The Preparation And Characterization Of Quantum Dots Modified With Lectin And Its Application In The Diagnosis Of Hepatocellular Carcinoma

objective: The preparation and characterization of quantum dots(QDs) modified with lectins and their applications in diagnosis of hepatocellular carcinoma(HCC).Methods: 1. The synthesis and characterization of QDs: Different particle sizes of water-soluble QDs were synthesized by two-step synthesis in the aqueous phase; UV-visible spectra and fluorescence spectrum were used to characterize the optical properties of QDs, and good optical properties of green QDs(QD550) and red QDs(QD618) were selected for the following research; MTT was used to evaluate the cytotoxicity of QDs and the suitable concentration of QDs was screened for subsequent experiments.2. The preparation and characterization of quantum dots modified with lectins: Using the carboxyl on the surface of QDs and the amino on the surface of lectins to prepare quantum dots modified with lectins; Gel electrophoresis technique were used to verify whether datura stramonium agglutinin(DSA) and lens culinaris agglutinin(LCA) were coupled with QD550 and QD618 respectively. The optical property of fluorescent probe was investigated by ultraviolet spectrum and fluorescence spectrum. The rabbit erythrocyte agglutination test and sugar inhibition experiment were used to investigate the biologic activity of DSA and LCA before and after being crosslinked with QDs. MTT method was used to test the toxic effects of quantum dots modified with lectins for Hep G2 cells and LO2 cells.3. The study of specificity targeted ability of the two kinds of fluorescent probes for hepatocellular carcinoma: Selecting Hep G2 cells as cell model, Hep G2 cells were labeled with the two kinds of fluorescent probes respectively which was characterizated qualitativly by inverted fluorescence microscope; Combining control experiments and lectin binding competition experiments to study the specificity targeting ability of the two kinds of fluorescent probes for the carbohydrates on the surface of Hep G2 cells; Selecting Hep G2 cells and LO2 cells as cell model, we analysed quantitatively expression levels of the two kinds of carbohydrates on surface of Hep G2 cells by using flow cytometry and compared the differences of the expression levels between Hep G2 cells and LO2 cells to evaluate the diagnosis efficacy of QD550-DSA and QD618-LCA for HCC.Results: The optimized synthesis conditions of QDs were that the best molar ratio of Cd2+: Te: Na BH4 was 1:0.5:10, reaction temperature was 98℃, and p H value was 10.5; Under the optimal reaction conditions, the quantum yield was 28.9% and uniform sizes, a diameter of about 2~3nm, good fluorescence stability of water-soluble QDs was synthesized; QD550 and QD618 were coupled respectively with DSA and LCA to obtain quantum dots modified with lectins(QD550-DSA and QD618-LCA); The results of ultraviolet spectrum and fluorescence spectrum verified that QD550-DSA and QD618-LCA kept the original optical properties of QD550 and QD618; What’s more, the rabbit blood agglutination experiment confirmed QD550-DSA and QD618-LCA kept the original protein activity of DSA and LCA and MTT results showed that the toxicity of QD550-DSA and QD618-LCA for LO2 cells and Hep G2 cells was low; The results of fluorescence imaging showed that QD550-DSA and QD618-LCA could effectively target the specific carbohydrates on surface of Hep G2 cells; By flow cytometry analysis, the combination with fluorescent probes was higher than bare QDs(p<0.01) and when the Hep G2 cells were saturated with lectin, the combination with fluorescent probe were much lower than before(p<0.05), showing that the combination of fluorescent probes and Hep G2 cells was specific; And the centents of N-acetylglucosamine on the surface of the Hep G2 cells was 1.64 times than that of LO2 cells(p<0.05) and the centents of mannose on the surface of the Hep G2 cells was 1.82 times than that of LO2 cells(p<0.01), showing that the expression levels of carbohydrates between LO2 cells and Hep G2 cells existed significant differences; And the two kinds of quantum dots modified with lectins could be used for the diagnosis of HCC.Conclusion: Under the optimal reaction conditions, two kinds of excellent optical performance, high optical stability and good biological activity of quantum dots modified with lectins were prepared. The two kinds of quantum dots modified with lectins could be used in the detection of carbohydrates of HCC qualitativly and quantitativly, and the expression levels of the two kinds of carbohydrates were higher in Hep G2 cells than in LO2 cells. Thus the two kinds of quantum dots modified with lectins could be used for diagnosis of HCC.