Methods for detecting target analytes and enzymatic reactions

• US 20030027126A1
• Filed: 03/23/2001
• Published: 02/06/2003
• Est. Priority Date: 03/14/1997
• Status: Abandoned Application

• Current Assignee: Trustees of Tufts College
• Original Assignee: Trustees of Tufts College
• Inventors: Michael, Karri Lynn, Walt, David R.
• Application Number:
  US09/816,651
• Publication Number:
  US 20030027126A1
• Time in Patent Office: Days
• US Class Current: 435/4
• 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 accomodating 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 detecting a target analyte in a sample comprising:

a) providing an array comprising;

i) an array substrate, wherein said array substrate is other than a fiber optic bundle; and

ii) at least first and second sites wherein first and second reaction components are immobilized at said first and second sites, respectively;

b) contacting said array substrate with said sample; and

c) detecting a change in an optical property around at least said first site as an indication of the interaction between said target analyte and at least said first reaction component.

2. The method according to claim 1, wherein said target analyte is an enzyme.

3. The method according to claim 2 wherein at least one of said first and second reaction components is a substrate of said enzyme.

4. The method according to claim 3, wherein said first and second reaction components are attached with a non-cleavable linker to first and second microspheres, respectively, wherein said first and second microspheres are randomly distributed on said array substrate.

5. The method according to claim 1 wherein said target analyte is an enzyme substrate.

6. The method according to claim 5 wherein at least one of said first and second reaction components is an enzyme.

7. The method according to claim 1, wherein said target analyte is an enzyme inhibitor.

8. The method according to claim 7, wherein at least one of said first and second reaction components is an enzyme.

9. The method according to claim 1, 2, 5, 6, 7 or 8, wherein said first and second reaction components are attached to first and second microspheres, respectively, said first and second microspheres randomly distributed on said array substrate.

10. The method according to claim 1 wherein said first and second reaction components are enzymes.

11. The method according to claim 1, wherein said array substrate is selected from the group consisting of glass, composite materials, ceramics, metals and plastic.

12. The method according to claim 1 or 11, wherein said array is a spotted array.

16. The method according to claim 1 or 13, wherein said contacting is in the presence of a diffusion retardant.

17. The method according to claim 16, wherein said diffusion retardant is glycerol.

13. A method of detecting a target analyte in a sample comprising:

a) providing an array comprising;

i) an array substrate comprising discrete sites; and

ii) a population of microspheres comprising at least first and second subpopulations comprising;

(a) first and second reaction components respectively; and

(b) a detection molecule;

b) contacting said array with said sample; and

c) detecting a change in an optical property around at least said first microsphere as an indication of the interaction between said target analyte and at least said first reaction component.

14. The method according to claim 13, wherein said first and second reaction components and said detection molecule are enzymes.

15. The method according to claim 14, wherein an enzymatic product from said first reaction component is an enzyme substrate for said detection molecule.

23. A method according to claim 13, 18 or 19, wherein said array substrate is a fiber optic bundle.

24. A method according to claim 13, 18 or 19, wherein said array substrate is selected from the group consisting of glass, composite materials, ceramics, metals and plastic.

25. A method according to claim 24, wherein said discrete sites are well.

18. A method of detecting an enzymatic reaction comprising:

a) providing an array comprising

i) an array substrate comprising discrete sites; and

ii) a population of microspheres randomly distributed on said array substrate, said microspheres comprising at least one enzyme;

b) contacting said array with a sample comprising a target analyte, wherein said target analyte is an enzyme substrate;

c) monitoring a signal in a region surrounding said microspheres, whereby detection of said signal provides an indication of the reaction between said enzyme and said enzyme substrate.

20. A method according to claim 18 or claim 19 further comprising:

d) including in said sample a solution to prevent the diffusion of said label away from said microsphere.

21. A method according to claim 20, wherein said solution comprises glycerol.

22. A method according to claim 18 or claim 19, wherein the product of said reaction comprises said label.

19. A method of detecting an enzymatic reaction comprising:

a) providing an array comprising;

i) an array substrate comprising discrete sites; and

ii) a population of microspheres randomly distributed on said array substrate, said population comprising first and second subpopulations, wherein said first and second subpopulations comprise first and second discrete oligonucleotides, respectively, attached to said microspheres;

b) contacting said array with a composition comprising an enzyme;

c) monitoring a signal in a region surrounding the microspheres, whereby detection of said signal provides an indication of the reaction between said enzyme and at least one of said discrete oligonucleotides.