Methods for applying small volumes of reagents

• US 6,440,669 B1
• Filed: 11/10/1999
• Issued: 08/27/2002
• Est. Priority Date: 11/10/1999
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies Europe BV (Agilent Technologies, Inc.)
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Tso, Jacqueline, Bass, Jay K.
• Primary Examiner(s): Ponnaluri, Padmashri
• Application Number:
  US09/437,990
• Publication Number:
  US 20020081236A1
• Time in Patent Office: 1,021 Days
• Field of Search: 435/701, 435/6, 435/DIG.1, 435/DIG.22, 435/DIG.34, 435/DIG.37, 435/DIG.43, 435/DIG.44, 435/DIG.46, 435/DIG.49, 536/25.3, 436/518, 422/131, 422/149.11, 422/111, 422/129
• 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 2219/0036 Nozzles
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00427 using masks
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00576 fluorophore
• 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 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B05B 17/0607 generated by electrical means, e.g. piezoelectric transducers
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Methods, devices and apparatus are disclosed for carrying out multiple chemical reactions, such as in situ synthesis of polynucleotides, on a surface comprising an array of discrete sites. Molecules are deposited at a predetermined number of the discrete sites on the surface for reaction at the discrete sites. The surface is positioned relative to an outlet of a fluid ejection device, which is activated to dispense a small volume of a fluid through the outlet to the surface to provide uniform coating of a continuous region of the surface comprising a multiple of the discrete sites. The fluid is dispensed as uniform particles having a diameter such that the uniform particles form a sheet to coat the continuous region of the surface. In one embodiment of the present invention, liquid is dispensed as uniform particles through a fluid ejection device activated by means of ultrasonic energy. The invention has particular application to the in situ synthesis of polynucleotides in arrays on a surface.

Claims

1. The method for forming an array of polynucleotides at discrete sites on a surface, said method comprising:

(a) applying reagents to predetermined discrete sites on said surface, said reagents being selected from the group consisting of nucleotides and polynucleotides, said discrete sites being activated for attachment of nucleotides or polynucleotides thereto,

(b) dispensing a volume of a liquid to uniformly coat the entire surface comprising said discrete sites with liquid, said volume of liquid being dispensed as particles of uniform diameter through a fluid ejection device activated by means of ultrasonic energy to provide a layer of liquid on said surface, said layer having a thickness of 20 to 500 microns, and

(c) optionally repeating step (a) or step (b) wherein step (a) is repeated at least once when said reagents are nucleotides.

2. The method according to claim 1 wherein said liquid comprises an agent selected from the group consisting of washing solutions, buffers, deblocking agents, blocking agents, oxidizing agents, reducing agents and phosphoramidite reagents.

3. The method according to claim 1 wherein said reagents are nucleotides and steps (a) and (b) are repeated for a number of times sufficient to form said array of polynucleotides.

4. The method according to claim 3 wherein phosphoramidite chemistry is employed.

5. The method according to claim 1 wherein said surface is essentially planar.

6. The method according to claim 1 wherein said liquid is dispensed from an outlet of said fluid ejection device as a mist.

7. The method according to claim 1 wherein step (a) is carried out by an application technology selected from the group consisting of printing technology, masking technology, ultrasonic technology and combinations thereof.

8. The method according to claim 1 wherein said particles of uniform diameter have a diameter on the average of about 10 microns to about 150 microns.

9. The method according to claim 1 wherein said ultrasonic energy has a frequency of about 5 kilohertz to about 300 kilohertz.

10. The method according to claim 1 wherein said volume of liquid is about 1 nanoliter to about 1000 nanoliters per square centimeter.

11. The method according to claim 1 wherein said surface is mounted on a linear stage and moved in position relative to said fluid ejection device above said stage.

12. The method according to claim 1 wherein said surface is rotated relative to said fluid ejection device and said fluid ejection device is moved radially relative to said surface.

13. The method according to claim 1 wherein said fluid ejection device is in fluid communication with a reservoir containing said liquid.

14. The method according to claim 1 wherein said fluid ejection device is in alternating fluid communication with a plurality of reservoirs each containing a different liquid.

15. The method for forming an array of polynucleotides at discrete sites on a surface, said method comprising:

(a) applying nucleotide reagents to predetermined discrete sites on said surface, said discrete sites being activated for attachment of nucleotides thereto,

(b) dispensing a volume of a liquid of about 1 nanoliter to about 1000 nanoliters per square centimeter to uniformly coat the entire surface comprising said discrete sites with liquid to provide a layer of liquid on said surface, said layer having a thickness of 20 to 500 microns, said volume of liquid being dispensed as particles of uniform diameter of about 10 microns to about 150 microns, wherein the diameter of each of the particles does not vary more than about 25%, through a fluid ejection device activated by means of ultrasonic energy at a frequency of about 5 kilohertz to about 300 kilohertz, and

(c) repeating step (a) wherein step (b) is optionally repeated prior to or after repeating step (a) and wherein step (a) is repeated until said array of polynucleotides is formed.

16. The method according to claim 15 wherein said liquids comprise agents selected from the group consisting of washing solutions, buffers, oxidizing agents, reducing agents, deblocking agents and blocking agents.

17. The method according to claim 15 wherein step (b) is repeated at least one time prior to repeating step (a) to dispense multiple layers of said liquid on said surface.

18. The method according to claim 17 wherein said liquid is dispensed as a layer of about 5 to 100 microns in thickness.