Enzyme immobilization and bioaffinity separations with perfluorocarbon polymer-based supports

• US 4,954,444 A
• Filed: 12/17/1987
• Issued: 09/04/1990
• Est. Priority Date: 03/02/1987
• Status: Expired due to Term

• Current Assignee: Arkion Life Sciences
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Eveleigh, John William d., Kobos, Robert K.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US07/134,028
• Time in Patent Office: 992 Days
• Field of Search: 435/7, 435/180, 435/181, 435/182, 435/803, 435/815, 435/817, 436/528, 436/531, 436/532, 436/824, 530/815, 530/816
• US Class Current: 435/181
• CPC Class Codes: B01J 20/261 obtained by reactions only involving carbon to carbon unsaturated bonds macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds per se C08F
• CPC Class Codes: B01J 20/28004 Sorbent size or size distribution, e.g. particle size
• CPC Class Codes: B01J 20/28016 Particle form
• CPC Class Codes: B01J 20/28033 Membrane, sheet, cloth, pad, lamellar or mat
• CPC Class Codes: B01J 20/286 Phases chemically bonded to a substrate, e.g. to silica or to polymers
• CPC Class Codes: B01J 20/3092 Packing of a container, e.g. packing a cartridge or column of chromatography columns B01D15/206
• CPC Class Codes: B01J 20/321 consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3246 having a well defined chemical structure
• CPC Class Codes: B01J 20/3251 comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/3425 of sorbents or filter aids comprising organic materials
• CPC Class Codes: B01J 20/345 using a particular desorbing compound or mixture elution or regeneration of stationary phases in liquid chromatography B01D15/08
• CPC Class Codes: B01J 2220/49 Materials comprising an indicator, e.g. colour indicator, pH-indicator
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: B01J 2220/58 Use in a single column
• CPC Class Codes: C12N 11/082 obtained by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/58 involving urea or urease
• CPC Class Codes: G01N 33/545 : Synthetic resin
• CPC Class Codes: Y10S 435/803 : Physical recovery methods, e.g. chromatography, grinding
• CPC Class Codes: Y10S 435/815 : by sorption
• CPC Class Codes: Y10S 435/817 : Enzyme or microbe electrode
• CPC Class Codes: Y10S 436/824 : Immunological separation techniques
• CPC Class Codes: Y10S 530/815 : Carrier is a synthetic polymer
• CPC Class Codes: Y10S 530/816 : Attached to the carrier via a bridging agent

Abstract

A bioaffinity separation method is provided along with a solid affinity support utilized in that method. Additionally, immobilized enzyme systems are provided for use as enzyme electrode systems. The support is based on an inert perfluorocarbon polymer carrier with ligands or binders attached to its surface through a highly fluorinated isocyanate anchor group. Methods for preparing such supports and their use in capturing target molecules from samples and in analytical applications are also provided.

Claims

1. A solid support containing an attached ligand or binder for the ligand consisting essentially of:

(A) a chemically inert, water immiscible solid perfluorocarbon polymer carrier having low nonspecific binding to a ligand or binder for the ligand;

(B) a highly fluorinated isocyanate-modified ligand or binder for the ligand securely but reversibly attached to the surface of said carrier through said highly fluorinated isocyanate; and

(C) a nonionic fluorosurfactant coating on said carrier.

2. The support of claim 1, wherein said ligand is selected from the group consisting of nucleic acid, and dye.

3. The support of claim 1, wherein said binder for the ligand is selected from the group consisting of antibody, enzyme and nucleic acid.

4. The solid support of claim 1 wherein the highly fluorinated isocyanate is a compound of the formula

5. The solid support of claim 4 wherein R_F is F(CF₂)₈.

6. The support of claim 1, wherein the carrier is selected from the group consisting of polytetrafluoroethylene, polyvinylfluoride and polyvinylidene difluoride.

7. The support of claim 1, wherein said ligand is a hapten.

8. The support of claim 1, wherein said ligand is an antigen.

9. The support of claim 1, wherein said ligand is selected from the group consisting of enzyme substrates, effectors and inhibitors.

10. The support of claim 1, wherein said binder for the ligand is a binding protein or a synthetic mimic of a binding protein.

11. A bioaffinity separation process comprising the steps of:

(A) forming a solid affinity support by

(a) attaching a highly fluorinated isocyanate-modified ligand or binder for the ligand to the surface of a solid perfluorocarbon carrier through said highly fluorinated isocyanate and

(b) contacting the product of step (a) with a nonionic fluorosurfactant to form an affinity support; and

(B) capturing a binder or ligand, complementary to the ligand or binder attached to the carrier from a mixture using said solid affinity support.

12. The process of claim 11, wherein said ligand is selected from the group consisting of nucleic acid, and dye.

13. The process of claim 11, wherein said binder for the ligand is selected from the group consisting of antibody, enzyme and nucleic acid.

14. The process of claim 11, wherein said ligand is a hapten.

15. The process of claim 11, wherein said ligand is an antigen.

16. The process of claim 11, wherein said ligand is selected from the group consisting of enzyme substrates, effectors and inhibitors.

17. The process of claim 11, wherein said binder for the ligand is a binding protein or a synthetic mimic of a binding protein.

18. An immobilized enzyme system consisting essentially of:

(A) a chemically inert, water immiscible solid perfluorocarbon polymer carrier having low nonspecific binding to a ligand or binder for the ligand;

(B) a highly fluorinated isocyanate-modified enzyme attached securely but reversibly to the surface of said carrier through said highly fluorinated isocyanate; and

(C) a nonionic fluorosurfactant coating on said carrier.

19. A process of preparing a solid support containing an attached ligand or binder for the ligand comprising the steps of:

(A) modifying a ligand or binder for the ligand by attaching a highly fluorinated isocyanate anchor group to said ligand or binder for the ligand;

(B) contacting a solid perfluorocarbon polymer with the highly fluorinated isocyanate-modified ligand or binder for the ligand to attach the ligand or binder to the polymer through said highly fluorinated isocyanate, and

(C) contacting the product formed the step (B) with a nonionic fluorosurfactant.

20. The process of claim 19, wherein the ligand is selected from the group consisting of nucleic acid, and dye.

21. The process of claim 19, wherein said binder for the ligand is selected from the group consisting of antibody, enzyme and nucleic acid.

22. The process of claim 19, wherein said ligand is a hapten.

23. The process of claim 19, wherein said ligand is an antigen.

24. The process of claim 19, wherein said ligand is selected from the group consisting of enzyme substrates, effectors and inhibitors.

25. The process of claim 19, wherein said binder for the ligand is a binding protein or a synthetic mimic of a binding protein.