Highly multiplexed particle-based assays

• US 20090131269A1
• Filed: 10/02/2008
• Published: 05/21/2009
• Est. Priority Date: 10/05/2007
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Panomics, Inc.
• Inventors: Martin, Jason, Nguyen, Quan Ngoc
• Granted Patent: US 8,114,681 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods are provided for detecting and optionally quantitating multiple analytes, including nucleic acid and/or polypeptide analytes, in particle-based assays that can be highly multiplexed. Compositions, systems, and kits related to the methods are also featured.

Claims

1. A method of detecting analytes of interest, the method comprising:

a) capturing a first analyte from a first sample on a first subset of particles, and separately capturing a second analyte from a second sample on a second subset of particles, wherein the particles of the second subset are distinguishable from those of the first subset;

b) after step a), combining the first and second subsets of particles; and

c) after step b), identifying at least a portion of the particles from each subset and determining whether those particles have analytes captured thereto.

2. A method of detecting analytes of interest, the method comprising:

a) providing a first sample comprising or putatively comprising a first group of one or more analytes;

b) providing a second sample comprising or putatively comprising a second group of one or more analytes;

c) providing a first population of particles, the first population comprising one or more subsets of particles;

wherein, when the first population comprises two or more subsets of particles, a plurality of the particles in each subset of the first population are distinguishable from a plurality of the particles in the other subsets of the first population; and

wherein the particles in each subset of the first population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the first group and, when the first population comprises two or more subsets of particles, which capture molecule is different from those on the other subsets of the first population;

d) providing a second population of particles, the second population comprising one or more subsets of particles;

wherein, when the second population comprises two or more subsets of particles, a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the other subsets of the second population;

wherein a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the subsets of the first population; and

wherein the particles in each subset of the second population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the second group and, when the second population comprises two or more subsets of particles, which capture molecule is different from those on the other subsets of the second population;

e) contacting the first sample and the first population of particles and capturing any analyte of the first group present in the first sample on a selected subset of the first population of particles, and separately contacting the second sample and the second population of particles and capturing any analyte of the second group present in the second sample on a selected subset of the second population of particles;

f) after step e), combining the first and second populations of particles; and

g) after step f), detecting which subsets of particles have an analyte of interest captured on the particles, thereby indicating which analytes were present in the first and second samples.

3. The method of claim 2, wherein:

the first sample comprises or putatively comprises a first group of two or more analytes;

the second sample comprises or putatively comprises a second group of two or more analytes;

the first population of particles comprises two or more subsets of particles, wherein a plurality of the particles in each subset of the first population are distinguishable from a plurality of the particles in the other subsets of the first population, and wherein the particles in each subset of the first population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the first group and which capture molecule is different from those on the other subsets of the first population; and

the second population of particles comprises two or more subsets of particles, wherein a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the other subsets of the second population and from a plurality of the particles in the subsets of the first population, and wherein the particles in each subset of the second population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the second group and which capture molecule is different from those on the other subsets of the second population.

4. The method of claim 2, wherein the capture molecule on a subset of the particles of the second population is substantially identical to the capture molecule on a subset of the first population.

5. The method of claim 3, wherein the capture molecules on each of the two or more subsets of the particles in the second population are substantially identical to the capture molecules on subsets of the first population.

6. The method of claim 2, wherein the analytes comprise polypeptides.

7. The method of claim 2, wherein the analytes comprise nucleic acids.

8. The method of claim 2, wherein the analytes of the first group and the analytes of the second group are the same group of target molecules derived from different sources.

9. The method of claim 2, wherein the analytes of the first group and the analytes of the second group are different target molecules.

10. The method of claim 2, wherein in step g) detecting which subsets of particles have an analyte of interest captured on the particles comprises:

i) associating a label with any of the analytes captured on the particles, by providing a detection reagent, which detection reagent comprises the label, and contacting the detection reagent with the combined populations of particles, whereby the detection reagent binds directly or indirectly to any analyte captured on the particles, thereby associating the label with any analyte captured on the particles,

ii) identifying at least a portion of the particles from each subset, and

iii) detecting the presence or absence of the label on those particles.

11. The method of claim 10, wherein the detection reagent is a labeled antibody.

12. The method of claim 10, wherein the detection reagent is a labeled polynucleotide.

13. The method of claim 10, wherein the detection reagent is a labeled avidin or a labeled streptavidin.

14. The method of claim 10, wherein detecting the presence or absence of the label on the particles comprises measuring an intensity of a signal from the label, the method comprising correlating the intensity of the signal for a given subset of particles with a quantity of the corresponding analyte of interest present.

15. The method of claim 2, wherein the capture molecules associated with the first and/or second populations of particles comprise antibodies.

16. The method of claim 2, wherein the capture molecules associated with the first and/or second populations of particles comprise polynucleotides.

17. The method of claim 2, wherein the analytes bind directly to the capture molecules.

18. The method of claim 2, wherein the analytes bind directly to molecules which are in turn bound directly to the capture molecules.

19. The method of claim 2, wherein the analytes are nucleic acids;

wherein the capture molecules are polynucleotide capture probes;

wherein in step e) capturing any analytes of the first group present in the first sample on a selected subset of the particles in the first population comprises i) providing one or more subsets of two or more capture extenders, wherein each subset of capture extenders is configured to hybridize to one of the nucleic acid analytes, and wherein the capture extenders in each subset are configured to hybridize to one of the capture probes, and ii) hybridizing any nucleic acid analyte of the first group present in the first sample to its corresponding subset of capture extenders and hybridizing the subset of capture extenders to its corresponding capture probe, whereby the nucleic acid analyte is captured on the selected subset of particles comprising that capture probe;

wherein in step e) capturing any analytes of the second group present in the second sample on a selected subset of the particles in the second population comprises i) providing one or more subsets of two or more capture extenders, wherein each subset of capture extenders is configured to hybridize to one of the nucleic acid analytes, and wherein the capture extenders in each subset are configured to hybridize to one of the capture probes, and ii) hybridizing any nucleic acid analyte of the second group present in the second sample to its corresponding subset of capture extenders and hybridizing the subset of capture extenders to its corresponding capture probe, whereby the nucleic acid analyte is captured on the selected subset of particles comprising that capture probe;

wherein in step g) detecting which subsets of particles have an analyte of interest captured on the particles comprises i) associating a label with any analyte captured on the particles by hybridizing one or more label extenders and a label probe system comprising the label to any nucleic acid analyte captured on the particles, ii) identifying at least a portion of the particles from each subset, and iii) detecting the presence or absence of the label on those particles.

20. The method of claim 2, wherein the analytes are biotinylated nucleic acids;

wherein the capture molecules are polynucleotides, each of which is complementary to one of the nucleic acid analytes;

wherein in step a) providing a first sample comprising or putatively comprising a first group of one or more analytes comprises isolating a first group of one or more biotinylated nucleic acids bound by one or more transcription factors;

wherein in step b) providing a second sample comprising or putatively comprising a second group of one or more analytes comprises isolating a second group of one or more biotinylated nucleic acids bound by one or more transcription factors;

wherein in step e) capturing any analytes of the first or second group present in the first or second sample on a selected subset of the first or second population of particles comprises hybridizing any biotinylated nucleic acid analyte present in the first or second sample to its complementary polynucleotide capture molecule;

wherein in step g) detecting which subsets of particles have an analyte of interest captured on the particles comprises i) binding labeled streptavidin or labeled avidin to the biotinylated nucleic acid analytes, ii) identifying at least a portion of the particles from each subset, and iii) detecting the presence or absence of the label on those particles.

21. The method of claim 2, wherein the analytes are tyrosine-phosphorylated polypeptides;

wherein the capture molecules comprise SH2 domains;

and wherein in step g) detecting which subsets of particles have an analyte of interest captured on the particles comprises i) binding a biotinylated anti-phosphotyrosine antibody to any tyrosine-phosphorylated polypeptide analyte captured on the particles, and binding labeled streptavidin or labeled avidin to the biotinylated anti-phosphotyrosine antibody, ii) identifying at least a portion of the particles from each subset, and iii) detecting the presence or absence of the label on those particles.

22. The method of claim 2, comprising after step e) and before step g), washing the particles to remove unbound material from the particles.

23. The method of claim 2, wherein the particles are microspheres, and wherein the microspheres of each subset are distinguishable from those of the other subsets on the basis of their fluorescent emission spectra, their diameter, or a combination thereof.

24. The method of claim 2, comprising:

providing a third sample comprising or putatively comprising a third group of one or more analytes;

providing a third population of particles, the third population comprising one or more subsets of particles;

wherein, when the third population comprises two or more subsets of particles, a plurality of the particles in each subset of the third population are distinguishable from a plurality of the particles in the other subsets of the third population;

wherein a plurality of the particles in each subset of the third population are distinguishable from a plurality of the particles in the subsets of the first and second populations; and

wherein the particles in each subset of the third population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the third group and, when the third population comprises two or more subsets of particles, which capture molecule is different from those on the other subsets of the third population;

wherein step e) comprises separately contacting the third sample and the third population of particles and capturing any analyte of the third group present in the third sample on a selected subset of the third population of particles;

wherein in step f) combining the first and second populations of particles comprises combining the first, second, and third populations of particles; and

wherein in step g) detecting which subsets of particles have an analyte of interest captured on the particles thereby indicates which analytes were present in the first, second, and third samples.

25. The method of claim 2, wherein the first, the second, and at least a third population of particles are provided, each of which populations comprises two or more subsets of particles;

wherein a plurality of the particles in each subset of each population are distinguishable from a plurality of the particles in the other subsets of that population and in the subsets of the other populations;

wherein the particles in each subset of each population comprise a capture molecule, which capture molecule is different from those on the other subsets of that population; and

wherein the capture molecules on the subsets are the same from population to population.

26. A method of detecting analytes of interest, the method comprising:

a) providing a first sample comprising or putatively comprising a first group of one or more analytes;

b) providing a second sample comprising or putatively comprising a second group of one or more analytes;

c) providing a first set of one or more reporter entities, each of which reporter entities is configured to capture a different one of the analytes of the first group;

wherein, when the first set comprises two or more reporter entities, the reporter entities of the first set are distinguishable from each other;

d) providing a second set of one or more reporter entities, each of which reporter entities is configured to capture a different one of the analytes of the second group;

wherein, when the second set comprises two or more reporter entities, the reporter entities of the second set are distinguishable from each other; and

wherein each of the reporter entities of the second set is distinguishable from the reporter entities of the first set;

e) contacting the first sample and the first set of reporter entities and capturing any analyte of the first group present in the first sample to a selected reporter entity of the first set, and separately contacting the second sample and the second population of reporter entities and capturing any analyte of the second group present in the second sample to a selected reporter entity of the second set;

f) after step e), combining the first and second sets of reporter entities; and

g) after step f), detecting which reporter entities have an analyte of interest captured thereto, thereby indicating which analytes were present in the first and second samples.

27. A composition comprising:

a mixture of at least a first population of particles and a second population of particles;

wherein the first population of particles comprises one or more subsets of particles, wherein, when the first population comprises two or more subsets of particles, a plurality of the particles in each subset of the first population are distinguishable from a plurality of the particles in the other subsets of the first population, and wherein the particles in each subset of the first population comprise a capture molecule, which capture molecule is different from those on the other subsets of the first population when the first population comprises two or more subsets of particles;

wherein the second population of particles comprises one or more subsets of particles, wherein a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the subsets of the first population, wherein, when the second population comprises two or more subsets of particles, a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the other subsets of the second population, and wherein the particles in each subset of the second population comprise a capture molecule, which capture molecule is different from those on the other subsets of the second population when the second population comprises two or more subsets of particles; and

wherein the capture molecule on a subset of the particles of the second population is substantially identical to the capture molecule on a subset of the first population.

28. The composition of claim 27, wherein the first population of particles comprises two or more subsets of particles and wherein the second population of particles comprises two or more subsets of particles.

29. The composition of claim 28, wherein the capture molecules on each of the two or more subsets of the particles in the second population are substantially identical to the capture molecules on subsets of the first population.

30. A composition comprising:

a first group of one or more analytes, which analytes originate from a first sample;

a second group of one or more analytes, which analytes originate from a second sample different from the first sample; and

a mixture of at least a first population of particles and a second population of particles,

wherein the first population of particles comprises one or more subsets of particles, wherein, when the first population comprises two or more subsets of particles, a plurality of the particles in each subset of the first population are distinguishable from a plurality of the particles in the other subsets of the first population, and wherein the particles in each subset of the first population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the first group and, when the first population comprises two or more subsets of particles, which capture molecule is different from those on the other subsets of the first population, and

wherein the second population of particles comprises one or more subsets of particles, wherein a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the subsets of the first population, wherein, when the second population comprises two or more subsets of particles, a plurality of the particles in each subset of the second population are distinguishable from a plurality of the particles in the other subsets of the second population, and wherein the particles in each subset of the second population comprise a capture molecule, which capture molecule is configured to capture one of the analytes of the second group and, when the second population comprises two or more subsets of particles, which capture molecule is different from those on the other subsets of the second population.

31. The composition of claim 30, wherein the first group comprises two or more analytes;

wherein the second group comprises two or more analytes;

wherein the first population of particles comprises two or more subsets of particles and wherein the second population of particles comprises two or more subsets of particles.

32. A composition comprising:

a first group of one or more analytes, which analytes originate from a first sample;

a second group of one or more analytes, which analytes originate from a second sample different from the first sample; and

a mixture of at least a first set of reporter entities and a second set of reporter entities,

wherein the first set of reporter entities comprises one or more reporter entities, each of which reporter entities is configured to capture a different one of the analytes of the first group, wherein, when the first set comprises two or more reporter entities, the reporter entities of the first set are distinguishable from each other, and

wherein the second set of reporter entities comprises one or more reporter entities, each of which reporter entities is configured to capture a different one of the analytes of the second group, wherein, when the second set comprises two or more reporter entities, the reporter entities of the second set are distinguishable from each other, and wherein each of the reporter entities of the second set is distinguishable from the reporter entities of the first set.