Porphyrinic compounds for use in flow cytometry
The present invention provides a method of detecting (e.g., by flow cytometry) a target compound, cell or particle, wherein the target is labelled with a detectable luminescent compound. The method comprises utilizing as the detectable luminescent compound a compound comprising a porphyrinic macrocycle such as a porphyrin, chlorin, bacteriochlorin, or isobacteriochlorin. In particular embodiments, the detectable luminescent compound comprises a compound of the formula A-A′-Z—B′—B, wherein: A is a targeting group or member of a specific binding pair that specifically binds the detectable luminescent compound to the target compound, cell or particle; A′ is a linker group or covalent bond; B′ is a linker group or covalent bond; B is a water-soluble group; and Z is the porphyrinic macrocycle.
1. In a method of detecting and distinguishing first and second target compounds, cells or particles, wherein said first target is labeled with a first detectable compound and said second target is labeled with a second detectable compound, the improvement comprising:
utilizing a compound comprising a first porphyrinic macrocycle as said first detectable compound and a compound comprising a second porphyrinic macrocycle as said second detectable compound;
wherein each of said first and second detectable compounds are excited at an excitation wavelength band;
wherein said first porphyrinic macrocycle comprises a porphyrin, chlorin, bacteriochlorin or isobacteriochlorin, and said second porphyrinic macrocycle comprises a porphyrin, chlorin, bacteriochlorin, or isobacteriochlorin;
and wherein:
(i) each of said first and second detectable compounds have a different emission wavelength band, said different emission wavelength bands characterized by peaks that are separated from one another by at least 5 nanometers; or
(ii) said second compound has a lifetime at least 20 percent greater than said first compound.
2. The method of claim 1 , wherein said first and second detectable luminescent compounds each comprise a compound of the formula A-A′-Z—B′—B, wherein:
A is a targeting group that specifically binds said detectable compound to said target compound, cell or particle;
A′ is a linking group or covalent bond;
B′ is a linking group or covalent bond;
B is a water-soluble group; and
Z is said porphyrinic macrocycle.
3. The method of claim 1 , wherein said detecting step is carried out by flow cytometry.
4. The method of claim 1 , wherein
both of said excitation wavelength bands have a peak between 350 and 900 nanometers; and
both of said emission wavelength bands have a peak between 550 and 1000 nanometers.
5. The method of claim 1 , wherein said different emission wavelength bands are characterized by peaks that are separated from one another by at least 50 nanometers.
6. The method of claim 1 , wherein said different emission wavelength bands are characterized by peaks that are separated from one another by 10 to 50 nanometers;
and wherein each of said different emission wavelength bands have a full width at half maximum peak of not more than 30 nanometers.
7. The method of claim 1 , wherein said first porphyrinic macrocycle is a chlorin or bacteriochlorin, and wherein said second porphyrinic macrocycle is a chlorin or bacteriochlorin.
8. The method of claim 7 , wherein said first and second detectable luminescent compounds each comprise a compound of the formula A-A′-Z—B′—B, wherein:
A is a targeting group that specifically binds said detectable compound to said target compound, cell or particle;
A′ is a linking group or covalent bond;
B′ is a linking group or covalent bond;
B is a water-soluble group; and
Z is said porphyrinic macrocycle.
9. The method of claim 7 , wherein said detecting step is carried out by flow cytometry.
10. The method of claim 7 , wherein
both of said excitation wavelength bands have a peak between 350 and 900 nanometers; and
both of said emission wavelength bands have a peak between 550 and 1000 nanometers.
11. The method of claim 7 , wherein said different emission wavelength bands are characterized by peaks that are separated from one another by at least 50 nanometers.
12. The method of claim 7 , wherein said different emission wavelength bands are characterized by peaks that are separated from one another by 10 to 50 nanometers;
and wherein each of said different emission wavelength bands have a full width at half maximum peak of not more than 30 nanometers.