Method of preparing fluorescently labeled microbeads

• US 4,609,689 A
• Filed: 01/28/1985
• Issued: 09/02/1986
• Est. Priority Date: 04/27/1984
• Status: Expired due to Fees

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Schwartz, Abraham, Williams, Joel, Stevens, Robert D.
• Primary Examiner(s): Wong, Jr., Harry
• Assistant Examiner(s): Teskin, Fred M.
• Application Number:
  US06/695,654
• Time in Patent Office: 582 Days
• Field of Search: 523/202, 523/205, 525/327.3, 525/330.3, 525/330.5, 525/343, 525/351, 525/379, 525/382, 525/384, 526/273, 526/320, 526/328, 526/328.5, 526/181, 524/461, 524/464, 524/558, 524/853, 428/407, 252/301.35
• US Class Current: 523/202
• CPC Class Codes: C08F 8/00 Chemical modification by after-treatment graft polymers, block polymers, crosslinking with unsaturated monomers or with polymers C08F251/00 - C08F299/00; of conjugated diene rubbers C08C
• CPC Class Codes: Y10S 526/909 Polymerization characterized by particle size of product
• CPC Class Codes: Y10T 428/2998 including synthetic resin or polymer

Abstract

Highly uniform polymeric microbeads of a predetermined size, preferably having an attached fluorescent label on the surface, are prepared by causing seed particles to swell by absorption of a plurality of low molecular weight substances, at least one of which is a monomer having a reactive functional group. After absorption, the monomer is internally polymerized, and the reactive functional group is preferably reacted with a polyfunctional spacing agent thereby to provide a second reactive functional group. The latter is then reacted with a fluorescent label to provide the labeled microbead.

Claims

1. A method for preparation of polymeric microbeads comprising:

(a) contacting a dispersion of swelled polymeric particles with an ethylenically unsaturated monomer containing an epoxy group, said monomer being absorbed into said particles;

(b) polymerizing said monomer to cause additional swelling of said particles, whereby said swelled particles provide a microbead with an epoxide group to serve as a first reactive functional group;

(c) reacting said epoxide with a polyfunctional spacing agent thereby to provide a second reactive functional group; and

(d) reacting said second reactive functional group with a suitable reagent which provides said microbead with a detectable label.

2. A method for preparation of polymeric microbeads comprising:

(a) contacting a dispersion of polymeric particles with a substance which is absorbed by said particles thereby to cause swelling of said particles;

(b) contacting said swelled particles with at least one monomer having the formula;

##STR6## wherein R and R₁ are independently selected from the group consisting of hydrogen or lower alkyl of 1 to 6 carbon atoms and A is selected from the group consisting of a methylene or carbonyl group whereby said monomers are absorbed into said particles thereby to provide said particles with an epoxide group to serve as a first reactive functional group;

(c) polymerizing said monomers to cause additional swelling of said particles whereby said swelled particles provide a microbead with an epoxide group to serve as a first reactive functional group;

(d) reacting said epoxide group with a polyfunctional spacing agent of the formula;

space="preserve" listing-type="equation">X--Q--Y

wherein X is selected from the group consisting of amino, hydroxyl or sulfhydryl, Q is selected from the group consisting of a straight or branched chain alkylene group of 2 to 10 carbon atoms, an aryl group or a heterocyclic group, and Y is selected from the group consisting of amino, hydroxyl, sulfhydryl, halo or carboxyl, thereby to provide said microbead with a second reactive functional group; and

(e) reacting said second reactive functional group with a fluorescent reagent selected from the group consisting of a derivative of fluorescein, a derivative of rhodamine or a derivative of phycobiliprotein.

4. The method in accordance with claim 2 wherein said substance absorbed by said particles has a molecular weight of from about 100 to about 5000 and a water solubility of from about 10^-6 to about 10^-2 g/l.

5. The method in accordance with claim 4 wherein said substance is selected from the group consisting of 1-chlorododecane, hexadecane or naphthalene.

6. The method in accordance with claim 2 wherein R and R₁ are independently selected from the group consisting of hydrogen or methyl.

7. The method in accordance with claim 2 further comprising contacting said swelled particles with a monomer of the formula:

##STR7## wherein R and R₁ are independently selected from the group consisting of hydrogen or lower alkyl of 1 to 6 carbon atoms and R₂ is lower alkyl of 1 to 6 carbon atoms.

8. The method in accordance with claim 2 further comprising stabilizing said dispersion of swelled particles and monomers with an ionic salt prior to said polymerizing step.

9. The method in accordance with claim 8 wherein said ionic salt is a halide of an alkali metal.

10. The method in accordance with claim 9 wherein said salt is potassium chloride.

11. The method in accordance with claim 2 wherein said spacing agent is a diamine having from 2 to 10 carbon atoms between the nitrogen atoms.

12. The method in accordance with claim 11 wherein said spacing agent is 1,3-diaminopropane.

13. The method in accordance with claim 2 wherein said fluorescein derivative is fluorescein isothiocyanate.

3. A method for the preparation of polymeric microbeads comprising the steps of:

(a) providing an emulsion of dichloroethylene, sodium lauryl sulfate and benzoyl peroxide;

(b) adding 1-chlorododecane to said emulsion and homogenizing said emulsion;

(c) adding acetone and polyvinyl toluene seed particles to said homogenized emulsion to provide a seed particle mixture;

(d) removing said acetone;

(e) adding to said seed particle mixture a potassium chloride solution and a monomer mixture containing glycidyl methacrylate, methyl methacrylate and sodium dilaurylsulfonate dispersed in a potassium chloride solution;

(f) polymerizing said monomer mixture to provide a uniform microbead having an epoxide group;

(g) stirring said microbeads with an aqueous solution of 1,3-diaminopropane; and

(h) stirring said microbeads with a solution of fluorescein isothiocyanate in dimethylformamide.