WO2002075027A1

WO2002075027A1 - Method and apparatus for making fibers for sectioned arrays

Info

Publication number: WO2002075027A1
Application number: PCT/US2002/003203
Authority: WO
Inventors: Norman G. Anderson; James A. Braatz
Original assignee: Large Scale Proteomics Corporation
Priority date: 2001-03-15
Filing date: 2002-02-05
Publication date: 2002-09-26
Also published as: US20020129889A1

Abstract

A method and apparatus for making a fiber, especially a fiber adapted for use in a sectioned array, are provided according to the invention. The method includes a step of supplying a composition into a mold or tubing wherein the composition solidifies in the mold or tubing. The method further includes a step of allowing the composition to solidify and form the fiber. The method further includes a step of placing a predetermined elongation force onto an end of the fiber, the predetermined elongation force causing an elongation and reduction in cross-section of the fiber and causing a separation of the fiber from an interior surface of the mold or tubing. The method further includes a step of substantially maintaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing.

Description

TITLE OF THE INVENTION

METHOD AND APPARATUS FOR MAKING FIBERS FOR SECTIONED ARRAYS

BACKGROUND OF THE INVENTION

[0001] The present invention relates generally to a method and apparatus for making fibers for sectioned arrays.

[0002] Analysis of the components of a particular substance or compound may be an important and difficult process. The substance may have many components, and the components may be present in widely varying quantities. There is a constant need for the ability to quickly, easily, and accurately determine the components of a test sample. One area for such analysis is medical diagnostics, where a patient sample may be tested for diseases, toxins, drug levels, hormone levels, etc. Another area in which component analysis is needed is, for example, detection of proteins, nucleic acids, etc. Additional uses include testing and analysis of any organic or inorganic compound.

[0003] Available test arrays reflect the ongoing need for small devices or test kits that test for a wide array of molecules or macromolecules. The need is for simple, high output testing, including the capability for automated testing and detection in a quick and reliable fashion.

[0004] The prior art has attempted to meet this need by creating arrays or microarrays that contain large groupings of binding agents of varying types. When exposed to a test sample, molecules or macromolecules of interest may bind to these various binding agents. The binding agents may then be detected in various ways, such as illumination by light and detection of light emitted in response (the binding agents may include dyes).

[0005] Microarrays are known in the art and are commercially available from a number of sources. Microarrays have been used for a number of analytical purposes, typically in the biological sciences. An array is essentially a two dimensional sheet where different portions or cells of the sheet have different biomolecule elements, such as, nucleic acids or peptides, 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.

[0006] The prior art methodology for preparation of protein chips requires preparation, use and reuse of large numbers of proteins in solution. Proteins in solution are unstable and deteriorate over time. Even if frozen, repeated use may involve repeated freeze-thaw cycles that denature certain proteins. By contrast, immobilized proteins have been shown to be stable over long periods of time. [0007] In the prior art, two-dimensional arrays of macromolecules are made either by depositing small aliquots on flat surfaces under conditions which allow the macromolecules to bind or be bound to the surface, or the macromolecules may be synthesized on the surface using light- activated or other reactions. Prior art array formation methods include using printing techniques to produce such arrays. Some methods for producing arrays have been described in "Gene- Expression Micro-Arrays: A New Tool for Genomics", Shalon, D., in Functional Genomics: Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S.R. & Savage, L.M., eds., International Business Communications, Inc., Southboro, Ma, 1997, pp 2.3.1.-2.3.8; "DNA Probe Arrays: Accessing Genetic Diversity, Lipshutz, R.J., in Functional Genomics: Drag Discovery from Gene to Screen, IBC Library Series, Gilbert, S.R. & Savage, L.M., eds., International Business Communications, Inc., Southboro, MA, 1997, pp 2.4.1-2.4.16; "Applications of High-Throughput Cloning of Secreted Proteins and High-Density Oligonucleotide Arrays to Functional Genomics:, Langer-Safer, P.R., in Functional Genomics: Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, S.R. & Savage, L.M., International Business Communications, Inc., Southboro, MA, 1997, pp 2.5.13; Jordan, B.R., "Large-scale expression measurement by hybridization methods: from high-densities to 'DNA chips'", J. Biochem. (Tokyo) 124: 251-8, 1998; Hacia, J.G., Brody, L.C. & Collins, F.S., "Applications of DNA chips for genomic analysis", Mol. Psychiatry 3: 483-92, 1998; and Southern, E.M., "DNA Chips: Analyzing sequence by hybridization to oligonucleotides on a large scale", Trends in Genetics 12: 110-5, 1996.

[0008] The terms "arrays" and "microarrays" are used somewhat interchangeably differing only in their general size. The present invention involves the same methods for making and using either. Each array typically contains many cells (typically 100-1,000,000+) wherein each cell is at a known location and contains a specific component of interest. Each array therefore contains numerous different components of interest.

[0009] The number of different cells and therefore the number of different biochemical molecules being tested simultaneously on one or more microarrays can range into the thousands.

Commercial icroarray plate readers typically measure fluorescence in each cell and can provide data on thousands of reactions simultaneously, thereby saving time and labor. A representative example of the dozens of patents in this field is U.S. Pat. No. 5,545,531.

[0010] Many biochemical analyses require that the analytical procedure have wide dynamic range. Enzyme and immunochemical assays are often done by determining the course of a reaction over a period of time, or by doing the analyses on a series of dilutions, h addition, parallel analyses using standards and blanks (controls) are required and are universally included. Large numbers of standardized inexpensive biochips will be required to meet these needs. These biochips may incorporate reactants of different classes that can, for example, detect and measure antigens, drugs, nucleic acids or other analytes simultaneously.

[0011] Regardless of the technique, each prior art microarray is individually and separately made, typically is used only once and cannot be individually precalibrated and evaluated in advance. Hence, the prior art depends on the reproducibility of the production system to produce error-free arrays. These factors have contributed to the high cost of currently produced biochips or microarrays, and have discouraged application of this technology to routine clinical use.

[0012] One of the advantages of microarrays is that they may be machine-read and the result may be digitally stored and/or processed. For reading or scanning microarrays, charged coupled device (CCD) cameras are widely used. However, in one proposed variation, an array is located at the end of a bundle of optical fibers with the nucleic acid or antibody/antigen attached to the end of the optical fiber. Detection of fluorescence may then be performed through the optical fiber.

[0013] Scanning or reading arrays are routinely produced in which glass or plastic fibers are arrayed in parallel in such a manner that all remain parallel, and an optical image may be transmitted through the array. Parallel arrays may also be made of hollow glass fibers, and the array sectioned normal to the axis of the fibers to produce channel plates used to amplify optical images. A microarray may be brought into proximity with a camera or scanner, and an image or images may be captured.

[0014] Arrays may have an entire set of antigens/antibodies, etc., in the various cells, along with controls to effectively screen blood samples for common bloodborne diseases before donated blood is provided for transfusion. Likewise, certain symptoms have a number of common causes that may be simultaneously screened for using arrays. For example, urinary tract infections are common and may be caused by a large number of different bacteria of varying sensitivity to various antibiotics. The simultaneous testing for numerous factors would save considerable time and expense.

[0015] In the prior art, biochemical molecules on microarrays have been synthesized directly at or on a particular cell on the microarray. Alternatively, preformed molecules have been attached to particular cells of the microarray by chemical coupling, adsorption or other means.

[0016] Regulatory approval of clinical tests and systems and methods for making them is required. When chips are fabricated using photolithography and other technology derived from electronic chip making, as has been done in the prior art, the cost of forming individual chips is extraordinarily high. In addition, the possibility of error when chips are individually made is very high. Since chips are individually made and used only once, quality control is difficult and there is no good way of proving that any given chip is satisfactory. The best that can be done is to test a large fraction of a batch at random.

[0017] In a recent development, fibers containing test materials are formed and used to make sectioned arrays. Immobilized enzymes have been prepared in fiber form from an emulsion as disclosed 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 have been used and are well known in the fields of solid phase imrnunoassays, nucleic acid hybridization assays and immobilized enzymes; see, for example, Hermanson, Greg, 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. Morgansen & E. Conldin, Pierce Chemical Company, Rockford IL, 1992, 467 pp.

[0018] In the prior art, a fiber is typically formed of a polymerizable material injected or inserted into a plastic tubing to form an encased fiber. In order to obtain a relatively dense array of a small physical size, a tubing may have an inner diameter as small as about 200 microns.

[0019] In the prior art, the fiber is not removed from the tubing. This has several drawbacks.

[0020] Due to the inclusion of the tubing and the fiber in the sectioned array, the size of the sectioned array may be larger than desired. Alternatively, the number of test sites may have to be reduced, h addition, the presence of the tubing surrounding the fiber may reduce contact between the binding agents and the fiber. Likewise, the sectioned array also suffers from a reduced surface area with which to interact with a test sample.

[0021] What is needed therefore are improvements in making fibers for sectioned arrays.

SUMMARY OF THE INVENTION

[0022] A first method for making a fiber, especially but not necessarily a fiber adapted for use in a sectioned array, is provided according to the invention. The method comprises a step of supplying a composition into a mold, the mold being of any desired shape, e.g., having a cross- section which is round, square or oval, and can be tubing. The composition is one which will solidify in the mold. The composition may be a polymerizable composition which polymerizes within the mold or it may be a composition which is liquid and then solidifies upon cooling, e.g., a wax. The composition may also be a polymer which is not yet solid but becomes a solid in the presence of a complexing agent such as a calcium, phosphate or other ion which can be diffused into the polymer solution within the mold or tubing. Examples of such polymers are alginates and other gels and gums. The method further comprises a step of allowing the composition to solidify (this term encompassing the formation of a solid via a polymerization step, a cooling step, a complexing step, etc.) and form the fiber. The method further comprises a step of placing a predetermined elongation force onto an end of the fiber, the predetermined elongation force causing an elongation and reduction in diameter of the fiber and causing a separation of the fiber from an interior surface of the mold or tubing. The method further comprises a step of substantially maintaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing. The method may optionally include a step of slitting the mold or tubing.

[0023] A second method for making a fiber adapted for use in a sectioned array is provided according to the invention. The method comprises a step of supplying a composition into a mold or tubing, wherein the composition is one which will solidify in the mold or tubing. The method further comprises a step of allowing the composition to solidify and fonn the fiber. The method further comprises a step of exposing an end of the fiber. The method further comprises a step of placing a predetermined elongation force onto the end of the fiber, the predetermined elongation force causing an elongation and reduction in diameter of the fiber and causing a separation of the fiber from an interior surface of the mold or tubing. The method further comprises an optional step of slitting the mold or tubing. The method further comprises a step of substantially maintaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing.

[0024] A third method for making a fiber adapted for use in a sectioned array is provided according to the invention. The method comprises a step of supplying a composition which can solidify into a mold or tubing using a plunger apparatus communicating with the mold or tubing. The method further comprises a step of allowing the composition to solidify and form the fiber in the mold or tubing and to solidify in the plunger apparatus. The method further comprises a step of placing a predetermined elongation force onto an end of the fiber using the plunger apparatus, the predetermined elongation force causing an elongation and reduction in diameter of the fiber and causing a separation of the fiber from an interior surface of the mold or tubing. The method further comprises a step of substantially mamtaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing. The method may optionally include a step of slitting the mold or tubing.

[0025] A fourth method for making a fiber and a sectioned array is provided according to the invention. The method comprises the steps of supplying a composition which can solidify into a mold or tubing; allowing the composition to solidify and form the fiber, and placing a predetermined elongation force onto an end of the fiber. The predetermined elongation force causes an elongation and reduction in diameter of the fiber and causes a separation of the fiber from an interior surface of the mold or tubing. The method further comprises a step of substantially maintaining the predetermined elongation force to propagate the separation through the mold or tubing until the fiber is completely separated from the interior surface of the mold or tubing. The method further comprises the steps of substantially aligning a plurality of fibers into a bundle, affixing fibers of the bundle, sectioning the bundle, and affixing a section piece to a substrate to form the sectioned array.

[0026] An apparatus for making a fiber adapted for use in a sectioned array is provided according to the invention. The apparatus comprises a knife block having a passage therethrough of a size to accommodate a tubing having a fiber therein. The apparatus further comprises at least one knife adjustably held in the knife block and positionable so that the at least one knife slits the tubing when the tubing is fed through the knife block. The apparatus further comprises a tensioning device capable of gripping the fiber and placing a predetermined elongation force on the fiber. The predetermined elongation force propagates an elongation and reduction in diameter of the fiber in the tubing and causes a separation of the fiber from an interior surface of the tubing. The predetermined elongation force also pulls the tubing and the fiber through the knife block, where the tubing is slit.

[0027] The above and other features and advantages of the present invention will be further understood from the following description of the preferred embodiments thereof, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows a tubing containing a fiber formed therein;

[0029] FIG. 2 shows a cut-away portion of the tubing that has been cut away to expose the end of the fiber;

[0030] FIG. 3 shows the fiber removal according to the present invention;

[0031] FIG. 4 shows a plunger apparatus such as a commonly available syringe; [0032] FIG. 5 shows an apparatus for forming an essentially continuous fiber; [0033] FIG. 6 shows detail of one knife block half;

[0034] FIG. 7 shows detail of one knife block half with one or more knives in place; and [0035] FIG. 8 shows a cut depth adjuster.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] The terms "binding component", "molecule of interest", "agent of interest", "ligand" or "receptor" 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 cell, site, or element of the array, and binds to an analyte being detected. Therefore, the location of an element or cell containing a particular binding component determines what analyte will be bound. Proteins, polypeptides, peptides, nucleic acids (oligonucleotides and polynucleotides), antibodies, ligands, polysaccharides, microorganisms, receptors, antibiotics, test compounds (particularly those produced by combinatorial chemistry), bacteria, viruses, or plant and animal cells and organelles or fractions of each may each be a binding component if immobilized in an element of a microarray. Each of the substances above may also be considered as analytes if they bind to a binding component on a chip.

[0037] When a molecule of interest has a high molecular weight, it is referred to as a "macromolecule". In terms of some biopolymers, the high molecular weight refers to greater than 100 amino acids or nucleotides or sugar molecules long.

[0038] The term "bind" includes any physical attachment or close association, which may be permanent or temporary. Generally, an interaction of hydrogen bonding, hydrophobic interactions, van der Waals forces, 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. This 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 invention.

[0039] The term "fiber" refers to a filament. A filament or rod may be a solid strand of monolithic, porous, or composite forms, or aggregate forms. Pluralities, typically a large number, of fibers are bound adjacent to each other in ribbons or bundles to form a "fiber bundle." The cross- section of the fibers may be of any shape, such as round, triangular, square, rectangular or polygonal. A bundle may be sectioned to produce a section piece containing a plurality of thin elements. The section piece may be bonded to a substrate to form an array (or a sectioned array or microarray). The term array is used here to denote both an array and a microarray, as they differ only in size. The term sectioned array is also used to denote how the array is formed by the use of a sectioned piece of a fiber bundle.

[0040] The term "particle" includes a large number of insoluble materials of any configuration, including spherical, thread-like, brush-like and many irregular shapes. Particles may be porous with regular or random channels inside them. Examples include silica, cellulose, Sepharose beads, polystyrene (solid, porous and derivitized) beads, controlled-pore glass, gel beads, sols, biological cells, viruses, subcellular particles, etc. Even certain high molecular weight materials, such as polymers and complexes, may serve as immobilizing structures that would constitute a "particle".

[0041] According to the invention, a plurality of fibers may be produced, with each containing a different agent of interest. The fiber may contain suspended binding agents or it may be coated or impregnated with the binding agents. A cut end of the fibers may be briefly treated with dilute solvents to expose active groups. In addition, a fiber may incorporate binding agents according to combinations of the above. The fibers and binding agents may be used to detect agents of interest in an analyte, with each fiber being capable of detecting a different agent of interest.

[0042] The agent of interest (i.e., a substance to be detected), may comprise a very broad range of chemicals, complexes, tissues, biological cells or fractions thereof. Nucleic acids or proteins, which may have been modified or are coated with detergents to make them more soluble in organic solvents, and a wide range of organic compounds, can be incorporated into mixtures which will solidify, e.g., 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. In the present invention, an agent of interest is extracted into an organic solvent which is miscible with either a thermosetting plastic mixture, or one which is polymerized chemically or by UN or ionizing radiation. This may be done by coating the agents with detergents or other reagents, which will make them soluble under the conditions chosen. The solvent may be miscible in the gelling material or it may be extractable or volatile so as to render a porous final product. Porous products are particularly preferred with solid filament fibers that are self-supporting.

[0043] Large numbers of different and potentially new active compounds may be simultaneously screened by immobilizing them in fibers, bundling, sectioning and forming a microarray. Peak fractions from separations, such as plant extracts, may be simultaneously collected and used to form a microarray. The microarrays may then 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.

[0044] Each fiber used to construct a sectioned array 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 fiber in some circumstances. Likewise, when fractionating a mixture, forming a fiber with a mixture of receptors may be acceptable, as total and complete isolation is difficult and time consuming.

[0045] It is equally a part of the present invention to immobilize microorganisms or other molecules of interest and use them to localize antibodies from a patient's sera, and then discover the location of the latter using a fluorescent anti-human antibody, thus diagnosing a disease which elicited antibody production in the first place.

[0046] When the immobilized macromolecules are antibodies, the microarray may be used to diagnose a variety of protein-based anomalies. A labeled second antibody to the protein of interest may be used to further highlight the cell. In addition, the array may be used to immobilize infectious agents which have been either previously stained or which are stained after immobilization. Thus, microbes from biological samples, e.g. serum or plasma, may be concentrated, stained with a fluorescent nucleic acid stain such as TOTO-1 or YOPRO-1, and then allowed to find their matching antibodies on the array. They may then be detected by scanning for fluorescence and identified by position.

[0047] Particularly preferred to be screened are the 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 a single fiber. Two dimensional electrophoresis gels from serum and other tissue and natural sources produce tens of thousands of different proteins separated on the gel. Each may be individually removed (e.g. cut, eluted, etc.) from the gel and used as the molecule of interest to form a single micro-fiber. In such a method, with different bundles being formed from different samples, protein differences between different samples may be readily compared.

[0048] The agent of interest may comprise a very broad range of chemicals, complexes, biological cells or fractions thereof. Nucleic acids, many proteins, proteins which have been modified or are coated with detergents such as sodium dodecyl sulfate are soluble in organic solvents, and thus can be incorporated into mixtures to be solidified, e.g., polymerizing mixtures such as those used to produce plastics. Hence it is technically feasible to produce long fibers of acrylic or other plastics each containing a different agent of interest using currently available extrusion technology for practice in the present invention.

[0049] Arrays have numerous uses other than determining bioactive properties. Chemical interactions and reactions may be tested as well, such as an array of different reactive chemicals being tested against a test substance or material to determine corrosion, electrochemical reaction, or other interaction. This 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.

[0050] FIG. 1 shows a tubing 100 containing a fiber 101 formed therein. The fiber 101 may comprise any composition which can solidify which contains binding agents. The tubing 100 may be, for example, polyethylene, polypropylene, or fluorocarbon (generic for TEFLON). The tubing

100 therefore functions as a mold into which a composition which can solidify may be inserted and allowed to solidify. In a solidified state, the composition has gelled or hardened, but remains elastic. In the past, removing such a fiber 101 from a tubing 100 presented a great problem in that the fiber

101 may be somewhat adhered to the inside surface of the tubing 100 and may be damaged or destroyed during removal.

[0051] Examples of compositions which can solidify include, but are not limited to, polymeric gels, coagulated materials and an IMMUNOBED™ (polymethacrylate, polymethylmethacrylate or polyglycolmethacrylate) material, available from Polysciences, Warrington, Pennsylvania. A number of polymerizing embedding agents have been developed for histological and histochemical studies, some of which are Durcupan, Nanoplast, Quetol 651, London Resin Gold, Lowicryl K4M Polar, Lowicryl Monostep, K4M Polar, Lowicryl Kl 1 Polar, JB-4, JB-4 Plus, IMMUNOBED™, and PolyFreeze, together with data on their composition, curing temperature, solvent used and viscosity. Polymers such as agarose, gelatin, collagen, xanthene, carrageenan, alginate, or a thermosetting, thermoplastic, chemosetting or UN polymerizing polymer may be used. Νon-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. These may be further modified by using thickeners, gums, hardening and crosslinking agents, plasticizers and various combinations of gelling materials. In general, the gelling material should be sufficiently inert to not interfere with an interaction between the binding component and an analyte. [0052] Immobilization may be accomplished by a number of techniques, known per se, such as entrapment in a matrix or chemically coupling, perhaps through a linking moiety through an amino, hydroxy, sulfhydryl or carboxyl moiety. Chemically attaching the chemical to a monomer to be solidified, e.g., polymerized, also effectively incorporates the component. Binding may also 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, or through a ligand that has a receptor such as digoxigenin-antidigoxigenin.

[0053] The binding between an analyte and the binding agents of the fiber 101 may be enhanced by etching the embedding matrix of each fiber, thereby exposing more of the surface area of particles in each fiber of the microarray.

[0054] In addition to methods by which a receptor or molecule of interest is immobilized and used to bind an analyte, general methods exist for arranging immobilized members of a class of reactants. For example, protein A or protein G may be immobilized and used to subsequently 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 covalently linked. This allows the production of surfaces to which a very wide variety of reactants can be readily and quickly attached (see Savage et al., Avidin-Biotin Chemistry: A Handbook, Pierce Chemical Company, 1992).

[0055] The immobilized binding components, e.g. nucleic acids, proteins, cells, etc., may be contained in a gel in the tubing 100, may be coated on the inside of the tubing 100, may be attached to structural elements embedded within a gel in the tubing 100, or may be coated on or impregnated in the fiber 101 after it is formed.

[0056] The fibers may contain embedded beads that are capable of attaching to agents or analytes of interest. The beads may be porous gel beads used in chromatography such as Sephadex, Biogels and others, or solid beads such as are used in chromatography. A variety of methods for derivitizing these and for attaching proteins, nucleic acids and polysaccharides and small molecules thereto have been developed and are well known to those skilled in the art.

[0057] Different dyes (fluorescent or non-fluorescent) may be incorporated into individual fibers, allowing their location in the two-dimensional array to be confirmed.

[0058] The fibers or their gelling material may also contain a dye or other optical absorber so that only analyte/binding components on the surface of each cell are visualized. Such an improvement reduces the effects of diffusion rates through a gel or porous material that may change with temperature, time, type of carrier liquid, etc. A dye that absorbs UN or emitted fluorescence will reduce fluorescence from non-surface analyte/binding component reactions.

[0059] The embedding matrix for the fibers may be black, opaque or otherwise adsorbent to emitted signals from a label in order to reduce cross talk between the cells in the chip. Additionally, any adhesive between the fibers may contain the same adsorbent material to reduce background between cells of the microarray. Optionally, a specific layer of this material may be placed between the fibers before they form the bundle.

[0060] In one embodiment, the composition which can solidify may be a reversible polymerizable material that is capable of later being liquefied. Therefore, a microarray containing such a reversible polymer element may be used with a test sample. Then the reversible polymer element may be liquefied and stored or further analyzed. In some applications this may be more feasible than reading the microarray or removing the element from the microarray for analysis.

[0061] As an alternative to a homogeneous fiber, a fiber may be prepared which incorporates polystyrene latex or other plastic particles (i.e., beads) onto which proteins or nucleic acids may be attached. The supporting plastic may be eroded to a depth of a few microns to reveal active subparticle surfaces, but without dissolving the supporting plastic latex beads. For example, proteins derivatized with fluorinated groups attach strongly to fluorocarbon microp articles. Such derivatized fluorocarbon particles in an acrylic plastic or other suitable embedding medium, for example, can be partially exposed at the plastic surface by a dilute acrylic solvent. The solvent may be composed of methylene chloride and ethyl alcohol, for example. 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.).

[0062] In another alternative non-homogeneous embodiment, the solidifying, polymerizing or gelling materials may also contain solid structural elements such as filaments, branched elements, etc., to further reinforce or strengthen the gel. Therefore, the fiber 101 may include embedded fibers, strings, threads, particles, objects, etc., to increase the strength of the fiber 101. The structural elements may also provide attachment sites for the agent of interest. Thus 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, etc. Threads or yarn-like and brush-like configurations of structural elements, etc., may be cast into the length of the fiber giving it strength and allowing the fiber to be more easily handled or dried. The structural elements may serve as the immobilizing component in the fiber for a desired binding component. A fiber 101 may therefore be formed with a substantially central string or thread incorporated therein to increase strength and to make the fibers 101 easier to handle.

[0063] Binding agents may be attached to the fibers after they are formed. For attachment of the ligands, the rods may be soaked h tubes containing the substance to be attached or the rods may be coiled up inside a hollow bowl centrifuge rotor having the general configuration of a zonal rotor (see Anderson, N.G., Natl. Cancer Inst. Monograph No. 21), but which may be centrifugally drained. The solution of the substance to be attached may be centrifuged into the mass and then out of it, followed by washing as necessary. The fibers 101 may then be dried.

[0064] In another alternative embodiment, a fiber 101 may include a second phase. This second phase may be in the form of, for example, hydrocarbon, aqueous or fluorocarbon microdroplets, particles of sugars or other water soluble materials, or inorganic particles such as calcium carbonate particles, which can be dissolved in dilute acids to reveal active groups. Brief exposure of the cut surface of a chip to a solvent will dissolve some of these inclusions, increasing the surface area of the support plastic containing the agents of interest.

[0065] The fibers may be identified by tags on the end of the fiber or by tags on the rolls carrying the fibers, and/or by incorporating different dyes in them. A barcode may also be printed directly near the end of fibers. Thermoplastic polymers may be used when the embedded product is sufficiently thermostable. Some of the fibers may be differently colored to assist in the localization of specific ligands in the array or to identify the array itself.

[0066] A wide variety of methods have been developed to detect reactions between immobilized molecules of interest and soluble reactants. These differ chiefly in the mechanism employed to produce a signal, and in the number of different reagents which must be sandwiched together directly or indirectly to produce that signal. These 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 whose fluorescence greatly increases after binding an analyte. The latter are chiefly used to detect nucleic acids. In more complex systems, including so-called sandwich assays, the end 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. Alternatively, the detection complex attached to the bound analyte may include a dendritic molecule, including branching DNA, to which are attached many fluorescent dye molecules.

[0067] Most immunochemical or competition assays depend on a signal produced by a reagent other than the analyte. However, methods for fluorescently labeling all proteins containing aliphatic amino groups in a complex mixture have been developed which are reproducible and quantitative. Of these, CyDyes supplied by Amersham Life Sciences, and particularly, Cy2, Cy3 and Cy5 have proven most useful. When the components of such labeled mixtures are reacted with an array of immobilized antibodies, with each being a specific antibody to one of the fluorescently labeled analytes, the presence of each of the specifically bound labeled analytes can be detected by fluorescence. This method can be further improved by exposing the bound antibody array to a solution containing known subsaturating quantities of each protein in a non-fluorescent form, washing the bound antibody array, and exposing it to a test mixture of labeled proteins, thus producing a multiple competition assay.

[0068] Not only can the chips of the present invention be used to identify infectious agents by identifying characteristic nucleic acid sequences, they can also be used for identifying intact bacteria, mycoplasmas, yeast, nanobacteria, and viruses using arrays of immobilized specific antibodies.

[0069] This system may be used for the identification of viruses or other infectious particles isolated by microbanding tubes, as described in WO99/46047. Thus, microbes from biological samples, e.g. serum or plasma, may be concentrated, stained with a fluorescent nucleic acid stain such as TOTO-1 or YOPRO-1, and then allowed to find their matching antibodies on the array. They may then be detected by scanning for fluorescence and identified by position. It is equally a part of the present invention to immobilize microorganisms or other molecules of interest in the described chips, to use them to localize antibodies from a patient's serum, and to then discover the location of the latter using a fluorescent anti-human antibody, thus diagnosing the disease which elicited antibody production.

[0070] FIG. 2 shows a cut-away portion 203 of the tubing 100 that has been cut away to expose the end of the fiber 101. The end of the fiber 101 may therefore be grasped in some manner.

[0071] FIG. 3 shows the fiber removal according to the present invention. A tensioning device 308, such as a clamp or jaws, for example, maybe used to grasp the end of the fiber 101 and place a predetermined elongation force onto it. The predetermined elongation force may be supplied by hand or by an appropriate mechanical device. The predetermined elongation force causes the fiber 101 to elongate and therefore reduce in diameter, leaving a separation region 305 around the elongated fiber portion. By mamtaining the predetermined elongation force on the fiber 101, the elongation and separation may be propagated through the tubing 100, causing the fiber 101 to eventually break completely free of the tubing 100. However, the predetermined elongation force must be substantially maintained until the elongation and separation propagates the full length of the tubing, at which time the fiber 101 may be removed from the tubing 100.

[0072] In a first step of a first method embodiment of forming a fiber according to the invention, the composition which can solidify is supplied into the tubing 100. This may be done, for example, by injecting with a plunger apparatus or by placing a vacuum or partial vacuum on one end of the tubing 100 and placing the other end in communication with the composition in order to draw it into the tubing 100. The tubing 100 may have any desired inner diameter.

[0073] In a second step, the composition is allowed to solidify inside the tubing 100 in order to form the fiber 101.

[0074] In a third step, a predetermined elongation force is placed on the fiber 101 in order to cause the elongation and reduction in diameter that separates the fiber 101 from the tubing 100. The predetermined elongation force may be supplied by hand or supplied by an appropriate mechanical device. The method may optionally include a step of slitting the tubing 100.

[0075] In a fourth step, the predetermined elongation force is substantially maintained until the elongation and separation is propagated throughout the tubing 100. Fibers thus formed may be used to create sectioned arrays of an advantageous size and with a simple and economical method of manufacture.

[0076] In a first step of a second method embodiment of forming a fiber according to the invention, the composition to be solidified is supplied into the tubing 100.

[0077] In a second step, the composition is allowed to solidify inside the tubing 100 to form the fiber 101.

[0078] In a third step, a portion of the tubing 100 is cut away to expose an end of the fiber 101. This is done to allow the end to be grasped in some manner.

[0079] In a fourth step, a predetermined elongation force is placed on the fiber in order to cause the fiber 101 to elongate and separate from the inner surface of the tubing 100. The predetermined elongation force may be supplied by hand or supplied by an appropriate mechanical device.

[0080] In an optional fifth step, the tubing 100 is slit in the separation region 305 (the region where the fiber 101 has separated from the tubing 100). This may be done in order to more easily remove the tubing 100 from around the fiber 101.

[0081] In a sixth step,, the predetermined elongation force is maintained on the fiber 101 in order to propagate the elongation and separation throughout the entire length of the tubing 100. This, of course, may be done by a machine or apparatus that continuously places a force on the fiber 101 to perform a continuous propagation of the elongation and separation of the fiber 101. In addition, the machine or device may continuously slit the tubing 100 in a region following the elongation and separation in order to remove the tubing 100. The tubing 100 may be slit in one or more circumferential positions on the tubing 100, preferably in two substantially opposite circumferential positions.

[0082] In a first step of a third method embodiment of forming a fiber according to the invention, the composition to be solidified is supplied into the tubing 100 using a plunger apparatus 400 (see FIG. 4). The composition may be supplied into the tubing 100 by injecting or by sucking or aspirating the composition into the tubing 100. The plunger apparatus 400 may be, for example, a syringe that is inserted into one end of the tubing 100 and that forces the composition into the tubing 100 or draws it in by aspiration, in which case the distal end of the tubing 100 is inserted into the composition. The plunger apparatus 400 may be suitable for forming non-continuous fibers 101.

[0083] h a second step, the composition is allowed to solidify inside the tubing 100 to form the fiber 101.

[0084] In a third step, the predetermined elongation force is placed on the fiber 101 using the plunger apparatus 400. This assumes that the plunger apparatus 400 is placed in and maintained in communication with the end of the tubing 100 while the composition solidifies. Therefore, the fiber 101 is formed not only in the tubing 100 but continuously into the plunger apparatus 400. The plunger apparatus 400 may therefore be grasped and used to place the predetermined elongation force on the fiber 101 (making it unnecessary to cut away a portion of the tubing 100 for the purpose of grasping the fiber 101). The method may optionally include a step of slitting the tubing 100.

[0085] In a fourth step, the predetermined elongation force is maintained in order to propagate the elongation and separation of the fiber 101 throughout the tubing 100.

[0086] In any of the method embodiments according to the invention, the fibers thus formed may be used to create one or more sectioned arrays. A plurality of fibers may be substantially aligned into a bundle and then the fibers may be affixed. The present invention makes microarrays or "chips" by sectioning assembled bundles of fibers containing immobilized binding components. The fibers may include, for example, biological molecules and entities such as nucleic acid fragments, antigens antibodies, proteins, peptides, carbohydrates, ligands, receptors, drug targets, biological cells or their subfractions, infectious agents or subtractions of them, drugs, toxic agents, or natural products. In addition, the fibers according to the invention may be composed of two different types of material in coaxial formation. [0087] There are two basic options for making two-dimensional arrays from these fibers.

The first is to make and evaluate ribbons, and then to form a set of ribbons into a long rectangular bar, while the second is to make the bar at the outset. The former option may be more advantageous, since the ribbons can be individually evaluated before being formed into a complete array.

[0088] An advantage of the invention is that very large numbers of arrays may be cut, and some fraction of them may be retained and used for standardization. For example, if a bar 100 cm in length were constructed, and if the bar were cut at 100 micron intervals, then 10,000 section pieces would be available. If the section pieces are 10 microns in thickness, then the number of section pieces would be 100,000.

[0089] If the individual fibers are 100 microns in diameter, and if there are 100 fibers per ribbon, there will be 10,000 fibers in a bar having a cross-sectional area of 1 cm square. If there are 330 fibers per ribbon, then the total number of fibers in a bar is 108,900, approximately the number of expressed genes postulated to be present in the human genome.

[0090] The present invention is the first array to have such a large number of different cells per unit area on a microarray without the binding agent being covalently attached to- the chip. It is preferred for the present invention to have at least 100, more preferably 250, 500, 1,000, 5,000, 10,000, 100,000 or a million or more cells per square centimeter of array. These are much higher concentrations than depositable cells formed by microfluidics in commercial microarrays.

[0091] To greatly increase the number of cells per square centimeter beyond even these high numbers, one may prepare a large fiber bundle with relatively large fibers and stretch or draw the bundle. While this makes the individual fibers thinner, it does not affect their basic composition or their orientation with respect to each other and cross-section geometry. This technique has the twin advantages of allowing one to make more microarrays and making them smaller. By using conventional 5 micron porous particles and a plastic embedding medium such as a low melting point wax, the result are deformable or ductile fibers which may be drawn to very thin fibers of less than 20 microns in diameter. The field of drawing thermoplastic materials is well known per se. Even if not truly drawable through a die, one can pull or extrude plastic materials between rollers to lengthen and reduce the diameter of the fibers. With the optional application of gentle heat, one need only pull the ends of the fiber bundle to generate the same lengthening and reducing of cross- sectional area. With smaller, porous particles, the fibers may be drawn to even thinner dimensions, thereby permitting microarrays of up to at least about 10 billion cells per square centimeter of microarray. [0092] The bundle may be affixed in a number of ways, hi a first method, the fibers are affixed by casting or embedding the bundle in a hardenable material. A variety of histological embedding media have been developed which preserve nucleic acids and antigens in a reactive form. These include, among others, Durcupan, Nanoplast, Quetrol 651 which may be cured by very mild heating, JB-4, IMMUNOBED™ which may be polymerized at room temperature, and the water soluble acrylic polymers London Resin Gold and Lowicryl which are polymerized 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.

[0093] The embedding material or adhesive used to hold the tubes in a bundled configuration may be opaque, while the tubes and preferably their contents will conduct light along their length. As a final check on the orientation of array elements, one element at a time at one end of the bundle may be illuminated, and the light detected and related to array position at the other end.

[0094] In a second method of affixing the fibers of a bundle, the fibers are affixed by bonding the fibers with an adhesive. A number of adhesives are known, including cyanoacrylate adhesives. The space between the fibers may be completely filled by adhesive or a monomer which is polymerized. Thermoplastic and gelling materials may also constitute the adhesive by causing a large number of fibers to be held together in a block.

[0095] In a third method of affixing the fibers of a bundle, the fibers are affixed by heating the bundle until the fibers soften and bond. Arrays of parallel fibers may be bonded together by many techniques, such as by the introduction of a heated solvent vapor . The vapor is allowed to interact with the array for a specified period of time, and is then removed by re-evacuation. Alternatively, in heat sintering, the bundle of fibers is placed under lateral compression and heated to the softening point of the fiber material. Bonding may further be accomplished by the use of low melting point metals, such as gallium, in an embedding matrix. The matrix may be heated and cooled in order to embed the fibers. By low melting point is meant temperatures at or about physiologic temperature of the binding component. Alternatively, the fibers may be bonded through non-chemical means, such as by passing an electrical current through the fibers to fuse them.

[0096] In a fourth method of affixing the fibers of a bundle, the bundle may be encased. This may include wrapping the bundle in a wrapper or placing the plurality of fibers in a tubing.

[0097] After the fibers are affixed, the bundle may be sectioned. The bundle may be cut transversely or at an angle into many thin disks and portions are optionally dissolved if desired. Microtomes and other sectioning or cutting instruments capable of cutting assembled bundles of tubes into thin sections, and of maintaining their orientation after sectioning, are known, hi general, blade cutting is preferred to sawing to reduce contamination of binding components between cells of the microarray. Microtomes for sectioning soft tissues in wax are commercially available, as are a variety of techniques and arrangements for attaching sections to glass or plastic slides, for treating them automatically to remove some or all of the embedding media, and for systematically exposing the slides to a series of reagents.

[0098] A section may be used to complete a sectioned array by bonding or affixing a section to a substrate. The sections (as microarray chips) may be attached directly to adhesive surfaces on flexible films or on solid surfaces, such as glass slides. It is also feasible to attach sections (the word "section" is used here in place of "chip") at intervals along a film strip, with others interleaved between them. Thus a set of about a dozen or more different sections may be placed in repeating order along the film, and the film then cut up to give one set. For sequencing studies, one DNA insert sample may be amplified, labeled, and its hybridization to a large set of sections examined.

[0099] With the present invention, a very large number of sections can be made from one composite assembly, and adjacent sections intercompared as well as those some distance apart. Statistical analyses will be able to predict the rate of errors that may occur. However, of even greater importance is the fact that since the sections can be made in large numbers and quite cheaply, it will be feasible to run duplicate analysis on clinical samples, and to run confirmatory analysis when important diagnostic results are obtained. The present invention therefore makes feasible widespread and routine application of genetic analyses hi the practice of medicine.

[0100] Essentially the same fiber may be used multiple times in the same microarray. This provides an internal quality control check and improves confidence in the binding assay. This also provides additional quantitative measurements if such an assay is performed to improve precision. Blank fibers with no molecule of interest bound thereto may be used to provide a good negative control, and should be used in every microarray.

[0101] Arrays may have two or more identical cells made from different fibers but containing identical binding agents. This provides an internal quality assurance check for the array. Additionally, it is preferred for some of the cells to provide different concentrations of the binding component for quantitative measurement of an analyte. These provide internal standards for the microarray for both qualitative detection and quantitative detection. For example, a series of cells may contain different concentrations of an antibiotic in their gels. When a sample microorganism is contacted with the cells and allowed to incubate, the absence of growth in one cell and the presence of growth in another cell provide an approximate minimal inhibitory concentration. The same can be done for determining mimmal bacteriocidal concentrations when stained with a vital dye such as trypan blue or fruorescein acetate. Since a microarray may contain thousands of cells, one can simultaneously 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.

[0102] In the course of using a chip of the instant invention, various known techniques and materials are used to reduce non-specific reaction. Thus, in the case of a protein-based assay, the non-specific sites on the chip contributed by the substance of the fiber or filament, the embedding material, and essentially everything aside from the binding component of interest are reacted with a blocking agent, such as albumin or milk, so that the blocking agent will bind to those areas not containing the binding component which could react with a ligand, analyte, reporter molecule or whatever would specifically bind to the binding component, as known in the art.

[0103] Since channels are reproducible between plates, the location of each channel or cell may be accurately determined by mechanical means. Reference markings on polished edges or other suitable locations may further identify each cell in the array. Current commercially available computer driven two-dimensional drives of sufficient accuracy are commercially available so that each cell may individually be visualized or tested, or material may be added thereto or withdrawn therefrom.

[0104] By using the present invention, one avoids the difficulties of individually depositing a different reagent on each cell on a solid phase or synthesizing a different compound at each cell. The former technique is limited by both the possibilities of spilling and mixing reagents, and by limitations in the accuracy of measurement of small fluid volumes. The latter technique is limited by the types of different compounds that can be synthesized on a solid phase surface. Both prior art techniques are expensive and require elaborate automated equipment or tedious labor to produce each array individually. By contrast, the present invention for producing microarrays is technically simple and quick, and the batch size may be in the thousands. The only individual effort required for each microarray is the step of cutting.

[0105] FIG. 4 shows a plunger apparatus 400 such as a commonly available syringe 400.

The syringe 400 includes a chamber 402 and a plunger 404. The syringe may optionally include a needle (not shown) that may be capable of fitting closely into the tubing 100. The chamber 402 may be filled with the composition to be solidified and the plunger apparatus 400 may be placed in communication with the tubing 100. The plunger 404 may be depressed, forcing the composition into the tubing 100. Alternatively, the opposite end of the tubing 100 can be inserted into a solution of the composition, which can be drawn into the tubing 100 and chamber 402 by aspiration caused by withdrawing the plunger 404. The plunger apparatus 400 may be left in communication with the tubing 100 until the material has solidified.

[0106] hi one embodiment, the plunger apparatus 400 may comprise a 16 gauge needle attached to a 3 cc (cubic centimeter) disposable syringe. The needle may be inserted into a length of polyethylene tubing 100 having a 1.5 millimeter internal diameter, for example. A pre-polymer solution may be prepared by mixing 25 parts of catalyzed IMMUNOBED™ Solution A with 1 part of IMMUNOBED™ Solution B (both available from Polysciences, Warrington, PA). The non- polymerized IMMUNOBED™ material may then be injected or aspirated into the tubing 100.

[0107] FIG. 5 shows an apparatus^' 500 for forming an essentially continuous fiber 101. A supply reel 501 includes a length of tubing 100 containing a fiber 101 therein. The supply reel 501 feeds into a knife block 523 , which is used to separate the fiber 101 from the tubing 100 and to slit the tubing 100. After passing through the knife block 523, the tubing 100 has been split into pieces 100' (preferably two pieces) which pass over the tensioning rollers 530 and onto the pick up reels 534.

[0108] The tubing 100 and encased fiber 101 may alternatively be fed into the knife block from a different source, such as, for example, a process or machinery for inserting or injecting the composition to be solidified into the tubing 100.

[0109] Before reaching the knife block 523, the tubing 100 passes over a first feed device 503 that includes a first roller 508 and a pair of second rollers 511. The first feed device 503 may be adjusted and moved in order to feed the tubing from the supply reel 501. The movement is performed by a screw device 515 which may include a motor 516 that can position the first feed device 503 (vertically, in the embodiment shown). A first pair of stationary rollers 519 maintains a constant feed height of the tubing into the knife block 523. The tubing passes through a hole in the knife block 523. The tubing 100 and encased fiber 101 are pulled through the knife block 523 by a tension applied to the finished fiber 101' and a tension applied to the split tubing pieces 100'. The pulling in a preferred embodiment is achieved mainly through tension applied to the finished fiber lo , although various tensions may be applied to either the finished fiber 101' or the split tubing pieces 100'.

[0110] After splitting in the knife block 523, the finished fiber 101' passes through a second pair of stationary rollers 537 and into a second feed device 539, which feeds the finished fiber 101' onto the take-up reel 560. [0111] The second feed device 539, like the first feed device 503, includes a first set of rollers 542 and a second roller 546. The second feed device 539 is moved by a screw device 551 and motor 552 that may be used to feed the finished fiber 101' onto the take-up reel 560 (vertically, in the embodiment shown).

[0112] The rollers 537 and 542 may be formed of fluorocarbon or may be fluorocarbon coated in order to prevent the finished fiber 101' from adhering to the rollers.

[0113] The knife block 523 may be comprised of two joined, symmetric knife block halves 523'. The two knife block halves 523' may be joined by fasteners 526 (such as by screws, bolts, etc.) and the adjustment knob or crank 527 may be used to control the slitting operation inside the assembled knife block 523, as will be explained below in conjunction with FIGS. 6-8.

[0114] The separation process may be started by hand, such as by cutting the tubing 100 and exposing an end of the fiber 101 before feeding it into the apparatus 500. Alternatively, the plunger apparatus 400 may be used to start the separation process. However, it should be understood that other starting procedures may be employed in order to initiate the separation process performed using the apparatus 500.

[0115] The apparatus 500 may be under the control of a computer (not shown) in order to control the feeding, splitting, and take-up operations. The computer may additionally perform tensioning of the various reels and rollers.

[0116] It should be understood that the finished fiber 101' produced by the methods and apparatus according to the invention may be sectioned and formed into sectioned arrays or microarrays. These sectioned arrays may be advantageously smaller and denser than a sectioned array formed according to the prior art (sectioning and using a fiber 101 still inside the tubing 100).

[0117] FIG. 6 shows detail of one knife block half 523'. The knife block half 523' includes a groove 604 of a predetermined size approximately equal to the outside diameter of the tubing 100.

The groove 604 may include bevels 605 at the ends to aid in the entry and exit of the tubing 100.

The tubing 100 is, therefore, fed through the hole made by the grooves 604 in the two knife block halves 523'.

[0118] The knife block half 523' also includes one or more milled cutouts 608. The one or more cutouts 608 accommodate one or more knife blades 714 used for the shtting operation, as will be shown in FIG. 7. The knife block half 523' also includes one or more recesses 612 and holes 616 for one or more cut-depth adjusters 527 (discussed in conjunction with FIG. 8). Holes 620 accommodate fasteners, such as bolts or screws, that are used to join the two knife block halves 523*. [0119] FIG. 7 shows detail of one knife block half 523' with one or more knives 714 in place. A knife 714 may be, for example, a single-edge razor blade. The at least one knife 714 has an edge 715 that may be positioned so that it protrudes into the groove 604. The amount of protrusion into the groove 604 will determine the depth of the cut into the tubing 100. It should be noted that the at least one knife 714 should preferably not cut into the fiber 101, but should only cut through the tubing 100. The at least one knife 714 , therefore, may be positioned so that when the at least one cut depth adjuster 527 is in place in the at least one cut depth adjuster recess 612, the at least one cut depth adjuster 527 can control how far into the groove 604 the at least one knife 714 protrudes, and can therefore control the cut depth.

[0120] FIG. 8 shows the cut depth adjuster 527. As illustrated in FIG. 5, the crank or knob portion 819 and the plate 816 are positioned on the outside of the assembled knife block 523. The shaft 805 fits into the hole 616 in the recess 612 of a knife block half 523', while the offset lobe 810 rests in the recess 612. When the knob or crank 819 is rotated, the offset lobe 810 displaces the knife 714, thereby adjusting how far the cutting edge 715 of the knife 714 protrudes into the groove 604.

[0121] The fiber produced according to the various methods and apparatus of the invention may then be grouped into a bundle and sectioned or cut into thin slices. The bundle may be substantially rectangular in cross-section, may be substantially circular, substantially ovoid, or even irregular. The slices may be bonded to substrates in order to form sectioned arrays or microarrays. The substrate may be a flexible film or a solid surface, such as glass, for example. The substrate may be transparent so that the sectioned array may be illuminated by light.

[0122] Various methods of array manufacturing currently exist. In one method, a plurality of fibers are created. The fibers are impregnated or coated with a variety of binding agents that bind to the molecules or macromolecules sought to be detected. The resulting fibers are grouped into a bundle and fastened together. Very thin slices of the bundle are sectioned (cut off) and glued or bonded to a substrate in order to form an array. The array and substrate are commonly referred to as a chip. The completed chip may be exposed to a test sample and then read, such as by an apparatus having a light source and a light detector that reads the pattern of light emitted or transmitted from the chip. The resulting light pattern will be modulated by the molecules or macromolecules that have bonded to the binding agents on the chip. The resulting light pattern will indicate the presence or absence of certain molecules or macromolecules.

[0123] For general clinical use it is important to have identifiers on the slide holding the chip, and identifiers may be integral with the chip itself. This may include a barcode printed along an edge or border of a chip in order to provide identification and orientation. In addition, small concentrations of dyes, usually non-fluorescent, may be incorporated into selected fibers to identify them or to present a pattern. It may also be useful to incorporate fluorescent dyes into selected cells or elements, and which serve to calibrate the fluorescence measurements.

[0124] Dyed fibers would be visible in such arrays to confirm identifications and orientation. In addition, the fibers can be dyed in such a manner that a visible pattern is formed if the array is correctly made, and the pattern may include a name or a number.

[0125] Additional fibers or ribbons may be added to the bundle as needed before sectioning additional arrays. Tins allows one to detect and measure newly discovered emerging diseases, new proteins, genes or compounds without recreating a completely new bundle.

[0126] This invention may be applied in an alternative fashion in which the bundles are stored at user sites, and the arrays only sliced off as needed. This 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.

[0127] Another alternative to slicing the bundle and using its sections as separate microarrays is to perform the assay with the end of the bundle directly. After the assay is performed wherein a first sample could be applied to the cut cross-sectional surface, and washed off, a detector could image the result. One may then mount the bundle in a microtome device, if the bundle were not already so mounted before the assay. A blade could then remove the used surface of the bundle, exposing a fresh surface for the next assay, which would repeat the same steps again. The bundle could thus be used in one machine for a series of up to 100,000 or more assays performed one after another. This arrangement has certain advantages as optical or electrical detection may be performed through the bundle itself with fiber optic fibers or conductive fibers. The detection system may be continuously attached to the bundle while a more general light or electrical energy is applied to the end being used for testing.

[0128] The invention allows different immobilization technologies, different classes of immobilized agents of interest, different classes of analytes, and different types of detection methodologies to be employed on a single chip.

[0129] In a first example, a fiber is produced according to the invention with the fiber containing non-covalently entrapped biological target molecules. In this example, an element of a microarray is formed by mixing a biological target molecule with a substance that can be subsequently removed from the element of the microarray, allowing the biological target molecule to then react with a component of a surface with which it is in contact, to provide a stable linkage between the biological target molecule and the surface. More specifically, a protein can be used for this purpose that has a recognition site for another molecule, such as biotin, which can be bound by streptavidin. This biotinylated-protein can be mixed with a reversible gelling system, such as, but not limited to, agarose. Fibers can be formed in which the elements are comprised of the reversible gel containing a biological target molecule. Once formed into an array, thin sections can be prepared and mounted on a surface that contains immobilized recognition factors, such as in this case streptavidin. The gel can then be dissolved or removed by any means to expose the biological target molecule, which is then free to diffuse and react with the immobilized recognition factor. This has the advantage of eliminating the support polymer as a barrier to reactants and can serve to increase the processing time for analyte detection. The recognition system can be comprised of many types of interactions, such as, but not limited to, antigen-antibody, lectin-carbohydrate, and, in general, any of the well-known ligand-receptor systems. Reversible gels can be comprised of, but are not limited to, heat-reversible agar or agarose systems, metal-dependent alginate systems, redox-dependent disulfide-containing polymeric systems, e.g., polymers formed by oxidation of sulfhydryl groups to disulfides that can be reduced back to free sulfhydryl groups. In addition, the support matrix may be one that can be degraded by any means to liberate the entrapped biological target molecule. The degradation process can consist of, but would not be limited to acid or base hydrolysis, enzymatic hydrolysis, photo degradation, temperature change such as with thermal responsive polymers which are solid or liquid, depending on the temperature, and other processors known in the art.

[0130] In a second example, a fiber is produced according to the invention with the fiber containing biological target molecules incorporated by diffusion after polymerization of the fibers. In this example, an element of a microarray is formed by incorporating a biological target molecule into the fiber by diffusion from a solution containing the biological target molecule. This example provides a means of incorporating labile biological target molecules into polymeric matrices in those cases where the conditions of polymerization, such as heat, presence of free radicals, etc., are capable of inactivating the biological target molecule. A fiber is prepared from a material such as, but not Umited to, IMMUNOBED™. The fiber is placed in a solution containing a biological target molecule of interest. The fiber is allowed to remain in contact with the solution for a period of time, at a temperature that is consistent with maintenance of biological activity, generally from, but not restricted to, 4°C to 40°C. The period of time of contact between the fiber and the solution for optimal incorporation of the biological target molecule will depend on many factors, including porosity of the fiber, molecular size of the biological target molecule, concentration of the biological target molecule, temperature, etc. After the fiber is loaded with biological target molecule it is washed with a buffer and incorporated into an array, as previously described.

[0131] In a third example, a fiber is produced according to the invention with the fiber containing biological target molecules incorporated by diffusion and entrapment after polymerization of the fibers. In this example, a biological target molecule is incorporated into a fiber by diffusion from a solution containing the biological target molecule. This example provides a means of incorporating labile biological target molecules into polymeric matrices in those cases where the conditions of polymerization, such as heat, presence of free radicals, etc., are capable of inactivating the biological target molecule. It also provides a means for entrapping the biological target molecule within the polymeric fiber to prevent the subsequent diffusion of the biological target molecule out of the fiber. A polymeric fiber of material is prepared from a material such as, but not limited to, IMMUNOBED™. The mixture of monomeric substances is mixed with an entrapping agent, such as, but not limited to, bovine serum albumin-biotin complex. The mixture is formed into a fiber, and the fiber is placed in a solution containing a biological target molecule of interest. In this case the biological target molecule of interest will be conjugated to a biotin- binding protein such as streptavidin. Other binding pairs, which are known in the art, can also be used for this purpose. The fiber is allowed to remain in contact with the solution for a period of time, at a temperature that is consistent with maintenance of biological activity, generally from, but not restricted to, 4°C to 40°C. The period of time of contact between the fiber and the solution for optimal incorporation of the biological target molecule into the fiber will depend on many factors, including porosity of the fiber, molecular size of the biological target molecule, concentration of the biological target molecule, temperature, etc. After the fiber is loaded with biological target molecule it is washed with a buffer and incorporated into an array, as previously described.

[0132] While the invention has been described in detail above, the invention is not intended to be limited to the specific embodiments as described. It is evident that those skilled in the art may now make numerous uses and modification's of and departures from the specific embodiments described herein without departing from the inventive concepts.

Claims

WHAT IS CLAIMED IS:

1. A method for removing an elastic temporarily deformable fiber from a mold, comprising the steps of: supplying a composition into a mold; allowing said composition to solidify in said mold and form said fiber; placing a predetermined elongation force onto an end of said fiber, said predetermined elongation force causing an elongation and reduction in cross-section of said fiber and causing a separation of said fiber from an interior surface of said mold; and substantially maintaining said predetermined elongation force to propagate said separation through said mold until said fiber is completely separated from said interior surface of said mold.

2. The method of claim 1 wherein said composition is a polymerizable composition.

3. The method of claim 1, wherein said supplying and placing steps are performed by a plunger apparatus communicating with said mold.

4. The method of claim 1, wherein said composition includes a binding agent.

5. The method of claim 1, wherein said composition includes non-polymer reinforcing elements.

6. The method of claim 1 , wherein said composition is a reversible polymerizable composition.

7. The method of claim 1 wherein said mold is tubing.

8. The method of claim 7, wherein said mold comprises fluorocarbon tubing.

9. The method of claim 7, wherein said mold comprises polyethylene tubing.

10. The method of claim 7, wherein said tubing comprises polypropylene tubing.

11. The method of claim 1 , wherein said composition comprises a gelling material.

12. The method of claim 1, wherein said composition comprises a coagulating material.

13. The method of claim 1, wherein said composition comprises polymethacrylate, polymethylmethacrylate or polyglycolmethacrylate.

14. The method of claim 1, further comprising the step of slitting said mold.

15. • The method of claim 1, further comprising the step of slitting said mold in at least two substantially opposite circumferential positions.

16. The method of claim 1, further comprising the step of cutting said end of said fiber to expose said end, said cutting occurring prior to placing said predetermined force onto said fiber.

17. The method of claim 1, wherein said composition is supplied into said mold by injecting said composition into said mold.

18. The method of claim 1, wherein said composition is supplied into said tubing by aspirating said composition into said tubing under a vacuum.

19. The method of claim 16 wherein said aspirating occurs by capillary action.

20. A method for making a fiber, comprising the steps of: supplying a composition into a mold; allowing said composition to solidify in said mold and form said fiber; exposing an end of said fiber; placing a predetermined elongation force onto said end of said fiber, said predetermined elongation force causing an elongation and reduction in cross-section of said fiber and causing a separation of said fiber from an interior surface of said mold; slitting said mold; and substantially maintaining said predetermined elongation force to propagate said separation through said mold until said fiber is completely separated from said interior surface of said mold.

21. The method of claim 20 wherein said composition is a polymerizable composition.

22. The method of claim 20, wherein said composition includes a binding agent.

23. The method of claim 20, wherein said composition includes non-polymer reinforcing elements.

24. The method of claim 20, wherein said composition is a reversible polymerizable composition.

25. The method of claim 20, wherein said mold is tubing.

26. The method of claim 25, wherein said tubing comprises fluorocarbon tubing.

27. The method of claim 25, wherein said tubing comprises polyethylene tubing.

28. The method of claim 25, wherein said tubing comprises polypropylene tubing.

29. The method of claim 20, wherein said composition comprises a gelling material.

30. The method of claim 20, wherein said composition comprises a coagulating material.

31. The method of claim 20, wherein said composition comprises polymethacrylate, polymethylmethacrylate or polyglycolmethacrylate.

32. The method of claim 20, wherein two substantially opposite sides of said mold are slit.

33. The method of claim 20, wherein said slitting is performed by feeding said mold and said fiber through a knife block containing at least one knife.

34. The method of claim 20, wherein said slitting is performed in a region of said mold where said fiber has separated from said mold.

35. The method of claim 20, wherein said composition is supplied mto said mold by inj ecting said composition into said mold.

36. The method of claim 20, wherein said composition is supplied into said mold by aspirating said composition into said mold under a vacuum.

37. The method of claim 36 wherein said aspirating is by capillary action.

38. A method for making a fiber, comprising the steps of: supplying a composition into a mold using a plunger apparatus communicating with said mold; allowing said composition to solidify and form said fiber in said mold and to solidify in said plunger apparatus; placing a predetermined elongation force onto an end of said fiber using said plunger apparatus, said predetermined elongation force causing an elongation and reduction in cross- section of said fiber and causing a separation of said fiber from an interior surface of said mold; and substantially maintaining said predetermined elongation force to propagate said separation through said mold until said fiber is completely separated from said interior surface of said mold.

39. The method of claim 38 wherein said composition is a polymerizable composition.

40. The method of claim 38, wherein said composition includes a binding agent.

41. The method of claim 38, wherein said composition includes non-polymer reinforcing elements.

42. The method of claim 38, wherein said composition is a reversible polymerizable composition.

43. The method of claim 38 wherein said mold is tubing.

44. The method of claim 43, wherein said tubing comprises fluorocarbon tubing.

45. The method of claim 43, wherein said tubing comprises polyethylene tubing.

46. The method of claim 43, wherein said tubing comprises polypropylene tubing.

47. The method of claim 38, wherein said composition comprises a gelling material.

48. The method of claim 38, wherein said composition comprises a coagulating material.

49. The method of claim 38, wherein said composition comprises polymethacrylate, polymethylmethacrylate or polyglycolmethacrylate.

50. The method of claim 38, further comprising the step of slitting said mold.

51. The method of claim 38, further comprising the step of slitting said mold in at least two substantially opposite circumferential positions.

52. The method of claim 38, wherein said composition is supplied into said mold by injecting said composition into said mold.

53. The method of claim 38, wherein said composition is supplied into said mold by asphating said composition into said mold under a vacuum.

54. The method of claim 53 wherein said aspirating is by capillary action.

55. A method for making a fiber and a sectioned array, comprising the steps of: supplying a composition into a mold; allowing said composition to solidify in said mold and form said fiber; placing a predetermined elongation force onto an end of said fiber, said predetermined elongation force causing an elongation and reduction in cross-section of said fiber and causing a separation of said fiber from an interior surface of said mold; substantially maintaining said predetermined elongation force to propagate said separation through said mold until said fiber is completely separated from said interior surface of said mold; substantially aligning a plurality of fibers into a bundle; affixing fibers of said bundle; sectioning said bundle; and affixing a section piece to a substrate to form said sectioned array.

56. The method of claim 55 wherein said composition is a polymerizable composition.

57. The method of claim 55, wherein said supplying and placing steps are performed by a plunger apparatus communicating with said mold.

58. The method of claim 55, further comprising the step of slitting said mold.

59. The method of claim 55, wherein said affixing comprises casting said bundle in a hardenable material.

60. The method of claim 55, wherein said affixing comprises bonding said fibers with an adhesive.

61. The method of claim 55, wherein said affixing comprises heating said bundle until said fibers bond.

62. The method of claim 55, wherein said affixing comprises encasing said bundle.

63. The method of claim 55, wherein said composition is supplied into said mold by injecting said composition into said mold.

64. The method of claim 55, wherein said composition is supplied into said mold by aspirating said composition into said mold under a vacuum.

65. The method of claim 64 wherein said aspiration occurs by capillary action.

66. A sectioned array produced by the method of claim 55.

67. A fiber made by the method of claim 20.

68. A bundle of fibers comprising fibers of claim 67.

69. A fiber made by the method of claim 38.

70. A bundle of fibers comprising fibers of claim 69.

71. An apparatus for making a fiber, comprising: a knife block having a passage therethrough of a size to accommodate a tubing having a fiber therein; at least one knife adjustably held in said knife block and positionable so that said at least one knife slits said tubing when said tubing is fed through said knife block; and a tensioning device capable of gripping said fiber and placing a predetermined elongation force on said fiber; wherein said predetermined elongation force propagates an elongation and reduction in cross-section of said fiber in said tubing and causes a separation of said fiber from an interior surface of said tubing, and said predetermined elongation force also pulls said tubing and said fiber through said knife block, where said tubing is slit.

72. The apparatus of claim 71, further comprising one or more rollers that guide said fiber after said separation.

73. The apparatus of claim 71 , further comprising one or more fluorocarbon-coated rollers that guide said fiber after said separation.

74. The apparatus of claim 71, wherein said tensioning device comprises a pair of jaws capable of gripping and pulling said fiber.

75. The apparatus of claim 71, wherein said tubing and said fiber are fed into said knife block from a supply reel.

76. The apparatus of claim 71, wherein said tensioning device comprises a take-up reel.

77. The apparatus of claim 71, wherein said fiber is accumulated on a take-up reel.

78. The apparatus of claim 71, wherein slit tubing is accumulated on at least one pick-up reel.

79. The apparatus of claim 71, wherein said knife block further comprises a pair of knife block halves capable of being fastened together, with each knife block half including a portion of said passage and at least one knife cutout to accommodate said at least one knife.

80. The apparatus of claim 71, wherein a cut depth of said at least one knife is adjustable.

81. The apparatus of claim 71 , wherein said knife block includes at least one rotatable cut depth adjuster including an offset lobe, and said at least one rotatable cut depth adjuster adjusts a cut depth when rotated in contact with said at least one knife.

82. The apparatus of claim 71, wherein said at least one knife comprises a single-edge razor blade.

83. The apparatus of claim 71, wherein said knife block includes two knives positioned to slit substantially opposite sides of said tubing.