Apparatus and method for spotting a substrate

• US 20020174831A1
• Filed: 07/30/2002
• Published: 11/28/2002
• Est. Priority Date: 03/15/1999
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PQ Corporation (Ecovyst, Inc.)
• Inventors: Vann, Charles S., Bryning, Zbigniew T.
• Granted Patent: US 6,579,367 B2
• Time in Patent Office: Days
• US Class Current: 118/401
• 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 2219/00376 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00527 Sheets
• 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 2219/00626 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2400/028 Pin is moved through a ring which is filled with a fluid
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/0251 : Pin and ring type or pin in tube type dispenser
• CPC Class Codes: B01L 3/0262 : using touch-off at substrate or container
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1034 : Transferring microquantities of liquid
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 2035/1039 : Micropipettes, e.g. microcapillary tubes
• CPC Class Codes: G01N 2035/1069 : by adjusting the spacing between multiple probes of a single transferring head
• CPC Class Codes: G01N 35/1016 : Control of the volume dispensed or introduced
• CPC Class Codes: G01N 35/1067 : for transfer to or from containers having different spacing
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention provides a method and apparatus for dispensing a small volume of a selected liquid, such as a biological sample or reagent, onto a substrate. The device includes a tube adapted to contain the liquid. An elongate fiber is disposed within the tube for axial movement therein between raised and lowered positions. Upon shifting or oscillating the fiber between its raised and lowered positions, a liquid spot can be formed at a selected position on the substrate. The device is readily adaptable for the production of micro-arrays having a great number of individual spots.

Claims

1. An apparatus for micro-spotting a predetermined volume of a liquid at a plurality of spaced regions on a substrate or substrates, comprising:

a tube adapted to contain such liquid, and having a lower end defining an orifice with an inner diameter less than about 200 μ

m;

a workpiece holder for holding such substrate(s),

an elongate, flexible fiber having a diameter of less than about 100 μ

m and a distal free end, said fiber being disposed within said tube for axial movement therein between raised and lowered positions at which the fiber'"'"'s free end is spaced from and in contact with the surface of such a substrate(s), respectively, with the substrate(s) held in the workpiece holder,

shifting means operatively connected to said fiber for shifting the same between its raised and lowered positions,

positioning means for positioning said tube and associated fiber laterally with respect to the holder, at selected deposition positions with respect to the substrate(s), and

a control unit operatively connected to the positioning means and shifting means for use in successively (i) positioning the tube and associated fiber at a selected deposition position with respect to a substrate, and (i) shifting the fiber to its lowered position, to deposit a selected volume of liquid upon such substrate, with variations in the distance between the tube'"'"'s lower end and the substrate being accommodated by flexing of the fiber.

2. The apparatus of claim 1, wherein said positioning means is operatively connected to said tube and said shifting means.

3. The apparatus of claim 1, for use in micro-spotting a predetermined volume of a liquid at a preselected position on each of a plurality of different substrates in the workpiece holder, wherein the control unit is operable to position the tube successively at such preselected position on each substrate.

4. The apparatus of claim 1, wherein the diameter of the orifice is between about 10-100 μ

m larger than that of the fiber.

5. The apparatus of claim 1, wherein the tube has a substantially uniform diameter, and further includes a larger-diameter upper reservoir for holding such liquid.

6. The apparatus of claim 1, wherein the tube'"'"'s inner diameter tapers on progressing downwardly to a defined-volume tube end region having the diameter of said orifice, the diameter of the tube end region is substantially the same as that of the fiber, and the fiber'"'"'s end, with such in its raised position, is disposed above the tube end region, such that shifting of the fiber from its raised to its lowered position is effective to expel from the tube a volume of liquid contained in said tube end region.

7. The apparatus of claim 1, for use in micro-spotting a predetermined volume of such a liquid simultaneously at selected deposition regions on a substrate, which further includes a plurality of such tubes, and associated fibers and shifting means.

8. An apparatus for micro-spotting a predetermined volume of a liquid on a substrate, comprising:

a tube adapted to contain such liquid, and having an inner diameter that tapers on progressing downwardly to a defined-volume tube end region having a substantially uniform diameter of less than about 500 μ

m,

an elongate fiber having a diameter substantially the same as that of said tube end region, said fiber being disposed within said tube for axial movement therein between raised and lowered positions at which the fiber'"'"'s free end is disposed above and below said tube end region, respectively, and

shifting means operatively connected to said fiber for shifting the same between its raised and lowered positions, whereby a defined volume of liquid contained in said tube end region is expelled from the tube onto such a substrate disposed below the tube.

9. The apparatus of claim 8, wherein the tube end region has a diameter of less than about 200 μ

m.

10. The apparatus of claim 9, wherein said fiber is a flexible fiber, and the fiber in its lowered position is adapted to make contact with such a substrate.

11. The apparatus of claim 10, for use in micro-spotting a predetermined volume of a liquid at a preselected position at each of a plurality of substrates, wherein said apparatus further includes positioning means for positioning said tube and associated fiber successively at said preselected position, and variations in the distance between the tube'"'"'s lower end and the different substrate positions is accommodated by fiber flexing.

12. The apparatus of claim 8, for use in micro-spotting a predetermined volume of such a liquid simultaneously at multiple selected deposition regions on a substrate, which further includes a plurality of such tubes, and associated fibers and shifting means.

13. The apparatus of claim 12, wherein the tube end regions have diameters of less than about 200 μ

m, said fibers are flexible fibers, and the fibers in their lowered positions are adapted to make contact with such a substrate, with variations in the distance between the tubes'"'"' lower ends and the associated positions on the substrate being accommodated by fiber flexing.

15. The apparatus of claim 14, wherein the spacing between adjacent upper-end openings is greater than that between adjacent lower-end openings.

16. The apparatus of claim 14, which further includes shifting means operatively connected to the support for shifting the same between its raised and lowered positions.

17. The apparatus of claim 14, wherein the diameter of the channels at their lower ends is less than about 200 μ

m, and from about 10 to about 100 μ

m larger than that of the associated fiber.

18. The apparatus of claim 17, wherein the fibers are adapted to contact such a substrate, with the support in its lowered position, and variations in the length of fiber extending between its associated channel end and its point of contact on the substrate is accommodated by fiber flexing.

19. The apparatus of claim 14, wherein each channel has a substantially uniform diameter extending along a lower end region that terminates at the channel'"'"'s lower end, the diameter of each channel end region is substantially the same as that of the associated fiber, and the fiber'"'"'s end, with such in its raised position, is disposed above the channel end region, such that shifting of the fiber from its raised to its lowered position is effective to expel from the channel the volume of liquid contained in said channel end region.

14. An apparatus for producing an array of liquid-reagent spots on a substrate, comprising:

a manifold having a plurality of capillary channels, each adapted to hold a selected liquid, said channels having opposite upper-end and lower-end openings, and inner diameters which decrease on progressing from the upper- to the lower-end openings, with said lower openings forming said array,

a support movable between raised and lowered positions with respect to said manifold, and

a plurality of fibers carried on said support for movement therewith, each fiber being adapted to move longitudinally within an associated channel, as the support is moved between its raised and lowered positions,

with movement of the fibers from their raised to lowered positions being effective to deposit a selected volume of liquid from each channel in the manifold.

20. Valving apparatus for use in metering a selected amount of liquid onto the surface of a substrate, comprising

a reservoir for holding such liquid,

a tube extending from the reservoir and terminating at a lower end orifice adjacent a plane adapted to be occupied by the surface of such a substrate,

a fiber disposed in said tube for axial oscillatory movement therein, with a lower portion of the fiber extending through the orifice, and the inner diameter of said tube and the diameter of said fiber being dimensioned to prevent fluid flow through said orifice in the absence of fiber oscillation,

oscillating means operatively connected to said fiber for oscillating the same, including a control unit for determining the oscillation amplitude, frequency and time applied to said fiber, and thereby the amount of liquid allowed to pass through the tube orifice.

21. The apparatus of claim 20, wherein said oscillation means is adapted to produce an oscillation frequency of at least about 10 Hertz.

22. The apparatus of claim 21, wherein said oscillation means is adapted to produce an oscillation frequency of at least about 100 Hertz.

23. The apparatus of claim 20, wherein said oscillation means is adapted to produce an oscillation amplitude of at least about 10 μ

m.

24. The apparatus of claim 23, wherein said oscillation means is adapted to produce an oscillation amplitude of at least about 100 μ

m.

25. The apparatus of claim 20, wherein said tube has a lower-end diameter of less than about 100 μ

m, and the clearance between the fiber and tube at its lower end is less than about 25 μ

m.

26. The apparatus of claim 20, wherein said fiber is adapted to contact such a substrate during at least a portion of its oscillation cycle.

27. The apparatus of claim 26, wherein said fiber is adapted to remain in contact with said substrate throughout its oscillation cycle.

28. The apparatus of claim 20, which further includes positioning means for positioning said tube and fiber with respect to the substrate, from one selected lateral position to another.

29. The apparatus of claim 28, wherein said positioning means is operatively connected to said tube, fiber and oscillating means.

30. A method of forming a reagent spot on a substrate, comprising:

reciprocally moving an elongate, flexible fiber longitudinally within a capillary tube holding a selected liquid at a frequency and amplitude sufficient to pump a portion of the liquid out through an orifice at a lower end of the tube, thereby forming a pendent drop; and

placing the pendent drop at a selected region on the substrate.

31. The method of claim 30, wherein the pendent drop is placed on the substrate by contacting an end of the fiber with the selected region of the substrate.

32. The method of claim 30, wherein the pendent drop is placed on the substrate by maintaining the fiber in spaced relation over the selected region and enlarging the pendent drop until it falls under the force of gravity.

33. An apparatus for micro-spotting a predetermined volume of a liquid at a plurality of spaced regions on a substrate or substrates, comprising:

a tube adapted to contain such liquid, and having a lower end defining an orifice with an inner diameter less than about 200 μ

m;

a workpiece holder for holding such substrate(s),

an elongate, flexible fiber having a diameter of less than about 100 μ

m and a distal free end, said fiber being disposed within said tube for axial movement therein between raised and lowered positions at which the fiber'"'"'s free end is spaced from and in contact with the surface of such a substrate(s), respectively, with the substrate(s) held in the workpiece holder,

an actuator movable between two positions, said actuator operatively connected to said fiber for shifting the same between its raised and lowered positions, and

an x-y positioner operatively connected to said tube for positioning said tube and associated fiber laterally with respect to the holder, at selected deposition positions with respect to the substrate(s), and

a control unit operatively connected to the x-y positioner and vertical actual for use in successively (i) positioning the tube and associated fiber at a selected deposition position with respect to a substrate, and (i) shifting the fiber to its lowered position, to deposit a selected volume of liquid upon such substrate, with variations in the distance between the tube'"'"'s lower end and the substrate being accommodated by flexing of the fiber.

34. An apparatus for micro-spotting a predetermined volume of a liquid on a substrate, comprising:

a tube adapted to contain such liquid, and having an inner diameter that tapers on progressing downwardly to a defined-volume tube end region having a substantially uniform diameter of less than about 500 μ

m,

an elongate fiber having a diameter substantially the same as that of said tube end region, said fiber being disposed within said tube for axial movement therein between raised and lowered positions at which the fiber'"'"'s free end is disposed above and below said tube end region, respectively, and

an actuator movable between two positions, said actuator operatively connected to said fiber for shifting the same between its raised and lowered positions, whereby a defined volume of liquid contained in said tube end region is expelled from the tube onto such a substrate disposed below the tube.

35. Valving apparatus for use in metering a selected amount of liquid onto the surface of a substrate, comprising

a reservoir for holding such liquid,

a tube extending from the reservoir and terminating at a lower end orifice adjacent a plane adapted to be occupied by the surface of such a substrate,

a fiber disposed in said tube for axial oscillatory movement therein, with a lower portion of the fiber extending through the orifice, and the inner diameter of said tube and the diameter of said fiber being dimensioned to prevent fluid flow through said orifice in the absence of fiber oscillation, and

an oscillating unit operatively connected to said fiber for oscillating the same, including a control unit for determining the oscillation amplitude, frequency and time applied to said fiber, and thereby the amount of liquid allowed to pass through the tube orifice.