Study On Degranulation Of Mast Cells By SERS Spectroscopy Based On Electrical Pulse And Ultrasound Assisted Perforation Technology

Surface enhanced Raman scattering（SERS）technology has a good application prospect in molecular level cell detection,drug cytotoxicity analysis and cancer cell screening.SERS is widely used in cell research,from extracellular to intracellular,from cell secretion to intracellular molecular substances,and then to the detection of organelles,from simple Raman scattering signal enhanced substrate to various functionalized SERS nanoprobes（including label-free or labeled SERS sensors）.Based on SERS detection,the structure and content of analytes can be obtained at the molecular level,so as to analyze the changes of biochemical components in life activities.Mast cells（MCs）,which are derived from hematopoietic stem cells and are widely distributed in the area in contact with the external environment.MCs will degranulate when activated by external stimuli,that is,some potential mediators in the body,such as histamine,β-hexosaminidase and so on,will be released outside the cell.Cell degranulation plays an important role in life process through humoral circulation,and is associated with inflammatory reaction,immediate allergic reaction and tumor formation and development.At present,there are many detection methods of MCs degranulation,such as histopathology,kit analysis,fluorescence detection and so on.These methods also have some shortcomings,such as long time-consuming,high requirements for detection instruments and equipment,can only detect single or specific components,rather than reflecting the overall degree of cell degranulation,etc.Based on the above shortcomings,this study combined SERS technology with electric pulse technology,ultrasonic technology and nuclear targeted nano probe to detect the degranulation of MCs from multiple dimensions,including the secretion of cell degranulation,the biochemical components inside the cell,and the targeted detection of nucleus.The specific research content includes the following four parts.1.The label-free SERS technology was used to detect and distinguish the degree of degranulation of MCs induced by low-intensity laser irradiation at different times（0 min,2 min,5 min and 10 min）.Based on the enhancement effect of silver nanoparticles,SERS technology could simultaneously reflect multiple components of degranulation.There was a significant difference in the SERS signal between the laser-irradiated group and the control group.Using the SERS technique combined with the PCA-LDA analysis method,the degree of MCs degranulation could be distinguished with high sensitivity,specificity and accuracy.This work indicates that,in the detection and analysis of the overall biochemical components of MCs degranulation secretion,the high-sensitivity SERS technology is better than the traditional single component detection technology,and SERS technique combined with the PCA-LDA algorithm has great potential in the detection of cell degranulation secretion.2.Biochemical detection of MCs treated with different concentrations（10μg/m L,20μg/m L and 30μg/m L）of MCs agonist（C48/80）was performed by electrical pulse-assisted SERS technology.The electrical pulse technology exploited its controllability to form reversible pores in the cell membrane to delivery SERS signal enhancement substrate（Ag nanoparticles）into cells while maintaining a high cell viability（93%）.The Ag nanoparticles delivered by electrical pulses were mainly distributed in the cytoplasm,therefore,more cytoplasmic biochemical information and less nuclear biochemical information would be obtained.The results of this study showed that with the increase of MCs agonist concentration,the effect of MCs degranulation was obvious,and the difference of SERS spectra between MCs agonist-treated group and control group（without drug treatment）was more obvious.This work will provide a reference for the screening of drug concentration.3.Biochemical detection of MCs treated with C48/80 at different concentrations（10μg/m L,20μg/m L and 30μg/m L）was performed by ultrasound-assisted SERS technology.The biochemical detection effect of C48/80-treated MCs was compared and analyzed for the first time by using the electrical pulse-assisted SERS technology and the ultrasound-assisted SERS technology.Electrical pulse technology utilized controllable electrical pulses to form reversible pores in the cell membrane to transport nanoparticles,which could deliver nanoparticles into the cytoplasm while maintaining a high cell viability（93%）.In contrast,ultrasound-assisted techniques based on cavitation were more damaging to cells.Nanoparticles delivered by electrical pulse were mainly distributed in the cytoplasm,while nanoparticles delivered by ultrasound could be distributed in the nucleus and cytoplasm.Therefore,the ultrasound-assisted SERS technique obtained more biochemical information of the nucleus.Both methods could obtain high-quality cellular SERS signals with high sensitivity,specificity,and accuracy in identifying drug-stimulated cells.This work provided a reference for real-time dynamic studies of intracellular biochemical components.At the same time,it also had great potential in the cytotoxicity analysis of antitumor drugs.4.In this work,a nuclear targeted SERS nanosensor(Au@^4-MBA-PEG-NLS)was developed.The sensors were rapidly introduced into MCs and nasopharyngeal carcinoma cell by electrical pulse technology.Under the guidance of the nuclear localization peptide on the surface of sensors,these sensors could target and connect to nucleus,so as to accurately locate the nucleus in SERS detection and obtain abundant SERS signals of biomolecules in the nucleus.From the SERS imaging and HE staining of CNE1,CNE2,C666 and NP69 cells,it could be seen that Au@^4-MBA-PEG-NLS has accurate nuclear localization ability and strong Raman scattering signal enhancement effect.Subsequently,these nuclear targeted SERS nanosensors were applied to analyze the biochemical changes of nucleus of MCs stimulated by drugs.The experimental results showed that the control group（without drug treatment）and the drug-treated group had significant discrimination sensitivity（80%）,specificity（93.3%）and accuracy（86.7%）.This study analyzed the difference of biochemical components of the nucleus between the drug treatment group and the control group at the molecular level,and reflected the effect of drug stimulation on the nucleus of mast cells.This method had great potential in drug cytotoxicity research,anti-cancer drug target screening,and real-time monitoring of cell stress response caused by exogenous molecules.Following the research hotspot of cell detection and combined with the urgent needs of clinical detection,this paper develops a series of new nondestructive detection methods based on cell SERS spectroscopy,which is expected to provide an important reference for nondestructive screening,prognosis evaluation and curative effect monitoring of clinical diseases related to mast cell degranulation.