Synthesis Of Rare Earth Fluorescent Complexes And Study On Their Protein Labeling

Rare earth elements have unique fluorescence properties,including the advantages of large Stokes shift and high fluorescence intensity.Homogeneous time-resolved fluorescence immunoassay(HTRF)has been widely used in biomarker immunoassay in recent years.The technique has been widely used in biochemical experiments and mass production because of its high sensitivity and convenient operation.To prepare rare earth fluorescent complexes labeled with protein,the paper synthesized a kind of Tb³⁺fluorescent organic complexes,determine the optimal synthetic route,the most convenient,the structure characterization,the spectrum properties,and a marked monoclonal antibodies,immune activity of protein markers before and after the test,to explore the best mark condition at the same time,indicates the synthetic experiment of Tb³⁺fluorescence complexes can be used for biological molecular markers.On the one hand,3-methyl-o-hydroxybenzoic acid was used as starting material to produce amide compounds with unique structure through a series of reactions such as acidification,acyl chloride and so on.On the other hand,dichloroethylamine hydrochloride was used as the starting material for another part of the reaction,the heterocyclic compounds were formed by ring-closing and ring-opening reactions,and then replaced by strong oxidant of hydrogen bromide to form hydrobromate.The polyamino compounds N,N,N',N'-Tetra(2-aminoethyl)ethylenediamine were synthesized by selective debromination using Silica Gel.A new organic Ligand,tetramethyl-4-H(2,2)-2-methoxyisophthalamide-tetrathiazoline,reacted with 5-amino-(2-aminoethyl)-H(2,2)-aminotert-butyl Ester in the presence of dichloromethane as solvent,a"cage"organic complex of tetramethyl-tert-butyloxycarbonyl protecting group-lysine-BH(2,2)IMA is formed.By adding and replacing the"arm"that modifies one end of the compound,a carboxyl end that can be firm Ly attached to the protein is formed.The Organic Ligand was bound to Tb³⁺,which made the complex have fluorescence effect,identify the compound by mass spectrometry and explore its spectral performance The maximum excitation wavelength of this complex is 375 nm,the emission wavelength is 583 nm,the fluorescence lifetime is 1023.12,and the quantum yield is 13.3%.It has good luminous power.The fluorescent complex of Tb³⁺was used to label AFP McAb,and activity before and after measurement.The optimal coating concentration of alpha-fetoprotein monoclonal antibody is 5?g/ml,the activity of antibody had no significant change before and after labeling,and the titer was 1:2400.Under the condition of p H 6,the optimal activation time is 10 h for Tb³⁺fluorescent complex,and the maximum labeling rate is reached when the incubation time of Tb³⁺fluorescent complex reacting with antibody is 1.5 h.These results indicate that Tb³⁺fluorescent complex has been synthesized successfully,and the feasibility of labeling with AFP McAb has been confirmed,which provides a new idea for immunoassay technology.