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Ratiometric Immunosensor for GP73 Detection Based on the Ratios of Electrochemiluminescence and Electrochemical Signal Using DNA Tetrahedral Nanostructure as the Carrier of Stable Reference Signal.

DNA tetrahedron nanostructure (DTNs) has the merits of simple synthesis, high yield, structural stability, and mechanical rigidity, and its three-dimensional structure provides a satisfactory biosensing interface to the improvement of the binding efficiency of antigenic proteins and antibodies. Electrochemiluminescence (ECL) reagent, tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)ruthenium(II) dichloride (Ru(dcbpy)3Cl2), was modified on the electrode through the formation of classical sandwich complex of antibody-antigen-antibody. ECL response of the system increased with the increment of the target (golgi protein 73 (GP73) in this study) with high selectivity. Besides, the composed double-stranded DNA (dsDNA) in each side of DTNs could act as an excellent carrier of methylene blue (MB), thus producing a stable electrochemical internal reference signal on the electrode surface to correct the potential interferences. Therefore, a highly selective and reproductive ratiometric immunosensor was developed on the basis of the ratio of ECL of Ru(dcbpy)3Cl2 and electrochemistry of MB. The ratio value of the ECL/electrochemistry had a linear relationship with GP73 concentration in the range of 15 pg/mL-0.7 ng/mL, and the limit of detection was 15 pg/mL. The proposed ratiometric ECL immunoassay has been applied to detect GP73 in real serum samples with satisfactory results.