Methods for performing multiple sequential reactions on a matrix

• US 5,847,105 A
• Filed: 08/13/1997
• Issued: 12/08/1998
• Est. Priority Date: 03/16/1994
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Gamble, Ronald C., Theriault, Thomas P., Baldeschwieler, John D.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/910,727
• Time in Patent Office: 482 Days
• Field of Search: 435/6, 435/91.1, 435/810, 436/501, 536/22.1, 536/23.1, 536/24.1, 536/24.3, 536/24.33, 536/25.3, 935/77, 935/78
• US Class Current: 536/25.3
• 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 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/00513 Essentially linear supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/0068 Means for controlling the apparatus of the process
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

A method and apparatus are provided for preparing a substrate upon which is located microdrop-sized loci at which chemical compounds are synthesized or diagnostic tests are conducted. The loci are formed by applying microdrops from a dispenser from which a microdrop is pulse fed onto the surface of the substrate.

Claims

1. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein said substrate is comprised of a porous material, comprised of a sheet having internal straight, essentially parallel pores and wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying through a single reagent dispenser to a first locus of said surface at least one microdrop of a first liquid activating reagent wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds;

(b) displacing said single reagent dispenser relative to said surface and applying at least one microdrop of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c to yield the second reagent covalently bound to said surface at said modified loci; and

(f) repeating step (e) with the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (d) until the synthesis of different compounds at each of at least 20 loci is completed.

2. The method of claim 1 wherein said porous material is paper.

3. The method of claim 1 wherein said first activating reagent is a deprotection reagent which removes protecting groups of said locus.

4. The method of claim 1 wherein said first activating reagent is an activation reagent which activates reactive groups of said locus.

5. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein said substrate is comprised a porous material, comprised of a sheet having internal straight, essentially parallel pores and wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying through a single reagent dispenser to a first locus of said surface at least one microdrop of a first liquid activating reagent wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds;

(b) displacing said single reagent dispenser relative to said surface and applying at least one microdrop of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at a third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c, wherein said second reagent comprises protected or unprotected amino acids, thereby forming polypeptides covalently attached to said surface at said modified loci; and

(f) repeating step (e) with the same the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (c) until the synthesis of polypeptides at each of at least 20 said loci is completed.

6. The method of claim 5 wherein said first activating reagent is a deprotection reagent which removes protecting groups of said locus.

7. The method of claim 5 wherein said first activating reagent is an activation reagent which activates reactive groups of said locus.

8. The method of claim 5 wherein said porous material comprises paper.

9. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein said substrate is comprised of a porous material, wherein said substrate is comprised a porous material, comprised of a sheet having internal straight, essentially parallel pores and wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying through a single reagent dispenser to a first locus of said surface at least one microdrop of a first liquid activating reagent wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds;

(b) displacing said single reagent dispenser relative to said surface and applying at least one microdrop of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c, wherein said second reagent comprises protected or unprotected nucleotides, thereby forming oligonucleotides covalently attached to said surface at said modified loci; and

(f) repeating step (e) with the same the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (c) until the synthesis of oligonucleotides at each of at least 20 said loci is completed.

10. The method of claim 9 wherein said porous material is paper.

11. The method of claim 9 wherein said first activating reagent is a deprotection reagent which removes protecting groups of said locus.

12. The method of claim 9 wherein said first activating reagent is an activation reagent which activates reactive groups of said locus.

13. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein said substrate is comprised of a porous material, and wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying through a single reagent dispenser to a first locus of said surface at least one microdrop of a first activating reagent of 0.8M zinc bromide in 9;

1 nitromethane;

isopropanol, wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds;

(b) displacing said single reagent dispenser relative to said surface and applying at least one microdrop of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c, wherein said second reagent comprises protected or unprotected nucleotides, thereby forming oligonucleotides covalently attached to said surface at said modified loci; and

(f) repeating step (e) with the same the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (c) until the synthesis of oligonucleotides at each of at least 20 said loci is completed.

14. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein said substrate is comprised a porous material, comprised of a sheet having internal straight, essentially parallel pores and wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying by a pulse through a piezoelectric jet head single reagent dispenser, to a first locus of said surface a quantity of a first liquid activating reagent wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds;

(b) displacing said single reagent dispenser relative to said surface and applying a quantity of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at a third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c, wherein said second reagent comprises protected or unprotected amino acids, thereby forming polypeptides covalently attached to said surface at said modified loci; and

(f) repeating step (e) with the same the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (c) until the synthesis of polypeptides at each of at least 20 said loci is completed.

15. The method of claim 14 wherein said first activating reagent is a deprotection reagent which removes protecting groups of said locus.

16. The method of claim 14 wherein said first activating reagent is an activation reagent which activates reactive groups of said locus.

17. The method of claim 14 wherein said porous material is paper.

18. A method of preparing a substrate having a plurality of non-contiguous microdrop-sized loci on a surface of said substrate, wherein at each of said loci a compound is covalently attached to or beneath said surface, comprising the steps of:

(a) applying by a pulse through a piezoelectric jet head single reagent dispenser to a first locus of said surface a quantity of a first liquid activating reagent wherein said reagent modifies said surface at said first locus to activate said surface for forming covalent bonds, wherein said piezoelectrectric jet head is positioned at a predetermined distance from said substrate, wherein said predetermined distance is less than the separation distance required for free droplet formation, whereby a smaller volume of liquid reagent is delivered to said substrate per microdrop as compared to the volume per microdrop when a droplet is allowed to form before contact with said substrate;

(b) displacing said single reagent dispenser relative to said surface and applying a quantity of said first activating reagent to said surface at a second locus;

(c) optionally repeating step (b) at a third and subsequent loci, each such locus thereby becoming a modified locus;

(d) washing the substrate to remove excess reagent;

(e) applying a quantity of a second reagent to the entire substrate, including loci modified by steps a, b and c, wherein said second reagent comprises protected or unprotected amino acids, thereby forming polypeptides covalently attached to said surface at said modified loci; and

(f) repeating step (e) with the same the same or a different second reagent where the same or different loci have been modified in accordance with steps (a) through (c) until the synthesis of polypeptides at each of at least 20 said loci is completed.