Method of making a membrane having hydrophilic and hydrophobic surfaces for adhering cells or antibodies by using atomic oxygen or hydroxyl radicals

• US 5,369,012 A
• Filed: 03/26/1992
• Issued: 11/29/1994
• Est. Priority Date: 03/26/1992
• Status: Expired due to Fees

• Current Assignee: United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration
• Original Assignee: United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration
• Inventors: Koontz, Steven L., Spaulding, Glenn F.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US07/857,901
• Time in Patent Office: 978 Days
• Field of Search: 435/7.92, 435/174, 435/180, 435/181, 436/531, 436/532, 530/815, 530/816
• US Class Current: 435/7.92
• CPC Class Codes: A61L 27/28 Materials for coating prostheses
• CPC Class Codes: A61L 33/0076 Chemical modification of the substrate
• CPC Class Codes: A61L 33/0094 Physical treatment, e.g. plasma treatment
• CPC Class Codes: B01D 15/3809 of the antigen-antibody type, e.g. protein A, G, L chromatography
• CPC Class Codes: B01D 2323/38 Graft polymerization
• CPC Class Codes: B01D 67/009 with wave-energy, particle-radiation or plasma
• CPC Class Codes: B01D 67/0093 Chemical modification
• CPC Class Codes: B01D 67/00931 by introduction of specific groups after membrane formation, e.g. by grafting
• CPC Class Codes: B01J 20/08 comprising aluminium oxide or hydroxide; comprising bauxite
• CPC Class Codes: B01J 20/103 comprising silica
• CPC Class Codes: B01J 20/321 consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3212 consisting of a polymer obtained by reactions otherwise than 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 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/328 Polymers on the carrier being further modified
• CPC Class Codes: B01J 20/3285 Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
• CPC Class Codes: B01J 20/3291 Characterised by the shape of the carrier, the coating or the obtained coated product
• CPC Class Codes: B29C 59/14 by plasma treatment plasma tubes per se H01J
• CPC Class Codes: B29L 2031/755 Membranes, diaphragms
• CPC Class Codes: C12N 2533/30 Synthetic polymers thermoreactive polymers, e.g. PNIPAm, C12N2539/10
• CPC Class Codes: C12N 5/0068 : General culture methods using substrates for specific animal cell type C12N5/06
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: G01N 2030/525 : surface properties, e.g. porosity
• CPC Class Codes: G01N 2030/528 : Monolithic sorbent material
• CPC Class Codes: G01N 33/54306 : Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/545 : Synthetic resin
• 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 portion of an organic polymer article such as a membrane is made hydrophilic by exposing a hydrophobic surface of the article to a depth of about 50 to about 5000 angstroms to atomic oxygen or hydroxyl radicals at a temperature below 100° C., preferably below 40° C., to form a hydrophilic uniform surface layer of hydrophilic hydroxyl groups. The atomic oxygen and hydroxyl radicals are generated by a flowing afterglow microwave discharge, and the surface is outside of a plasma produced by the discharge. A membrane having both hydrophilic and hydrophobic surfaces can be used in an immunoassay by adhering antibodies to the hydrophobic surface. In another embodiment, the membrane is used in cell culturing where cells adhere to the hydrophilic surface. Prior to adhering cells, the hydrophilic surface may be grafted with a compatibilizing compound. A plurality of hydrophilic regions bounded by adjacent hydrophobic regions can be produced such that a maximum of one cell per each hydrophilic region adheres.

Claims

1. An organic polymer membrane having first and second opposing surfaces, wherein said first surface is hydrophilic and has an adjacent absorbent layer and said second surface is hydrophobic and contains adhered antibodies, said hydrophilic surface is prepared by a method comprising:

exposing a hydrophobic exterior surface of said membrane to a dosage of atomic oxygen or hydroxyl radicals at a temperature below about 40°

C., said dosage being sufficient to form said hydrophilic surface having a uniform surface layer of hydrophilic functional hydroxyl groups, wherein said dosage is generated by a flowing afterglow microwave discharge and said surface is outside of a plasma produced by said discharge, wherein said surface exposed to said dosage has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage.

2. An organic polymer membrane having a surface comprised of a plurality of hydrophilic regions bounded by adjacent hydrophobic regions, said hydrophilic regions containing adhered cells at a maximum density of one cell per each hydrophilic region and said hydrophobic regions are substantially free of adhered cells, said hydrophilic regions having a uniform surface layer of hydrophilic functional hydroxyl groups created by exposing an exterior hydrophobic surface of said membrane at a temperature below about 40°

C. to a dosage of atomic oxygen or hydroxyl radicals, said dosage being generated by a flowing afterglow microwave discharge and said surface being outside of a plasma produced by said discharge, wherein said exterior surface exposed to said atomic oxygen has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage.

3. The membrane of claim 2 wherein said hydrophilic regions are grafted with a compatibilizing compound selected from the group consisting of phosphorylcholines, peptides, lipids, proteins, nucleic acids, poly(ethylene oxide), poly(vinyl alcohol) and acyl terminated upper alkyl radicals.

4. The membrane of claim 3 wherein said compound is selected from the group consisting of one or more phosphorylcholines, peptides, lipids, proteins, and nucleic acids.

5. The membrane of claim 3, wherein said compatibilizing compound is selected from the group consisting of poly(vinyl alcohol) and poly(ethylene oxide).

6. The membrane of claim 2 in the form of a tube.

7. An assay method using a permeable organic polymer membrane having a hydrophilic surface formed opposite a hydrophobic surface, said hydrophilic surface having a uniform surface layer of hydrophilic functional hydroxyl groups created by exposing an exterior hydrophobic surface of said membrane at a temperature below about 40°

C. to a dosage of atomic oxygen or hydroxyl radicals, said dosage being generated by a flowing afterglow microwave discharge and said surface being outside of a plasma produced by said discharge, wherein said surface exposed to said atomic oxygen has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage, said assay comprising the steps of;

(a) adhering known antibodies to said hydrophobic surface;

(b) placing said hydrophilic surface in contact with an adjacent absorbent material;

(c) covering said antibodies with a test solution, wherein antigens specific to said antibodies adhere thereto to form an antibody-antigen complex and non-adhered antigens migrate to said hydrophilic surface and are absorbed by said absorbent material;

(d) covering said hydrophobic surface with a solution of said antibodies conjugated to an indicating enzyme, wherein said conjugated antibodies and said antigen-antibody complex form an antibody-antigen-antibody/enzyme complex and excess fluid is absorbed by said absorbent material; and

(e) developing said antibody-antigen-antibody/enzyme complex with a solution of developing compound reactive with said enzyme to indicate presence of said antibody-antigen-antibody/enzyme complex as a positive result or an absence of said complexes as a negative result, wherein excess developing solution is absorbed by said absorbent material.

8. A cell culturing method using a permeable organic polymer membrane having a hydrophilic surface comprising a plurality of hydrophilic regions bounded by adjacent hydrophobic regions, said hydrophilic regions having a uniform surface layer of hydrophilic functional hydroxyl groups created by exposing an exterior hydrophobic surface of said membrane at a temperature below about 40°

C. to a dosage of atomic oxygen or hydroxyl radicals, said dosage being generated by a flowing afterglow microwave discharge and said surface being outside of a plasma produced by said discharge, wherein said exterior surface exposed to said atomic oxygen has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage, said cell culturing method comprising the steps of;

(a) distributing a group of cells on said membrane in a growth medium, wherein said cells adhere to said hydrophilic regions at a maximum density of one cell per each hydrophilic region and said hydrophobic regions are substantially free of adhered cells;

(b) growing said cells on said membrane; and

(c) obtaining a count of malignant cells growing on said membrane.

9. A cell culturing method using a permeable organic polymer membrane having first and second opposing surfaces, said first surface comprising a hydrophilic region bounded by a hydrophobic region, said hydrophilic region having a uniform surface layer of hydrophilic functional hydroxyl groups created by exposing an exterior hydrophobic surface of said membrane at a temperature below about 40°

C. to a dosage of atomic oxygen or hydroxyl radicals, said dosage being generated by a flowing afterglow microwave discharge and said surface being outside of a plasma produced by said discharge, wherein said exterior surface exposed to said atomic oxygen has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage, second said cell culture method comprising the steps of;

(a) forming cell culturing and product compartments on opposite sides of said membrane, wherein said first membrane surface is adjacent to said cell culturing compartment and said second membrane surface is adjacent to said product compartment, said cell culturing compartment comprising cells in a growth medium, said cells having a gene for expression of a desired product;

(b) inducing said cells to adhere to said hydrophilic region of said first surface of said membrane, wherein said cells polarize with a basal cell wall adjacent said first surface; and

(c) inducing migration of said nutrient from said nutrient compartment to said cells compartment by maintaining nutrient in said cell culturing compartment, wherein said cells absorb said nutrient and in response thereto secrete a product preferentially through said basal cell wall and said membrane into said product compartment.

10. A cell culturing method using a permeable organic polymer membrane, said membrane having first and second opposing surfaces, said first surface comprising a hydrophilic surface and said second surface comprising a hydrophobic surface, said hydrophilic surface having a uniform surface layer of hydrophilic functional hydroxyl groups created by exposing an exterior hydrophobic surface of said membrane at a temperature below about 40°

C. to a dosage of atomic oxygen or hydroxyl radicals, said dosage being generated by a flowing afterglow microwave discharge and said surface being outside of a plasma produced by said discharge, wherein said exterior surface exposed to said atomic oxygen has a depth of from about 50 to about 5000 angstroms and said dosage over said surface is within about 10 percent of an average dosage, said cell culture method comprising the steps of;

(a) forming a nutrient compartment and a cells compartment on opposite sides of said membrane, wherein said first surface is adjacent said nutrient compartment and said second surface is adjacent to said cells compartment, said nutrient compartment comprising a solution of nutrient and said cells compartment comprising cells in a growth medium; and

(b) inducing migration of said nutrient from said nutrient compartment to said cells compartment by maintaining a higher concentration of said nutrient in said nutrient compartment.