Hence, the transmission from your Ru complex is usually expected to be 80-fold smaller than that for fluorescein

Hence, the transmission from your Ru complex is usually expected to be 80-fold smaller than that for fluorescein. of high-molecular-weight analytes. The use of such metalligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2C5 ns decay time of the previously used fluorophores. Fluorescence-based assays are widely used in analytical and clinical chemistry (1C3). Many of these applications of fluorescence are based on the use of immunological methods which provide the specificity needed to selectively detect a wide variety of antigens (4C6). Fluorescence immunoassays can be based on a variety of mechanisms which impact the observed spectral properties, including fluorescence quenching, enhancement, polarization, energy transfer, and the use of fluorogenic substrates (6). Fluorescence polarization immunoassays (FPI)1 is one of the more versatile and widely used methods. The assays are based on polarization measurements of antigens labeled with fluorescence probes, typically fluorescein derivatives. This method was launched by several groups (7C11) and is now in widespread commercial use in an instrument marketed by Abbott Laboratories. A serious limitation of present immunoassays is usually that they are limited to low-molecular-weight antigens. This limitation is the result of the use of fluorophores, such as fluorescein, which display lifetimes near 4 ns. A FPI requires that this emission from your unbound labeled antigen be depolarized, so that an increase in polarization may be observed upon binding to antibody. For depolarization to occur the antigen must display a rotational Cefpodoxime proxetil correlation time much shorter than 4 ns, which limits the FPI to antigens with molecular excess weight less than several thousand daltons. In the present paper we demonstrate that this Ru complex [Ru(bpy)2 (dcbpy)] displays high polarization in viscous answer and when bound to proteins. This is Nfia a amazing result because one may expect such symmetric complexes to have low polarization due to randomization of the excited state among the three ligands. Importantly, the lifetime of these complexes is near 400 ns. Consequently, the use of such metal-ligand complexes Cefpodoxime proxetil in FPI should allow immunoassays of antigens with molecular weights up to one million daltons. THEORY The fluorescence polarization (is the Boltzmann constant, is the absolute temperature (is the viscosity, and is the molecular volume (12). The molecular volume of the protein Cefpodoxime proxetil is related to the molecular weight (is the ideal gas constant, is the specific volume of the protein, and is the hydration, typically 0.2 g H2O/g of protein. Generally, the observed correlation times are about twofold longer than those calculated for an anhydrous sphere (Eq. [3] with = 0) due to the effects of hydration and the nonspherical shapes of most proteins. Hence, in aqueous solution at 20C (= 1 cP) one can expect a protein such as HSA (and are the vertically and horizontally polarized components of the emission (13). The values of and can be interchanged using and are both in common use. The values of are used more often in FPI because they are entrenched by tradition and slightly larger than the anisotropy values. The parameter is preferred on the basis of theory. The anisotropy of a labeled macromolecule is given by for the proteins in aqueous solution at 20C with a viscosity of 1 1 cP. The arrows and bars indicate = 0.029), which should be observed on total replacement of Ru-HSA by HSA. The most evident explanation for this effect would have been nonspecific binding of the Ru-HSA to other proteins present in solution. As the polarization of Ru-HSA.