Metal chelates and methods of using them for time-resolved fluorescence

Abstract

β-diketone fluorescent tags are disclosed, particularly those enabling the use of excitation energy in the near visible or visible spectrum. In some cases, these tags allow the use of cost-effective excitation devices such as LED's. The compounds form fluorescent chelates (complexes) with lanthanide(III) rare earth metal ions (such as Eu3+). The fluorescent complex may be included in a latex microparticle, such as a styrene latex particle. Ideally, the complex has an absorption maximum λ equal to or greater than 360 nm, and the compound is characterized by a pKa <9.0. Kits and methods for detecting target molecules (e.g. immunoassays) are also disclosed. Such methods and kits typically use a ligand for binding to the target molecule and a labeling agent attached to the ligand. The fluorescent complexes described above are at least part of the labeling agent. Apparatus for detecting fluorescence from a sample includes an irradiating energy source that produces irradiating energy λ equal to or greater than 360 nm; a detector positioned to detect fluorescence from the sample; and a sample holder for holding the sample in position to be irradiated by the energy source. A light-emitting diode is preferably used as the irradiating energy source.

1 - 32 . (canceled)  
     
     
         33 . Apparatus for detecting fluorescence from a sample comprising 
 an irradiating energy source that produces irradiating energy λ equal to or greater than 360 nm;    a detector positioned to detect fluorescence from the sample; and    a sample holder for holding the sample in position to be irradiated by the energy source.    
     
     
         34 . The apparatus of  claim 33  in which the irradiating energy source is a semiconductor light source.  
     
     
         35 . The apparatus of  claim 34  in which the irradiating energy source is a light-emitting diode.  
     
     
         36 . The apparatus of  claim 34  in which the irradiating energy source is a laser diode.  
     
     
         37 . The apparatus of  claim 34  in which the detector comprises circuitry to allow instantaneous or time-resolved detection of the fluorescence.  
     
     
         38 . The apparatus of  claim 37  in which the detector comprises circuitry to allow time-resolved detection of the fluorescence.  
     
     
         39 . The apparatus of  claim 34  in which the sample holder is sized and designed for a detection process selected from the group consisting of: cell imaging, flow cytometry, immunohistochemistry, and immunoassay.  
     
     
         40 . The apparatus of  claim 34  further comprising an aqueous sample position in said sample holder.  
     
     
         41 . The apparatus of  claim 40  in which said aqueous sample comprises a fluorescent Europium chelate in aqueous solvent.