Multiplex microparticle system

• US 20060240411A1
• Filed: 04/14/2006
• Published: 10/26/2006
• Est. Priority Date: 04/20/2005
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Mehrpouyan, Majid, Varro, Rudolf, Recktenwald, Diether J.
• Granted Patent: US 7,507,588 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/110833 Utilizing a moving indicator strip or tape
• CPC Class Codes: Y10T 436/112499 with sample on test slide

Abstract

Arrays of microparticle populations, each population labeled with a single fluorescent dye, are provided for use in multiplex assays. The populations form a virtual multidimensional array wherein each microparticle is identified by fluorescence intensity in two different fluorescence detection channels. The arrays are useful in a variety of assays, including multiplex, multi-analyte assays for the simultaneous detection of two or more analytes by, for example, flow cytometry, and a labeling reagents in, for example, microscopy. The use of singly-dyed microparticles to form multidimensional arrays greatly simplifies the creation of multiplex assays.

Claims

1. An array of populations of microparticles, comprising a plurality of sets,

wherein each set contains a plurality of populations of microparticles,

wherein microparticle populations within a set are labeled with different levels of a single fluorophore such that each population exhibits a measurably distinct fluorescence intensity;

wherein microparticles in different sets are labeled with different fluorophores;

and wherein all of said fluorophores emit light that is detectable using a single pair of detection channels, and wherein the relative emission in each of the two detection channels is distinguishably distinct between different fluorophores.

2. The array of claim 1, wherein said array comprises at least 3 sets of microparticle populations.

3. The array of claim 1, wherein said array comprises at least 4 sets of microparticle populations.

4. The array of claim 1, wherein said array comprises at least 5 sets of microparticle populations.

5. The array of claim 2, wherein said array comprises at least 20 populations of microparticles.

6. The array of claim 2, wherein said array comprises at least 30 populations of microparticles.

7. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 1;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

8. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 2;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

9. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 3;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

10. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 4;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

11. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 5;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

12. An array of microparticles for detecting multiple analytes in a sample, comprising:

a) an array of microparticles of claim 6;

b) a plurality of analyte-specific reagents, wherein reagents of the same specificity are coupled to microparticles in the same population, and reagents of different specificity are coupled to microparticles in different populations.

13. The array of claim 7, wherein at least one of said analyte-specific reagents is an antibody that binds specifically to an analyte that is an antigen, or at least one of said analyte-specific reactants is an antigen that binds specifically to an analyte that is an antibody.

14. The array of claim 7, wherein said analytes are selected from the set consisting of a protein, peptide, hormone, hapten, antigen, antibody, receptor, enzyme, nucleic acid, polysaccaride, chemical, polymer, pathogen, toxin, organic drug, inorganic drug, cell, tissue, microorganism, virus, bacteria, fungi, algae, parasite, allergen, pollutant, or a combination thereof.

15. A method of measuring a plurality of analytes in a sample comprising:

a) contacting a sample with an array of microparticles of claim 7 for a time sufficient to allow for the formation of microparticle-analyte complexes;

b) exposing said microparticle-analyte complex to excitation light;

c) measuring fluorescence of said microparticles in each of said pair of detector channels;

d) identifying the specificity of said analyte-specific reagent coupled to said microparticle from the fluorescence measured in step (c), thereby identifying the analyte present in said microparticle-analyte complex;

e) measuring the amount of analyte present in said microparticle-analyte complex.

16. The method of claim 15, wherein said sample is further contacted with a set of fluorescently labeled analyte-specific reagents, wherein said set contains an analyte-specific reagent specific for each analyte to be measured.

17. The method of claim 15, wherein said detecting is carried out by flow cytometry.