Method and apparatus for detection of fluorescently labeled materials

• US 6,741,344 B1
• Filed: 10/30/2000
• Issued: 05/25/2004
• Est. Priority Date: 02/10/1994
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Stern, David, Fiekowsky, Peter
• Primary Examiner(s): EVANS, FANNIE L
• Application Number:
  US09/699,852
• Time in Patent Office: 1,303 Days
• Field of Search: 356/317, 356/318, 356/417, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 436/518, 435/5
• US Class Current: 356/317
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• 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 21/645 Specially adapted constructive features of fluorimeters
• 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 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16

Abstract

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Claims

1. An apparatus comprising:

an optical scanning system comprising a light source;

an optical train for directing excitation light from said light source toward a substrate including a surface having biological polymers with fluorescently marked regions;

a translation system co-operatively arranged with said optical train and coupled to said substrate;

a focusing system for focusing said light onto said surface including said biological polymers with fluorescently marked regions; and

a confocal detector for detecting said fluorescent light from said fluorescently marked regions in response to said excitation light; and

a flow cell comprising a mounting surface and a cavity, wherein said mounting surface is constructed to receive said substrate in a sealed relationship with respect to said cavity, whereby said surface having biological polymers is in fluid communication with said cavity.

2. The apparatus as recited in claim 1, wherein said focusing system comprises means for determining a focal plane of the light passing through the optical train and means for providing data for locating said surface of said substrate at said focal plane.

3. The apparatus of claim 1, further comprising:

a photodiode for detecting light reflected from said substrate;

a focusing lens for focusing said reflected light from said substrate, from said optical train at said photodiode; and

a drive connected to said substrate.

4. The apparatus of claim 1, wherein the detector comprises:

a photomultiplier tube; and

a lens for focusing said fluoresced light separated by said optical train, at said photomultiplier tube.

5. The apparatus as recited in claim 1, wherein said flow cell includes a temperature sensor disposed adjacent to or within the cavity of said flow cell and a control system for controlling temperature in said cavity of said flow cell.

6. The apparatus of claim 5, wherein the flow cell defines one or more channels for directing heat exchanging fluid therethrough to control temperature in said cavity.

7. The apparatus of claim 1, wherein said substrate is light transparent.

8. The apparatus of claim 1, wherein said surface has said biological polymers immobilized thereon prior to mounting said substrate to said flow cell.

9. The apparatus of claim 1, wherein said translation system includes an x-y-z translation table.

10. The apparatus of claim 1, wherein said translation system includes an x-y-z translation table, and wherein said flow cell is attachable to said translation table.

11. The apparatus of claim 10, wherein said flow cell is attached to said translation table, and wherein said attached flow cell is constructed to receive fluid for hybridization of said biological polymers to targets thereby creating said fluorescently marked regions.

12. The apparatus of claim 1, wherein said scanning system is constructed to store intensities of said fluorescent light emitted from said fluorescently marked regions over at least a portion of said surface said having biological polymers in order to create a corresponding image.

13. An apparatus comprising:

an optical scanning system comprising a light source;

an optical train for directing excitation light from said light source toward a substrate including a surface having biological polymers with fluorescently marked regions;

a translation system co-operatively arranged with said optical train and coupled to said substrate;

a focusing system for focusing said light onto said surface including said biological polymers with fluorescently marked regions; and

a confocal detector for detecting said fluorescent light from said fluorescently marked regions in response to said excitation light; and

a flow cell comprising a mounting surface and a cavity, wherein said mounting surface is constructed to receive said substrate in a sealed relationship with respect to said cavity, whereby said surface having biological polymers is in fluid communication with said cavity.

14. The apparatus of claim 13, wherein said confocal detector includes a confocal, pinhole and an associated lens.

15. The apparatus of claim 13, wherein said substrate is light transparent.

16. The apparatus of claim 13, wherein said substrate is removably attachable to said flow cell.

17. The apparatus of claim 13, wherein said surface has said biological polymers immobilized thereon prior to mounting said substrate to said flow cell.

18. The apparatus of claim 13, wherein said translation system includes an x-y-z translation table.

19. The apparatus of claim 13, wherein said translation system includes an x-y-z translation table, and wherein said flow cell is attachable to said translation table.

20. The apparatus of claim 19, wherein said flow cell is attached to said translation table, and wherein said attached flow cell is constructed to receive fluid for hybridization of said biological polymers to targets thereby creating said fluorescently marked regions.

21. The apparatus of claim 13, wherein the detector comprises a photomultiplier tube;

and a lens for focusing said fluoresced light, separated by said optical train, to said photomultiplier tube.

22. The apparatus of claim 13, wherein said scanning system is constructed to store intensities of said fluorescent light emitted from said fluorescently marked regions over at least a portion of said surface having biological polymers in order to create a corresponding image.