Methods for detecting target analytes and enzymatic reactions

• US 10,107,804 B2
• Filed: 12/22/2017
• Issued: 10/23/2018
• Est. Priority Date: 03/23/2001
• Status: Expired due to Fees

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Walt, David R., Michael-Ballard, Karri Lynn
• Primary Examiner(s): Lam, Ann Y
• Application Number:
  US15/853,157
• Publication Number:
  US 20180136203A1
• Time in Patent Office: 305 Days
• Field of Search: None
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00524 in the shape of fiber bundles
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00704 integrated with the reactor apparatus
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/0074 : Biological products
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/00 : Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 15/1456 : without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/1468 : with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1438 : Using two lasers in succession
• CPC Class Codes: G01N 2021/6441 : with two or more labels
• CPC Class Codes: G01N 2021/6484 : Optical fibres
• CPC Class Codes: G01N 2021/7786 : Fluorescence
• CPC Class Codes: G01N 2035/0097 : monitoring reactions as a function of time
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6456 : Spatial resolved fluorescence measurements; Imaging
• CPC Class Codes: G01N 21/7703 : using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: G01N 21/78 : producing a change of colour
• CPC Class Codes: G01N 2201/0833 : Fibre array at detector, resolving
• CPC Class Codes: G01N 33/54313 : the carrier being characterised by its particulate form

Abstract

A microsphere-based analytic chemistry system and method for making the same is disclosed in which microspheres or particles carrying bioactive agents may be combined randomly or in ordered fashion and dispersed on a substrate to form an array while maintaining the ability to identify the location of bioactive agents and particles within the array using an optically interrogatable, optical signature encoding scheme. A wide variety of modified substrates may be employed which provide either discrete or non-discrete sites for accommodating the microspheres in either random or patterned distributions. The substrates may be constructed from a variety of materials to form either two-dimensional or three-dimensional configurations. In a preferred embodiment, a modified fiber optic bundle or array is employed as a substrate to produce a high density array. The disclosed system and method have utility for detecting target analytes and screening large libraries of bioactive agents.

Claims

1. A method of making an array, said method comprising:

(a) providing a substrate with a surface comprising a plurality of discrete sites wherein said substrate is not a fiber optic bundle;

(b) providing a population of beads comprising a first subpopulation of beads having nucleic acids associated therewith and a second subpopulation of beads having enzymes associated therewith, said beads of the first subpopulation being a different size from the beads of the second subpopulation; and

(c) randomly distributing said population of beads on said surface such that at least one discrete site of said plurality of discrete site has a single bead from said first subpopulation associated therewith and such that beads from said second subpopulation end up at said site(s) that have a single bead from the first subpopulation of beads.

2. The method of claim 1, wherein said first subpopulation and said second subpopulation are sequentially distributed on said surface.

3. The method of claim 1, further comprising making said first subpopulation of beads by attaching a digest of a eukaryotic or prokaryotic genome to said first subpopulation of beads.

4. The method of claim 1, further comprising making said first subpopulation of beads by attaching copies of fragments of a prokaryotic or eukaryotic genome to said first subpopulation of beads.

5. The method of claim 1, wherein at least one discrete site of said plurality of discrete sites lacks a single bead from said first population.

6. The method of claim 1, further comprising optically coupling a fiber optic bundle to said substrate.

7. The method of claim 1, wherein said plurality of discrete sites comprises a plurality of wells.

8. The method of claim 1, wherein said first subpopulation comprises at least 10 beads having copies of the same nucleic acid associated therewith.

9. A method of making an array, said method comprising:

(a) providing a substrate comprising a plurality of wells wherein said substrate is not a fiber optic bundle;

(b) randomly distributing beads of a first subpopulation of beads into wells of said plurality of wells such that at least one well of the plurality of wells contains a single bead from the first subpopulation of beads, wherein beads of the first subpopulation of beads have nucleic acids associated therewith; and

(c) distributing beads of a second subpopulation of beads into the well(s) containing a single bead from the first subpopulation of beads, wherein beads of the second subpopulation of beads have enzymes associated therewith, and wherein beads of the second subpopulation of beads are smaller than beads of said first subpopulation of beads.

10. The method of claim 9, further comprising making said first subpopulation of beads by attaching a digest of a prokaryotic or eukaryotic genome to said first subpopulation of beads.

11. The method of claim 9, further comprising making said first subpopulation of beads by attaching copies of fragments of a prokaryotic genome to said first subpopulation of beads.

12. The method of claim 9, further comprising making said first subpopulation of beads by attaching copies of fragments of a eukaryotic genome to said first subpopulation of beads.

13. The method of claim 9, wherein at least one well of said plurality of wells lacks a single bead from said first subpopulation.

14. The method of claim 9, further comprising optically coupling a fiber optic bundle to said substrate.

15. The method of claim 9, wherein said first subpopulation comprises at least 10 beads having copies of the same nucleic acid associated therewith.

16. A method of detecting a nucleic acid on an array, said method comprising:

(a) providing a substrate with a surface comprising a plurality of discrete sites wherein said substrate is not a fiber optic bundle;

(b) providing a population of beads comprising a first subpopulation of beads having nucleic acids associated therewith and a second subpopulation of beads having enzymes associated therewith, said beads of the first subpopulation being a different size from the beads of the second subpopulation;

(c) randomly distributing said population of beads on said surface such that at least one discrete site of said plurality of sites has a single bead from said first subpopulation associated therewith and such that beads from said second subpopulation end up at said site(s) that have a single bead from the first subpopulation of beads;

(d) contacting said substrate with a sample comprising target nucleic acids, whereby said target nucleic acids hybridize to the nucleic acids associated with said first subpopulation of beads; and

(e) determining the presence of said target nucleic acids hybridized to said probe nucleic acids.

17. A method of detecting a nucleic acid on an array, said method comprising:

(a) providing a substrate comprising a plurality of wells wherein said substrate is not a fiber optic bundle;

(b) randomly distributing beads of a first subpopulation of beads into wells of said plurality of wells such that the wells contain a single bead from the first subpopulation of beads, wherein beads of the first subpopulation of beads have nucleic acids associated therewith; and

(c) distributing beads of a second subpopulation of beads into the wells containing a single bead from the first subpopulation of beads, wherein beads of the second subpopulation of beads have enzymes associated therewith, and wherein beads of the second subpopulation of beads are smaller than beads of said first subpopulation of beads

(d) contacting said substrate with a sample comprising target nucleic acids, whereby said target nucleic acids hybridize to the nucleic acids associated with said first subpopulation of beads; and

(e) determining the presence of said target nucleic acids hybridized to said probe nucleic acids on said first subpopulation of beads.

18. The method of claim 17, wherein said enzymes are attached to the beads of the second subpopulation.

19. A method of making an array comprising:

(a) providing a population of beads comprising a first subpopulation of beads having attached nucleic acids and a second subpopulation of beads having attached enzymes, said beads of the first subpopulation being a different size from the beads of the second subpopulation; and

(b) providing a substrate having a surface comprising a random pattern of beads of said first subpopulation, said first subpopulation of beads are located at discrete sites,

wherein each discrete site can have only a single associated bead from said first subpopulation, and

wherein at least one discrete site having a single associated bead from said first subpopulation further comprises beads from said second subpopulation.