Surface assembly for immobilizing DNA capture probes in genetic assays using enzymatic reactions to generate signal in optical bio-discs and methods relating thereto

• US 20030059803A1
• Filed: 05/16/2002
• Published: 03/27/2003
• Est. Priority Date: 05/18/2001
• Status: Active Grant

• Current Assignee: Vindur Technologies, Inc.
• Original Assignee: Nagaoka & Co., Ltd.
• Inventors: Gordon, John Francis, Valencia, Ramoncito Magpantay, Werner, Martina Elisabeth
• Granted Patent: US 7,083,920 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00578 electrophoric
• 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 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• 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 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• 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 2219/00689 using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 : separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 2300/0806 : Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2400/0409 : centrifugal forces
• CPC Class Codes: B01L 3/5025 : for parallel transport of multiple samples
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502707 : characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/545 : for laboratory containers
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, 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
• CPC Class Codes: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
• CPC Class Codes: Y10S 977/924 : using nanostructure as support of dna analysis

Abstract

The present invention relates to methods and systems for the detection of specific sequences of nucleic acids or oligonucleotides. It relates more particularly to a conjugated enzyme based assay system utilizing reflective and/or transmissive optical bio-discs for detection of specific sequences of nucleic acids. In the assay according to the present invention, an enzyme reaction is used to detect the presence of an analyte (DNA or RNA) in a microchannel in an optical bio-disc. The analyte is immobilized by hybridization with a specific capture probe on a capture layer on the surface and the signal that is generated is localized and specific. The signal can be in the form of a pellet, a fluorescent product, and/or a colored product, and can be detected and quantified by an optical bio-disc reader utilized in conjunction with the inventions hereof. This assay is thus quantitative in nature.

Claims

1. An optical bio-disc, comprising:

a substantially circular substrate having a center and an outer edge;

an active layer associated with the substrate;

a target zone disposed between the center and the outer edge; and

at least one strand of DNA having an affinity for the active layer such that the DNA is immobilized on the active layer in the target zone, wherein the active layer is formulated to immobilize a pellet formed by an enzyme reaction.

2. The optical bio-disc according to claim 1 wherein the active layer comprises nitrocellulose.

3. The optical bio-disc according to claim 1 wherein the substrate includes encoded information associated therewith, the encoded information being readable by a disc drive assembly to control rotation of the bio-disc.

4. The optical bio-disc according to claim 1 further comprising a reflective layer formed on a surface of the substrate.

5. The optical bio-disc according to claim 1 further comprising an enzyme, wherein the enzyme, when exposed to an enzyme substrate, produces a signal detectable by an incident beam of electromagnetic radiation.

6. The optical bio-disc according to claim 1 further comprising a flow channel in fluid communication with the target zone and an input site in fluid communication with the flow channel.

7. A method of testing for the presence of a target-nucleic acid in a test sample, said method comprising the steps of:

providing a bio-disc, the bio-disc comprising a substantially circular substrate having a center and an outer edge, an active layer associated with the substrate, a target zone disposed between the center and the outer edge, at least one strand of capture-DNA having an affinity for the active layer such that the capture-DNA is immobilized on the active layer in the target zone, the capture-DNA and the target-nucleic acid having at least some complementary sequence;

depositing the test sample on the target zone;

allowing any target-nucleic acid present in the test sample to hybridize with the capture-DNA;

providing a plurality of enzyme molecules;

binding the enzyme molecules to the target-nucleic acid such that enzyme molecules bound to the target-nucleic are immobilized within the target zone;

washing the target zone to remove any unbound enzyme molecules;

depositing onto the target zone at least one enzyme substrate that reacts with the enzyme molecules to produce at least one detectable signal; and

detecting any signal in the target zone to thereby determine whether target-nucleic acid is present in the test sample.

8. The method of claim 7, further comprising the step of amplifying the test sample with at least one DNA primer set specific to the target-nucleic acid, to thereby amplify any target-nucleic acid present in the test sample.

9. The method of claim 8, wherein the DNA primer is labeled with an affinity agent.

10. The method of claim 9, wherein the affinity agent is biotin.

11. The method of claim 9, wherein the enzyme molecules are conjugated with a binding agent that interacts with the affinity agent.

12. The method of claim 11, wherein the binding agent is selected from the group consisting of streptavidin and neutravidin.

13. The method of claim 7, wherein the enzyme is horseradish peroxidase.

14. The method of claim 13, wherein the enzyme substrate is selected from the group consisting of 3,3′

,5,5′

-tetramethylbenzidine (TMB), 4-chloronaphthol/3,3′

-diaminobenzidine, tetrahydrochloride (CN/DAB), 4-chloro-1-napthol (4-CN), 3-amino-9-ethyl carbazol (AEC), and 3,3′

-diaminobenzidine tetrahydrochloride (DAB).

15. The method of claim 7, wherein the detectable signal is a precipitate, which adheres to the active layer.

16. The method of claim 7, further comprising the steps of:

providing a signal DNA, wherein the signal DNA has a portion of sequence complementary to the target-nucleic acid but not to the capture-DNA;

binding the enzyme molecules to the signal DNA; and

hybridizing the signal DNA to the target-nucleic acid, such that the enzyme molecules are bound to the target-nucleic acids via the signal DNA.

17. A method of making an optical bio-disc, said method comprising the steps of:

providing a substrate having a center and an outer edge;

encoding information on an information layer associated with the substrate, the encoded information being readable by a disc drive assembly to control rotation of the disc;

forming a target zone on the substrate between the center and the outer edge;

applying an active layer in the target zone, wherein the active layer is formulated to immobilize a precipitate formed by an enzyme reaction; and

depositing within the target zone, a plurality of strands of capture-DNA, at least some of the capture-DNA attaching to the active layer to thereby become immobilized within the target zone.

18. The method of claim 17, further comprising the step of forming a flow channel in fluid communication with said target zone.

19. The method of claim 18, further comprising the step of forming a chamber in fluid communication with the flow channel, the chamber having an input port.

20. The method of claim 19, further comprising the step of providing a pad on which an enzyme has been dried, wherein the pad is disposed with the chamber.