Use of restriction enzymes and other chemical methods to decrease non-specific binding in dual bead assays and related bio-discs, methods, and system apparatus for detecting medical targets

• US 20060068409A1
• Filed: 02/28/2005
• Published: 03/30/2006
• Est. Priority Date: 11/27/2000
• Status: Abandoned Application

• Current Assignee: Nagaoka & Co., Ltd.
• Original Assignee: Nagaoka & Co., Ltd.
• Inventors: Coombs, James Howard, Phan, Brigitte Chau, Lam, Amethyst Hoang, Virtanen, Jorma Antero, Yeung, KaYuen
• Application Number:
  US11/069,760
• Publication Number:
  US 20060068409A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• 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 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0633 with moving parts
• CPC Class Codes: B01L 2400/0683 mechanically breaking a wall or membrane within a channel or chamber
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/545 for laboratory containers
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/288 disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 2201/18 Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 for use in medical applications
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: F16K 2099/008 Multi-layer fabrications
• CPC Class Codes: F16K 2099/0084 Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0023 : with ball-shaped valve members
• CPC Class Codes: F16K 99/0034 : Operating means specially adapted for microvalves
• CPC Class Codes: G01N 27/745 : for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 33/54313 : the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• 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: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C

Abstract

Methods for decreasing non-specific bindings of beads in dual bead assays and related optical bio-discs and disc drive systems. Methods include identifying whether a target agent is present in a biological sample and mixing capture beads each having at least one transport probe affixed thereto, reporter beads each having at least one signal probe affixed thereto, and a biological sample. Mixing is performed under binding conditions to permit formation of a dual bead complex if the target agent is present in the sample. The reporter bead and capture bead each are bound to the target agent. Denaturing the target agent and keeping it in the denatured form by use of a specialized hybridization buffer is also provided. A denaturing agent is guanidine isothiocynate. Methods further include isolating the dual bead complex from the mixture to obtain an isolate, exposing the isolate to a capture field on a disc, and detecting the presence of the dual bead complex in the disc to indicate that the target agent is present in the sample. The methods may further include selectively breaking up non-specific binding between capture beads and reporter beads employing a digestion agent. Also employed is a method for selectively breaking up non-specific binding between capture beads and reporter beads using a wash buffer containing a chemical agent. The methods are applied to detecting medical targets.

Claims

1. An optical bio-disc, comprising:

a substrate having encoded information associated therewith, said encoded information being readable by a disc drive assembly to control rotation of the disc;

a target zone associated with said substrate, said target zone disposed at a predetermined location relative to said substrate;

an active layer associated with said target zone;

a plurality of capture agents attached to said active layer so that when said substrate is rotated, said capture agents remain attached to said active layer to thereby maintain a number of said capture agents within said target zone; and

a dual bead complex attached to said capture agents, wherein said dual bead complex has been pre-washed in a buffer containing a dissociation agent.

2. The optical bio-disc according to claim 1, wherein said capture agent is selected from the group consisting of a single stranded oligonucleotide sequence, a double stranded oligonucleotide sequence, an antibody, an antigen, biotin, and streptavidin.

3. The optical bio-disc according to claim 1, wherein said active layer is disposed between said substrate and a cap.

4. The optical bio-disc according to claim 1, wherein said active layer is selected from the group consisting of nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, and modified polystyrene.

5. The optical bio-disc according to claim 1, wherein said dissociation agent is an enzyme.

6. The optical bio-disc according to claim 5, wherein said enzyme is a DNase.

7. The optical bio-disc according to claim 1, wherein said dissociation is a chaotropic agent.

8. The optical bio-disc according to claim 7, wherein said chaotropic agent is urea.

9. The optical bio-disc according to claim 7, wherein said chaotropic agent is guanidine isothiocyanate.

10. An optical bio-disc assay system, comprising:

a substrate having encoded information associated therewith, said encoded information being readable by a disc drive assembly to control rotation of the disc;

a target zone associated with said substrate, said target zone disposed at a predetermined location relative to said substrate;

an active layer associated with said target zone;

a plurality of capture agents attached to said active layer so that when said substrate is rotated, said capture agents remain attached to said active layer to thereby maintain a number of said capture agents within said target zone; and

a dual bead complex which interacts with said capture agents, wherein said dual bead complex has been pre-washed in a buffer containing a dissociation agent.

11. The optical bio-disc bio-disc according to claim 10, wherein said capture agent is selected from the group consisting of a single stranded oligonucleotide sequence, a double stranded oligonucleotide sequence, an antibody, an antigen, biotin, or and streptavidin.

12. The optical bio-disc according to claim 10, wherein said active layer is disposed between said substrate and a cap.

13. The optical bio-disc according to claim 10, wherein said active layer is selected from the group consisting of nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, and modified polystyrene.

14. The optical bio-disc according to claim 10, wherein said dissociation agent is an enzyme.

15. The optical bio-disc according to claim 14, wherein said enzyme is a DNase.

16. The optical bio-disc according to claim 10, wherein said dissociation is a chaotropic agent.

17. The optical bio-disc according to claim 16, wherein said chaotropic agent is urea.

18. The optical bio-disc according to claim 16, wherein said chaotropic agent is guanidine isothiocyanate.