WO2001051207A1 - Linear probe carrier - Google Patents

Linear probe carrier Download PDF

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Publication number
WO2001051207A1
WO2001051207A1 PCT/US2001/001026 US0101026W WO0151207A1 WO 2001051207 A1 WO2001051207 A1 WO 2001051207A1 US 0101026 W US0101026 W US 0101026W WO 0151207 A1 WO0151207 A1 WO 0151207A1
Authority
WO
WIPO (PCT)
Prior art keywords
probe
probes
substrate
carrier
probe carrier
Prior art date
Application number
PCT/US2001/001026
Other languages
English (en)
French (fr)
Inventor
Shiping Chen
Yuling Luo
Anthony C. Chen
Original Assignee
Genospectra, 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
Application filed by Genospectra, Inc. filed Critical Genospectra, Inc.
Priority to IL15043401A priority Critical patent/IL150434A0/xx
Priority to KR1020027008921A priority patent/KR20020089323A/ko
Priority to AU27868/01A priority patent/AU2786801A/en
Priority to EP01902020A priority patent/EP1248678A1/en
Priority to JP2001551617A priority patent/JP2003519784A/ja
Priority to CA002397069A priority patent/CA2397069A1/en
Publication of WO2001051207A1 publication Critical patent/WO2001051207A1/en

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    • 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
    • B01L3/54—Labware with identification means
    • B01L3/545—Labware with identification means for laboratory containers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N35/00009—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 a sample supporting tape, e.g. with absorbent zones
    • G—PHYSICS
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    • G01N35/00584—Control arrangements for automatic analysers
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    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • 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
    • B01L3/505—Containers for the purpose of retaining a material to be analysed, e.g. test tubes flexible containers not provided for above
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • 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
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00108—Test strips, e.g. paper
    • G01N2035/00118—Test strips, e.g. paper for multiple tests
    • 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
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00732—Identification of carriers, materials or components in automatic analysers
    • G01N2035/00742—Type of codes
    • G01N2035/00752—Type of codes bar codes
    • 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
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00732—Identification of carriers, materials or components in automatic analysers
    • G01N2035/00742—Type of codes
    • G01N2035/00762—Type of codes magnetic code

Definitions

  • This invention relates generally to the field of target analysis by binding to probes, as is commonly found in DNA sequence identification.
  • This invention also relates to arrangements of immobilized nucleic acid probes on a solid substrate. More particularly, the invention relates to packaging of probe carrier threads wherein probes are immobilized in an array alone a flexible carrier.
  • Such probes may be ohgonucleotides, proteins, antibodies, or cell-binding molecules and the choice of probes is theoretically limited only by the possibilities of specific binding to or reaction with sample.
  • the binding of a sample to a probe is detected, and the probe identified, thereby identifying the sample.
  • Technology has primarily developed around the use of these two-dimensional, planar arrays, especially in the area of arrays of ohgonucleotides, which have become small and dense enough to be termed microarrays.
  • microarrays in an efficient and cost-effective manner is of considerable interest to researchers worldwide and of significant commercial value.
  • N microarray is capable of dramatically boosting the efficiency of traditional biochemical experiments. Tests that would have taken years can now be completed in hours or even minutes.
  • the applications of this technology affect more than the healthcare sector including gene profiling, disease diagnostics, drug discovery, forensics, agronomics, biowarfare and even biocomputers.
  • Various types of microarray manufacturing devices and technologies have been described.
  • Chemical ink jetting has an inaccuracy similar to in-situ synthesis and test site size limits similar to mechanical spotting.
  • processing time for each scan also increases with increasing density of the two- dimensional probe array.
  • microarray In addition, the basic, operating principle of microarray involves a probe immobilized on a substrate to react with specific molecules in sample fluid. Hybridization requires providing probes with sufficient chances to meet their complementary molecules. In existing systems, this is achieved through diffusion or driving the sample fluid across the microarray. The former is a random process and the later requires complex microfluidic systems.
  • the present invention provides a new direction and approach in making a probe carrier or probe configuration that does not require dense two-dimensional symmetrical arrays built upon a rigid substrate and also does not inherently limit the size of the probes that can be attached to a substrate.
  • the present invention can be relatively easily fabricated through use of assembly-line-like techniques.
  • the invention provides a probe carrier in which a plurality of probes are immobilized in discrete areas, one probe per area, on an elongated flexible substrate with a length.width ratio of at least about 5:1, at least 50:1, at least 500:1, at least 10,000:1, or at least 100,000:1.
  • the length of each probe-containing area does not exceed 1000 micrometers, in another embodiment the length of each probe-containing area does not exceed 500 micrometers, in another embodiment the length of each probe-containing area does not exceed 100 micrometers, in still another embodiment the length of each probe- containing area does not exceed 50 micrometers, and in yet a further embodiment the length of each probe-containing area does not exceed 20 micrometers.
  • the invention also provides a probe carrier in which a plurality of probes are immobilized in discrete areas, one probe per area, on a flexible substrate with a length:width ratio of at least about 5:1, where the substrate has layer on its surface, and where the probes are immobilized on the surface of the layer.
  • the invention also has a second layer between the first layer and the substrate.
  • the first layer comprises silica and the second layer comprises a metallic material.
  • the invention also provides a linear one-dimensional arrangement of probes immobilized in a single file on the surface of a flexible substrate, in which the linear density of the probes exceeds 10 probes per linear cm, or preferably 50 probes per linear cm. In another aspect of the invention, the linear density of the probes exceeds 100 probes per linear cm, in a further aspect of the invention, the linear density of the probes exceeds 200 probes per linear cm, and in yet a further aspect of the invention, the linear density of the probes exceeds 500 probes per linear cm.
  • the invention provides a plurality of probes immobilized on discrete areas of the surface of a flexible tape substrate, one probe per area, where the tape has a thickness not exceeding 500 micrometers.
  • the tape does not exceed 100 micrometers in thickness, and in yet another aspect the tape does not exceed 20 micrometers in thickness.
  • the invention also provides a plurality of probes immobilized on discrete areas of the surface of a flexible fiber substrate, one probe per area, where the fiber has a diameter not exceeding 500 micrometers.
  • the fiber does not exceed 200 micrometers in diameter, in yet another aspect the fiber does not exceed 100 micrometers in diameter, and in still another aspect the fiber does not exceed 20 micrometers in diameter.
  • All of the above aspects of the invention may further include a first marker which conveys information about a first set of probes, and a second marker which conveys information about a second set of probes.
  • the markers maybe optical markers, such as optical bar codes or fluorescent markers, in another embodiment the markers may be magnetic.
  • the probes are polynucleotides, in another embodiment which includes markers, the probes are polypeptides, in yet another embodiment which includes markers, the probes are antibodies, and in still another embodiment which includes markers, the probes are selected from the group consisting of cell surface receptors, oligosaccharides, polysaccharides, and lipids.
  • the apparatus also includes a first layer, with the probes immobilized on that layer; the layer may be composed of silica.
  • the apparatus includes both a first layer and a second layer; the first layer may be silica and the second layer may be a metallic material. If the second layer is metallic, it may also be magnetizable.
  • the probes may be arranged as a linear configuration of spots, or as a linear configuration of stripes with the stripes being at an angle to the long axis of the substrate.
  • the probes may be polynucleotides, or polypeptides, or antibodies, or ligands, or be selected from the group consisting of cell surface receptors, oligosaccharides, polysaccharides, and lipids. If the probes are polynucleotides, they may be DNA, and if DNA, they may be single-stranded DNA.
  • the substrate for the invention may be silica glass, or plastic, or a metallic material, or a polymer, and if a polymer, the polymer may be selected from the group consisting of polyimide and polytetrafluoroethylene.
  • a preferred substrate is an optical fiber. If the substrate is a metallic material, it may also have a layer between the substrate and the probes, so that the probes are immobilized on the layer; the lay may be silica. Furthermore, the metallic substrate may be magnetizable.
  • the invention may be wound about a drum or a plurality of drums. It may also be wound upon itself in a flat spiral, with or without a flat backing, and it may be further attached to a spool at the center of the flat spiral. In addition, the outermost end of the substrate in the spiral may be extended and attached to a second spool.
  • the invention comprises an apparatus for transporting a plurality of probe fluids to a substrate to print a probe array.
  • the apparatus includes a reservoir with a plurality of wells, and a set of capillaries, where the capillaries are arranged so that one end of each capillary is connected to a well in the reservoir, such that the contents of the well may enter the capillary, and the second end of the capillaries are arranged in a flat single file row.
  • the reservoirs are wells in a microtiter plate.
  • probe fluids may be moved from the wells in the reservoir into the capillary tubing by applying a pressure differential between the reservoir and the tubing, and/or by providing a voltage between the reservoir and the substrate.
  • the capillary may be positioned parallel to and may move across the longitudinal axis of the elongated probe substrate to deposit a set of probes on the substrate. Other methods of probe deposition are described below in further detail.
  • a second probe transport apparatus has a row of probe containers configured in a fashion similar to a conveyer belt.
  • the row of containers is moved at one speed and direction to intersect with the substrate, which is moving at another speed and direction, to deposit probes, one by one, onto the substrate.
  • the row of probe containers is moved to intersect a moving row of spotters, which are made of a flexible strand of material, such that each spotter intersects a container to transfer probe from the container to the spotter.
  • a conveyor for instance, also moves a substrate so that it intersects the row of probe-carrying spotters such that each spotter deposits its probe onto the substrate after it has picked up the probe from a container.
  • the row of spotters may be configured as a loop, and further the spotters may be washed in a washing station after they have deposited probe on the substrate and before they return to the containers.
  • the invention also includes methods of depositing probes from a fluid transportation apparatus onto the substrate surface.
  • probes are painted as strips on the substrate.
  • the probe may be carried on a thin, flexible and elastic spotter, which contacts the substrate surface in a brushing action to paint the strip.
  • the spotter can be a silica capillary or fiber.
  • the capillary or fiber can be made of other materials such as metal, ceramics, polymer, or other material that is capable of transporting the probe- containing fluid.
  • the probes may be deposited in a non-contact fashion either as strips or dots. These methods include magnetic, electric, thermal, acoustic and inkjet deposition, a magnetic deposition method, the probe is attached to magnetic beads.
  • An electromagnet placed underneath the substrate is activated as the spotter carrying a probe immobilized on magnetic beads intersects the substrate.
  • the magnetic field generated by the electromagnet pulls the probe from the fluid transportation apparatus to deposit the probe onto the surface of the substrate, hi an electric deposition method, a voltage of appropriate polarity is applied between the substrate and the delivery device to establish an electric field to push the electrically charged probes (such as ohgonucleotides) onto the substrate surface, hi a thermal deposition method, rapid, localized heating is introduced into the path of fluid, producing a rapid local volume expansion (a bubble) that propels probe fluid onto substrate. Rapid heating can be introduced either electrically by a resistance heating wire or optically using suitable laser light.
  • a piezoelectric actuator is built in the probe fluid container. Activated by a voltage signal, the piezoelectric actuator rapidly reduces the volume of the container thus pushing out the probe fluid onto the substrate surface.
  • painting the probes in strips on the substrate may be accomplished by a probe deposition apparatus in which a matrix of fibers is dipped into a corresponding matrix of reservoirs, each reservoir containing probe, then the matrix of fibers is moved across a first section of substrate, with the fibers and the substrate positioned so that each fiber deposits a separate line of probe across the substrate with the desired substrate between lines.
  • the fiber matrix may be washed and dipped into another matrix of reservoirs then moved across another section of substrate to deposit another set of strips of probes.
  • the substrate may be a plurality of fibers arranged in parallel, so that several fibers receive probe with one pass of the probe-deposition instrument, or the substrate may be a tape, where, after probe deposition, the tape is optically cut along its long axis to produce a plurality of probe-carrying tapes.
  • the probes may be covalently linked to the substrate.
  • the further step of adding markers to the substrate may be included.
  • the invention is based in the technical finding that a probe carrier having a one-dimensional configuration of probes on a flexible substrate provides a simple, economical, reliable, and classification-specific way to identify the presence of target molecules in a sample. Further, probes on a probe carrier of this invention are not limited in size.
  • This invention encompasses a new way of improving the efficiency of hybridization to target or reaction with target.
  • This method involves moving the probe carrier through sample fluid to enhance the chance for the probes immobilized on the carrier to mix with their target molecules in the sample fluid.
  • the carrier may take a variety of forms including thread, tape, slide, coil, drum or pin. The movement can involve translation, rotation or vibration of the probe carrier, either alone or in combination.
  • This invention also includes several designs of hybridization device, where the probe carrier is inserted into a chamber containing the sample fluid.
  • the gap between the probe carrier and the inner wall of the chamber is minimized to reduce the volume of sample fluid.
  • the probe carrier is driven to move within the chamber to improve the efficiency of probe/target interaction.
  • An additional method of hybridization enhancement involves applying a voltage between the probe carrier and the wall of the hybridization chamber. At one polarity, the electric field pulls target molecules towards the probes on the carrier, which increases the local concentration of target molecules and improves the likelihood of hybridization. At the opposite polarity, the electric field repels target molecules away from the probes, which can help to increase the specificity of hybridization. By alternating the polarities at suitable frequencies, the hybridization efficiency between the target and probe can be improved.
  • the invention may be used in the analysis of known point mutations, expression analysis, genomic fingerprinting, polymorphism analysis, linkage analysis, characterization of mRNAs and mRNA populations, sequence determination, sequence confirmation, disease diagnosis, and other uses which will be apparent to those of skill in the art.
  • Figure 1 illustrates one embodiment of the probe carrier, in which probes are immobilized as spots on a substrate, which also carries markers in the form of optical bar codes. Probes may also be immobilized as stripes or as rows of spots, and markers may be optical, magnetic, or any other identifiable marking;
  • Figure 2a is a cross-sectional view of a probe-carrier, in which the probes are immobilized in a notch in the carrier; and Figure 2b is a cross-sectional view of two layers of a probe-carrier in which the probes are immobilized in a notch on the carrier, and illustrates how the position of the immobilized probes in the notch protects them from friction with the next layer.
  • Figure 3 illustrates an apparatus and method of fabricating probe carriers in which a plurality of tubes transports probes from reservoirs to the substrate and "paints" the probes in stripes on the substrate.
  • Figure 4 schematically represents a method of fabricating probe carriers in which individual probe containers pass across a substrate or plurality of substrates and deposit probe on the substrate as each probe container passes over the substrate.
  • Figure 5 illustrates various types of probe containers which may be used in the preceding fabrication technique, and the means that each employs to deposit probe from the carrier onto the substrate.
  • Figures 6a - 6e illustrate methods of fabricating probe carriers.
  • Figure 6a illustrates a method of fabricating probe carriers in which probe is contained in liquid in individual reservoirs.
  • Figure 6b illustrates a method of fabricating probe carriers in which a moving belt of spotters intersects the reservoirs so that each spotter picks up a separate probe.
  • Figure 6c illustrates a method of fabricating probe carriers in which the moving belt of spotters, with probe associated, intersects a substrate or plurality of substrates and deposits the probe thereon.
  • Figure 6d illustrates a method of fabricating probe carriers in which the spotters can be arranged in a continuous loop in which individual spotters are washed and reused for spotting new probes which are provided from a reservoir array.
  • Figure 6e depicts a method of transferring probe from a reservoir to a spotter. In this configuration, the spotter moves under the substrate and the substrate surface is positioned face down to allow the spotter to deposit the probe from underneath the subsfrate.
  • Figure 7a and 7b illustrate another method of constructing probe carriers, in which a matrix of spotters dips into a corresponding matrix of wells, each well of which contains a probe, then the spotter matrix is brushed across a substrate or plurality of substrates at such an angle that each spotter deposits a separate line, then the spotter array is washed and moves to a new matrix of probe containing wells, and repeats the dipping-brushing- washing cycle on a new section of substrate.
  • Figure 8 illustrates configurations of a probe carrier pin and a probe carrier rod.
  • Figure 9 illustrates fabrication methods for a probe carrier pin and a probe carrier rod.
  • Figure 10a illustrates a top view of a flexible probe carrier in a coil configuration.
  • Figure 10b illustrates a side view of a flexible probe carrier in a coil configuration.
  • Figure 10c is a cross-sectional view of two adjacent turns of a probe-carrier thread in which the probes are immobilized within notches on the carrier.
  • Figure 11a illustrates a flexible probe carrier in a spool configuration packaged in a mini cassette.
  • Figure 1 lb is a cross-sectional view of two layers of a probe-carrier in the spool in which the probes are immobilized in a notch on the carrier, and illustrates how the position of the immobilized probes in the notch protects them from friction with the next layer.
  • Figure 12 illustrates a method of using an electric field to control hybridization to a probe carrier.
  • Figure 13 illustrates a method of hybridization to a probe carrier pin.
  • Figure 14 illustrates a method of parallel hybridization of multiple target samples in standard microtiter plate format using probe carrier pins.
  • Figure 15 is a view of hybridization equipment for a probe carrier rod as viewed along the axis of the rod.
  • Figure 16 is a side view of hybridization equipment for a probe carrier coil.
  • Figure 17a and 17b illustrate hybridization equipment for a probe carrier spool.
  • Figure 18 illustrates a reader for scanning a probe carrier pin or a probe carrier rod.
  • Figure 19 illustrates a reader for scanning a probe carrier coil.
  • Figure 20 illustrates a reader for scanning a probe carrier spool.
  • the previous techniques utilize the optical fiber on which probes are immobilized to conduct light both to and from the markers of hybridization, which are typically fluorophores.
  • This detection technique relies on evanescent illumination from the optical fiber, which is inherently limited to the area immediately adjacent to the fiber surface, does not provide discrimination among groups of probes, and is limited in sensitivity.
  • the use of the optical fiber itself to conduct the excitation and emission light limits one to the use of optical fibers alone as substrates on which to immobilize probes and precludes the use of other substrates, such as metal wire or polymer, which may offer other advantages such as the ability to carry information about individual probes or groups of probes, as well as advantages in hybridization.
  • a probe carrier thread immobilizes the probes in an one dimensional array along a single length of thin, flexible thread.
  • a probe carrier thread system is comprised of three essential elements: probe carrier thread configuration and fabrication, hybridization and readout. Improved packaging of a probe carrier thread by use of probe carrier pin, probe carrier rod, probe carrier coil and probe carrier spool technologies increases the density of probes and enhances the inherent advantages of the probe carrier thread technology.
  • “Flexible,” as used herein, means capable of being bent, wound, coiled or otherwise flexed to the degree necessary for the operation of the invention without breaking. As illustrated in Fig.
  • probes are immobilized as spots (110) at the center or as narrow stripes (see Fig. 4, 404) across the width of a long, thin and flexible substrate (100).
  • probes can be immobilized as successive rows of spots, said rows being at an angle to the long axis of the substrate.
  • the length:width ratio of the substrate is at least about 5:1, preferably at least 50:1, more preferably at least 500:1, and most preferably at least 10,000: 1.
  • the length:width ratio of the probe-containing portion of the substrate is at least about 5:1, preferably at least 50:1, more preferably at least 500:1, and most preferably at least 10,000:1.
  • a "probe,” as used herein, is a set of copies of one type of molecule or one type of molecular structure which is capable of specific binding to or specific reaction with a particular sample or portion of a sample.
  • the set may contain any number of copies of the molecule or multimolecular structure.
  • "Probes,” as used herein, refers to more than one such set of molecules or multimolecular structures.
  • the molecules or multimolecular structures may be polynucleotides, polypeptides, oligosaccharides, polysaccharides, antibodies, cell receptors, ligands, lipids, cells, small molecules as are used to e.g. screen drugs as are used in screening pharmaceuticals, or combinations of these structures, or any other structures to which samples of interest or portions of samples of interest will bind or react with specificity.
  • the probes may be immobilized on the substrate by either covalent or noncovalent attachment.
  • “Flexible,” as used herein, means capable of being bent, wound, coiled or otherwise flexed to the degree necessary for the operation of the invention without breaking.
  • “Width” of the substrate is defined as the length of the longest perpendicular to the long axis of the subsfrate which is entirely contained within the substrate.
  • “Width” of the probe- containing portion of a cylindrical such as a thread substrate is defined as the linear distance of the longest arc, contained within the probe-containing portion of the substrate, which is perpendicular to the long axis of the probe-containing portion of the substrate.
  • Length of the probe-containing portion of the substrate is the linear distance along the long axis of the substrate from the first probe to the last probe of the probes on the substrate or, if there is a substantially larger gap between probes that form groups of probes, the length is the linear distance from the first to last probe in the group.
  • the present apparatus is used to analyze samples by 1) distinguishing the probes which have bound or react with sample or sample fragment from those that have not bound sample, then 2) establishing the identity of the probe(s) which have bound sample.
  • a further way to identify probes is by markers which serve to identify individual probes or sets of probes. Such markers may be used to convey more information than simply the identity of the probe.
  • the long, thin, and flexible nature of the probe carrier lends itself to numerous novel means of containment and use.
  • the probe carrier may be packaged in a number of formats including but not limited to a pin, a rod, a coil and a spool. Hybridization methods are considerably enhanced by requiring less hybridization fluid and enhanced mixing.
  • a flexible probe carrier packaged in a spool is especially advantageous in applications that require high volume, low to medium scale microarrays, such as those involved in disease diagnostics and management in major hospitals. In these applications, the required number of probes in the array is small (in the range of several hundreds to several thousands) but a very large number of the same type of arrays may be consumed every day.
  • a fully automated system integrates equipment for every stage of the analytical process, such as a hybridization station and a scanner. The machine takes in patients' DNA samples and feed the flexible probe carrier through the entire process and produces analysis and results in a fully automated manner without human intervention.
  • the substrate of the invention can be made of various materials.
  • the requirements of the substrate are that it have sufficient flexibility to withstand the conformational changes necessary to the manufacture and use of the apparatus, and that it be capable of immobilizing the particular probes to be used, or be capable of modification (for example, by coating) so that it is capable of such immobilization.
  • the substrate may also comprise various layers made of different materials, each of which has a function in the apparatus.
  • Flexibility may be measured by the ability to withstand winding to a certain diameter, for example a diameter of 10 cm, 5 cm, 2 cm, 1 cm, 0.5. cm, or 0.1 cm.
  • Preferred materials for the substrate of the present invention are silica glass, metallic materials, plastics, and polymers of sufficient strength to withstand the processes of manufacture and use.
  • silica i.e. pure glass
  • silica is a preferred material because polynucleotides and polypeptides can be covalently attached to a treated glass surface and silica gives out a minimum fluorescent noise signal.
  • the silica may be a layer on another material, or it may be the substrate, core or base material of the apparatus, or both.
  • One embodiment of the present invention comprises a metal wire as the core substrate, with a coating of silica on it for probe immobilization.
  • Another embodiment comprises a plastic or polymer tape as a base substrate, with a coating of silica for probe embodiment.
  • a further layer of metallic material maybe added, either on the opposite side of the tape from the silica layer, or sandwiched between the silica layer and the polymer or plastic.
  • the probe carrier thread can be made of different materials.
  • a preferred material is silica because DNA can be covalently attached onto a treated glass surface and silica emits minimum fluorescent noise.
  • fibers made of silica are flexible and have great elastic strength.
  • the optical fiber currently mass-produced for the telecommunication industry is made of silica.
  • Optical fiber is a substrate material which is made of primarily of silica and provides the necessary requirements. Although such fibers are manufactured for the purpose of transmitting light, the present invention does not require this feature of the fibers (although it may be used in some embodiments). Rather, it is other features of the optical fiber which make it particularly advantageous for the present invention.
  • the mechanical strength of optical fibers has been measured at 7 GPa, about 4 times that of the strongest steel while only 1/6 of its weight. Optical fibers are also highly flexible.
  • Standard 125 ⁇ m diameter fibers can be coiled in loops down to 5mm in diameter without breakage.
  • optical fibers are made from preforms, typically 1 meter long and 3 cm in diameter, fabricated using silica.
  • the center portion of the preform is doped with Germanium to create a core with higher refractive index to guide light through.
  • the perform is installed on a fiber draw tower in clean room environment, which heats it to the melting point and pulls out the fiber on to a large drum.
  • the cross- section shape of the fiber generally resembles that of the preform and the diameter of the fiber can be controlled through the pulling speed.
  • Most optical fibers on the market have
  • the cross-section of the fiber can be adjusted using a notched, D-shaped preform so that the fiber (200) has a notch, or groove (202) , in which probes (110) are immobilized.
  • This design protects the probes of one layer from friction with the substrate of a succeeding layer, as shown in Fig. 2b, where the cross-sections of two successive layers are shown one on top of the other.
  • optical fibers have very few structural defects. Also, optical fibers have excellent dimensional precision. Diameters are controlled to within ⁇ l ⁇ m. Finally, the cost of optical fibers is very low, at about l ⁇ 2jz. per meter. This is because the fabrication process of fibers is fairly straightforward and a single preform can produce up to 100 km of standard telecommunication fibers.
  • This detection technique relies on evanescent illumination from the optical fiber, which is inherently limited to the area immediately adjacent to the fiber surface, does not provide discrimination among groups of probes, and is limited in sensitivity. Furthermore, the use of the optical fiber itself to conduct the excitation and emission light limits one to the use of optical fibers as substrates on which to immobilize probes and precludes the use of other substrates, such as metal wire or polymer, which may offer other advantages such as the ability to carry information about individual probes or groups of probes, as well as advantages in hybridization, as discussed below.
  • Commercial telecom fibers are coated with a layer of non-porous polymer, which is not optimal for probe immobilization. The coating can be removed by techniques known in the art, such as those described in U.S.
  • Patent #5,948,202 which is incorporated by reference herein in its entirety.
  • bare fiber without this coating is prone to attack by water vapor, which generates micro-cracks on the fiber surface and degrades its strength.
  • the bare silica fiber may not survive the very tough environment during the hybridization stage.
  • One approach is to wind the fiber into a spiral coil along an elongated cylinder or drum after probe immobilization.
  • the fibers sit side-by-side on the drum and are attached to its solid surface.
  • the probes are aligned along a side of the fiber that is distal to the side of the fiber attached to the drum.
  • the drum provides mechanical support to the fiber during hybridization of sample and detection of the hybridization pattern.
  • a second approach is to strengthen the fiber substrate by applying one or several layers of coating to the silica fiber, which protect the fiber from the onslaught of water vapor and at the same time maintain good binding to probes.
  • the strengthened fiber can then be wound, for example, on a specially designed spool and assembled in a sealed cassette for transportation and handling.
  • An example of a substrate strengthening method is to coat the fiber with a metallic material, then an additional layer of silica.
  • Suitable coating materials including gold, silver and titanium due to their relative inertness in chemical solutions. Carbon coating is also widely used in the fiber optic telecommunications industry for hermetic sealing.
  • This invention in one embodiment further provides an additional layer of silica coating over the hermetic layer(s) to provide covalent binding with DNA probes.
  • a coating can be implemented through low cost sol-gel process and provides a surface for immobilization of probes, especially by covalent binding.
  • silica In addition to silica, other materials can also be used as the main body of the substrate. These include thin metal wires or strong polymer (polyimide or polytetrafluoroethylene (PTFE), for example) tapes. Again a sol-gel silica coating can be applied to the substrate to facilitate probe binding. For the polymer tape substrate, one can add a layer of metallic material sandwiched between the tape and the silica.
  • PTFE polytetrafluoroethylene
  • the metallic material element in all the substrate designs described above not only protects and/or strengthens the substrate, it may provide additional benefits during fabrication of the probe carrier and during binding of samples which carry a charge, which are described below.
  • the substrate is elongated. "Elongated,” as used herein, means that the length:width ratio of the substrate exceeds about 5:1, preferably exceeds 100:1, more preferably exceeds 1000: 1 , and most preferably exceeds 10,000: 1. It is contemplated that the length: width ratio can be even greater, such as at least 100,000:1 or at least 1,000,000:1. As defined above, "width" of the substrate is defined as the length of the longest perpendicular to the long axis of the substrate which is entirely contained within the substrate.
  • the width to be used to calculate the length:width ratio is the longest width.
  • "Width" of the probe-containing portion of the substrate is defined as the longest arc (for an arc shaped probe-containing area, as is typically found on a cylindrical thread-like substrate) or the large lineal distance for a flat substrate, contained within the probe-containing portion of the substrate, which is perpendicular to the long axis of the probe-containing portion of the substrate.
  • “Length” of the substrate is defined as the length of the long axis of the substrate. If the substrate has more than one length, the shortest of the lengths is used to calculate the length: width ratio.
  • the cross-section of the substrate can be of any shape.
  • cross-section is defined as the planar section through the substrate perpendicular to the long axis of the substrate.
  • cross-section can be any shape, two particular shapes represent different embodiments of the invention.
  • a “tape” refers to an embodiment utilizing a tape- , ribbon-, or strip-like substrate, whose cross-section is rectangular or nearly rectangular, or in the shape of a parallelogram.
  • Such a tape will have a thickness, corresponding to the width of the cross-sectional area. In various embodiments of the invention, this thickness does not exceed 500 micrometers, or 100 micrometers, or 50 micrometers, or 20 micrometers.
  • a "fiber” is an embodiment which utilizes a fiber-, thread-, or wire-like substrate, whose cross-section is rounded.
  • the cross- section may be circular, elliptical, or partially circular, for instance as with a fiber with a D- shaped cross-section.
  • the cross-section has a diameter, defined herein as the longest linear dimension of the cross section. In various embodiments of the invention, the diameter of the fiber does not exceed 500 micrometers, or 200 micrometers, or 100 micrometers, or 50 micrometers, or 20 micrometers.
  • the terms “tape” and "fiber” are intended to represent two parts of a spectrum of cross-sectional shapes. The invention can, however, have a cross- section of any shape.
  • the substrate may incorporate a groove or grooves running approximately parallel to the long axis of the fiber, in which probes are immobilized, as illustrated in Figure 2a.
  • a groove can be seen as an indentation in the cross-section.
  • the use of such a groove or indentation reduces or eliminates friction between probes immobilized in the groove and other surfaces; for example, when the substrate is wound on itself in a spiral, probes immobilized on one winding would be protected from the substrate on the next winding due to being recessed in the groove.
  • a D-shaped cross section incorporating such an indentation facilitates stacking of one layer of a winding on the next, as well as protection of probes.
  • Other embodiments may utilize different cross-sections, which will be useful in the use of the apparatus, and will be apparent to one of skill in the art.
  • a “probe,” as used herein, is a set of copies of one type of molecule or one type of multimolecular structure which is capable of specific binding to a particular sample or portion of a sample. "Probes,” as used herein, refers to more than one such set of molecules.
  • a probe may be immobilized on the substrate by either covalent or noncovalent attachment. Probes may be polynucleotides, polypeptides, oligosaccharides, polysaccharides, antibodies, cell receptors, ligands, lipids, cells, or combinations of these structures, or any other structures to which samples of interest or portions of samples of interest will bind with specificity. The set of probes chosen depends on the use of the apparatus.
  • polynucleotide means a polymeric form of nucleotides of any length, which contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • polynucleotide and nucleotide as used herein are used interchangeably. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes double- or single-stranded, and triple-helical molecules.
  • any embodiment of the invention described herein that includes a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form. Relatively shorter lengths of polynucleotides (less than about 100 nucleotides) are also refe ⁇ ed to as ohgonucleotides.
  • polynucleotides a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinylcytosine, 4- acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudo-uracil, 5- pentynyl-uracil and 2,6-diaminopurine.
  • uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
  • modification to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • modifications included in this definition are, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates,
  • any of the hydroxyl groups ordinarily present in the sugars may be replaced by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides or to solid supports.
  • the 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping groups moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls may also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, but not limited to, 2'-O-methyl-, 2'-O-allyl, 2'- fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, ⁇ -anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • one or more phosphodiester linkages may be replaced by alternative linking groups.
  • linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate”), P(S)S ("dithioate”), "(O)NR 2 ("amidate"), P(O)R, P(O)OR', CO or CH (“formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing and ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. Substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in' designing a final product, as can alternative backbone structures like a polyamide backbone.
  • polypeptide oligopeptide
  • peptide protein
  • polymers of amino acids of any length may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino ⁇ acids, etc.
  • Polypeptides can occur as single chains or associated chains.
  • a "ligand,” as used herein, is a molecule which binds to a particular receptor.
  • the receptor may be a cell receptor or it may be a portion of another molecule, for example, a receptor for an allosteric modifier of an enzyme.
  • ligands include, but are not limited to, enzyme cofactors, substrates and inhibitors, allosteric modifiers of enzymes, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, haptens, hormones, lectins, and drugs such as opiates and steroids.
  • a "cell receptor,” as used herein, is a cellular molecule, which may be normally located either intracellularly or in association with the cell membrane, which has an affinity for a given ligand. Examples include, but are not limited to, hormone receptors, cellular transporters, cytokine receptors, and neurotransmitter receptors.
  • Oligonucleotide probes of the invention are affixed, immobilized, provided, and/or applied to the surface of the solid support using any available means to fix, immobilize, provide and/or apply ohgonucleotides at a particular location on the solid support.
  • the various species may be placed at specific sites using inkjet printing (U.S. Pat. No. 4,877,745), photolithography (See, U.S. Pat. Nos.
  • oligonucleotide probes (20-25-mers) or peptide nucleic acids (PNAs) are produced either in situ during microa ⁇ ay fabrication, or offline using traditional methods and spotted on the microarrays.
  • PNAs peptide nucleic acids
  • the probes are long complementary DNAs (cDNAs) 500-5000 bases long, synthesized by fraditional methods before immobilization. Deficiencies of such technologies as quill-based spotters include imprecise sample uptake and delivery as well as lack of durability.
  • Martinsky et al. (U.S. Pat. No. 6,101,946) describe the use of an electronic discharge machine (EDM) which can be attached to a motion control system for precise and automated movement in three dimensions.
  • EDM electronic discharge machine
  • the oligonucleotide primers may also be applied to a solid support as described in Brown and Shalon, U.S. Pat. No. 5,807,522 (1998). Additionally, the primers may be applied to a solid support using a " robotic system, such as one manufactured by Genetic MicroSystems (Woburn, MA), GeneMachines (San Carlos, CA) or Cartesian Technologies (Irvine, CA).
  • chemically reactive solid substrates one may provide for a chemically reactive group to be present on the nucleic acid, which will react with the chemically active solid substrate surface.
  • silicon functionalities one may use organic addition polymers, e.g. styrene, acrylates and methacrylates, vinyl ethers and esters, and the like, where functionalities are present which can react with a functionality present on the nucleic acid.
  • Amino groups, activated halides, carboxyl groups, mercaptan groups, epoxides, and the like may also be provided in accordance with conventional ways.
  • the linkages may be amides, amidines, amines, esters, ethers, thioethers, dithioethers, and the like. Methods for forming these covalent linkages may be found in U.S. Pat. No. 5,565,324 and references cited therein.
  • One may prepare nucleic acids with ligands for binding and sequence tags by primer extension, where the primer may have the ligand and/or the sequence tag, or modified NTPs may be employed, where the modified dNTPs have the ligand and/or sequence tag.
  • RNA For RNA, one may use in vitro transcription, using a bacteriophage promoter, e.g. T7, T3 or SP6, and a sequence tag encoded by the DNA, and transcribe using T7, T3 or SP6 polymerase, respectively, in the presence of NTPs including a labeled NTP, e.g. biotin-16-UTP, where the resulting RNA will have the oligonucleotide sequence tag at a predetermined site and the binding ligand relatively randomly distributed in the chain.
  • a bacteriophage promoter e.g. T7, T3 or SP6, and a sequence tag encoded by the DNA
  • T7, T3 or SP6 polymerase e.g. T7, T3 or SP6 polymerase
  • probes may be synthesized in situ on the substrate or may be manufactured then immobilized on the subsfrate. This technique has been described for polynucleotides in U.S. Patent No. 5,419,966, incorporated herein by reference.
  • polymeric probes such as polynucleotides, may be synthesized in a stepwise fashion from individual monomers or from smaller polynucleotides or other subunits.
  • probes are immobilized in discrete areas of the substrate.
  • more than one probe can be immobilized in a particular area, with individual probe molecules of a particular type being distinguishable from other probe molecules by differential labeling, for example, with differently colored fluorescent tags.
  • Immobilize means to attach a probe to the substrate by covalent or non-covalent means, with sufficient affinity to withstand manufacturing, sample-binding, sample analysis steps, and, if necessary, re-use.
  • Methods and materials for derivatization of solid phase supports for the purpose of immobilizing polynucleotides and polypeptides are well-known in the art and are described 1 in, for example, U.S. Patent Nos. 5,744,305 and 5,919,523, which are hereby incorporated by reference in their entirety.
  • the prefe ⁇ ed method is by biotin- streptavidin attachment, but any method of non-covalent attachment that provides the necessary affinity is possible with the present invention.
  • assemblages of molecules may also be used as in the case of organelles, e.g. nuclei, mitochondria, plastids, liposomes, etc., or cells, both prokaryotic and eukaryotic.
  • the bound component may be directly bound to a solid substrate or indirectly bound, using one or more intermediates, which serve as bridges between the bound component and the solid substrate.
  • the surface may be activated using a variety of functionalities for reaction, depending on the nature of the bound component and the nature of the surface of the solid substrate.
  • chemically reactive solid substrates one may provide for a chemically reactive group to be present on the nucleic acid, which will react with the chemically active solid substrate surface.
  • silicon functionalities one may use organic addition polymers, e.g. styrene, acrylates and methacrylates, vinyl ethers and esters, and the like, where functionalities are present which can react with a functionality present on the nucleic acid.
  • Amino groups, activated halides, carboxyl groups, mercaptan groups, epoxides, and the like may also be provided in accordance with conventional ways.
  • the linkages may be amides, amidines, amines, esters, ethers, thioethers, dithioethers, and the like. Methods for forming these covalent linkages may be found in U.S. Pat. No. 5,565,324 and references cited therein.
  • One may prepare nucleic acids with ligands for binding and sequence tags by primer extension, where the primer may have the ligand and/or the sequence tag, or modified NTPs may be employed, where the modified dNTPs have the ligand and/or sequence tag.
  • RNA For RNA, one may use in vitro transcription, using a bacteriophage promoter, e.g. T7, T3 or SP6, and a > sequence tag encoded by the DNA, and transcribe using T7, T3 or SP6 polymerase, respectively, in the presence of NTPs including a labeled NTP, e.g. biotin- 16-UTP, where the resulting RNA will have the oligonucleotide sequence tag at a predetermined site and the binding ligand relatively randomly distributed in the chain.
  • NTPs including a labeled NTP, e.g. biotin- 16-UTP
  • markers may be used with conventional two-dimensional a ⁇ ays as well was with the present, one-dimensional configurations.
  • Markers are any type of identifiable marking, a ⁇ angement, or other structure or pattern on, in, or associated with the substrate and/or probes which conveys information about a particular probe or set of probes.
  • One type of marker can be optical. These can be space markers (i.e., breaks in the row of probes on the substrate, as described above) and/or bar codes , fluorescent markers, chemilluminescent markers, or any other marker capable of being detected with light.
  • a further type of marker is magnetic markers. The present invention lends itself to such markers because the substrate may contain metallic elements which are magnetizable.
  • One method for space marking and/or additional information is to coat the reverse side of the substrate from the probes with a layer of magnetic thin film. Then spatial or probe identification can be recorded during the fabrication process, by magnetic means.
  • An important advantage of this approach is that additional information regarding the target can be written on to the substrate itself during the hybridization stage. And at the scanning stage, scanning parameters and other digital outputs may also be written on to the same tape for further reference. Other types of markers will be apparent to one of ordinary skill in the art. 1.5 Sets of probes.
  • Probes may be immobilized in sets on the substrate, each set sharing some common characteristic. For example, if probes are nucleotides, a group of nucleotides requiring common hybridization conditions maybe immobilized along a certain length of substrate, and another group requiring a different set of hybridization conditions may be immobilized along another length of substrate. In this manner, each set of probes may be exposed to sample under a different set of conditions, optimizing sample binding.
  • a single probe carrier can carry different sets of probes for diagnosing different diseases.
  • one set of probes located along one stretch of a carrier might be used to diagnose HIV, which another set could be used to diagnose herpes, etc.
  • a carrier or portion of a carrier could be devoted to the HER-2/neu gene.
  • the HER- 2 gene also known as HER-2/neu and c-erbB2
  • the amplified HER- 2 gene results in the over-production of protein receptors found on the surface of tumor cells. These special proteins bind with other circulating growth factors to cause uncontrolled tumor growth.
  • the probe carrier could contain probes for the HER-2/neu gene(s) and variations.
  • the sets of probes may be redundant, allowing a single carrier to be used repeatedly for the same assay, with a new set of probes used for each successive assay.
  • Probes may be immobilized on the substrate in any configuration that allows one to distinguish and identify probes which have bound sample from those which have not. The simplest way to do this is by placing probes in discrete areas, one probe per area. The areas may be spots, as shown in Fig. 1, or lines, as shown in Fig. 4, 404. The areas may be configured as a single row running along or parallel to the long axis of the substrate. The probes may be directly attached to the substrate, or, in an alternative embodiment, the probes may be attached to beads which are then attached to the substrate. Methods of attaching probes to beads of various materials are well-known in the art and are described in, for example, WO 99/60170, which is incorporated herein by reference in its entirety.
  • DNA probes 100 are immobilized as spots at the center or as na ⁇ ow stripes across the width of a long, thin and flexible thread subsfrate 100.
  • Probe identification is achieved through space markers and/or bar codes 120 printed in the space 130 between groups of probes. Alternatively, these markers can be printed on the other side of the thread substrate.
  • the thread 100 can have a special cross-sectional shape.
  • the cross-section of the fiber can be adjusted so that the fiber 200 has a notch or groove 202 in which probes 110 are immobilized. This design protects the probes of one layer from friction with the substrate of a succeeding layer, as shown in Fig. 1 lb, where the cross-sections of two successive layers are shown one on top of the other.
  • the long, thin, and flexible nature of the probe carrier lends itself to numerous novel means of containment, arrangement and use.
  • the probe carrier may be packaged in a number of formats including but not limited to a pin, a rod, a coil and a spool. Hybridization methods are considerably enhanced by requiring less hybridization fluid and enhanced mixing.
  • a flexible probe carrier packaged in a spool is especially advantageous in applications that require high volume, low to medium scale microa ⁇ ays, such as those involved in disease diagnostics. In these applications, the required number of probes in the a ⁇ ay may be small (in the range of several hundreds to several thousands) but a very large number of the same type of a ⁇ ays may be available for consumption every day.
  • a pin or rod package is made by spirally winding a certain length of fabricated flexible probe carrier thread around a section of solid cylinder or tube. The thread sits tightly side-by-side on the outer surface of the supporting cylinder in the prefe ⁇ ed embodiment and may be permanently attached to it by glue, cement or other means.
  • the difference between the probe carrier pin and the probe carrier rod is the size.
  • a probe carrier pin normally has a diameter less than 10mm while a probe carrier rod is larger and can have a much larger diameter thus accommodating many more probes.
  • a 1.5 meter long, 50 ⁇ m diameter thread occupies only a short 5mm section after being wound on a 5mm diameter probe carrier pin, which may carry approximately 15,000 probes, presuming a lOO ⁇ m probe space along the thread.
  • a probe carrier rod of 30mm wide and 40mm in diameter can accommodate as many as 700,000 probes along a 70 meter long, 50 ⁇ m diameter thread.
  • the fabricated flexible probe carrier thread is wound into a flat, disc shape coil.
  • the probes on the thread are exposed on one side of the disc while the other side is permanently attached to a solid discshaped planar support by epoxy, cement or other suitable means.
  • probes are deposited in a notch on the surface of the probe carrier thread.
  • the planar support can be pre- coated with a conductive layer to facilitate hybridization control. Assuming a 50 ⁇ m diameter thread, a probe carrier coil of 40mm in diameter can accommodate up to 24 meters of probe carrier thread, carrying 240,000 probes.
  • the configuration of a probe carrier spool can be very similar to that of the probe carrier coil.
  • the probe carrier thread is not permanently attached to a supporting surface, thus allowing the thread to unwind from the spool for hybridization, reading and other purposes.
  • the cross-section shape of the probe carrier thread can be designed to avoid friction between DNA probes and the probe carrier thread in an adjacent turn.
  • a cassette can be constructed to protect the probe carrier spool and facilitate its winding and unwinding.
  • multiple spools can be stacked up in one cassette.
  • a fiber- or tape-like substrate is intrinsically suitable for continuous, high speed, mass production.
  • Figures 3 through 7 show examples of fabrication systems designs.
  • conventional spotting techniques may be used to produce discrete areas containing one probe each
  • one aspect of the present invention is the use of brushing or painting of probes on substrates.
  • Such techniques coupled with the essentially one-dimensional nature of the substrate, lend themselves to fabrication systems in which multiple copies of the same tape or fiber may be manufactured at once at high speed and with great precision.
  • Multi-stranded brush An exemplary embodiment of this apparatus and a method of manufacture is presented in Fig. 3.
  • probes which are either in a suitable liquid or are liquid themselves (for example, some lipids which are liquid at room temperature or can be liquefied at suitable temperatures), into tubing and depositing the probe from the tubing onto the substrate by moving the tubing relative to the substrate while at the same time driving the probe-liquid from the tube onto the subsfrate.
  • movements are relative and can be accomplished by moving the tubing assembly, moving the substrate, or both.
  • the tip of the capillary tube may contact the substrate surface. Alternatively, the tip may move a short distance above or underneath the subsfrate surface.
  • the probe fluids are deposited onto the subsfrate using one of the non-contact deposition methods.
  • These include attaching probes to magnetic beads suspended in the probe fluid and placing an electromagnet under the substrate.
  • the magnet is activated during when the capillary and substrate intersect (i.e. the capillary passes in proximity to the subsfrate), which pulls the magnetic beads and their associated probe onto the substrate surface.
  • Another non-contact deposition method is to coat a metal layer on both the end facet of the capillary tubing and either the substrate surface or the support under the substrate, then apply a high voltage between the capillary and the substrate or substrate support. The electric field will pull the electrically charged probes (such as ohgonucleotides) onto the substrate surface.
  • probe will be deposited on the substrate as a dot. If the signal is on for much or all of the time that the capillary and substrate intersect, the result will be a stripe of probe on the substrate. Conditions may be selected to ensure immobilization of the probe on the substrate.
  • a plurality of tubes may be joined together to create a "brush" capable of depositing multiple probe stripes simultaneously. Further, a plurality of such brushes may be a ⁇ anged to multiply the number of probes which may be deposited, either simultaneously or sequentially as different brushes move relative to the substrate and deposit probe stripes.
  • the substrate is a fiber
  • several fiber substrates may be positioned so that one stroke of a brush deposits probes stripes on all of the substrates.
  • a wide tape substrate may be used to receive the probe stripes, then the tape may be cut lengthwise into a plurality of individual, thinner, tapes. It can be appreciated that such a method of manufacture greatly multiplies the number of probe carriers which may be produced simultaneously, increasing throughput and reducing cost. It can also be appreciated that standard mass production methods, such as the use of conveyor belts, can be readily adapted to automate and control this and other methods of manufacture presented herein.
  • a set of flexible capillaries 300 are glued into a hole under each well of a standard microtifre plate 302.
  • a capillary 300 can also be inserted into the well from the top.
  • the capillary 300 is then lined up into a linear a ⁇ ay to form a "brush" 304.
  • Each individual DNA probe is stored in a separate well in the plate and is driven into the capillary 300 linked to the well by pressure differentiation or by applying a voltage between the well and the tip of the brush 304. Because DNA molecules are negatively charged, a negative polarity should be applied to the well end. Multiple such capillary brushes can be constructed.
  • the capillary a ⁇ ay is moved to "brush" across a stationary probe carrier tape subsfrate 306 and deposit an array of DNA probes 110, then the tape substrate 306 will move forward to a new position to enable a second capillary "brush” 304 to deposit more probes 110 on subsequent positions.
  • the same brush can be used to deposit more copies of the same probe array along the tape substrate 306.
  • a large number of thread substrates can be laid in parallel under the brush so that each "brushing" action can produce multiple copies of the 1-dimensional probe a ⁇ ay on different threads or tapes.
  • markers for the probes 110 are also deposit or establish markers for the probes 110 (see, for example, Fig. 1).
  • markers may be spaces between or around probes, or they may be optical bar codes (Fig. 1, 120), or fluorescent markers, or magnetic markers encoded on a metallic element in the subsfrate, or any other means that would serve to identify a particular probe or group of probes. They may be established on the same side of the substrate that the probes are deposited on, on the opposite side, or both. It will be understood that a substrate may have only one surface (for example, a fiber with a circular cross-section), and that the term "side" in this context refers to the particular area of the surface on which probe is deposited.
  • top or bottom surfaces In the case of a tape, with a more defined top surface and bottom surface, "side" means one of these top or bottom surfaces.
  • a variety of means may be used to provide the force to drive probe from the reservoir, into the tubing, and onto the substrate. For example, a pressure differential may be established. Alternatively, if the probe is charged— as is the case with, for example, DNA ⁇ a voltage may be established between the reservoir and the substrate, such that the probe moves from the reservoir and tubing onto the substrate.
  • the substrate may contain a metallic element such as a metal layer which forms an electrode.
  • Figure 4 shows a second design of a probe carrier thread fabrication system, where each probe is stored in its own "printing head" 410 of a print system 408.
  • a large number of such printing heads 410 are a ⁇ anged into a one-dimensional a ⁇ ay on a conveyer belt 400 moving at a constant speed V h .
  • the belt can wind across pulleys or capstans 412 into a spools 406 to conserve space.
  • the spacing between the heads can be as large as several millimeters and be sufficient to accommodate a reservoir for each probe.
  • a probe carrier tape substrate 402 is placed under the printing heads a ⁇ ay, also moving at a constant, albeit slower speed V t .
  • the line may be diagonal across the substrate, since the substrate is moving, or the subsfrate and conveyor may intersect at an angle that results in a line of probe that is perpendicular to the long axis of the substrate.
  • the substrate may instead be stopped while the print head prints, then advanced to the next printing position before the next probe is applied.
  • Each printing head in the system consists of a reservoir that holds a certain quantity of the probe sample and a means to transfer the probe onto probe carrier or tape thread substrate.
  • Probes are dispersed in a liquid (or are themselves liquid) which provides the necessary conditions for transfer and immobilization of the probes on the substrate, the exact nature of which depends on the particular probe and the particular subsfrate.
  • Figure 5 shows some possible designs for the printing heads.
  • a very thin, flexible fiber 500 is attached to a small opening 502 under a probe reservoir 504.
  • the fiber 500 is hydrophilic so it draws the probe fluid onto its surface through surface tension or
  • the fiber 500 has to be thin ( ⁇ 80 ⁇ m), flexible and yet not deform plastically.
  • a solid or hollow silica fiber coated with metal or nylon is a good candidate. When intersected with the probe carrier tape substrate 402, it "draws" a stripe on the surface of the probe carrier tape substrate 402 using the probe as "ink”. In the case of metal coated fiber, a negative voltage can be applied to the fiber to push the DNA sample on to the probe carrier thread or tape.
  • Figures 5(b)-(h) show different designs based on the inkjet principle, where a pin hole is produced on the bottom of the probe reservoir. Pulse energy is introduced into the reservoir, which ejects droplets out of the pinhole on to the probe carrier thread underneath.
  • a piezo ring 506 is glued on the wall of the reservoir tube, which squeezes the tube under a voltage and ejects a droplet.
  • a piezo-film 506 is coated on a diaphragm 508 on top of the reservoir 504, which will have the same function as the piezo ring but could be less expensive when using a large number of reservoirs 504.
  • a cu ⁇ ent pulse through a resister wire 510 generates a bubble through localized heating, which in turn pushes out the droplet.
  • the ejecting energy is introduced through an external ultrasound transducer 512.
  • the reservoir tube is transparent and the heating is realized by focusing a laser 514 to a light absorption patch inside the tube.
  • the reservoir 516 is made of metal. A high voltage is applied between the reservoir and the probe carrier tape substrate 402 (or object underneath the probe carrier tape substrate 402) with the negative polarity on the reservoir. Since DNA carries negative charge, the electric field will eject the sample on to the probe carrier tape subsfrate 402 surface.
  • probe molecules are attached to magnetic beads suspended in fluid 520 within reservoir 518.
  • a current pulse is applied to the electromagnet 519 underneath the substrate, which attracts the probe from the small opening under the reservoir onto the substrate surface 402.
  • the actuators are external and do not move with the printing head. Since each reservoir (504 or 516 or 518) only intersects the probe carrier tape subsfrate 402 once at a fixed location, only one such actuator is needed in the system. Presuming a reservoir spacing of 2mm, a 150,000 reservoirs a ⁇ ay is 300m long and can be accommodated in a spool less than 80cm in diameter. 2.3 Spotters and reservoirs
  • Fig. 6 illustrates another probe carrier fabrication system design, where the printing head configuration of the previous design is separated into a "spotter configuration" and a "reservoir configuration.” Each probe has its own reservoir, the structure of which is kept simple to reduce cost.
  • Figure 6a illustrates one of the possible reservoir designs, where the combination of liquid internal pressure and surface tension causes the liquid 600, which contains the probe 110, to bulge up a little at the opening 612.
  • a large number of reservoirs 602 are assembled on a conveyer belt to form a linear a ⁇ ay.
  • the spotter configuration 604 can be fabricated by, for example, shaping a thin metal tape into a linear configuration of miniature teeth or gluing short silica fibers 606 to a flexible metal tape 608.
  • the tip of the spotter is suspended a short distance above the reservoir opening that allow the tip to contact the probe fluid.
  • the spotter is made of highly elastic materials, such as silica fiber, the spotter tip can actually slightly lower the opening so that the spotter can tip into the opening to collect the probe fluid.
  • Both the spotter and reservoir configurations are driven to travel at e.g. a constant speed in different directions as indicated by a ⁇ ows 614 and 616.
  • a spotter intersects opening 612 of a reservoir 602
  • a droplet of probe liquid 600 will be transfe ⁇ ed from the reservoir to form droplet 610 on the spotter.
  • the amount of liquid in the droplet can be controlled by the duration of the intersection and the shape and size of the spotter.
  • the movement pattern of the spotter and reservoir configurations is designed in such a way (to be described later) that each consecutive spotter will intersect with co ⁇ esponding consecutive reservoir so that each spotter now carries a different probe droplet.
  • the spotter configuration 604 moves on to intersect with a substrate 618 moving in a direction such that the spotter configuration and the substrate intersect.
  • the tip of each spotter 606 may physically contact the substrate or may be suspended a very short distance (several tens of micrometers) above the subsfrate, at a distance that allows droplet 610 to contact the substrate 618.
  • the probe droplet will be transfe ⁇ ed from the spotter configuration to the subsfrate to form a linear probe configuration 630 on the subsfrate. If the probe is charged, it is possible to charge a particular spotter by electronic means when it intersects a probe reservoir with a charge that will attract probe, then switch the charge on the spotter to the opposite charge when it later intersects the subsfrate, in order to repel the probe from the spotter and onto the substrate. This refinement allows the size of the droplet on the spotter to be controlled precisely with good consistency. Such a method to transfer probe material to the substrate is termed "spotting", and is widely used manually in laboratories.
  • the spotter configuration 604 may move in a circle and the number of spotters in the configuration can be far fewer than the total number of probes.
  • the spotter After the spotter leaves the substrate 618, it can be washed in a washing area 620, dried in a drying area 622 and reused by circling around to intersect 626 the probe reservoir configuration 624 again. Since the washing is carried out in parallel with spotting, it does not affect fabrication throughput.
  • a spotter according to this invention is easy to clean
  • the probe reservoir can be a straight tubing 632 or a well 634 with a small opening 636 at its bottom 638.
  • the probe fluid 600 bulges downward at opening due to the combination of gravity and capillary force.
  • the spotter 606 intersects the probe reservoir from underneath and collects some probe fluid 600 at its tip. Then the spotter moves on to intersect the probe carrier substrate 610 and paints a na ⁇ ow stripe on the substrate in a configuration similar to Figure 6c, except the spotter carrier 406 now passes under the subsfrate instead of above it.
  • the substrate is positioned face down to be painted by spotters.
  • the configuration of the entire fabrication system including probe collection, spotting, washing and drying is similar to that shown in Figure 6d.
  • every component can move at a predefined, constant speed. This reduces the complexity of the motion control and precision requirement.
  • a large number of probe carriers 100 can be manufactured continuously without manual intervention. As a result, the manufacturing throughput can be very high.
  • silica fiber or thin wire is adapted as the probe carrier subsfrate, many fibers can be attached in parallel to a wide carrier tape for the fabrication stage. So, multiple copies of the same probe carrier thread can be manufactured at the same time.
  • a wide tape substrate can be used on the fabrication station, with probe droplets being deposited in a line across the tape as illustrated in area 628 of Fig. 6d.
  • the wide tape can be cut after the probe deposition to produce many copies of the same probe carrier threads 100, as illustrated in Fig. 1. In either case, the throughput can be further dramatically increased.
  • spacing between probes on the thread and that between reservoirs (and spotters) can be lOO ⁇ m and 5mm, respectively. Assuming the thread substrate moves at a speed of lcm/s, the reservoir and spotter a ⁇ ays travel at 50cm/s, which is easily to achieve. Further, where the carrier tape 618 is separated into 20 thread substrates, the above two system designs should be capable of manufacturing a 150,000 probe array every 7.5 seconds. 2.4 Spotter matrix
  • Figure 7 shows a fourth fabrication system design, which has more flexibility and is particularly suitable for custom fabrication of smaller scale probe carriers.
  • Fig. 7a is an overhead view and Fig. 7b is a frontal view of the system.
  • probes are stored in standard microtifre plates or similar matrix containers 700.
  • a matching spotter matrix 702 has the same spacing as the well matrix in the plate. Different from conventional spotting pins, each spotter is a thin, flexible hydrophilic fiber 704, as used in the preceding system. The spotter matrix is first dipped into the well matrix, then moved to intersect the probe carrier tape subsfrate 706, which is temporarily held stationary.
  • the direction that the spotter moves is pe ⁇ endicular to the probe carrier tape subsfrate 706, but the direction of the matrix rows is tilted at a small angle of ⁇ .
  • the spotter matrix a ⁇ ay is washed and cleaned in a cleaning area 710 and dried at a drying station 712.
  • the probe carrier tape substrate 706 advances to a fresh section and a new well matrix 712 is loaded, ready for the next "dipping and spotting" cycle.
  • the spacing between probes on the probe carrier tape subsfrate 706 is given by
  • Lc/(R+1) can be about 250 ⁇ m. Such a density is useful for smaller scale custom a ⁇ ays, as a probe carrier tape substrate 706 carrying 10,000 probes at a 259 um probe spacing is 2.5 meters long, which can be wound into a spool less than 3 cm in diameter.
  • the flexibility of the probe carrier thread platform enables the fabricated probe carrier thread to be packaged into a wide variety of different formats, which includes, but is not limited to, probe carrier pin, probe carrier rod, probe carrier coil and probe carrier spool.
  • the superior strength, precision and flexibility of the probe carrier thread substrate are ideal for the precise probe positioning and transportation required in the probe carrier thread fabrication and reading process. Assuming both the probe spacing and thread thickness being
  • probe carrier thread packaging is prefe ⁇ ed for greater compaction of probes.
  • probe carrier pin 810 and probe carrier rod 820 are made by spirally winding a certain length of fabricated probe carrier thread 100 around a section of an elongated support member 804 such as a solid cylinder or tube.
  • the tightly- wound thread 100 sits side-by-side 806 on a section 802 of the outer surface of the supporting cylinder 804 and may be permanently attached to it by glue, cement or other means.
  • the cylinder 804 may be coated with conductive material before the winding process for hybridization control.
  • the probes 110 are located on a side of the probe carrier thread 100 distal from the side of the probe carrier thread 100 which is contact with the support member 804.
  • a probe carrier pin 810 normally has a diameter less than 10mm while a probe carrier rod 820 is larger and thus accommodates many more
  • a 1.5 meter long, 50 ⁇ m diameter thread occupies only a short 5mm section after being wound on a 5mm diameter pin 810, which may carry approximately
  • a probe carrier rod 820 of 30mm wide and 40mm in diameter can accommodate as many as 700k probes along a 70 meter long, 50 ⁇ m diameter thread 100.
  • the probe carrier rod 820 of 30mm wide and 40mm in diameter can accommodate as many as 700k probes along a 70 meter long, 50 ⁇ m diameter thread 100.
  • flexible probe carrier may be a tape substrate carrying probes immobilized in a 2-dimensional a ⁇ ay. Fabrication of such a ⁇ ays is illustrated in Figs. 3 and 4 for instance.
  • Such a flexible probe carrier tape can be wrapped around a pin 810 or a rod 820 instead of winding a probe carrier thread 100.
  • the probe carrier pin 810 and probe carrier rod 820 described above can be manufactured efficiently at a high throughput.
  • a certain length of "blank" space 904 is introduced between any two sets of probe a ⁇ ays along the probe carrier thread or tape 100 during thread fabrication and prior to placing the thread or tape on the supporting cylinder. Then the probe carrier thread 100 is wound continuously along a long supporting cylinder 804.
  • the cylinder 804 is pre-coated with epoxy or other adhesive at certain positions, where sections of the probe carrier thread 100 carrying probes 110 will be attached. After the epoxy is cured, the long cylinder 804 with thread 100 on can be cut at appropriate intervals to produce multiple probe carrier pins 810 or probe carrier rods 820 with probe carrier thread 100, with probes 110 attached, wound around the cylinder and at certain sections 902. Because the section 904 of the supporting cylinder 804, where the blank thread is attached to, is not pre-coated with epoxy, the blank thread will come loose and break off the cylinder after cutting, thus exposing a section 904 of the original supporting cylinder, which can be used to fit into adapters during the hybridization process. 3.2 Probe Carrier Coil
  • a probe carrier coil shown in Figure 10 the fabricated probe carrier thread 100 is wound into a flat, disc shape coil 1012.
  • Fig. 10a shows a top view
  • Fig. 10b shows a side view of a probe carrier coil 1012 assembly.
  • the probes 110 on the thread 100 are exposed on one side of the disc 1012 while the other side is permanently attached to a solid planar support disc 1010 by epoxy, cement or other suitable means.
  • Figure 10c which illusfrates an enlarged view of a cross-section 1000 of aprobe carrier coil 1012, probes 110 are deposited in a notch 202 on the probe carrier thread 100 surface. This feature is optional in this packaging format.
  • the planar support 1010 can be pre-coated with a conductive layer
  • a probe carrier coil 1012 of 40mm in diameter can accommodate up to 24 meters of thread, carrying 240,000 probes.
  • the configuration of a probe carrier spool 1110 is very similar to that of the probe carrier coil. However, unlike probe carrier coil 1012, the probe carrier thread 100 is not permanently attached to a supporting surface 1010, thus allowing the thread to unwind from the spool for hybridization, reading and other purposes (although the end of the thread may be attached to the substrate).
  • the cross-sectional shape of the probe carrier thread 100 can be designed to avoid friction between DNA probes and the thread in adjacent turns.
  • the cross-section of the substrate used to manufacture the probe carrier thread 100 can be selected such that the fiber 200 has a notch or groove 202 in which probes 110 are immobilized.
  • a cassette 1100 can be constructed to protect the spool 1110 and facilitate its winding and unwinding, hi addition, multiple spools 1110 can be stacked up in a single cassette 1100.
  • an apparatus involves: 1) preparation of the sample; 2) formation of a probe-sample complex; and 3) analysis of the binding pattern in order to identify the individual probes to which sample has bound.
  • sample preparation protocols for analysis of polynucleotides including labeling of samples with fluorescent tags in order to facilitate step 3), analyzing the binding pattern, are well-known in the art. See, for example, U.S. Patent No. 5,800,992, which is hereby inco ⁇ orated by ⁇ reference in its entirety.
  • the sample is fragmented, using known techniques such as restriction endonuclease digestion, converted to single-stranded form, and the single-stranded fragments are labeled with an appropriate fluorescent tag.
  • sample or sample fragments which have an affinity for particular probes bind with those probes.
  • Present microarrays generally utilize hybridization of complementary strands of DNA as the binding method. DNA hybridization is highly dependent upon hybridization conditions, which have been extensively studied and described; see, for example, U.S. Patent Nos. 6,054,270 and 5,700,637, which are hereby inco ⁇ orated by reference in their entirety.
  • the present invention also encompasses any sort of sample-probe binding which will allow one to derive information from determining which probes have bound to sample or sample fragments. Examples include determining the identity of antigens or antibodies in a sample by using various antibodies or antigens, respectively, as probes, or identifying hormones in a sample by the receptors to which they bind, etc.
  • the list of sample/probe pairs extends to any sets of pairs which bind with each other with a sufficient degree of affinity and specificity to be identified, and further examples of sample/probe pairs will be readily apparent to those of skill in the art.
  • Nucleic acid hybridization generally involves the detection of small numbers of target nucleic acids (DNA and RNA) among a large amount of non-target nucleic acids with a high degree of specificity. Stringent hybridization conditions are necessary to maintain the required degree of specificity and various combinations of agents and conditions such as salt, temperature, solvents, denaturants and detergents are used for the piupose. Nucleic acid hybridization has been conducted on a variety of solid support formats, (see, e.g., Beltz, G.A., • et al., Methods in Enzymology, Vol. 100, part B, 19: 266-308, Academic Press, NY (1985)).
  • a DNA chip including an oligonucleotide a ⁇ ay is comprised of a number of individual ohgonucleotides linked to a solid support in a regular pattern such that each oligonucleotide is positioned at a known location.
  • samples containing the target sequences are exposed to the a ⁇ ay, hybridized to the complementing ohgonucleotides bound to the a ⁇ ay, and detected using a wide variety of methods, most commonly radioactive or fluorescent labels.
  • U.S. Pat. No. 5,837,832 (Chee et al.) and related patent applications describe immobilizing an a ⁇ ay of oligonucleotide probes for hybridization and detection of specific nucleic acid sequences in a sample.
  • This invention also provides some specially designed equipment for the hybridization of probe carrier thread based microa ⁇ ays.
  • hybridization is achieved by either natural diffusion or forced fluid circulation. The format is slow and the latter system is complicated to fabricate.
  • the hybridization chambers are designed to ensure that there is only a very thin layer of target fluid between the probe carrier thread or its packaged form and the inner wall of the hybridization chamber. In this way, only a very small volume of the target fluid is required for the hybridization, improving contact between probe molecules and target molecules.
  • Hybridization acceleration is thus achieved by e.g. moving the probe carrier thread 100 or its packaged format through the target fluid.
  • hybridization process can be further controlled by applying a voltage between the support of probe carrier thread and the inner wall of the hybridization chamber.
  • hybridizations may also be accelerated by adding cations, volume exclusion or chaotropic agents.
  • cations volume exclusion or chaotropic agents.
  • the array is washed sequentially with a low stringency wash buffer and then a high stringency wash buffer.
  • an AC oscillation voltage 1220 can be applied between the probe carrier thread 100 or its support 1200 and the wall of the hybridization chamber 1210 to improve the efficiency of the process.
  • the support 1200 and the wall of the hybridization chamber 1210 have conductive coating 1212 in order to facilitate the process.
  • a brush slip ring can be used to conduct voltage on to the moving electrode. The design of such electric slip ring is well known in the art.
  • a probe carrier pin 810 can be hybridized by directly plugging into a well 1300 containing target fluid 1310.
  • the diameter of the well 1300 is only slightly larger than the outer diameter of the probe carrier pin 810.
  • the required volume for the target fluid is minimal. For example, presuming the well is
  • section is 5mm high, 3 ⁇ l target fluid would be sufficient to cover the entire effective section
  • the probe carrier pin can undergo an up-and-down translational 1330 or back-and-forth 1332 rotational motion, or the combination of the two, in order to increase the hybridization speed. Because of the spiral winding pattern on probe carrier pin 810, probe carrier rod 820 and probe carrier coil 1012, rotational motion drives the target fluid along not only the circular direction but also the axial or radial directions of the package. It efficiently moves the molecules in the target over the entire surface area covered by probe carrier thread 100. Multiple probe carrier pins 810 can be plugged into an adapter frame 1400 to form a matrix that is compatible with the spatial pitch and pattern of a standard microtiter plate 1420.
  • multiple hybridization processes can be carried out in parallel directly in a standard microtiter plate 1420 by dipping each probe carrier pin 810 into a co ⁇ esponding well 1410 of the standard microtiter plate 1420, as shown in Figure 14, and optionally translating the adapter plate up and down or rotating the individual probe carrier pins.
  • a probe carrier rod 820 can be hybridized in a similar, albeit larger hybridization chamber as that of probe carrier pin. Alternately, a chamber design 1500 shown in Figure 15 can be used. A probe carrier rod 820 is rotated 1520 to move the target fluid 1510 over the probes. Because of the spiral winding pattern of the probe carrier thread 100 on the probe carrier rod 820, target fluid 1510 can be moved not only along the circular but also axial direction of the rod 820, thus covering all probe positions on the probe carrier rod 820. An AC oscillation voltage 1530 can be applied between the probe carrier thread 100 and the wall of the hybridization chamber 1500 to improve the efficiency of the process.
  • a hybridization chamber design 1600 for probe carrier coil 1012 is shown in Figure 16. Again, a back and forth rotational motion 1620 is introduced to the coil through a mechanical drive or a magnetic drive and AC oscillation voltage alteration 1630 is applied between the coil support and the chamber to enhance the hybridization efficiency.
  • Figure 17a shows a chamber design 1700 for the hybridization of probe carrier thread 100 that is unwound from a probe carrier spool 1110, in which a mostly water-tight capillary 1760 is formed by closing a lid 1770 on a na ⁇ ow slot produced on a subsfrate 1780.
  • the cross-sectional size of the slot is slightly larger than the probe carrier thread as shown in Fig. 17b.
  • Target fluid 1750 is introduced into the middle section of slot before closing the lid or it is introduced through a small opening 1790 in the lid after the lid is closed onto the slot.
  • the probe carrier thread 100 is moved back-and-forth through the chamber to enhance the efficiency of the hybridization. As the thread is hydrophobic, the target fluid is retained inside the slot by the capillary force.
  • the hybridization efficiency can be further improved by applying an alternating voltage 1730 between a metal layer on the probe carrier thread 100 and the inner wall of the capillary 1760 of chamber 1700.
  • Reader All probe carrier thread packaging formats described above can be read using a scanning microscope with laser or broadband excitation. Scanning can be carried out by scanning electron microscopy, confocal microscopy, a charge-coupled device, scanning tunneling electron microscopy, infrared microscopy, atomic force microscopy, electrical conductance, and fluorescent or phosphor imaging. However, special scanning motions may preferably be provided in the readout instrument for various probe carrier thread formats.
  • both probe carrier pin 810 and probe carrier rod 820 can be plugged into an adapter in the readout instrument designed to hold the ends of the pin or rod and rotate 1810 and/or translate 1812 them along the longitudinal axis at a predetermined ratio of speeds.
  • This motion brings all probes distributed along the probe carrier thread 100 under the optical excitation and readout lens 1800.
  • the probe carrier pin 810 or probe carrier rod 820 may rotate 1810 while the optical head 1800 translates along the axis of the pin or rod to scan the length of the probe carrier thread 100 mounted on a probe carrier pin 810 or a probe carrier rod 820.
  • probe carrier coil 1012 can be scanned by introducing a rotation 1910 of the coil 1012 and a relative translation 1912 motion between the coil 1012 and the optical read head 1900 along a radial direction of the coil.
  • a probe carrier spool 1110 contained in a cassette 1100 unwinds a stretch of unwound probe carrier thread 100 which is passed under an optical read head 2002 and a marker reader 2004.
  • the unwound probe carrier thread 100 carries the entire set of probes moving under the optical read head 2002, which can remain stationary.
  • the unwound probe carrier thread 100 can be collected in a second spool 2012.
  • the apparatus lends itself to use in a number of fields.
  • An apparatus which uses polynucleotides as probes maybe used for analysis of known point mutations, genomic finge ⁇ rinting, linkage analysis, characterization of mRNAs and mRNA populations, sequence determination, disease diagnosis, and polymo ⁇ hism analysis.
  • An apparatus which uses antibodies as probes would be especially useful in diagnostics. Other uses involving other probes will be apparent to those of skill in the art.
  • the use of the apparatus involves: 1) Preparation of the sample, if necessary; 2) Formation of probe-sample complex; 3) Analyzing the binding pattern in order to identify the individual probes to which sample has bound. 6.1. Preparation of the sample.
  • sample preparation protocols for analysis of polynucleotides including labeling of samples with fluorescent tags in order to facilitate step 3), analyzing the binding pattern, are well-known in the art. See, for example, U.S. Patent No. 5,800,992, which is hereby inco ⁇ orated by reference in its entirety.
  • the sample is fragmented, using known techniques such as restriction endonuclease digestion, converted to single-stranded form, and the single-stranded fragments are labeled with an appropriate fluorescent tag.
  • Formation of the probe-sample complex Upon contact of sample with the apparatus, sample or sample fragments which have an affinity for particular probes bind with those probes.
  • the present invention also encompasses any sort of sample-probe binding which will allow one to derive information from determining which probes have bound to sample or sample fragments.
  • the sample may be composed of a number of molecules, some of which are enzymes.
  • the probes of the apparatus to be used to analyze this sample could be substrates for various enzymes (here the word "substrate” is used in the sense of a reactant upon which an enzyme works as a catalyst), and the identity of the enzymes in the samples may be obtained by determining which substrate-probes have bound enzymes after contact.
  • Other examples include determining the identity of antibodies in a sample by using various antigens as probes, or identifying hormones in a sample by the receptors to which they bind, etc.
  • the list of sample/probe pairs extends to any sets of pairs which bind with each other with a sufficient degree of affinity and specificity to be identified, and further examples of sample/probe pairs will be readily apparent to those of skill in the art.
  • One aspect of the present invention can greatly enhance binding of charged sample. This is the ability to supply a voltage across the substrate, where the substrate contains a metallic element or is otherwise electrically conductive. For example, if DNA is the sample to be analyzed, an oscillating voltage across the substrate will alternately attract the negatively charged DNA to the probe carrier, then repel it. The attraction will facilitate the binding of complementary strands, while the repulsion cycle will expedite the release of non- specifically bound or incompletely hybridized sample. The same principle holds true for any type of charged sample, and increases the efficiency and fidelity of sample binding. 6.3 Analysis of the binding pattern.
  • Distinguishing probe-sample pairs from probes which have not bound sample may be done in any manner that allows localization. Many such techniques are well-established in the art. Detecting labeled sample polynucleotides, for example, can be conducted by standard methods used to detect the type of label used. Thus, for example fluorescent labels or radiolabels can be detected directly. Other labeling techniques may require that a label such as biotin or digoxigenin that is inco ⁇ orated into the sample during preparation of the sample and detected by an antibody or other binding molecule (e.g. streptavidin) that is either labeled or which can bind a labeled molecule itself, for example, a labeled molecule can be e.g.
  • an antibody or other binding molecule e.g. streptavidin
  • a fluorescent molecule e.g. fluorescein isothiocyanate, Texas red and rhodamine
  • conjugated to an enzymatically active molecule e.g. fluorescein isothiocyanate, Texas red and rhodamine
  • the labels e.g. fluorescent, enzymatic, chemilluminescent, or colorimetric
  • the labels can be detected by a laser scanner or a CCD camera, or X-ray film, depending on the label, or other appropriate means for detecting a particular label.
  • sample polynucleotides are fragmented, then each sample fragment is tagged with a fluorescent label.
  • the sample fragments which have hybridized with complementary probe polynucleotides may be located by the fluorescent tag on the sample. Probes which have not bound sample fragments have no such fluorescent label. Similar fluorescent tagging may be done for other types of molecules, such as antibodies, enzymes, etc. Other types of tags, such as radioactive labels, chemilluminescent labels, phosphorescent labels, magnetic labels, etc., will be readily apparent to one of skill in the art.
  • light detectable means are prefe ⁇ ed, although other methods of detection may be employed, such as radioactivity, atomic spectrum, and the like.
  • light detectable means one may use fluorescence, phosphorescence, abso ⁇ tion, chemilluminescence, or the like. The most convenient will be fluorescence, which may take many forms.
  • Illustrative fluorescers include fluorescein, rhodamine, Texas red, cyanine dyes, phycoerythrins, thiazole orange and blue, etc.
  • pairs of dyes one may have one dye on one molecule and the other dye on another molecule which binds to the first molecule. The important factor is that the two dyes when the two components are bound are close enough for efficient energy fransfer.
  • the present invention provides opportunities for greatly streamlining and expanding the second step in the analysis, that is, the step of identifying the specific probes to which samples or sample fragments have bound.
  • the identity of a probe is established by determining its x-y position in the a ⁇ ay; the x-y position of every probe is known. Determining the position of the probe by known techniques requires complex and expensive imaging equipment. Because the probes in the present invention are a ⁇ anged in a one-dimensional row, positional analysis is much easier and requires much less complex equipment, because only one dimension need be tracked (as is the case for a spooled thread), rather than two.
  • markers associated with probes or groups of probes provides a means for keeping track of probes in any of the embodiments of the invention. This has been discussed previously. Markers may be simple or complex, may be on the same side of the substrate as the probes or a different side, may be of more than one type, and may contain more information than just the identity of the probe or probes.
  • Probe carriers of the present invention can be used to construct very large probe a ⁇ ays packaged in minimal volume which are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip provides an immediate finge ⁇ rint identification of the target nucleotide sequence. Patterns can be manually or computer analyzed, but it is clear that positional sequencing by hybridization lends itself to computer analysis and automation. Algorithms and software have been developed for sequence reconstruction which are applicable to the methods described herein (R. Drmanac et al., J. Biomol. Struc. & Dyn. 5:1085-1102, 1991; P. A. Pevzner, J. Biomol. Struc. & Dyn.
  • Flexible probe carriers containing immobilized nucleic acid sequences prepared in accordance with the invention can be used for large scale hybridization assays in numerous genetic applications, including analysis of known point mutations, genomic finge ⁇ rinting, linkage analysis, characterization of mRNAs and mRNA populations, sequence determination, disease diagnosis, and polymo ⁇ hism analysis.
  • An apparatus which uses antibodies as probes would be especially useful in diagnostics. Other uses involving other probes will be apparent to those of skill in the art.
  • a gene or a cloned DNA fragment is hybridized to an ordered array of DNA fragments, and the identity of the DNA elements applied to the a ⁇ ay is unambiguously established by the pixel or pattern of pixels of the a ⁇ ay that are detected.
  • One application of such a ⁇ ays for creating a genetic map is described by Nelson, et al., Nature Genetics 4:11-18 (1993).
  • a ⁇ ays of immobilized cloned DNA fragments are hybridized with other cloned DNA fragments to establish whether the cloned fragments in the probe mixture overlap and are therefore contiguous to the immobilized clones on the a ⁇ ay.
  • Flexible probe carriers of immobilized DNA fragments may also be used for genetic diagnostics.
  • a probe carrier containing multiple forms of a mutated gene or genes can be probed with a labeled mixture of a patient's DNA which will preferentially interact with only one of the immobilized versions of the gene. The detection of this interaction can lead to a medical diagnosis. Also, detection of expression levels of certain genes are diagnostic of certain medical conditions. For example, amplification of the HER- 2/neu (c-erbB-2) gene resulting in overexpression of the pi 85HER-2 growth factor receptor occurs in approximately 25% of early stage breast cancers.
  • HER-2/neu has been established as an important independent prognostic factor in early stage breast cancer in large cohorts of patients and in cohorts with very long (30 year) follow-up duration. New data has emerged to suggest that HER-2/neu may be useful not only as a prognostic factor but also as a predictive marker for projecting response to chemotherapeutics, antiestrogens, and therapeutic anti- HER-2/neu monoclonal antibodies.
  • ⁇ ER-2/neu codes for a 185 kD transmembrane oncoprotein which is amplified and/or over-expressed in some breast cancer patients, a feature generally associated with a poorer prognosis than that for women with unamplified HER-2/neu.
  • Flexible probe carriers of immobilized DNA fragments can also be used in DNA probe diagnostics.
  • identity of a pathogenic microorganism can be established unambiguously by hybridizing a sample of the unknown pathogen's DNA to a probe carrier containing many types of known pathogenic DNA.
  • a similar technique can also be used for unambiguous genotyping of any organism.
  • Other molecules of genetic interest, such as cDNAs and RNAs can be immobilized on the probe carrier or alternately used as the labeled probe mixture that is applied to the probe carrier.
  • a probe carrier of cDNA clones representing genes is hybridized with total cDNA from an organism to monitor gene expression for research or diagnostic pu ⁇ oses. Labeling total cDNA from a normal cell with one color fluorophore and total cDNA from a diseased cell with another color fluorophore and simultaneously hybridizing the two cDNA samples to the same a ⁇ ay of cDNA clones allows for differential gene expression to be measured as the ratio of the two fluorophore intensities. This two-color experiment can be used to monitor gene expression in different tissue types, disease states, response to drugs, or response to environmental factors.
  • Such a procedure could be used to simultaneously screen many patients against all known mutations in a disease gene.
  • This invention could be used in the form of, for example, 96 identical probe carrier pins in a matrix where each probe carrier pin could contain, for example, 1500 DNA fragments representing all known mutations of a given gene.
  • the region of interest from each of the DNA samples from 96 patients could be amplified, labeled, and hybridized to the 96 individual a ⁇ ays with each assay performed in 10 microliters of hybridization solution.
  • the adapter matrix containing all 96 identical probe carrier pins assayed with the 96 patient samples is incubated, rinsed, detected and analyzed as a single sheet of material using standard radioactive, fluorescent, or colorimetric detection means (Maniatis, et al., 1989). Previously, such a procedure would involve the handling, processing and tracking of 96 separate membranes in 96 separate sealed chambers. By processing all 96 patient samples in a single step with minimal hybridization liquid, significant time and cost savings are possible.
  • the assay format can be reversed where the patient or organism's DNA is immobilized as the probe elements and each probe carrier is hybridized with a different mutated allele or genetic marker.
  • a probe carrier matrix can also be used for parallel non- DNA ELISA assays. Furthermore, the invention allows for the use of all standard detection methods.
  • One aspect of this invention involves the detection of nucleic acid sequence differences using coupled ligase detection reaction (LDR) and polymerase chain reaction (PCR) as disclosed in U.S. Pat. No. 6,027,889 entitled “Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions” to Baranyi, et al. which is inco ⁇ orated herein by reference in its entirety.
  • LDR coupled ligase detection reaction
  • PCR polymerase chain reaction
  • a ⁇ ays of whole cells, peptides, enzymes, antibodies, antigens, receptors, ligands, phospholipids, polymers, drug cogener preparations or chemical substances can be fabricated by the means described in this invention for large scale screening assays in medical diagnostics, drug discovery, molecular biology, immunology and toxicology.
PCT/US2001/001026 2000-01-10 2001-01-10 Linear probe carrier WO2001051207A1 (en)

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IL15043401A IL150434A0 (en) 2000-01-10 2001-01-10 Linear probe carrier
KR1020027008921A KR20020089323A (ko) 2000-01-10 2001-01-10 선형 프로브 캐리어
AU27868/01A AU2786801A (en) 2000-01-10 2001-01-10 Linear probe carrier
EP01902020A EP1248678A1 (en) 2000-01-10 2001-01-10 Linear probe carrier
JP2001551617A JP2003519784A (ja) 2000-01-10 2001-01-10 線形プローブキャリア
CA002397069A CA2397069A1 (en) 2000-01-10 2001-01-10 Linear probe carrier

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US17522500P 2000-01-10 2000-01-10
US60/175,225 2000-01-10
US19049500P 2000-03-20 2000-03-20
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US22787400P 2000-08-25 2000-08-25
US60/227,874 2000-08-25
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JP2002181819A (ja) * 2000-09-25 2002-06-26 Olympus Optical Co Ltd 立体基体を用いた検出用アレイ
WO2002061438A1 (fr) * 2001-01-29 2002-08-08 Commissariat A L'energie Atomique Procede et systeme permettant de realiser en flux continu un protocole biologique, chimique ou biochimique.
WO2002083293A2 (en) * 2001-04-18 2002-10-24 3 D Molecular Sciences Limited Chemical libraries based on coded particles
WO2003042697A1 (en) * 2001-11-14 2003-05-22 Genospectra, Inc. Biochemical analysis system with combinatorial chemistry applications
FR2846339A1 (fr) * 2002-10-28 2004-04-30 Ipsogen Procede d'analyse d'un etat biologique complexe d'un biosysteme et support permettant son interpretation directe
WO2004046231A1 (en) * 2002-11-20 2004-06-03 Bio-Molecular Holdings Pty Limited Dna isolation material and method
JP2005517959A (ja) * 2002-02-21 2005-06-16 コミツサリア タ レネルジー アトミーク 生物又は生化学分析マイクロシステム用のコンポーネント
GB2412730A (en) * 2004-03-31 2005-10-05 Toshiba Res Europ Ltd A flexible carrier and methods of monitoring molecular binding and reactions
EP1585958A2 (en) * 2001-10-18 2005-10-19 Vitra Bioscience, Inc. Coded particles for multiplexed analysis of biological samples
JP2006501441A (ja) * 2002-08-02 2006-01-12 グリコマインズ リミテッド 多発性硬化症を診断するための方法
WO2009014613A2 (en) * 2007-07-20 2009-01-29 Visigen Biotechnologies, Inc. An automated synthesis or sequencing apparatus and method for making and using same
US7625746B2 (en) 2006-07-24 2009-12-01 Nanosphere, Inc. Method of denaturing and fragmenting DNA or RNA using ultrasound
US7888107B2 (en) 2006-07-24 2011-02-15 Nanosphere, Inc. System using self-contained processing module for detecting nucleic acids
CN101980023A (zh) * 2010-10-29 2011-02-23 中国人民解放军军事医学科学院野战输血研究所 蓖麻毒素的新型生物条形码检测方法
US7906291B2 (en) 2005-01-31 2011-03-15 Glycominds Ltd. Method for diagnosing multiple sclerosis
US8048639B2 (en) 2009-02-12 2011-11-01 Glycominds Ltd. Method for evaluating risk in multiple sclerosis
US8216791B2 (en) 2005-01-31 2012-07-10 Glycominds Ltd. Method for diagnosing multiple sclerosis
EP2823889A1 (en) * 2013-07-10 2015-01-14 Roche Diagniostics GmbH Device and method for biological sample collection and inspection
US10753927B2 (en) 2006-09-22 2020-08-25 ALERE TECHNOLOGIES GmbH Methods for detecting an analyte
CN111610173A (zh) * 2020-05-27 2020-09-01 中国水利水电科学研究院 三维流体浓度场标定装置及标定方法

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US20080200575A1 (en) * 2004-03-31 2008-08-21 Universal Bio Research Co., Ltd. Immobilization Vehicle, Method of Treating Vehicle and Continuous Vehicle Treating Apparatus
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US8349616B2 (en) * 2006-11-22 2013-01-08 Clondiag Gmbh Assays
JP5154186B2 (ja) 2007-10-01 2013-02-27 電気化学工業株式会社 テープ状フィルムの巻き取り状態評価方法およびテープ状フィルムの巻き取り方法
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JP5163999B2 (ja) * 2007-11-14 2013-03-13 国立大学法人富山大学 類似植物体および生薬同定用ｄｎａマイクロアレイ
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TWI458962B (zh) * 2012-05-14 2014-11-01 Univ Nat United 檢測元件及其製造方法與用途
CN107192844B (zh) * 2017-04-20 2019-10-15 燕山大学 一种磁探针阵列装置、电磁推射速度测量装置及方法
CN110951829B (zh) * 2018-09-26 2022-03-29 中国农业大学 一种喷施雾滴飘失或沉积特性检测试剂盒及检测方法

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EP0837320A2 (en) * 1996-10-21 1998-04-22 Bayer Corporation System for the optical indentification of coding on a diagnostic test strip
WO1999039186A1 (en) * 1998-02-02 1999-08-05 Crystal Medical Products Detection system

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002181819A (ja) * 2000-09-25 2002-06-26 Olympus Optical Co Ltd 立体基体を用いた検出用アレイ
WO2002061438A1 (fr) * 2001-01-29 2002-08-08 Commissariat A L'energie Atomique Procede et systeme permettant de realiser en flux continu un protocole biologique, chimique ou biochimique.
US7404930B2 (en) 2001-01-29 2008-07-29 Commissariat A L'energie Atomique Method and system for performing in continuous flow a biological, chemical or biochemical protocol
WO2002083293A2 (en) * 2001-04-18 2002-10-24 3 D Molecular Sciences Limited Chemical libraries based on coded particles
WO2002083293A3 (en) * 2001-04-18 2003-01-09 3 D Molecular Sciences Ltd Chemical libraries based on coded particles
EP1585958A2 (en) * 2001-10-18 2005-10-19 Vitra Bioscience, Inc. Coded particles for multiplexed analysis of biological samples
EP1585958A4 (en) * 2001-10-18 2007-07-25 Millipore Corp CODED PARTICLES FOR THE MULTIPLEX ANALYSIS OF BIOLOGICAL SAMPLES
WO2003042697A1 (en) * 2001-11-14 2003-05-22 Genospectra, Inc. Biochemical analysis system with combinatorial chemistry applications
JP2005517959A (ja) * 2002-02-21 2005-06-16 コミツサリア タ レネルジー アトミーク 生物又は生化学分析マイクロシステム用のコンポーネント
JP4727148B2 (ja) * 2002-02-21 2011-07-20 コミッサリア ア レネルジー アトミーク エ オ ゼネルジ ザルタナテイヴ 生物又は生化学分析マイクロシステム用のコンポーネント
JP2009258138A (ja) * 2002-08-02 2009-11-05 Glycominds Ltd 多発性硬化症を診断するための方法
JP4721703B2 (ja) * 2002-08-02 2011-07-13 グリコマインズ リミテッド 多発性硬化症を診断するための方法
JP2006501441A (ja) * 2002-08-02 2006-01-12 グリコマインズ リミテッド 多発性硬化症を診断するための方法
WO2004040015A3 (fr) * 2002-10-28 2004-06-24 Ipsogen Procede d'analyse d'un etat biologique complexe d'un biosysteme et support permettant son interpretation directe
WO2004040015A2 (fr) * 2002-10-28 2004-05-13 Ipsogen Procede d'analyse d'un etat biologique complexe d'un biosysteme et support permettant son interpretation directe
FR2846339A1 (fr) * 2002-10-28 2004-04-30 Ipsogen Procede d'analyse d'un etat biologique complexe d'un biosysteme et support permettant son interpretation directe
WO2004046231A1 (en) * 2002-11-20 2004-06-03 Bio-Molecular Holdings Pty Limited Dna isolation material and method
GB2412730A (en) * 2004-03-31 2005-10-05 Toshiba Res Europ Ltd A flexible carrier and methods of monitoring molecular binding and reactions
GB2412730B (en) * 2004-03-31 2006-08-23 Toshiba Res Europ Ltd An encoded carrier and a method of monitoring a carrier
US8119408B2 (en) 2004-03-31 2012-02-21 Kabushiki Kaisha Toshiba Encoded carrier and a method of monitoring an encoded carrier
US8216791B2 (en) 2005-01-31 2012-07-10 Glycominds Ltd. Method for diagnosing multiple sclerosis
US8129128B2 (en) 2005-01-31 2012-03-06 Glycominds, Ltd. Immunoassay reagent compositions for diagnosing multiple sclerosis
US7906291B2 (en) 2005-01-31 2011-03-15 Glycominds Ltd. Method for diagnosing multiple sclerosis
US7625746B2 (en) 2006-07-24 2009-12-01 Nanosphere, Inc. Method of denaturing and fragmenting DNA or RNA using ultrasound
US7888107B2 (en) 2006-07-24 2011-02-15 Nanosphere, Inc. System using self-contained processing module for detecting nucleic acids
US10753927B2 (en) 2006-09-22 2020-08-25 ALERE TECHNOLOGIES GmbH Methods for detecting an analyte
WO2009014613A3 (en) * 2007-07-20 2009-05-14 Visigen Biotechnologies Inc An automated synthesis or sequencing apparatus and method for making and using same
WO2009014613A2 (en) * 2007-07-20 2009-01-29 Visigen Biotechnologies, Inc. An automated synthesis or sequencing apparatus and method for making and using same
US8048639B2 (en) 2009-02-12 2011-11-01 Glycominds Ltd. Method for evaluating risk in multiple sclerosis
CN101980023A (zh) * 2010-10-29 2011-02-23 中国人民解放军军事医学科学院野战输血研究所 蓖麻毒素的新型生物条形码检测方法
CN101980023B (zh) * 2010-10-29 2013-06-19 中国人民解放军军事医学科学院野战输血研究所 蓖麻毒素的新型生物条形码检测方法
EP2823889A1 (en) * 2013-07-10 2015-01-14 Roche Diagniostics GmbH Device and method for biological sample collection and inspection
US9939429B2 (en) 2013-07-10 2018-04-10 Roche Molecular Systems, Inc. Device and method for biological sample collection and inspection
US11067565B2 (en) 2013-07-10 2021-07-20 Roche Molecular Systems, Inc. Device and method for biological sample collection and inspection
CN111610173A (zh) * 2020-05-27 2020-09-01 中国水利水电科学研究院 三维流体浓度场标定装置及标定方法

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JP2003519784A (ja) 2003-06-24
CA2397069A1 (en) 2001-07-19
IL150434A0 (en) 2002-12-01
CN1406153A (zh) 2003-03-26
AU2786801A (en) 2001-07-24
EP1248678A1 (en) 2002-10-16
KR20020089323A (ko) 2002-11-29

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