| The intracellular environment is the place where cells carry out physiological activities,biosynthesis,substance metabolism and other processes.The disturbance of the intracellular environment will directly lead to the degeneration,senescence and even death of cell functions.Due to cell heterogeneity,intracellular molecular expression and genome are different in the process of cell division and differentiation.Therefore,it is of great significance to analyze and detect the internal environment at the single-cell level.In recent years,single cell analysis has made great progress.The traditional fluorescence analysis,mass spectrometry（MS）,microfluidics and other single-cell analysis methods have high sensitivity and accuracy,but at the same time there are some defects,such as photobleaching phenomenon,difficult in situ real-time monitoring,weak response of complex systems.Nano tip technology has significant advantages in single cell analysis,which can puncture cells to reach different locations in the cell or different organelles,realizing microlocation and non-destructive analysis.Surface-enhanced Raman scattering（SERS）probes are a powerful tool for cell analysis due to their advantages of high sensitivity,multiple markers and anti-biological matrix interference.Specific active substances in cells can cause the structure change of reporting molecules adsorbated on the surface of SERS probes,and the resulting changes in probe signals can dynamically reflect the temporal and spatial changes of active substances in real time,indicating the occurrence of molecular events.The combination of nano tips and SERS probes is expected to further improve the level of single cell detection.In this academic paper,the development of SERS nano tips and their application in the analysis of intracellular environment were carried out.In the research and development of SERS nano tips,precious metal nanomaterials with high SERS enhanced ability were firstly screened.By comparing the SERS intensity of Au nanospheres,Au nanostars and single particles of core-satellite Au nanoparticles with similar particle sizes,the SERS enhanced factor（AEF）was calculated,and finally the independently developed core-satellite Au nanoparticles were selected as the modified particle of nano tips.The high boric acid glass capillary was drawn into a conical tip with a diameter of about 200nm by a needle pulling instrument.Then,amphiphilic polymer PS-b-P4VP was adsorbed on the surface of nano tips,and core-satellite Au nanoparticles were adsorbed on the surface of the tips by electrostatic binding,and SERS nano tips were obtained.Intracellular pH and hypoxia state were selected as detection objects to explore and verify the application of SERS nano tips.4-mercaptobenzoic acid（4-MBA）and 4-nitrophenthiol（4-NT）reporter molecules were respectively modified on the surface of SERS tips.The increase of pH resulted in the enhancement of-COOH peak in 4-MBA,and anoxia caused the reduction of-NO2 in 4-NT to azo bond,resulting in a new peak in SERS spectrum.Based on this principle,the coupling module of SERS nano tips and micro-injection 3D control platform was used to realize accurate positioning and non-destructive detection of the nano tips in a single cell,as well as the detection of cytoplasmic pH of human hepatocytes（HL 7702）and the monitoring of hypoxia state.Further explore the mechanism of action of drugs on cells by using SERS nano tips.Through PH-sensitive SERS nano tips,the pH changes of the nuclear and cytoplasmic pH of human cervical cancer cells（HeLa）affected by vincristine sulfate（VCR）at different concentrations were monitored.The results showed that both nucleus and cytoplasm acidified after the action of VCR.The distribution and transport process of doxorubicin（DOX）in HeLa cells was tracked,and quantitative analysis of intracellular DOX was completed.DOX was found to be decreased in the cytoplasm but increased in the nucleus,these results indicate that DOX is transported from the cytoplasm to the nucleus and exerts its pharmacological effect.In conclusion,this academic paper provides a new method for single cell detection.SERS nano tips technology dispensed with the negative impact of incubation on cells and ensured that cells were analyzed and detected in the natural state.Core-satellite Au nanoparticles as SERS nano tips significantly improved the sensitivity of analysis and detection,and combined the nano tip technology with Raman,realized the DXR laser confocal micro-Raman spectrum-nano tip detection mode.Compared with traditional analysis techniques,it has the advantages of in situ real-time monitoring,high accuracy,high sensitivity and non-destructive.The analysis of intracellular environment changes after the action of drugs and the study on the action process of drugs in a single cell were completed by using SERS nano tips,which provided a new analytical method for the quantitative analysis of drugs in a single cell and the study on the mechanism of action. |
