Diagnosis Of Tumor And Study On Metabolic Activity Of Single Cell By Raman Spectroscopy

The death of 90%cancer patients is caused by tumor metastasis.Circulating tumor cells（CTCs）are rare tumor cells released from primary tumors focus into the peripheral blood circulation,which have been used as important biomarkers in blood for early diagnosis and evaluation of cancer metastasis.However,the rapid and accurate detection of rare CTCs in human patient blood is still a challenge due to their extremely low abundance compared to blood cells.At the same time,the measurement of tumor cell metabolic activity is of great significance for the screening of anti-tumor drugs,but there is still a lack of single cell level measurement methods for tumor cell metabolic activity.In this study,efficient capture and specific detection of CTCs,as well as the measurement of metabolic activity of tumor cells at the single cell level,were investigated.Single-cell analysis system based on surface-enhanced Raman spectroscopy（SERS）and stable isotope-Raman spectroscopy（SIP-Raman）was established.1.Detection of single tumor cells by microporous membrane capture combined with aptamer-SERS nanoprobeThe system combining aptamer-SERS nanoprobes with microporous filter membrane for the capture and detection of tumor cells was established based on parlyene microporous filter membrane for efficient cell capture and aptamer-SERS nanoprobes for specific identification of tumor cells,which can quickly detect non-small cell lung cancer A549 tumor cells in blood at the single cell level.Three kinds of parlyene filter membranes（poly-p-xylene）with pore sizes of 13μm,16μm,and 18μm were designed and prepared,which could remove red blood cells in 1 m L of blood within less than 10 seconds only under gravity and retain tumor cells,leaving only very few white blood cells（WBCs）.Compared with the traditional"salt aging"synthesis method,the optimized"ethyl acetate dehydration method"can rapidly synthesize SERS nanoprobes（Au NPs@4-MBA-Aptamer）within 2 hours,and without Raman signal interference from dehydration solvent.Among the seven reported A549 specific aptamers,the aptamer with the be the highest specific recognition ability for A549 tumor cells in blood was selected.The specificity of the SERS nanoprobes was well demonstrated by using human cervical cancer cell line He La as a negative control.Based on the comparison of the Raman signal intensity of Raman report 4-mercaptobenzoic acid（4-MBA）at 1075 cm^-1,A549tumor cells can be effectively distinguished from the residual WBCs on the membrane（P<0.001）.The system can detect 100 A549 cells in 1m L blood sample,with a recovery rate and identification rate>90%,（RSD=2.20%）.2.Detection of single tumor cell by Raman spectroscopy of heavy water metabolism.To realize a simpler tumor cell detection,without the use of antibodies or aptamers to synthesize biological probes,a method of heavy water-single cell Raman spectroscopy（D₂O-SCRS）was established,and single tumor cells were identified by the characteristic"C-D"Raman peak formed by deuterium（D）metabolism in D₂O.The"D-H"exchange of D₂O with the compounds in tumor cells with high metabolism generates the"C-D"Raman characteristic peak with signal range of 2100～2300 cm^-1.The CCK-8 assay and ATP-bioluminescent cell viability method were used to detect the cytotoxicity of D₂O of different concentrations.After incubation in 30%D₂O cell culture medium for 48 hours,the cell activity remained above 95%.WBCs obtained by lysing blood cells from human fingertip blood were co-cultured with A549 tumor cells in 30%D₂O cell culture medium for24 hours for single-cell Raman analysis.D₂O-SCRS method can distinguish normal cells from tumor cells,and can be used as a new method to detect circulating tumor cells in blood.3.Detection of single cell activity by Raman spectroscopy of heavy water metabolism.Raman spectroscopy combined with stable isotope probing（Raman-SIP,Stable Isotope Probing）can detect single-cell metabolic activity in situ,and quantify cell activity based on the characteristic Raman signals intensity of C-D peak.As H₂O is involved in most of cellular metabolic processes,D₂O can generate D-H exchange with the compounds in the tumor cells during metabolism,forming C-D bonds in the cells.Due to the distinct changes of C-D peak signal at 2100～2300cm^-1easy to observe,the ratio of Raman peak areas（C-D）/Raman peak areas（C-D+C-H）can be used to quantify the metabolic activity of cells,presenting cellular metabolic heterogeneity of single cell.The addition of anticancer drugs can inhibit cell metabolic activity,and by measuring the inhibition of drugs on single-cell activity,the IC50 values of each drug on single cell can be obtained for drug screening.The IC50 values obtained by the D₂O-SCRS method were almost the same as the IC50value of the traditional cell viability methods based on ATP-bioluminescent cell viability method and the CCK-8 assay.The results showed that the relative standard deviation（RSD）of relative metabolic activities values of cisplatin and taxol on A549 tumor cells were 3.79%～10.36%and 3.32%～11.25%,respectively,and the RSD of IC50 for repeated testing on different days was 9.06%,showing good reproducibility and daytime reproducibility,it can meet the requirements of drug screening.The established perelin microporous filter membrane capture aptamer-SERS probe system could easily and specifically detect single non-small cell lung cancer circulating tumor cells in peripheral blood.Based on the C-D peak of D₂O metabolism characteristic of tumor cells,the D₂O-SCRS method achieves the low-cost detection of single tumor cells in blood without the need of biological probe labeling.Based on the inhibition of D₂O metabolism in tumor cells by antitumor drugs,the Raman intensity of C-D peaks was determined to achieve quantitative detection of metabolic activity at the single cell level.Compared with traditional population cell activity assay methods,this method is equivalent and can be used for screening anticancer drugs.