Lanthanide fluorophores labelling

Keywords Amplification Fluorescent reporter Fluorophore FRET In vitro In vivo Labeling Lanthanide chelate Multiplexing Nanoparticle Quantum dot Transition metal complex... 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Examples of fluorophores that are used as labels in fluo-roimmunoassay and their properties are listed in Table 9-3. Initially, background fluorescence from drugs, drug metabolites, and protein-bound substances, such as bilirubm, limited the utility of this technique. However, this problem has largely been overcome by the use of rare earth (lanthanide) chelates and background rejection (time-resolved)... 

Among nonisotopic techniques, fluorescence (both intrinsic and extrinsic) offers a convenient mode of detection, and the sensitivity of some fluorescent labels is comparable to that of radiolabeled iodine. Recent innovations include the use of polarized light for excitation, such that the degree of polarization of the emission as well as its intensity can provide information about the concentration and size-related behavior (e.g., rotational diffusion) of the fluorescent-labeled molecule. One disadvantage of steady-state fluorescence techniques is that many analytical samples either autofluoresce or quench the fluorescence of the substance of interest. A recent development that circumvents this problem utilizes long-lived fluorophores such as the lanthanide metal ions as labels. Detection is time resolved and data are collected after the decay of spurious or otherwise unwanted fluorescence, i.e., after 100-200 psec. 

These systems are promising as potential labels due to high emission quantum yields and excited-state lifetimes that can be as long as several tenths of a millisecond (108). A cyclen (12-ane-N4) unit connected to a phenanthridine moiety in fluorophore-spacer-receptor conhguration (Fig. 26) exhibit strong Tb(III) based luminescence (109) in the absence of protons and oxygen. Few other luminescent lanthanide complexes are available in the literature (110,... 

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