Heavy Water Combined With Raman Spectroscopy To Explore A New Method Of Breast Cancer Diagnosis

Background:Breast cancer is a common malignant tumor in women with an increasing incidence.Early detection and definite diagnosis are essential to improve prognosis.The current gold standard for breast cancer diagnosis,histopathology,has limitations such as time consuming,high cost and inability to make rapid diagnosis.Raman spectroscopy has been widely used in clinical diagnosis,tissue engineering and toxicology experiments due to its advantages of no label,low destruction,high sensitivity and strong specificity.However,due to the complexity of Raman spectra of biomolecules and confounding factors,its application is limited.Heavy water is composed of two deuterium（D）atoms and one oxygen（O）atom.Its chemical formula is D₂O.Heavy water is free to diffuse into cells and tissues where it binds to carbon（C）atoms in biomolecules such as lipids and proteins to form C-D bonds.Using Raman spectroscopy to detect cells,the heavy-water labeled C-D bond produces a specific C-D peak in the"silent region"of the spectrum(wavelength2040-2300cm^-1),which can measure metabolic activity of cells in a non-destructive manner at the single-cell level.Objective:This study intends to use heavy water as a new tracer for Raman spectroscopy detection of breast cancer cells,optimize the optimal detection parameters,and explore a new method for breast cancer diagnosis.As an auxiliary detection technology,it complements the current diagnostic methods to reduce the clinical limitations of traditional methods,further improve the accuracy,sensitivity and safety of breast cancer diagnosis,and reduce the false negative rate.Methods:Raman spectroscopy was used to detect the labeling of breast cancer cells and normal breast epithelial cells by heavy water.Origin software was used to analyze Raman spectroscopy,and cell metabolic indices was calculated by C-D peak generated by heavy water labeled cells.The influence of heavy water concentration and incubation time on cells was studied,and appropriate detection parameters were optimized.The data were statistically analyzed and graphed by Graph Pad Prime 8.0software,and the metabolic differences and associations between normal breast epithelial MCF-10A cells and three different types of breast cancer cells,such as MCF-7,MDA-MB-231 and SKBR-3,were compared.One-way ANOVA was used to determine whether the results were different.P<0.05 indicated significant statistical difference,and P<0.01 indicated extremely significant statistical difference.Results:At 30%concentration and 24H time range,heavy water had no effect on the important biological molecules in cells.The intensity of C-D peak in Raman spectrum and the metabolic indices of cells increased with the increase of heavy water concentration and incubation time.The optimal detection parameters were 30%concentration of heavy water and 24H incubation time.The metabolic indices of different types of breast cancer cells MCF-7,MDA-MB-231 and SKBR-3 were higher than those of normal breast epithelial cells MCF-10A（P<0.05）.The metabolic difference between breast cancer SKBR-3 cells and normal breast epithelial MCF-10A cells was the most significant among the three breast cancer cells（P<0.01）.Conclusion:In this study,heavy water labeled cells combined with Raman spectroscopy were used to detect the metabolic activity of breast cancer cells and normal mammary epithelial cells at the single-cell level.The optimal detection parameter was incubated in 30%heavy water for 24 hours.The single cell Raman spectra under the optimal detection parameters showed that the metabolic indices of different types of breast cancer cells labeled with heavy water were higher than those of normal breast epithelial cells,confirming the potential of heavy water combined with Raman spectroscopy as a new method for breast cancer diagnosis.