Multianalyte molecular analysis using application-specific random particle arrays

• US 20040132122A1
• Filed: 02/19/2003
• Published: 07/08/2004
• Est. Priority Date: 06/21/2000
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Seul, Michael, Banerjee, Sukanta, Chau, Chiu W., Li, Alice X., Podual, Kairali
• Granted Patent: US 7,892,854 B2
• Time in Patent Office: Days
• US Class Current: 435/7.92
• 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 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00576 fluorophore
• 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 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/155 : Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: G01N 2035/00564 : Handling or washing solid phase elements, e.g. beads
• CPC Class Codes: G01N 2035/00762 : magnetic code
• CPC Class Codes: G01N 2446/20 : the magnetic material being present in the particle core
• CPC Class Codes: G01N 2446/64 : Magnetic material dispersed in oil drop
• CPC Class Codes: G01N 27/745 : for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 33/5434 : using magnetic particle immunoreagent carriers which constitute new materials per se
• CPC Class Codes: G01N 33/54346 : Nanoparticles
• CPC Class Codes: G01N 33/587 : Nanoparticles
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis

Abstract

The present invention provides methods and apparatus for the application of a particle array in bioassay format to perform qualitative and/or quantitative molecular interaction analysis between two classes of molecules (an analyte and a binding agent). The methods and apparatus disclosed herein permit the determination of the presence or absence of association, the strength of association, and/or the rate of association and dissociation governing the binding interactions between the binding agents and the analyte molecules. The present invention is especially useful for performing multiplexed (parallel) assays for qualitative and/or quantitative analysis of binding interactions of a number of analyte molecules in a sample.

Claims

1. A method of determining elements of a co-affinity matrix which describes pair-wise analyte-binding interactions in a competitive multiconstituent equilibrium reaction comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate;

determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith;

contacting the binding agents with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex;

detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed; and

determining the identity of the binding agents involved in the complex formation by the chemically or physically distinguishable characteristic associated with the corresponding particles,

wherein said change in optical signature for each type of the analyte-binding agent complexes determines affinities characterizing said analyte-binding agent interaction and said affinities providing elements of a co-affinity matrix which describes pairwise analyte-binding agent interactions in a competitive multiconstituent equilibrium reaction.

3. The method of claim 1 or 2, wherein the co-affinity matrix obtained is used to characterize the analyte.

4. The method of claim 3, wherein a combination of the coefficients of the co-affinity matrix and known concentrations of analyte and binding agents participating in the formation of analyte-binding agent complexes serves to define a competitive binding interaction descriptor.

5. The method of claim 1 or 2, further comprising the determination of the affinity constant for each type of the analyte-binding agent interaction, wherein each affinity constant is calculated by determining the number of analyte-binding agent complexes formed from said change in the optical signature of each type of the analyte-binding agent complexes formed.

6. A method of determining the presence or absence of an impurity in a sample comprising:

preparing a co-affinity matrix of a reference analyte with a set of binding agents according to the method of claim 1 or 2, and recording said co-affinity matrix,

introducing a sample containing said reference analyte and possibly an impurity analyte, and determining the co-affinity matrix subsequent to the addition of the sample; and

detecting for the deviation in the coefficients of the co-affinity matrix of the reference analyte subsequent to the addition of the sample that might contain an impurity, wherein the deviation in the co-affinity matrix indicates the presence of an impurity in said sample.

9. The method of any of claims 1 to 8, wherein the change in the optical signature comprises a change in the fluorescent intensity associated with the particles involved in the binding interaction.

10. The method of any one of claims 1 to 8, wherein the determining of the identity of the binding agents in the array comprises taking a decoding image of the array that detects the chemically or physically distinguishable characteristic of each bead in the array, and wherein the optical signature detecting step comprises taking an assay image of the array that detects the change in the optical signature associated with the beads.

11. The method of claim 10, wherein the assay image and the decoding image are compared using a template matching algorithm.

12. The method of any one of claims 1 to 8, wherein the planar particle array is immobilized on the substrate.

13. The method of any one of claims 1 to 8, wherein the particles are associated with a chemically distinguishable characteristic.

14. The method of claim 13, wherein the chemically distinguishable tag comprises a fluorophore tag.

33. The method of claim 5, wherein the analyte specifically binds to one binding agent but not to other binding agent(s) present.

34. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], given [L₀], [R₀], and n_B, where [L₀] is the initial analyte concentration, [R₀] is the number of binding agents per bead and n_B is the number of beads.

35. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], for at least two measurements involving different bead numbers, n_B and given initial analyte concentration [L₀].

36. The method of claim 33, wherein, using the law of mass action, affinity constant K is determined from said change in optical signature following conversion to yield [LR], for at least two measurements involving different number of binding agents per bead, [R₀] and given initial analyte concentration [L₀].

37. The method of claim 33, wherein, using the law of mass action, concentration of analyte [L], is determined from said change in optical signature following conversion to yield [LR], given K, n_B and [R₀]

38. The method of claim 33, wherein in a two-step assay involving the use of a first labeled analyte of interest in the first binding reaction, followed by injection of a second analyte with known affinity constant K′

, the affinity constant K is determined from said the optical signature before and after injection of the second analyte, following conversion to yield [LR]₁ and [LR]₂ given the initial concentrations of the analytes, [L₀] and [L₀]′ and

[R₀].

39. The method of claim 33, wherein in a two-step assay involving the use of a first labeled analyte of interest in the first binding reaction, followed by injection of a second analyte with known affinity constant K′

, the affinity constant K is determined from said the optical signature before and after injection of the second analyte, following conversion to yield [LR]₁ and [LR]₂ given the initial concentrations of the analytes, [L₀] and [L₀]′ and

n_B.

40. The method of claim 33, wherein:

using the law of mass action, the initial analyte concentration [L₀] is determined from said change in optical signature following conversion to yield [LR], given K, [R₀] and n_B.

2. A method of determining elements of a co-affinity matrix which describes pair-wise analyte-binding interactions in a competitive multiconstituent equilibrium reaction comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles,

contacting the binding agents with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex;

forming a planar array of the particles on a substrate;

detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed; and

determining the identity of the binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith, the determining step occurring either before or after the detecting step, and

wherein said change in optical signature for each type of the analyte-binding agent complexes determines affinities characterizing said analyte-binding agent interaction and said affinities providing elements of a co-affinity matrix which describes pairwise analyte-binding agent interactions in a competitive multiconstituent equilibrium reaction.

7. A method of analyzing the kinetics of molecular binding interactions governing the association of analyte-binding agent complexes formed between one or more analytes and one or more binding agents, comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate;

determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith;

contacting, at a preset initial time, the binding agents in the array with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex;

detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed at preset intervals following said preset initial time; and

determining, for each type of analyte-binding agent complex, the association rate constants from the time dependence of said changes in optical signature associated with said particles.

8. A method of analyzing the kinetics ofmolecular binding interactions governing the dis-association of analyte-binding agent complexes formed between one or more analytes and one or more binding agents, comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate;

determining the identity of said binding agents on each particle in the array by the chemically or physically distinguishable characteristic associated therewith;

contacting the binding agents in the array with an analyte molecule so as to allow the analyte to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a change in an optical signature associated with the particles whose binding agent is involved in the formation of the complex;

detecting the change in the optical signature associated with said particles for each type of the analyte-binding agent complexes formed;

exchanging, at a preset exchange time, the analyte solution with a second solution containing no analyte to allow the bound analytes to unbind from the analyte-binding agent complexes, said unbinding resulting in a change in the optical signature associated with the particles having analyte-binding agent complexes, and

recording, at preset intervals following said preset exchange time, said changes in optical signature and deterining, for each type of analyte-binding agent complex, the dis-association rate constants from the time dependence of said changes in optical signature associated with said particles.

15. A method of performing a bioassay comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles, and wherein the particles are arranged in a planar array on a substrate;

generating a de-coding image of the array showing the location of each binding agent in the array;

contacting the binding agents with a sample that may contain an analyte so as to allow the analyte, if present in the sample, to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a corresponding or a proportional change in the optical signature associated with the particles whose binding agent is involved in the formation of the complex;

generating an assay image of the array which detects the change in the optical signature associated with said particles, and deriving from the change in the optical signature the number of analyte-binding agent complexes formed; and

determining the identity of the analyte in the analyte-binding agent complex by comparing the decoding image with the assay image.

17. The method of claim 15 or 16, wherein the comparing of the assay and the decoding image comprises use of optical microscopy apparatus including an imaging detector and computerized image capture and analysis apparatus.

18. The method of claim 15 or 16, wherein the analyte comprises a ligand and the binding agents comprise receptors.

19. The method of claim 15 or 16, wherein the analyte and the binding agents comprise nucleotide fragments, and wherein the number of hybridization complexes formed between the nucleotide fragment and its complementary sequence fragments is determined.

20. The method of claim 15 or 16, wherein the particles comprise magnetic particles distinguishable by a chemical or physical characteristic that uniquely identifies the binding agent attached thereto.

21. The method of claim 20, wherein the chemical or physical characteristic comprises one or more fluorophore dyes spectrally distinguishable by excitation wavelength, emission wavelength, excited-lifetime or emission intensity.

22. The method of claim 21, wherein the array of magnetic particles is assembled by application of a magnetic field to said particles.

23. The method of claim 15 or 16, further comprising calculating the affinity constant of a binding interaction between an analyte and its binding agent from the number of the analyte-binding agent complexes formed.

24. A method of integrating sample preparation and bioassay using magnetic particles comprising:

providing a plurality of magnetic particles comprising at least two different particle populations, each population being distinguishable by a recognition molecule attached thereto, wherein the particles are attached to a chemical characteristic that uniquely identifies a biomolecule of interest that selectively binds to the recognition molecule;

providing a biological fluid containing biomolecules and allowing said biomolecules to interact with the recognition molecules on the magnetic particles;

removing the fluid along with unbound components thereof;

transforming the biomolecules bound to the magnetic particles to produce transformed biomolecules, wherein the transformed biomolecules remain attached to the magnetic particles on which they are synthesized;

performing a bioassay according to the method of claim 15 or 16, wherein the binding agents comprise the transformed biomolecules.

25. The method of claim 24, wherein the biomolecules of interest comprises mRNA and the transforming comprises reverse transcribing said mRNA to produce cDNA, which is attached to the magnetic particles.

26. The method of claim 24, wherein the sample preparation and bioassay occur in the same compartment.

16. A method of performing a bioassay comprising:

providing a plurality of particles comprising at least two different particle populations, each population being distinguishable by a binding agent attached thereto, wherein the particles are associated with a chemically or physically distinguishable characteristic that uniquely identifies the binding agents on said particles,

contacting the binding agents with a sample that may contain an analyte so as to allow the analyte, if present in the sample, to form an analyte-binding agent complex with one or more binding agents, the formation of each complex resulting in a corresponding or proportional change in the optical signature associated with the particles whose binding agent is involved in the formation of the complex;

forming a planar array of the particles on a substrate;

generating an assay image of the array which detects the change in the optical signature associated with said particles, and deriving from the change in the optical signature the number of analyte-binding agent complexes formed;

generating a decoding image of the array showing the location of each binding agent in the array, wherein the decoding image is generated either before or after generating the assay image; and

determining the identity of the analyte in the analyte-binding agent complex formed by comparing the decoding image with the assay image.

27. A method for performing a bioassay involving integration of sample preparation and parallel molecular interaction assay analysis, comprising

providing an apparatus comprising at least a sample preparation compartment and an assay compartment, and means for fluidically connecting the sample and the assay compartments;

providing, in the sample preparation compartment, a biological fluid containing a biomolecule of interest and a plurality of magnetic particles capable of binding to the biomolecule of interest, and allowing the magnetic particles to bind the biomolecules of interest;

removing the biological fluid along with unbound components of said fluid, while retaining the magnetic particles and the biomolecules bound to said particles;

releasing said biomolecules from said magnetic particles and transporting said biomolecules from the sample preparation compartment to the assay compartment through the fluidic means; and

performing a bioassay according to the method of 15 or 16, wherein the analyte in the bioassay comprises transported biomolecules of interest.

28. The method of claim 27, further comprising transforming the biomolecule of interest, which is then used as an analyte in the bioassay.

29. The method of claim 27, wherein the biomolecule of interest comprises mRNA and the transformation comprises reverse transcription of said mRNA to produce cDNA, and wherein the analyte in the bioassay comprises said cDNA and the binding agents comprises oligonucleotides or other DNA probes.

30. The method of claim 29, wherein the reverse transcription occurs in the sample preparation compartment, while said mRNA is bound to the magnetic particles, and the cDNA is released from said magnetic particles after the reverse transcription and transported to the assay compartment and used as an analyte in the bioassay.

31. A method of preparing monodisperse magnetic fluorescent particles comprising providing polymeric microparticles and swelling said microparticles in an organic solvent containing one or more and magnetic nanoparticles to produce magnetic particles, wherein the fluorescent dyes and the magnetic nanoparticles are distributed throughout the magnetic particles without being localized at specific locations with the particle.

32. An array comprising a plurality of magnetic particles comprising at least two different particle populations distinguishable by a chemical characteristic associated therewtih, wherein the magnetic particles are assembled on a planar array in a compositionally random manner.