Electroporation Assisted Surface-enhanced Raman Spectroscopy For Cell Detection And Screening

Surface-enhanced Raman scattering(SERS)is a promising tool for cell biochemical detection and cancer screening.The SERS nano-probes(labeled/label-free Au/Ag nanoparticles or various functional nanoparticles)were first delivered into live cells,followed by the intracellular SERS measurements achieved by the confocal microscopic Raman spectrometer.With the capability of detecting molecular vibrations,SERS was widely used to analyze biochemical changes in life activities.In this study,to overcome the shortcomings of the existing nanoparticle delivery methods,we presented an optimized electroporation method to transfer nanoparticles into living cells.We also applied this method to detect cell biology and screen cancers.The main research results were as followed:1.We presented an optimized electroporation method for transferring nanoparticles into living cells.In the optimized scheme,we changed the direction and location of cells in the electric field,and this method can help form induced pores in different positions of the cell membrane.The nanoparticles then entered into cells from more induced-pores and diffused over the cytoplasm.The distribution of intracellular nanoparticles was monitored by the SERS signal of nano-probes and validated by TEM images.Compared with the previous electroporation method,the optimized electroporation achieved a quick delivery of nanoparticles into living cells(<10min)with a much more uniform distribution of nanoparticles in the cytoplasm.Results demonstrated the great potential for the optimized electroporation-based SERS analysis in the intracellular biochemical study.2.The electroporation-based SERS technique was used to detect the intracellular pH value.The pH-dependent SERS spectra of 4-mercaptobenzoic acid(4-MBA)adsorbed on gold nanoparticles were used to create a pH:eter working over the range of 6.0-8.0 pH units.The 4-MBA-coated nanoparticles were delivered into living cells by electroporation.SERS imaging indicated the intracellular pH distribution of a single live CNE1 cell,which was imaged according to the intensity ratio of the SERS peaks at 1423 cm-1 and 1074 cm-1.This work demonstrated the potential of electroporation-based SERS techniques in live cell biochemical detection.3.The electroporation-based SERS technique was employed to differentiate the human myeloid leukemia cells from the normal human bone marrow mononuclear cells with the aim to develop a fast and label-free method for leukemia cell screening.The Ag nanoparticles were delivered into living cells by electroporation,and then high-quality SERS spectra were successfully obtained from acute promyelocytic leukemia cells(HL60 cell line),chronic myelogenous leukemia cells(K562 cell line)and normal human bone marrow mononuclear cells(BMC).There were significant differences in SERS spectra between the myeloid leukemia cells and the normal bone marrow mononuclear cells.Tentative assignments of the SERS bands in the measured spectra showed specific biomolecular differences,including an increase in the relative amounts of proteins and a decrease in the percentage of nucleic acid in leukemia cells as compared to normal cells.Using SERS spectroscopy combined with PCA-LDA analysis,we were able to differentiate the leukemia cells(HL60 cells and K562 cells)from the normal cells with high diagnostic sensitivity(98.3%)and specificity(98.3%).Moreover,a diagnostic model developed by PLS successfully predicted the unidentified subjects with a diagnostic accuracy of 96.7%(29/30).The results of this study demonstrated that electroporation-based SERS spectroscopy in conjunction with PCA-LDA and PLS diagnostic algorithms has great potential for high-throughput cell screening applications.4.The electroporation-based SERS technique was applied to detect and differentiate the clinical nasopharyngeal cell samples obtained by nasopharyngeal swab.Nasopharyngeal cell samples from nasopharyngeal cancer patients(n=30)and non-tumor subjects(n=30)were analyzed.Tentative assignments of the Raman bands in the measured SERS spectra suggest eancer specifie biomolecular differences,including an increase in the relative amounts of nucleic acid and coenzyme and a decrease in the percentage of aminoacid and collagen in the cell sample of nasopharyngeal cancer patients as compared to non-tumor subjects.Principal component analysis(PCA)and linear discriminate analysis(LDA)of the measured SERS spectra separated the spectral features of the two groups into two distinct clusters with high sensitivity(100%)and specificity(96.67%).The results of this exploratory study demonstrated great potentials for developing swabbing-cell electroporation-SERS analysis into a novel clinical tool for non-invasive detection of nasopharyngeal cancers.