Stable biologically active fluorochemical emulsions

• US 5,401,634 A
• Filed: 03/18/1993
• Issued: 03/28/1995
• Est. Priority Date: 08/07/1986
• Status: Expired due to Term

• Current Assignee: 3M Company
• Original Assignee: 3M Company
• Inventors: Milbrath, Dean S.
• Primary Examiner(s): BICKEL, MINDY B
• Application Number:
  US08/033,131
• Time in Patent Office: 740 Days
• Field of Search: 435/6, 435/7.1, 436/518, 436/528, 436/804, 264/4.1, 428/402.2, 210/656
• US Class Current: 435/6.15
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/544 the carrier being organic
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/804 Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Fluorochemical emulsions comprised of a fluorochemical droplet discontinuous phase and aqueous continuous phase with at least one specific binding species immobilized on the fluorochemical droplets are shown. The emulsions may include a primer material to couple to specific binding species to the fluorochemical droplets. The emulsions may be used in diagnostic procedures or biochemical reactors where binding of the immobilized specific binding species to its binding partner is desired. The droplets may also incorporate a species which is detectable by spectrophotometric, fluorometric or colormetric means or a precursor to a detectable species.

Claims

1. A method to bind a first member of a two member specific binding pair contained in an aqueous solution to a second member of the Specific binding pair which is immobilized on an accessible surface of a liquid fluorochemical droplet comprising the steps of

i) forming a stable fluorochemical emulsion having an aqueous phase and a discontinuous liquid fluorochemical phase by mixing a perfluorinated liquid and a fluorinated surfactant in a buffered aqueous solution containing a water-soluble nonionic or amphoteric surfactant to give a liquid fluorochemical droplet, wherein the fluorochemical droplet consists essentially of a perfluorinated liquid and 0.01-5.0 wt. % of a fluorinated surfactant and wherein the fluorochemical droplet is substantially stable to centrifugation at 30,000×

g for fifteen minutes, wherein the perfluorinated liquid is selected from the group consisting of straight chain, branched chain and cyclic perfluorocarbons, straight chain, branched chain and cyclic perfluoro-tertiary amines and straight chain, branched chain and cyclic perfluoroethers and wherein the fluorinated surfactant is selected from the group consisting of perfluorinated alkanoic acids, perfluorinated amidoamines, 1,1-dihydroperfluoroalcohols, and perfluoroether polymers with terminal ester functional groups;

ii) contacting the liquid fluorochemical droplet with the second member of the specific binding pair, wherein the second member of the specific binding pair is immobilized on the liquid fluorochemical droplet at the continuous-discontinuous interface without a significant loss of activity to give an activated fluorochemical droplet;

iii) contacting the activated fluorochemical droplet with an aqueous solution containing the first member of the specific binding pair; and

iv) binding the first member to the second member of the specific binding pair on the fluorochemical droplet.

2. The method of claim 1 wherein the liquid fluorochemical droplet consists of a perfluorinated liquid and 0.1-1.0 wt. % of a fluorinated surfactant.

3. The method of claim 1 wherein the fluorinated surfactant is selected from the group consisting of perfluorohexanoic acid, perfluoroctanoic acid, 1,1-dihydroperfluoro-n-octanol, CH₃ O₂ CCF₂ O--(CF₂ CF₂ O)₇ --(CF₂ O)₁₄ --CF₂ CO₂ CH₃ and C₇ F₁₅ CONH(CH₂)₄ N(CH₃)₂.

4. The method of claim 1 wherein the first member of the specific binding pair is an antibody and wherein the second member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids.

5. The method of claim 1 wherein the first member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids and the second member of the specific binding pair is an antibody.

17. The method of claim 4 wherein the antigen is a therapeutic substance.

18. The method of claim 5 wherein the antigen is a therapeutic substance.

6. A method to bind a first member of a two member specific binding pair contained in an aqueous solution to a second member of the specific binding pair which is immobilized on an accessible surface of a liquid fluorochemical droplet comprising the steps of

i) forming a stable fluorochemical emulsion having an aqueous phase and a discontinuous liquid fluorochemical phase by mixing a perfluorinated liquid and a fluorinated surfactant in a buffered aqueous solution containing a water-soluble nonionic or amphoteric surfactant to give a liquid fluorochemical droplet, wherein the fluorochemical droplet consists essentially of a perfluorinated liquid and 0.01-5.0 wt. % of a fluorinated surfactant and wherein the fluorochemical droplet is substantially stable to centrifugation at 30,000×

g for fifteen minutes, wherein the perfluorinated liquid is selected from the group, consisting of straight chain, branched chain and cyclic perfluorocarbons, straight chain, branched chain and cyclic perfluoro-tertiary amines and straight chain, branched chain and cyclic perfluoroethers and wherein the fluorinated surfactant is selected from the group consisting of perfluorinated alkanoic acids, perfluorinated amidoamines, 1,1-dihydroperfluoroalcohols, perfluoroether polymers with terminal ester functional groups;

ii) contacting the liquid fluorochemical droplet with a primer material selected from the group consisting of proteins, sera, fibrinogen, collagen, polylysine-phenylalanine and polylysine to give a primed liquid fluorochemical droplet;

iii) contacting the primed liquid fluorochemical droplet with a second member of a specific binding pair, wherein the second member of the specific binding pair is immobilized on the primed liquid fluorochemical droplet at the continuous-discontinuous interface without a significant loss of activity to give an activated fluorochemical droplet,

iv) contacting the activated fluorochemical droplet with an aqueous solution containing the first member of the specific binding pair; and

v) binding the first member to the second member of the specific binding pair on the fluorochemical droplet.

7. The method of claim 6 wherein the liquid fluorochemical droplet consists of a perfluorinated liquid and 0.1-1.0 wt. % of a fluorinated surfactant.

8. The method of claim 6 wherein the fluorinated surfactant is selected from the group consisting of perfluorohexanoic acid, perfluoroctanoic acid, 1,1-dihydroperfluoro-n-octanol, CH₃ O₂ CCF₂ O--(CF₂ CF₂ O)₇ --(CF₂ O)₁₄ --CF₂ CO₂ CH₃ and C₇ F₁₅ CONH(CH₂)₄ N(CH₃)₂.

9. The method of claim 6 wherein the first member of the specific binding pair is an antibody and wherein the second member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids.

10. The method of claim 6 wherein the first member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids and the second member of the specific binding pair is an antibody.

11. The method of claim 6 further comprising the step of coupling the second member of the specific binding pair to the primer material with a coupling agent.

19. The method of claim 9 wherein the antigen is a therapeutic substance.

20. The method of claim 10 wherein the antigen is a therapeutic substance.

23. The method of claim 11 wherein the coupling agent is selected from the group consisting of 1-ethyl-3-(3-N,N-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate, acrolein, methacrolein, 2-butenal, glutaraldehyde, propanedial, butanedial, 2-iminothiolane hydrochloride, disuccinimidyl suberate, disuccinimidyl tartrate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl propionate, ethylene glycolbis(succinimidyl succinate), N-(5-azido-2-nitrobenzoyloxy)succinimide, p-azidophenylbromide, p-azidophenylglyoxal, 4-fluoro-3-nitrophenylazide, N-hydroxysuccinimidyl-4-azidobenzoate, m-maleimidobenzoyl N-hydroxysuccinimide ester, methyl-4-azidophenylglyoxal, 4-fluoro-3-nitrophenyl azide, N-hydroxysuccinimidyl-4-azidobenzoate hydrochloride, p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate, N-succinimidyl-6-(4'"'"'-azido-2'"'"'-nitrophenylamino)hexanoate, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, succinimidyl 4-(p-maleimido-phenyl)butyrate, N-succinimidyl(4-azidophenyldithio)-propionate, N-succinimidyl 3-(2-pyridyldithio)propionate, N-(4-azidophenylthio)phthalamide, 1,5-difluoro-2,4-dinitrobenzene, 4,4'"'"'-difluoro-3,3'"'"'-dinitrodiphenylsulfone, 4,4'"'"'-diisothiocyano-2,2'"'"'-disulfonic acid stilbene, p-phenylenediisothiocyanate, carbonylbis(L-methionine p-nitrophenyl ester), 4,4'"'"'-dithiobisphenylazide, erythritolbiscarbonate, dimethyl adipimidate hydrochloride, dimethyl suberimidate, and dimethyl 3,3'"'"'-dithiobispropionimidate hydrochloride.

12. A method to bind a first member of a two member specific binding pair contained in an aqueous solution to a second member of the specific binding pair which is immobilized on an accessible surface of a liquid fluorochemical droplet comprising the steps of

i) forming a stable fluorochemical emulsion having an aqueous phase and a discontinuous fluorochemical phase by mixing a perfluorinated liquid and a fluorinated surfactant in a buffered aqueous solution containing a water-soluble nonionic or amphoteric surfactant to give a liquid fluorochemical droplet, wherein the fluorochemical droplet consists essentially of a perfluorinated liquid and 0.01-5.0 wt. % of a fluorinated surfactant and wherein the fluorochemical droplet is substantially stable to centrifugation at 30,000×

g for fifteen minutes, wherein the perfluorinated liquid is selected from the group consisting of straight chain, branched chain and cyclic perfluorocarbons, straight chain, branched chain and cyclic perfluoro-tertiary amines and straight chain, branched chain and cyclic perfluoroethers and wherein the fluorinated surfactant is selected from the group consisting of perfluorinated alkanoic acids, perfluorinated amidoamines, 1,1-dihydroperfluoroalcohols, and perfluoroether polymers with terminal ester functional groups;

ii) contacting the liquid fluorochemical droplet with a primer material selected from the group consisting of proteins, sera, fibrinogen, collagen, polylysine-phenylalanine and polylysine to give a primed liquid fluorochemical droplet;

iii) contacting the primed liquid fluorochemical droplet with a dye selected from the group consisting of perfluoroalkyl phthalein dye, perfluoroalkyl phthalocyanine dye, perfluoroalkyl rhodamine dye, perfluoroalkyl quinothaline dye and perfluoroalkyl-beta-diketone lanthanide complexes, and with the second member of the specific binding pair, wherein the second member of the specific binding pair is immobilized on the primed fluorochemical droplet at the continuous-discontinuous interface to give an activated fluorochemical droplet;

iv) contacting the activated fluorochemical droplet with an aqueous solution containing the first member of the specific binding pair; and

v) binding the first member to the second member of the specific binding pair on the fluorochemical droplet.

13. The method of claim 12 wherein the liquid fluorochemical droplet consists of of a perfluorinated liquid and about 0.1-1.0 wt. % of a fluorinated surfactant.

14. The method of claim 12 wherein the fluorinated surfactant is selected from the .group consisting of perfluorohexanoic acid, perfluoroctanoic acid, 1,1-dihydroperfluoro-n-octanol, CH₃ O₂ CCF₂ O--(CF₂ CF₂₀)₇ --(CF₂ O)₁₄ --CF₂ CO₂ CH₃ and C₇ F₁₅ CONH(CH₂)₄ N(CH₃)₂.

15. The method of claim 12 wherein the first member of the specific binding pair is an antibody and wherein the second member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids.

16. The method of claim 12 wherein the first member of the specific binding pair is an antigen selected from the group consisting of natural and synthetic proteins, polysaccharides, and nucleic acids and the second member of the specific binding pair is an antibody.

21. The method of claim 15 wherein the antigen is a therapeutic substance.

22. The method of claim 16 wherein the antigen is a therapeutic substance.