibited good water solubility,strong fluorescence stability and good biocompatibility.In tumor cells,MASPB showed a nucleoli-targeting feature whereas showed no obvious fluorescent signals in the nuclear chromatin area.Interestingly,in the cells which were relatively mature（eg.mature white blood cells）,the nuclear chromatin could be targeted by MASPB and emited strong fluorescence.Experiments of DNase and RNase in tumor cells and white blood cells showed that the difference of fluorescence distribution was not dependent on DNA or RNA.Further,through transmission electron microscopy（TEM）analysis of chromatin density and fluorescent imaging of chromatin（chromosome）in different cell cycle,we proposed that the degree of the chromatin density is the key of whether MASPB could cause restricted intramolecular motion and then inspired its AIE effect.Namely,whether MASPB could give rise to the fluorescence "turn on" effect depended on whether or not the chromatin was dense enough.Based on the above,we propose that MASPB can be used as a new AIE fluorescent probe for chromatin density analysis.In the second chapter of this thesis,we attempted to explored the application of MASPB in the tumor pathological tissue slices.Compared with the traditional hematoxylin and eosin staining（HE staining）,MASPB could achieve rapid fluorescence staining of tumor pathological tissue slices in only 1 min,and the whole staining process required only one step without washing,which significantly improved the staining efficiency.In tumor tissue area,MASPB could target the nucleoli of tumor cells,while in normal tissue cells（such as fibroblasts）,their nuclear chromatin could be targeted by MASPB.Based on the difference of fluorescence distribution of MASPB in the nucleus,the tumor cells and normal tissue cells in the tumor pathological tissue slices could be identified.In summary,a novel fluorescence "turn-on" and "rinse-free" AIE probe called MASPB was successfully designed and developed.MASPB may serve as a novel tool for analysis of chromatin density and for determination of different stages in cell cycle,and can be applied to clinical tumor pathological tissue slices to distinguish tumor cells with normal tissue cells,providing new insights into designing and developing other AIEgens for biomedical applications.