Fiber Optic SERS Tip Exploring Metabolic Events At Single Cell Level

Cell metabolism is the main way to maintain the operation of life.Cancer cells have a special metabolic mechanism different from normal cells,namely aerobic glycolysis.In the process of metabolism,cancer cells produce a series of biomarkers.Due to the heterogeneity between cancer cells,detection or recognition of such biomarkers at the single-cell level is of great significance for understanding the metabolic pathways.In the process of exploring the emerging techniques of single-cell analysis,researchers have developed a large number of high spatial and temporal resolution,nondestructive and rapid detection methods from light,electricity,force,and mass spectroscopy.The development of technology brings an opportunity to study the dynamic process of single cell.Surface-enhanced Raman scattering(SERS)has great advantages in single-cell analysis: The high sensitivity of SERS allows label-free fingerprint analysis;The active molecules of Raman can be detected indirectly by labeling,and the activity is stable and free from laser interference.At the beginning,the nanotip technique was used for electrophysiological analysis with patch clamp and scanning probe of near-field optical microscope.After combining with the spectrum detection platform,the nanotip technique played a greater role.The nanotip technology could realize the study of dynamic biological processes inside single cell in a natural state,and gradually became a research hotspot.In order to realize the light physiological analysis of single cell,we first built a micromanipulation-Raman spectral detection instrument to provide the controllable positioning function of the micromanipulation technology and Raman spectral detection technology.Furthermore,the end of the quartz fiber was treated with tip of less than 500 nm by means of tubular etching to ensure that the tip could avoid damage caused by physical machinery after insertion into the cell.By laser-induced deposition method,SERS substrate and Raman active molecules were modified on the surface of tips,and the deposition process of single molecule layer was discussed and the deposition time was optimized to achieve the best SERS enhancement.In the performance test,SERS spectral intensity detected by different fiber SERS probes was basically the same.After examining dozens of groups of cells,the probe molecules remained stable in strength.After the cells underwent insertion and extraction,they were stained with living and dead cells.The green fluorescence under the fluorescence microscope showed that the cells were still alive.In the discussion of different excitation collection methods,we compared the influence of fiber excitation and lens excitation on the detection efficiency,and calculated the enhancement factor,and optimized the detection method.The repeatability,stability and cytotoxicity of the fiber SERS probe indicated that the probe could be used in cell system.We prepared SERS fiber optic probes,using one normal cell and three cancer cell lines as models,to realize the optical physiological detection of intracellular and extracellular,as well as subcellular areas.The final expression of aerobic glycolysis in cancer cells is the release of lactate,and glycolysis involves many REDOX reactions.Therefore,in exploring the metabolic events of single cells,we detected pH and oxidative stress levels respectively.In pH detection,4-mercaptopyridine was used as a response probe,which presented different SERS spectra at different pH levels.Based on this principle,the pH data of the single-cell microenvironment were characterized,and we further analyzed the pH difference between intracellular and extracellular of normal cells and cancer cells.Compared with normal cells,cancer cells showed higher internal environment and lower external environment,which was consistent with the general research.In the study of the metabolic characteristics of the three types of cancer cells,the pH levels of the nucleus and cytoplasm were detected respectively,and the differences of the nuclear and cytoplasmic regions were calculated by using the p value(significance analysis).Furthermore,single cell data were processed into scatter plots,and the pH distribution characteristics of single cell were analyzed from multiple angles.In the detection of oxidative stress,4-hydroxyphenylthiophenol was used as a REDOX probe to obtain the oxidation curve and reduction curve,and to test the cycling performance of the probe and the universality of its application in cells.Finally,the oxidative stress events of the extracellular changing with time under the stimulation of PMA were explored.