Fluorescent microspheres and methods of using them

• US 5,132,242 A
• Filed: 11/13/1989
• Issued: 07/21/1992
• Est. Priority Date: 07/15/1987
• Status: Expired due to Fees

• Current Assignee: Sau W. Cheung
• Original Assignee: Sau W. Cheung
• Inventors: Cheung, Sau W.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US07/434,892
• Time in Patent Office: 981 Days
• Field of Search: 435/7.5, 435/6, 436/533, 436/800, 436/501, 436/503, 436/532, 436/546, 436/805, 436/823
• US Class Current: 436/501
• CPC Class Codes: B01J 13/20 After-treatment of capsule walls, e.g. hardening
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se

Abstract

Highly fluorescent latex microspheres have a diameter of less than five hundred angstroms and have more than five thousand fluorescent markers per sphere. The microspheres are prepared by reacting an acrylic latex bead with a diamine and a fluorescent amine at elevated pH to attach a spacer arm and a marker, respectively, to the bead by transacylation of ester terminations on the surface of the bead. A protein such as avidin or an immunoglobulin may then be conjugated to the spacer arm. A single fluorescent microsphere is visible using standard fluorescent microscopy. Therefore the microspheres may be utilitzed not only to visualize cell surface antigens but also DNA encoding for single genes, by means of a biotinylated DNA probe.

Claims

1. A fluorescent marker system comprising a plurality of fluorescent microspheres, each fluorescent microsphere comprising an acrylic latex bead having a diameter of less than 500 angstroms, said bead having conjugated, by transacylation of ester terminations on the surface of said bead, (a) at least 5,000 fluorescent molecules, and (b) spacer arms and specific binding means attached to said spacer arms for attaching said microspheres to biological analytes.

2. The marker system of claim 1 wherein said beads have a diameter of from 200 to 400 angstroms.

3. The marker system of claim 1 wherein said beads have at least 10,000 fluorescent molecules conjugated per bead.

4. The marker system of claim 3 wherein said fluorescent molecules are conjugated to said beads by carbon chains having a length of from two to ten carbons.

5. The marker system of claim 1 wherein said beads are primarily in single suspension in an aqueous medium.

6. The marker system of claim 5 wherein said spacer arms comprise carbon chains having a length of from eight to twenty carbons.

7. A dyed microsphere comprising an acrylic latex bead having a diameter of less than 500 angstroms, at least 5,000 dye marker means conjugated to the surface of said bead by transacylation of ester terminations on the surface of said bead for rendering said bead individually visible by microscopy, a plurality of spacer arms attached to said bead by transacylation of ester terminations on the surface of said bead, and specific binding means on said spacer arms for binding specifically with a biological analyte to label said biological analyte.

8. The microsphere of claim 7 wherein said specific binding means is chosen from the group consisting of avidin and biotin.

9. The microsphere of claim 7 wherein said specific binding means comprise antibodies.

10. A method of identifying a site on a biological analyte, said method comprising a step of reacting with said site a fluorescent acrylic latex microsphere having a diameter of less than 500 angstroms and at least 5,000 fluorescent markers conjugated to the microsphere by transcylation of ester terminations on the surface of said microsphere, and at least one species conjugated to the microsphere through a spacer arm by transacylation of ester terminations on the surface of said microsphere, said species having a specific affinity for said binding site, and a step of detecting said individual microsphere by fluorescence microscopy.

11. The method of claim 10 wherein said step of detecting said individual microsphere is by human observation.

12. The method of claim 10 wherein the fluorescent markers are conjugated to said microsphere by carbon chains having a length of from two to ten carbons.

13. The method of claim 10 wherein said spacer arms comprise carbon chains having a length of from eight to twenty carbons.

14. The method of claim 10 wherein the species having a specific affinity for said binding site is chosen from the group consisting of avidin and biotin.

15. The method of claim 10 wherein the species having a specific affinity for said binding site is an antibody.