EP1585957A2 - Bio-disques magneto-optiques et systemes utilisant des techniques en rapport - Google Patents

Bio-disques magneto-optiques et systemes utilisant des techniques en rapport

Info

Publication number
EP1585957A2
EP1585957A2 EP02795687A EP02795687A EP1585957A2 EP 1585957 A2 EP1585957 A2 EP 1585957A2 EP 02795687 A EP02795687 A EP 02795687A EP 02795687 A EP02795687 A EP 02795687A EP 1585957 A2 EP1585957 A2 EP 1585957A2
Authority
EP
European Patent Office
Prior art keywords
disc
beads
capture
magneto
bio
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02795687A
Other languages
German (de)
English (en)
Inventor
James Howard Coombs
Brigitte Chau Phan
Ramoncito Magpantay Valencia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nagaoka Co Ltd
Nagaoka KK
Burstein Technologies Inc
Original Assignee
Nagaoka Co Ltd
Nagaoka KK
Burstein Technologies Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/997,741 external-priority patent/US20030003464A1/en
Priority claimed from US10/099,266 external-priority patent/US20030082568A1/en
Application filed by Nagaoka Co Ltd, Nagaoka KK, Burstein Technologies Inc filed Critical Nagaoka Co Ltd
Publication of EP1585957A2 publication Critical patent/EP1585957A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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 characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures 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 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B03C1/031—Component parts; Auxiliary operations
    • B03C1/033—Component parts; Auxiliary operations characterised by the magnetic circuit
    • B03C1/0332—Component parts; Auxiliary operations characterised by the magnetic circuit using permanent magnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/30—Combinations with other devices, not otherwise provided for
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G11—INFORMATION STORAGE
    • G11B—INFORMATION STORAGE BASED ON RELATIVE MOVEMENT BETWEEN RECORD CARRIER AND TRANSDUCER
    • G11B13/00—Recording simultaneously or selectively by methods covered by different main groups among G11B3/00, G11B5/00, G11B7/00 and G11B9/00; Record carriers therefor not otherwise provided for; Reproducing therefrom not otherwise provided for
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B01L2300/0806—Standardised forms, e.g. compact disc [CD] format
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical or biological applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N35/00069—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • This invention relates in general to molecular and cellular biomagnetic assays • and, in particular, to molecular and cellular biomagnetic assays conducted on optical bio-discs.
  • the invention further relates to magneto-optical (MO) analysis discs and MO drive systems, hereinafter referred to as magneto-optical bio-disc systems (MOBDS). More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention relates to biomagnetic methods, including immunomagnetic methods, for detection and selective manipulation of specific target cells in cell populations and solutions of cell populations, using magnetic particles or beads, and to magnetically guided neurite growth and nerve regeneration.
  • the present invention relates to performing biomagnetic assays and laboratory analysis, and particularly to using magnetic, paramagnetic, or superparamagnetics particles, herein referred to as bio-magnetic or magnetic particles or beads, on optical bio-discs, including, but not limited to, CD, CD-R, DVD, DVD-R, and MO discs.
  • the biomagnetic assays of the present invention may include, for example, immunomagnetic assays, and molecumagnetic assays such as assays using DNA and RNA, implemented on non-magnetic and magnetic platforms.
  • the non-magnetic platforms may include, for example, microtiterplates and non-magneto-optical bio- discs systems.
  • the magnetic platform includes, for example, the magneto-optical bio- disc system (MOBDS).
  • MO bio-magnetic assays MOBMA
  • ODBMA optical disc bio-magnetic assays
  • the ODBMA may be carried out, for example, using a modified optical disc drive having a controllable electromagnet associated therewith.
  • the invention includes methods for preparing assays, methods for performing assays, methods for performing laboratory or clinical analysis, discs for performing assays or analysis, and related detection systems.
  • the biological sample can include blood, serum, plasma, cerebrospinal fluid, breast aspirate, synovial fluid, pleural fluid, perintoneal fluid, pericardial fluid, urine, saliva, amniotic fluid, semen, mucus, a hair, feces, a biological particulate suspension, a single-stranded or double-stranded nucleic acid molecule, a cell, an organ, a tissue, or a tissue extract, or any other sample that includes a target that may be bound to a magnetic particle through chemical or biological processes. Further details relating to other aspects associated with the selection and detection of various targets is disclosed in, for example, commonly assigned co-pending U.S. Provisional Patent Application Serial No.
  • the target of interest can include tumor cells, bacteria, virus, or a target agent molecule such as a nucleic acid characteristic of a disease, or a nucleotide sequence specific for a person, or a nucleotide sequence or an antigenic determinant specific for an organism or cell type, which may be a bacterium, a virus, a mycoplasm, a fungus, a plant, or an animal.
  • the target agent can include a nucleic acid molecule or antigenic determinant associated with cancer.
  • the target nucleic acid molecule can include a nucleic acid, which is at least a portion of a gene selected from the group consisting of HER2neu, p52, p53, p21, and bcl-2.
  • the target agent can be an antibody that is present only in a subject infected with HIV-1 , a viral protein antigen, or a protein characteristic of a disease state in a subject.
  • the methods and apparatus of the present invention can be used for determining whether a subject is infected by a virus, whether nucleic acid obtained from a subject exhibits a single nucleotide mutation (SNM) relative to corresponding wild-type nucleic acid sequence, or whether a subject expresses a protein of interest, such as a bacterial protein, a fungal protein, a viral protein, an HIV protein, a hepatitis C protein, a hepatitis B protein, or a protein known to be specifically associated with a disease.
  • SNM single nucleotide mutation
  • multiplexing methods wherein more than one target agent (e.g., tens, hundreds, or even thousands of different target agents) can be identified on one optical analysis disc.
  • Multiple capture agents can be provided in a single chamber together in capture fields, or separately in separate capture fields.
  • Different reporter beads can be used to be distinguishable from each other, such as beads that fluoresce at different wavelengths or different size reporter beads. Experiments were performed to identify two different targets using the multiplexing technique. An example of one such assay is discussed below in Example 2.
  • the invention includes an optical disc with a substrate, a capture layer associated with the substrate, and a capture agent bound to the capture layer, such that the capture agent binds to a dual bead complex.
  • the disc can be designed to allow for some dual bead processing on the disc with appropriate chambers and fluidic structures, and can be pre-loaded with reporter and capture beads so that only a sample needs to be added to form the dual bead complex structures.
  • a disc and disc drive system for performing bead or bead-cell assays.
  • the disc drive can include an electromagnet for performing the isolation process, and may include appropriate light source control and detection for the type of reporter beads used.
  • the disc drive can be optical or magneto-optical.
  • the drive may advantageously include an electromagnet, and the disc preferably has a mixing chamber, a waste chamber, and capture area.
  • the sample is mixed with beads in the mixing chamber, a magnetic field is applied adjacent the mixing chamber, and the sample not held by the magnet is directed to the waste chamber so that all magnetic beads, whether bound into a dual bead complex or unbound, remain in the mixing chamber.
  • the magnetic beads are then directed to the capture area.
  • One of a number of different valving arrangements can be used to control the flow.
  • the Magneto-Optical Bio-Disc System (MOBDS)
  • a MOBMA is performed using MO discs made for use with biological samples and used in conjunction with a disc drive, such as a magneto-optical (MO) disc drive, that can selectively form magnetic domains or regions on a disc.
  • a disc drive such as a magneto-optical (MO) disc drive
  • magnetic domains can be formed in the MO disc in a highly controllable and precise manner. These domains may be employed advantageously to magnetically bind biomagnetic particles like magnetic beads, including unbound magnetic capture beads, and magnetic complexes including dual bead complexes with magnetic capture beads, magnetic bead-cell complexes, or any biological or chemical complex having at least one magnetic particle or a magnetic property associated therewith so that these complexes may be bound using a magnet or magnetic domains.
  • the MO disc drive can write to selected locations on the disc, and then use an optical reader to detect features located at those domains or regions.
  • the domains can be selectively erased, thereby allowing individual beads and complexes to be selectively released. Biomedical applications related to using the MOBDS for analysis is described below.
  • a method of using a bio-disc and drive including forming magnetic domains or regions on the optical bio- disc or medical CD.
  • This method includes providing magnetic beads to the discs so that the beads bind at the magnetic domains.
  • the method preferably further includes detecting at the locations where the magnetic beads bind biological samples, preferably using reporter beads that are detectable, such as by fluorescence or optical event detection.
  • the method can be formed in multiple stages in terms of time or in terms of location through the use of multiple chambers.
  • the domains are selectively written at pre-determined locations on the MO bio-disc and a sample is moved over the magnetic domains in order to capture magnetic beads or magnetic complexes.
  • the regions can then be selectively erased and the magnetic beads or magnetic complexes released individually and relocated if desired.
  • This method allows many different tests to be performed at one time, and can allow a level of interactivity between the user and the disc drives such that additional tests can be created during the testing process.
  • Further details relating to magneto-optical recording, precise creation of magnetic regions on a magneto-optical disc, and magneto-optical detection methods are disclosed in, for example, U. S. Patent No. 6,212,136 to Maeda at al., U. S. Patent No. 5,329,503 to Ohmori et al., U. S. Patent No. 4,985,881 to Saito et al., U. S. Patent No. 4,843,604 to Fujiwara et al., and U. S. Patent No. 4,748,606 to Naito et al. All of which are hereby incorporated by reference in their entireties.
  • the MO bio-disc may also be optimized for different types of MOBMA applications including, for example, optimization of the optical properties of the magneto-optic stack of the MO bio-disc so that an incident tracking and detection beam of electromagnetic radiation is allowed to partially pass through the reflective layer and a transmitted beam detected using a separate detector and components of the operative layer of the MO bio-disc may be modified such that the magnetic field and strength of the magnetic field generated on the MO bio-disc is large and strong enough to capture and distinguish between magnetic complexes having different types of magnetic beads attached thereto.
  • different types of electromagnetic radiation sources may be used in conjunction with the MOBDS including, but not limited to, infra-red, red, blue, and fluorescent type optical sources.
  • the magnetic beads and magnetic complexes may be detected, for example, optically by analysis of the characteristics of the reflected and/or transmitted beam, as discussed below, for example, in conjunction with Figs. 28A, 28B, 29A and 29B, or by fluorescence using a fluorescent type optical source in conjunction with a fluorescent detector and fluorescent labelled target molecules or cells.
  • the bio-magnetic bead assays according to the present invention may be implemented, in a genetic assay, herein referred to as the molecumagnetic assay, with magnetic capture beads and fluorescent reporter beads. These bio-magnetic beads or particles are coated with capture probes and reporter probes respectively. The capture probes and reporter probes are complementary to a target sequence but not to each other. The capture beads are mixed with varying quantities of target DNA. Unbound target is removed from the solution by magnetic concentration of the magnetic beads. Fluorescent reporter beads are then allowed to bind to the captured target DNA. Unbound reporter beads are removed by magnetic concentration of the magnetic beads. Thus, only in the presence of the target sequence, the magnetic capture beads bind to fluorescent reporter beads, resulting in a dual bead assay.
  • a number of different surface chemistries and different methods for binding the probes to the beads were investigated including covalently conjugating the capture or reporter probe onto carboxylated capture beads and reporter beads, respectively, via EDC conjugation.
  • One observed result using the EDC conjugation method of attaching the probes on the beads was non-covalent attachment of the probes.
  • This limitation was overcome by the development of a method for attaching the probes using partially double stranded DNA probes and by selection of an appropriate bead type with high conjugation efficiency.
  • the use of double stranded probes in the conjugation process reduces the non-covalent attachment of probes to beads significantly.
  • the covalent conjugation efficiency was as high as 95%.
  • each target molecule may hybridize to one reporter bead. Due to its size, a single target molecule is not detectable by any existing technologies. However, a 1 ⁇ m or larger reporter bead can be easily detected and quantified by various methods. Therefore, the bead assay increases the sensitivity of the target capture tremendously.
  • the present invention also involves implementing the methods recited above on an analysis disc, modified optical disc, MOBDS, or a bio-disc.
  • a bio-disc drive assembly may be employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the test samples in a flow channel of the bio-disc.
  • the bio-disc drive is thus provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return and/or transmitted signals form the disc, and an analyzer for analyzing the processed signals.
  • the rotation rate of the motor is controlled to achieve the desired rotation of the disc.
  • the bio-disc drive assembly may also be utilized to write information to the bio-disc either before or after the test material in the flow channel and target zones is interrogated by a read beam of the drive and analyzed by an analyzer.
  • the bio-disc may include encoded information for controlling the rotation rate of the disc, providing processing information specific to the type of test to be conducted, and for displaying the results on a monitor associated with the bio-drive.
  • the various embodiments of the apparatus and methods of the present invention can be designed for use by an end-user, inexpensively, without specialized expertise and expensive equipment.
  • the system can be made portable, and thus usable in remote locations where traditional diagnostic equipment may not generally be available.
  • Other related aspects applicable to components of this assay system and signal acquisition methods are disclosed in commonly assigned and co-pending U.S. Patent Application Serial No. 10/038,297 entitled “Dual Bead Assays Including Covalent Linkages For Improved Specificity And Related Optical Analysis Discs" filed January 4, 2002; U.S. Provisional Application Serial No. 60/272,525 entitled "Biological Assays Using Dual Bead Multiplexing Including Optical Bio-Disc and Related Methods” filed March 1 , 2001 ; and U.S.
  • Provisional Application Serial Nos. 60/275,643, 60/314,906, and 60/352,270 each entitled "Surface Assembly for Immobilizing Capture Agents and Dual Bead Assays Including Optical Bio-Disc and Methods Relating Thereto” respectively filed March 14, 2001 , August 24, 2001 , and January 30, 2002. All of these applications are herein incorporated by reference in their entirety.
  • Fig. 1 is a perspective view of an optical disc system according to the present invention
  • Fig. 2 is a block and pictorial diagram of an optical reading system according to embodiments of the present invention
  • Figs. 3A, 3B, and 3C are respectively exploded, top, and perspective views of a reflective disc according to embodiments of the present invention.
  • Figs. 4A, 4B, and 4C are respectively exploded, top, and perspective views of a transmissive disc according to embodiments of the present invention.
  • Fig. 5A is a partial longitudinal cross sectional view of the reflective optical bio- disc shown in Figs. 3A, 3B, and 3C illustrating a wobble groove formed therein;
  • Fig. 5B is a partial longitudinal cross sectional view of the transmissive optical bio-disc illustrated in Figs. 4A, 4B, and 4C showing a wobble groove formed therein and a top detector;
  • Fig. 6A is a partial radial cross-sectional view of the disc illustrated in Fig. 5A
  • Fig. 6B is a partial radial cross-sectional view of the disc illustrated in Fig. 5B;
  • Figs. 7A, 8A, 9A, and 10A are schematic representations of a capture bead, a reporter bead, and a dual bead complex as utilized in conjunction with genetic assays;
  • Figs. 7B, 8B, 9B, and 10B are schematic representations of a capture bead, a reporter bead, and a dual bead complex as employed in conjunction with immunochemical assays;
  • Fig. 11A is a pictorial representation of one embodiment of a method for producing genetic dual bead complex solutions
  • Fig. 11 B is a pictorial representation of one embodiment of a method for producing immunochemical dual bead complex solutions
  • Fig. 12A is a pictorial representation of another embodiment of a method for producing genetic dual bead complex solutions
  • Fig. 12B is a pictorial representation of another embodiment of a method for producing immunochemical dual bead complex solutions
  • Fig. 13 is a longitudinal cross sectional view illustrating the disc layers in combination with a mixing or loading chamber
  • Fig. 14 is a view similar to Fig. 13 showing the mixing chamber loaded with dual bead complex solution
  • Figs. 15A and 15B are radial cross sectional views of the disc and target zone illustrating one embodiment for binding of reporter beads to capture agents in a genetic assay
  • Figs. 16A and 16B are radial cross sectional views of the disc and target zone showing another embodiment for binding of reporter beads to capture agents in a genetic assay;
  • Fig. 17 is radial cross sectional view of the disc and target zone illustrating one embodiment for binding of capture beads to capture agents in a genetic assay
  • Fig. 18 is radial cross sectional view of the disc and target zone depicting another embodiment for binding of capture beads to capture agents in a genetic assay
  • Figs. 19A, 19B, and 19C are partial cross sectional views illustrating one embodiment of a method according to this invention for binding the reporter bead of a dual bead complex to a capture layer in a genetic assay
  • Figs. 20A, 20B, and 20C are partial cross sectional views showing one embodiment of a method according to the present invention for binding the reporter bead of a dual bead complex to a capture layer in a immunochemical assay;
  • Figs. 21 A, 21 B, and 21 C are partial cross sectional views illustrating another embodiment of a method according to this invention for binding the reporter bead of a dual bead complex to a capture layer in a genetic assay
  • Figs. 22A, 22B, and 22C are partial cross sectional views presenting another embodiment of a method according to the invention for binding the reporter bead of a dual bead complex to a capture layer in a immunochemical assay
  • Figs. 23A and 23B are partial cross sectional views depicting one embodiment of a method according to the present invention for binding the capture bead of a dual bead complex to a capture layer in a genetic assay;
  • Figs. 24A and 24B are partial cross sectional views showing another embodiment of a method according to this invention for binding the capture bead of a dual bead complex to a capture layer in a genetic assay;
  • Figs. 25A-25D illustrate a method according to the present invention for detecting the presence of target DNA or RNA in a genetic sample utilizing an optical bio-disc;
  • Figs. 26A-26D illustrate another method according to this invention for detecting the presence of target DNA or RNA in a genetic sample utilizing an optical bio-disc
  • Figs. 27A-27D illustrate a method according to the present invention for detecting the presence of a target antigen in a biological test sample utilizing an optical bio-disc
  • Fig. 28A is a graphical representation of an individual 2.1 urn reporter bead and a 3 urn capture bead positioned relative to the tracks of an optical bio-disc according to the present invention
  • Fig. 28B is a series of signature traces derived from the beads of Fig. 28A utilizing a detected signal from the optical drive according to the present invention
  • Fig. 29A is a graphical representation of a 2.1 urn reporter bead and a 3 urn capture bead linked together in a dual bead complex positioned relative to the tracks of an optical bio-disc according to the present invention;
  • Fig. 29B is a series of signature traces derived from the dual bead complex of Fig. 29A utilizing a detected signal from the optical drive according to this invention
  • Fig. 30A is a bar graph showing results from a dual bead assay according to the present invention.
  • Fig. 30B is a graph showing a standard curve demonstrating the detection limit for fluorescent beads detected with a flourimeter
  • Fig. 30C is a pictorial representation demonstrating the formation of the dual bead complex
  • Fig. 31 is a bar graph showing the sensitivity of disc drive detection using a dual bead complex
  • Fig. 32 is a schematic representation of combining beads for dual bead assay multiplexing according to embodiments of the present invention.
  • Fig. 33A is a schematic representation of a fluidic circuit according to the present invention utilized in conjunction with a magnetic field generator to control movement of magnetic beads;
  • Figs. 33B-33D are schematics of a first fluidic circuit that implements the valving structure of FIG. 33A according to one embodiment of fluid transport aspects of the present invention
  • Figs. 34A-34C are schematics of a second fluidic circuit that implements the valving structure of FIG. 33A according to another embodiment of the fluid transport aspects of this invention
  • Fig. 35 is a perspective view of the magnetic field generator and a disc including one embodiment of a fluidic circuit employed in conjunction with magnetic beads according to this invention
  • Figs. 36A, 36B, and 36C are plan views illustrating a method of separation and detection for dual bead assays using the fluidic circuit shown in Fig. 35;
  • Fig. 37 is a perspective view of a magneto-optical bio-disc showing magnetic domains or regions, magnetically bound capture beads, and the formation of dual bead complexes according to another aspect of the present invention;
  • Fig. 38 shows the use of ligation to form a covalent bond between the capture and reporter probes;
  • Fig. 39 is a bar graph showing the results from a genetic test detected by an enzyme assay in a ligation experiment
  • Fig. 40 is a bar graph comparing the number of beads bound as a function of target concentration using 2.1 ⁇ m reporter beads with and without ligation
  • Fig. 41 is a bar graph comparing the number of beads bound as a function of target concentration using a 39mer bridge with and without ligation;
  • Fig. 42A is schematic representation of various probe structures including DNA sequences for use in a dual bead complex employing cleavable or displaceable spacers according to the present invention
  • Fig. 42B is pictorial diagrammatic representation showing a cleavable spacer connecting a dual bead complex prior to binding of a target;
  • Fig. 42C is a view similar or Fig. 42B illustrating the cleavable spacer including a Notl connecting the dual bead complex after target binding;
  • Fig. 42D is a view similar to Fig. 42C depicting the dual bead complex after target binding and after cleavage by Notl;
  • Fig. 43A is pictorial diagrammatic representation showing a displaceable spacer connecting a dual bead complex prior to binding of a target
  • Fig. 43B is a view similar to Fig. 43A illustrating initial binding of a displacement probe to the displaceable spacer connecting the dual bead complex after target binding;
  • Fig. 43C is a view similar to Fig. 43B depicting complete displacement of the displacement probe connecting the dual bead complex in the presence of target mediated binding;
  • Fig. 44 is a pictorial representation of cleavable spacers covalently attached to a capture bead according to the present invention.
  • Fig. 45 is a view similar to Fig. 44 showing thiol groups attached to the cleavable spacers binding covalently to a metallic reporter bead;
  • Fig. 46A is a pictorial representation of a pair of dual bead complexes bound together by a cleavable spacer before target binding;
  • Fig. 46B is a view similar to Fig. 46A showing the dual bead complexes bound together by the cleavable spacer after target binding and without target binding;
  • Fig. 46C is a view similar to Fig. 46B showing one of the dual bead complexes dissociated after enzyme cleavage and the other held together by the presence of the target;
  • Fig. 47A is a pictorial presentation of a dual bead complex formed by a pair of cleavable spacers and use of a bridge bound to a target;
  • Fig. 47B is a view similar to Fig. 47A after target binding including the bridge resulting in a double helix containing two breaks;
  • Fig. 47C is a view similar to Fig. 47B after restriction digestion of the cleavable spacers and ligation of the breaks in the double helix;
  • Fig. 48A is a pictorial representation of two dual bead complexes each joined together by a pair of cleavable spacers as implemented in an immunochemical assay prior to target antigen binding;
  • Fig. 48B is a view similar to Fig. 48A showing the dual bead complexes bound together by the cleavable spacer with and without target binding
  • Fig. 48C is a view similar to Fig. 48B illustrating one of the dual bead complexes dissociated after enzyme digestion and the other held together by the presence of the target;
  • Fig. 49 is a schematic presenting a method for evaluating a solid phase for covalent conjugation of a probe
  • Fig. 50 is a schematic detailing various steps in the quantification of covalently- bound and non-covalently bound probes to a solid substrate
  • Fig. 51 A is a graphic presentation of experimental results of various tests of magnetic bead carriers for covalent linkage of a probe
  • Fig. 51 B is a graphic presentation of experimental results of various tests of fluorescent bead carriers for covalent linkage of a probe
  • Fig. 52A is a pictorial representation illustrating the structural differences between single-stranded and double-stranded DNA that are relevant to their use as probes;
  • Fig. 52B is a graphic presentation of results of an experiment designed to evaluate the binding properties of single-stranded and double-stranded DNA to a solid phase;
  • Fig. 53A is graphic presentation of enzyme assay results of a screen of two different capture beads for use in a dual bead assay, these results indicating that both of the tested beads bind a similar amount of target regardless of whether the probe is bound covalently or non-covalently;
  • Fig. 53B is a graphic presentation of results of a dual bead assay designed to examine the number of reporter beads captured by two different capture beads, these results indicate that covalent bonding of the probe to the capture bead greatly improves assay sensitivity;
  • Fig. 54 is a graphic presentation demonstrating that the introduction of PEG linkers into probes significantly improves target mediated binding
  • Fig. 55 is a bar graph presentation illustrating probe density determination employing 3 ⁇ m beads
  • Fig. 56 is a bar graph presentation demonstrating the pretreatment of the beads with various detergents including salmon sperm DNA which reduced nonspecific binding by over 10 fold;
  • Fig. 57 is a bar graph presentation showing the range of detection of the dual bead assay
  • Fig. 58 is a bar graph illustrating the use of NaCl in varying concentrations and the related non-specific binding
  • Fig. 59 is a bar graph presentation showing increasing EDTA concentration and the related non-specific binding
  • Fig. 60 is a bar graph presentation depicting an increasing NaCl concen-tration and the related non-specific binding
  • Figs. 61 A and 61 B are bar graph presentations illustrating an increasing concentration of MgCI 2 and related non-specific binding
  • Fig. 62 is a pictorial schematic representation showing the use of probe blocking agents to increase the sensitivity of the bead assay
  • Fig. 63 is a bar graph presentation illustrating the effect of incubation time during a hybridization reaction
  • Fig. 64 is a bar graph presentation showing a mixing method directed to increasing efficiency in dual bead binding
  • Figs. 65A and 65B together comprise a pictorial representation of another embodiment of a method for producing genetic dual bead complex solutions related to the method discussed in connection with Fig. 11 A
  • Figs. 66A and 66B taken together form a pictorial representation of another embodiment of a method for producing immunochemical dual bead complex solutions similar to that shown in Fig 11 B;
  • Figs. 67A and 67B together show a pictorial representation of yet another embodiment of a method for producing genetic dual bead complex solutions, this method being related to the method illustrated in Fig. 12A;
  • Figs. 68A and 68B taken together illustrate a pictorial representation of still another embodiment of a method for producing immunochemical dual bead complex solutions which is similar to that shown in Fig. 12B;
  • Fig. 69 is a bar graph presentation demonstrating the effect of DNAsel digestion in absence of reporter beads;
  • Fig. 70 is a bar graph presentation showing the efficiency of dual bead assay by the effect of DNAsel enzymes digestion
  • Fig. 71 is a schematic representation of separation of reporter beads from capture beads by enzyme digestion and physical or chemical treatments
  • Fig. 72 is a bar graph presentation showing dual bead complexes prior to and after washing in a basic solution
  • Fig. 73A is a bar graph presentation illustrating dual bead complexes prior to and after washing in a 7M urea solution
  • Fig. 73B is a bar graph presentation representing dual bead complexes prior to and after washing in a 7M urea solution including the detection of dissociated reporter beads after the urea wash;
  • Fig. 74 is a bar graph presentation demonstrating the use of 1.5M guanidine isothiocyanate as a denaturing agent during dual bead assay; and Fig. 75 is a bar graph presentation showing the varying concentrations of guanidine isothiocyanate employed as a denaturing agent during dual bead assay;
  • Fig. 76 is a top plan view of a portion of a magneto-optical bio-disc having fluidic circuits.
  • Figs. 77A-77E are plan views illustrating a method of separating and testing cells using the fluidic circuit shown in Fig. 76. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the following description of the present invention relates to optical analysis discs, disc drive systems, optical disc biomagnetic assays, and assay chemistries and techniques.
  • the invention further relates to alternate magneto-optical drive systems, MO bio-discs, MO bio-disc systems, MO biomagnetic assays, and related processing methods.
  • FIG. 1 there is shown a perspective view of an optical analysis disc, optical bio-disc, or medical CD 110 for use in an optical disc drive 112.
  • Drive 112 in conjunction with software in the drive or associated with a separate computer, can cause images, graphs, or output data to be displayed on display monitor 114.
  • discs and drives there are different types of discs and drives that can be used including, but not limited to, magneto-optical discs and magneto-optical disc drives.
  • the disc drive can be in a unit separate from a controlling computer, or provided in a bay within a computer.
  • the device can be made as portable as a laptop computer, and thus usable with battery power and in remote locations not generally served by advanced diagnostic equipment.
  • the drive is preferably a conventional drive with minimal or no hardware modification, but can be a dedicated bio-disc or medical CD drive. Further details regarding these types of drive systems and related signal processing methods are disclosed in, for example, commonly assigned and co- pending U.S. Patent Application Serial No. 09/378,878 entitled “Methods and Apparatus for Analyzing Operational and Non-operational Data Acquired from Optical Discs” filed August 23, 1999; U.S. Provisional Patent Application Serial No. 60/150,288 entitled “Methods and Apparatus for Optical Disc Data Acquisition Using Physical Synchronization Markers” filed August 23, 1999; U.S. Patent Application Serial No.
  • Optical bio-disc 110 for use with embodiments of the present invention may have any suitable shape, diameter, or thickness, but preferably is implemented on a round disc with a diameter and a thickness similar to those of a compact disc (CD), a recordable CD (CD-R), CD-RW, a digital versatile disc (DVD), DVD-R, DVD-RW, a magneto-optical disc, or other standard optical disc format.
  • the disc may include encoded information, preferably in a known format, for performing, controlling, and post-processing a test or assay, such as information for controlling the rotation rate and direction of the disc, timing for rotation, stopping and starting, delay periods, locations of samples, position of the light source, and power of the light source. Such encoded information is referred to generally here as operational information.
  • the disc may be a reflective disc, as shown in Figs. 3A-3C, a transmissive disc, Figs. 4A-4C, or some combination of reflective and transmissive.
  • a reflective disc an incident light beam is focused onto the disc (typically at a reflective surface where information is encoded), reflected, and returned through optical elements to a detector on the same side of the disc as the light source.
  • a transmissive disc light passes through the disc (or portions thereof) to a detector on the other side of the disc from the light source.
  • some light may also be reflected and detected as reflected light.
  • Fig. 2 shows an optical disc reader system 116.
  • This system may be a conventional reader for CD, CD-R, DVD, MO, or other known comparable format, a modified version of such a drive, or a completely distinct dedicated device.
  • the basic components are a motor for rotating the disc, a light system for providing light, and a detection system for detecting light.
  • a light source 1 18 provides light to optical components 120 to produce an incident light beam 122.
  • a return beam 124 is reflected from either reflective surface 156, 174, or 186, Figs. 3C and 4C. Return beam 124 is provided back to optical components 120, and then to a bottom detector 126.
  • the return beam may carry operational information or other encoded data as well as characteristic information about the investigational feature or test sample under study.
  • transmissive disc 180 For transmissive disc 180, Figs. 4A-4C, some of the energy from the incident beam 122 will undergo a light/matter interaction with an investigational feature or test sample and then proceed through the disc as a transmitted beam 128 that is detected by a top detector 130.
  • a transmissive disc including a semi-reflective layer 186 (Fig. 4C) as the operational layer some of the energy from the incident beam 122 will also reflect from the operational layer as return beam 124, which carries operational information or stored data.
  • Optical components 120 can include a lens, a beam splitter, and a quarter wave plate that changes the polarization of the light beam so that the beam splitter directs a reflected beam through the lens to focus the reflected beam onto the detector.
  • An astigmatic element such as a cylindrical lens, may be provided between the beam splitter and detector to introduce astigmatism in the reflected light beam.
  • the light source can be controllable to provide variable wavelengths and power levels over a desired range in response to data introduced by the user or read from the disc. This controllability is especially useful when it is desired to detect multiple different structures that fluoresce at different wavelengths.
  • a computer 132 including a processor 134 and an analyzer 136.
  • An image or output results can then be provided to a monitor 114.
  • Computer 132 can represent a desktop computer, programmable logic, or some other processing device, and also can include a connection (such as over the Internet) to other processing and/or storage devices.
  • a drive motor 140 and a controller 142 are provided for controlling the rotation rate and direction or rotation of disc the 144 or 180. Controller 142 and the computer 132 with processor 134 can be in remote communication or implemented in the same computer. Methods and systems for reading such a disc are also shown in Gordon, U.S. Patent No. 5,892,577, which is incorporated herein by reference.
  • the detector can be designed to detect all light that reaches the detector, or light only at specific wavelengths, through its design or an external filter. By making the detector controllable in terms of the detectable wavelength, beads or other structures that fluoresce at different wavelengths can be separately detected.
  • a hardware trigger sensor 138 may be used with either a reflective disc 144 or transmissive disc 180. Triggering sensor 138 provides a signal to computer 132 (or to some other electronics) to allow for the collection of data by processor 134 only when incident beam 122 is on a target zone or inspection area. Alternatively, software read from a disc can be used to control data collection by processor 134 independent of any physical marks on the disc. Such software or logical triggering is discussed in further detail in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/352,625 entitled "Logical Triggering Methods And Apparatus For Use With Optical Analysis Discs And Related Disc Drive Systems" filed January 28, 2002, which is herein incorporated by reference in its entirety.
  • the substrate layer of the optical analysis disc may be impressed with a spiral track that starts at an innermost readable portion of the disc and then spirals out to an outermost readable portion of the disc.
  • this track is made up of a series of embossed pits with varying length, each typically having a depth of approximately one-quarter the wavelength of the light that is used to read the disc.
  • the varying lengths and spacing between the pits encode the operational data.
  • the spiral groove of a recordable CD-like disc has a detectable dye rather than pits.
  • the data is stored in magnetic domains created on the MO disc. This is where the operation information, such as the rotation rate, is recorded.
  • the rotation rate may be variable with intervening or consecutive periods of acceleration, constant speed, and deceleration. These periods may be closely controlled both as to speed and time of rotation to provide, for example, mixing, agitation, or separation of fluids and suspensions with agents, reagents, antibodies, or other materials.
  • Different optical analysis disc, medical CD, and bio-disc designs that may be utilized with the present invention, or readily adapted thereto, are disclosed, for example, in commonly assigned, co-pending U.S. Patent Application Serial No. 09/999,274 entitled "Optical Bio-discs with Reflective Layers" filed on November 15, 2001 ; U.S. Patent Application Serial No.
  • the disc drive assembly is thus employed to rotate the disc, read and process any encoded operational information stored on the disc, and analyze the liquid, chemical, biological, or biochemical investigational features in an assay region of the disc.
  • the disc drive assembly may be further utilized to write information to the disc either before, during, or after the material in the assay zone is analyzed by the read beam of the drive.
  • the disc drive assembly is implemented to deliver assay information through various possible interfaces such as via Ethernet to a user, over the Internet, to remote databases, or anywhere such information could be advantageously utilized. Further details relating to this type of disc drive interfacing are disclosed in commonly assigned co-pending U.S. Patent Application Serial No. 09/986,078 entitled “Interactive System For Analyzing Biological Samples And Processing Related Information And The Use Thereof " filed November 7, 2001 , which is incorporated herein by reference in its entirety.
  • the reflective disc 144 is shown with a cap 146, a channel layer 148, and a substrate 150.
  • the channel layer 148 may be formed by a thin-film adhesive member.
  • Cap 146 has inlet ports 152 for receiving samples and vent ports 154.
  • Cap 146 may be formed primarily from polycarbonate, and may be coated with a cap reflective layer 156 on the bottom thereof.
  • Reflective layer 156 is preferably made from a metal such as aluminum or gold.
  • Channel layer 148 defines fluidic circuits 158 by having desired shapes cut out from channel layer 148. Each fluidic circuit 158 preferably has a flow channel 160 and a return channel 162, and some have a mixing chamber 164.
  • a mixing chamber 166 can be symmetrically formed relative to the flow channel 160, while an off-set mixing chamber 168 is formed to one side of the flow channel 160.
  • Fluidic circuits 158 are rather simple in construction, but a fluidic circuit can include other channels and chambers, such as preparatory regions or a waste region, as shown, for example, in U.S. Patent No. 6,030,581 entitled "Laboratory in a Disc” which is incorporated herein by reference. These fluidic circuits can include valves and other fluid control structures such as those alternatively employed herein and discussed in further detail in connection with Figs. 33A-33D, 34A-34C, 35, and 36A-36C.
  • Channel layer 148 can include adhesives for bonding to the substrate and to the cap.
  • Substrate 150 has a plastic layer 172, and has target zones 170 formed as openings in a substrate reflective layer 174 deposited on the top of layer 172.
  • reflective layer 174 is used to encode operational information.
  • the reflective layer 174 is not limited to a single layer but may also be several stacks of reflective layers on the substrate 150 such as optical stacks on an MO disc, for example.
  • Plastic layer 172 is preferably formed from polycarbonate.
  • Target zones 170 may be formed by removing portions of the substrate reflective layer 174 in any desired shape, or by masking target zone areas before applying substrate reflective layer 174.
  • the substrate reflective layer 174 is preferably formed from a metal, such as aluminum, gold, or magnetic alloys, and can be configured with the rest of the substrate to encode operational information that is read with incident light, such as through a wobble groove or through an arrangement of pits. Light incident from under substrate 150 thus is reflected by layer 174, except at target zones 170, where it is reflected by layer 156. Target zones are where investigational features are detected. If the target zone is a location where an antibody, strand of DNA, or other material that can bind to a target is located, the target zone can be referred to as a capture zone.
  • optical disc 144 is cut away to illustrate a partial cross-sectional perspective view.
  • An active layer 176 is formed over substrate reflective layer 174.
  • Active layer 176 may generally be formed from nitrocellulose, polystyrene, polycarbonate, gold, activated glass, modified glass, or a modified polystyrene such as, for example, polystyrene-co-maleic anhydride.
  • channel layer 148 is situated over active layer 174.
  • samples can be introduced through inlet ports 152 of cap 146. When rotated, the sample moves outwardly from inlet port 152 along active layer 176.
  • detectable features referred to as investigational features
  • Examples of such processes are shown in the incorporated U.S. Patent No. 6,030,581 and in commonly assigned, co-pending U.S. Patent Application No. 09/988,728 entitled “Methods And Apparatus For Detecting And Quantifying Lymphocytes With Optical Biodiscs” filed November 16, 2001 ; and U.S. Patent Application No.
  • the investigational features captured within the target zones, by the capture layer with a capture agent may be designed to be located in the focal plane coplanar with reflective layer 174, where an incident beam is typically focused in conventional readers. Alternatively, the investigational features may be captured in a plane spaced away from the focal plane.
  • the former configuration is referred to as a "proximal” type disc, and the latter a “distal” type disc.
  • the transmissive optical disc 180 includes a clear cap 182, a channel layer 148, and a substrate 150.
  • the clear cap 182 includes inlet ports 152 and vent ports 154 and is preferably formed mainly from polycarbonate.
  • Trigger marks 184 may be included on the cap 182.
  • Channel layer 148 has fluidic circuits 158, which can have structure and use similar to those described in conjunction with Figs. 3A, 3B, and 3C.
  • Substrate 150 may include target zones 170, and preferably includes a polycarbonate layer 172.
  • Substrate 150 may, but need not, have a thin semi-reflective layer 186 deposited on top of layer 172.
  • Semi-reflective layer 186 is preferably significantly thinner than substrate reflective layer 174 on substrate 150 of reflective disc 144 (Figs. 3A-3C).
  • the semi-reflective layer 186 is not limited to a single layer but may also be several stacks of semi-reflective layers on the substrate 150 such as optical stacks on an MO disc, for example.
  • Semi-reflective layer 186 is preferably formed from a metal, such as aluminum, gold, or magnetic alloys, but is sufficiently thin to allow a portion of an incident light beam to penetrate and pass through layer 186, while some of the incident light is reflected back.
  • a gold film layer for example, is 95% reflective at a thickness greater than about 700 ⁇ , while the transmission of light through the gold film is about 50% transmissive at approximately 100 A.
  • FIG. 4C is a cut-away perspective view of transmissive disc 180.
  • the semi- reflective nature of layer 186 makes its entire surface potentially available for target zones, including virtual zones defined by trigger marks or encoded data patterns on the disc.
  • Target zones 170 may also be formed by marking the designated area in the indicated shape or alternatively in any desired shape. Markings to indicate target zone 170 may be made on semi-reflective layer 186 or on a bottom portion of substrate 150 (under the disc). Target zones 170 may be created by silk screening ink onto semi-reflective layer 186.
  • An active layer 176 is applied over semi-reflective layer 186. Active layer 176 may be formed from the same materials as described above in conjunction with layer 176 (Fig.
  • transmissive disc 180 there is no reflective layer, on the clear cap 182, comparable to reflective layer 156 in reflective disc 144 (Fig. 3C).
  • FIG. 5A there is shown a cross sectional view taken across the tracks of the reflective disc embodiment 144 (Figs. 3A-3C) of the bio-disc 110 (Fig. 1) according to the present invention. As illustrated, this view is taken longitudinally along a radius and flow channel of the disc.
  • Fig. 5A includes the substrate 150 that is composed of a plastic layer 172 and a substrate reflective layer 174.
  • the substrate 150 includes a series of grooves 188.
  • the grooves 188 are in the form of a spiral extending from near the center of the disc toward the outer edge.
  • the grooves 188 are implemented so that the interrogation or incident beam 122 may track along the spiral grooves 188 on the disc.
  • This type of groove 188 is known as a "wobble groove".
  • the groove 188 is formed by a bottom portion having undulating or wavy side walls. A raised or elevated portion separates adjacent grooves 188 in the spiral.
  • the reflective layer 174 applied over the grooves 188 in this embodiment is, as illustrated, conformal in nature.
  • Fig. 5A also shows the active layer 176 applied over the reflective layer 174.
  • the target zone 170 is formed by removing an area or portion of the reflective layer 174 at a desired location or, alternatively, by masking the desired area prior to applying the reflective layer 174.
  • the plastic adhesive member or channel layer 148 is applied over the active layer 176.
  • Fig. 5A also shows the cap portion 146 and the reflective surface 156 associated therewith. Thus, when the cap portion 146 is applied to the plastic adhesive member 148 including the desired cut-out shapes, the flow channel 160 is thereby formed.
  • Fig. 5B is a cross sectional view, similar to that illustrated in Fig. 5A, taken across the tracks of the transmissive disc embodiment 180 (Figs. 4A-4C) of the bio- disc 110 (Fig. 1) according to the present invention. This view is taken longitudinally along a radius and flow channel of the disc.
  • Fig. 5B illustrates the substrate 150 that includes the thin semi-reflective layer 186. This thin semi-reflective layer 186 allows the incident or interrogation beam 122, from the light source 118 (Fig. 2), to penetrate and pass through the disc to be detected by the top detector 130, while some of the light is reflected back in the form of the return beam 124.
  • the thickness of the thin semi-reflective layer 186 is determined by the minimum amount of reflected light required by the disc reader to maintain its tracking ability.
  • the substrate 150 in this embodiment like that discussed in Fig. 5A, includes the series of grooves 188.
  • the grooves 188 in this embodiment are also preferably in the form of a spiral extending from near the center of the disc toward the outer edge.
  • the grooves 188 are implemented so that the interrogation beam 122 may track along the spiral.
  • Fig. 5B also shows the active layer 176 applied over the thin semi-reflective layer 186.
  • the plastic adhesive member or channel layer 148 is applied over the active layer 176.
  • Fig. 5B also shows the clear cap 182.
  • Fig. 6A is a view similar to Fig. 5A but taken perpendicularly to a radius of the disc to illustrate the reflective disc and the initial refractive property thereof when observing the flow channel 160 from a radial perspective.
  • Fig. 6B is a similar view to Fig.
  • FIG. 5B but taken perpendicularly to a radius of the disc to represent the transmissive disc and the initial refractive property thereof when observing the flow channel 160 from a radial perspective.
  • Grooves 188 are not seen in Figs. 5A and 5B since the sections are cut along the grooves 188.
  • Figs. 6A and 6B show the presence of the narrow flow channel 160 that is situated perpendicular to the grooves 188 in these embodiments.
  • Figs. 5A, 5B, 6A, and 6B show the entire thickness of the respective reflective and transmissive discs.
  • the incident beam 122 is illustrated initially interacting with the substrate 150 which has refractive properties that change the path of the incident beam as shown to provide focusing of the beam 122 on the reflective layer 174 or the thin semi-reflective layer 186.
  • the fluidic circuits 158 may also be configured in the equi-radial or "e-rad" format disclosed in commonly assigned and copending U.S. Provisional Application
  • Capture bead 190 can be used in conjunction with a variety of different assays including biological assays such as immunoassays (Figs. 7B-10B), molecular assays, and more specifically genetic assays (Figs. 7A-10A).
  • biological assays such as immunoassays (Figs. 7B-10B), molecular assays, and more specifically genetic assays (Figs. 7A-10A).
  • antibody transport probes 196 are conjugated onto the beads.
  • Antibody transport probes 196 include proteins, such as antigens or antibodies, implemented to capture protein targets.
  • oligonucleotide transport probes 198 would be conjugated onto the beads.
  • Oligonucleotide transport probes 198 include nucleic acids such as DNA or RNA implemented to capture genetic targets.
  • a target agent such as target DNA or RNA 202, obtained from a test sample, is added to a capture bead 190 coated with oligonucleotide transport probes 198.
  • transport probes 198 are formed from desired sequences of nucleic acids.
  • a target agent such as target antigen 204 from a test sample is added to a capture bead 190 coated with antibody transport probes 196.
  • the transport probes 196 are formed from proteins such as antibodies.
  • Capture bead 190 has a characteristic that allows it to be isolated from a material suspension or solution.
  • the capture bead may be selected based upon a desired size, and a preferred way to make it isolatable is for it to be magnetic.
  • Fig. 8A illustrates the binding of target DNA or RNA 202 to complementary transport probes 198 on capture bead 190 in the genetic assay implementation of the present invention.
  • Fig. 8B shows an immunoassay version of Fig. 8A, transport probes 196 can alternatively include antibodies or antigens for binding to a target protein 204.
  • Fig. 9A shows a reporter bead 192 coated with oligonucleotide signal probes 206 complementary to target agent 202 (see Fig. 8A).
  • Reporter bead 192 is selected based upon a desired size and the material properties for detection and reporting purposes. In one specific embodiment a 2.1 urn polystyrene bead is employed.
  • Signal probes 206 can be strands of DNA or RNA to capture target DNA or RNA.
  • Fig. 9B illustrates a reporter bead 192 coated with antibody signal probes 208 that bind to the target agent 204 as shown in Fig. 8B.
  • Reporter bead 192 is selected based upon a desired size and the material properties for detection and reporting purposes. This may also preferably include a 2.1 urn polystyrene bead.
  • Signal probes 208 can be antigens or antibodies implemented to capture protein or glycoprotein targets.
  • Fig. 10A is a pictorial representation of a dual bead complex 194 that can be formed from capture bead 190 with probe 198, target agent 202, and reporter bead 192 with probe 206.
  • Probes 198 and 206 conjugated on capture bead 190 and reporter bead 192, respectively, have sequences complementary to the target agent 202, but not to each other. Further details regarding target agent detection and methods of reducing non-specific binding of target agents to beads are discussed in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/278,106 entitled “Dual Bead Assays Including Use of Restriction Enzymes to Reduce Non-Specific Binding" filed March 23, 2001 ; and U.S. Provisional Application Serial No. 60/278,110 entitled “Dual Bead Assays Including Use of Chemical Methods to Reduce Non-Specific Binding” also filed March 23, 2001 , which are both incorporated herein by reference in their entirety.
  • Fig. 10B is a pictorial representation of the immunoassay version of a dual bead complex 194 that can be formed from capture bead 190 with probe 196, target agent 204, and reporter bead 192 with probe 208.
  • the efficiency and specificity of target agent binding may be enhanced by using a cleavable spacer or a displaceable spacer that temporarily links the reporter bead 192 and capture bead 190.
  • the dual bead complex formed by the cleavable spacer or displaceable spacer essentially places the transport probe and the signal probe in close proximity to each other thus allowing more efficient target binding to both probes. Once the target agent is bound to the probes, the spacer may then be cleaved permitting the bound target agent to retain the dual bead structure.
  • cleavable spacers and displaceable spacers are also described below in conjunction with Figs. 42A-42D and 43A-43C.
  • Fig. 11 A there is illustrated a method of preparing a molecular assay using a "single-step hybridization" technique to create dual bead complex structures in a solution according to one aspect of the present invention.
  • This method includes 5 principal steps identified consecutively as Steps I, II, III, IV, and V.
  • Step I of this method a number of capture beads 190 coated with oligonucleotide transport probes 198 are deposited into a test tube 212 containing a buffer solution 210.
  • the number of capture beads 190 used in this method may be, for example, on the order of 10E+07 and each on the order of 1 urn or greater in diameter.
  • Capture beads 190 are suspended in hybridization solution and are loaded into the test tube 212 by injection with pipette 214.
  • the preferred hybridization solution is composed of 0.2M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris-HCI, pH 7.5, and 5X Denhart's mix.
  • a desirable hybridization temperature is 37 degrees Celsius.
  • transport probes 198 are conjugated to 3 urn magnetic capture beads 190 by EDC conjugation. Further details regarding conjugation methods are disclosed in commonly assigned U.S. Provisional Application Serial No.
  • target DNA or RNA 202 is added to the solution.
  • Oligonucleotide transport probes 198 are complementary to the DNA or RNA target agent 202.
  • the target DNA or RNA 202 thus binds to the complementary sequences of transport probe 198 attached to the capture bead 190 as shown in Fig. 8A.
  • reporter beads 192 coated with oligonucleotide signal probes 206 are complementary to the target DNA or RNA 202.
  • signal probes 206 which are complementary to a portion of the target DNA or RNA 202, are conjugated to 2.1 urn fluorescent reporter beads 192.
  • Signal probes 206 and transport probes 198 each have sequences that are complementary to the target DNA 202, but not complementary to each other.
  • the target agent 202 and signal probe 206 are preferably allowed to hybridize for three to four hours at 37 degrees Celsius.
  • intermittent mixing i.e., periodically mixing and then stopping
  • the disc drive motor 140 and controller 142, Fig. 2 may be advantageously employed to periodically rotate the disc to achieve the desired intermittent mixing. This may be implemented in mixing protocols encoded on the disc that rotate the disc in one direction, then stop the disc, and thereafter rotate the disc again in the same direction in a prescribed manner with a preferred duty cycle of rotation and stop sessions.
  • the encoded mixing protocol may rotate the disc in a first direction, then stop the disc, and thereafter rotate the disc again in the opposite direction with a preferred duty cycle of rotation, stop, and reverse rotation sessions.
  • Step IV of Fig. 11 A after hybridization, the dual bead complex 194 is separated from unbound reporter beads in the solution.
  • the solution can be exposed to a magnetic field to capture the dual bead complex structures 194 using the magnetic properties of capture bead 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension.
  • Step IV includes the removal of supernatant containing free-floating particles. Wash buffer is added into the test tube and the bead solution is mixed well.
  • the preferred wash buffer for the one step assay consists of 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM, and 10mM EDTA. Most of the unbound reporter beads 182, free-floating DNA, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target sequences, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Further details relating to other aspects associated with methods of decreasing non-specific binding of reporter beads to capture beads are disclosed in, for example, commonly assigned U.S. Provisional Application Serial No. 60/272,134 entitled “Reduction of Non-Specific Binding in Dual Bead Assays by Selection of Bead Type and Bead Treatment” filed February 28, 2001 ; and U.S. Provisional Application Serial No. 60/275,006 entitled “Reduction of Non-Specific Binding in Dual Bead Assays by Selection of Buffer Conditions and Wash Conditions” filed March 12, 2001. Both of these applications are herein incorporated by reference in their entirety.
  • Fig. 11 A The last principal step shown in Fig. 11 A is Step V.
  • Step V The last principal step shown in Fig. 11 A is Step V.
  • the assay mixture may be loaded into the disc and ready to be analyzed.
  • Fig. 11 B illustrates an immunoassay using a "single-step antigen binding" method, similar to that in Fig. 11 A, to create dual bead complex structures in a solution.
  • This method similarly includes 5 principal steps. These steps are respectively identified as Steps I, II, III, IV, and V in Fig. 11 A.
  • capture beads 190 e.g., on the order of 10E+07 in number and each on the order of 1 urn or above in diameter, which are coated with antibody transport probes 196 are added to a buffer solution 210.
  • This solution may be that same as that employed in the method shown in Fig. 11 A or alternatively may be specifically prepared for use with immunochemical assays.
  • the antibody transport probes 196 have a specific affinity for the target antigen 204.
  • the transport probes 196 bind specifically to epitopes within the target antigen 204 as also shown in Fig. 8B.
  • antibody transport probes 196 that have an affinity for a portion of the target antigen may be conjugated to 3 urn magnetic capture beads 190 via EDC conjugation.
  • conjugation of the transport probes 196 to the capture bead 190 may be achieved by passive adsorption.
  • the target antigen 204 is added to the solution.
  • the target antigen 204 binds to the antibody transport probe 196 attached to the capture bead 190 as also shown in Fig. 8B.
  • reporter beads 192 coated with antibody signal probes are illustrated in Step III.
  • Antibody signal probes 208 specifically binds to the epitopes on target antigen 204 as also represented in Figs. 9B and 10B.
  • signal probes 208 are conjugated to 2.1 urn fluorescent reporter beads 192.
  • Signal probes 208 and transport probes 196 each bind to specific epitopes on the target antigen, but not to each other.
  • the dual bead complex 194 is formed such that the target antigen 204 links capture bead 190 and reporter bead 192. With specific and thorough washing, there should be minimal non-specific binding between reporter bead 192 and capture bead 190.
  • Step IV after the binding in Step III, the dual bead complex 194 is separated from unbound reporter beads in the solution.
  • the solution can be exposed to a magnetic field to capture the dual bead complex structures 194 using the magnetic properties of capture bead 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that capture beads not bound to reporter beads will also be isolated. Alternatively, as indicated above, this magnetic removal step may also be performed on-disc as shown in Figs. 33A, 35, and 36A-36C.
  • the purification process of Step IV includes the removal of supernatant containing free-floating particles.
  • Wash buffer is added into the test tube and the bead solution is mixed well. Most of the unbound reporter beads 182, free-floating protein samples, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target antigen, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step V The last principal step in Fig. 11 B is Step V. In this step, once the dual bead complex has been washed approximately 3-5 times with wash buffer solution, the assay mixture is loaded into the disc and is thereby in condition to be analyzed.
  • Fig. 12A shows an alternative genetic assay method referred to here as a "two- step hybridization" to create the dual bead complex which has 6 principal steps.
  • capture beads are coated with oligonucleotide transport probes 198 complementary to DNA or RNA target agent and placed into a buffer solution.
  • transport probes that are complementary to a portion of target agent are conjugated to 3 um magnetic capture beads via EDC conjugation.
  • Other types of conjugation of the oligonucleotide transport probes to a solid phase may be utilized. These include, for example, passive adsorption or use of streptavidin-biotin interactions.
  • capture beads 190 suspended in hybridization solution, are loaded from the pipette 214 into the test tube 212.
  • the preferred hybridization solution is composed of 0.2M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris-HCI, pH 7.5, and 5X Denhart's mix.
  • a desirable hybridization temperature is 37 degrees Celsius.
  • target DNA or RNA 202 is added to the solution and binds to the complementary sequences of transport probe 198 attached to capture bead 190.
  • target agent 202 and the transport probe 198 are allowed to hybridize for 2 to 3 hours at 37 degrees Celsius. Sufficient hybridization, however, may be achieved within 30 minutes at room temperature. At higher temperatures, hybridization may be achieved substantially instantaneously.
  • target agents 202 bound to the capture beads are separated from unbound species in solution by exposing the solution to a magnetic field to isolate bound target sequences by using the magnetic properties of the capture bead 190.
  • the magnetic field can be enclosed in a magnetic test tube rack 216 with a built-in magnet permanent 218 or electromagnet to draw out the magnetic beads and remove any unbound target DNA 202 free-floating in the suspension via pipette extraction of the solution.
  • this magnetic removal step may be performed as shown in Figs. 33A, 35, and 36A-36C.
  • a wash buffer is added and the separation process can be repeated.
  • the preferred wash buffer after the transport probes 198 and target DNA 202 hybridize consists of 145mM NaCl, 50mM Tris, pH 7.5, and 0.05% Tween.
  • Hybridization methods and techniques for decreasing non-specific binding of target agents to beads are further disclosed in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/278,691 entitled "Reduction of Non-Specific Binding of Dual Bead Assays by Use of Blocking Agents” filed March 26, 2001. This application is herein incorporated by reference in its entirety.
  • reporter beads 192 are added to the solution as discussed in conjunction with the method shown in Fig. 11 A.
  • Reporter beads 192 are coated with signal probes 206 that are complementary to target agent 202.
  • signal probes 206 which are complementary to a portion of target agent 202, are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 206 and transport probes 198 each have sequences that are complementary to target agent 202, but not complementary to each other.
  • the dual bead complex structures 190 are formed. As would be readily apparent to one of skill in the art, the dual bead complex structures are formed only if the target agent of interest is present.
  • target agent 202 links magnetic capture bead 190 and reporter bead 192.
  • Target agent 202 and signal probe 206 are preferably allowed to hybridize for 2-3 hours at 37 degrees Celsius. As with Step II discussed above, sufficient hybridization may be achieved within 30 minutes at room temperature. At higher temperatures, the hybridization in this step may also be achieved substantially instantaneously.
  • Step V shown in Fig. 12A after the hybridization in Step IV, the dual bead complex 194 is separated from unbound species in solution. The solution is again exposed to a magnetic field to isolate the dual bead complex 194 using the magnetic properties of the capture bead 190. Note again that the isolate will include capture beads not bound to reporter beads. As with Step III above in the on- disc counterpart hereto, this magnetic separation step may be performed as shown in Figs. 33A, 35, and 36A-36C.
  • a purification process to remove supernatant containing free-floating particles includes adding wash buffer into the test tube and mixing the bead solution well.
  • the preferred wash buffer for the two-step assay consists of 145mM NaCl, 50mM Tris, pH 7.5, 0.1 % SDS, 0.05% Tween, 0.25% NFDM, and 10mM EDTA. Most unbound reporter beads, free-floating DNA, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target agents, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step VI The final principal step shown in Fig. 12A is Step VI.
  • the assay mixture is loaded into the disc and analyzed.
  • the oligonucleotide signal and transport probes may be ligated to prevent breakdown of the dual bead complex during the disc analysis and signal detection processes. Further details regarding probe ligation methods are disclosed in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/278,694 entitled "Improved Dual Bead Assays Using Ligation" filed March 26, 2001 , which is herein incorporated in its entirety by reference.
  • Fig. 12B shows an immunoassay method, similar to those discussed in connection with the immunoassay method of Fig. 11 B and following the steps of the genetic assay of Fig. 12A.
  • This method is also referred to here as a "two-step binding" to create the dual bead complex in an immunochemical assay.
  • this method includes 6 main steps.
  • capture beads coated with antibody transport probes that specifically bind to epitopes on target antigens are placed into a buffer solution.
  • antibody transport probes are conjugated to 3 um magnetic capture beads. Different sized magnetic capture beads may be employed depending on the type of disc drive and disc assembly utilized to perform the assay.
  • These 6 main steps according to this alternative method of the invention are respectively identified as Steps I, II, III, IV, V and VI in Fig. 12B.
  • Step I With specific reference now to Step I shown in Fig. 12B, capture beads 190, suspended in buffer solution 210, are loaded into a test tube 212 via injection from pipette 214.
  • target antigen 204 is added to the solution and binds to the antibody transport probe 196 attached to capture bead 190.
  • Target antigen 204 and the transport probe 196 are preferably allowed to bind for 2 to 3 hours at 37 degrees Celsius. Shorter binding times are also possible.
  • target antigen 204 bound to the capture beads 190 is separated from unbound species in solution by exposing the solution to a magnetic field to isolate bound target proteins or glycoproteins by using the magnetic properties of capture bead 190.
  • the magnetic field can be enclosed in a magnetic test tube rack 216 with a built-in magnet permanent 218 or electromagnet to draw out the magnetic beads and remove any unbound target antigen 204 free-floating in the suspension via pipette extraction of the solution.
  • a wash buffer is added and the separation process can be repeated.
  • reporter beads 192 are added to the solution as discussed in conjunction with the method shown in Fig. 11B.
  • Reporter beads 192 are coated with signal probes 208 that have an affinity for the target antigen 204.
  • signal probes 208 which bind specifically to a portion of target agent 204, are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 208 and transport probes 196 each bind to specific epitopes on the target agent 204, but do not bind to each other.
  • the dual bead complex structures 190 are formed.
  • target antigen 204 links magnetic capture bead 190 and reporter bead 192.
  • target antigen 204 and signal probe 208 are allowed to hybridize for 2-3 hours at 37 degrees Celsius. As with Step II discussed above, sufficient binding may be achieved within 30 minutes at room temperature. In the case of immunoassays temperatures higher than 37 degrees Celsius are not preferred because the proteins will denature. Turning next to Step V as illustrated in Fig. 12B, after the binding shown in Step
  • the dual bead complex 194 is separated from unbound species in solution. This is achieved by exposing the solution to a magnetic field to isolate the dual bead complex 194 using the magnetic properties of the capture bead 190 as shown. Note again that the isolate will include capture beads not bound to reporter beads.
  • a purification process to remove supernatant containing free-floating particles includes adding wash buffer into the test tube and mixing the bead solution well. Most unbound reporter beads, free-floating proteins, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target agents, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step VI The final main step shown in Fig. 12B is Step VI.
  • the assay mixture is loaded into the disc and analyzed.
  • the magnetic removal or separation steps in the method shown in Fig. 12B may be alternatively performed on- disc using the disc, fluidic circuits, and apparatus illustrated in Figs. 33A-33D, 34A- 34C, 35, and 36A-36C.
  • FIG. 13 there is shown a cross sectional view illustrating the disc layers (similar to Fig. 6) of the mixing or loading chamber 164. Access to the loading chamber 164 is achieved by an inlet port 152 where the dual bead assay preparation is loaded into the disc system.
  • Fig. 14 is a view similar to Fig. 13 showing the mixing or loading chamber 164 with the pipette 214 injection of the dual bead complex 194 onto the disc.
  • the complex includes reporters 192 and capture bead 190 linked together by the target DNA or RNA 202.
  • the signal DNA 206 is illustrated as single stranded DNA complementary to the capture agent.
  • Fig. 15A shows the flow channel 160 and the target or capture zone 170 after anchoring of dual bead complex 194 to a capture agent 220.
  • the capture agent 220 in this embodiment is attached to the active layer 176 by applying a small volume of capture agent solution to the active layer 176 to form clusters of capture agents within the area of the target zone 170.
  • the capture agent includes biotin or BSA-biotin.
  • reporter beads 192 and capture beads 190 are attached to the reporter beads 192.
  • the anchor agent 222 may include streptavidin or Neutravidin. So when the reporter beads 192 come in close proximity to the capture agents 220, binding occurs between the anchor probe 222/206 and the capture agent 220, via biotin-streptavidin interactions, thereby retaining the dual bead complex 194 within the target zone 170. At this point, an interrogation beam 224 directed to the target zone 170 can be used to detect the dual bead complex 194 within the target zone 170.
  • Figs. 15A and 15B may alternatively be implemented on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • Fig. 15B is a cross sectional view similar to Fig. 15A illustrating the entrapment of the reporter bead 192 within the target zone 170 after a subsequent change in disc rotational speed.
  • the change in rotational speed removes the capture beads 190 from the dual bead complex 194, ultimately isolating the reporter bead 192 in the target zone 170 to be detected by the interrogation or read beam 224.
  • Fig. 16A is a cross sectional view, similar to Fig. 15A, that illustrates an alternative embodiment to Fig. 15A wherein the signal probes 206 or anchor agents 222, on the reporter beads 192, directly hybridize to the capture agent 220.
  • Fig. 16A shows the flow channel 160 and the target or capture zone 170 after anchoring of dual bead complex 194 with the capture agent 220.
  • the capture agent 220 in this embodiment is attached to the active layer 176 by applying a small volume of capture agent solution to the active layer 176 to form clusters of capture agents within the area of the target zone 170.
  • the capture agent 220 may be attached to the active layer using an amino group that covalently binds to the active layer 176.
  • the capture agent includes DNA.
  • Fig. 16A also shows reporters 192 and capture beads 190 as components of a dual bead complex 194 as employed in the present invention.
  • anchor agents 222 are attached to the reporter beads 192.
  • the anchor agent 222 in this embodiment may be a specific sequence of nucleic acids that are complimentary to the capture agent 220 or the oligonucleotide signal probe 206 itself. So when the reporter beads 192 come in close proximity to the capture agents 220, hybridization occurs between the anchor agent 222 and the capture agent 220 thereby retaining the dual bead complex 194 within the target zone 170.
  • the signal probe 206 serves the function of anchor agent 222. At this point, an interrogation beam 224 directed to the target zone 170 may be used to detect the dual bead complex 194 within the target zone 170.
  • Fig. 16B illustrates the embodiment in Fig. 16A after a subsequent change in disc rotational speed.
  • the change in rational speed removes the capture bead 190 from the dual bead complex 194, ultimately isolating the reporter bead 192 and the target DNA sequence 202 in the target zone 170 to be detected by an interrogation beam 224.
  • Figs. 16A and 16B may alternatively be implemented on the transmissive disc illustrated in Figs. 4A-4C, 5B, and 6B.
  • anchor agents 222 are attached to the capture beads 190 instead of the reporter beads.
  • the anchor agent 222 in this embodiment may include streptavidin or Neutravidin.
  • the target zone 170 is coated with a capture agent 220.
  • the capture agent may include biotin or BSA- biotin.
  • Fig. 17 also shows reporters 192 and capture beads 190 as components of a dual bead complex 194 as employed in the present invention.
  • Fig. 18 is an alternative to the embodiment illustrated in Fig. 16A.
  • anchor agents 222 are attached to the capture beads 190 instead of the reporter beads.
  • the transport probes 198, or an anchor agent 222 on the capture bead 190 directly hybridizes to the capture agent 220.
  • the capture agent 220 includes specific sequences of nucleic acid.
  • the anchor agent 222 in this embodiment may be a specific sequence of nucleic acids that are complimentary to the capture agent 220 or the oligonucleotide signal transport probe 198 itself. So when the capture beads 190 come in close proximity to the capture agents 220, hybridization occurs between the anchor agent 222 and the capture agent 220 thereby retaining the dual bead complex 194 within the target zone 170.
  • an interrogation beam 224 directed to the target zone 170 can be used to detect the dual bead complex 194 within the target zone 170.
  • the embodiment of the present invention illustrated in Fig. 18, may alternatively be implemented on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • Figs. 19A-19C are detailed partial cross sectional views showing the active layer 176 and the substrate 174 of the present bio-disc 110 as implemented in conjunction with the genetic assays discussed herein.
  • Figs. 19A-19C illustrates the capture agent 220 attached to the active layer 176 by applying a small volume of capture agent solution to the active layer 176 to form clusters of capture agents within the area of the target zone. The bond between capture agent 220 and the active layer 176 is sufficient so that the capture agent 220 remains attached to the active layer 176 within the target zone when the disc is rotated.
  • Figs. 19A and 19B also depict the capture bead 190 from the dual bead complex 194 binding to the capture agent 220 in the capture zone.
  • the capture agent 220 includes biotin and BSA-biotin.
  • the reporter bead 192 anchors the dual bead complex 194 in the target zone via biotin/streptavidin interactions.
  • the target zone may be coated with streptavidin and may bind biotinylated reporter beads.
  • Fig. 19C illustrates an alternative embodiment which includes an additional step to those discussed in connection with Figs. 19A and 19B.
  • a variance in the disc rotations per minute may create a centrifugal force great enough to break the capture beads 190 away from the dual bead complex 194 based on the differential size and/or mass of the bead.
  • the reporter bead 192 remains anchored to the target zone.
  • the reporter beads 192 are maintained within the target zone and detected using an optical bio-disc or medical CD reader.
  • connection Figs. 19A- 19C may be implemented on the reflective disc illustrated in Figs. 3A-3C, 5A, and 6A or on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • Figs. 20A, 20B, and 20C illustrate an alternative embodiment to the embodiment discussed in Figs. 19A-19C.
  • Figs. 20A-20C show detailed partial cross sectional views of a target zone implemented in conjunction with immunochemical assays.
  • Figs. 20A and 20B also depict the capture bead 190 from the dual bead complex 194 binding to the capture agent 220 in the capture zone.
  • the capture agent 220 includes biotin and BSA-biotin. These dual bead complexes may be prepared according to methods such as those discussed in Figs. 11 B and 12B.
  • the reporter bead 192 anchors the dual bead complex 194 in the target zone via biotin/streptavidin interactions.
  • Figs. 20A-20C may be implemented on the reflective disc depicted in Figs. 3A-3C, 5A, and 6A or on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • FIGs. 21A, 21 B, and 21 C there is shown detailed partial cross sectional views of a target zone including the active layer 176 and the substrate 174 of the present bio-disc 110 as implemented in conjunction with the genetic assays discussed herein.
  • Figs. 21A-21C illustrate the capture agent 220 attached to the active layer 176 by use of an amino group 226 that is an integral part of the capture agent 220. As indicated, the capture agent 220 is situated within the target zone. The bond between the amino group 226 and the capture agent 220, and the amino group 226 and the active layer 176 is sufficient so that the capture agent 220 remains attached to the active layer 176 within the target zone when the disc is rotated.
  • the preferred amino group 226 is NH 2 .
  • a thiol group may alternatively be employed in place of the amino group 226.
  • the capture agent 220 includes the specific sequences of amino acids that are complimentary to anchor agent 222 or oligonucleotide signal probe 206 which are attached to the reporter bead 192.
  • Fig. 21 B depicts the reporter bead 192 of the dual bead complex 194, prepared according to methods such as those discussed in Figs. 11A and 12A, binding to the capture agent 220 in the target zone.
  • the dual bead complex 194 flows towards the capture agent 220 and is in sufficient proximity thereto, hybridization occurs between the anchor agent 222, or oligonucleotide signal probe 206, and the capture agent 220.
  • the reporter bead 192 anchors the dual bead complex 192 within the target zone.
  • Fig. 21 C illustrates an alternative embodiment that includes an additional step to those discussed in connection with Figs. 21A-21 B.
  • a variance in the disc rotations per minute may create enough centrifugal force to break the capture beads 190 away from the dual bead complex 194 based on the differential size and/or mass of the bead.
  • the reporter bead 192 with the target DNA sequence 202 remains anchored to the target zone. In either case, the reporter beads 192 are maintained within the target zone as desired.
  • Figs. 21A-21C may be implemented on the reflective disc shown in Figs. 3A-3C, 5A, and 6A or on the transmissive disc illustrated in Figs. 4A-4C, 5B, and 6B.
  • Figs. 22A, 22B, and 22C illustrate an alternative embodiment to the embodiment discussed in Figs. 21A-21C.
  • Figs. 22A-22C show detailed partial cross sectional views of a target zone implemented in conjunction with immunochemical assays.
  • Figs. 22A and 22B also depict the reporter bead 192 from the dual bead complex 194, prepared according to methods such as those discussed in Figs. 11 B and 12B, binding to the capture agent 220 in the capture zone.
  • the capture agent 220 includes antibodies bound to the target zone by use of an amino group 226 that is made an integral part of the capture agent 220.
  • the capture agents 220 may be bound to the active layer 176 by passive absorption, and hydrophobic or ionic interactions.
  • the reporter bead 192 anchors the dual bead complex 194 in the target zone via specific antibody binding.
  • Fig. 22C shows an alternative embodiment that includes an additional step to those discussed in connection with Figs. 22A-22B.
  • a variance in the disc rotations per minute may create enough centrifugal force to break the capture beads 190 away from the dual bead complex 194 based on the differential size and/or mass of the bead.
  • the reporter bead 192 with the target antigen 204 remains anchored to the target zone. In either case, the reporter beads 192 are maintained within the target zone as desired.
  • the embodiment of the present invention described in conjunction with Figs. 22A-22C may be implemented on the reflective disc illustrated in Figs. 3A-3C, 5A, and 6A or on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • Figs. 23A and 23B are detailed partial cross sectional views showing the active layer 176 and the substrate 174 of the present bio-disc 110 as implemented in conjunction with the genetic assays.
  • Figs. 23A and 23B illustrate an alternative embodiment to that discussed in Figs. 19A and 19B above.
  • the anchor agent 222 is attached to the capture bead 190 instead of the reporter bead 192.
  • Fig. 23B illustrates the capture bead 190, from the dual bead complex 194, binding to the capture agent 220 in the capture zone.
  • the capture agent 220 includes biotin and BSA-biotin.
  • the capture bead 190 anchors the dual bead complex 194 in the target zone via biotin/streptavidin interactions.
  • Figs. 23A and 23B may be implemented on the reflective disc illustrated in Figs. 3A-3C, 5A, and 6A or on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B.
  • FIGs. 24A and 24B there is presented detailed partial cross sectional views showing the active layer 176 and the substrate 174 of the present bio-disc 110 as implemented in conjunction with the genetic assays.
  • Figs. 23A and 23B illustrate an alternative embodiment to that discussed in Figs. 21 A and 21 B above.
  • the anchor agent 222 is attached to the capture bead 190 instead of the reporter bead 192.
  • Fig. 23B illustrates the capture bead 190, from the dual bead complex 194, binding to the capture agent 220 in the capture zone.
  • the capture agent 220 is attached to the active layer 176 by use of an amino group 226 that is made an integral part of the capture agent 220. As indicated, the capture agent 220 is situated within the target zone. The bond between the amino group 226 and the capture agent 220, and the amino group 226 and the active layer 176 is sufficient so that the capture agent 220 remains attached to the active layer 176 within the target zone when the disc is rotated.
  • the capture agent 220 includes the specific sequences of amino acids that are complimentary to the anchor agent 222 or oligonucleotide transport probe 198 which are attached to the capture bead 190.
  • the capture bead 190 anchors the dual bead complex 194 in the target zone via hybridization between the capture agent 220 and the anchor agent or the transport probe 198.
  • Figs. 24A and 24B may be implemented on the reflective disc illustrated in Figs. 3A-3C, 5A, and 6A or on the transmissive disc depicted in Figs. 4A-4C, 5B, and 6B.
  • FIGs. 25A-25D there is shown the target zones 170 set out in Figs. 21A-21C and Figs. 24A-24B in the context of a disc, using as an input the solution created according to methods such as those shown in Figs. 11A and 12A.
  • Fig. 25A shows a mixing/loading chamber 164, accessible through an inlet port 152, and leading to a flow channel 160.
  • Flow channel 160 is pre-loaded with capture agents 220 situated in clusters.
  • Each of the clusters of capture agents 220 is situated within a respective target zone 170.
  • Each target zone 170 can have one type of capture agent or multiple types of capture agents, and separate target zones can have one and the same type of capture agent or multiple different capture agents in multiple capture fields.
  • the capture agent can include specific sequences of nucleic acids that are complimentary to anchor agents 222 on either the reporter 192 or capture bead 190.
  • a pipette 214 is loaded with a test sample of DNA or RNA that has been sequestered in the dual bead complex 194.
  • the dual bead complex is injected into the flow channel 160 through inlet port 152.
  • the dual bead complex 194 begins to move down flow channel 160 as the disc is rotated.
  • the loading chamber 164 can include a break-away retaining wall 228 so that complex 194 moves down the flow channel at one time.
  • anchor agents 222 attached to reporter beads 192, bind to the capture agents 220 by hybridization, as illustrated in Fig. 25C.
  • reporter beads 192 are retained within target zone 170. Binding can be further facilitated by rotating the disc so that the dual bead complex 194 can slowly move or tumble down the flow channel. Slow movement allows ample time for additional hybridization. After hybridization, the disc can be rotated further at the same speed or faster to clear target zone 170 of any unattached dual bead complex 194, as illustrated in Fig. 25D.
  • An interrogation beam 224 can then be directed through target zones 170 to determine the presence of reporters, capture beads, and dual bead complex, as illustrated in Fig. 25D.
  • a small amount of background signal may be detected in the target zones from non-specific binding.
  • the interrogation beam 224 is directed into the target zone 170, a zero or low reading results, thereby indicating that no target DNA or RNA was present in the sample.
  • the speed, direction, and stages of rotation such as one speed for one period followed by another speed for another period, can all be encoded in the operational information on the disc.
  • Figs. 25A-25D may also be performed on the transmissive disc illustrated in Figs. 4A-4C, 5B, and 6B using a system with the top detector 130.
  • Figs. 26A-26D show the target zones 170 including the capture chemistries discussed in Figs. 19A-19C and Figs. 23A-23B. This method uses as an input the solution created according to methods shown in Figs. 11A and 12A.
  • Figs. 26A-26D illustrate an alternative embodiment to that discussed in Figs. 25A-25D showing a different bead capture method described in further detail below.
  • Fig. 26A shows a mixing/loading chamber 164, accessible through an inlet port
  • Flow channel 160 is pre-loaded with capture agents 220 situated in clusters.
  • Each of the clusters of capture agents 220 is situated within a respective target zone 170.
  • Each target zone 170 can have one type of capture agent or multiple types of capture agents, and separate target zones can have one and the same type of capture agent or multiple different capture agents in multiple capture fields.
  • the capture agent can include specific biotin and BSA-biotin that has affinity to the anchor agents 222 on either the reporter 192 or capture bead 190.
  • the anchor agents may include streptavidin and Neutravidin.
  • a pipette 214 is loaded with a test sample of DNA or RNA that has been sequestered in the dual bead complex 194.
  • the dual bead complex is injected into the flow channel 160 through inlet port 152.
  • the dual bead complex 194 begins to move down flow channel 160 as the disc is rotated.
  • the loading chamber 164 can include a break-away retaining wall 228 so that complex 194 moves down the flow channel at one time.
  • anchor agents 222 attached to reporter beads 192, bind to the capture agents 220 by biotin-streptavidin interactions, as illustrated in Fig. 26C.
  • reporter beads 192 are retained within target zone 170. Binding can be further facilitated by rotating the disc so that the dual bead complex 194 can slowly move or tumble down the flow channel. Slow movement allows ample time for additional binding between the capture agent 220 and the anchor agent 222. After binding, the disc can be rotated further at the same speed or faster to clear target zone 170 of any unattached dual bead complex 194, as illustrated in Fig. 26D.
  • An interrogation beam 224 can then be directed through target zones 170 to determine the presence of reporters, capture beads, and dual bead complex, as illustrated in Fig. 26D.
  • no target DNA is present in the test sample, there will be no dual bead complex structures beads bound to the target zones 170.
  • a small amount of background signal may be detected in the target zones from non-specific binding.
  • the interrogation beam 224 is directed into the target zone 170, a zero or low reading results, thereby indicating that no target DNA or RNA was present in the sample.
  • the speed, direction, and stages of rotation, such as one speed for one period followed by another speed for another period can all be encoded in the operational information on the disc. The method discussed in conjunction with Figs.
  • FIG. 26A-26D was illustrated on a reflective disc such as the disc shown in Figs. 3A-3C, 5A, and 6A. This method may also be performed on the transmissive disc shown in Figs. 4A-4C, 5B, and 6B using a system with the top detector 130.
  • Figs. 27A-27D there is shown a series of cross sectional side views illustrating the steps of yet another alternative method according to the present invention.
  • Figs. 27A-27D show the target zones 170 including the capture mechanisms discussed in connection with Figs. 22A-22C. This method uses an input the solution created according to the preparation methods shown in Figs. 11 B and 12B.
  • Figs. 27A-27D illustrate an immunochemical assay and an alternative bead capture method.
  • Fig. 27A shows a mixing/loading chamber 164, accessible through an inlet port 152, and leading to a flow channel 160.
  • Flow channel 160 is pre-loaded with capture agents 220 situated in clusters.
  • Each of the clusters of capture agents 220 is situated within a respective target zone 170.
  • Each target zone 170 can have one type of capture agent or multiple types of capture agents, and separate target zones can have one and the same type of capture agent or multiple different capture agents in multiple capture fields.
  • the capture agent can include antibodies that specifically bind to epitopes on the anchor agents 222 on either the reporter 192 or capture bead 190.
  • the capture agent can directly bind to epitopes on the target antigen 204 within the dual bead complex 194.
  • the anchor agents 222 can include the target antigen, antibody transport probe 196, the antibody signal probe 208, or any antigen, bound to either the reporter bead 192 or the capture bead 190, that has epitopes than can specifically bind to the capture agent 220.
  • a pipette 214 is loaded with a test sample of target antigen that has been sequestered in the dual bead complex 194.
  • the dual bead complex is injected into the flow channel 160 through inlet port 152.
  • the dual bead complex 194 begins to move down flow channel 160 as the disc is rotated.
  • the loading chamber 164 may include a break-away retaining wall 228 so that complex 194 moves down the flow channel at one time.
  • anchor agents 222 attached to reporter beads 192, bind to the capture agents 220 by antibody-antigen interactions, as illustrated in Fig. 27C.
  • reporter beads 192 are retained within target zone 170. Binding can be further facilitated by rotating the disc so that the dual bead complex 194 can slowly move or tumble down the flow channel. Slow movement allows ample time for additional binding between the capture agents 220 and the anchor agent 222. After binding, the disc can be rotated further at the same speed or faster to clear target zone 170 of any unattached dual bead complex 194, as illustrated in Fig. 27D.
  • An interrogation beam 224 can then be directed through target zones 170 to determine the presence of reporters, capture beads, and dual bead complex, as illustrated in Fig. 27D.
  • no target antigen is present in the test sample, there will be no dual bead complex structures, reporters, or capture beads bound to the target zones 170, but a small amount of background signal may be detected in the target zones from non-specific binding.
  • the interrogation beam 224 is directed into the target zone 170, a zero or low reading results, thereby indicating that no target was present in the sample.
  • the speed, direction, and stages of rotation such as one speed for one period followed by another speed for another period, can all be encoded in the operational information on the disc.
  • Figs. 25A-25D, 26A-26D, and 27A-27D are implemented using the reflective disc system 144. As indicated above, it should be understood that these methods and any other bead or sphere detection may also be carried out using the transmissive disc embodiment 180, as described in Figs. 4A-4C, 5B, and 6B. It should also be understood that the methods described in Figs. 11A- 11 B, 12A-12B, 25A-25D, 26A-26D, and 27A-27D are not limited to creating the dual bead complexes outside of the optical bio-discs but may include embodiments that use "in-disc” or "on-disc” formation of the dual bead complexes.
  • the dual bead complex is formed within the fluidic circuits of the optical bio-disc 110.
  • the dual bead formation may be carried out in the loading or mixing chamber 164.
  • the beads and sample are added to the disc at the same time, or nearly the same time.
  • the beads with the probes can be pre-loaded on the disc for future use with a sample so that only a sample needs to be added.
  • the beads would typically have a long shelf life, with less shelf life for the probes.
  • the probes can be dried or lyophilized (freeze dried) to extend the period during which the probes can remain in the disc. With the probes dried, the sample essentially reconstitutes the probes and then mixes with the beads to produce dual bead complex structures can be performed.
  • the basic process for on-disc processing includes: (1) inserting the sample into a disc with beads with probes; (2) causing the sample and the beads to mix on the disc; (3) isolating, such as by applying a magnetic field, to hold the dual bead complex and move the non-held beads away, such as to a region referred to here as a waste chamber; and (4) directing the dual bead complexes (and any other material not moved to the waste chamber) to the capture fields.
  • the detection process can be the same as one of those described above, such as by event detection or fluorimetry.
  • the disc surface capturing techniques and the linking techniques for forming the dual bead complexes illustrated in Figs. 25A-25D, 26A-26D, and 27A-27D may be interchanged to create alternate variations thereof.
  • the capture agents 220 as implemented to include specific sequences of nucleic acids may be used to capture dual bead complexes formed by either DNA hybridization as illustrated in Fig. 10A or the antibody-antigen interactions shown in Fig. 10B.
  • capture agents 220 as implemented to include antibodies may be employed to capture dual bead complexes formed by either the DNA hybridization method shown in Fig. 10A or the antibody-antigen interactions illustrated in Fig.
  • capture agents 220 as implemented to include biotin or BSA-biotin may be similarly utilized to capture dual bead complexes formed by either the DNA hybridization techniques illustrated in Fig. 10A or the antibody-antigen interactions depicted in Fig. 10B.
  • Other combinations including different anchor agents to perform the binding function with the capture agent, are readily apparent from the present disclosure and are thus specifically provided for herein.
  • the number of reporter beads, target cells, or particles bound in the capture field or zone can be detected in a qualitative manner, and may also be quantified by the optical disc reader.
  • the test results of any of the test methods described above can be readily displayed on monitor 114 (Fig. 1).
  • the disc according to the present invention preferably includes encoded software that is read to control the controller, the processor, and the analyzer as shown in Fig. 2. This interactive software is implemented to facilitate the methods described herein and the display of results.
  • Fig. 28A is a graphical representation of an individual 2.1 um reporter bead 192 and a 3 um capture bead 190 positioned relative to tracks A, B, C, D, and E of an optical bio-disc or medical CD according to the present invention.
  • Fig. 28B is a series of signature traces, from tracks A, B, C, D, and E, derived from the beads of Fig. 28A utilizing a detected signal from the optical drive according to the present invention. These graphs represent the detected return beam 124 of the reflective disc illustrated in Figs. 5A and 6A for example, or the transmitted beam 128 of the transmissive disc illustrated in Figs, 5B and 6B. As shown, the signatures for a
  • 2.1 um reporter bead 190 are sufficiently different from those for a 3 um capture bead 192 such that the two different types of beads can be detected and discriminated.
  • This detection method is not limited to beads or bead complexes but may also be used to detect other objects such as cells or bead-cell complexes.
  • a sufficient deflection of the trace signal from the detected return beam as it passes through a bead is referred to as an event.
  • Fig. 29A is a graphical representation of a 2.1 um reporter bead and a 3 um capture bead linked together in a dual bead complex positioned relative to the tracks A, B, C, D, and E of an optical bio-disc or medical CD according to the present invention.
  • Fig. 29B is a series of signature traces, from tracks A, B, C, D, and E, derived from the beads of Fig. 29A utilizing a detected signal from the optical drive according to the present invention. These graphs represent the detected return beam 124 of a reflective disc 144 or transmitted beam 128 of a transmissive disc 180. As shown, the signatures for a 2.1 um reporter bead 190 are sufficiently different from those for a 3 um capture bead 192 such that the two different types of beads can be detected and discriminated. A sufficient deflection of the trace signal from the detected return beam or transmitted as it passes through a bead is referred to as an event.
  • reporter beads can be fluorescent or phosphorescent. Detection of these reporters can be carried out in fluorescent or phosphorescent type optical disc readers. Other signal detection methods are described, for example, in commonly assigned co-pending U.S. Patent Application Serial No. 10/008,156 entitled “Disc Drive System and Methods for Use with Bio-Discs” filed November 9, 2001 , which is expressly incorporated by reference; U.S. Provisional Application Serial Nos.
  • Fig. 30A is a bar graph of data generated using a fluorimeter showing concentration-dependent target detection using fluorescent reporter beads. This graph shows the molar concentration of target DNA versus the number of detected beads. The dynamic range of target detection shown in the graph is 10E-16 to 10E- 10 Molar (moles/liter). While the particular graph shown was generated using data from a fluorimeter, the results may also be generated using a fluorescent type optical disc drive.
  • Fig. 30B presents a standard curve demonstrating that the sensitivity of a fluorimeter is approximately 1000 beads in a fluorescent dual bead assay.
  • the sensitivity of any assay depends on the assay itself and on the sensitivity of the detection system.
  • Figs. 30A-30C various studies were done to examine the sensitivity of the dual bead assay using different detection methods, e.g., a fluorimeter, and bio-disc or medical CD detection according to the present invention.
  • a fluorimeter As stated above and shown in Fig. 30B, the sensitivity of a fluorimeter is approximately 1000 beads in a fluorescent dual bead assay.
  • Fig. 30A shows that even at 10E-16 Molar (moles/liter), a sufficient number of beads over zero concentration can be detected to sense the presence of the target.
  • a dual bead assay represents a very sensitive detection method for DNA that does not require DNA amplification (such as through PCR) and can be used to detect even a single bead.
  • a medical CD or bio- disc coupled with a CD-reader or optical bio-disc drive improves the sensitivity of detection.
  • detection with a fluorimeter is limited to approximately 1000 beads (Fig. 30B)
  • use of a bio-disc coupled with CD-reader may enable the user to detect a single bead with the interrogation beam as illustrated in Figs. 29A, 29B, and 30C.
  • the bioassay system provided herein improves the sensitivity of dual bead assays significantly.
  • the detection of single beads using an optical bio-disc or medical CD is discussed in detail in conjunction with Figs. 28A and 28B. Fig.
  • Dual bead complexes may also be identified by the bio-disc reader using the unique signature traces collected from the detection of a dual bead complex as shown in Figs. 29A and 29B.
  • Different optical bio-disc platforms including but not limited to the reflective and the transmissive disc formats illustrated respectively in Figs. 3C and 4C, may be used in conjunction with the reader device for detection of beads.
  • Fig. 30C is a pictorial representation demonstrating the formation of the dual bead complex linked together by the presence of the target in a genetic assay. Sensitivity to within one reporter molecule is possible with the present dual bead assay quantified with a bio-CD reader shown in Figs. 1 and 2 above. Similarly, the dual bead complex formation may also be implemented in an immunochemical assay format as illustrated above in Figs. 7B, 8B, 9B, 10B, 11 B, and 12B.
  • Fig. 31 shows data generated using a fluorimeter illustrating the concentration- dependent detection of two different targets.
  • Target detection was carried out using two different methods (the single and the duplex assays).
  • the capture bead contains a transport probe specific to a single target and a reporter probe coated with a signal probe specific to the same target is mixed in a solution together with the target.
  • the capture bead contains two different transport probes specific to two different targets. Experimental details regarding the use of the duplex target detection method are discussed in further detail in Example 2. Mixing different reporter beads (red and green fluorescent or silica and polystyrene beads, for example) containing signal probes specific to one of the two targets, allows the detection of two different targets simultaneously.
  • Detection of the dual bead duplex assay may be carried out using a magneto- optical disc system described below.
  • Figs. 32 and 37 illustrate the formation and binding of various dual bead complexes onto an optical disc which may be detected by an optical bio-disc drive (Fig. 2), a magneto-optical disc system, a fluorescent disc system, or any similar device.
  • Unique signature traces of a dual bead complex collected from an optical disc reader are shown in Fig. 29B above. The traces from Fig. 29B further illustrate that different bead types can be detected by an optical disc reader since different beads show different signature profiles.
  • a dual bead assay in the capture of targets allows for the use in multiplexing assays.
  • This type of multiplexing is achieved by combining different sizes of magnetic beads with different types and sizes of reporter beads.
  • different target agents can be detected simultaneously.
  • four sizes of magnetic capture beads, and four sizes of three types of reporter beads produce up to 48 different types of dual bead complex.
  • probes specific to different targets are thus conjugated to capture beads.
  • Reporter beads having different physical and/or optical properties, such as fluorescence at different wavelengths, allow for simultaneous detection of different target agents from the same biological sample.
  • small differences in size can be detected by detecting reflected or transmitted light.
  • Fig. 33A is a general representation of an optical disc according to another aspect of the present invention.
  • the disc 110 illustrated in Fig. 33A may be employed to practice the methods corresponding generally to the single-step methods of Figs. 11 A and 11 B illustrated above or the related single step methods described below in connection with Figs. 65A-65B and 66A-66B.
  • the sample and beads can be added at one time or successively but closely in time. Alternatively, the beads can be preloaded into a portion of the disc.
  • These materials can be provided to a mixing chamber 164 that can have a breakaway wall 228 (see Fig. 25A), which holds in the solution within the mixing chamber 164. Mixing the sample and beads on the disc would be accomplished through rotation at a rate insufficient to cause the wall to break or the capillary forces to be overcome.
  • the disc can be rotated in one direction, or it can be rotated alternately in opposite directions to agitate the material in a mixing chamber.
  • the mixing chamber is preferably sufficiently large so that circulation and mixing is possible.
  • the mixing can be continuous or intermittent.
  • Fig. 33B shows one embodiment of a rotationally-directionally-dependent valve arrangement that uses a movable component for a valve.
  • the mixing chamber leads to an intermediate chamber 244 that has a movable component, such as a ball 246.
  • the ball 246 In the non-rotated state, the ball 246 may be kept in a slight recessed portion, or chamber 244 may have a gradual V-shaped tapering in the circumferential direction to keep the ball centered when there is no rotation.
  • Figs. 33C and 33D in addition to Figs. 33A and 33B, when the disc is rotated clockwise (Fig. 33C), ball 246 moves to a first valve seat 248 to block passage to detection chamber 234 and to allow flow to waste chamber 232, shown in Fig. 33A.
  • Fig. 33D ball 246 moves to a second valve seat 250 to block a passage to waste chamber 232 and to allow flow to detection chamber 234.
  • Figs. 34A-34C show a variation of the prior embodiment in which the ball is replaced by a wedge 252 that moves one way or the other in response to acceleration of the disc.
  • the wedge 252 can have a circular outer shape that conforms to the shape of an intermediate chamber 244.
  • the wedge is preferably made of a heavy dense material relative to chamber 244 to avoid sticking.
  • a coating can be used to promote sliding of the wedge relative to the chamber.
  • a magnetic field from a magnetic field generator or field coil 230 can be applied over the mixing chamber 164 to hold the dual bead complexes and unbound magnetic beads in place while material without magnetic beads are allowed to flow away to a waste chamber 232.
  • This technique may also be employed to aid in mixing of the assay solution within the fluidic circuits or channels before any unwanted material is washed away.
  • only magnetic capture beads, unbound or as part of a dual bead complex remain. The magnetic field is released, and the dual bead complex with the magnetic beads is directed to a capture and detection chamber 234.
  • the process of directing non-magnetic beads to waste chamber 232 and then magnetic beads to capture chamber 234 can be accomplished through the microfluidic construction and/or fluidic components.
  • a flow control valve 236 or some other directing arrangement can be used to direct the sample and non-magnetic beads to waste chamber 232 and then to capture chamber 234.
  • a number of embodiments for rotationally dependent flow can be used. Further details relating to the use of flow control mechanisms are disclosed in commonly assigned co-pending U.S. Patent Application Serial No. 09/997,741 entitled "Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto" filed November 27, 2001 , which is herein incorporated by reference in its entirety.
  • Fig. 35 is a perspective view of a disc including one embodiment of a fluidic circuit employed in conjunction with magnetic beads and the magnetic field generator 230 according to the present invention.
  • Fig. 35 also shows the mixing chamber 164, the waste chamber 232, and the capture chamber 234.
  • the magnetic field generator 230 is positioned over disc 110 and has a radius such that as disc 1 10 rotates, magnetic field generator 230 remains over mixing chamber 164, and is radially spaced from chambers 232 and 234.
  • a magnetic field from the magnetic field generator 230 can be applied over the mixing chamber 164 to hold the dual bead complexes and/or unbound magnetic beads in place while additional material is allowed to enter the mixing chamber 164.
  • the method of rotating the disc while holding magnetic beads in place with the magnetic field generator 230 may also be employed to aid in mixing of the assay solution within the mixing chamber 164 before the solution contained therein is directed elsewhere.
  • Figs. 36A-36C are plan views illustrating a method of separation and detection for dual bead assays using the fluidic circuit shown in Fig. 35.
  • Fig. 36A shows an unrotated optical disc with a mixing chamber 164 shaped as an annular sector holding a sample with dual bead complexes 194 and various unbound reporter beads 192.
  • the electromagnet is activated and the disc is rotated counter-clockwise (Fig. 36B), or it can be agitated at a lower rpm, such as 1X or 3X.
  • Dual bead complexes 194, with magnetic capture beads remain in mixing chamber 164 while the liquid sample and the unbound reporter beads 192 move in response to angular acceleration to a rotationally trailing end of mixing chamber 164.
  • the disc is rotated in the counterclockwise direction illustrated in Fig. 36B with sufficient speed to overcome capillary forces to allow the unbound reporter beads in the sample to move through a waste fluidic circuit 238 to waste chamber 232.
  • the liquid will not move down the capture fluidic circuit 240 because of the physical configuration of the fluidic circuit as illustrated.
  • Fig. 36C the magnet is deactivated and the disc is rotated clockwise.
  • Dual bead complexes 194 move to the opposite trailing end of the mixing chamber 164 in response to angular acceleration and then through a capture fluidic circuit 240 to the capture chamber 234.
  • the dual bead solution will not move down the waste fluidic circuit 238 due to the physical layout of the fluidic circuit, as shown.
  • the embodiment shown in Figs. 36A-36C thus illustrates directionally-dependent flow as well as rotational speed dependent flow.
  • a plurality of regions can be formed and distributed about the disc, for example, in a regular manner to promote balance.
  • instructions for controlling the rotation can be provided on the disc. Accordingly, by reading the disc, the disc drive can have instructions to rotate for a particular period of time at a particular speed, stop for some period of time, and rotate in the opposite direction for another period of time.
  • the encoded information can include control instructions such as those relating to, for example, the power and wavelength of the light source. Controlling such system parameters is particularly relevant when fluorescence is used as a detection method.
  • a passage can have a material or configuration that can seal or dissolve either under influence from a laser in the disc drive, or with a catalyst pre-loaded in the disc, or such a catalyst provided in the test sample.
  • a gel may solidify in the presence of a material over time, in which case the time to close can be set sufficiently long to allow the unbound capture beads to flow to a waste chamber before the passage to the waste chamber closes.
  • the passage to the waste chamber can be open while the passage to the detection chamber is closed. After the unbound beads are directed to the waste chamber, the passage to the direction chamber is opened by energy introduced from the laser to allow flow to the detection chamber.
  • magneto-optic recording is an optical storage technique in which magnetic domains or areas are written into a thin film by heating it with a focused laser in the presence of an external magnetic field. The presence of these domains is then detected by the same laser from differences in the polarization of the reflected light between the different magnetic domains in the layer (Kerr rotation). By switching either the magnetic field for constant high laser power, or modulating the laser power with a constant magnetic field, a data pattern can be written into the layer.
  • Many magneto-optic storage systems have been introduced into the market, including both computer data storage systems and audio systems (most notably MiniDisc).
  • a disc such as one used with a magneto-optical drive, has magnetic domains or regions 242 that can be selectively written and erased with a magnetic head.
  • this type of disc will generally be referred to as a "magneto-optical bio-disc” or an "MO bio- disc”.
  • a magneto-optical disc drive can create magnetic domains 242 as small as 1 um by 1 um square. The close-up section of the magnetic domain 242 shows the direction of the magnetic field with respect to adjacent regions.
  • the ability to selectively write to small areas in a highly controllable manner to make them magnetic allows capture areas to be created in desired locations. These magnetic capture areas or domains can be formed in any desired configuration or location in one fluidic chamber or in multiple fluidic chambers. These areas capture and hold magnetic beads when applied over the disc. The domains can be selectively erased if desired, thereby allowing them to be made non-magnetic and allowing the beads to be released.
  • a set of three radially oriented magnetic capture regions 243 are shown, by way of example, with no beads attached to the magnetic capture regions in the columns illustrated therein.
  • Fig. 37 there is shown a set of four columns in Section A with individual magnetic beads magnetically attached to the magnetic areas in a magnetic capture region.
  • Another set of four columns arrayed in Section B is shown after binding of reporter beads to form dual bead complexes attached to specific magnetic domains or areas, with different columns having different types of reporter beads.
  • some of the reporter beads utilized vary in size to thereby achieve the multiplexing aspects of the present invention as implemented on a magneto-optical bio-disc or MO medical disc.
  • Section C a single column of various dual bead complexes is shown as another example of multiplexing assays employing various bead sizes individually attached at separate magnetic areas.
  • the write head in an MO drive is employed to create magnetic domains, and then a sample can be directed over that domain to capture magnetic beads provided in the sample. After introduction of the first sample set, other magnetic domains may also be created and another sample set can be provided to the newly created magnetic capture region for detection. Thus detection of multiple sample sets may be performed on a single disc at different time periods.
  • the magneto-optical drive also allows the demagnetization of the magnetic domains or capture regions to thereby release and isolate the magnetic beads if desired.
  • this system provides for the controllable capture, detection, isolation, and release of one or more specific target molecules from a variety of different biochemical, chemical, or biological samples.
  • a sample can be provided to a fluidic chamber on a disc.
  • a sample could be provided to multiple chambers that have sets of different beads.
  • a series of chambers can be created such that a sample can be moved by rotational motion from one chamber to the next, and separate tests can then be performed in each chamber.
  • a large number of tests can be performed at one time and can be performed interactively.
  • the system can be instructed to create a new set of magnetic regions for capturing the dual bead complex. Regions can be created one at a time or in large groups, and can be performed in successive chambers that have different pre- loaded beads.
  • Other processing advantages can be obtained with an MO bio-disc that has writeable magnetic regions.
  • the "capture agent" is essentially the magnetic field created by the magnetic region on the disc and therefore there is no need to add an additional biological or chemical capture agent.
  • the MO bio-disc illustrated in Fig. 37 may include any of the fluidic circuits, mixing chambers, flow channels, detection chambers, inlet ports, or vent ports employed in the reflective and transmissive discs discussed above. Illustrative examples of the use of the MO bio- disc according to this aspect of the present are provided below in Examples 5 and 6.
  • a method of performing a genetic dual bead assay in association with a magneto-optical bio-disc includes the steps providing a plurality of magnetic capture beads having covalently attached transport probes, providing a plurality of reporter beads having covalently attached specific sequences of DNA, preparing a sample containing target DNA molecules to be tested for DNA sequences complementary to the specific DNA sequences, and loading the capture beads into a magneto-optical bio-disc via an inlet port provided therein.
  • the magneto-optical bio-disc has a magnetic capture layer.
  • This method further includes loading the sample and the plurality of reporter beads into the bio- disc, rotating the bio-disc to facilitate hybridization of any target DNA present in the sample to the specific sequences of DNA on the reporter beads and to the transport probes to form dual bead complexes, interrogating a number of the magnetic capture beads with an incident beam of radiant energy to determine whether each of the number of magnetic capture beads has formed a dual bead complex, magnetizing specific regions of the magnetic capture layer to bind thereto a plurality of the dual bead complexes, and quantitating the plurality of the dual bead complexes.
  • the method may include the further steps of rotating the disc to direct any unbound beads into a waste chamber and then de-magnetizing the specific regions of the magnetic capture layer to thereby release a number of the plurality of the dual bead complexes. Thereafter the disc may be rotated to direct the released number of dual bead complexes to an analysis area for further processing so that the released number of dual bead complexes are sequestered in the analysis area.
  • the analysis area may be an analysis chamber having agents that react with the sequestered dual bead complexes.
  • a second embodiment of the magneto-optical aspects of the present invention there is provided another method of performing a dual bead assay in association with a magneto-optical bio-disc.
  • This other method includes the steps of providing a plurality of magnetic capture beads having attached transport probes, providing a plurality of reporter beads having attached signal probes, and loading the capture beads into a magneto-optical bio-disc via an inlet port provided therein.
  • the magneto-optical bio-disc has a magnetic capture layer.
  • This second method further includes loading a sample containing a target and the plurality of reporter beads into the bio-disc, rotating the bio-disc to facilitate binding of the target and the reporter beads to the magnetic capture beads to form dual bead complexes, interrogating a number of the magnetic capture beads with an incident beam of radiant energy to determine whether each of the number of magnetic capture beads has formed a dual bead complex, magnetizing specific regions of the magnetic capture layer to bind thereto a plurality of the dual bead complexes, and quantitating the plurality of the dual bead complexes.
  • This method may similarly include the further step of rotating the disc to direct any unbound beads into a waste chamber and then de-magnetizing the specific regions of the magnetic capture layer to thereby release a number of the plurality of the dual bead complexes. It is also an aspect of this method to then rotate the disc to direct the released number of dual bead complexes to an analysis area for further processing so that the released number of dual bead complexes are sequestered in the analysis area.
  • the analysis area may include a reaction chamber having agents that react with the sequestered dual bead complexes.
  • a method of performing a multiplexed dual bead assay in association with a magneto-optical bio-disc includes the steps of (1) providing at least two groups of differently sized magnetic capture beads, each group having magnetic capture beads of the same size and having a different specific type of transport probe associated with each group; (2) providing a plurality of reporter beads having attached at least two different types of signal probes; and (3) loading the capture beads into a magneto-optical bio-disc via an inlet port provided therein.
  • this magneto-optical bio-disc has a magnetic capture layer.
  • the method also includes (4) loading a sample containing at least one target and the plurality of reporter beads into the bio-disc; (5) rotating the bio-disc to facilitate binding of the target and the reporter beads to the magnetic capture beads to form dual bead complexes; (6) interrogating a number of the magnetic capture beads with an incident beam of radiant energy to determine whether each of the number of magnetic capture beads has formed a dual bead complex; and (7) determining the size of the magnetic bead in the dual bead complex.
  • This particular method concludes with the steps of (8) magnetizing specific regions of the magnetic capture layer to bind thereto a plurality of the dual bead complexes; and (9) quantitating the plurality of the dual bead complexes.
  • the step of quantitating may advantageously include quantitating the plurality of the dual bead complexes according to the size of the magnetic capture bead.
  • the method may include the further step of rotating the disc to direct any unbound beads into a waste chamber and then de-magnetizing the specific regions of the magnetic capture layer to thereby release a number of the plurality of the dual bead complexes containing same-sized magnetic capture beads.
  • the method may further include rotating the disc to direct the released number of same-sized dual bead complexes to an analysis area for further processing so that the released number of same-sized dual bead complexes are sequestered in the analysis area.
  • the analysis area may include a reaction chamber having agents that react with the sequestered same-sized dual bead complexes.
  • the signal probe is a specific sequence of DNA.
  • This additional dual bead multiplexing method includes the steps of (1) providing at least two groups of different types of reporter beads, each group having reporter beads of the same type and having a different specific type of signal probe associated with each group; (2) providing a plurality of magnetic capture beads having different types of transport probes attached thereto; and (3) loading the capture beads into a magneto-optical bio-disc via an inlet port provided therein.
  • this particular magneto-optical bio-disc has a magnetic capture layer.
  • the method continues with the additional steps of (4) loading a sample to be tested for at least one target and the plurality of reporter beads into the bio-disc; (5) rotating the bio-disc to facilitate binding of any target present in the sample to the reporter beads and to the magnetic capture beads to form dual bead complexes; and (6) interrogating a number of the reporter beads with an incident beam of radiant energy to determine whether each of the number of reporter beads has formed a dual bead complex.
  • This particular embodiment of the present method then concludes with (7) determining the type of the reporter bead in the dual bead complex; (8) magnetizing specific regions of the magnetic capture layer to bind thereto a plurality of the dual bead complexes; and (9) quantitating the plurality of the dual bead complexes.
  • the step of quantitating includes quantitating the plurality of the dual bead complexes according to the type of reporter bead.
  • the method may further include the further step of rotating the disc to direct any unbound beads into a waste chamber and then, if desired, de-magnetizing the specific regions of the magnetic capture layer to thereby release a number of the plurality of the dual bead complexes containing same-type reporter beads.
  • the further step of rotating the disc to direct the released number of same-type dual bead complexes to an analysis area for further processing so that the released number of same-type dual bead complexes are sequestered in the analysis area may also be performed.
  • the analysis area may include a reaction chamber having agents that react with the sequestered same-type dual bead complexes.
  • the present invention further contemplates an optical bio-disc used to perform any of the above methods, and an optical bio-disc used to analyze any the dual bead complexes prepared according to the methods discussed above in connection with Figs. 11 A, 11 B, 12A, 12B or those methods discussed in detail below in conjunction with Figs. 65A and 65B, 66A and 66B, 67A and 67B as well as 68A and 68B.
  • the MO bio-disc may be implemented in other biomagnetic assays including immunomagnetic and molecumagnetic assays such as cellular capture and analysis methods using the MO biodisc as discussed below.
  • Fig. 38 there is shown the dual bead complex 194 held together by the target DNA 202 through the covalently bound transport probes 198 and signal probes 206 on the capture bead 190 and the reporter bead 192, respectively.
  • the 5' end of the signal probe 206 is held right next to the 3' end of the transport probe 198.
  • This configuration allows the ligation of the 3' and 5' ends of the probes upon addition of ligase. Ligation of both probes only occurs in the presence of the target and it enhances the sensitivity of the assay by increasing the bond strength between the reporter and capture beads preventing the dissociation of the dual bead complex.
  • Fig. 39 there is a bar graph illustrating the results from a genetic test detected by an enzyme assay.
  • a 3 ⁇ m capture bead bound to transport probes was used to capture the target in this test.
  • a biotinylated reporter probe was introduced and allowed to bind to the target.
  • the capture beads were then washed to remove unbound reporter probes.
  • Ligase is then added to the solution to ligate the ends of the reporter and transport probes, as shown in Fig. 38.
  • streptavidinated-alkaline phosphatase is added to the bead solution and allowed to bind with the biotin on the reporter probe.
  • the beads are again washed and a chromagen alkaline phosphatase substrate is added to the bead solution.
  • the intensity of the color formed by the alkaline phosphatase and substrate reaction is then quantified using a spectrophotometer. ' The results from this quantification are shown in Fig. 39.
  • the data presented in this figure indicates that there is approximately a 50% increase in signal when the probes are ligated. Thus the assay sensitivity is significantly increased by the ligation step in this experiment.
  • Examples 3 and 4 discuss in detail the procedures followed in carrying out a similar experiment.
  • Fig. 40 shows a bar graph from a genetic test using a ligation step implemented in a dual bead assay instead of an enzyme assay.
  • the enzyme assay as discussed in Fig. 39 is used to verify the activity of ligase in a non-dual bead format, which serves as a control in the dual bead experiment.
  • the same 3um capture beads bound to transport probes were used in the dual bead assay.
  • the reporter beads used in the dual bead assay were 2.1 um fluorescent beads.
  • the dual beads were formed as discussed in either Fig. 11A or 12A.
  • the ligation step is implemented in Step V in Fig. 11A or Step VI in Fig.
  • ligase is added to the dual bead complex solution and allowed to ligate the transport probes to the signal probes.
  • the data shown in Fig. 40 indicates that ligation significantly increases the signal and sensitivity of the assay relative to the non-ligated control treatment in Set 1 but not in Set 2.
  • Fig. 41 is a bar graph showing the number of reporter beads bound in a dual bead complex using a 39mer bridge employing the same ligation step as discussed in Fig. 40.
  • the data in Fig. 41 indicates that ligation significantly increases the sensitivity of the dual bead assay in both Sets 1 and 2.
  • This data demonstrates that the use of a 39mer bridge aids in the ligation process thus enhancing the signal from both Sets as implemented in the dual bead assay.
  • cleavable spacers in dual bead assay increases the specificity of the assay. Indeed, in addition to complementary sequences to the target DNA, the capture probes and reporter probes contain sequences that are complementary to each other. This additional requirement enhances specificity to target capture. Furthermore, additional bonding between the capture bead and reporter beads via the hydrogen bonds between capture and reporter probes strengthen the interactions between the dual beads. In this embodiment of the present invention, in the absence of a target, the capture probe hybridizes to the reporter probes, resulting in the formation of the dual bead complexes as shown in 42B and 43A. As illustrated in Figs. 42B and 42C, the dual bead complexes are subjected to selective restriction enzyme digestion after target capture.
  • the sequence specific digestion will selectively cleave the hydrogen bonds between the capture probes and reporter probes as depicted in Fig. 42D.
  • the dual beads dissociate from each other.
  • the capture and reporter beads remain bound via the target-mediated hydrogen bonds (Fig. 42D).
  • the amount of target captured therefore is correlated with the number of dual beads remaining after enzyme digestion.
  • the bond holding the capture probes and reporter probes can be unraveled by the use of a displaceable linker.
  • the linker is detached using a displacement probe.
  • the reporter probe contains a sequence that is partially complementary to the capture probe resulting in a mismatched overhang as depicted in Fig. 43A.
  • the complex is subjected to heat treatment that will initiate the melting of the reporter probe from the capture probe, followed by addition of a large excess of displacement probe.
  • the higher concentration of displacement probe and the tighter interactions between the displacement probe and the mismatched overhang which will result in the unraveling of the reporter probe from the capture probe as illustrated in Figs 43B and 43C. This will result in the dissociation of reporter beads from capture beads in the absence of target DNA.
  • the dual bead assay according to the present invention may be implemented using 3 ⁇ m magnetic capture beads and 2.1 ⁇ m fluorescent reporter beads. These beads are coated with transport probes and signal probes respectively.
  • the transport probes and signal probes in addition to being complementary to a target sequence, pUC19 for example, contain sequences that are complementary to each other, as illustrated in Figs. 42A, 42B, 42C, and 43D.
  • the sequences that bind the transport probe and the signal probes together are designed such that they are susceptible to the cleavage of very rare restriction enzymes including Not 1.
  • the use or rare restriction enzymes and restriction sites prevents the accidental cleavage of the target DNA.
  • the capture beads and reporter beads are mixed with varying quantities of target DNA.
  • the DNA complex is subjected to restriction digestion by a rare restriction enzyme including Not 1.
  • the restriction digestion by this enzyme will cleave the DNA sequence connecting the reporter beads to the capture beads.
  • the reporter beads will be dissociated from the capture beads and removed by magnetic concentration of the magnetic beads.
  • the magnetic capture beads bind to fluorescent reporter beads, resulting in a dual bead assay.
  • the introduction of cleavable spacers into the capture and reporter probes improves the specificity and the sensitivity of the dual bead significantly.
  • a shorter overlap and a mismatched overhang between the complementary sequences of probes on the reporter bead and the capture bead (probe 1 and probe 2B), resulting in the formation of a displaceable linker is used in conjunction with a displacement probe as illustrated in Figs. 43A and 43B.
  • the mismatched overhang on probe 2B is the site for initial binding of the displacement probe as shown in Fig. 42B. Once the displacement probe binds to the overhang, the displacement probe proceeds to displace the overlaping sequences between probe 1 and probe 2B which is depicted in Fig. 43C.
  • the reporter beads In the absence of target DNA, the reporter beads will be dissociated from the capture beads by the actions of the displacement probe and consequently removed by magnetic concentration of the magnetic beads. Thus, only in the presence of the target sequence, the magnetic capture beads bind to fluorescent reporter beads, resulting in the non-dissociation of the dual bead complex.
  • a capture bead is provided with a derivatized surface to which is attached a plurality of cleavable spacer molecules 256.
  • Each spacer 256 including a cleavage site 258, a signal probe 206, and a transport probe 198.
  • the transport probes include a thiol group which reacts to form a covalent bond with metallic elements as discussed in conjunction with Fig. 45.
  • the capture bead which may be porous or solid, can be selected from a variety of materials such as plastics, glass, mica, silicon, and the like.
  • the surface of the capture bead 190 or reporter bead 192 can be conveniently derivatized to provide covalent bonding to each of the probes including the cleavable spacer molecule 256.
  • metallic reporter beads that provide a convenient reflective signal-generating means for detecting the presence of a target.
  • Typical materials used in creating metallic beads are gold, silver, nickel, chromium, platinum, copper, and the like, with gold being presently preferred for its ability readily and tightly to bind e.g. via dative binding to a free SH group at the signal responsive end of the cleavable spacer.
  • the metal beads may be solid metal or may be formed of plastic, or glass beads or the like, on which a coating of metal has been deposited. Also, other reflective materials can be used instead of metal.
  • the presently preferred gold spheres bind directly to the thio group of the signal probe 206.
  • the transport probe 198 is attached covalently at the amino end via an amide linkage.
  • the cleavable spacer molecule includes the cleavage site 258 that is susceptible to cleavage during the assay procedure, by chemical or enzymatic means, heat, light or the like, depending on the nature of the cleavage site.
  • Chemical means are presently preferred with a siloxane cleavage group, and a solution of sodium fluoride or ammonium fluoride, exemplary, respectively, of a chemical cleavage site and chemical cleaving agent.
  • Other groups susceptible to cleaving such as ester groups or dithio groups, can also be used.
  • Dithio groups are especially advantageous if gold spheres are added after cleaving the spacer.
  • the cleavage site may be a restriction site for cleavage using restriction enzymes. Restriction cleavage is the preferred method when performing genetic or immunochemical assays.
  • Spacers may contain two or more cleavage sites to optimize the complete cleavage of all spacers.
  • the transport and signal probes are adapted to bind complementary strands of nucleic acids that may be present in a test sample.
  • the complementary oligonucleotides comprise members of a specific binding pair, i.e., one oligonucleotide will bind to a second complementary oligonucleotide.
  • cleavable spacer molecules 256 including the transport probes 198 and signal probes 206 located at different sites on the surface of the capture bead 190 and reporter bead 192.
  • oligonucleotide target agents 202 are located in close proximity to the transport probes 198 and signal probes 206. In the event these target agents are complimentary to both probes, hybridization occurs between the target agent 202, transport probe 198, and the reporter probes 206 to form a double helix as is shown in Fig. 46B. If there is no complementarity between the target agent 202 and the probes, there is no binding between those groups as is further illustrated in Fig. 46B where no double helix if formed.
  • the reporter beads 192 When the cleavage site 258 is cleaved, but for the binding by the double helix- coupled oligonucleotides, the reporter beads 192 will be free of the capture bead 190 and dissociated therefrom. This is illustrated more fully in Fig. 46C.
  • the presence or absence of dual bead complexes 194 may then be detected by an incident light, particularly an incident laser light.
  • the bridging agent 260 may include a relatively short oligonucleotide sequence for binding to a portion of a target such that when the target binds to the transport 198 and signal probes 206, the bridging agent 260 acts as a bridge between the ends on the transport probe 198 and the signal probe 206. This results in the formation of a double helix with two breaks as depicted in Fig. 47B.
  • Fig. 47C there is shown a schematic representation of the use of DNA ligase in conjunction with the cleavable spacer in a further embodiment of the nucleic acid detection embodiment of the present invention.
  • the ligation procedure links the breaks in the double helix covalently. This covalent linkage increases the strength with which analyte-specific binding adheres the dual bead complex thus permitting, in this embodiment, increased stringency of wash affording increased specificity of the assay.
  • cleavable reflective signal elements of the present invention are particularly well suited for detecting amplified nucleic acids of defined size, particularly nucleic acids amplified using the various forms of polymerase chain reaction (PCR), ligase chain reaction
  • the cleavable spacer 258 includes modified antibodies to permit an immunoassay.
  • the modified antibodies may be attached non-covalently to the cleavable spacer 258 mediated by oligonucleotides that are covalently attached to the antibodies.
  • Use of complementary nucleic acid molecules to effectuate non-covalent, combinatorial assembly of supramolecular structures is described in further detail in co-owned and co-pending U.S. Patent Applications Serial No. 08/332,514, filed October 31 , 1994; 08/424,874, filed April 19, 1995; and 08/627,695, filed March 29, 1996, incorporated herein by reference.
  • antibodies can be attached covalently to the cleavable spacer using conventional cross-linking agents, either directly or through linkers.
  • the antibody probes include an antibody transport probe 196 bound to the capture bead 190 and an antibody signal probe 208 bound to the reporter bead 192. Both beads and probes are held together by the cleavable spacer 258.
  • the antibody transport probe 196 and the antibody signal probe 208 have affinity to different epitopic sites of an antigen of interest.
  • target antigen 204 binds to the antibody transport probe 196 and the antibody signal probe 208 as shown in Fig. 48B.
  • This binding prevents decoupling of the dual bead complex 194 when the cleavage site 258 is cleaved, such as, for example, by contact with a chemical cleaving agent.
  • the second cleavable signal element which was not bound by the non-specific target agent 200 because the lack of binding affinity of the antibodies to the target agent 200, allow the dual bead complexes to dissociate as illustrated in Fig. 48C.
  • Presence and absence of the dual bead complex 194 may then be detected as reflectance or absence of reflectance of incident light, particularly incident laser light.
  • coupling of antibodies as depicted permits the adaptation of standard immunoassay chemistries and immunoassay geometries for use with the cleavable spacers in the dual bead assay of the present invention.
  • Some of these classical immunoassay geometries are further described in U.S. Pat. No. 5,168,057, issued Dec. 1 , 1992, incorporated herein by reference.
  • Other immunoassay geometries and techniques that may usefully be adapted to the present invention are disclosed in Diamandis et al.
  • the present invention will prove particularly valuable in immunoassays screening for human immunodeficiency viruses, hepatitis a virus, hepatitis B virus, hepatitis C virus, and human herpes viruses.
  • antibodies are exemplary of the broader concept of specific binding pairs, wherein the antibody may be considered the first member of the specific binding pair, and the antigen to which it binds the second member of the specific binding pair.
  • a specific binding pair may be defined as two molecules, the mutual affinity of which is of sufficient avidity and specificity to permit the practice of the present invention.
  • the cleavable spacer of the present invention may include other specific binding pair members as side members.
  • the first side member of the cleavable signal element includes a first member of a first specific binding pair
  • the second side member of the cleavable spacer includes a first member of a second specific binding pair
  • said second member of said first specific binding pair and said second member of said second specific binding pair are connectably attached to one another, permitting the formation of a tethering loop of the general formula: first member of first specific binding pair- second member of first specific binding pair-second member of second specific binding pair-first member of second specific binding pair.
  • biologic receptors and their natural agonist and antagonist ligands, proteins and cofactors, biotin and either avidin or streptavidin, alpha spectrin and beta spectrin monomers, and antibody Fc portions and Fc receptors.
  • probes are attached covalently to the beads. Efficiency of the covalent conjugation depends on the type of bead utilized and the specific conjugation method employed. As illustrated in Fig. 49, a systematic method to evaluate the use of a solid phase for probe conjugation is presented. The methodology identifies covalent linkages that improve specificity of a dual bead assay.
  • probes are tagged with an appropriate molecule for detection and measurement of the amount of probe bound at a later time.
  • a biotin moiety B
  • the probe is conjugated in the presence or absence of a cross- linking agent, e.g., EDC (1 -Ethyl 3-3 dimethylaminopropyl carbodiimide-HCI).
  • a probe In the presence of a cross-linking agent, a probe will be conjugated both covalently and non- covalently. Alternatively, in the absence of the cross-linking agent, a probe will only be absorbed to the bead non-covalently.
  • a detection agent is added that binds specifically to the biotin molecule previously tagged to the probe. For example, streptavidin-alkaline phosphatase (S- AP) is added to the probe-bound beads, and the S-AP binds specifically to the biotinylated probes. Next, alkaline phosphatase substrate is added to the sample.
  • S- AP streptavidin-alkaline phosphatase
  • This substrate develops color upon loss of a phosphate group, and the intensity of the color correlates with the amount of probes bound to the beads.
  • the solution is isolated and the optical density of the solution at an appropriate wavelength is determined with a spectrophotometer or microtiter plate reader.
  • Fig. 50 there is illustrated conjugation of an oligonucleotide probe onto a carboxylated bead. Conjugation of probes may be carried out covalently or non-covalently. In a dual bead assay, covalent probe conjugation is preferred over non-covalent conjugation as discussed in further detail in connection with Figs. 51 A, 51 B, 53A, and 53B. This conjugation process is performed prior to Step I of the dual bead assay as presented in Figs. 11 A, 11 B, 12A, and 12B.
  • the amount of probe covalently bound to the solid surface may be evaluated by determining the amount of probe that binds to the solid phase covalently and non-covalently, i.e., non-specifically, in the presence and absence of a crosslinking agent (e.g., EDC).
  • the percentage of non-covalently bound probe can be determined according to the formula 100% * N/T, and the percentage of covalently bound probe can be determined by the formula 100% * (T-N)/T, wherein "T” represents the total amount of signal obtained in the presence of a cross-linking agent (i.e., the total amount of covalently and noncovalently bound probe) and "N" represents the total amount of signal obtained when no crosslinking agent is used.
  • the amount of probes conjugated covalently can be obtained directly if all non-covalently bound probes are removed prior to the addition of the S-AP. This can be conveniently achieved by heating the beads to 70°C prior to the step of adding the S-AP. If the percentage of non- covalently bound probe is less than 20%, the beads being tested can be used as solid phase for covalent conjugation. Results of an application of this methodology are presented in Figs. 51 A, 51 B, and 55 (see Example 7 for details). As depicted in Figs. 51 A and 51 B, the 1.8 ⁇ m, 2.1 ⁇ m, and 3 ⁇ m beads provide suitable solid phase for covalent probe conjugation with at least 75% conjugation efficiency. The 1 -2 ⁇ m beads, however, may not be suitable for covalent conjugation of probes due to their low covalent conjugation efficiency of less than 21%.
  • Fig. 52A shows the structural differences between single stranded and double stranded DNA in order to illustrate how the single stranded DNA can more readily bind non-covalently to a solid phase.
  • Single-stranded DNA has hydrophobic base side chains that can readily absorb to a solid phase non-covalently.
  • hydrophobic base interaction with a solid phase does not generally occur and non-covalent or non-specific binding is limited in comparison to a single-stranded DNA molecule.
  • double stranded DNA can be utilized in place of single-stranded DNA, thereby enhancing DNA binding to a solid phase by covalent linkage (Fig. 52B).
  • the non-covalently bound strand may be removed by heating the sample to 70°C in the appropriate buffer. Under these conditions, the double stranded DNA are separated, and only single strand DNA probe that is covalently attached to the bead ramain and is used to capture the target.
  • Experimental details regarding the use of double stranded DNA for covalent probe conjugation is described in further detail below in Example 8.
  • heat treatment can be used to selectively remove non-covalently bound probe(s) from a solid phase.
  • This method is useful when, for example, despite all optimizations with respect to the type of the solid phase, treatment of the solid phase, and the use of double stranded DNA, non- covalent binding to the solid phase is still problematic.
  • the conditions for the heat treatment have been optimized; the optimal buffer consists of: 2%BSA, 50 mM Tris- HCI, 145 mM NaCl, 1 mM MgCI2, 0.1 mM ZnCI2.
  • the treatment is done at a temperature less than or equal to approximately 70°C, since at higher temperatures, the magnetic beads can lose their magnetic properties.
  • the methodology presented herein to determine optimal conditions to obtain covalent linkages that improve specificity of a dual bead assay can be applied to a disc surface that is used as a solid phase.
  • the invention provides in other embodiments analogous to those described herein above to evaluate solid surfaces for protein binding. For example, such an application would be useful where the probe utilized is an antigen or antibody.
  • Fig. 53A there is shown a bar graph of results collected from an enzyme assay detecting targets bound to probes on two different capture beads for use in a dual bead assay.
  • the 1-2 ⁇ m beads have a covalent binding efficiency up to 20% and the rest of the probes bind non-covalently and the covalent binding efficiency of the 3 ⁇ m beads is between 75-85%.
  • the data shown in Fig. 53A indicates that both of the tested beads bind a similar amount of target regardless of whether the probe is bound covalently or non-covalently. This suggests that covalent binding is not necessary in an enzyme assay format.
  • Fig. 53B represents results of a dual bead assay designed to examine the number of reporter beads captured by the same capture beads used in Fig. 53A.
  • the results shown in Fig. 53B indicate that covalent binding of the probe to the capture bead is necessary to enhance the sensitivity of the assay.
  • the 3 ⁇ m capture bead contains more covalently bound probes than the 1-2 ⁇ m beads, as mentioned above. This allows the retention of the reporter bead in the dual bead complex since covalently tethered probes on the capture bead have higher bond strength than non-covalently bound probes.
  • Probe linkers may be used to extend the length of the probes to increase probe target accessibility as discussed with reference to Fig. 52A.
  • Linkers may increase the assay sensitivity by approximately 50% or more.
  • the use of linkers also decreases non-specific reporter bead binding to the capture beads.
  • probes are attached to a solid phase by way of a linker molecule.
  • the use of a linker molecule makes the probe longer and more rigid. These two properties increase the accessibility of the probe(s), and, therefore, maximize the efficiency of target capture and the sensitivity of the dual bead assay.
  • various linker molecules can be used that satisfy the criteria described herein.
  • bovine serum albumin BSA
  • PEG polyethylene glycol
  • the linker can be a series of 3 to 10 PEG molecules that are attached covalently to the 5' end of a DNA probe. Details relating to the use of PEG as a linker molecule are described below in Example 9. With reference now to Fig. 55, there is shown a bar graph demonstrating determination of percent covalent probe density on 3 ⁇ m Spherotech beads. These graphs represent signals generated from an enzyme assay using biotinylated probes and streptavidin-linked alkaline phosphatase enzyme reactions. As discussed with reference to Fig.
  • the covalent conjugation efficiency can be calculated by determining the total amount of probes bound to non heat-treated beads. A separate aliquot of the beads is then heated to remove the non-covalently bound probes and the amount of covalent probes is then determined using the enzyme assay as described in Example 7 below. With these data, the percentage of covalent probe binding can then be determined using the following formula: H/T*100 where H represents signal from heat treated beads and T is the total signal from the non-heat treated beads.
  • Fig. 56 is a bar graph presentation demonstrating the pretreatment of the beads with various blocking agents including detergents. Decreasing non-specific bead binding is critical in the dual bead assay since the assay sensitivity is inversely related to the baseline signal which is the non-specific binding of the reporter beads to the capture beads. Thus the lower the baseline, the more sensitive the assay becomes.
  • Salmon sperm DNA blocking reduced non-specific binding by approximately 10 fold. Salmon sperm DNA is, therefore, a preferred method for blocking non-specific bead binding in one aspect of the present invention.
  • Other blocking agents may also be used including BSA, Denhardt's solution, and sucrose.
  • beads should be blocked by an appropriate blocking agent after conjugation and heat treatment as shown in Fig. 50 or prior to Step I in Figs. 11A, 11 B, 12A, and 12B above to increase the dual bead assay sensitivity.
  • Fig. 56 is a bar graph presentation demonstrating the pretreatment of the beads with various blocking agents including detergents to decrease non-specific binding of the beads. Decreasing non-specific bead binding is critical in the dual bead assay since the assay sensitivity is inversely related to the baseline signal which is the non-specific binding of the reporter beads to the capture beads. Thus the lower the baseline the more sensitive the assay becomes. As illustrated, the use of salmon sperm DNA worked best in reducing the nonspecific binding relative to the other blocking agents tested in this experiment. Salmon sperm DNA blocking reduced non-specific binding by approximately 10 fold.
  • Salmon sperm DNA is, therefore, a preferred method for blocking non-specific bead binding in one aspect of the present invention.
  • Other blocking agents may also be used including BSA, Denhardt's solution, and sucrose.
  • beads should be blocked by an appropriate blocking agent prior to Step I in Figs. 11 A, 11 B, 12A, and 12B, above, to increase the dual bead assay sensitivity.
  • Fig. 57 is a bar graph of data generated using a fluorimeter showing a concentration dependent target detection using fluorescent reporter beads in a dual bead assay.
  • This graph shows the picomolar concentration of target DNA versus number of beads bound in a dual bead complex.
  • the dynamic range of target detection shown in this graph is 0.25 pM to 2500 pM (picomoles/liter). While this particular graph was generated using data from a fluorimeter, the results may also be generated using a fluorescent type optical disc drive. Experimental details from an experiment related to detection of a range of target concentrations is discussed in detail in Example 10. Referring now to Figs.
  • 58, 59, 60, 61 A, and 61 B here are shown data from experiments performed to determine the optimal concentration or ionic strength of various salts in the hybridization buffer or assay buffer.
  • the salt concentration in the assay buffer needs to be optimized in order to increase hybridization efficiency or binding efficiency and decrease non-specific bead binding between capture and reporter beads resulting in lower signal to noise ratio which increases the sensitivity of the assay.
  • the data presented in these figures show that 40mM EDTA, 300mM NaCl, 30mM MgCI 2 are the optimal salt concentrations for use in one embodiment of the dual bead assay.
  • Fig. 58 there is a bar graph presentation showing data collected from an experiment using various concentrations of NaCl in the bead buffer and the related non-specific binding as a result of changes in the ionic strength of the buffer. Based on the results presented in Fig. 58, the optimal bead buffer concentration of sodium chloride for use in the dual bead assay is 0.2M since nonspecific bead binding is minimal at this NaCl concentration.
  • Fig. 59 there is shown a bar graph illustrating the effect of increasing EDTA concentration on the dual bead assay sensitivity using different target concentrations. Fig. 59 also shows the related non-specific binding as affected by the concentration of EDTA in the assay buffer. The optimal EDTA buffer concentration, based on the data presented, for use in the hybridization buffer is 40mM since signal generated from the dual bead assay was highest at this concentration.
  • Fig. 60 presents a bar graph presentation showing the effect of increasing NaCl concentration on the dual bead assay sensitivity using different target concentrations.
  • the non-specific bead binding data related to optimization of the buffer concentration of NaCl is represented in Fig. 58.
  • the optimal NaCl concentration for hybridization as implemented in a dual bead assay is 0.3M NaCl. A detailed description of the experimental procedure used to generate this data is discussed below in Example 11.
  • Figs. 61 A and 61 B are bar graph presentations showing the effect of increasing the concentration of MgCI 2 in the assay buffer on the dual bead assay sensitivity and an enzyme assay sensitivity, respectively.
  • Data from these figures indicate that a concentration of 30mM MgCI 2 in the hybridization buffer is optimal for increasing the signal generated and the assay sensitivity.
  • the enzyme assay appears to be more sensitive than the dual bead assay in the 30mM MgCI 2 treatment. This conclusion is based on the difference in signal within the treatment group from the various target concentrations.
  • the slope of the concentration curve in the 30mM MgCI 2 group of the enzyme assay of Fig. 61 B is steeper than the corresponding curve in Fig. 61 A.
  • Example 12 describes in detail the procedure for carrying out an experiment relating to Fig. 61 A.
  • Fig. 62 there is shown a pictorial representation of the use of probe blocking agents to increase the sensitivity of the bead assay.
  • the probe blocking agent used in this particular example is a biotinylated DNA that is complimentary to the probe on the bead.
  • the amount of probe blocking agent used to block excess probes on the bead is such that a pre-determined fraction of probes are blocked by the blocking agent.
  • the use of the probe blocking agent in dual bead assay increases the sensitivity of the assay in that it enhances the probability of target binding to a single capture and reporter bead in a dual bead assay. This may increase the sensitivity of the dual bead assay up to one target per dual bead complex.
  • biotinylated probe blocking agent allows for the quantitation of the blocking efficiency of the probe blocking agent for optimization of the assay.
  • the amount of biotinylated probes bound to the beads may be quantitated by an enzyme assay using streptavidinated or neutravidinated enzymes including streptavidin-alkaline phosphatase (S-AP) and their appropriate substrates.
  • streptavidinated or neutravidinated enzymes including streptavidin-alkaline phosphatase (S-AP) and their appropriate substrates.
  • S-AP streptavidin-alkaline phosphatase
  • the choice of enzyme and substrate for use in this test is dictated by the type of detection desired.
  • a colorimetric test is performed wherein the enzyme-substrate reaction produces color that is quantified by a spectrophotometer.
  • streptavidinated or neutravidinated fluorescent tags may also be used which may be quantified using a fluorimeter or a Fluorimager. Both the colorimetric and fluorescent quantitation may also be carried out using the appropriate optical disc reader as shown in Figs. 1 and 2.
  • Fig. 63 shows a bar graph presentation of data illustrating the effect incubation time on the signal generated and the assay sensitivity using different target concentrations during a hybridization reaction in a dual bead assay.
  • the data shows that 2 hours is the minimum incubation time required to generate the maximum signal and sensitivity for the dual bead assay and that a 4 hour or overnight hybridization is not necessary.
  • Fig. 64 shows a bar graph of data collected illustrating the effect of incubation time and mixing on the hybridization efficiency and the assay sensitivity using different target concentrations in a hybridization reaction as implemented on a dual bead assay.
  • Fig. 64 also shows that 2 hours is an optimal time for hybridization and extending the hybridization time does not increase the signal generated.
  • mixing significantly increased the hybridization efficiency after 2 hours of hybridization relative to control.
  • Example 14 presented below, explains the details regarding the experiment performed to generate the data shown in Figs. 63 and 64.
  • Figs. 65A and 65B there is illustrated a method of preparing a molecular assay using a "single-step hybridization" technique to create dual bead complex structures in a solution according to one aspect of the present invention.
  • This method is similar to the method discussed above in connection with Fig. 11 A.
  • the present method includes eight principal steps identified consecutively as Steps I, II, III, IV, V, VI, VII, and VIII.
  • Step I of this method a number of capture beads 190 coated with oligonucleotide transport probes 198 are deposited into a test tube 212 containing a buffer solution 210.
  • the number of capture beads 190 used in this method may be, for example, on the order of 10E+07 and each on the order of 1 um or greater in diameter.
  • Capture beads 190 are suspended in hybridization solution and are loaded into the test tube 212 by injection with pipette 214.
  • the preferred hybridization solution is composed of 0.2M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris-HCI, pH 7.5, and 5X Denhart's mix.
  • a desirable hybridization temperature is 37 degrees Celsius.
  • transport probes 198 are conjugated to 3 um magnetic capture beads 190 by EDC conjugation. Further details regarding conjugation methods are disclosed in commonly assigned U.S. Provisional Application Serial No. 60/271 ,922 entitled, “Methods for Attaching Capture DNA and Reporter DNA to Solid Phase Including Selection of Bead Types as Solid Phase” filed February 27, 2001 ; and U.S. Provisional Application Serial No. 60/277,854 entitled “Methods of Conjugation for Attaching Capture DNA and Reporter DNA to Solid Phase” filed March 22, 2001 , both of which are herein incorporated by reference in their entirety.
  • target DNA or RNA 202 is added to the solution.
  • Oligonucleotide transport probes 198 are complementary to the ( DNA or RNA target agent 202.
  • the target DNA or RNA 202 thus binds to the complementary sequences of transport probe 198 attached to the capture bead 190 as shown in Fig. 8A.
  • reporter beads 192 coated with oligonucleotide signal probes 206 there is added to the solution 210 reporter beads 192 coated with oligonucleotide signal probes 206.
  • signal probes 206 are complementary to the target DNA or RNA 202.
  • signal probes 206 which are complementary to a portion of the target DNA or RNA 202, are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 206 and transport probes 198 each have sequences that are complementary to the target DNA 202, but not complementary to each other. After adding reporter beads 192, the dual bead complex 194 is formed such that the target DNA 202 links capture bead 190 and reporter beads 192. With specific and thorough washing, there should be minimal non-specific binding between reporter bead 192 and capture bead 190.
  • the target agent 202 and signal probe 206 are preferably allowed to hybridize for three to four hours at 37 degrees Celsius.
  • the disc drive motor 140 and controller 142, Fig. 2 may be advantageously employed to periodically rotate the disc to achieve the desired intermittent mixing.
  • This may be implemented in mixing protocols encoded on the disc that rotate the disc in one direction, then stop the disc, and thereafter rotate the disc again in the same direction in a prescribed manner with a preferred duty cycle of rotation and stop sessions.
  • the encoded mixing protocol may rotate the disc in a first direction, then stop the disc, and thereafter rotate the disc again in the opposite direction with a preferred duty cycle of rotation, stop, and reverse rotation sessions.
  • the dual bead complex 194 is separated from unbound reporter beads in the solution.
  • the solution can be exposed to a magnetic field to capture the dual bead complex structures 194 using the magnetic properties of capture bead 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that capture beads not bound to reporter beads will also be isolated. Alternatively, this magnetic removal step may be performed on-disc as shown in Figs. 33A, 35, and 36A-36C.
  • the purification process illustrated in Step IV includes the removal of supernatant containing free-floating particles.
  • Wash buffer is added into the test tube and the bead solution is mixed well.
  • the preferred wash buffer for the one step assay consists of 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM, and 10mM EDTA.
  • Most of the unbound reporter beads 182, free-floating DNA, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target sequences, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • the capture beads 190 are separated from the, now unbound, reporter beads 192 in the solution, as shown in Step VII.
  • the solution can be exposed to a magnetic field to capture the magnetic capture beads 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that non-dissociated dual bead complexes not separated during Step VI will also be removed from the solution.
  • the supernatant containing the released reporter beads are collected using a pipette 214.
  • the assay mixture may then be loaded into the disc 144 or 180 and analyzed using an optical bio-disc or medical CD reader, as illustrated in Step VIII.
  • Either a transmissive bio- disc 180 or a reflective bio-disc 144 may be used to analyze the reporter beads. Details relating to the reflective and transmissive optical bio-discs are discussed above in conjunction with Figs. 3A-3C and 4A-4C, respectively.
  • the optical bio-disc reader and other alternative disc readers that may be used to analyze optical bio-discs are described in detail above in connection with Figs. 1 and 2. If the reporter beads are fluorescent, the reporter beads isolated in Step VII may also be quantified using a fluorimeter or any similar fluorescent type analyzer including fluorescent optical disc readers.
  • Figs. 66A and 66B taken together illustrate an immunoassay using a "single- step antigen binding" method, similar to that in Figs. 65A and 65B, to create dual bead complex structures in a solution.
  • This method similarly includes eight principal steps and is related to the method discussed above in connection with Fig. 11 B. The eight steps of this method are respectively identified as Steps I, II, III, IV, V, VI, VII, and VIII in Figs. 66A and 66B.
  • Fig. 66A capture beads 190, e.g., on the order of 10E+07 in number and each on the order of 1 um or above in diameter, which are coated with antibody transport probes 196 are added to a buffer solution 210.
  • This solution may be the same as that employed in the method shown in Figs. 65A and 65B or alternatively may be specifically prepared for use with immunochemical assays.
  • the antibody transport probes 196 have a specific affinity for the target antigen 204.
  • the transport probes 196 bind specifically to epitopes within the target antigen 204 as also shown in Fig. 8B.
  • antibody transport probes 196 which have an affinity for a portion of the target antigen may be conjugated to 3 um magnetic capture beads 190 via EDC conjugation.
  • conjugation of the transport probes 196 to the capture bead 190 may be achieved by passive adsorption.
  • the target antigen 204 is added to the solution.
  • the target antigen 204 binds to the antibody transport probe 196 attached to the capture bead 190 as also shown in Fig. 8B.
  • reporter beads 192 coated with antibody signal probes 208 are added to the solution.
  • Antibody signal probes 208 specifically binds to the epitopes on target antigen 204 as also represented in Figs. 9B and 10B.
  • signal probes 208 are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 208 and transport probes 196 each bind to specific epitopes on the target antigen, but not to each other.
  • the dual bead complex 194 is formed such that the target antigen 204 links capture bead 190 and reporter bead 192. With specific and thorough washing, there should be minimal non-specific binding between reporter bead 192 and capture bead 190.
  • Step IV after the binding in Step III, the dual bead complex 194 is separated from unbound reporter beads in the solution.
  • the solution can be exposed to a magnetic field to capture the dual bead complex structures 194 using the magnetic properties of capture bead 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that capture beads not bound to reporter beads will also be isolated. Alternatively, as indicated above, this magnetic removal step may also be performed on-disc as shown in Figs. 33A, 35, and 36A-36C.
  • the purification process of Step IV includes the removal of supernatant containing free-floating particles. Wash buffer is added into the test tube and the bead solution is mixed well. Most of the unbound reporter beads 182, free-floating protein samples, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target antigen, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step V The next step in Fig. 66A is Step V.
  • Urea, acids, or bases may be added to the dual bead solution as dissociation agents, using a pipette 214 as illustrated.
  • the acids or bases employed herein are preferably strong acids or bases, respectively.
  • the dual bead complexes are thus dissociated by the actions of these dissociation agents thereby releasing the reporter beads 192 from the capture beads 190 as shown in Fig. 66B, Step VI.
  • the capture beads 190 are separated from the, now unbound, reporter beads 192 in the solution, as shown in Step VII.
  • the solution can be exposed to a magnetic field, either on-disc or off-disc, to capture the magnetic capture beads 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that non-dissociated dual bead complexes not separated during Step VI will also be removed from the solution.
  • the supernatant containing the released reporter beads are collected using a pipette 214.
  • the assay mixture may then be loaded directly into the disc and analyzed using an optical bio-disc reader, as illustrated in Step VIII.
  • Either a transmissive bio-disc 180 or a reflective bio-disc 144 may be used to analyze the reporter beads. Details relating to the reflective and transmissive optical bio-discs are discussed in detail in connection with Figs. 3A-3C and 4A-4C, respectively.
  • the optical bio-disc reader and other alternative disc readers that may be used to analyze optical bio-discs are described in detail above with reference to Figs. 1 and 2. If the reporter beads are fluorescent, the reporter beads isolated in Step VII may also be quantified using a fluorimeter or any similar fluorescent type analyzer including fluorescent optical disc readers.
  • Figs. 67A and 67B taken together show an alternative genetic assay method referred to here as a "two-step hybridization".
  • This method is a modified embodiment of the method discussed above in connection with Fig. 12A.
  • the present method has nine principal steps directed to creating the dual bead complex.
  • capture beads are coated with oligonucleotide transport probes 198 complementary to DNA or RNA target agent and placed into a buffer solution.
  • transport probes which are complementary to a portion of target agent are conjugated to 3 um magnetic capture beads via EDC conjugation.
  • Other type of conjugation of the oligonucleotide transport probes to a solid phase may be utilized. These include, for example, passive adsorption or use of streptavidin-biotin interactions.
  • the nine main steps according to this method of the present invention are consecutively identified as Steps I, II, III, IV, V, and VI in Fig. 67A, and Steps VII, VIII, and IX in Fig. 67B.
  • capture beads 190 suspended in hybridization solution, are loaded from the pipette 214 into the test tube 212.
  • the preferred hybridization solution is composed of 0.2M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris-HCI, pH 7.5, and 5X Denhart's mix.
  • a desirable hybridization temperature is 37 degrees Celsius.
  • target DNA or RNA 202 is added to the solution and binds to the complementary sequences of transport probe 198 attached to capture bead 190.
  • target agent 202 and the transport probe 198 are allowed to hybridize for 2 to 3 hours at 37 degrees Celsius. Sufficient hybridization, however, may be achieved within 30 minutes at room temperature. At higher temperatures, hybridization may be achieved substantially instantaneously.
  • target agents 202 bound to the capture beads are separated from unbound species in solution by exposing the solution to a magnetic field to isolate bound target sequences by using the magnetic properties of the capture bead 190.
  • the magnetic field can be enclosed in a magnetic test tube rack 216 with a built-in magnet permanent 218 or electromagnet to draw out the magnetic beads and remove any unbound target DNA 202 free-floating in the suspension via pipette extraction of the solution.
  • this magnetic removal step may be performed as shown in Figs. 33A, 35, and 36A-36C.
  • a wash buffer is added and the separation process can be repeated.
  • the preferred wash buffer after the transport probes 198 and target DNA 202 hybridize consists of 145mM NaCl, 50mM Tris, pH 7.5, and 0.05% Tween.
  • Hybridization methods and techniques for decreasing non-specific binding of target agents to beads are further disclosed in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/278,691 entitled "Reduction of Non-Specific Binding of Dual Bead Assays by Use of Blocking Agents" filed March 26, 2001. This application is herein incorporated by reference in its entirety.
  • Step IV illustrated in Fig. 67A reporter beads 192 are added to the solution as discussed in conjunction with the method shown in Fig. 65A.
  • Reporter beads 192 are coated with signal probes 206 that are complementary to target agent 202.
  • signal probes 206 which are complementary to a portion of target agent 202, are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 206 and transport probes 198 each have sequences that are complementary to target agent 202, but not complementary to each other.
  • the dual bead complex structures 190 are formed. As would be readily apparent to one of skill in the art, the dual bead complex structures are formed only if the target agent of interest is present. In this formation, target agent 202 links magnetic capture bead 190 and reporter bead 192.
  • Target agent 202 and signal probe 206 are preferably allowed to hybridize for 2-3 hours at 37 degrees Celsius. As with Step II discussed above, sufficient hybridization may be achieved within 30 minutes at room temperature. At higher temperatures, the hybridization taking place in this step may also be achieved substantially instantaneously.
  • Step V shown in Fig. 67A after the hybridization in Step IV, the dual bead complex 194 is separated from unbound species in solution. The solution is again exposed to a magnetic field to isolate the dual bead complex 194 using the magnetic properties of the capture bead 190. Note again that the isolate will include capture beads not bound to reporter beads. As with Step III above in the on- disc counterpart hereto, this magnetic separation step may be performed as shown in Figs. 33A, 35, and 36A-36C.
  • a purification process to remove supernatant containing free-floating particles includes adding wash buffer into the test tube and mixing the bead solution well.
  • the preferred wash buffer for the two-step assay consists of 145mM NaCl, 50mM Tris, pH 7.5, 0.1 % SDS, 0.05% Tween, 0.25% NFDM, and 10mM EDTA. Most unbound reporter beads, free-floating DNA, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target agents, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step VI The next step shown in Fig. 67A is Step VI.
  • the assay mixture is loaded into the disc and analyzed.
  • the oligonucleotide signal and transport probes may be ligated to prevent breakdown of the dual bead complex during the disc analysis and signal detection processes. Further details regarding probe ligation methods are disclosed in commonly assigned and co-pending U.S. Provisional Application Serial No. 60/278,694 entitled "Improved Dual Bead Assays Using Ligation" filed March 26, 2001 , which is herein incorporated in its entirety by reference.
  • restriction enzymes, urea, acids, or bases may be added using a pipette 214 to the dual bead solution, as illustrated in Step VI of Fig. 67A.
  • the dual bead complexes are dissociated by the actions of these dissociation agents thus releasing the reporter beads 192 from the capture beads 190 as shown in Fig. 67B, Step VII.
  • the acids or bases utilized herein are preferably strong acids or bases, respectively.
  • the capture beads 190 are separated from the, now unbound, reporter beads 192 in the solution, as shown in Step VIII.
  • the solution can be exposed to a magnetic field to capture the magnetic capture beads 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that non-dissociated dual bead complexes not separated during Step VIII will also be removed from the solution.
  • the supernatant containing the released reporter beads are collected using a pipette 214.
  • the assay mixture may then be loaded into the disc and analyzed using an optical bio-disc reader, as illustrated in Step IX.
  • Either a transmissive bio-disc 180 or a reflective bio- disc 144 may be used to analyze the reporter beads. Details relating to the reflective and transmissive optical bio-discs are discussed in detail in conjunction with Figs. 3A- 3C and 4A-4C, respectively.
  • the optical bio-disc reader and other alternative disc readers that may be used to analyze optical bio-discs are described in detail above with reference to Figs. 1 and 2.
  • the reporter beads are fluorescent
  • the reporter beads isolated in Step VIII may also be quantified using a fluorimeter or any similar fluorescent type analyzer including fluorescent optical disc readers. Experiments performed using dissociation agents to release the reporter beads from the dual bead complex are described in detail below in Examples 15 and 16.
  • Figs. 68A and 68B taken together show an immunoassay method, similar to those discussed in connection with Figs. 66A and 66B, and following the techniques of the genetic assay in Figs. 67A and 67B.
  • This method is also referred to here as a "two-step binding" to create the dual bead complex in an immunochemical assay.
  • This method is a related and more specific embodiment of the method illustrated above in Fig. 12B. As with the method shown in Figs. 67A and 67B, this method includes nine main steps. In general, capture beads coated with antibody transport probes which specifically binds to epitopes on target antigen are placed into a buffer solution.
  • antibody transport probes are conjugated to 3 um magnetic capture beads. Different sized magnetic capture beads may be employed depending on the type of disc drive and disc assembly utilized to perform the assay.
  • the nine main steps according to this alternative method of the invention are respectively identified as Steps I, II, III, IV, V and VI in Fig. 68A and Steps VII, VIII, and IX in Fig. 68B.
  • Step I shown in Fig. 68A capture beads 190, suspended in buffer 210 solution, are loaded into a test tube 212 via injection from pipette 214.
  • target antigen 204 is added to the solution and binds to the antibody transport probe 196 attached to capture bead 190.
  • Target antigen 204 and the transport probe 196 are preferably allowed to bind for 2 to 3 hours at 37 degrees Celsius. Shorter binding times are also possible.
  • target antigen 204 bound to the capture beads 190 are separated from unbound species in solution by exposing the solution to a magnetic field to isolate bound target proteins or glycoproteins by using the magnetic properties of the capture bead 190.
  • the magnetic field can be enclosed in a magnetic test tube rack 216 with a built-in magnet permanent 218 or electromagnet to draw out the magnetic beads and remove any unbound target antigen 204 free-floating in the suspension via pipette extraction of the solution.
  • a wash buffer is added and the separation process can be repeated.
  • reporter beads 192 are added to the solution as discussed in conjunction with the method shown in Fig. 66A. Reporter beads 192 are coated with signal probes 208 that have an affinity for the target antigen 204.
  • signal probes 208 which bind specifically to a portion of target agent 204, are conjugated to 2.1 um fluorescent reporter beads 192.
  • Signal probes 208 and transport probes 196 each bind to specific epitopes on the target agent 204, but do not bind to each other.
  • the dual bead complex structures 190 are formed. As would be readily apparent to those skilled in the art, these dual bead complex structures are formed only if the target antigen of interest is present. In this formation, target antigen 204 links magnetic capture bead 190 and reporter bead 192. Using the preferred buffer solution, with specific and thorough washing, there is minimal nonspecific binding between the reporter beads and the capture beads.
  • Target antigen 204 and signal probe 208 are allowed to hybridize for 2-3 hours at 37 degrees Celsius. As with Step II discussed above, sufficient binding may be achieved within 30 minutes at room temperature. In the case of immunoassays temperatures higher than 37 degrees Celsius are not preferred because the proteins will denature.
  • Step V as illustrated in Fig. 68A, after the binding shown in Step IV, the dual bead complex 194 is separated from unbound species in solution. This is achieved by exposing the solution to a magnetic field to isolate the dual bead complex
  • the isolate will include capture beads not bound to reporter beads.
  • a purification process to remove supernatant containing free-floating particles includes adding wash buffer into the test tube and mixing the bead solution well. Most unbound reporter beads, free-floating proteins, and non-specifically bound particles are agitated and removed from the supernatant.
  • the dual bead complex can form a matrix of capture beads, target agents, and reporter beads, wherein the wash process can further assist in the extraction of free floating particles trapped in the lattice structure of overlapping dual bead particles.
  • Step VI The last step shown in Fig. 68A is Step VI.
  • urea, acids (preferably strong acids), or bases (preferably strong bases) may be added to the dual bead solution using a pipette 214, as illustrated in Step VI of Fig. 68A.
  • the dual bead complexes are dissociated by the actions of these dissociation agents thus releasing the reporter beads 192 from the capture beads 190 as shown next in Step VII of Fig. 68B.
  • the capture beads 190 are separated from the, now unbound, reporter beads 192 in the solution, as shown in Step VIII of Fig. 68B.
  • the solution can be exposed to a magnetic field to capture the magnetic capture beads 190.
  • the magnetic field can be encapsulated in a magnetic test tube rack 216 with a built-in magnet 218, which can be permanent or electromagnetic to draw out the magnetic beads and remove any unbound reporter beads in the suspension. Note that non-dissociated dual bead complexes not separated during Step VII will also be removed from the solution.
  • Step VIII in this method the supernatant containing the released reporter beads 192 are collected using a pipette 214.
  • the assay mixture may then be loaded into the disc and analyzed using an optical bio-disc reader, as illustrated in Step IX.
  • Either a transmissive bio-disc 180 or a reflective bio-disc 144 may be used to analyze the reporter beads. Details relating to the reflective and transmissive optical bio-discs are discussed in detail with reference to Figs. 3A-3C and 4A-4C, respectively.
  • the optical bio-disc reader and other alternative disc readers that may be used to analyze optical bio-discs or medical CDs are described in detail above in conjunction with Figs. 1 and 2.
  • the reporter beads isolated in Step VIII may also be quantified using a fluorimeter or any similar fluorescent type analyzer including fluorescent optical disc readers.
  • the magnetic removal or separation steps in the method shown in Figs. 68A and 68B may be alternatively performed on-disc using the disc, fluidic circuits, and apparatus illustrated in Figs. 33A-33D, 34A-34C, 35, 36A-36C, and 37.
  • Fig. 69 there is shown a bar graph presentation demonstrating the DNAsel digestion efficiency in the absence of reporter beads.
  • biotinylated target DNA 202 was hybridized to transport probes 198 on the magnetic capture bead 190 as illustrated above in Fig. 8A.
  • streptavidinated alkaline phosphatase (S-AP) was added to the assay mix and allowed to bind with the biotin on the target DNA.
  • FIG. 70 there is shown a bar graph illustration of data collected from an experiment similar to that discussed in Fig. 69.
  • dual bead complexes were used instead of the magnetic capture bead alone as described in Fig. 69.
  • the target 202 is situated between the capture bead 190 and reporter bead 192 as shown above in Fig. 10A.
  • Steps l-V the amount of reporter beads in the supernatant collected in Step IV (Fig. 65A) and the reporter beads bound to the capture beads in the assay mix shown in Step V (Fig. 65A) were quantitated using a fluorimeter.
  • DNAsel was then added to the assay mix and allowed to cleave the target bound to the probes in the dual bead complex, as represented in Step VI in Fig. 65B, thereby releasing the reporter bead from the magnetic capture beads.
  • the next step involved the isolation of the capture beads and collection of the supernatant containing newly released fluorescent reporter beads as shown in Step VII in Fig. 65B.
  • the signal from the reporter beads in the supernatant was quantified using a fluorimeter.
  • the undissociated reporter beads were also quantified by a fluorimeter. Data collected from this experiment shows the DNAsel enzyme digestion was not as efficient in a dual bead complex relative to that in a single bead set-up as described in Fig. 69.
  • the decrease in DNAse digestion activity may be due to steric hindrance from the beads in the dual bead complex blocking DNAse access to the target.
  • Experimental details regarding the use of restriction enzyme digestion as implemented in a dual bead assay is discussed in further detail below in Example 15.
  • the fluorescent reporter beads collected in this assay may also be quantified using an optical bio-disc or medical CD with a fluorescent type optical disc reader or any similar device as discussed above.
  • the sensitivity of any assay depends on the ratio of signal over noise.
  • the sensitivity of the dual bead assay relies on the minimization of non-specific binding between capture beads and reporter beads. The non-specific interactions between the dual beads in the absence of targets are so stable that stringent washing cannot eliminate them.
  • the contribution of the non-specific dual beads can be negated by the exclusive detection and quantification of target-mediated dual beads.
  • the dual bead complexes can be separated by enzyme digestion (DNAse, restriction enzymes) or by chemical and physical treatments (heat, urea, base, or acid treatment).
  • the quantification of reporter beads in the presence of a large excess of unbound capture beads may reduce the sensitivity of the detection assay. Therefore, the separation of reporter beads from capture beads will facilitate the quantification of reporter beads by the bio-disc reader and thus increase the sensitivity of the assay.
  • Fig. 71 there is a schematic representation of separation of reporter beads from capture beads in a dual bead complex by enzyme digestion and physical or chemical treatments.
  • Fig. 71 shows a summary of the dual bead formation and dissociation using various dissociation agents as discussed in conjunction with Figs. 65A, 65B, 67A, and 67B.
  • the beads are quantified by any one of several methods including a fluorimeter, a fluorimager, a fluorescent type optical disc reader system, a CD-R type optical disc system or any device capable of detecting micro- spheres or fluorescence.
  • the current preferred method of detection is the use of an optical bio-disc or medical CD system as discussed in detail in connection with Figs. 1 , 2, 3A, 3B, 3C, 4A, 4B, and 4C.
  • Fig. 72 there is a bar graph presentation showing separation of reporter beads from capture beads using high pH washes at various target concentrations similar to that described in Fig. 70.
  • the dual bead complex is formed as described in Steps l-VI in Fig. 67A.
  • a strong base is added to the dual bead solution as shown in Step VI (Fig. 67A).
  • the dual beads are dissociated by the actions of the base that disrupts the hydrogen bonding between the target and the probes thereby releasing the fluorescent reporter beads and the magnetic capture beads from the dual bead complex as shown in Step VII (Fig. 67B).
  • the next step is to isolate the capture beads as described in Step VIII of Fig.
  • the isolated capture beads will also contain non-dissociated dual bead complexes. This isolate was quantified by a fluorimeter in this particular experiment. The data shown in Fig. 72 illustrate 100% dissociation of dual bead complexes at target concentrations from 1 x 10 '16 M to 1 x 10 "14 M. Details of an experiment performed using base as a dissociation agent is described in detail below in Example 16.
  • the reporter beads may also be quantified by any one of several methods including a fluorimager, a fluorescent type optical disc reader system, a CD-R type optical disc system or any device capable of detecting microspheres or fluorescence. The preferred method of detection is the use of an optical bio-disc or medical CD system as discussed in detail in conjunction with Figs. 1 , 2, 3A, 3B, 3C, 4A, 4B, and 4C.
  • Fig. 73A there is a bar graph illustration of data collected from an experiment using urea as a denaturing or dissociation agent. Details of this experiment are similar to those discussed in conjunction with Fig. 69.
  • biotinylated target DNA 202 was hybridized to transport probes 198 on the magnetic capture bead 190 as illustrated above in Fig. 8A.
  • streptavidinated alkaline phosphatase (S-AP) was added to the assay mix and allowed to bind with the biotin on the target DNA 202.
  • an S-AP chromagen substrate was added to an aliquot of the assay solution and the amount of bound target was quantified colorimetrically using a spectrophotometer.
  • an aliquot of equal volume to that taken above was incubated in buffer containing 7M urea.
  • the assay mix was washed and S-AP was added to the solution and allowed to bind to residual targets, still bound to transport probes on the capture bead, that were not denatured by the 7M urea.
  • the results thereof showed a relatively efficient denaturation activity as revealed by the difference in signal between the control and the 7M urea treatments. Details regarding an experiment similar to the one discussed here is described in detail below in Example 16.
  • Fig. 73B there is a bar graph illustrating data collected from an experiment using urea as a dissociation agent in a dual bead assay. Details of this experiment are similar to that discussed in conjunction with Fig. 70. As in Fig. 70, dual bead complexes were used instead of the magnetic capture bead alone as described in Fig. 73A. In this case, the target is situated between the capture and reporter beads as shown above in Fig. 10A. After formation of the dual bead complex, as described in Fig. 67A, Steps l-VI, the amount of reporter beads 192 in the supernatant collected in Step V (Fig.
  • the undissociated reporter beads were also quantified by a fluorimeter. Data collected from this experiment shows the 7M urea denaturation was more efficient in a dual bead complex assay relative to using DNAsel as a dissociation agent as described in Fig. 70. The increase in dissocaition may be due to the lack of steric hindrance from the beads in the dual bead complex since urea is a significantly smaller molecule than DNAse.
  • Experimental details regarding the use of 7M urea denaturation as implemented in a dual bead assay is discussed in further detail in Example 16.
  • the fluorescent reporter beads collected in this assay may also be quantified using an optical bio-disc or medical CD with a fluorescent type optical disc reader or any similar device as discussed above.
  • DNA Denaturing Agents to Improve DNA Target Detection It is a principal aspect of the invention to further modify the dual bead assays to detect medical targets.
  • the DNA targets are double-stranded and very long.
  • the ability of the dual bead assay, as well as for any other DNA diagnostic assays, to detect sequences of clinical interest within the whole genome relies first on the specificity of the probes for the sequence of interest and second on the use of very strong detergent to keep the DNA target in the denatured, single-stranded, form for capture.
  • the success of the dual bead assays in detecting sequences of clinical interest relies primarily on the design of the probes. Given the complexity and degeneracy of the human genome, the probes designed to detect sequences of clinical interest have to be unique to the diagnostic sequence and yet common enough to recognize mutants of the sequences. The design of the probes using computer software allows comparison of sequences to existing sequences in the data bank such as Blast search. Once probes specific for the sequence of interest have been designed, the major modification introduced to the dual bead assays includes the use of a denaturing agent in the hybridization buffer to prevent re-annealing of complementary sequences of the target DNA. This allows hybridization between the target and probes.
  • a bio- disc or medical CD drive assembly such as those discussed above with reference to Figs, 1 and 2, may be employed to rotate the disc, read and process any encoded information stored on the disc, and analyze the DNA samples in the flow channel of the bio-disc or medical CD.
  • the bio-disc drive is thus provided with a motor for rotating the bio-disc, a controller for controlling the rate of rotation of the disc, a processor for processing return signals form the disc, and an analyzer for analyzing the processed signals.
  • the rotation rate of the motor is controlled to achieve the desired rotation of the disc.
  • the bio-disc drive assembly may also be utilized to write information to the bio-disc either before, during, or after the medical test material in the flow channel and target zones is interrogated by the read beam of the drive and medically diagnosed by the analyzer.
  • the analyzer may advantageously include specialized diagnostic software to thereby provide a medical expert system.
  • the bio-disc may similarly include corresponding medical expert system software and encoded information for controlling the rotation rate of the disc, providing processing information specific to the type of DNA test to be conducted, and for displaying the results on a monitor associated with the bio-drive.
  • guanidine isothiocyanate is used as a typical denaturing agent.
  • Data collected from an experiment using 1.5M guanidine isothiocyanate as denaturing agent is illustrated in Fig. 74. The experimental procedure followed for this experiment is described in detail below in Example 17.
  • Fig. 74 further illustrates that the assay sensitivity is significantly increased if an appropriate amount of denaturing agent is used in a hybridization experiment.
  • biotinylated target DNA 202 was hybridized to transport probes 198, in the presence of 1.5M guanidine isothiocyanate, on the magnetic capture bead 190 as illustrated above in Fig. 8A.
  • S-AP streptavidinated alkaline phosphatase
  • guanidine isothiocyanate An appropriate amount of guanidine isothiocyanate is necessary to prevent re- annealing of the complementary sequences of the target DNA while allowing hybridization between the target and the probes. At high concentrations, however, guanidine isothiocyanate prevents any hybridization.
  • a titration of guanidine isothiocyanate was performed. The data from this titration experiment is shown in Fig. 75. As illustrated in Fig. 75, the optimal hybridization buffer concentration of guanidine isothiocyanate is 1.5M since the addition of 1.5M guanidine isothiocyanate showed the highest difference in signal between the 0.0M and 1.0x1O "1o M target concentration.
  • MO Bio-Magnetic Assays for Selecting. Detecting, and
  • MOBDS MO Bio-Disc System
  • a further aspect of the present invention relates to a magnetic method for detection of specific target cells in cell populations and solutions of cell populations, using magnetic particles or beads as described above and referred to as the MOBMA.
  • the magnetic beads are coated with one or more binding agents or capture probes to thereby form bio-magnetic particles or beads. Further details related to attaching binding agents onto a solid support including magnetic particles is disclosed in commonly assigned and co-pending U.S. Patent Application Serial No. 10/194,396 entitled “Multi-Purpose Optical Analysis Disc For Conducting Assays and Various Reporting Agents for use Therewith” filed July 12, 2002, which is incorporated by reference in its entirety herein.
  • the binding agents may include, for example, antibodies recognizing Fc portions of target cell associating antibodies directed to specific antigen determinants on cell membranes. Capture, detection, manipulation, and quantitation of target cells are carried out using the MOBDS of the present invention.
  • the captured cells may also be magnetically manipulated or moved from one analysis, separation, or testing chamber to another, on the MO bio-disc, to facilitate cellular testing.
  • captured target cancer cells may be magnetically aliquoted into equal cell numbers to different analysis chambers containing cancer therapeutic agents to determine the effect of these agents on a cellular level. Specific examples of applications of this method are described below.
  • the method can further be used to dynamically isolate, purify, manipulate, and quantify target cells magnetically captured on magnetic capture zones on the MO bio-disc. Further details relating to a method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations is disclosed in, for example, U.S. Patent Serial No. 6,184,043 to Fodstad and Kvalheim, which is herein incorporated by reference in its entirety.
  • the binding agents may bind one or more chemical/biochemical entities together by affinity.
  • affinity binding a pair of binding partners, which for example are attached to the substances to be linked, bind to each other, when brought in contact.
  • a molecule on a cell surface may represent one of the binding partners, in such a linkage.
  • binding partner systems are known, such as, for example, antigen-antibody, enzyme-receptor, ligand-receptor interactions on cells, biotin-avidin binding, hapten/anti-hapten binding pairs, and oligonucleotide complementary sequence binding, of which antigen-antibody binding is most frequently used.
  • one of the binding partners is attached to an insoluble support, such as magnetic particles or beads, and by which isolation of target cells in a mixed cell population is performed as negative isolation or positive isolation.
  • an insoluble support such as magnetic particles or beads
  • isolation of target cells in a mixed cell population is performed as negative isolation or positive isolation.
  • the unwanted cells can be removed from the cell preparation by incubating the cells with antibody-coated magnetic beads (biomagnetic beads or particles) specific for the unwanted cells. Following the incubation the resulting cell-bead complex can be removed or isolated magnetically, leaving the wanted target cells behind.
  • antibody-coated magnetic beads biomagnetic beads or particles
  • the wanted target cells are removed from the mixed cell population using magnetic beads coated with antibodies (bio-magnetic beads) directed to the cells of interest.
  • An object of the MOBMA aspect of the present invention is to detect and study specific target cells for diagnostic purposes.
  • the test samples may include blood, bone marrow, ascites fluid, and cells from various tissues or tumors.
  • the present invention represents a sensitive detection system and method for detecting a variety of cell types, such that a high number of cells can be readily screened using the MOBDS and the procedure is rapid and simple.
  • the present MOBDS may be used for isolation of cells for biochemical, biological and immunological examination, and for studying of specific genes at the nucleotide or protein level.
  • the isolated or captured cell-bead complexes may be released by disrupting or removing the magnetic field within the magnetic capture zone, on the MO bio-disc, where cells of interest are located, using the associated MO drive and the cells cultured or used for in vitro cytotoxicity studies without the need for cleaving the cell- bead complex.
  • Another embodiment of the MOBMA aspect of the present invention includes immunomagnetic positive isolation of target cells, normal or pathogenic, in a mixed cell population or physiological solution.
  • a linkage is created between a specific target cell and an insoluble support such as a magnetic bead or particle.
  • the particle is either coated with an anti-cell antibody directed to specific antigen determinants on the membranes of the target cells, or the particles are coated with a polyclonal anti-mouse or anti-human antibody capable of binding to the Fc-portions of a specific anti-cell antibody (primary antibody) directed to the antigen determinants on the membrane of the target cell.
  • a monoclonal rat anti-mouse/anti-human antibody may be used instead of using the polyclonal anti-mouse/anti-human antibody for coating the particles.
  • the use of a monoclonal antibody may reduce the risk for possible cross-reactions with non-target cells in solutions.
  • incubation of the cell suspension with a mild detergent and/or second set of antibodies or antibody fragments that bind to antigenic determinants on one or more cell types among the captured cells in a mixed cell capture, pre-labeled with fluorescent agents, magnetic particles, metallocolloids, bio-magnetic particles, radioisotopes, biotin-complexes or certain enzymes allowing visualization, will dramatically increase the specificity of the method of this embodiment.
  • cancer cells as the target cells for detection and isolation.
  • This embodiment is, however, not limited to cancer cells and the disclosure shall not limit the method to this particular field of use since the method is suitable for use with a range of cytological applications including assays directed to, for example, cells of the immune system such as natural killer cells, monocytes, T- cells, B-cells, and subtypes thereof including CD4+ and CD8+ cells; bacterial cells (Chandler, et al., Int. J. of Food Micr., 70: 143-154, 2001 , and Yu, J. of Immuno. Mthds., 218: 1-8, 1998); and peroxisomes (Luers, et al., Electrophoresis, 19: 1205- 1210, 1998).
  • the staging of the disease with regards to whether it is localized or if metastatic spread has occurred to other tissues is of utmost importance for the choice of therapeutic regimens for the individual patient.
  • Malignant cells spread by direct invasion into the surrounding tissue, through the lymphatics, or by the distribution of tumor cells in the blood to distant organs, including the bone marrow the central nervous system, and the cerebrospinal fluid.
  • Detection of metastatic tumor cells has, until recently, relied on morphological methods using light and electron microscopy on biopsied tumor specimens, on smears of bone marrow and peripheral blood, and on slides prepared after cytocentrifugation of various body fluids.
  • Flow cytometry may provide better sensitivity than the methods involving the use of a microscope, but requires the availability of a high number of cells, and also involves several technical difficulties. Furthermore, aggregation of cells may cause problems with flow cytometry, and this method does not provide possibilities to distinguish between labeled tumor cells and non-specifically fluorescing normal cells.
  • the present invention allows for a very sensitive detection of, for example, metastatic tumor cells, since a high number of cells can readily be screened and the cell-bead complexes are easily detected and quantitated by the MOBDS.
  • the monoclonal antibodies used for the detection of target cells in the present invention are selected such that these antibodies bind with sufficient specificity to, for example, tumor cells and not to non-target cells present in mixed cell suspensions or test sample.
  • the test sample may include, for example blood, bone marrow, and other solutions containing tumor manifestations, such that all cells with attached beads represent the target cells.
  • the target cells are then captured on specific magnetized areas or magnetic domains in a magneto-optical (MO) bio-disc and quantitated using the associated magneto-optical drive and software.
  • MO magneto-optical
  • the immunomagnetic embodiment of the MOBMA aspect of the present invention involves the binding of capture antibodies including monoclonal antibodies, e.g. of murine or human origin, that specifically recognize antigens present on tumor cells, but not antigens on normal cells, or for other purposes to specified sub-populations of normal cells, to magnetic particles or beads to thereby form bio-magnetic particles.
  • the capture antibodies may bind directly to antigens on the target cell or the Fc-portion of primary antibodies or cell binding antibodies that bind to the tumor cells.
  • the cell binding antibodies may be of the IgG or IgM type or a fragment of IgG or IgM antibodies.
  • the capture or primary antibodies may be antibodies directed against groups of antigen determinants including, for example, tumor associated glycoprotein 72 (TAG 72) antigen (CC49 antibody) (Barjer et al., Gynec. Onco. 82: 57-63, 2001), CD56/NCAM antigen (MOC-1 antibody) (Speirs et al., J. Histochem. Cytochem., 41 (9):1303-10, 1993), epithelial cell surface antigen (BER-EP4 antibody) (Borgen et al., J. Hematother., 6(2):103-114, 1997, and Ziggeuner et al. J.
  • TAG 72 tumor associated glycoprotein 72
  • MOC-1 antibody CD56/NCAM antigen
  • BER-EP4 antibody epithelial cell surface antigen
  • HMW-melanoma-associated antigen 225.28S antibody
  • TP-1 & TP-3 antibodies 80 kD
  • Sarcoma-associated antigen TP-1 & TP-3 antibodies
  • cytokeratin antigens pan-anti-CK antibody
  • mucin antigens TAG 12 2E11
  • EGF-receptor antigen 425.3 antibody
  • the 425.3 antibody is directed towards antigens in both normal and malignant cells.
  • the capture antibodies can furthermore be directed against growth factor receptors, for example EGF-receptor, PDGF (A and B) receptor, insuline receptor, insuline-like receptor, transferrin receptor, NGF and FGF receptors, group of integrins, other adhesion membrane molecules and MDR proteins in both normal cells and abnormal cells, and antigens present on subpopulations of normal cells, in addition to oncogenic products, expressed on the membranes of normal and malignant cells and on malignant cells alone, for example Neu/erb B2/HER2.
  • the malignant cells these may be breast, ovarian and lung carcinoma cells, melanoma, sarcoma, glioblastoma, cancer cells of the gastrointestinal tract and the reticuloendothelial system, or the target cells may be associated with non-neoplastic diseases, such as cardiovascular, neurological, pulmonary, autoimmune, gastrointestial, genitourinary, reticuloendothelial and other disorders.
  • non-neoplastic diseases such as cardiovascular, neurological, pulmonary, autoimmune, gastrointestial, genitourinary, reticuloendothelial and other disorders.
  • the malignant cell population may be located in bone marrow, peripheral blood, come from pleural and peritoneal effusions and other body fluid compartments, such as urine, cerebrospinal fluid, semen, lymph or from solid tumors in normal tissues and organs, for example liver, lymph nodes, spleen, lung, pancreas, bone tissue, the central nervous system, prostatic gland, skin and mucous membranes.
  • body fluid compartments such as urine, cerebrospinal fluid, semen, lymph or from solid tumors in normal tissues and organs, for example liver, lymph nodes, spleen, lung, pancreas, bone tissue, the central nervous system, prostatic gland, skin and mucous membranes.
  • the target cells can be positively separated from non-target cells and pre- concentrated using a magnet prior to analysis in the MOBDS.
  • the isolated target cells can then be enumerated using the MOBDS and the fraction of target cells relative to the total number of cells in the initial cell suspension can be calculated.
  • the MOBDS may be used to isolate, manipulate, and study target cells in vitro.
  • One assay that can be performed on target cells is a cytotoxicity, chemosensitivity, drug sensitivity, or drug resistance assay to measure drug-induced cell death following drug exposure.
  • the target cells may be, for example, collected from a malignant tumor biopsy, bacteria, plant, virus, and unicellular organisms.
  • the target cells are immunomagnetically isolated in one or more chambers in the MO bio-disc, as described above, to thereby constitute an isolate. Once the target cells are isolated, a pre-determined number of cells, from the isolate, are magnetically moved or manipulated to various chambers within the MO bio-disc including test chambers.
  • chemotherapeutic agents may include, for example, breast cancer, lung cancer, brain cancer, liver cancer, and ovarian cancer drugs such as Cisplatin, Topotecan, Taxol, Gemcitabine (1), Mitomycin-C, Navelbine, Nitrogen Mustard, 5-Fluorouracil, Doxorubicin, Etoposide, Trimetrexate, and various combinations thereof including, but not limited to, Cisplatin and Topotecan, Cisplatin and Taxol, Cisplatin and Gemcitabine (1), Cisplatin and Nitrogen Mustard, and Cisplatin and 5-Fluorouracil.
  • the isolated cells are then incubated at pre-determined conditions in the various concentrations and combinations of drugs. After incubation, the number of live cells are counted, using the MOBDS, and the IC50 (drug concentration in which 50% of the cells die as a result of exposure a drug or a combination of drugs) from all the drugs are calculated to determine the best therapy regimen for the patient.
  • This test is very important since drugs found active in vitro are approximately 10 times more likely to be clinically effective relative to drugs found inactive in vitro.
  • In vivo drug sensitivity assays are well know in the art (Bosanquet et al., Leukemia 16(6): 1035-44, 2002; Nagoumey et al., J. Clin. Onco.
  • the assays of the prior art are mostly performed using a microtiter plate and the cell counting and analysis done using a microscope or colorimetrically using a microtiter plate reader by preferential staining of the live or dead cells, to determine the number of live cells.
  • the present invention automates the process in that the cell selection, manipulation, incubation, analysis, and IC50 determination is performed using the MOBDS without the need for a separate analysis device such as a microscope or a microtiter plate reader.
  • the automated process increases the efficiency and the accuracy of these drug sensitivity assays.
  • Steps for performing the above described immunomagnetic embodiment of the present invention may differ depending on type of tissues to be examined including, for example: a) cells from solid tissue or needle tumor biopsies are separated mechanically or with mild enzymatic treatment to generate a suspension of single cells, to which the primary, specific antibodies or antibody fragments are added directly or after washing the cell suspension in phosphate buffered saline or culture medium with or without serum, such as fetal calf serum, bovine, horse, pig, goat, or human serum; b) cells in pleural or ascitic effusion, cerebrospinal fluid, urine, lymph or body fluids such as effusions in the joints of patients with various forms of arthritis, the specific capture antibodies or antibody fragments are either added to the samples directly, or after centrifugation with or without washings before or after the cells in the samples are spun down and brought back into suspension; and c) mononuclear cells from blood or bone marrow aspirate are isolated by gradient centrifugation and the capture antibodies are added to
  • Fibronectin receptor ( ⁇ 5 ⁇ 1 integrin) Pierce 36114, BTC 21/22
  • CD44-variants 11.24, 1 1.31 , 1 1 .10
  • E-cadherin BTC 111 HECD-1 , 6F9
  • Gastrointestinal cancer associated antigen (MW 200 kD) CA 19-9
  • MOC-31 epitope cluster 2 epithelial antigen MOC-31 , NrLu10
  • MUC-1 antigens such as DF3-epitope (gp290 kD)
  • Ovarian carcinoma OC125 epitope (MW 750 kD) OC125
  • Colon antigen Co17-1 A (MW 37000) 17-1 A G9-epitope (colon carcinoma) G9
  • Hepatocellular carcinoma antigen MW 900 kD KM-2
  • Hepernal epitope (gp43) Hepatocellular care, ag Hepema-1 O-linked mucin containing N-glycolylneuraminic acid 3E1.2
  • CAK1 (ovarian cancers) K1 Colon specific antigen p Mu-1 , Mu-2
  • ANTIGENS MONOCLONAL ANTIBODIES gp85 bladder carcinoma antigen T43 gp25 bladder carcinoma antigen T138 Neuroblastoma antigens
  • Neuroblastoma-associated such as UJ13A epitope UJ13A Glioma antigens
  • Apo-1 epitope Apo 1 Various Plasminogen activator antigens & receptors Rabbit polyclonal p-glycoprotein C219, MRK16, JSB-1 , 265/F4 cathepsin D CIS-Diagnostici, Italy biliary epithelial antigen HEA 125 neuroglandular antigen (CD63) ME491. NKI-C3, LS62 CD9 TAPA-1.
  • target cells may be separated by a two dimensional or dual parameter immunomagnetic cell separation method such as that described in, for example, Partington, et al., A Novel Method of Cell Separation Based on Dual Parameter Immunomagnetic Cell Selection, J. of Immuno. Mthds., 223: 195-205, 1999, which is incorporated by reference in its entirety herein.
  • the tagging and separation of target cells with biomagnetic particles may be performed within the fluidic circuits in the MO bio-disc.
  • the first step of separation may be the positive selection of cells utilising 50nm beads.
  • the strength of the magnetic field during the second separation step is modulated such that the magnetic force generated on the MO disc magnetic domain is sufficient to attract only the larger Dynabeads but not the 50nm beads.
  • This dual parameter embodiment thus provides a better method for isolating and purifying cells of interest.
  • target cells may be labeled or tagged with magnetic particles by ballistic transfer.
  • the ballistic transfer technology employs a cold gas shock wave to accelerate microprojectiles that carry matter into cells by mechanical force.
  • This embodiment of the present invention relates to a method that enables separation of target cells rendered magnetically susceptible and can thus be separated by retaining them in a strong magnetic field.
  • the tagged cells may then be captured, analyzed, manipulated, and quantitated using the MOBDS. Further details relating to ballistic tagging of cells with magnetic particles is disclosed in, for example, U.S. Patent Serial No. 6,348,338 to Wittig, which is herein incorporated in its entirety by reference.
  • a further embodiment of the current invention is using the MOBDS to perform cellular analysis, manipulation, and quantitation by immunomagnetic cell selection, as described above, and aligning the target cells in various pre-determined configurations on the MO bio-disc such as alignment of cells on the tracks of the MO bio-disc.
  • cells are magnetically sorted and aligned on the MO bio-disc, and analyzed using the MO disc drive and associated software.
  • Related methods and systems for immunomagnetic cellular analysis are described in, for example, Tibbe et al., Cytometry, 43:31-37, 2001 , and Tibbe et al., Cytometry, 47:163-172, 2002, both of which are herein incorporated by reference in their entireties.
  • the isolated target cells may be characterized for the presence of specific biochemical and biological features. Of particular importance will be the use of such cells for studies in molecular biology.
  • the present method allows studies and growth of the target cells without performing a cleavage of the magnetic particle-target cell linkage.
  • tumor biopsies for example urine, cerebrospinal fluid, semen, lymph, or from otherwise normal tissues and organs, for example liver, lymph nodes, spleen, lung, pancreas, bone tissues, central nervous system, prostatic gland, skin and mucous membranes, and in other areas of cytological assays.
  • body fluids for example urine, cerebrospinal fluid, semen, lymph, or from otherwise normal tissues and organs, for example liver, lymph nodes, spleen, lung, pancreas, bone tissues, central nervous system, prostatic gland, skin and mucous membranes, and in other areas of cytological assays.
  • signals obtained on Southern, Northern and Western blots represent the normal cells as well as the tumor cells in the biopsy. If a single cell suspension is first prepared from the tumor material, and the tumor cells are then positively immunomagnetically detected and separated using the MOBDS of the present invention, any gene studies performed on this material would represent the target cells only.
  • This also relates to, for example, malignant cells present in mammalian tissues including bone marrow, peripheral blood, pleural and peritoneal effusions, and other body fluids, for example urine, cerebrospinal fluid, semen and lymph. Studies involving polymerase chain reaction (PCR) and microarray methodologies will also gain increased specificity and reliability when performed on pure target cell populations obtained using the methods and apparatus of the present invention.
  • neurons may be isolated and stimulated to grow and regenerate in biological matrix or solution in a MO bio-disc.
  • the biological matrix may be formed from a gel or any biologically compatible material that provides a solid support for the cells while allowing passage of essential nutrients and gases to enable survival of the cells.
  • the biological matrix also allows growth and movement of the cells and its components such as axon and dendrite movement within the matrix. The direction and rate of growth of the axons, dendrites, and cells may be controlled by a magnetic field on the MO bio-disc, generated by the MO drive.
  • neurites may be provided with magnetic nanoparticles or beads that are absorbed or actively incorporated into the neurons and their axons. Arrays of these neurons are then aligned in predetermined locations within the biological matrix on the MO bio-disc. The aligned neurons or neurites are then exposed to a magnetic field that is displaced to thereby physically move the magnetic particle-loaded neurons and their axons along a desired axis for bridging the gap or gaps between the neurons in the array. Since magnetic regions generated on the MO disc are relatively small and can be precisely controlled, use of the magnetically directed neurite growth embodiment of the present invention allows a more precise control of neurite growth relative to methods of the prior art.
  • Magnetically directed neurite growth in vitro and nerve regeneration in vivo are well known in the art (Moorman et al., Brain Res. Bulletin, 35(5-6):419-422, 1994, Dubey et al., Exp. Neuro., 158:338-350, 1999, Macias et al., Bioelectromagnetics, 21 :272-286, 2000, Shah et al., Bioelectromagnetics, 22:267-271 , 2001 , and U.S. Patent No. 6,132,360 to Halpern, all of which are herein incorporated by reference in their entireties).
  • This embodiment of the present invention is directed at using the MOBDS to study precise magnetic control of neurite growth in vitro and at creating neural networks in vitro.
  • Fig. 76 there is illustrated a top plan view of a portion of a magneto-optical bio-disc having fluidic circuits including an inlet port 152, a mixing chamber 164, a separation or analysis chamber 300, and testing chambers 302.
  • the magneto-optical bio-disc may include one or more components of the bio-discs described above in conjunction with Figs. 3A to 3C, 4A to 4C, 33A to 33C, 35, 36A- 36C, and 37.
  • test cells 306 are loaded into the mixing chamber 164 through the inlet port 152 using a pipette 214 (Fig. 77A).
  • Bio-magnetic particles 308 are then loaded into the mixing chamber 164 using a pipette 214.
  • the bio-magnetic particles are coated with binding agents specific for surface markers on the cells of interest in the sample.
  • the binding agents may include, for example, antibodies outlined above in Table 1.
  • the cells 306 and the bio-magnetic particles are then incubated for a sufficient time to allow binding of the bio-magnetic particles 308 to the cells of interest through binding of the binding agents and the cell surface markers. After incubation, cells of interest or target cells are thus labeled with bio-magnetic particles resulting in a labeled cell 310. This renders the labeled cells 310 susceptible to magnetic manipulation.
  • Magnetic domains or regions 246 may also be formed in the separation chamber 300 before of after the incubation using a magneto-optical drive. Details relating to the formation of magnetic domains 246 on magneto-optical bio-discs are discussed above in conjunction with Fig. 37.
  • the disc is rotated at a pre-determined speed and duration, using the magneto-optical drive, to move the suspension containing unlabeled cells 306, biomagnetic particles 308, and labeled cells 310 into the separation chamber 300 (Fig. 77B).
  • the labeled cells 310 and free or unattached bio-magnetic particles 308 magnetically bind to the magnetic domains 243 within separation chamber 300.
  • the disc is then rotated at another pre-determined speed and time to move unbound and unlabeled cells 306 in the suspension to the bottom of the separation chamber 300 (Fig. 77C).
  • the magnetic domains 246 are then sequentially erased and formed to selectively release the magnetically bound labeled cells 310 (Fig. 77D).
  • the labeled cells 310 are then guided magnetically to one or more testing chambers 302 by sequentially erasing and forming the magnetic domains 246 (Fig. 77E).
  • the testing chamber 302 may be preloaded with a test solution having a test agent including, but not limited to, chemotherapeutic agents, antibiotics, and anti-viral medications.
  • the labeled cells 310 may then be incubated with the test agent.
  • a beam of electromagnetic radiation may then be scanned through the testing chamber 302 to quantify the live and apototic cells and thereby determine the sensitivity of the cells when exposed to the test agent.
  • Apototic cells are characterized by membrane blebbing, cell shrinkage, protein fragmentation, chromatin condensation and DNA degradation, all of which alter the optical properties of the cell rendering it distinguishable from non-apototic cells using an optical disc reader.
  • 09/988,850 entitled “Methods and Apparatus for Blood Typing with Optical Bio-discs” filed on November 19, 2001
  • U.S. Patent Application Serial No. 09/989,684 entitled “Apparatus and Methods for Separating Agglutinants and Disperse Particles” filed November 20, 2001
  • U.S. Patent Application Serial No. 09/997,741 entitled “Dual Bead Assays Including Optical Biodiscs and Methods Relating Thereto” filed November 27, 2001
  • U.S. Patent Application Serial No. 09/997,895 entitled “Apparatus and Methods for Separating Components of Particulate Suspension” filed November 30, 2001
  • the dual assay in carried out to detect the gene sequence DYS that is present in male but not in female.
  • the assay is comprised of 3 ⁇ magnetic and capture beads coated with covalently attached capture probe; 2.1 ⁇ fluorescent reporter beads coated with a covalently attached sequence specific for the DYS gene, and target DNA molecule containing DYS sequences.
  • the target DNA is a synthetic 80 oligonucleotide sequence.
  • the capture probe and reporter probes are 40 nucleotides in length and are complementary to DYS sequence but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 microgram per milliliter Salmon Sperm DNA for 1 hr. at room temperature. This pretreatment will reduce non-covalent binding between the capture and reporter beads in the absence of target DNA as shown in Fig. 38.
  • the capture beads were concentrated magnetically with the supernatant being removed.
  • a 100 microliter volume of the hybridization buffer (0.2 M NaCl, 1 mM EDTA, 10 mM MgCI 2 , 50 mM Tris HCI, pH 7.5, and 5X Denhart's mixture, 10 microgram per milliliter denatured salmon sperm DNA) were added to the capture beads and the beads were re-suspended.
  • Various concentrations of target DNA ranging from 1, 10, 100, 1000 femtomoles were added while mixing at 37°C for 2 hours.
  • the beads were magnetically concentrated and the supernatant containing target DNA was removed.
  • a 100 microliter volume of wash buffer (145mM NaCl, 50 mM Tris pH 7.5, 0.1 % SDS, 0.05 % Tween, 0.25 % NFDM, 10 mM EDTA) was added and the beads were re-suspended. The beads were magnetically concentrated and the supernatant was again removed. The wash procedure was repeated twice.
  • a 2x10 7 amount of reporter beads in 100 microliter hybridization buffer ( 0.2 M NaCl, 1 mM EDTA, 10 mM MgCI 2 , 50 mM Tris HCI, pH 7.5, and 5X Denhart's mixture, 10 microgram per milliliter denatured salmon sperm DNA) were then added to washed capture beads.
  • the beads were re-suspended and incubated while mixing at 37°C for an additional 2 hours. After incubation the capture beads were concentrated magnetically, and the supernatant containing unbound reporter beads were removed.
  • wash buffer 145mM NaCl, 50 mM Tris pH 7.5, 0.1 % SDS, 0.05 % Tween, 0.25 % NFDM, 10 mM EDTA
  • the beads were re-suspended in 20 microliters of binding buffer (50 mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1% BSA). A 10 microliter volume was loaded on to the disc that was prepared as described below in Part B of this example.
  • binding buffer 50 mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1% BSA.
  • a gold disc was coated with maleic anhydride polystyrene.
  • An amine DNA sequence complementary to the reporter probes (or capture agent) was immobilized on to the discrete reaction zones on the disc.
  • the channels Prior to sample injection, the channels were blocked with a blocking buffer (50mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1 % BSA, 1% sucrose) to prevent non-covalent binding of the dual bead complex to the disc surface.
  • a blocking buffer 50mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1 % BSA, 1% sucrose
  • a capture zone could be created with the capture agent such as BSA Biotin which could be immobilized on to the disc (pretreated with Polystyrene) by passive absorption.
  • a perspective view of the disc assembly showing the use of biotin capture agents is presented in Figs. 26A-26D.
  • Various methods for use in this type of anchoring of beads onto the disc are also shown in Figs. 15A-15B, 17, 19A-19C, and 23A-23B.
  • a 10 microliter volume of the dual bead mixture prepared as described in Part A above was loaded in to the disc chamber and the injection ports were sealed.
  • the disc was centrifuged at low speed (less than 800 rpm) upto 15 minutes.
  • the disc was read in the CD reader at the speed 4X (approx. 1600 rpm) for 5 minutes. Under these conditions, the unbound magnetic capture beads were centrifuged away from the capture zone.
  • the magnetic capture beads that were in the dual bead complex remained bound to the reporter beads in the capture zone.
  • the steps involved in using the disc to capture and analyze dual bead complexes are presented in detail in Figs. 25A-25D, 26A-26D, and 27A-27D.
  • the amount of target DNA captured could be enumerated by quantifying the number of capture magnetic beads and the number of reporter beads since each type of bead has a distinct signature.
  • the dual bead assay is carried out to detect two DNA targets simultaneously.
  • the assay is comprised of 3 ⁇ m magnetic capture bead.
  • One population of the magnetic capture bead is coated with capture probes 1 which are complementary to the DNA target 1
  • another population of magnetic capture beads is coated with capture probes 2 which are complementary to the DNA target 2.
  • two different types of magnetic capture beads may be used.
  • Various combinations of beads that may be used in a multiplex dual bead assay format are depicted in Fig. 32.
  • One type of reporter bead is coated with reporter probes 1 , which are complementary to the DNA target 1.
  • the other reporter beads are coated with reporter probes 2, which are complementary to the DNA target 2. Again the capture probes and the reporter probes are complementary to the respective targets but not to each other.
  • the specific methodology employed to prepare the dual bead assay multiplexing involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pretreatment will reduce non-covalent binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads were concentrated magnetically with the supernatant being removed.
  • a 100 microliter volume of the hybridization buffer (0.2 M NaCl, 1 mM EDTA, 10 mM MgCI 2 , 50 mM Tris HCI, pH 7.5, and 5X Denhart's mixture, 10 microgram per milliliter denatured salmon sperm DNA) were added and the beads were re-suspended.
  • Various concentrations of target DNA ranging from 1 , 10, 100, 1000 femto moles were added to the capture beads suspension. The suspension was incubated while mixing at 37°C for 2 hours. The beads were magnetically concentrated and the supernatant containing target DNA was removed. A 100 microliter volume of wash buffer (145mM NaCl, 50 mM Tris pH 7.5, 0.1 % SDS, 0.05 % Tween, 0.25 % NFDM, 10 mM EDTA) was added and the beads were re-suspended. The beads were magnetically concentrated and the supernatant was again removed. The wash procedure was repeated twice. A 2x10 7 amount of reporter beads in 100 microliter hybridization buffer (0.2 M
  • a gold disc was coated with maleic anhydride polystrene as described. Distinct reaction zones were created for two types of reporter beads. Each reaction zone consisted of amine DNA sequences complementary to the respective reporter probes (or capture agents). Prior to sample injection, the channel were blocked with a blocking buffer (50mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1 % BSA, 1 % sucrose) to prevent non-covalent binding of the dual bead complex to the disc surface.
  • a blocking buffer 50mM Tris, 200mM NaCl, 10 mM MgCI 2 , 0.05% Tween 20, 1 % BSA, 1 % sucrose
  • magnetic beads employed in a multiplexing dual bead assay format may be detected using a magneto-optical disc and drive.
  • the chemical reaction zones, in the magnetic disc format are replaced by distinctly spaced magnetic capture zones as discussed in conjunction with Fig. 37, see below Examples 5 and 6.
  • the disc was centrifuged at low speed (less than 800 rpm) for up to 15 minutes.
  • the disc was read in the CD reader at the speed 4X (approx. 1600 rpm) for 5 minutes. Under these conditions, the unbound magnetic capture beads were centrifuged to the bottom of the channels.
  • the reporter beads bound to the capture zone via hybridization between the reporter probes and their complementary agent.
  • the sensitivity of the dual bead assay depends on the strength of the target mediated-bonds holding the dual beads together.
  • the dual beads are held together by hydrogen bonds. The strength of the bond would increase significantly if the bond holding the dual beads is covalent.
  • a ligation reaction is carried out to create a covalent bond between the capture and reporter probes as illustrated above in Fig. 38.
  • the 5' end of the reporter probe carries a phosphate group which is required in the ligation reaction.
  • the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL) coated with covalently attached capture probes; 2.1 ⁇ m fluorescent reporter beads (Molecular Probes, Eugene, OR) coated with a covalently attached sequence specific for the DYS gene, and target DNA molecules containing DYS sequences.
  • the target DNA is a synthetic 80 oligonucleotide long.
  • the capture probes and reporter probes are 40 nucleotides in length and are complementary to the DYS sequence but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads were concentrated magnetically with the supernatant being removed. Then 100 ⁇ l of the hybridization buffer (0.2M NaCl, 1 mM EDTA, 10 ⁇ mM MgCI 2 , 50mM Tris- HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) was added and the beads were resuspended.
  • Various concentration of target DNA ranging from 1 , 10, 100, and 1000 femtomoles were added to the capture bead suspensions.
  • the beads suspension was incubated while mixing at 37 degrees Centigrade for 2 hours.
  • the beads were magnetically concentrated and the supernatant containing unbound target DNA was removed.
  • One hundred microliters of wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) was added and the beads were resuspended.
  • the beads were magnetically concentrated and the supernatant was again removed.
  • the wash procedure was repeated twice.
  • a 2x10 7 amount of reporter beads in 100 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) was then added to washed capture beads.
  • the beads were resuspended and incubated while mixing at 37 degrees Centigrade for an additional 2 hours. After incubation, the capture beads were concentrated magnetically, and the supernatant containing unbound reporter beads were removed.
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • the beads were resuspended in 20 ⁇ l of binding buffer (50mM Tris, 200mM NaCl, 10mM MgCI 2 , 0.05% T-20, 1% BSA). Then 10 ⁇ l was loading onto the bio-disc which was prepared as described above in Example 2, Part B.
  • binding buffer 50mM Tris, 200mM NaCl, 10mM MgCI 2 , 0.05% T-20, 1% BSA.
  • Capture Beads The specific methodology employed to prepare the above assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA. The capture beads were concentrated magnetically with the supernatant being removed. Then 100 ⁇ l of the hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) was added and the beads were resuspended.
  • the hybridization buffer 0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA
  • a 2x10 7 amount of reporter beads or 100 pmoles of reporter probes in 100 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA ) was then added to washed capture beads. The beads were resuspended and incubated while mixing at
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1 % SDS, 0.05% Tween, 0.25% NFDM
  • the beads were magnetically concentrated and the supernatant was again removed.
  • the amount of reporter probe was directly correlated with the amount of target DNA captured. Therefore, one way to quantify the target captured was to quantify the amount of reporter probe.
  • the rationale for this assay is that the reporter probe was biotinylated. The concentrations of the reporter probe therefore could be determined by an enzyme assay wherein the enzyme Streptavidin-Alkaline phosphatase binds to the biotin moiety.
  • a chromogenic substrate for Alkaline phosphatase, p-nitrophenyl phosphate was used as reporter. This colorless substrate is hydrolyzed by alkaline phosphatase to a yellow product which has an absorbance at 405nm.
  • the beads were washed with 100 ⁇ l of CDB (2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, 1.0 mM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ) and incubated with 100 ⁇ l of 250ng/ml Streptavidin-Phosphatase for 1 hour at 37°C.
  • the beads were washed 3 times with wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.05% Tween) to get rid of unbound S- AP.
  • the beads were incubated with 100 ⁇ l of the S-AP substrate p-nitrophosphate at 3.7mg/ml in 0.1 M Tris, pH 10, 2mM MgCI 2 for 5-15 minutes at room temperature.
  • the color development of the supernatant was monitored at 405nm. The intensity of the color is directly correlated with the amount of the biotinylated reporter probe and thus the amount of target captured.
  • cleavable spacers in dual bead assay increases the specificity of the assay.
  • the following example is directed to a dual bead assay using cleavable spacers.
  • the design of capture probes and reporter probes is critical in the success of the dual bead assay using cleavable spacers.
  • the capture probes and reporter probes contain 3 branches as illustrated above in Fig.
  • One branch of the reporter or capture probes participates in the target capture.
  • Several linkers PEG groups
  • the second branch of the capture or reporter probes contains 3 linkers followed by a biotin at the end.
  • Other functional groups such as carboxyl or amine could also be used.
  • the biotin participates in the conjugation of the capture or reporter probes onto the solid phase.
  • the third branch of the capture probe hybridizes to the reporter probe.
  • restriction enzyme digestion is the method of choice for cleaving the capture and reporter probes
  • a restriction site is introduced into the sequences of the probes.
  • the choice of restriction site is important in that it has to be unique (not common) so that only the sequence holding the capture and reporter probes (and not the target DNA) is cleaved.
  • the formation of the capture and reporter probes in the presence of the target is shown above in Fig. 42C.
  • the first step in the assay is the conjugation of the capture probe onto a solid phase.
  • 2.8 ⁇ m magnetic beads coated with streptavidin from Dynal were used as the solid phase.
  • 6.7x10 7 Dynal beads were used per conjugation.
  • the beads were resuspended in 200 ⁇ l of binding and washing buffer (10mM Tris-HCI, pH 7.5, 1 mM EDTA, 2M NaCl). The beads were magnetically concentrated and the supernatant was removed. The wash procedure was repeated twice.
  • the magnetic beads were washed 3 times with binding and washing buffer (10mM Tris-HCI, pH 7.5, 1 mM EDTA, 2M NaCl) and resuspended in 1000 ⁇ l hybridization buffer (0.2M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris, pH 7.5).
  • Amount of biotinylated capture probe 1 bound to 1 mg of bead 127 pmoles or 8 x 10 13 molecule
  • Hybridization Out of the 1000 ⁇ l bead suspension, 400 ⁇ l was mixed with 400 ⁇ l TE buffer containing 1 nanomole of reporter probe 2A, 400 ⁇ l was mixed with 400 ⁇ l TE buffer containing 1 nanomole of reporter probe 2B, 200 ⁇ l was mixed with 200 ⁇ l TE (Tris-EDTA) as a negative control. The hybridization was carried out at 37°C for 2 hours.
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.05% Tween
  • the beads were washed with 100 ⁇ l of CDB (2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, LOmM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ) and incubated with 100 ⁇ l of 250ng/ml Streptavidin-Phosphatase for 1 hour at 37°C.
  • the beads were washed 3 times with wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.05% Tween) to get rid of unbound S-AP.
  • the beads were incubated with 100 ⁇ l of the S-AP substrate p-nitrophosphate at 3.7mg/ml in 0.1 M Tris, pH 10, 2mM MgCI 2 for 5-15 minutes at room temperature.
  • the color development of the supernatant was monitored at 405nm.
  • the intensity of the color is directly correlated with the amount of the biotinylated reporter probe 2A or 2B hybridized.
  • the reporter probes could be attached to another solid phase via their biotin moiety.
  • a different type of streptavidin coated beads i.e. polystyrene or fluorescent, is added to the bead suspension, resulting in the formation of the dual bead complexes. If the solid phase is the surface of the bio-disc, then the mixture of capture and reporter probes is incubated on a streptavidin coated disc surface.
  • Hybridization In this example, the target DNA was a single stranded 80mer oligonucleotide. Various concentrations of target DNA ranging from 0, 1 , and 1000 picomoles were added to the bead suspensions. The beads suspensions were incubated while mixing at 37 degrees Centigrade for 2 hours. 2. Washing: The beads were magnetically concentrated and the supernatant containing unbound target DNA was removed. One hundred microliters of wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) was added and the beads were resuspended. The beads were magnetically concentrated and the supernatant was again removed. The wash procedure was repeated twice.
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • Restriction enzyme digestion The restriction enzyme site that was introduced in the capture and reporter probes was NOT1. This restriction enzyme site is rare and in this model system is not found in any other sites.
  • the beads were resuspended in 400 ⁇ l CDB (2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, 1.0 mM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ).
  • the bead suspension was aliquoted into seven tubes, one control and 6 digestion tubes.
  • the enzyme NOT1 was prepared according to the manufacturer's specifications. Then 5 units of enzyme were added to the each digestion tubes in a total volume of 100 ⁇ l. Water was added to the control tube. The digestion was carried out for 3-4 hours at 37°C.
  • Displacement of the reporter probe by the displacement probe The beads were resuspended in 400 ⁇ l CDB (2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, 1.0 mM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ). The bead suspension was aliquoted into two tubes, one control and one displacement tube. The beads were heated for 5 minutes at 55°C in 200 ⁇ l of 6xSSC, 1 mM EDTA. The heat treatment was used to induce the melting of the reporter probe 2B from the capture probe. At this point, a 10 fold excess of displacement probe was added to the bead suspension and the mixture was incubated at 37°C for several hours Water was added to the control tube.
  • CDB 2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, 1.0 mM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ).
  • the amount of reporter probe remaining after the restriction enzyme digestion or probe displacement was directly correlated with the amount of target DNA captured. Therefore, one way to quantify the target captured was to quantify the amount of remaining reporter probe.
  • the rationale for this assay is that the reporter probes 2A and 2B were biotinylated. The concentrations of these probes therefore could be determined by an enzyme assay wherein the enzyme Streptavidin-Alkaline phosphatase binds to the biotin moiety.
  • the beads were washed with 100 ⁇ l of CDB (2% BSA, 50mM Tris-HCI, pH 7.5, 145mM NaCl, 1.0 mM MgCI 2 , 0,1 mM ZnCI 2 , 0.05% NaN 3 ) and incubated with 100 ⁇ l of 250ng/ml Streptavidin-Phosphatase for 1 hour at 37°C.
  • the beads were washed 3 times with wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.05% Tween) to get rid of unbound S-AP.
  • the beads were incubated with 100 ⁇ l of the S-AP substrate p-nitrophosphate at 3.7mg/ml in 0.1 M Tris, pH 10, 2mM MgCI 2 for 5-15 minutes at room temperature.
  • the color development of the supernatant was monitored at 405nm. The intensity of the color is directly correlated with the amount of the biotinylated reporter probe 2A or 2B hybridized.
  • the amount of target captured could be quantified by dual bead assay.
  • the number of reporter beads remaining following restriction enzyme digestion or probe displacement could be enumerated by the fluorimeter (for fluorescent beads) or by the bio-CD reader since each type of bead has a distinct signal signature.
  • EXAMPLE 5 The following example illustrates a dual bead assay carried out on a magnetically writable and erasable analysis disc such as the magneto-optical bio-disc 110 discussed in conjunction with Fig. 37.
  • the dual bead assay is carried out to detect the gene sequence DYS which is present in male but not female.
  • the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL) coated with covalently attached transport probes; 2.1 ⁇ m fluorescent reporter beads (Molecular Probes, Eugene, OR) coated with a covalently attached sequence specific for the DYS gene, and target DNA molecules containing DYS sequences.
  • the target DNA is a synthetic 80 oligonucleotides long.
  • the transport probes and reporter probes are 40 nucleotides in length and are complementary to the DYS sequence but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA. After pretreatment with salmon sperm DNA, the capture beads are loaded inside the MO bio-disc via the injection port. The MO bio-disc contains magnetic regions created by the magneto optical drive. The capture beads thus are held within specific magnetic regions on the MO bio-disc.
  • the sample containing target DNA and reporter beads in 200 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) is then added to the MO bio- disc via the injection port.
  • the injection port is then sealed.
  • the magnetic field is released.
  • the disc is rotated at very low speed (less than 800rpm) in the drive to facilitate hybridization of target DNA and reporter beads to the capture beads.
  • the temperature of the drive is kept constant at 33 degrees Centigrade. After 2 hours of hybridization, the magnetic field is created by the magneto optical drive.
  • Unbound target and reporter beads are directed to a waste chamber by any of the mechanisms described above.
  • Two hundred microliters of wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) is then added.
  • the magnetic field is released and the disc is rotated at low speed (less than 800rpm) for 5 minutes to remove any non-specific binding between the capture beads and reporter beads.
  • the magnetic field is then reapplied.
  • the wash buffer is directed to the waste chamber by any of the mechanisms described above. The wash procedure is repeated twice.
  • a dual bead assay using the multiplexing techniques described above in connection with Figs. 32 and 37 is carried out on a magnetically writable and erasable analysis disc such as the MO bio-disc 110 discussed with reference to Fig. 37.
  • the dual bead assay is carried out to detect 2 or more DNA targets simultaneously.
  • the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL).
  • One population of the magnetic capture beads is coated with transport probes 1 which are complementary to the DNA target 1.
  • Another population of the magnetic capture beads is coated with transport probes 2 which are complementary to the DNA target 2.
  • 2 or more different types of magnetic capture beads may be used.
  • the reporter beads may differ by chemical composition (for example silica and polystyrene) and/or by size.
  • One type of reporter beads is coated with reporter probes 1 , which are complementary to the DNA target 1.
  • the other reporter beads are coated with reporter probes 2, which are complementary to the DNA target 2.
  • the transport probes and reporter probes are complementary to the respective targets but not to each other.
  • the specific methodology employed to prepare the dual bead assay multiplexing involved treating 1x10 7 capture beads and 2x10 7 reporter beads in
  • the capture beads are loaded in the MO bio-disc.
  • the magnetic field is applied to create distinct magnetic zones for specific capture beads.
  • the capture beads can be held on the MO bio-disc at a density of 1 capture bead per 10 ⁇ m 2 .
  • the surface area usable for bead deposition on the MO bio-disc is approximately 3 x 10 9 ⁇ m 2 .
  • the capacity of the MO bio-disc for 3 ⁇ m beads at the given density is about 3 x 10 8 beads.
  • the sample containing the targets DNA of interest is mixed with different types of reporter beads in 200 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) and added to the MO bio-disc via the injection port.
  • the injection port is then sealed.
  • the magnetic field is released.
  • the disc is rotated at very low speed (less than 800rpm) in the drive to facilitate hybridization of targets DNA and reporter beads to the different types of capture beads.
  • the temperature of the drive is kept constant at 33 degrees Centigrade. After 2 to 3 hours of hybridization, the magnetic field is regenerated by the magneto optical drive.
  • the magnetic field is released and the dual bead complexes are directed to a detection chamber.
  • the amount of different types of target DNA can be enumerated by quantifying the number of corresponding capture magnetic beads and reporter beads since each type of bead has a distinct signature as shown above in Figs. 28A, 28B, 29A, and 29B.
  • EXAMPLE 7 This experiment was performed to determine the amount of covalently conjugated probe on different beads to determine which bead type is best for covalent probe linking.
  • the probes were allowed to conjugate for 2-3 hours at room temperature on a rotating mixer.
  • the beads were then magnetically concentrated and the supernatant was collected.
  • the optical density (at 260nm) of the supernatant was measured before and after the conjugation.
  • the beads were resuspended in 100 ⁇ l CDB containing 550ng/ml streptavidin-alkaline phosphatase (S-AP) and incubated for 1 hour at 37° C to allow sufficient time for the streptavidin to bind to the biotin on the probe. Following incubation with S-AP, the beads were magnetically concentrated, and the supernatant containing unbound S-AP was removed. The beads were washed three times in wash buffer.
  • S-AP streptavidin-alkaline phosphatase
  • pNPP p-nitrophenylphosphate
  • a substrate for alkaline phosphatase at a concentration of 3.7 mg/ml in 0.1 M Tris- HCI, pH 10 was added to the beads at fixed time intervals to minimize the variation due to difference in incubation time.
  • the incubation time with the substrate was varied (from 2 min upto 30 min) as needed to obtain reliable OD at 405 nm since time for color development varies depending upon the concentration of probe.
  • the optical density obtained from a spectrophotometer at 405nm wavelength was proportional to the amount of probes bound to the beads.
  • the results of the experiment are presented in Figs. 51 A and 51 B. As indicated, 87% of the probes that bound to the 1-2 ⁇ m magnetic beads from Polysciences were non covalently bound, as compared to 15% of non-covalently bound probes on the 3 ⁇ m Spherotech beads.
  • Figs. 53A and 53B data showing a correlation between the covalent conjugation efficiency and the sensitivity of the dual bead assay is presented. These results indicate that with higher covalent conjugation efficiency, the more sensitive and specific the dual bead assay is.
  • the amount of covalenty bound probes may be calculated by repeating steps in this Part B after performing the steps in Part C below. The calculation of the amount of covalent binding is presented in Fig. 55.
  • the steps in Parts A and B above may be repeated using non-biotinylated probes and the appropriate bead type for use in a dual bead assay.
  • the non-covalently bound probes could be selectively removed by heat treatment of the beads. For this purpose, up to 3x10 7 beads were resuspended in 100 ⁇ l of CDB solution heated at 70°C for 10 minutes. The beads were then immediately magnetically concentrated and the supernatant was removed. The beads were washed twice in wash buffer and once in CDB and re-suspended in 10O ⁇ l CDB. At this point the beads are now ready for use in a dual beads test.
  • the capture probe utilized was 40 nucleotides in length and contained an aminogroup (NH 2 ) at the 5' end and several chains of PEG (polyethylene glycol) linker.
  • the strand complimentary to the aminated probe used in this experiment was 40 nucleotides in length and contained a biotin group at the 5' end.
  • a hybridization reaction was carried out with an excess of complementary probes under stringent conditions at 37°C.
  • the beads were resuspended with 100 ⁇ l of buffer containing 550ng/ml streptavidin-alkaline phosphatase (S-AP) and incubated for 1 hour at 37° C. Following incubation with S-AP, the beads were magnetically concentrated, and the supernatant containing unbound S-AP was removed. The beads were washed three times in wash buffer. Next 100 ⁇ l of p- nitrophenylphosphate (pNPP), a substrate for alkaline phosphatase at a concentration of 3.7 mg/ml, was added to the beads at fixed time intervals to minimize the variation due to difference in incubation time. The time for color development varies depending upon the concentration of probe.
  • pNPP p- nitrophenylphosphate
  • the incubation time with the substrate was varied from 2 min up to 30 min as needed to obtain reliable OD at 405 nm.
  • the optical density at 405nm was proportional to the amount of probes bound to the beads.
  • the beads were washed three times in wash buffer (0.145M NaCl, 50 mM Tris-HCI pH 7.5, 0.5% Tween-20), once with hybridization buffer ( 50mM Tris-HCI pH 7.5, 0.1 M NaCl, 10mM MgCI, 1 mM EDTA pH 7.5) and re-suspended in hybridization buffer containing 100 ⁇ g/ml DNA, and 5X Denhart's mixture.
  • wash buffer 0.145M NaCl
  • hybridization buffer 50mM Tris-HCI pH 7.5, 0.1 M NaCl, 10mM MgCI, 1 mM EDTA pH 7.5
  • hybridization buffer 50mM Tris-HCI pH 7.5, 0.1 M NaCl, 10mM MgCI, 1 mM EDTA pH 7.5
  • Different concentrations of a single target were used including Control (0 femtomole), 10 femtomole, 1 femtomole, 0.1 femtomole, 0.01 femtomole, 0.001 femtomole, 0.0001 femtomole diluted in hybridization buffer containing 100 ⁇ g/ml of salmon sperm DNA and 5X Denhart's solution.
  • the various target solutions were then mixed with the capture beads and incubated at 37° C for 2 hours to allow ample time for target hybridization to the capture probe on the beads.
  • hybridized capture beads were washed three times with wash buffer, once with hybridization buffer, and resuspended in 100 ⁇ l hybridization buffer including 100 ⁇ g/ml DNA, and 5X Denhart's mixture.
  • the capture bead solution, containing hybridized target was then mixed with 100 ⁇ l of reporter beads and incubated at 37°C for 2 hours while continuously mixing. Then washed 6 times with new wash buffer (145mM NaCl, 50mM Tris-HCI pH 7.5, 05 % Tween 20, 0.1 % SDS, 0.25% NFDM) and once with PBS.
  • the washed solution containing the dual bead complexes was then re-suspended with 250 ⁇ l PBS.
  • the fluorescent signal from the reporter beads were then quantified using a fluorimeter.
  • the beads used in this experiment were magnetic capture beads (3um Spherotech) and yellow-fluorescent reporter beads (1 um Polysciences) each covalently conjugated with DNA transport probes and DNA signal probes, respectively. Approximately 1x10 7 capture beads and 2x10 7 reporter beads were used for this experiment. These beads were washed 3X with PBS and resuspended in 1 ml water containing 100 ⁇ g/ml digested salmon sperm DNA. The bead solutions in the salmon sperm DNA mixture were then incubated for 1 hour at room temperature.
  • hybridization buffer 0.1M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris, pH 7.5. The beads were then resuspended in hybridization buffer (containing 100 ⁇ g/ml of DNA).
  • Dual Bead Assay A serial dilution of DNA target agents were prepared containing: 100 femtomole, 10 femtomole, 1 femtomole, 0.1 femtomole, 0.01 femtomole, and 0 femtomoles (negative control). Equal amounts of capture beads were then mixed with the various solutions of target and incubated at 37°C for 2 hours to let target hybridized to the 5' capture probe on the beads.
  • hybridization buffer 0.1 M NaCl, 10mM MgCI 2 , 1 mM EDTA, 50mM Tris, pH 7.5, then resuspended in hybridization buffer (containing 100 ⁇ g/ml salmon sperm DNA).
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.05% Tween, 0.1 % SDS, 0.25% NFDM
  • EXAMPLE 11 This study was performed to determine the optimal salt concentration in the hybridization buffer for use in a genetic dual bead type assay.
  • the beads used in this experiment were magnetic capture beads (3um Spherotech) and yellow-fluorescent reporter beads beads (2.1 um from Molecular Probes) each covalently conjugated with DNA transport probes and DNA signal probes, respectively.
  • the beads were washed 1X with hybridization buffer (0.1 M NaCl, 10 mM MgCI 2 , 1 mM EDTA, 50mM tris, pH 7.5).
  • the beads were pretreated with 0.1% CHAPS and salmon sperm DNA for 1 hour at room temp.
  • the beads were then washed 3X with wash buffer (145mM NaCl, 50 mM Tris, pH 7.5, 0.1 % SDS, 0.05% Tween, 0.25% NFDM), 1X with respective hybridization buffer (145mM NaCl, 10mM MgCI 2 , I mMEDTA, 100 ⁇ g/ml salmon sperm DNA, 50 mM Tris, pH 7.5). After washing, the beads were resuspended in hybridization buffer.
  • wash buffer 145mM NaCl, 50 mM Tris, pH 7.5, 0.1 % SDS, 0.05% Tween, 0.25% NFDM
  • respective hybridization buffer 145mM NaCl, 10mM MgCI 2 , I mMEDTA, 100 ⁇ g/ml salmon sperm DNA, 50 mM Tris, pH 7.5.
  • the magnetic capture beads prepared in section A, were divided into 24 10ul aliquots. Six sets of aliquots were diluted in hybridization solution containing various concentrations of NaCl (OmM, 145mM, 300mM, and 400mM).
  • the target DNA mixture was made in the hybridization buffer, of varying salt concentrations, in the following concentrations: l OOfmole, l Ofmole, Ifmole, O.lfmole, O.OIfmole, 0 femtomole (negative control).
  • the various target solutions were then mixed with their respective bead solutions according to the salt concentration of the hybridization buffer and incubated at 37°C for 2 hours to let target to hybridize to the 5' transport probe on the capture beads.
  • the assay solutions were washed 3X with wash buffer containing the appropriate amount of NaCl required for each treatment group (OmM, 0.145mM, 0.3mM, 0.4mM NaCl), 1X with the appropriate hybridization buffer.
  • the beads were then resuspended in their respective hybridization buffers containing the appropriate amount of NaCl.
  • a 100 ⁇ l volume of reporter beads in hybridization buffer containing various NaCl concentrations (OmM, 0.145mM, 0.3mM, 0.4mM NaCl), were added to the appropriate assay solution, so that the same concentration of NaCl was maintained within the different treatment groups.
  • These assay solutions were then incubated at 37°C for 2 hours in a rotating mixer.
  • Fig. 2. a magneto-optical disc system, a fluorescent disc system, or any similar device.
  • Unique signature traces of a dual bead complex collected from an optical disc reader are shown above in Fig. 29B.
  • the magnetic capture beads and yellow-fluorescent reporter beads were each covalently conjugated with DNA transport probes and DNA signal probes, respectively. After conjugation, the beads were washed 1X with hybridization buffer (0.1 M NaCl, 10 mM MgCI 2 , 1 mM EDTA, 50mM Tris, pH 7.5). The beads were pretreated with 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temp. The beads were then washed 3X with wash buffer (145mM NaCl, 50 mM Tris, pH 7.5, 0.1% SDS,
  • the magnetic capture beads as prepared in Part A, were divided into 24 10ul aliquots. Six sets of aliquots were diluted in hybridization solution containing various concentrations of MgCI 2 (OmM, 10mM, 20mM, and 30mM).
  • the target DNA mixture was made in hybridization buffer containing varying amounts MgCI 2 , in the following concentrations: lOOfmole, 10f mole, Ifmole, O.lfmole, 0.01 f mole, Ofmole (negative control).
  • the various target solutions were then mixed with their respective bead solutions according to the salt concentration of the hybridization buffer and incubated at 37°C for 2 hours to let target to hybridize to the 5' transport probe on the capture beads.
  • the assay solutions were washed 3X with wash buffer containing the appropriate amount of MgCI 2 required for each treatment group (OmM, 10mM, 20mM, 30mM MgCI 2 ), 1X with the appropriate hybridization buffer.
  • the beads were then resuspended in their respective hybridization buffers containing the appropriate amount of MgCI 2 .
  • the magnetic capture beads (3um Spherotech) and yellow-fluorescent reporter beads (2.1 um Polysciences) were each covalently conjugated with DNA transport probes and DNA signal probes, respectively. After conjugation, the beads were washed 1X with hybridization buffer (0.1 M NaCl, 10 mM MgCI 2 , 1 mM EDTA, 50mM tris, pH 7.5). The beads were pretreated with 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temp.
  • hybridization buffer 0.1 M NaCl, 10 mM MgCI 2 , 1 mM EDTA, 50mM tris, pH 7.5.
  • the beads were then washed 3X with wash buffer (145mM NaCl, 50 mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM), 1X with hybridization buffer (100mM NaCl, 10mM MgCI 2 , I mMEDTA, 100 ⁇ g/ml salmon sperm DNA, 50 mM Tris, pH 7.5). After washing, the beads were resuspended in hybridization buffer.
  • wash buffer 145mM NaCl, 50 mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM
  • hybridization buffer 100mM NaCl, 10mM MgCI 2 , I mMEDTA, 100 ⁇ g/ml salmon sperm DNA, 50 mM Tris, pH 7.5.
  • a biotinylated transport blocking probe was diluted to the following final concentrations: 500pmole, 50pmole, 35pmole, 30pmoles.
  • a 13 ⁇ l (2x10 7 ) volume of magnetic beads were used for each tube (5 tubes total).
  • a 5 ⁇ l amount of blocking probe, as prepared above, and 32 ⁇ l hybridization buffer was added to each tube. The blocking probes and the transport probes were then hybridized for 2 hours at 37°C.
  • the beads were washed 3X with wash buffer (145nM NaCl, 50nM Tris, pH 7.5, 0.05% Tween) and resuspended in 100 ⁇ l CDB (2% BSA, 50mM Tris-HCI, pH7.5, 145mM NaCl, LOmM MgCI 2 , 0.1 mM ZnCI 2 , 0.05% NaN 3 ).
  • the reporter beads were prepared in a similar fashion using a biotinylated reporter blocking probe.
  • D. Dual Bead Assay The beads, as prepared in Part B, were washed and resuspended in hybridization buffer containing 100 ⁇ g/ml salmon sperm DNA and 5X Denhart's solution.
  • a solution target DNA mixture was prepared in the hybridization buffer with the following concentrations: Of mole-control, lOfmole, Ifmole, O.lfmole, O.OIfmole, 0.001 fmole, 0.0001 fmole.
  • the target solutions were then mixed with equal amounts of capture and reporter beads and incubated at 37°C for 2 hours.
  • the capture and reporter beads having been blocked with the same amount of probe blocking agent for each set of assay mixture, i.e., add 10ul l Ofmole target to 100ul reporter and capture bead solution each having been blocked with 50pmole blocking probe.
  • the assay solution was washed 3X with wash buffer and 1X with hybridization buffer and resuspended in hybridization buffer (containing 100 ⁇ g/ml salmon sperm DNA and 5X Denhart's Mix).
  • the assay solution was concentrated and resuspended in 250 ⁇ l water.
  • the beads used in this experiment were 25 ⁇ l of capture beads (3 ⁇ m carboxylated magnetic particles at a concentration of 1.5x10 7 beads/ ⁇ l) with 5' transport probes attached by covalent conjugation and 400 ⁇ l of reporter beads (2 ⁇ m YF beads at a concentration of 6.6x10 6 beads/ ⁇ l). These beads were washed 3X with
  • hybridization buffer 50mM Tris, pH 7.5, 0.05% Tween
  • hybridization buffer 50mM Tris-HCI pH 7.5, 10mM MgCI 2 , 0.1 M NaCl, 1 mM EDTA.
  • the capture beads were then resuspended in 250 ⁇ l hybridization buffer, and the reporter beads in 400 ⁇ l hybridization buffer.
  • a set of target DNA solutions are made in hybridization solution with the following concentrations: 0 picomole, 1 picomole, 10 picomole, 100 picomole target.
  • a test sample was prepared containing 10 ⁇ l capture beads, 15 ⁇ l of reporter beads, 1 ⁇ l salmon sperm DNA and 74 ⁇ l target solution all in hybridization buffer. Aliquots of this test sample were analyzed at various incubation times 30min, 1 hr, 2hr, 3hr, 4hr, and overnight. One set was incubated at 37° C without mixing and the other set was mixed on a rotating mixer.
  • the reporter beads can be separated from the capture beads in a DNA dependent procedure.
  • the dual bead complexes are subjected to DNAases (enzymes that specifically cut DNA). This treatment separates the reporter beads from the capture beads by cutting the DNA that holds them together. Thus, the non- target mediated dual beads will not be affected.
  • the reporter beads that are released after the DNAse treatment are indicative of the amount of target DNA present in the sample.
  • the DNAsel effect in a dual bead assay was evaluated. The results from this experiment is shown in Figs. 69 and 70.
  • the dual bead assay was carried out as described previously in Example 1 , Part A. Briefly, the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL) coated with covalently attached transport probes; 2.1 ⁇ m fluorescent reporter beads (Molecular Probes, Eugene, OR) coated with a covalently attached reporter probes, and target DNA molecules of interest.
  • the target DNA is a synthetic 80 oligonucleotides long.
  • the transport probes and reporter probes are 40 nucleotides in length and are complementary to the target DNA but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads were concentrated magnetically with the supernatant being removed.
  • a 100 ⁇ l volume of the hybridization buffer (0.2M NaCl, 1mM EDTA, 10mM MgCI 2 , 50mM Tris- HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) were added and the beads were resuspended.
  • Various concentration of target DNA ranging from 1 , 10, 100 and 1000 femtomoles were added to the capture bead suspensions.
  • the beads suspension was incubated while mixing at 37 degrees Centigrade for 2 hours.
  • the beads were magnetically concentrated and the supernatant containing unbound target DNA was removed.
  • One hundred microliters of wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) was added and the beads were resuspended.
  • the beads were magnetically concentrated and the supernatant was again removed.
  • the wash procedure was repeated twice.
  • a 2x10 7 amount of reporter beads in 100 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10g/ml denatured salmon sperm DNA) were then added to washed capture beads.
  • the beads were resuspended and incubated while mixing at 37 degrees Centigrade for an additional 2 hours. After incubation, the capture beads were concentrated magnetically, and the supernatant containing unbound reporter beads were removed.
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • DNAsel was selected for this purpose because it is not sequence specific. Following washing, the dual bead complexes were resuspended in 87.5 ⁇ L water. Ten (10) units of DNAsel (2.5 ⁇ L) and 10 ⁇ L of DNAsel reaction buffer (40mM Tris-HCI, 10mM MgS0 , 1 mM CaCI 2 ) were added to the re-suspended beads. The digestion reaction was carried out for 1 hour at 37°C. After digestion, the capture beads were concentrated magnetically and the supernatant containing reporter beads was removed. The magnetic capture beads were washed 2 times with 10O ⁇ l water. The washed water was combined with the supernatant.
  • the number of reporter beads was quantified by the fluorimeter Fluoromax-2 at excitation 500nm, emission 530nm and slit sizes 2.0. See Figs. 69 and 70 for results from this experiment. Alternatively, the number of fluorescent reporter beads can be quantified by the bio-disc reader as described above.
  • the dual bead complexes are separated by physical or chemical treatments.
  • the dual bead assay was carried out as described above in
  • Example 15 Following washing, the bead products were washed 5 times with the wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) and divided into four sets.
  • the wash buffer 145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA
  • the target DNA is single stranded.
  • the hybridization buffer requires a denaturing reagent such as guanidine isothiocyanate.
  • concentration of the denaturing reagent used in the assay is a major contributor in the specificity and sensitivity of the dual bead assay.
  • the dual bead assay to detect HSV was carried out in the presence of 1.5M guanidine isothiocyanate.
  • the dual bead assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL) coated with covalently attached 5' HSV transport probes and 2.1 ⁇ m fluorescent reporter beads from Molecular Probes (Eugene, OR) conjugated to the 3' HSV reporter probes and target DNA molecules of interest.
  • the target was a double-strand PCR product containing the HSV gene sequence, amplified for 30 cycles and Qiagen column purified.
  • the transport probes and reporter probes are 40 nucleotides in length and are complementary to the target DNA but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads were concentrated magnetically with the supernatant being removed.
  • the capture beads were resuspended in 600 ⁇ L of hybridization buffer (1.5 GuSCN, 8mM EDTA, 40mM Tris, pH7.5) containing 5X Denhart's mix, and 10 ⁇ g/ml salmon sperm DNA.
  • the target was a double-strand PCR product, amplified for 30 cycles and Qiagen column purified.
  • the target was diluted to appropriate concentrations, and heated at 95°C for 5 minutes to denature the double strand then quickly chilled on ice.
  • a total of 12.5 ⁇ L of the chilled target was added to the 100 ⁇ L of the pretreated capture beads.
  • Various concentrations of target DNA ranging from 0, 10 "16 , 10 "15 , 10 " 14 , 10 "13 , and 10 "12 moles were added to the capture bead suspensions.
  • the beads suspension was incubated while mixing at 37 degrees Centigrade for 2 hours.
  • the beads were magnetically concentrated and the supernatant containing unbound target DNA was removed.
  • One hundred microliters of wash buffer (145mM NaCl, 50mM Tris, pH 7.5, 0.1% SDS, 0.05% Tween, 0.25% NFDM (Non Fat Dried Milk), 10mM EDTA) was added and the beads were resuspended.
  • the beads were magnetically concentrated and the supernatant was again removed.
  • the wash procedure was repeated twice.
  • the number of fluorescent reporter beads can be quantified by use of the optical bio-disc reader as described above.
  • the following example illustrates a dual bead assay carried out on a magnetically writable and erasable analysis disc such as the magneto-optical bio-disc 110 discussed in conjunction with Fig. 37.
  • the dual bead assay is carried out to detect the gene sequence
  • the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL) coated with covalently attached transport probes; 2.1 ⁇ m fluorescent reporter beads (Molecular Probes, Eugene, OR) coated with a covalently attached sequence specific for the DYS gene, and target DNA molecules containing DYS sequences.
  • the target DNA is a synthetic 80 oligonucleotides long.
  • the transport probes and reporter probes are 40 nucleotides in length and are complementary to the DYS sequence but not to each other.
  • the specific methodology employed to prepare the assay involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 100 ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads are loaded inside the MO bio-disc via the injection port.
  • the MO bio-disc contains magnetic regions created by the magneto optical drive. The capture beads thus are held within specific magnetic regions on the MO bio-disc.
  • the sample containing target DNA and reporter beads in 200 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI 2 , 50mM Tris-HCI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) is then added to the MO bio- disc via the injection port.
  • the injection port is then sealed.
  • the magnetic field is released.
  • the disc is rotated at very low speed (less than 800rpm) in the drive to facilitate hybridization of target DNA and reporter beads to the capture beads.
  • the temperature of the drive is kept constant at 33 degrees Centigrade. After 2 hours of hybridization, the magnetic field is created by the magneto optical drive.
  • a dual bead assay using the multiplexing techniques described above in connection with Figs. 32 and 37 is carried out on a magnetically writable and erasable analysis disc such as the MO bio-disc 110 discussed with reference to Fig. 37.
  • the dual bead assay is carried out to detect 2 or more DNA targets simultaneously.
  • the assay is comprised of 3 ⁇ m magnetic capture beads (Spherotech, Libertyville, IL).
  • One population of the magnetic capture beads is coated with transport probes 1 which are complementary to the DNA target 1.
  • Another population of the magnetic capture beads is coated with transport probes 2 which are complementary to the DNA target 2.
  • 2 or more different types of magnetic capture beads may be used.
  • the reporter beads may differ by chemical composition (for example silica and polystyrene) and/or by size.
  • One type of reporter beads is coated with reporter probes 1 , which are complementary to the DNA target 1.
  • the other reporter beads are coated with reporter probes 2, which are complementary to the DNA target 2.
  • the transport probes and reporter probes are complementary to the respective targets but not to each other.
  • the specific methodology employed to prepare the dual bead assay multiplexing involved treating 1x10 7 capture beads and 2x10 7 reporter beads in 10O ⁇ g/ml salmon sperm DNA for 1 hour at room temperature. This pre-treatment will reduce the non-specific binding between the capture and reporter beads in the absence of target DNA.
  • the capture beads are loaded in the MO bio-disc.
  • the magnetic field is applied to create distinct magnetic zones for specific capture beads.
  • the capture beads can be held on the MO bio-disc at a density of 1 capture bead per 10 ⁇ m 2 .
  • the surface area usable for bead deposition on the MO bio-disc is approximately 3 x 10 9 ⁇ m 2 .
  • the capacity of the MO bio-disc for 3 ⁇ m beads at the given density is about 3 x 10 8 beads.
  • the sample containing the targets DNA of interest is mixed with different types of reporter beads in 200 ⁇ l hybridization buffer (0.2M NaCl, 1 mM EDTA, 10mM MgCI , 50mM Tris- ⁇ CI, pH 7.5 and 5X Denhart's mix, 10 ⁇ g/ml denatured salmon sperm DNA) and added to the MO bio-disc via the injection port.
  • the injection port is then sealed.
  • the magnetic field is released.
  • the disc is rotated at very low speed (less than 800rpm) in the drive to facilitate hybridization of targets DNA and reporter beads to the different types of capture beads.
  • the temperature of the drive is kept constant at 33 degrees Centigrade. After 2 to 3 hours of hybridization, the magnetic field is regenerated by the magneto optical drive.
  • the magnetic field is released and the dual bead complexes are directed to a detection chamber.
  • the amount of different types of target DNA can be enumerated by quantifying the number of corresponding capture magnetic beads and reporter beads since each type of bead has a distinct signature as shown above in
  • Figs. 28A, 2 ⁇ B, 29A, and 29B are identical to Figs. 28A, 2 ⁇ B, 29A, and 29B.
  • EXAMPLE 20 A. Separation of T-Helper Cells in AIDS Patients using Monoclonal CD4 Antibodies Attached to Paramagnetic Beads
  • a sample (whole blood or mononuclear cells) is injected into the mixing/loading chamber where it is mixed with anti-CD4 coated paramagnetic beads (bio-magnetic particles).
  • anti-CD4 coated paramagnetic beads bio-magnetic particles.
  • magnetic domains are created within the flow channel or analysis chamber using the laser in the MO drive.
  • the tagged CD4+ cells will then bind to these magnetic domains such that when the disc is rotated at a pre-determined speed and time untagged cells and cellular components will move down the flow channel toward a waste chamber.
  • the number of CD4+ cells bound to the magnetic domains is then quantified by the MO reader. The number of CD4+ cells thus determined will be indicative of the heath status of the patient.
  • the magnetic domains are then erased.
  • the disc is then rotated at a pre-determined speed and direction to cause the newly released CD4+ cells to move to a flow channel into different testing chambers where the cells could be exposed to different drug treatments that could reduce their susceptibility to HIV destruction.
  • the design of the fluidic circuits and the speed and direction of rotation of the disc can be determined by one of skill in the art by no more than routine experimentation.
  • a sample (single cell suspension prepared for example from the biopsied tissue) is loaded into the mixing chamber of the MO bio-disc or MO analysis disc. Paramagnetic beads are then coated with MOC-31 capture antibodies to thereby form bio-magnetic beads. These bio-magnetic beads are then loaded in to the mixing chamber containing the sample. After a 15-minute incubation, magnetic domains are created within the mixing chamber in the MO disc to bind magnetically tagged or labeled cells. The disc is then rotated at a pre-determined speed and duration to remove unlabelled cells. The labeled or tagged carcinoma cells, which are bound to the paramagnetic fields, will be immobilized in the magnetic field whereas other cells will move down the flow channel toward a waste chamber.
  • the number of cancer cells can be quantified by the reader. Further details related to quantification of particles and cells within fluidic circuits are disclosed in, for example, commonly assigned and co-pending U.S. Patent Application Serial No. 10/241 ,512 entitled “Methods for Differential Cell Counts Including Related Apparatus and Software Performing Same” filed September 11 , 2002; and U.S. Patent Application Serial No. 10/279,677 entitled “Segmented Area Detector for Biodrive and Methods Relating Thereto” filed October 24, 2002. Both of which are herein incorporated by reference in their entireties.
  • the magnetic field is turned off and the rotation of the disc will cause the identified cancels cells to move to various testing chambers on the MO disc where the cancer cells will be exposed to different anticancer drugs.
  • the efficacy of the drug can be determined by quantifying the number of live cells or dead cells since apoptotic cells have a different signature trace than live cells.
  • a sample containing DNA fragments is injected into the mixing/loading chamber where it is mixed with paramagnetic beads or bio-magnetic particles coated with DNA probes for one or more species of TB.
  • the sample and the probes are then allowed to hybridize for about 1 hour in the disc with intermittent mixing by rotating the disc clockwise then counter clockwise in the reader.
  • Magnetic domains are then created in the mixing chamber where the magnetic beads are captured.
  • the mixing chamber is then washed to remove unbound DNA fragments.
  • the magnetic domains are then erased and the disc is rotated to cause the magnetic particles to be released and move to an analysis chamber where the captured specific TB DNA sequence could be amplified for downstream applications.
  • EXAMPLE 23 The interactions of different neurons in the brain to generate specific action potentials are still unclear. Interactions of specific neurons in the brain can be studied on the MO disc. In various incubation chambers, neurons carrying different cellular markers can be isolated using paramagnetic beads coated with antibodies directed against specific cellular surface markers.
  • the neurons isolated in one chamber can then be manipulated, moved and mixed with the neurons isolated in a different chamber using magnetic field on the disc. To determine whether there is "communication" between the 2 types of neurons, the production of an action potential (K + or Ca 2+ ) could be monitored.
  • any of the off-disc preparation procedures may be readily performed on-disc by use of suitable fluidic circuits employing the methods described herein.
  • any of the fluidic circuits discussed in connection with the reflective and transmissive discs may be readily adapted to the MO bio-disc.
  • the scope of the present invention is not solely limited to the formation of only dual bead complexes.
  • the methods and apparatus hereof may be readily applied to the creation of multi-bead assays.
  • a single capture bead may bind multiple reporter beads.
  • a single reporter bead may bind multiple capture beads.
  • linked chains of multi-bead or dual bead complexes may be formed by target mediated binding between capture and reporter beads. The linked chains may further agglutinate to thereby increase detectability of a target agent of interest.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Fluid Mechanics (AREA)
  • General Engineering & Computer Science (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

Cette invention a trait, d'une manière générale, à des dosages moléculaires et cellulaires bio-magnétiques et, plus particulièrement, à des dosages moléculaires et cellulaires bio-magnétiques réalisés sur des bio-disques magnéto-optiques. Elle porte également sur des systèmes à bio-disques magnéto-optiques comprenant ces bio-disques ainsi que des lecteurs magnéto-optiques. Plus précisément, mais sans se restreindre aux modes de réalisation particuliers décrits ci-après conformément à la meilleure façon de pratiquer, cette invention, qui concerne des techniques bio-magnétiques, y compris des techniques immuno-magnétiques, aux fins de la détection et la manipulation sélective de cellules cibles spécifiques dans des populations cellulaires et dans des solutions de populations cellulaires, au moyen de particules ou de billes magnétiques, concerne aussi la croissance de neurites et la régénération nerveuse commandées par voie magnétique ainsi que des réseaux neuronaux constitués par voie magnétique au moyen des systèmes à bio-disques magnéto-optiques (MOBDS).
EP02795687A 2001-11-27 2002-11-27 Bio-disques magneto-optiques et systemes utilisant des techniques en rapport Withdrawn EP1585957A2 (fr)

Applications Claiming Priority (17)

Application Number Priority Date Filing Date Title
US997741 2001-11-27
US09/997,741 US20030003464A1 (en) 2000-11-27 2001-11-27 Dual bead assays including optical biodiscs and methods relating thereto
US35564402P 2002-02-05 2002-02-05
US355644P 2002-02-05
US35698202P 2002-02-13 2002-02-13
US356982P 2002-02-13
US35847902P 2002-02-19 2002-02-19
US358479P 2002-02-19
US99266 2002-03-14
US10/099,266 US20030082568A1 (en) 2000-11-27 2002-03-14 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
US37200702P 2002-04-11 2002-04-11
US372007P 2002-04-11
US38813202P 2002-06-12 2002-06-12
US388132P 2002-06-12
US40822702P 2002-09-04 2002-09-04
US408227P 2002-09-04
PCT/US2002/038021 WO2003046511A2 (fr) 2001-11-27 2002-11-27 Bio-disques magneto-optiques et systemes utilisant des techniques en rapport

Publications (1)

Publication Number Publication Date
EP1585957A2 true EP1585957A2 (fr) 2005-10-19

Family

ID=27574728

Family Applications (1)

Application Number Title Priority Date Filing Date
EP02795687A Withdrawn EP1585957A2 (fr) 2001-11-27 2002-11-27 Bio-disques magneto-optiques et systemes utilisant des techniques en rapport

Country Status (6)

Country Link
EP (1) EP1585957A2 (fr)
JP (1) JP2005530127A (fr)
CN (1) CN1636138A (fr)
AU (1) AU2002360433A1 (fr)
CA (1) CA2468245A1 (fr)
WO (1) WO2003046511A2 (fr)

Families Citing this family (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006006281A (ja) * 2004-06-29 2006-01-12 Toyo Univ 細胞固定化基板ならびに細胞固定化方法
EP1650297B1 (fr) * 2004-10-19 2011-04-13 Samsung Electronics Co., Ltd. Procédé et appareil pour la destruction rapide de cellules ou de virus à l'aide de billes micro magnétiques et d'un laser
EP1815252B1 (fr) 2004-11-05 2012-10-10 Imec Procede de transport de particules magnetiques et dispositifs correspondant
JP2007155484A (ja) * 2005-12-05 2007-06-21 Rohm Co Ltd マイクロチップ
JP4670015B2 (ja) * 2006-05-02 2011-04-13 独立行政法人産業技術総合研究所 光検出型分子センサ及び分子検出方法
CA2657970A1 (fr) * 2006-05-17 2007-11-29 Luminex Corporation Systemes de type cytometres en flux sur circuits integres pour analyser des particules marquees de maniere fluorescente
US8296088B2 (en) 2006-06-02 2012-10-23 Luminex Corporation Systems and methods for performing measurements of one or more materials
WO2007143615A2 (fr) * 2006-06-02 2007-12-13 Luminex Corporation Systèmes et procédés pour effectuer des mesures sur un ou plusieurs matériaux
EP1894617B1 (fr) * 2006-08-31 2013-08-14 Samsung Electronics Co., Ltd. Procédé de mélange d'au moins deux types de fluides dans un substrat de traitement de microfluide à centrifuge
KR100818274B1 (ko) * 2006-09-05 2008-04-01 삼성전자주식회사 미세유동 시스템 제어장치 및 그 방법, 및 미세유동 시스템
US8273310B2 (en) * 2006-09-05 2012-09-25 Samsung Electronics Co., Ltd. Centrifugal force-based microfluidic device for nucleic acid extraction and microfluidic system including the microfluidic device
MY174446A (en) 2010-06-25 2020-04-19 Petroliam Nasional Berhad Petronas A method and system for validating energy measurement in a high pressure gas distribution network
EP3644058A1 (fr) * 2010-10-14 2020-04-29 Meso Scale Technologies, LLC Stockage de réactifs dans un dispositif de dosage
US8510239B2 (en) * 2010-10-29 2013-08-13 International Business Machines Corporation Compact cognitive synaptic computing circuits with crossbar arrays spatially in a staggered pattern
DE102010043276A1 (de) * 2010-11-03 2012-05-03 Siemens Aktiengesellschaft Magnetische Zelldetektion
CN103076416B (zh) * 2011-11-15 2014-08-06 南昌大学 一种富集养殖家畜尿样中克伦特罗、莱克多巴胺和沙丁胺醇的方法
WO2016012449A1 (fr) * 2014-07-22 2016-01-28 Tbdiadirect Ab Anticorps monoclonal, procédé, kit et utilisation
EP3213071B1 (fr) 2014-10-30 2019-07-10 GE Healthcare Bio-Sciences AB Méthode pour établir les courbes de correction solvant
EP3023150A1 (fr) * 2014-11-19 2016-05-25 F. Hoffmann-La Roche AG Mélange de particules
WO2016085841A1 (fr) * 2014-11-24 2016-06-02 Nanostring Technologies, Inc. Procédés et appareils pour la purification et l'imagerie de gènes
WO2018008779A1 (fr) * 2016-07-07 2018-01-11 서강대학교 산학협력단 Procédé d'orientation de la direction de croissance de dendrites neuronales
CN109804235B (zh) * 2016-11-30 2022-05-13 国立研究开发法人产业技术综合研究所 目标物质检测装置及目标物质检测方法
JP7028455B2 (ja) * 2016-11-30 2022-03-02 国立研究開発法人産業技術総合研究所 標的物質検出装置及び標的物質検出方法
GB2561173A (en) * 2017-04-03 2018-10-10 Univ Dublin City Microfluidic device for detection of analytes
EP3400983B1 (fr) * 2017-05-09 2019-11-27 Miltenyi Biotec B.V. & Co. KG Système de colonne rechargeable
CN107607384B (zh) * 2017-09-13 2021-05-25 北京智康博药肿瘤医学研究有限公司 一种细胞纯化方法
CN109395875B (zh) * 2018-12-11 2023-10-20 苏州英赛斯智能科技有限公司 一种磁珠分离机构、装置以及磁珠分离方法
CN114026423A (zh) * 2019-05-24 2022-02-08 康涅狄格大学 流体分析装置

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6342349B1 (en) * 1996-07-08 2002-01-29 Burstein Technologies, Inc. Optical disk-based assay devices and methods
JP2002521666A (ja) * 1998-07-21 2002-07-16 バースタイン テクノロジーズ，インコーポレイティド 光ディスクをベースとするアッセイ装置および方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03046511A2 *

Also Published As

Publication number Publication date
JP2005530127A (ja) 2005-10-06
WO2003046511A2 (fr) 2003-06-05
CN1636138A (zh) 2005-07-06
AU2002360433A1 (en) 2003-06-10
CA2468245A1 (fr) 2003-06-05
WO2003046511A3 (fr) 2006-12-07

Similar Documents

Publication Publication Date Title
EP1585957A2 (fr) Bio-disques magneto-optiques et systemes utilisant des techniques en rapport
US20030054376A1 (en) Dual bead assays using cleavable spacers and/or ligation to improve specificity and sensitivity including related methods and apparatus
US20020168663A1 (en) Methods for DNA conjugation onto solid phase including related optical biodiscs and disc drive systems
US20030003464A1 (en) Dual bead assays including optical biodiscs and methods relating thereto
US20030082568A1 (en) 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
US20020172980A1 (en) Methods for decreasing non-specific binding of beads in dual bead assays including related optical biodiscs and disc drive systems
US7033747B2 (en) Multi-parameter assays including analysis discs and methods relating thereto
US20060068409A1 (en) 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
US9575081B2 (en) Device and methods for detecting analytes in saliva
US20050266476A1 (en) Dual bead assays including covalent linkages for improved specificity and related optical analysis discs
US20070166835A1 (en) Multiplex assays using magnetic and non-magnetic particles
JP5161218B2 (ja) 薄膜化学分析装置及びこれを用いた分析方法
WO2002042498A2 (fr) Analyses a l'aide de complexe a deux billes, biodisques optiques, et procedes d'analyse
US20040248093A1 (en) Magneto-optical bio-discs and systems including related methods
US20050003459A1 (en) Multi-purpose optical analysis disc for conducting assays and related methods for attaching capture agents
US20050032126A1 (en) Methods and apparatus for use in detection and quantitation of various cell types and use of optical bio-disc for performing same
JP2002521666A (ja) 光ディスクをベースとするアッセイ装置および方法
KR100985603B1 (ko) 분자 검출을 위한 방법 및 장치
WO2002071929A2 (fr) Utilisation de procedes mettant en oeuvre des enzymes de restriction et d'autres composes chimiques pour diminuer la fixation non specifique dans des essais sur double bille et biodisques apparentes, procedes et dispositifs servant a detecter des cibles medicales
JP2006505766A (ja) 分析ディスクを含むマルチパラメータ検定とその関連方法
WO2002073605A2 (fr) Essai biologique a deux perles utilisant des espaceurs clivables et/ou la ligature pour ameliorer la specificite et la sensibilite, procedes et dispositifs associes
US20050069923A1 (en) Dual bead assays using cleavable spacers and/or ligation to improve specificity and sensitivity including related methods and apparatus
WO2002068697A2 (fr) Procedes visant a reduire les liaisons non specifiques de billes dans des techniques a deux billes incluant des biodisques optiques et des systemes d'entrainement de disques associes
WO2002068696A2 (fr) Procedes de conjugaison d'adn sur une phase solide y compris biodisques optiques et systemes d'entrainement de disque connexes
US6489120B1 (en) System and method for a parallel immunoassay system

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20040604

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LI LU MC NL PT SE SK TR

PUAK Availability of information related to the publication of the international search report

Free format text: ORIGINAL CODE: 0009015

RIC1 Information provided on ipc code assigned before grant

Ipc: C12Q 1/68 20060101ALI20070213BHEP

Ipc: C07H 21/02 20060101AFI20070213BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20110531