WO2003064997A2 - Microreseaux produits par la formation de parties transversales dans des plaques comportant plusieurs echantillons - Google Patents

Microreseaux produits par la formation de parties transversales dans des plaques comportant plusieurs echantillons Download PDF

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Publication number
WO2003064997A2
WO2003064997A2 PCT/US2003/002149 US0302149W WO03064997A2 WO 2003064997 A2 WO2003064997 A2 WO 2003064997A2 US 0302149 W US0302149 W US 0302149W WO 03064997 A2 WO03064997 A2 WO 03064997A2
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Prior art keywords
channels
array
block
channel
analyte
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PCT/US2003/002149
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English (en)
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WO2003064997A3 (fr
Inventor
Leigh N. Anderson
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Large Scale Proteomics Corporation
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Priority to AU2003209361A priority Critical patent/AU2003209361A1/en
Publication of WO2003064997A2 publication Critical patent/WO2003064997A2/fr
Publication of WO2003064997A3 publication Critical patent/WO2003064997A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements

Definitions

  • the instant invention relates to microarrays containing bioreactive molecules, uses thereby and methods for manufacture thereof.
  • the arrays are constructed by sectioning stackable molds or blocks comprising channels, each channel containing unique reactants. The block is then sliced many times to produce large numbers of identical arrays having intrinsically addressable locations.
  • a microarray is essentially a two-dimensional support or sheet wherein different portions or cells (sectors) of the support or sheet carry different biomolecules or elements, such as, nucleotides, polynucleotides, peptides, polypeptides, saccharides or polysaccharides, bound thereto.
  • Microarrays are similar in principle to other solid phase arrays except that assays involving such microarrays are performed on a smaller scale, allowing many assays to be performed in parallel. Microarrays have been used for a number of analytical purposes, typically in the biological sciences.
  • Biochemical molecules on microarrays have been synthesized directly at or on a particular cell (sector) on the microarray, or preformed molecules have been attached to particular cells (sectors) of the microarray by chemical coupling, adsorption or other means.
  • the number of different cells (sectors) and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands.
  • Commercial microarray plate readers typically measure fluorescence in each cell (sector) and can provide data on thousands of reactions simultaneously thereby saving time and labor.
  • a representative example of a patent in the field is U.S. Pat. No. 5,545,531.
  • each microarray is individually and separately made, typically is used only once and cannot be individually precalibrated and evaluated in advance. Hence, one depends on the reproducibility of the production system to produce error-free arrays. Those factors have contributed to the high cost and variability of currently produced biochips or microarrays, and have discouraged application of the technology to routine clinical use.
  • Immobilized enzymes have been prepared in fiber form from an emulsion as disclosed, for example, in Italy Pat. No. 836,462. Antibodies and antigens have been incorporated into solid phase fibers as disclosed in U.S. Pat. No. 4,031,201.
  • a large number of other different immobilization techniques are known in the fields of solid phase immunoassays, nucleic acid hybridization assays and immobilized enzymes, see, for example, Hermanson, G.T., Bioconjugate Techniques. Academic Press, New York. 1995, 785 pp; Hermanson, G.T., Mallia, A.K. & Smith, P.K. Immobilized Affinity Ligand Techniques. Academic Press, New York, 1992, 454 pp; and Avidin-Biotin Chemistry: A Handbook. D. Savage, G. Mattson, S. Desai, G. Nielander, S.
  • biochips include only one class of immobilized reactant, and perform only one class of reactions. For many types of clinical and other analyses, there is a need for chips that can incorporate reactants immobilized in different ways in one chip.
  • the instant invention relates to a method for producing devices comprising a plurality of channels, each channel containing a different entrapped or attached biological agent of interest.
  • the device has walls between the channels for keeping the channels in an intrinsically fixed addressable location with respect to other channels.
  • Stacks of the devices are sectioned to produce large numbers of identical arrays or chips; and for performing a variety of different quantitative biochemical analyses on individual arrays or chips based on, for example, enzymatic activities, immunochemical activities, nucleic acid hybridization and small molecule binding under conditions yielding, for example, fluorescence, optical absorbance or chemiluminescence signals, for acquiring images of the signals which can be processed electronically and compared to produce clinically and experimentally useful data.
  • the invention relates to devices that contain or are coated within with agents of interest and methods for manufacture thereof.
  • the invention further relates to means and methods for constructing devices of the invention, in which channels are present, are stacked to form blocks, which are then bonded to form a multilayer channel device.
  • immobilized agents of interest are within or on the inner surface of the channels comprising the blocks.
  • the invention also relates to methods for constructing devices where the channels therein intrinsically form addressable locations in which the position of each channel relative to all others is retained throughout the block comprising said channels. Upon sectioning, such a slice, preferably perpendicular to the path of the channels, would afford microarray products that retain addressable locations by virtue of said construction.
  • the invention relates to the preparation of microarrays wherein the channel devices are cut transversely many times at short intervals to yield cross sectional slices thereof to form microarrays and a microarray so prepared.
  • the instant invention relates to forming a channel containing an agent of interest, or means for immobilizing at least one or a class of agents of interest thereto.
  • the channels contain different immobilized agents of interest.
  • the invention relates to means for embedding or attaching whole or fragments of biological cells, tissues or infectious agents to channels in such a manner that the biologicals are exposed on the cut end of each channel.
  • the array consists of channels containing gel or other polymerizing materials that adhere to the channel inner surface.
  • agents of interest are attached to the polymerizing or suspending medium in the inner surface of the channels.
  • the agents of interest are attached to particles that are suspended in a polymerizing medium, which suspension is used to fill channels comprising the arrays.
  • the invention further relates to a method for the large scale production of identical flat two-dimensional arrays of immobilized nucleic acid-based agents for use in nucleic acid sequencing, in the analysis of complex mixtures of ribonucleic acids (RNA's) and deoxyribonucleic acids (DNA's), and in the detection and quantitation of other analytes including proteins, polysaccharides, organic polymers and low molecular mass analytes, by sectioning the block of devices.
  • RNA's ribonucleic acids
  • DNA's deoxyribonucleic acids
  • the invention relates to exploiting microarrays for mass screening of large numbers of samples from one to a large number of agents of interest.
  • the invention relates to the development of sets of tests on different chips or microarrays done in optionally branching sequence, which reduces the cost, delay and inconvenience of diagnosing human diseases, and other conditions, while providing complex data ordinarily obtained by time-consuming sequential batteries of conventional tests.
  • the invention relates to the fabrication of identical arrays that are sufficiently inexpensive to allow several identical arrays to be mounted on the same slide or test strip, and cross-compared for quality control purposes.
  • the invention relates to the incorporation of a non- fluorescent dye, carbon particles, or other light absorbing material into the devices to prevent signals from passing from one location on the microarray to another.
  • a dye or light adsorbing material may also be in the substance of the array to control the depth to which light used to excite fluorescence penetrates the array, thereby controlling the depth to which fluorescence analytes are detected, and insuring that fluorescent analytes which diffuse too deeply into the content of the channels, and therefore do not diffuse out, are not detected.
  • the devices may be made of opaque material.
  • the invention relates to the reproducible manufacture of biochips or microarrays for bioanalysis.
  • the invention relates to increasing the dynamic range of multiple-parallel assays by providing means for making serial measurements of fluorescence or absorbance over time, and for determining the rate of change of fluorescence or absorbance in each element of the array over time.
  • the dynamic range of the assay may be increased by having multiple locations on the microarray have the same agent of interest in substantially different concentrations. It is an additional aspect of the invention to produce biochips that are inexpensive and sufficiently standardized to allow more than one to be used for each analysis, and for controls and standards to be run routinely and simultaneously in parallel.
  • the invention relates to the production of chips in which the array elements or channels may differ from one another in the composition of the immobilizing matrix or substrate, or the class of agent of interest may be different in different channels (sectors).
  • the invention relates to the production of chips in which different types of reactions may be carried out at each channel of the array, with the reactions including immunological, enzymatic or hybridization reactions.
  • FIG. 1 is a perspective view of a block having a plurality of channels formed therein for receiving a biological detection substance or analyte in accordance with the present invention
  • FIG. 2 A is a perspective view of a plurality of the blocks depicted in FIG. 1, shown stacked to form an elongated array where each of the many channels are able to retain a plurality of biological detection substances in accordance with the present invention
  • FIG. 2B is a perspective view similar to FIG. 2A, showing a plurality of stacks of elongated blocks in accordance with the present invention
  • FIG. 2C is a side view of the stacks of blocks depicted in FIG. 2B and a blade for sectioning the stacks of blocks to form a sliced microarray in accordance with the present invention
  • FIG. 2D is a perspective view showing the stack of blocks depicted in FIG. 2C with a sliced microarray in accordance with the present invention
  • FIG. 3 A is a front view of an alternate embodiment of the block depicted in
  • FIGS. 1 and 2A-2D wherein each block is formed with channels formed on one side of the block and grooves formed on either side of the block for receiving portions of adjacent blocks in accordance with the present invention
  • FIG. 3B is a front view of yet another embodiment of the block wherein each block is formed with channels on opposite sides thereof in accordance with the present invention
  • FIG. 3C is a front view of yet another embodiment of the present invention wherein the channels in the block are V-shaped;
  • FIG. 3D is a front view of yet another embodiment of the present invention wherein the channels in the block are semi-circular shaped;
  • FIG. 3E is a front view of yet another embodiment of the present invention wherein channels are formed on opposite sides of the block and are offset from one another;
  • FIG. 3F is a front view of yet another embodiment of the present invention wherein grooves are formed on opposite sides of the block and are have a semi-circular shape;
  • FIG. 3G is a front view of another embodiment of the present invention, showing a plurality of blocks stacked in a staggered orientation, where each block is formed with a single rectangular shaped channel open at one corner of the rectangular shaped channel;
  • FIG. 31 is a front view of another embodiment of the present invention, showing a plurality of stacked blocks, where each block has an L-shape forming a single channel;
  • FIG. 3J is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks, interlocking with one another, each block having a plurality of channels with non-parallel sides;
  • FIG. 4A is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks with rectangular channels, interlocking with one another, each block having dove-tail interlocking portions;
  • FIG. 4B is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks with oval channels, interlocking with one another, each block having dove-tail interlocking portions;
  • FIG. 4C is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks with a combination of oval and rectangular channels, interlocking with one another, each block having dove-tail interlocking portions;
  • FIG. 4D is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks with channels having non-parallel sides and interlocking with one another;
  • FIG. 4E is a front view of another embodiment of the present invention, showing a plurality of staggered stacked blocks with channels having non-parallel sides, each block having dove-tail interlocking portions;
  • FIG. 5 A is a perspective view of a block in accordance with another embodiment of the present invention, the block having a plurality of channels, where each channel includes a portion parallel to a portion of all other channels, and each channel having a funnel-like portion that extends outward and away from adjacent channels;
  • FIG. 5B is a perspective view of a plurality of the blocks depicted in FIG. 5 A, the plurality of blocks stacked on one another to form an array of channels in accordance with the present invention;
  • FIG. 5C is a perspective view of the plurality of stacked blocks depicted in FIG. 5B turned on end showing openings of the funnel-like portions of the channels in accordance with the present invention
  • FIG. 6A is a perspective view of a block in accordance with another embodiment of the present invention, wherein the block is formed with metallic powder which renders the block susceptible to magnetic fields;
  • FIG. 6B is a perspective view of a plurality of blocks depicted in FIG. 6A, each the stacked blocks having metallic powder therein;
  • FIG. 7A is a perspective view of a block in accordance with another embodiment of the present invention, wherein the block is formed with a metallic layer on portions of the block that subsequently contact other blocks, the metallic layer being conductive such that upon being subjected to electric current, heat and fuse to portions of blocks in contact with the metallic layer;
  • FIG. 7B is a perspective view of a plurality of the blocks depicted in FIG. 7A, the stacked blocks being fused together by heat generated by passing current through the metallic layer material; and
  • FIG. 8 is a perspective view of a plurality of stacked blocks in accordance with yet another embodiment of the present invention, wherein a surface of each channel in each block is provided with a layer of material that assists in retention of biological material within the channel.
  • FIG. 9 A is a front view of a plurality of blocks, wherein the lower block presents a layer of precious metal to be pressed into the inner surfaces of said presenting (lower) block, wherein the upper block presses said layer onto the presenting block thus channels formed on one side of the block and grooves are formed on either side of the block for receiving portions of adjacent blocks in accordance with the present invention
  • FIG. 9B is a front view of a plurality of blocks, wherein the blocks of 9A have been pressed together to deposit the layer of precious metal onto the inner surfaces of the lower (receiving) block, to include layering of said metal onto the interstices of lower block channel surfaces.
  • FIGS. 10A and 10B are front views of another embodiment of the present invention, showing a plurality of stacked devices, each device formed with semicircular or with oval channels, each channel having a predetermined material adhered therein such that when the devices are stacked, the channels in one device combined with adjacent channels in an adjacent device define single channels, each channel having opposing surfaces with separate and distinct materials on each opposing surface;
  • FIG. IOC is an end view showing the stacked devices depicted in FIGS. 10A and 10B, along with a cutting blade for making slices of the stacked devices, where the stacked devices are rotated 90 degrees such that the cutting occurs parallel to the joints between the stacked devices;
  • FIG. 10D is a side view of the cutting blade and stacked devices showing the blade at an angle with respect to the vertical such that subsequent slices of the stacked devices are longer than the height of the stacked devices and the surface of the channels are not perpendicular to the face of the sliced block;
  • FIG. 10E is an end view of the slice formed by the blade depicted in FIGS. IOC and 10D depicting, in black, the coated surfaces of the channels;
  • FIG. 11 A is a perspective view showing yet another embodiment of the present invention, where a device is formed with square channels, and as shown, the horizontal surface of each channel is coated with a unique first biological material for attracting a specific target molecule(s) and at least one vertical surface of each channel is coated with a separate biological material different from the first biological material;
  • FIG. 1 IB is a perspective view showing a plurality of the devices depicted in FIG. 11 A stacked to form an elongated bundle, in accordance with the present invention
  • FIG. 11 C is a side view showing the bundle depicted in FIG. 1 IB turned 45 degrees for cutting by a blade;
  • FIG. 1 ID is an end view of the blade and the bundle depicted in 11C;
  • FIG. 1 IE is an end view of a slice made by the blade from the bundle depicted in FIGS. 11C and 1 ID;
  • FIG. 12A is a perspective view of a device in accordance with still another embodiment of the present invention that is formed with V-shaped grooves or channels, where one surface of the channel is coated with a unique first biological material for attracting a specific target molecule(s) and the other surface of the channel is coated with a separate biological material different from the first biological material;
  • FIG. 12B is a perspective view showing a plurality of the devices depicted in FIG. 12A stacked to form a bundle in accordance with the present invention;
  • FIG. 12C is a side view showing the bundle depicted in FIGS. 12A and 12B, along with a cutting blade for making slices of the stacked devices; and FIG. 12D is an end view of the slice formed by the blade depicted in FIG. 12C in accordance with the present invention.
  • binding component may be any of a large number of different molecules, biological cells or aggregates, and the terms are used interchangeably.
  • Each binding component is immobilized at a channel of the array and binds to an analyte being detected. Therefore, the location of a channel containing a particular binding component determines what analyte will be bound.
  • the high molecular weight refers to greater than 100 amino acids, nucleotides or sugar molecules long.
  • binding includes any physical attachment or close association, which may be permanent or temporary. Generally, an interaction of hydrogen bonding, hydrophobic forces, van der Waals forces, covalent and ionic bonding etc., facilitates physical attachment between the molecule of interest and the analyte being measured.
  • the "binding" interaction may be brief as in the situation where binding causes a chemical reaction to occur. That is typical when the binding component is an enzyme and the analyte is a substrate for the enzyme. Reactions resulting from contact between the binding agent and the analyte are also within the definition of binding for the purposes of the present invention.
  • matrix or "substrate” means an inert material that serves as a solid phase.
  • channel herein means a usually longitudinal passage or conduit.
  • Pluralities, typically a large number, of channels are adjacent to each other in the form of an array within a block or a solid.
  • the channel or block can have any shape, such as having angular walls or arcuate walls.
  • the channel is not enclosed.
  • the superior aspect of a channel can be open, exposing said channel, and samples can be introduced into the channel through said longitudinal aperture.
  • the bottom aspect of a device placed atop another device can serve as the enclosing side of a channel.
  • particle includes a large number of insoluble materials of any configuration, including spherical, thread-like, brush-like and many irregular shapes.
  • Particles are frequently porous with regular or random channels inside. Examples include silica, cellulose, Sepharose beads, polystyrene (solid, porous and derivitized) beads, controlled-pore glass, gel beads, sols, biological cells, subcellular particles, microorganisms (protozoans, bacteria, yeast, viruses, etc.) micelles, liposomes, cyclodextrins, two phase systems (e.g. agarose beads in wax) etc. and other structures which entrap or encapsulate a material. Particularly preferred are recombinant hosts and viruses that express the protein of interest. Even certain high molecular weight materials, such as, polymers and complexes, may serve as immobilizing structures that would constitute a "particle".
  • arrays and “microarrays” are used somewhat interchangeably differing only in general size. The instant invention involves the same methods for making and using either.
  • a microarray means an article of manufacture resulting from the stacking of multiple channel devices containing a plurality of channels.
  • a microarray would comprise a section from a block, said section comprising a surface having an inert solid phase surrounding addressable reactive surfaces, where such surfaces contain agents of interest.
  • a positive reaction can be comprehended as a signal within the field (i.e., the whole reactive area or a sub-region therein can be visualized or otherwise detected) or as a halo outlining the reactive area.
  • Each channel device typically contains many channels (typically 10 to a 100 or more) wherein each channel is at an intrinsically addressable location, separated from other channels and contains a specific component of interest. Each resulting array therefore contains numerous different components of interest.
  • the number of channels in a block is a multiple of the number of channels per device and depends on the number of such devices stacked together to form the block.
  • the devices and blocks may be stacked in any direction, horizontally, vertically, diagonally, rolled (spiral) and concentricly.
  • the instant invention makes microarrays, "chips” or “biochips” by sectioning channel devices, where each channel contains an immobilized binding component, including biological molecules and entities such as nucleic acid fragments, nucleotides, antigens, antibodies, proteins, peptides, carbohydrates, ligands, receptors, drug targets, biological cells or subtractions thereof (e.g. ground-up cells, organelles, solvent extract, etc.), infectious agents or subtractions thereof, drugs, toxic agents or natural products.
  • Each channel device, or block may have a barcode, orientation marker or other identifying indicia for easy identification and manipulation.
  • embedding/immobilizing media may be cast in said channels prior to assembly into a block.
  • the channels are sealed prior to casting with embedding/immobilizing media and prior to block formation.
  • the channel can be modified after block formation.
  • the devices may be of materials such as glass, metal, ceramic or plastic.
  • the immobilized binding components e.g. nucleic acids, proteins, cells etc., may be coated on the inside of the channels, contained in a gel cast in said channels or attached to or embedded in small particles or beads which fill the channels.
  • the particles or beads may be a component of a gelling material or can be separate components such as latex beads made of a variety of synthetic plastics (polystyrene etc.).
  • Each channel device section cut constitutes at least part of a microarray for use in various binding assays.
  • Typical methods for making the devices include injection molding, blow molding, pressing, cutting a recess into a surface, melting a groove with a hot wire or the like, passing through a knife block, die etc. These and other techniques for fabricating such devices are readily apparent to those skilled in the art.
  • a representative individual device is shown by Figure 1. It is essentially a solid block with grooves. When a second individual device is stacked on top, the bottom of the second one completes the wall and thereby forms channels and the combination with closed and separate channels. Additional devices stacked on top form channels by the grooves in the device immediately below it.
  • the stack is referred to as a block and is shown by Figure 2a.
  • Blocks may optionally be stacked or placed sidewise to form an even larger block and the process continued to generate a block of any size having any desired number of channels. By attaching blocks on the sides having open channels, the channels may effectively be lengthened to any length so desired. This larger block is shown by figure 2b.
  • the grooves may be on any side and may form any shape, a number of possibilities are shown as figures 3a-j.
  • Two individual devices may fit snugly together by having a projection, which partially fills a recess to form a channel. This is shown in figure 4a. By having a slight non-parallel portion to the projection and/or recess, the two devices may be pressure fit together. Alternatively, the recess in one device may match a recess in another device to form the channel as shown in Figures 4b-d.
  • a first device may be stacked and bonded to a second channel-containing device to form the block. Bonding may occur by placing an adhesive on one or more surfaces of the devices or by a sealing material that maintains two devices in position, such as, an adhesive tape, (black carbon double sided adhesive tape worked well) a clamp or a devise that affixes the devices in position.
  • the devices can be made with a profiled surface that locks with adjacent devices by pressure fit or by a dovetail-like lock.
  • FIG. 1 A first embodiment of the present invention is shown in FIG. 1.
  • a device (also known as a black) 10 shown in FIG. 1 is formed with a plurality of generally parallel channels 15, each channel for receiving a predetermined binding component.
  • FIGS. 2A and 2B a plurality of the devices 20a and 20b depicted in FIG. 1 are stacked on one another to form an elongated array devices with an array of channels, each channel able to retain at least one binding component.
  • the stacked devices are fixed to one another mechanically, via adhesive or resinous material, embedding, welding or sintering together.
  • the stacked devices 20a and 20b, with each of the channels filled with a biological detecting substance, may then be sliced to form thin microarrays 25, as shown in FIGS. 2C and 2D.
  • a blade 27, such as the one depicted in FIG. 2C may be used to slice the elongated array of stacked devices to form a plurality of the thin microarrays 25.
  • each device 30 is formed with channels 15 on one surface thereof and grooves 32 on an opposite side for receiving portions of adjacent devices in accordance with the present invention.
  • the grooves 32 mate with wall portions 33 of the device that define the channels 15.
  • the wall portions 33 fit into the grooves 32 to provide greater stability in the formation of the array of devices.
  • FIG. 3B shows yet another embodiment of the device where each device 35 is fo ⁇ ned with channels 15a and 15b on opposite sides thereof.
  • the channels 15a and 15b in the device in FIG. 3B are sized such that with opposing faces of adjacent devices mated, full channels are formed.
  • the channels 15c in the devices 37 have a V-shape and in FIG. 3D the channels 15d have a semi-circular shape.
  • channels 15e and 15f are formed on opposite sides of the device 40 and are offset from one another. When the devices 40 are arrayed together, adjacent to one another, the devices can be staggered providing intercontacting surfaces that provide greater strength when adhered together to form an array 42.
  • Another embodiment depicted in FIG. 3F includes grooves 15d and 15g formed on opposite sides of the device having a semi-circular shape.
  • One advantage of this configuration is simplicity in formation of the block 45 and of the device 44 itself.
  • Devices 48 shown in FIG. 3G are stacked in a staggered orientation to provide maximum contact between adjacent devices.
  • Each device 48 is formed with a single rectangular shaped channel 50 open at one corner. The opposite corner of each device
  • the embodiment of the devices 70 in FIG. 31 also provides an increase in surface area contact between stacked devices.
  • Each of the devices 70 in FIG. 31 is formed from a rectangular shape, with one corner removed to form a channel
  • each device 70 in FIG. 31 is formed with an L-shape that partially defines a channel.
  • Another embodiment of the device is depicted in FIG. 3J and is shown with a plurality of stacked devices 75 that are staggered, thereby interlocking with one another.
  • Channels 76 formed in each device have non-parallel sides.
  • each device76 is formed with a plurality of small protrusions 77 that mate with the upper portion of each channel 76 in adjacent devices to help ensure spatial stability of the array 80 formed by the stacked devices 75.
  • FIG. 4A shows yet another embodiment of the present invention, where a plurality of staggered stacked devices 82a are formed with rectangular channels 85 on an upper surface 86 thereof, each chamiel having angled upper wall portions 87 that interlock with dove-tail shaped portions 90 formed on a lower surface 88 of each device 82a.
  • the devices 82b shown in FIG. 4B interlock in a manner similar to the devices 82a in FIG. 4A, but in FIG. 4B the channels 95 have an oval shape, the upper portion of each channel is able to receive a corresponding dovetail interlocking portion 90 formed on the lower surface 88 of adjacent devices 82b.
  • the devices 82c in the embodiment depicted in FIG. 4C include a plurality of channels 95 and 98 that are oval (95) and rectangular (98) shaped, and interlock with one another via dovetail interlocking portions 90.
  • a plurality of staggered stacked devices 100 are formed on an upper surface 101 thereof with two types of channels, one type of channel 102 having non-parallel sides and the second type of channel 103 having an oval shape that interlocks with oval protrusions 104 formed on the lower side 105 of each device.
  • a plurality of staggered stacked devices 108 are formed again with two types of channels, one set of channels 102 having non-parallel sides, and a second set of channels 103 formed with an oval shape that receives a dove-tail interlocking portions 110 formed on a lower surface of the devices.
  • devices 115 are formed with a plurality of channels, where each channel includes two portions. First portions 117a of the channels are parallel to one another and second portions 117b of the channels diverge away from one another such that the second portions 117b of the channels define funnels. Funnel openings are visible in the depiction of the same array 120 of stacked devices shown from a different angle in FIG. 5C. The funnel openings provide a simple and easy means for inserting biological detecting substances into the array of channels of the array devices 115 in FIGS. 5A and 5B. With the divergence of the second channel portions 117b, the tiny channels are spaced apart from one another making insertion of material easier and simpler.
  • a device 125 is formed with metallic powder 128 which renders the device 125 susceptible to magnetic fields for simplified manipulation by robotic and/or automated manufacturing devices.
  • a plurality of these devices 125 are depicted in FIG. 6B
  • the devices 130 may also be formed with a metallic layer 132 on portions of the device that contact other devices, as shown in FIGS. 7 A and 7B. Resistance from current passed through these metallic layers generates heat thereby fusing stacked devices.
  • the devices 135 of the present invention may also be formed with a layer of material 138 at the bottom of each channel, as shown in FIG. 8, where the layer of material 138 assists in retention of biological material within the channel.
  • the layer of material maybe precious metal, to include but not limited to gold, that is deposited in the channel.
  • the layer of material 138 may be a binding component adsorptive material or the like.
  • the devices 30 depicted in FIG. 3 A are provided with a layer of foil or precious metal layer 140 between adjacent devices 30.
  • the layer of material 140 is slightly deformed such that the first portions 140a of the material 140 are trapped between adjacent portions 32 and 33 of the devices 30.
  • Second portions 140b of the material 140 contact the underside 145 of the upper device 30, as shown in FIG. 9B.
  • the foil material can include, but are not limited to, precious metals such as silver and gold for binding component attachment or any suitable metal for heat fusion.
  • current passed through the portions 140a generates heat thereby fusing stacked devices 30 and adhering metallic layers 140a to said surfaces 32 and 33.
  • the various configurations of stacked plates are formed into bundles that are sliced at an angle relative to the end face of the bundle such that the slices are elongated with respect to the height of each bundle, thereby exposing at least two surfaces of the channels.
  • the two separately coated surfaces of the channels of the devices are visible from a point of view normal to the flat end face of the sliced chip, as is described in greater detail below and shown in FIGS. 10A through 12D.
  • devices 150 are formed with semi-circular or with oval channels 152.
  • Each channel 152 has a predetermined material adhered therein.
  • biological materials 155a, 155b, 155c, 155d and 155e are adhered.
  • completely different biological materials 156a, 156b, 156c, 156d and 156e are adhered.
  • each channel in array 158 has the capability of capturing at least two separate agents, two different molecules or different types of molecules.
  • the array 158 depicted in FIG. 10B is rotated 90 degrees, as shown in FIG. IOC.
  • the array 158 is sliced by a cutting blade 160 to make slices of the stacked devices, to form sliced microarrays 165.
  • the array 158 is rotated 90 degrees so that the cut occurs parallel to the joints between the stacked devices.
  • the blade 160 slices the array 158 at an angle of anywhere from 20 degrees to 70 degrees with respect to the end face 159 of the block array 158. In FIG. 10D, the blade 160 is shown cutting at an angle of approximately 60 degrees with respect to the end face of the array 158.
  • the sliced microarrays 165 are elongated when compared to the height of the end face 159 of the array 158, as shown in FIG. 10E. Further, as shown in FIG. 10E, the cutting angle of the blade 160 is such that the biological materials 155a-155e and 156a-156e within the channels of each sliced microarray 165 are more visually pronounced because more surface area of the channels are included in each sliced microarray 165. Further, due to the exposure of the biological materials 155a-155e and 156a-156e, activity of the biological materials and molecules attracted to these biological materials are more easily observed visually.
  • each channel in a microarray has two different biological agents
  • the channels may be square, as shown in FIG. 11 A, where a device 170 is formed with square channels having at least two surfaces, 171a and 171b.
  • the horizontal surface 171b of each channel is coated with a unique first biological material for attracting a specific target molecule(s) and the vertical surfaces 171a of each channel are coated with a separate biological material different from the first biological material.
  • a plurality of the devices 170 are stacked to form an elongated bundle 175, that is cut to form sliced microarrays 180 shown in FIG. 1 IE.
  • the diamond shape of the sliced microarrays 180 is made possible by rotating the bundle 175 such that a blade 185 cuts the bundle 175 at an angle with respect to the end face 182 of the bundle 175.
  • the blade 185 cuts the bundle 175 from corner 175a to corner 175b and as shown in FIG. 1 IC, further cuts at an angle of about 60 degrees with respect to the end face 182.
  • the blade may cut at an angle of anywhere from about 20-70 degrees with respect to the end face 182. As shown in FIG.
  • a device 200 is formed with V-shaped grooves or channels having two surfaces 201a and 201b, where one surface 201a of the channel is coated with a unique first biological material for attracting a specific target molecule(s) and the other surface 201b of the channel is coated with a separate biological material different from the first biological material.
  • FIG. 12B shows a plurality of the devices 200 depicted in FIG. 12A stacked to form a bundle 210.
  • the bundle 210 is cut at an angle in a manner similar to the embodiments described above, with a blade 230 angled with respect to the end face of the bundle 210, as shown in FIG. 12C.
  • the subsequently sliced microarrays 215 are elongated as compared to the height of the uncut bundle 210, thereby exposing a greater amounts of the biological agents 220a, 220b, 220c, 220d and 220e, and 221a, 221b, 221c, 221d and 221e disposed on the surfaces 201a and 201b of the channels in the devices 200.
  • Different inner surfaces of the channels may be coated with different materials by a variety of methods. Particularly preferred is by coating the surfaces with a photosensitive material and a binding component one wishes to immobilize and apply light or other wave energy to one surface but not another surface. The process may be repeated with a different binding component by applying the light etc. to a different surface. Instead of immobilizing the entire binding component to the surface by this method, one may synthesize by photosynthesis part or all of the binding component by using a number of different solutions sequentially in a manner similar to the photosynthesis of peptides and oligonucleotides on commercially available microarrays.
  • Illumination of the imier surfaces of the channels may be done with any wavelength of light from the same detection as the detection means, from an angle with respect to the detection means or from behind the microarray.
  • Detected emitted radiation may be the same (e.g. light scatter or adsorption measurements) or different wavelength from that applied (e.g. fluorescent measurements).
  • the devices comprising the channels fall into several classes of invention, with subdivisions of each.
  • a fluid containing the binding component is allowed to contact with the channel surface. This may be done by pumping fluids through channels, tilting to allow the fluid to flow, or by treating devices before they are stacked to form a block.
  • a first class is composed of channels containing immobilizing matrices with the immobilized binding component being part of the composition of the matrix.
  • the agent of interest in the instant invention may comprise a very broad range of chemicals, complexes, tissues, biological cells or fractions thereof. Nucleic acids, sugars, proteins, which may be modified or coated with detergents to enhance solubility in organic solvents, and a wide range of organic compounds can be incorporated into polymerizing mixtures such as those used to produce plastics. Oligonucleotides and nucleic acids are soluble in methylene chloride, for example, and hence may be included in acrylics during polymerization.
  • Materials for forming the device include but are not limited to melamine, epoxy, acrylics, methacrylate, gycol methacrylate, methyl methacrylate, butyl methacrylate, styrenes, waxes, polyols etc.
  • Other methods for casting agents of interest into the channels include pipetting liquids into the channels in a preformed block and mobilizing the agent of interest through the matrix of the immobilizing agent using an electromotive force.
  • Various microfluidic methods may also be used such as Bernard et al, Anal Chem Jan 1;73(1):8-12 (2001).
  • a second class consists of channels comprising immobilizing reagent where the reagent is not homogeneous and the polymerizing, solidifying or gelling material also may contain solid structural elements such as filaments, branched elements etc., to further strengthen the gel and also may provide attachment sites for the agent of interest.
  • the added components serve to strengthen the gel and may provide attachment sites for inclusions including dendrimer branched polynucleic acids, branched or crosslinked polymeric materials, metal or glass fibers. Threads, yarn-like configurations and brush-like configurations of structural elements maybe cast into the length of the channel to provide enhanced strength.
  • the structural elements may serve as the immobilizing component in the channel for a desired binding component.
  • channels of acrylic or other plastics each containing a different agent of interest using currently available technology in the instant invention.
  • the cut end of the channels may be treated briefly with dilute solvents to expose active groups.
  • Such beads may be attached together by many techniques.
  • a preferred one is by vapor sintering.
  • the vapor perhaps a hot solvent, is allowed to interact with the beads for a specified period of time and then is removed by evacuation, h heat sintering, the beads are placed under lateral compression and the array heated to the softening point of the plastic.
  • Another means is the use of low melting point metals, such as gallium. By low melting point is meant temperatures acceptable to retaining the binding abilities of the binding component.
  • cast channels described herein can be filled with a matrix comprising string or thread through the center thereof to increase strength.
  • the internal surface of the channels may be modified so that the gel or polymerizing mixture introduced into the channels will adhere to said surface, preferably by covalent attachment.
  • Acrylamide derivatives may be linked to the channel wall to make an acrylamide gel adhere, while gelatin, agar, or agarose derivatives may be attached similarly to link with the respective gels.
  • Methods for linking agents of interest, such as, proteins and nucleic acids, to linear acrylamide, gelatin and agarose are well known, and the derivitized molecules can be incorporated into the gels used for casting.
  • Acrylamide can be made to gel at room temperature either chemically or using photoactivation, while low temperature-gelling Sepharose is available. Gelatin sets slowly and at temperatures below ambient.
  • “Ambient” herein means an encompassing atmosphere.
  • the polymers used to fill the channels are typically homogeneous, but may contain agents of interest, which become attached to the polymerizing medium. Examples include covalent attachment of proteins to short acrylamide chains that become incorporated into acrylamide gels and proteins covalently linked to gelatin. Thus, gels are available or can be produced which contain labile biomolecules without exposing them to denaturing temperatures.
  • Arrays from the instant device may have the interior of the channels coated with biomolecules either covalently or in suitable polymer coatings.
  • Isocyanate polymers such as oxyethylene-based diols or polyols wherein most if not all of the hydroxyl groups thereof carry polyisocyanate groups are suitable. Some such polymers can be comprised of polyurea/urethane polymers. The polymers are well hydrated and fall in the category of hydrogels. Suitable, starting materials include triols, such as glycerol, trimethylolpropane and triethanolamine, tetrols and polyethylene glycols. Suitable polyisocyanates include diisocyanates and such. The polyisocyanates can be aromatic, aliphatic or cycloaliphatic. (Braatz et al., U.S. Patent 5,169,720 and Braatz, J. Biomaterials Applications 9:71- 96 (1994)).
  • the inside surface of the channels described may be modified chemically to allow attachment of polynucleotides, polypeptides, polysaccharides or other molecules either directly or through linkers.
  • DNA and RNA are conventionally synthesized on small polystyrene beads
  • one approach to generating a nucleic acid array is to synthesize oligonucleotides on said beads and then to pack the channels with the beads, until completely filled.
  • different batches of beads having different sequences attached could be used.
  • the beads may be kept in place by careful heating thereof to sinter it or residual latex is added to the channels and dried in place with air pumped through the channels.
  • a variety of histological embedding media has been developed that preserves biological molecules in reactive form.
  • Durcupan, Nanoplast and Quetrol 651 can be cured by very mild heating; methyl methacrylate, styrenes, butyl methacrylate, JB-4 and Immunobed can be polymerized at room temperature; and the water soluble acrylic polymers, London Resin Gold and Lowicryl, polymerize at below freezing temperatures by ultraviolet light (all are available from Polysciences Inc.).
  • Conventional embedding media use solvents and waxes, and the waxes must be at least partially removed before analysis.
  • Embedding and sectioning methods therefore are available to identify and localize specific biological molecules, h the case of nucleic acids, specific nucleic acid targets can be detected by, for example, in situ hybridization and amplification of specific sequences by the polymerase chain reaction (PCR) and other nucleic acid amplification techniques (Ligase Chain Reaction, Rolling Circle Amplification, Strand Displacement Amplification, etc).
  • PCR polymerase chain reaction
  • other nucleic acid amplification techniques Liigase Chain Reaction, Rolling Circle Amplification, Strand Displacement Amplification, etc.
  • the immobilizing and attaching method and means are those that retain the configuration of the candidate molecules that allows recognition and binding by the hormone receptor.
  • many protein or carbohydrate antigens may be detected using immunological reagents. Detection is generally by incorporation of a fluorescent dye or other label into the analyte or into the second layer of a sandwich assay, or by coupling an enzyme to an analyte or a second or third layer of a sandwich assay that produces an insoluble dye, which may be fluorescent.
  • the surfaces of the channel-containing blocks may be used directly to immobilize reactants; others must be modified to allow such additions.
  • Antibodies will adhere to clean polystyrene surfaces, as will many other proteins (Van Oss, C.J., & Singer, J.M. The binding of immune globulins and other proteins by polystyrene latex particles. J. Reticuloendothelial Society 3: 29040, 1966.) Polystryene, either in the form of microtiter plates or beads, has been modified to bind biological molecules, such as, polynucleotides, polypeptides and polysaccharides.
  • Perfluorocarbon such as fluorocarbon polymers known as Teflon®
  • Teflon® polytefrafiuoroefhylene
  • PTFE polytefrafiuoroefhylene
  • polyvinylfluoride polyvinylidene ' difluoride and perfluorodecalin
  • surfaces bind proteins or other biological molecules (U.S. Pat. No. 5,270,193).
  • Such surfaces can be made to include fluorinated surfactants, which may render the surface hydrophilic, or positively or negatively charged.
  • Glass including controlled pore glass, can be used to passively adsorb biological molecules or may be modified to allow covalent attachment of antibodies, antigens, polysaccharides, polynucleotides, nucleic acids and the like.
  • Plastic surfaces may be modified non-specifically using corona plasma discharge or electron beam radiation and then may be coated with a variety of coatings or adhesives to which macromolecules may be attached.
  • More specific covalent attachment of biological molecules maybe achieved by a variety of modifications, which attach reactive groups to polystyrene, or acrylic surfaces, which groups, with or without extending linkers, then will couple under mild conditions to the biopolymers. Also, gold or silver surfaces may be modified (e.g. with a thiol) to attach the biological molecules.
  • a variety of chromatographic media also has been adapted to support immobilized bioreactants. Such media include soft gel beads, generally composed of acrylamide, agarose, Sepharose, which maybe chemically cross-linked, and less compressible beads designed for high-pressure chromatography.
  • a natural product useful as an immobilization support is cellulose, which is readily available in powdered form.
  • the supports may be modified chemically to allow covalent bioreactant attachment, or may be purchased in modified form ready for attachment.
  • Long DNA or RNA molecules may be immobilized by being polymerized in a gel and are retained purely by physical entanglement. An example is the retention of DNA in agar or acrylamide gels.
  • other biological molecules such as polypeptides, proteins, polysaccharides or nucleic acids may be linked covalently to long polymers so that, when embedded in a gel, diffusion does not occur and the biological molecule remains available for reaction with soluble reactants. Examples include the attachment of proteins or nucleic acids to polyethylene glycol (so-called PEGylation) or to linear acrylamide chains.
  • a receptor or molecule of interest is immobilized and used to bind an analyte
  • general methods exist for immobilizing members of a class of reactants.
  • protein A or protein G may be immobilized and used subsequently to bind specific immunoglobulins, which in turn will bind specific analytes.
  • a more general approach is built around the strong and specific reaction between other ligands and receptors such as avidin and biotin.
  • Avidin may be immobilized on a solid support or attached to a gel and used to bind antibodies or other reactants to which biotin has been linked covalently.
  • Immobilization may be either directly on the surface, through a linker moiety or compound, by adsorption, chemical binding, physical entrapping or simply loosely held by an adhesive or in a matrix stuck to the surface.
  • the surface may be derivitized to possess tethered biotin moieties capable of binding subsequently added avidin.
  • the device and block may be handled under protein denaturing or other harsh conditions until the biotinylated binding partner and avidin (or avidin bound binding partner) is added.
  • Other examples of binding pairs may be used to indirectly immobilize the binding partner to the surface.
  • a wide variety of methods has been developed to detect reactions between immobilized molecules of interest and soluble reactants. The methods differ chiefly in the mechanism employed to produce a signal and in the number of different reagents that must be sandwiched together directly or indirectly to produce that signal.
  • labels and detection systems are known per se in the filed of inding reactions and any of these may be sues.
  • Common preferred ones include fluorescence (including delayed fluorescence) with the fluorescent tag covalently attached to the analyte, fluorescence involving soluble dyes, which bind to an analyte, and similar dyes wherein the fluorescence thereof greatly increases after binding an analyte.
  • the latter can be used to detect nucleic acids.
  • sandwich assays the result is the immobilization in the detection complex of an enzyme that, in combination with a soluble substrate, produces a preferably insoluble dye that may be fluorescent.
  • the detection complex attached to the bound analyte may include a dendritic molecule, including branching DNA, to which is attached many fluorescent dye molecules.
  • Methods for making dental floss having attached short transverse fibers to give a brush-like configuration may be modified to allow attachment of reactants.
  • the art of detecting bubbles or voids in liquid filled tubing is known and may depend on differences in refraction, light absorption or fluorescence as measured along individual channels. Such methods serve as quality control means for said channels.
  • the binding partners may be added to the channels after a block is formed. This may be done by a number of techniques, the simplest being to pipette liquids directly into individual channels or a group of channels. A small funnel may be advantageous to use.
  • the art of using centrifugal force to fill short lengths of tubing with viscous media can be modified to fill the channel containing blocks .
  • Microtomes for sectioning tissue blocks which may contain samples ranging from soft tissues to bone, often in blocks of embedding material (e.g. wax, paraffin, OCT, etc.), are commercially available, as are a variety of techniques and arrangements for attaching sections to glass or plastic slides, for treating the slide automatically to remove some or all of the embedding media, and for systematically exposing the slides to a series of reagents.
  • embedding material e.g. wax, paraffin, OCT, etc.
  • Microtomes and other sectioning or cutting instruments capable of cutting assembled bundles of tubes into thin sections, and of maintaining the orientation of the component tubes after sectioning are known. Blade cutting may reduce contamination of binding components between sites of the microarray.
  • the microarrays can be of any thickness as required by the anticipated use thereof. Another determining factor might be the rigidity of the blocks. In one embodiment, the sections will be less than 1 cm in thickness. In another embodiment, the sections will be less than 50 ⁇ m in thickness. As will be exemplified in further detail herein below, sections can be on the order of microns in thickness.
  • That method can be improved further by exposing the bound antibody array to a solution containing known saturating or subsaturating quantities of each analyte protein in a non-fluorescent form, washing the array, and exposing the array to a test mixture of labeled proteins, thus producing a multiple competition assay.
  • Other labeling systems known per se may be used.
  • Any of the conventional binding assay formats involving an immobilized binding partner may be used with the microarray systems of the instant invention.
  • the microarray may have either plural ligands or plural receptors and the analyte may be either plural ligands or plural receptors. Competing elements that bind to either the analytes or the microarray reactive surfaces may be added.
  • the sample may be labeled and/or the competing element may be labeled and/or the microarray cell may be labeled.
  • the labels may be interacting with each other to make a detectable signal or product, or to quench a signal or product.
  • the number of different combinations is in the dozens and any may be used in the instant invention as well as different combinations for different cells of the microarray assay. Often several different clinical tests are required to define a particular disease.
  • Arrays have numerous uses other than determining bioactive properties. Chemical interactions and reactions may be tested as well. Such an assay can, for example, enable testing different reactive chemicals simultaneously against a test substance or material to determine corrosion, electrochemical reaction or other interaction. That is particularly advantageous in the chemical formulations of plural substances such as in cosmetics, paints, lubricants etc. Alternatively, one may assay for desirable interactions between the analyte and all of the molecules of interest in the array.
  • microarray may be mounted on a porous membrane and passing the ligand and or ligand solution through the microarray by hydrodynamic, electrophoretic or mechanical means.
  • fluid may be flowed through the microarray by pressure difference on each side of a membrane. Fluid also may be drawn through by simply applying a stack of paper towels on the backside of a membrane to draw fluid through the microarray.
  • electrophoretic means a potential is applied across the microarray either across the entire microarray or using single point electrodes located on both sides of a single or group of sites of the microarray.
  • Mechanical means may involve a pump of various configurations to mechanically push or pull fluid through the microarray by providing a pressure differential.
  • the slice When the block has hollow channels and is cut, the slice is porous and permits fluid flow through the pores defined by the channels. Should the channel be filled with a porous matrix, the slice will also be porous. Structural support may be given by mounting the slice on a porous solid support to form the microarray.
  • the instant invention By using the instant invention, one avoids the difficulties of individually spotting each cell on a solid phase or forming a compound at each cell.
  • the former method is limited by human intervention and apparatus, as well as the ability to measure quantitatively small amounts of liquid.
  • the latter technique is limited by the types of compounds that can be synthesized on the solid phase.
  • Both prior art techniques are expensive and require elaborate automated equipment or tedious labor as each array is produced individually.
  • the instant invention is technically simple and quick where the "batch" is in the thousands to millions of microarrays. The only individual effort required for each microarray is the step of cutting.
  • the present invention may also use the entire block instead of a section as the microarray.
  • microarrays Large numbers of different and potentially new active compounds may be screened simultaneously by immobilization, casting in channels, sectioning and forming a microarray. Peak fractions from separations, such as plant extracts, may be collected simultaneously and used to form a microarray. The microarrays then may be used in a large number of assay systems simultaneously, dramatically reducing the time and effort to screen all of the compounds present for whatever activity one chooses. Particularly preferred are large numbers of proteins or peptides generated by mass techniques. Different fractions from a separation technique from a natural source provide a resource of many different proteins and peptides. A number of fractionation procedures are known to separate mixtures of many compounds. Different fractions or specific compositions may be used to form separate channels.
  • Each channel may contain a mixture of molecules of interest. For example, during chemical synthesis, a number of isomers are prepared. It is convenient to not separate the isomers before forming a channel in some circumstances. Likewise, when fractionating a mixture, forming a channel with a mixture of receptors may be acceptable, as total and complete isolation is difficult and time consuming.
  • the embedding matrix for the channels may be black, opaque or otherwise adsorbent to emitted signals of a label to reduce cross talk between the reactive surfaces in the array. Additionally, any adhesive for forming the stacked blocks may contain the same adsorbent material to reduce background between sites of the microarray.
  • a microarray may contain thousands of sites, one can determine the antibiotic sensitivity to numerous antibiotics simultaneously. Quantitative determination of other biological activities with either ligand or receptor immobilized in the gel may be used. Essentially the same channel binding partner may be used multiple times in the same microarray. That provides an internal quality control check and improves confidence in the binding assay. That also provides additional quantitative measurements if such an assay is performed to improve precision. Blank channels, channels with no molecule of interest bound thereto, provide a good negative control and should be used in every microarray.
  • Particles may also be "chemically sintered" after casting in a channel.
  • a blocking agent may be added to block any remaining active sites or adsorption areas on the particle.
  • the beads are then cast into a channel formed after stacking of a second device.
  • a chemically reactive compound which crosslinks or couples either the blocking agent and/or the molecule of interest and/or unreacted sites on the beads then is added and at the locations where the beads touch, chemical adhesion results.
  • the molecules of interest in the internal pores of the beads are not touching and thus are not altered significantly.
  • the pores of the beads may be filled with a hydrophilic solution and held by capillary action while the spaces between the beds are filled with a hydrophobic adhesive or setting liquid.
  • a representative example of chemical sintering is to adsorb Protein G on porous beads and then to add a gelatin blocking agent.
  • the resulting beads are filled in, for example, 1 mm (height) x 1 mm (width) x 10 cm (length) channels comprising a rectangular block and then a protein crosslinking agent added, e.g. carbodiimide. After the reaction is complete, unreacted reagents are washed free and then any suitable antibody of interest is added thereto to bind to Protein G, thereby forming a square channel device suitable for cleaving to make a microarray.
  • a channel first is filled with both beads in dry form, the device shaken and then fluid is pumped there through permitting a reaction to occur thereby forming a solid matrix of beads.
  • the beads may be added first (with or without fluid) and the second set added later so that the beads filter down through the spaces between the larger beads and react accordingly.
  • the reaction between the beads may be through specific binding moieties or of a non-specific binding reaction to form a crosslinking of the beads into a sliceable solid.
  • the second beads may be black to reduce stray light in the fluorescence detection.
  • a fixed pattern becomes apparent and the device is cut transversely or at an angle into many thin sections containing a linear arrangement of sites. If a number of blocks are stacked, a two-dimensional matrix of sites is obtained wherein one dimension is the number of sites in a device and the other dimension is the number of stacked devices.
  • Each sectioned two-dimensional array would contain relatively large numbers of binding components, such as DNA, RNA, or protein molecules.
  • a solution which can erode the plastic, or other material surface of the array very slowly, without affecting the surrounding matrix (inert) of the block, is washed over the surface.
  • each block has multiple molecules of interest in the same arrangement as will be present in the microarray, one can perform a quality control check on all members of the array by examining a single section. That is particularly important when the microarray is used for diagnostic purposes. Sampling microarrays from a batch may be a quality control check but it does not actually check the microarrays being sold. By contrast, small slices of the blocks themselves are being used in the instant invention. Assaying the block members represents an actual test of every microarray in that block.
  • the key agent of interest of the channel is retained by the channel by being immobilized therein.
  • Immobilization may be accomplished by a number of techniques, known per se, such as entrapment in a matrix and chemical coupling, perhaps through a linking moiety through an amino, hydroxy, sulfhydryl or carboxyl moiety. Chemically attaching the chemical to a monomer or being used as a monomer to be polymerized also effectively incorporates the component. Binding also may be accomplished by a number of affinity techniques such as protein A or protein G for antibody attachment, ligand/receptor pairs such as biotin-avidin, HIV-CD4, sugar-lectin or through a ligand that has a receptor such as digoxigemn-antidigoxigenin.
  • a gel or a non-gel, gelling matrix such as wax, silicone polymers and silicone emulsions may be used.
  • Liquid wax or a gelling agent simply is mixed with the key component and cooled to form a cast within the channel.
  • identifiers may be integral with the array itself.
  • Optical, magnetic, physical or other machine readable indicia may be printed along one border to provide identification and orientation. A region for recording information about the sample, tests performed and/or results etc. may also be present.
  • small concentrations of dyes or other easily recognizable indicia may be incorporated into the polymers from which selected channels are made such that they present a pattern for orientation, for example, of one or more numbers, or one or more letters. It is also useful to have a few sites or elements which do incorporate fluorescent dyes and which serve to calibrate the fluorescence measurements. It is further feasible to introduce dyes into the contents of selected channels to additionally identify them.
  • the device material and adhesive used to hold the separate blocks may be opaque, while the channels and preferably, the contents thereof may conduct light along the entire length.
  • An arrangement for detection using epifluorescence as shown diagrammatically in applicants previous application WO/USOl/09607 may be used. Different systems for detecting fluorescence patterns on chips are known to those skilled in the arts.
  • the channel may be comprised of material that is preferably glass, metal, plastic or other polymeric material. Each material may be a composite of two or more components.
  • the channels may act as light pipes or total internal reflection fiber optics to transmit information regarding chemical and biological reactions occurring on the surface or coating on or condition of the surface.
  • the channel material preferably is chosen to support attachment of cells and molecules of interest such as oligonucleotides, peptides and polysaccharides.
  • Light and electrons emitted directly or indirectly from a reaction or component inside the site may be amplified and easily detected when the channel material from which the site is derived is made of glass or other transparent or translucent material.
  • the channel material may contain a component to react with, detect or convert into another form, the light, electrons or other chemical components emitting from the components or reactions occurring in the square. Detection of chemiluminescent reactions on the reactive surface is a suitable method.
  • Surfaces of the channel walls may be coated to enhance binding of the binding partner or coated to give desired chemical moieties on the surface.
  • Such techniques include vapor deposition, gold surfaces (e.g. leaf), liquid suspension, reduction deposition, etc. nickel, sputtering, etc. Different coatings may be added depending on the desired surface. To increase surface area, a roughened or three dimensional surface is helpful. For a smooth monolyaer of binding partner, gold and other surfaces may be used. Hydrophilic and hydrophobic surfaces may also be preferred depending on the chemical nature of the binding partner that is being immobilized.
  • the field or surface treatments is known per se and its use for the microarrays of the present invention, particularly when the block is cut at an angel. Other material facilitating attachment also known per se and may be used.
  • Coatings of silica, and the like, may be applied to the device first by chemical vapor deposition or wet silane chemistry to make a thin (e.g. 150 angstroms) coating for gold attachment if so desired. Binding partners may also be directly attached to the silica or other material coating. Binding partners may be attached to gold surfaces by a number of techniques. For proteinaceous binding partners, one may use the following chemistries: A. bind 11-mecaptoundecanoic acid to gold coating activate-COOH with carbodiimide (can bind protein) bind aminobutyl nitrilotriacetic acid bind His-tag proteins
  • the binding agent may be conjuncated to nitrocellulose strips by well known techniques. These thin strips of protein (or other binding agent)-coated nitrocellulose are placed in the grooves of the square channel devices and sealed in place, e.g. with an appropriate adhesive. Multiple units are stacked, and sections are then evaluated directly with fluorescent-labeled antigens, by chemiluminescence using HRP conjugates or other techniques. The thin strip may also be threaded into a preformed block if so desired. A number of other configuration for the carrier may be used in lieu of a thin strip, such as a thread, a porous rod, tube etc.
  • Gelling materials used in the present invention may be selected from a large number of such known materials. Polymers such as agarose, gelatin, collagen, xanthene, carrageenan, alginate, or a thermosetting, thermoplastic, chemosetting or UV polymerizing polymer may be used. Non-polymeric gelling materials including waxes and clays may be used. Hydrogels are particularly preferred when a reaction occurring between the agent of interest and an added substance for interrogation requires an aqueous environment.
  • Hydrogels have many desirable features such as variable gel porosity, ability to bind proteins during or after polymerization, low non-specific binding, transparency, harmless polymerization byproducts, controllable polymerization open time, usable with a variety of solvents and so on. Isocyanate polyurethane liquid prepolymers are preferred.
  • gelling material should be sufficiently inert to not interfere with an interaction between the binding components and/or analytes.
  • the gelling materials may also contain a dye or other optical absorber so that only analyte/binding components on the surface of each site are visualized.
  • a dye or other optical absorber so that only analyte/binding components on the surface of each site are visualized.
  • a dye that adsorbs UV or emitted fluorescence will reduce fluorescence from non-surface analyte/binding component reactions.
  • Different dyes may be incorporated into individual channels. This permits the location of the individual sites in the two- dimensional array to be confirmed.
  • the devices forming the channel device may be adhered to each other by a variety of techniques. If the components are sufficiently heat stable, the devices may be sintered together. Alternatively, a number of adhesives are known, including cyanoacrylate adhesives microwave and RF heating. Thermoplastic and gelling materials also may constitute the adhesive causing the devices to be held together. Non- chemical means, such as passing an electrical current through the devices, heat sintering, ultrasonic welding to fuse them may also be used.
  • the open ends of the channels may be sealed against a flat plate, by pressing a deformable material against the surface, evaporating a plastic (e.g. paralene) on the surface, or by sealing with a chemical such as a thermoplastic or thermosetting plastic material.
  • An advantage of the instant system is that very large numbers of arrays may be cut, and some fraction used for standardization. For example, if a block containing ten side-by-side 10 cm long channels is cut at 100-micron intervals, then 1000 sections, each containing 10 sites would be available. If the sections were 10 microns in thickness, then the number of sections would be 10,000.
  • the instant invention it is preferred for the instant invention to have at least 100, more preferably 250, 500, 1,000, 5,000, 10,000, 100,000 or a million or more reactive channel surfaces per square centimeter of array. That is a much higher concentration than depositable cells formed by microfluidics in commercial microarrays. To increase greatly the number of reactive surfaces per square centimeter beyond even such high numbers, one may prepare a large block with relatively longer channels and increase the number of channels in a block.
  • the embedding medium may be incompatible with the molecule of interest or use in a binding assay, yet still be useable.
  • an aqueous solution may be used to protect proteins and a low melting point wax used to embed the porous particles.
  • microarrays of the instant invention be used to identify infectious agents by identifying characteristic nucleic acid sequences
  • the microarrays also can be used for identifying intact bacteria, mycoplasmas, yeast, nanobacteria and viruses using arrays of immobilized specific antibodies.
  • the invention may be applied in such a fashion that the blocks or devices are stored at user sites assembled as desired, and the arrays sliced as needed. That arrangement may be useful for research purposes where identical arrays are required over the long term, but only a few are required at any one time.
  • An electrical detection may be used by incorporating a conductor into the passage or block which is in communication with and can detect events happening at the specific pre-defined location on the microarray or block.
  • the microarray is preferably mounted on a surface with electrical contacts which align with those in the microarray for transferring electrical signals to a suitable detection means.
  • a separate electrode may be in the microarray or block itself or it may be positioned in another location such as directly above as has been previously suggested for conventional DNA microarrays.
  • Cut surfaces of each block may be polished so that matching blocks may be opposed to each other with little possibility of cross leakage if the block material is not inherently hydrophobicor vice versa.
  • the binding partner attachment surfaces may be derivatized to enhance binding partner attachment or the hydrophilicity may be changed in general such as by plasma discharge treatment, the binding partner.
  • Surface treatment with a material repellant to the fluid to be eventually located inside each square further reduces cross leakage.
  • fluorinating (Teflonizing) or silanizing agents repel water thereby generating sufficient surface tension to reduce cross leakage of squares filled with an aqueous solution.
  • Devices and blocks may be arranged side-by-side, end by end and/or on top of each other.
  • the sections After sections have been cut from a device, the sections generally are bound to a solid backing to provide structural support and ease of handling.
  • the solid backing is typically a sheet of plastic or metal although other materials may be used.
  • the attachment generally is done by a permanent adhesive or heat fusion.
  • the backing may be porous which is preferred with a porous microarray.
  • Individual sites in the array may be detected or visualized by scanning the entire array or portions thereof (e.g., one or a few sites) with a charged coupled device (CCD) or by illuminating one or a few sites at a time, such as by a condenser lens and objective lens. The absorbance and emission of light thus may be detected.
  • An optical fiber bundle aligned and registering with the microarray may be used for optically detecting differences between the sites of the microarray.
  • a positive reaction can be detected as a signal or lack of a signal within the field (i.e., the entire site can be visualized or a sub-region therein) or as a halo outlining the site.
  • Detection may be based on a large number of detectable labels including radioactive, enzyme, luminescent, optically absorbent dye, magnetic, spin-labeled, oxidizers or reducers, chemiluminescence, electroluminescent or an electrical sensitive reaction may also be used by having electrodes on the microarray and at least one electrode at a particular site or groups of sites or movable to the same.
  • Indirect labels which interact with a detectable component interacting with the agents of interest in the microarray, may be used.
  • Signal amplifying systems such as generating peroxidase/antiperoxidase complexes may be used..
  • a suitable detectable labeling system is based on fluorescence, usually epifluorescence.
  • the interrogating sample be labeled with one or more fluorescent dyes.
  • the amount of test material required is very small since the dye would be applied to the arrays as a thin dilute film. Hybridization of nucleic acids would be done under conditions of carefully controlled stringency.
  • one either may seal off the selected sites and/or fill the sites with an easily detectable substance.
  • Different colored inks, dyes and colored materials are particularly well suited as well as detectable components similar to or opposite from the detectable component(s) being detected in other sites.
  • Printing methods with drying inks or plastics, sublimation, solvent containing an ink, or ink-jet printing may be used. The indicia so formed permits better alignment or easily detectable marking when the array is in use. That permits easy optical alignment.
  • the microarray has been used in a binding assay and the ligands are bound to the receptors, in certain instances it may be useful to provide further identification of the ligand.
  • the ligand is a cell, a macromolecular complex or a derivitized molecule with the derivitized portion acting as the ligand, etc.
  • further analysis may be desirable.
  • one may elute the ligands from the microarray and collect the ligand for further analysis.
  • a pH 2-3 environment or other conditions should strip the ligands.
  • nucleic acid hybridization raising the temperature should strip the ligands.
  • a variety of other chemical, physical and electrical techniques for breaking such bonds are known per se.
  • the substrate can be configured to enable maintaining a charge that would enhance trapping the biological agent of interest at a particular site.
  • the agent of interest is a nucleic acid
  • each site can be configured to carry a positive charge.
  • a counterelectrode carries the opposite charge. Then, if necessary, a particular medium is placed into the site and the charges in the electrodes reversed thereby releasing the ligands, in the example, nucleic acids, at that location.
  • the counterelectrode also may be part of or contain appended thereto a micropipette to collect the elements released from the site, see U.S. Pat. No. 5,434,049.
  • one uses a porous membrane and applies a current on opposite sides of the membrane.
  • the method used for analysis of the eluate may be capillary electrophoresis, mass spectrometry or a second binding assay.
  • the microarray itself may be introduced into a laser-matrix desorption system incorporated into a mass spectrometry system wherein bound molecules are desorbed and analyzed.
  • the receptor is attached to the matrix of the microarray by a cleavable linker, one can isolate the analyte by cleaving the linker.
  • Different sites of the microarray may have different linkers or the same linker and subsequent purification may be needed before additional analysis.
  • the previous methodology for preparation of protein chips requires preparation, use and reuse of large numbers of proteins in solution. Proteins, nucleic acids, biological cells, other chemicals and complexes in solution are unstable and deteriorate over time. Even if frozen, repeated use may involve repeated freeze-thaw cycles that denature certain proteins as well. By contrast, immobilized proteins have been shown to be stable over long periods of time.
  • EXAMPLE 1 A mold for preparing a square channel array was formed from Teflon®. The mold was designed to produce a device 0.9 x 1.0 cm, with 1.0 mm channels as shown in Figure 1. The mold was filled with 200 ml of a solution of ImmunoBed monomer (Polysciences, Inc., Warrington, PA), prepared according to the manufacturer's directions. The solution in the mold was covered with a flat block of Teflon® and kept at room temperature for 1 hour. After polymerization, the resulting device appeared as shown in Figure 1. The device was placed in a microtome, and 10 mm sections were cut. EXAMPLE 2:
  • Another mold was prepared similar to that shown in Figure 1 but was 0.9 X 8 cm with 1.0 mm channels. To this mold was added a mixture of ImmunoBed monomer solution prepared as in Example 1, but containing 1% graphite (General's Graphite, General Pencil Co. Inc., Jersey City, NJ). A flat Teflon® block was placed on top of " " the solution in the mold. The device was allowed to stand overnight at room temperature to polymerize. After polymerization, the device was removed and viewed under a fluorescence microscope. The background fluorescence of the device was significantly reduced by the incorporation of graphite.
  • graphite General's Graphite, General Pencil Co. Inc., Jersey City, NJ
  • a 0.9 X 1.0 cm square channel device with 1.0 mm channels was prepared using black polystyrene in the same mold used in Example 1. Small particles of black polystyrene were placed in the mold and heated until molten. A flat Teflon® block was then placed on top of the molten polystyrene and compressed to form the device. The resulting square channel device was rigid and exhibited no background fluorescence when viewed under a fluorescence microscope.
  • EXAMPLE 4 A 0.9 X 8.0 cm square channel device with 1.0 mm channels was prepared using polymethylmethacrylate in the same mold used in Example 2. Methacrylate monomer was prepared by mixing 2.5 ml of a solution containing 8 parts methylmethacrylate, 2 parts butyl methacrylate, and 1.5% benzoyl peroxide with 0.5 ml of a solution containing 2 gm benzoyl peroxide and 18 gm methylmethacrylate. This solution was used to fill the mold, which was then covered with a flat Teflon® block, and then place in an oven at 60oC for 24 hours. The resulting square channel device was clear and hard.
  • EXAMPLE 5 Alternate channels in the channel device of Example 3 are filled with 10 microliters of phage display antibodies (Omniclonal, Biosite) directed to human serum albumin. The remaining alternate channels are filled with 10 microliters of phage display antibodies (Omniclonal, Biosite) directed to human alpha 2 macroglobulin. The solutions are allowed to contact the channels of the device for 10 minutes at room temperature and then washed repeated in phosphate buffered saline, pH 7.2. A thin film of toluene is applied to the underside of a channel device with no antibody attached and pressed on top of the channel device with adsorbed antibodies.
  • the block formed by two devices adhered together is placed in a microtome and 20 micron slices taken at a 45 degree angle and glued on a glass slide. 100 microliters of FITC labeled human serum albumin is applied to the slice, incubated for 10 minutes at room temperature in a humidified chamber and then repeatedly washed in phosphate buffered saline, pH 7.2. The slice is visualized under a fluorescent microscope with alternate channels showing fluorescence.
  • the black polystyrene devices of Example 3 were coated with a thin layer of gold by the electroless deposition of gold on polymeric substrates as described by Menon et al, Anal. Chem. 67:1920-1928 (1995) and Jirage et al., Anal Chem. 71:4913- 4918 (1999). Briefly, tin was first applied to the surface, then treated with a solution of silver nitrate. This causes a redox reaction in which Sn(II) is oxidized to Sn(IV), and the Ag + was reduced to elemental Ag. The silver-coated device was then immersed in an Au + plating solution containing formaldehyde. The Ag was displaced by Au , which was reduced to Au(0) by the formaldehyde. The device was then immersed in nitric acid to dissolve residual Sn and Ag.
  • FITC-labeled anti-human serum albumin antibody high concentration FITC-labeled anti-human serum albumin antibody, low concentration
  • the liquids were allowed to adsorb overnight at 4°C in a humidified chamber. Methylene chloride was applied to the underside of the devices to cause them to adhere and the devices were stacked and sectioned at a 45 angle and mounted on a slide. Slices of varying thickness were taken. In each case, the bottom portion of the groove as represented by the channel wall was visualized from atop. The thicker the slices, the greater the amount of the chamiel wall was observable until no light could directly pass through the channel when observed from on top. Sections cut at 90 degrees were also taken.

Abstract

L'invention concerne des microréseaux préparés par le tranchage d'un bloc contenant plusieurs canaux séparés, chaque canal contenant un partenaire de liaison pour des analytes à tester. Les blocs sont assemblés à partir de piles de dispositifs rainurés, de sorte que des canaux se forment à partir de la rainure et d'une surface du second dispositif.
PCT/US2003/002149 2002-01-25 2003-01-24 Microreseaux produits par la formation de parties transversales dans des plaques comportant plusieurs echantillons WO2003064997A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006023323A1 (fr) * 2004-08-19 2006-03-02 Biocept, Inc. Microreseaux utilisant des hydrogels
WO2011129710A1 (fr) * 2010-04-15 2011-10-20 Digital Sensing Limited Microréseaux
WO2011153962A1 (fr) 2010-06-11 2011-12-15 Industrial Technology Research Institute Appareil de détection de molécule unique
US9482615B2 (en) 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
US9778188B2 (en) 2009-03-11 2017-10-03 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object

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US20020110839A1 (en) * 2000-04-28 2002-08-15 David Bach Micro-array evanescent wave fluorescence detection device
US20030073228A1 (en) * 2000-11-08 2003-04-17 David Duffy Peelable and resealable devices for arraying materials

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110839A1 (en) * 2000-04-28 2002-08-15 David Bach Micro-array evanescent wave fluorescence detection device
US20030073228A1 (en) * 2000-11-08 2003-04-17 David Duffy Peelable and resealable devices for arraying materials

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006023323A1 (fr) * 2004-08-19 2006-03-02 Biocept, Inc. Microreseaux utilisant des hydrogels
US9778188B2 (en) 2009-03-11 2017-10-03 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US10996166B2 (en) 2009-03-11 2021-05-04 Industrial Technology Research Institute Apparatus and method for detection and discrimination molecular object
US9482615B2 (en) 2010-03-15 2016-11-01 Industrial Technology Research Institute Single-molecule detection system and methods
US9777321B2 (en) 2010-03-15 2017-10-03 Industrial Technology Research Institute Single molecule detection system and methods
WO2011129710A1 (fr) * 2010-04-15 2011-10-20 Digital Sensing Limited Microréseaux
WO2011153962A1 (fr) 2010-06-11 2011-12-15 Industrial Technology Research Institute Appareil de détection de molécule unique
CN102713572B (zh) * 2010-06-11 2015-11-25 财团法人工业技术研究院 单分子侦测装置
EP2580576B1 (fr) 2010-06-11 2017-04-05 Industrial Technology Research Institute Appareil de détection de molécule unique
US9995683B2 (en) 2010-06-11 2018-06-12 Industrial Technology Research Institute Apparatus for single-molecule detection

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