Fluorescent microparticles with controllable enhanced stokes shift

• US 5,326,692 A
• Filed: 05/13/1992
• Issued: 07/05/1994
• Est. Priority Date: 05/13/1992
• Status: Expired due to Term

• Current Assignee: BIOSITE INCORPORATED (Abbott Laboratories Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Brinkley, John M., Haugland, Richard P., Singer, Victoria L.
• Primary Examiner(s): Kepplinger, Esther L.
• Assistant Examiner(s): Green, Lora M.
• Application Number:
  US07/882,299
• Time in Patent Office: 783 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/7.92, 436/518, 436/528, 436/529, 436/520, 436/531, 436/546, 436/800, 427/213.34, 427/157, 428/40 R, 428/402.24, 428/407, 252/301.34, 252/301.35
• US Class Current: 435/6.12
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

The invention relates to microparticles incorporating a series of two or more fluorescent dyes having overlapping excitation and emission spectra allowing efficient energy transfer from the excitation wavelength of the first dye in the series, transfer through the dyes in the series and re-emitted as an optical signal at the emission wavelength of last dye in the series, resulting in a desired effective Stokes shift which is controlled through selection of appropriate dyes.

Claims

1. A fluorescent microparticle made by the process comprising:

a) selecting a series of dyes comprising an initial donor dye with a desired excitation peak and a final acceptor dye with a desired emission peak, wherein the desired excitation peak and the desired emission peak are determined in a polymeric material, and wherein said polymeric material is made up of polymerizable monomers, and wherein each dye in the series has a spectral overlap sufficient to allow for significant energy transfer of excitation energy to the final acceptor dye, b) incorporating said series of dyes in a polymeric microparticle comprising said polymeric material, wherein said series of dyes are randomly incorporated into said polymeric microparticle.

2. A microparticle, as claimed in claim 1, wherein the spectral overlap allows for energy transfer that is greater than about 90%.

3. A microparticle, as claimed in claim 1, wherein the polymeric microparticle is polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, or polydivinylbenzene.

4. A microparticle, as claimed in claim 1, wherein the fluorescent dyes are polyazaindacene;

coumarin;

oxazole or oxadiazole;

aryl- or heteroaryl-substituted polyolefin wherein the olefin portion has 2-8 carbons;

carbocyanine;

phthalocyanine;

oxazine;

carbostyryl;

or porphyrin dyes;

or combinations thereof.

5. A microparticle, as claimed in claim 4, wherein at least one of the fluorescent dyes is a polyazaindacene dye of the formula:

##STR2## wherein R₁ -R₆, which may be the same or different, are hydrogen, halogen, or alkyl, alkoxy, alkenyl, cycloalkyl, arylalkyl, acyl, wherein the alkyl portions each contain fewer than about 20 carbons, or aryl, heteroaryl, alone or in combination; and

R₇ is nitrogen, methine, or halogen-, alkyl-, alkyl-, alkoxy-, alkenyl-, cycloalkyl-, arylalkyl-, acyl-, (the alkyl portions of which each contain fewer than about 20 carbons) aryl-, or heteroaryl-substituted methine.

6. A microparticle, as claimed in claim 1, wherein at least one of the fluorescent dyes is a polyazaindacene dye of the formula:

##STR3## wherein R₁ -R₆, which may be the same or different, are hydrogen, halogen, or alkyl, alkoxy, alkenyl, cycloalkyl, arylalkyl, acyl, wherein the alkyl portions each contain fewer than about 20 carbons, or aryl, heteroaryl, alone or in combination; and

R₇ is nitrogen, methine, or halogen-, alkyl-, alkoxy-, alkenyl-, cycloalkyl-, arylalkyl-, acyl-, (the alkyl portions of which each contain fewer than about 20 carbons) aryl-or heteroaryl-substituted methine.

7. A microparticle, as claimed in claim 6, wherein the spectral overlap allows for energy transfer that is greater than about 95%.

8. A microparticle, as claimed in claim 1, wherein the series of fluorescent dyes contains less than about 6 dyes in a total dye concentration that is between about 0.5% and 2% weight to weight.

9. A microparticle, as claimed in claim 1, wherein the ratio of initial donor to ultimate acceptor is between about 1:

5 and about 10;

1.

10. A microparticle, as claimed in claim 1, wherein the polymeric microparticle is polystyrene, polymethylmethacrylate, polyacrylonitrile, polyacrylamide, or polyacrolein;

the series of fluorescent dyes contains less than about 6 dyes which are different polyazaindacene dyes of the formula;

##STR4## wherein R₁ -R₆, which may be the same or different, are hydrogen, halogen, or alkyl, alkoxy, alkenyl, cycloalkyl, arylalkyl, acyl, (the alkyl portions of which each contain fewer than about 20 carbons) or aryl, heteroaryl, alone or in combination; and

R₇ is nitrogen, methine, or halogen-, alkyl-, alkoxy-, alkenyl-, cycloalkyl-, arylalkyl-, acyl-, (the alkyl portions of which each contain fewer than about 20 carbons) aryl- or heteroaryl-substituted methine;

for which the total dye concentration is between about 0.8 and 1.2% (w/w); and

the ratio between initial donor and ultimate acceptor dyes is between about 4;

1 and about 6;

1.

11. A fluorescent microparticle comprising:

a series of fluorescent dyes having an initial donor dye with a desired excitation peak and an ultimate acceptor dye with a desired emission peak, wherein the desired excitation peak and the desired emission peak are determined in a polymeric material, wherein said polymeric material is made up of polymerizable monomers, and wherein each dye in the series has a spectral overlap sufficient to allow for significant energy transfer of excitation energy to the ultimate acceptor dye, wherein said series of dyes is randomly incorporated in a polymeric microparticle comprising said polymeric material such that the total dye concentration is less than about 10% w/w and the ratio of initial donor to ultimate acceptor is between about 1;

5 and about 1;

10.

12. A microparticle, as claimed in claim 11, wherein the microparticle is polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, or polydivinylbenzene and is less than about 15 micrometers in diameter.

13. A microparticle, as claimed in claim 12, wherein the series of dyes contains less than about 6 polyazaindacene dyes of the formula:

##STR5## wherein R₁ -R₆, which may be the same or different, are hydrogen, halogen, or alkyl, aldoxy, alkenyl, cycloalkyl, arylaklyl, acryl, wherein the alkyl portions each contain fewer than about 20 carbons, or aryl, heteroaryl, alone or in combination; and

R₇ is nitrogen, methine, or halogen-, alkyl-, alkoxy-, alkenyl-, cycloalkyl-, arylalkyl-, acyl-, (the alkyl portions of which each contain fewer than about 20 carbons) aryl-or heteroarylsubstituted methine.

14. A microparticle, as claimed in claim 13, further comprising a bioreactive substance, wherein said bioreactive substance has been either bound covalently or passively adsorbed.

15. A microparticle, as claimed in claim 14, wherein said bioreactive substance is a biomolecule that is biotin, avidin, streptavidin, digoxigenin, or a nucleic acid.