Quantitative Detection And Imaging Of Sialic Acid On Cell Surface With Cyano-modified SERS Probes

Sialic acid(SA)is a special monosaccharide widely distributed at the termini of glycan chains on the cell surface.At present,a lot of evidence indicates that the overexpression of SA on the cell surface is closely related to the deterioration or metastasis of many kinds of tumors.Therefore,it is important for the diagnosis and treatment of tumors to accurately monitor the expression of SA on cell surface.Available approaches for SA detection are mainly based on electrochemistry,mass spectrometry,fluorescence,and surface-enhanced Raman scattering(SERS).SERS is a spectroscopic technique to obtain the fingerprint spectrum of molecular vibration.Because of its high sensitivity,and nondestructive and non-invasive characteristics,it has become a new tool for glycan research.Cell is the basic unit of structure and function of organism.It is composed of several biomacromolecules,such as carbohydrate,protein,nucleic acid and so on.And these biomolecules have complex fingerprint information.When the SERS nanoprobes bind to the cell surface,some biomolecules will be adsorbed on the substrate surface.They will produce strong Raman responses in the 200-1800 cm-1 region,and Raman signals of the classical Raman reporter molecules are also in the fingerprint region.The overlapping of Raman signals between reporters and biomolecules often has a significant impact on the quantitative analysis and imaging.The groups of alkynyl,azide,cyanide,deuterium and metal carbonyl show sharp and strong Raman scattering peaks in the Raman silent region of 1 800-2800 cm-1.These peaks can avoid the background interference of cellular endogenous substances.Therefore,the substance producing SERS signals in the Raman silent region can be used as the Raman reporter molecules,to realize the quantitative analysis and imaging without background interference in the cells.In this work,a novel SERS nanoprobe of silver nanoparticles functionalized with 4-mercaptophenylboric acid and 4-mercaptobenzenitrile was developed to quantitate the SA on the tumor cell surface.4-mercaptobenzonitrile was selected as the Raman reporter molecule.The C=N group exhibited a distinct Raman scattering peak at 2232 cm-1,which avoided overlapping with the Raman signals of the biomolecules.The boric acid groups have high selectivity to SA molecules under physiological conditions.When 4-mercaptophenylboric acid combined to SA molecules,the SERS nanoprobes were drawn close to the cell surface.The intensity of C?N Raman signal is closely related to the number of cells and the amount of sialic acid on the cell surface.The expression level of SA can be accurately quantified on the basis of the C?N Raman signal collected from the cell surface.The results suggest that the Raman signal at 2232 cm-1 can completely avoid the background interference from endogenous substances(such as protein and nucleic acid).The probe has high sensitivity and good biocompatibility,and the detection limit is 0.079 pmol·L-1.The number of sialic acid molecule is about 4.6×107 on the surface of single HeLa cell.In addition,the SERS probes were used for Raman imaging of sialic acid on cell surface,and were also used to dynamically monitor the changes of sialic acid expression level on tumor cell surface under the influence of drugs.It is of great significance to reveal the relationship between tumors and cell surface glycosylation,to further understand the role of glycosylation in the formation and migration of malignant tumors.