Methods and gel compositions for encapsulating living cells and organic molecules

• US 7,172,866 B2
• Filed: 04/02/2002
• Issued: 02/06/2007
• Est. Priority Date: 04/03/2001
• Status: Expired due to Term

• Current Assignee: Biocept, Inc.
• Original Assignee: Biocept, Inc.
• Inventors: Tsinberg, Pavel, Edman, Carl F., Dong, Xiaofan, Hahn, Soonkap, Fagnani, Roberto
• Primary Examiner(s): Lankford, Jr.; Leon B
• Application Number:
  US10/398,725
• Publication Number:
  US 20040029241A1
• Time in Patent Office: 1,771 Days
• Field of Search: 435/6, 435/7.1, 435/325, 435/395, 435/287.2, 527/301, 527/204, 436/518
• US Class Current: 435/6.19
• CPC Class Codes: A61K 9/1641 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00367 capillary
• 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 2219/00644 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 2219/00677 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: B01J 2219/0074 Biological products
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C08G 18/10 Prepolymer processes involving reaction of isocyanates or isothiocyanates with compounds having active hydrogen in a first reaction step
• CPC Class Codes: C08G 18/3876 containing mercapto groups
• CPC Class Codes: C08G 2210/00 Compositions for preparing hydrogels
• CPC Class Codes: C08G 65/33348 containing isocyanate group
• CPC Class Codes: C08G 65/3342 having sulfur bound to carbon and hydrogen
• CPC Class Codes: C08L 2203/02 for biomedical use
• CPC Class Codes: C12N 11/04 : entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• 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 33/5436 : with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386

Abstract

A method for encapsulating biologics within a hydrogel by using an aqueous solution of an isocyanate-functional hydrogel prepolymer which is mixed with an amount of biologics and an aqueous solution containing a dithiol crosslinking agent under physiological pH conditions. An additional bidentate crosslinking agent may be included. The product of such method may be a bioreactor or an assay device having a plurality or different biologics encapsulated at predetermined locations in a substrate.

Claims

1. A bioassay comprising the steps of:

(a) providing a microarray which comprises a substrate having a plurality of discrete spots of a stabilized hydrogel composition bound thereto which composition is the reaction product of a prepolymer of polyethylene glycol, polypropylene glycol, or a copolymer thereof having isocyanate end groups, and a thio-functional crossliniking agent, which results in thio-urethane groups wherein said stabilized hydrogel composition further includes biologics encapsulated therewithin which remain bioactive;

(b) contacting the microarray with an analyte solution, and

(c) detecting the interactions of the microarray with the analyte solution.

2. The bioassay of claim 1 wherein said hydrogel composition is exposed to a second analyte solution as a part of said detecting step.

3. The bioassay of claim 1 wherein said analyte solution contains a marker which binds to said biologic which is a target and wherein the step of detecting comprises detecting the marker bound to the target.

4. The bioassay of claim 3 wherein the marker is capable of fluorescence and wherein the step of detecting the bound target comprises detecting fluorescence from the marker.

5. A microarray which comprises:

a substrate, and

a plurality of discrete spots of a hydrogel biologic composition attached to said substrate, which hydrogel biologic composition comprises;

(a) the reaction product of a prepolymer of polyethylene glycol, polypropylene glycol, or a copolymer thereof having isocyanate end groups, and a thio-functional crossliniking agent, which results in thio-urethane groups and provides a stabilized hydrogel, and

(b) a biologic which is encapsulated within said stabilized hydrogel and which remains bioactive.

6. The microarray of claim 5 wherein the thiol-functional crossliniking agent comprises a dithiol-functional crossliniker and a bidentate crossliniking agent having one thiol group and another different isocyanate-reactive group in a molar amount from about 1 to 3 times the molar amount of said dithiol crossliniker.

7. The microarray of claim 6 wherein said dithiol-functional crossliniking agent comprises a backbone of polyethylene glycol, polypropylene glycol, or a copolymer thereof and has a molecular weight between about 2,000 and about 6,000.

8. The microarray of claim 5 wherein said spots are spatially arranged to form a regular array and wherein the plurality of spots include some at known locations within said array wherein different biologics are entrapped.

9. The microarray of claim 5 wherein said biologics are living eukaryotic cells.

10. The microarray of claim 5 wherein said biologics are proteins or other organic molecules having a molecular weight between 100 and 2000 which have been derivatized to include a thiol group.

11. The microarray of claim 5 wherein said stabilized hydrogel is optically transparent.