Fluorescent latex containing at least two fluorochromes, process for producing it and application thereof

• US 5,716,855 A
• Filed: 03/01/1996
• Issued: 02/10/1998
• Est. Priority Date: 07/12/1993
• Status: Expired due to Term

• Current Assignee: Societe prolufab
• Original Assignee: Societe prolufab
• Inventors: Vaslin, Sophie, Teychenne, Dominique, Ricchiero, Frederic, Richard, Joel, Lerner, Dan
• Primary Examiner(s): YOON, TAE H
• Application Number:
  US08/586,673
• Time in Patent Office: 711 Days
• Field of Search: 436/533, 436/172, 436/800, 523/200, 523/201, 252/301.35, 424/7.1
• US Class Current: 436/533
• CPC Class Codes: C08K 5/0041 Optical brightening agents, organic pigments
• CPC Class Codes: C08K 9/10 Encapsulated ingredients
• CPC Class Codes: C08L 21/02 Latex
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/582 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

Abstract

A fluorescent latex with particles containing at least one encapsulated hydrophobic fluorochrome A (acceptor) and at least one encapsulated hydrophobic fluorochrome D (donor), wherein A is different from D and exposure to light radiation enables the emission from fluorochrome D to excite fluorochrome A, and a method for producing said latex and its use as a marker, particularly a biological marker, are also disclosed.

Claims

1. Fluorescent latex the particles of which contain at least one hydrophobic fluorochrome A (acceptor) and at least one hydrophobic fluorochrome D (donor) which are encapsulated, A being different from D and the emission of the fluorochrome D making it possible, under the action of light radiation, to excite the fluorochrome A.

2. Fluorescent latex of claim 1, wherein the emission of the fluorochrome D makes it possible to excite the fluorochrome A directly, the emission spectrum of the fluorochrome D partially overlapping the excitation spectrum of the fluorochrome A.

3. Fluorescent latex of claim 2, wherein for at least 10% by weight of the particles, the A:

D molar ratio is greater than 1.

4. Fluorescent latex of claims 2 or 3, wherein the fluorochromes A and D are chosen from condensed, polyaromatics, tetraphenylporphine and one of the organometallic complexes thereof.

5. Fluorescent latex of claim 4, wherein the fluorochromes A and D are chosen from 9,10 diphenyl-anthracene (9,10 DPA), 9,10 bis-phenyl-ethynyl-anthracene (9,10 BPEA), 1,8 dichloro 9,10 bis-phenyl-ethynyl-anthracene (1,8 Cl₂ -9,10 BPEA), 5-12 bis-phenyl-ethynyl-naphthacene (5,12 BPEN), 6,13-bis (phenyl-ethynyl) pentacene, tetrabenzo(de,hi,op,st)pentacene, Coumarin 153, Nile red, 1,4-di 2(5-phenyloxazolyl)!-benzene (POPOP), 1,4-di 2-(4-methyl-5-phenyloxazolyl)!-benzene 1,4-di 4-methyl-5-phenyl-2-oxazolyl!benzene, and tetraphenylporphyrine (TPP).

6. Fluorescent latex of claim 5, wherein the fluorochromes A and D are chosen from the following pairs:

space="preserve" listing-type="tabular">______________________________________ D A ______________________________________ 9,10 DPA 9,10 BPEA 1,8 Cl.sub.2 -9,10 BPEA 5,12-BPEN 1,8 Cl.sub.2 -9,10 BPEA TPP Coumarin 153 1,8 Cl.sub.1 -9,10 BPEA 9,10 DPA Coumarin 153 1,8 Cl.sub.2 -9,10 BPEA Nile red 9,10 BPEA Nile red POPOP Coumarin 153 9,10 BPEA TPP ______________________________________

7. Fluorescent latex of claim 1, wherein the emission of the fluorochrome D makes it possible to excite the fluorochrome A indirectly, the particles of the latex containing at least one intermediate fluorochrome I whose excitation spectrum partially overlaps the emission spectrum of D and the emission spectrum partially overlaps the excitation spectrum of A.

8. Fluorescent latex of claim 7, wherein the molar concentration ratios A:

I and I;

D are greater than 1.

9. Fluorescent latex of claims 7 or 8, wherein the fluorochromes are chosen from the following trios:

space="preserve" listing-type="tabular">______________________________________ D I A ______________________________________ POPOP Coumarin 153 1,8 Cl.sub.2 -9,10 BPEA 9,10 DPA 9,10 BPEA Nile red Coumarin 153 1,8 Cl.sub.2 -9,10 BPEA TPP Coumarin 153 1,8 Cl.sub.2 -9,10 BPEA 5,12-BPEN ______________________________________

10. Fluorescent latex of claims 7 or 8, wherein when they are subjected to an excitation using a light radiation, the emission spectrum of the particles does not overlap the excitation spectrum of the said particles.

11. Fluorescent latex of claim 1, wherein when they are subjected to a light radiation, the detection threshold of the particles is less than or equal to 10^-12 mol of particles per liter.

12. Fluorescent latex of claim 1, wherein its particles are between 0.01 micron and 20 microns in size.

13. Fluorescent latex of claim 1, wherein the particles constitute between 0.05 and 10% of its weight.

14. Fluorescent latex of claim 1, wherein the polymer constituting its particles is a homopolymer or copolymer containing units derived from vinylaromatic or ethylenic monomers or from alkanoic or ethylenic acids or esters, which are optionally functionalized.

15. Fluorescent latex of claim 1, wherein it is functionalized with at least one immunoreactive species.

16. A method of using the fluorescent latex of claim 15 as a reagent in immunological tests, in reverse immunofluorescence, as a scintillator, as a calibration standard in flow cytofluorimetry and/or as cell markers.

17. Process for the preparation of a latex of claim 1, comprising at least one of the following steps:

introducing, during the polymerization of the monomer(s) intended to constitute the latex particles, of one or more of the fluorochromes D, A and optionally I in suspension in a fraction of the monomer or of one of the monomers, and

mixing of one or more of the fluorochromes D, A and I, solubilized in a non-aqueous solvent, with an aqueous dispersion of latex particles.

18. Fluorescent latex of claim 3, wherein the A:

D molar ratio is greater than 1.2.

19. Fluorescent latex of claim 7, wherein the molar concentration ratios A:

I and I;

D are greater than 1.2.