Three dimensional format biochips

• US 7,638,464 B2
• Filed: 10/25/2001
• Issued: 12/29/2009
• Est. Priority Date: 04/26/1999
• Status: Expired due to Term

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Hahn, Soonkap, Tsinberg, Pavel, Fagnani, Roberto, Dong, Xiaofan, Matsumoto, Sandra, Pircher, Tony
• Primary Examiner(s): Lundgren; Jeffrey S.
• Application Number:
  US10/054,728
• Publication Number:
  US 20050037343A1
• Time in Patent Office: 2,987 Days
• Field of Search: 435/4, 435/7.1, 435/174, 435/180, 435/181, 436/518, 436/528, 260/2.5 AD, 260/2.5 AY, 260/77.5 AM, 260/77.5 AP, 552/114, 552/116, 527/200, 527/300
• US Class Current: 506/13
• CPC Class Codes: B01J 2219/00351 Means for dispensing and evacuation of reagents
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00587 High throughput 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 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00644 the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/544 the carrier being organic
• CPC Class Codes: G01N 33/545 : Synthetic resin

Abstract

A biochip is formed with a plurality of optically clear hydrogel cells attached to the top surface of a solid substrate in the form of an array. Each of the cells is formed of a hydrogel of polyethylene glycol, polypropylene glycol or a copolymer thereof having reactive isocyanate groups. Binding entities are immobilized in these cells, which entities are effective to selectively hybridize to or sequester a target biomolecule. Different binding entities are immobilized in different cells in an array to create a biochip that can be used to assay for a number of target biomolecules.

Claims

1. A biochip comprising:

a) a solid substrate having a flat top surface;

b) a plurality of optically clear, individual, three-dimensional hydrogel cells at least 20 μ

m thick attached to the flat surface of the substrate at discrete locations to form an array of discrete individual three-dimensional cells protruding from said otherwise flat top surface, which hydrogel cells are formed from an isocyanate-functional prepolymer with urethane linkages; and

c) a different binding entity immobilized within or upon various of said hydrogel cells by covalent linkage of said binding entity or an intermediate agent with reactive isocyanate groups of said hydrogel, which entity is effective to selectively hybridize to or sequester a target molecule.

2. The biochip of claim 1 wherein the hydrogel comprises polyethylene glycol, polypropylene glycol, or copolymers thereof having a molecular weight of about 5000.

3. The biochip according to claim 2, wherein the three-dimensional individual hydrogel cells are between about 30 μ

m and about 100 μ

m thick.

4. The biochip according to claim 1, wherein said binding entity is directly covalently bound to and within the hydrogel cell through reaction with the isocyanate groups.

5. The biochip of claim 1 wherein about 15% to about 5% of the reactive isocyanates in said prepolymer that forms said cell have reacted to immobilize said binding entities or said intermediate agents.

6. The biochip of claim 1 wherein each said binding entity comprises DNA, RNA or PNA.

7. The biochip of claim 1 wherein each said binding entity comprises an immunoglobin, an enzyme, a receptor, an enzyme inhibitor, an enzyme substrate, or a peptide.

8. The biochip of claim 7 wherein each said binding entity is immobilized within the hydrogel through an interaction with an intermediate agent.

9. The biochip of claim 1 wherein the substrate is optically transparent and has reactive molecules on its top surface to which the hydrogel is covalently bound through some of said isocyanate groups of the polymer.

10. A hydrogel biochip comprising:

a) a solid substrate having a flat top surface;

b) a plurality of optically clear, individual, three-dimensional hydrogel cells of an isocyanate-functional hydrogel at least about 20 μ

m thick, comprising polyethylene glycol, polypropylene glycol, or copolymers thereof having urethane linkages, bound to the top surface of said substrate at discrete locations to form an array of discrete individual three-dimensional cells protruding from said otherwise flat top surface;

c) intermediate agents immobilized within or upon said hydrogel cells by covalent binding to reactive isocyanate groups of said hydrogel; and

d) different protein binding entities bound to said intermediate agents respectively within at least several of said hydrogel cells by interaction therewith in a manner so that said protein binding entities assume their native conformations.

11. A biochip comprising:

a) a solid substrate having a flat top surface;

b) a plurality of optically clear, individual, three-dimensional hydrogel cells at least about 20 μ

m thick attached to the surface of the substrate at discrete locations to form an array of discrete individual three-dimensional cells protruding from said otherwise flat top surface, each hydrogel cell being a polymer formed from an isocyanate-functional urethane prepolymer; and

c) different protein binding entities immobilized via linkage to isocyanate groups of said hydrogel within or upon different said hydrogel cells, each protein binding entity being effective to selectively hybridize to or sequester a target molecule.

12. The biochip of claim 11 wherein the hydrogel comprises polyethylene glycol, polypropylene glycol, or copolymers thereof having a molecular weight of at least about 5000 with urethane linkages to polyisocyanates.

13. The biochip of claim 12 wherein each hydrogel cell is between about 20 μ

m and about 100 μ

m thick.

14. The biochip of claim 11 wherein each said protein binding entity is directly covalently bound to and within the hydrogel cells through reaction with isocyanate groups of said prepolymer that forms said hydrogel.

15. The biochip of claim 11 wherein with said protein binding entities comprise immunoglobins or aptamers.

16. A biochip comprising:

a) a solid substrate having a flat top surface;

b) a plurality of optically clear, individual, three-dimensional hydrogel cells at least 20 μ

m thick comprising urethane polymers of(i) polyethylene glycol, polypropylene glycol, or copolymers thereof and (ii) polyisocyanates, which polymers are isocyanate-functional, and which cells are bound to the flat top surface of said substrate at discrete locations to form an array of discrete individual three-dimensional cells protruding from said otherwise flat top surface;

c) intermediate agents immobilized within or upon said hydrogel cells by covalent linkage to reactive isocyanate groups of said hydrogel; and

d) different protein binding entities bound to said intermediate agents within at least several of said hydrogel cells by interaction therewith in a manner so that said protein binding entities can assume their native conformations.

17. The biochip of claim 16 wherein said hydrogel is a urethane-based polymer formed from a prepolymer with excess isocyanate groups in an amount of about 0.2 meq/g to about 0.8 meq/g, and wherein said protein binding entities are bound through pairs of intermediate coupling agents.

18. The biochip of claim 16 wherein said intermediate agent is nitrilotriacetic acid.

19. A biochip comprising:

a) a solid substrate having a flat top surface that is derivatized with groups reactive with isocyanate;

b) a plurality of optically clear, individual, three-dimensional hydrogel cells at least 20 μ

m thick bound to the flat surface of the substrate at discrete locations which form an array of discrete individual three-dimensional cells protruding from said otherwise flat top surface, said hydrogel cells being formed from an isocyanate-functional urethane prepolymer wherein up to about 5% of its isocyanate groups are covalently bound to said derivatized groups of said substrate; and

c) a different binding entity immobilized within or upon various of said hydrogel cells by covalent linkage of said binding entity or an intermediate agent with reactive isocyanate groups of said prepolymer that forms said hydrogel, said entity being effective to selectively hybridize to or sequester a target molecule.