Surface-Enhanced Raman Spectroscopy For Investigations Of Tumor Cell Membrane Proteins Under External Stimulations

Tumor is a major disease plaguing human beings at present.With the advancement of technology,the research on tumors has become more in-depth.Metformin is a common drug for patients with diabetes.It has been reported that metformin combined with heme has a good effect in treating triple-negative breast cancer patients.Studies have shown that under low glucose/metformin treatment,intracellular glycogen synthase kinase 3?(GSK3?)is overactivated and promotes apoptosis.In patients with advanced colorectal cancers,mutations in KRAS,one of the RAS genes that may lead to cancer,allow metformin to accumulate in large amounts in cancer cells,ultimately inhibiting cancer cell proliferation Clinical and laboratory studies suggest that metformin plays a positive role in tumor therapy.However,the specific mechanism is unclear,so the role of metformin in treatment can be further understood by observing tumor cell-associated proteins.When N-cadherin is on the surface of tumor cells,it suggests that the metastatic ability of such tumor cells is enhanced.Aberrant expression of N-cadherin is found in a large number of tissues of cancer,such as lung,breast,prostate and squamous cell carcinomas.Aberrant expression of N-cadherin is closely associated with aspects of human malignancy progression,transformation,adhesion,apoptosis,angiogenesis,invasion and metastasis,suggesting that N-cadherin may contribute to metastatic and therapeutic targets for invasion.By observing the effect of metformin on N-cadherin,the role of metformin in tumor metastasis can be obtained.Various previous studies have suggested that electrical stimulation can induce tumor cell apoptosis.However,the demarcation between tumor tissues and cells and normal tissues and cells is often unclear in clinical practice.Under electrical stimulation,it may lead to apoptosis of normal cells and cause local damage.While weak electrical stimulation does not lead to cell apoptosis,it can still cause cell damage,expose proteins with specific immunogenicity,and induce the immune system in vivo to clear tumor cells.Along with the positive effect of metformin on the tumor treatment process,through the changes of cell surface proteins and cell nuclei,these two treatment methods are sought to achieve the therapeutic effect of tumors in the case of less impact on normal cells.Develop a gentle,less invasive entry point for the treatment of tumors.Raman spectroscopy is a fingerprint for substances.Surface-enhanced Raman technology is used to enhance Raman spectral signals.The method of modifying antibodies and beacon molecules of related proteins on the surface of precious metals can qualitatively analyze specific proteins and indirectly reflect the number of proteins through the Raman signal intensity of beacon molecules.The paper analyzes the effects of metformin and electrical stimulation in the tumor treatment process at the subcellular level through the detection results of labeled and label-free surface-enhanced Raman spectroscopy.The main contents are as follows:(1)for understanding the change of the N-cadherin by the metformin.construct an immuno-Raman probe to detect the signal of tumor cells and normal cells coincubated with metformin and compare the changes of cell membrane surface proteins after co-incubation with metformin by the SERS intensity of the labeled immunoprobe,and detect by label-free SERS.The labeled immunoprobe is composed of a silver nanosphere as a base,an antibody to N-cadherin on the surface of the assembled cell membrane and 4-mercaptobenzoic acid as a labeled probe molecule.The substrate and probe molecules are anchored on the surface of the cell membrane by an antibody against N-cadherin,and the difference in the total amount of surface proteins is indirectly reflected by detecting the intensity of the labeled probe molecules in the Raman spectrum.Label-free nuclear probes were constructed by assembling cell penetrating peptides(RGD,RGDRGDRGDRGDPGC)and nuclear targeting peptides(NLS,CGGGPKKKRKVGG)on the surface of silver nanospheres.The effect of metformin on the expression of N-cadherin on the cell membrane surface of tumor and normal cells was analyzed by the change of the characteristic signal intensity of 4-mercaptobenzoic acid detected by SERS.The effect of metformin on the nucleus at the molecular level was obtained by comparing the changes of the SERS spectra of the nucleus obtained by the labeled probes before and after the metformin co-incubation.(2)Based on the immune probe technology,the effect of electrical stimulation on the immune exposure of tumor cells was obtained.By comparing on the immune escape proteins of the cell surface before and after the electrical stimulation,it found that under weak electrical stimulation,although no apoptosis occurred,due to the change of programmed cell death-ligand 1(PD-L1)of the cell membrane surface,To achieve the purpose of making tumor cells lose the function of immune escape.The maximal voltage at electrical stimulation causing less apoptosis was first identified by live/dead staining.The constructed surface was assembled with signal molecules of 4-mercaptobenzoic acid and silver nanospheres of the anti-PD-L1 antibody as substrates.The intensity changes of the signal molecules before and after electrical stimulation were detected,it was confirmed that the immune evasion ability of tumor cells was weakened.Through the SERS spectrum analysis of label-free modified silver nanospheres as substrates,the molecular effects on cell membranes after electrical stimulation were found,and the reasons for tumor immune exposure after electrical stimulation were obtained.(3)The effect of metformin on cell electrical stimulation was obtained by detecting cell nuclei.Comparing the changes in cell death rate caused by electrical stimulation after co-incubation with metformin,by comparing the SERS results of the nuclei of such cells,the additive effect after simultaneous incubation with metformin and electrical stimulation was obtained.Comparing the detection results of WST-1 in electrical stimulation before and after co-incubation with metformin,the additive effect of metformin and electrical stimulation was obtained.It proved that the amidopurine descended and the thymidine conformation changed by two conditions.