Cyclometalated transition metal complexes for multiplex analyte detection

A kit for labeling proteins, said kit comprising:

• a plurality of complexes, each complex comprising a transition metal ion;
• a plurality of donor ligands, each said donor ligand fully coordinated to said transition metal ion and each said donor ligand being either a nitrogen donor ligand or a cyclometalated donor ligand, such that at least one of said donor ligands is a cyclometalated donor ligand;
• and a reactive group substituted on at least one of said donor ligands through a linker, said reactive group reactive with a functional group to form a covalent bond, and said linker comprising a chain of 4 to 10 atoms;
• said complexes differentiable from each other by luminescent emissions emitted by said cyclometalated transition metal complexes upon excitation.

A method for individually detecting a plurality of proteins in a single liquid sample, said method comprising:

• (a) incubating said sample with a plurality of cyclometalated transition metal complexes that are equal in number to said proteins and that are selected such that said cyclometalated transition metal complexes are differentiable from each other by luminescent emissions emitted by said cyclometalated transition metal complexes upon excitation, each said cyclometalated transition metal complex further having bonded thereto an immunological binding member having selective binding affinity toward one of said proteins, to cause each protein to bind to a different cyclometalated transition metal complex by affinity binding through said immunological binding member;
• and
• (b) with said proteins so bound, detecting cyclometalated transition metal complexes bound to said proteins while differentiating said cyclometalated transition metal complexes so detected by luminescent emissions, thereby individually detecting said analytes;
• each said cyclometalated transition metal complex comprising:
• a transition metal ion,
• a plurality of donor ligands, each said donor ligand fully coordinated to said transition metal ion and each said donor ligand being either a nitrogen donor ligand or a cyclometalated donor ligand, such that at least one of said donor ligands is a cyclometalated donor ligand, and
• a reactive group substituted on at least one of said donor ligands through a linker, said reactive group reactive with a functional group to form a covalent bond, and said linker comprising a chain of 4 to 10 atoms.

A method for comparing a plurality of samples for their contents of selected proteins defined as proteins of interest, said method comprising:

• (a) preparing an extract of proteins from each of said samples;
• (b) reacting each said extract with a different dye from a set of matched luminescent dyes to form dye-labeled proteins, wherein each said luminescent dye comprises:
• a transition metal ion,
• a plurality of donor ligands, each said donor ligand fully coordinated to said transition metal ion and each said donor ligand being either a nitrogen donor ligand or a cyclometalated donor ligand, such that at least one of said donor ligands is a cyclometalated donor ligand, each dye differing from the other dyes in said set by a difference in a donor ligand, said difference causing each dye of said set upon excitation to emit luminescent light at a spectrum that is sufficiently different from the spectra of emitted luminescent light from the remaining dyes of said set to provide each dye with a detectably distinct signal, and
• a reactive group substituted on at least one of said donor ligands through a linker, said reactive group reactive with one of said proteins to bind said protein to said donor ligand, optionally through an affinity binding member, said linker comprising a chain of 4 to 10 atoms;
• (c) mixing said extracts resulting from step (b) to form a single combined mixture of dye-labeled proteins;
• (d) separating dye-labeled proteins of interest from other dye-labeled proteins in said single combined mixture;
• and
• (e) irradiating said separated dye-labeled proteins of interest with excitation energy and detecting differences in luminescence intensity between the dye-labeled proteins of interest labeled with one dye and the dye-labeled proteins of interest labeled with other dyes as an indication of differences in amounts of said proteins of interest among said samples.

A method of detecting an analyte in a sample, said method comprising

• (a) contacting said sample with a luminescent complex comprising:
• a transition metal ion,
• a plurality of donor ligands, each said donor ligand fully coordinated to said transition metal ion and each said donor ligand being either a nitrogen donor ligand or a cyclometalated donor ligand, such that at least one of said donor ligands is a cyclometalated donor ligand, and
• a binding member selected from the group consisting of an antibody, an oligonucleotide, and an affinity-type binding reagent other than antibodies and oligonucleotides, and covalently attached to one of said donor ligands through a linker comprising a chain of 4 to 10 atoms, said binding member being interactive with said analyte upon contact to bind thereto by either a covalent bond or an affinity-type interaction;
• and
• (b) isolating components of said sample to which said luminescent complex is bound in step (b) from unbound molecules of said luminescent complex, and
• (c) irradiating said sample with excitation energy and detecting luminescence emitted therefrom as an indication of the presence of said analyte in said sample.