Fluorescence Polarization Immunoassay of a High-Molecular-Weight Antigen Based on a Long-Lifetime Ru-Ligand Complex

Abstract

We describe a new class of fluorescence polarization immunoassays based on the luminescence from an asymmetrical Ru-ligand complex. We found that such a complex displays larger polarization values than those of comparable symmetrical complexes and appear to be highly photostable in aqueous solution. We synthesized a conjugatable Ru-ligand complex, which was used to label human serum albumin (HSA) as the antigen. The Ru-ligand complex displays a long decay time near 400 ns when covalently linked to proteins. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format using unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high-molecular-weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2-5 ns decay time of the previously used fluorophores.

Long-Lifetime Metal–Ligand pH Probe

“…In this case the pK a was found we observed that the emission spectrum of to be 7.14, somewhat lower than the value of 7.5 ob-[Ru(dcbpy)(bpy) 2 ] 2/ , where dcbpy is 4,4-dicarboxyserved by fluorescence. This small difference in pK a 2,2-bipyridine, displays a red shift relative to Ru(bpy) 3 values is not surprising, as metal-ligand complexes (38,39). The dcbpy ligand is probably a better electron with ionizable diimine ligands often display different acceptor than bpy.…”

Synthesis, Electrochemical and Electrogenerated Chemiluminescence Studies of Ruthenium(II) Bis(2,2′‐bipyridyl){2‐(4‐methylpyridin‐2‐yl)benzo[d]‐X‐azole} Complexes

“…It must be noted that, besides the similarity in shape, the photoluminescent signal of the solution of 4 alone and 4-BSA equimolar in fluorophore (determined on the basis of the dye-to-protein ratio reported below) exhibited almost the same intensity, indicating that 4 maintains its Φ em when linked to the protein. The dye-to-protein ratio of the 4-BSA conjugate and the whole protein concentration were estimated by measuring the absorbance of 4-BSA at 460 nm (ε 460 = 12200  -1 /cm) [38,39] and by the Bradford method, [40] respectively. The molar dye-to-protein conjugation ratio of 4-BSA was 1.2.…”

Multilabeling Biomolecules at a Single Site. 1. Synthesis and Characterization of a Dendritic Label for Electrochemiluminescence Assays

“…The reason for choosing [Ru(bpy) 3 ] 2+ as a signal-generating unit of the prototype multilabeling reagent is 3-fold. First, [Ru(bpy) 3 ] 2+ derivatives are used not only as valuable probes [11][12][13][14] in biological systems such as nucleic acids, peptides, and proteins but also as labels for various bioanalytical assays such as fluorescence polarization 15 and electrochemiluminescence (ECL) immunoassays and DNA 28,29 a concern about the feasibility of the prototype label is still associated with the possible interaction among the very closely packed [Ru(bpy) 3 ] 2+ units. In this preliminary account, we report the synthesis, characterization, and ECL of the prototype label and demonstrate multilabeling a model protein with the prototype label at a single NH 2 position.…”