Method for immobilizing molecules onto surfaces

• US 7,687,437 B2
• Filed: 07/12/2002
• Issued: 03/30/2010
• Est. Priority Date: 07/13/2001
• Status: Expired due to Fees

• Current Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Original Assignee: Nanosphere Incorporated (DiaSorin S.p.A.)
• Inventors: Garimella, Viswanadham
• Primary Examiner(s): Steele; Amber D.
• Application Number:
  US10/194,138
• Publication Number:
  US 20030082588A1
• Time in Patent Office: 2,818 Days
• Field of Search: 436/518, 436/524, 436/527, 436/106, 436/109, 436/127, 435/4, 435/6, 435/174, 506/32
• US Class Current: 506/32
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

A method for immobilizing amino-group containing molecules onto surfaces and devices having immobilized isocyanate-group containing molecules prepared by the method are disclosed. The method comprises reacting a surface (i.e., glass surface) having free hydroxyl groups with a silyl isocyanate derivatizing agent to provide immobilized reactive moieties, the agent having a formula:
(R₁O)(R₂O)(R₃O)Si—X—NCO
wherein R₁, R₂ and R₃ are independently represents C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy; X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comprising oxygen, nitrogen, and sulfur and reacting the immobilized reactive moieties with the amino group-containing molecule so as to immobilize said molecule on the surface. Devices having a surface with immobilized molecules such as nucleic acids or proteins are useful for detection of target analytes in a sample.

Claims

1. A method for immobilizing a probe on a glass surface comprising the steps of:

(a) reacting the glass surface with a solution at room temperature to provide immobilized reactive moieties, the solution comprising (3-isocyanatopropyl) triethoxysilane, an organic solvent, and an acid; and

(b) reacting the immobilized reactive moieties with an amino-group containing molecule so as to immobilize said molecule on the glass surface, wherein the amino-group containing molecule further comprises one or more functional groups;

(c) reacting the immobilized amino-group containing molecule on the surface with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) that reacts with a functional group of the immobilized amino-group containing molecule so as to produce second immobilized reactive moieties; and

(d) reacting the second immobilized reactive moieties with a second molecule having a functional group capable of reacting with the second immobilized reactive moieties.

2. The method of claim 1 wherein the second molecule is a probe.

3. The method of claim 2, wherein the probe is selected from the group consisting of a protein, a peptide, a nucleic acid, a peptide nucleic acid, a linked nucleic acid, an amino acid, a nucleoside triphosphate, a carbohydrate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, and a whole cell.

4. The method of claim 2, wherein the probe has been derivatized to contain one or more amino and/or hydroxyl groups.

5. The method of claim 4, wherein the probe has been derivatized to contain one or more amino groups.

6. The method according to claim 1 wherein the (3-isocyanatopropyl) triethoxysilane is present in solution at a concentration ranging between about 20 mM and about 200 mM.

7. The method of claim 1 wherein the amino-group containing molecule immobilized on the surface has at least one carboxyl group.

8. The method of claim 7 wherein the second molecule has at least one amino group.

9. The method of claim 7 wherein the second immobilized reactive moieties are the reaction products of the carboxyl group with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

10. The method according to claim 1, wherein the amount of acid in the solution is between about 0.01 % (v/v) to about 0.1 % (v/v).

11. A method for immobilizing an amino-group containing molecule on a surface having free hydroxyl groups comprising the steps of:

(a) reacting said surface with a solution at room temperature to provide immobilized reactive moieties, the solution comprising a silyl isocyanate derivatizing agent, an organic solvent, and an acid, the silyl isocyanate agent having a formula;

(R₁O)(R₂O)(R₃O)Si—

X—

NCO

wherein R₁R₂ and R₃ independently represents C₁-C₆ alkyl, phenyl, or aryl substituted with one or more groups selected from the group consisting of C₁-C₆ alkyl and C₁-C₆ alkoxy;

X represents linear or branched C₁-C₂₀ alkyl or aryl substituted with one or more groups selected from the group consisting of C₁C₆ alkyl and C₁-C₆ alkoxy, optionally substituted with one or more heteroatoms comrrising oxygen, nitrogen, and sulfur;

(b) reacting the immobilized reactive moieties with the amino-group containing molecule so as to immobilize the amino-group containing molecule on the surface, wherein the amino-group containing molecule further includes one or more functional groups and comprises a carboxyl group;

(c) reacting the immobilized amino-group containing molecule on the surface with a chemical agent that reacts with a functional group of the amino-group containing molecule so as to produce second immobilized reactive moieties; and

(d) reacting the second immobilized reactive moieties with a second molecule having a functional group capable of reacting with the second immobilized reactive moieties.

12. The method of claim 11, wherein the chemical agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and the second immobilized reactive moieties are the reaction products of the carboxyl group with EDC.

13. The method of claim 12, wherein the second molecule contains or has been derivatized to contain one or more amino groups.