Method of shielding biosynthesis reactions from the ambient environment on an array

• US 6,387,636 B1
• Filed: 10/22/1999
• Issued: 05/14/2002
• Est. Priority Date: 10/22/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Perbost, Michel G. M., Lefkowitz, Steven M.
• Primary Examiner(s): Celsa, Bennett
• Assistant Examiner(s): Prasthofer, Thomas
• Application Number:
  US09/426,823
• Time in Patent Office: 935 Days
• Field of Search: 435/7.1, 435/DIG.49, 536/22.1, 536/23.1
• US Class Current: 506/16
• 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 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase 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 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00698 Measurement and control of process parameters
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/08 Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

A method of fabricating an array of biopolymers provides a shield for biochemical reactions and biochemical reactants and is particularly useful for those reactions and reactants that are susceptible to reaction with a component of the ambient environment during the fabrication of the array. The method is applicable to the conventional fabrication and synthesis methods used to fabricate a biopolymer array, such as in situ synthesis of biopolymers on an array and the attachment of pre-synthesized biopolymers on to an array. The method comprises applying a non-miscible fluid (NMF) to the array surface where the biopolymers are being synthesized or attached. The NMF is inert and insoluble with the biochemical reactants and other ancillary materials in solution used in conventional synthesis or attachment of biopolymers. The NMF provides a shield between the ambient atmosphere and the biopolymer synthesis materials or the deprotected pre-synthesized biopolymer at the surface of the array during the synthesis or attachment processes. The NMF may be applied as droplets over each feature location on the surface or may be applied by flooding the surface of the array to fully cover the features. Biomonomer or biopolymer solutions are deposited into or through the NMF to the feature locations on the surface of the array where the synthesis or attachment reactions are to take place using conventional deposition equipment to eject the solutions into the NMF. The NMF provides a shield for activated biomonomers that are susceptible to reaction with a component in the ambient environment, such as moisture in the air. Moreover, the NMF provides a shield for pre-synthesized biopolymers that are susceptible to evaporation when deprotected for attachment to the array surface. The method provides a means by which the potential reactivity of the activated biomonomer or deprotected biopolymer with an ambient atmosphere component can be kept low. As a result, biopolymer arrays can be more accurately fabricated.

Claims

1. A method of fabricating a biopolymer array from biomonomers comprising the steps of:

(a) depositing a biomonomer in solution onto a substrate for linking the biomonomer to a surface of the substrate in an array pattern of features;

(b) deprotecting the linked biomonomer, such that the linked biomonomer can react with subsequent biomonomers in solution;

(c) depositing a subsequent biomonomer in solution onto the features, the subsequent biomonomer being activated for attachment to the linked biomonomer on the array feature by an activation reagent, the subsequent biomonomer becoming the linked biomonomer upon attachment;

(d) applying a non-miscible fluid (NMF) over the features on the surface of the substrate before or after any of the above steps (a) to (c), the NMF being inert and insoluble with the biomonomer solution, the activation reagent, the activated biomonomer and the linked biomonomer; and

(e) repeating at least step (c) until the biopolymer is synthesized at each feature.

2. The method of claim 1, wherein the biopolymer array comprises multiple different array features and steps (a) through (d) are repeated at each of multiple different features on the same substrate, to produce the array of multiple different biopolymers.

3. The method of claim 1, wherein the step of applying (d) the NMF comprises the step of depositing an amount of NMF so as to cover an area greater than the area of each feature.

4. The method of claim 3, wherein the step of depositing an amount of NMF comprises the step of depositing a droplet of NMF on each feature that is large enough to completely cover the feature, and wherein the steps (a) and (c) of depositing the biomonomers comprise the steps of:

loading the biomonomer in solution into a pulsejet of a deposition system;

positioning the pulsejet over the NMF droplet on the surface of the array;

ejecting the biomonomer from the pulsejet into the NMF droplet and to the feature;

moving the pulse jet to a next feature; and

repeating the steps of positioning, ejecting and moving for each applicable feature location until the biomonomer is deposited on all applicable features.

5. The method of claim 3, wherein the step of depositing an amount of NMF comprises the step of covering the entire array surface and completely covering the features with the NMF.

6. The method of claim 5, wherein the steps (a) and (c) of depositing the biomonomers comprise the steps of:

loading the biomonomer in solution into a pulsejet of a deposition system;

immersing the pulsejet into the NMF over a feature on the surface of the array;

ejecting the biomonomer from the pulsejet to the feature;

moving the immersed pulse jet to a next applicable feature location and ejecting the biomonomer to the applicable feature; and

repeating the step of moving and ejecting until the biomonomer is deposited on all applicable features.

7. The method of claim 5, wherein the steps (a) and (c) of depositing the biomonomers comprise the steps of:

loading the biomonomer in solution into a pulsejet of a deposition system;

immersing the pulsejet into the NMF over a feature on the surface of the array;

ejecting the biomonomer from the pulsejet to the feature;

removing the pulse jet from the NMF and moving the pulse jet to a next applicable feature location; and

repeating the steps of immersing, ejecting and removing over each applicable synthesis site until the biomonomer is deposited on all applicable features.

8. The method of claim 5, wherein the steps (a) and (c) of depositing the biomonomers comprise the steps of:

loading the biomonomer in solution into a pulsejet of a deposition system;

positioning the pulsejet above the NMF over a feature location on the surface of the array;

ejecting the biomonomer from the pulsejet and into the NMF to the feature;

moving the pulse jet to a next applicable feature location; and

repeating the steps of positioning, ejecting and moving over each applicable feature location until the biomonomer is deposited on all applicable features.

9. The method of claim 5, wherein the steps (a) and (c) of depositing the biomonomers comprise the steps of:

loading the biomonomer in solution into a pulsejet of a deposition system;

immersing the array surface into the NMF and aligning a feature on the array surface with the pulsejet;

ejecting the biomonomer from the pulsejet to the feature;

moving the array such that a next applicable feature location is aligned with the pulse jet; and

repeating the steps of ejecting and moving for each applicable feature location until the biomonomer is deposited on all applicable features.

10. The method of claim 1, wherein the NMF has a density that is different from the density of the biomonomer solution.

11. The method of claim 10, wherein the NMF has a lower density than the density of the biomonomer solution.

12. The method of claim 10, wherein the NMF has a higher density than the density of the biomonomer solution.

13. The method of claim 1 further comprising the steps of:

(d′

) deactivating unreacted activation reagent, the deactivation reagent having a density that is different from the NMF; and

(d″

) removing the NMF, reagents, and unreacted biomonomer solution from the array surface before the step (e).

14. The method of claim 13, wherein the step of deactivating (d′

) comprises hydrolyzing the activation reagent and unreacted biomonomer.

15. The method of claim 13, wherein the biopolymer is selected from a group consisting of an oligonucleotide and polynucleotide;

the biomonomers are selected from a group consisting of phosphoramidites, phosphites and H-phosphonates;

the solution comprises a solvent selected from a group consisting of acetonitrile, propylene carbonate and adiponitrile;

the activation reagent is selected from a group consisting of tetrazole, ethylthiotetrazole, dicyanoimidazole and benzimidazolium triflate;

the deactivation reagent is selected from a group consisting of methanol, water, an alkyl alcohol, a halogenalkylalcohol, and trichloroethanol; and

wherein the NMF is selected from a group consisting of heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, cycloheptane, cyclooctane, cyclononane, and cyclodecane.

16. The method of claim 1, where the NMF is applied after the biomonomer is linked to the surface of the substrate, but before the subsequent biomonomer and activation reagent are deposited.

17. The method of claim 1, where the NMF is applied before the biomonomer is deposited and linked to the surface of the substrate.

18. In a method of fabricating a biopolymer array from biomonomers synthesized on the array by depositing and linking a biomonomer from a biomonomer solution to a feature location on a surface of the array, and depositing a subsequent biomonomer in solution onto the feature location that is suitably activated with an activation reagent to couple with the linked biomonomer, wherein the above steps are repeated to form a biopolymer sequence at multiple feature locations on the array, the improvement comprising the step of:

applying a non-miscible fluid (NMF) over the feature locations on the surface of the array, the NMF being inert and insoluble with the biomonomer solution and activation reagent.

19. The method of claim 18, where the NMF is applied after the biomonomer is linked to the surface of the array, but before the subsequent biomonomer and activation reagent are deposited.

20. The method of claim 18, where the NMF is applied before the biomonomer is deposited and linked to the surface of the array.

21. The method of claim 18, wherein the step of applying a NMF comprises the step of depositing an amount of NMF so as to cover an area greater than the area of each feature location.

22. The method of claim 21, wherein the step of depositing an amount of NMF comprises the step of covering the entire array surface and feature locations with the NMF.

23. The method of claim 18, wherein the NMF has a density that is different from the density of the biomonomer solution.

24. The method of claim 23, wherein the NMF has a lower density than the density of the biomonomer solution.

25. The method of claim 23, wherein the NMF has a higher density than the density of the biomonomer solution.

26. The method of claim 18 further comprising the steps of:

deactivating unreacted activation reagent with a deactivation reagent, the deactivation reagent having a density that is different from the NMF, the NMF being inert and insoluble in the deactivation reagent; and

removing the NMF, reagents and unreacted biomonomer solution from the array surface before another biomonomer is deposited onto the array.

27. The method of claim 26, wherein the step of deactivating comprises hydrolyzing the activation reagent and unreacted biomonomer.

28. The method of claim 26, wherein the biopolymer is selected from a group consisting of an oligonucleotide and polynucleotide;

the biomonomers are selected from a group consisting of phosphoramidites, phosphites and H-phosphonates;

the solution comprises a solvent selected from a group consisting of acetonitrile, propylene carbonate and adiponitrile;

the activation reagent is selected from a group consisting of tetrazole, ethylthiotetrazole, dicyanoimidazole and benzimidazolium triflate;

the deactivation reagent is selected from a group consisting of methanol, water, an alkyl alcohol, a halogenalkylalcohol, and trichloroethanol; and

wherein the NMF is selected from a group consisting of heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, cycloheptane, cyclooctane, cyclononane, and cyclodecane.