Micro-array and its manufacturing method

Description: TECHNICAL FIELD [Detailed description of the invention]

This application is a partial continuation application of patent application No. 60 / 146,653 filed on July 30, 1999, and a partial continuation application of patent application No. 09 / 482,460 filed on January 13, 2000. is there. The entire contents of these patents are cited herein.

【0001】

[Technical field to which the invention belongs]

The present invention relates to a microarray containing a bioreactive molecule, its use, and a method for producing the same. This array can be produced by slicing a bundle of thin tubes or rods each containing a unique reactant to produce a large number of identical arrays.

【0002】

[Conventional technology]

Microarrays are essentially two-dimensional supports or sheets, with different biomolecules or components such as nucleotides, polynucleotides, peptides, polypeptides, sugars or polysaccharides in different parts or cells of the support or sheet. Is bonded and supported. Microarrays are in principle the same as other solid phase arrays, except that many assays can be performed in parallel by performing assays using microarrays on a small scale. Microarrays have been used for many analytical purposes in conventional biological sciences.

【0003】

In conventional techniques, biochemical molecules on a microarray are synthesized directly on a microarray or on a specific cell (sector) of the microarray, or preformed molecules are chemically bonded or by other means to a specific cell (sector) of the microarray. ) Is attached. The number of different types of cells (sectors) and associated different types of biochemical molecules tested simultaneously on one or more microarrays can be in the thousands. A commercially available microarray plate reader is usually used to measure the fluorescence of each cell (sector) to obtain data on thousands of reactions at the same time, thereby saving time and labor. A representative example of a patent in this area is US Pat. No. 5,545,531.

【0004】

Currently, two-dimensional arrays of macromolecules are subjected to photoactive reactions or other synthetic reactions by attaching a small amount of aliquots to a flat surface under conditions where macromolecules are attached or bonded to the surface. It is made by using it to allow macromolecules to be synthesized on the surface. As an example of the method so far, the production of an array by using a printing technique can be mentioned. Some examples of array fabrication methods are "Gene-Expression Micro Arrays: A NewTool for Genomics" by Shalon, D., Functional Genomics; Drug Discovery from Gene to Screen, IBC Library Series, Gilbert, SR & Savage, LM, International Business Communications, Inc., Southborough, Massachusetts, pp. 2.3.1.-2.3.8; "DNA Probe Arrays: Accessing Genetic Diversity", Lipshutz , RJ, Gilbert, SR & Savage, LM, same as above, 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, PR, Gilbert , SR & Savage, LM, same as above; Jordan, BR, "Large-Scale" expression measurement by hybridization methods: from high-densities to "DNA chips", J. Biochem. (Tokyo) 124: 251-8, 1998; by Hacia, JG, Brody, LC & Collins, FS, "Applications of DNA chips for" "Genomic analysis", Mol. Psychiatry 3: 483-92, 1998; and Southern, EM, "DNA chips: Analyzing sequence by hybridization to oligonucleotides on a large Scale", Trends in Genetics 12: 110-5, 1996. It is published in.

【0005】

With any of these techniques, each microarray is created individually and separately, and usually cannot be pre-calibrated for numerical values with only one use. Therefore, it depends on the reproducibility of the manufacturing system for producing an error-free array. Due to these factors, the biochips or microarrays currently manufactured are expensive, and this technology has not yet been used in daily clinical practice.

【0006】

A charge-coupled device (CCD) camera can be used to scan the array. The prices of these devices have been steadily declining, and purpose-built cameras and software are now widely available. This device generally detects a light source or absorbance. In one proposed application, the array is placed at the other end of a fiber optic bundle bearing a nucleic acid or antibody / antigen at one end. This makes it possible to detect fluorescence via an optical fiber. See U.S. Pat. No. 5,837,196.

【0007】

A fiber optical array can be made by aligning glass or plastic fibers in parallel so that optical images can be transmitted through the array while holding all the fibers in parallel. Parallel arrays can also be made of hollow glass fiber, which can be sliced perpendicular to the axis of the fiber to make channel plates used for the amplification of optical images. Such devices are used as night vision and other optical signal amplification equipment. The channel plate is made by slicing a bundle of channel plates, immobilizing separate proteins or nucleic acids separated for each group of holes, and then adapting them so that the binding reaction with each filled hole can be detected. Yes (US Pat. No. 5,843,767).

【0008】

Hollow porous fibers are used for dialysis of biological samples such as kidney dialysis machines and water purifiers. Methods have been described for aligning fibers in parallel arrays, impregnating fibers containing plastic with a constant amount, and cutting the ends of the array (see, eg, US Pat. No. 4,289,623). .. For example, as disclosed in Italian Patent No. 836,462, the immobilized enzyme is formed from the emulsion in fiber form. Antibodies and antigens are incorporated within solid phase fibers as disclosed in US Pat. No. 4,031,201. Various other immobilization techniques are well known in the fields of solid phase immunoassays, nucleic acid hybridization assays and immobilized enzymes. For example, Hermanson, GT, "Bioconjugate Techniques" Academic Press, New York, 1995, p. 785; Hermanson, GT, Mallia, AK and Smith, PK, "Immobilized Affinity Ligand Techniques", Academic Press Press, New York, 1992, p. 454; and "Avidin-Biotin Chemistry: A Handbook", by D. Savage, G. Mattson, S. Desai, G. Nielander, S. Morgansen and E. Conklin, Pierce Chemical, Rockford, Illinois. See Company, 1992, p. 467.

【0009】

Currently available biochips contain only one type of immobilized reactant and only one type of reaction can be performed. For many types of clinical and other analyses, a chip that can embed various reactants in one chip is required.

【0010】

[Means for solving problems]

In the present invention, rods or thin tubes in which different types of target biological substances are enclosed or adhered are prepared, and the rods or thin tubes are arranged and held in a parallel bundle state. Then, the bundle is optionally impregnated with a machinable adhesive material or the bundle is embedded with a machinable adhesive material, and after impregnation, all the components of the bundle are in the longitudinal direction thereof. Arbitrarily confirm that a constant composition or pattern is maintained throughout, and slice this into thin slices to produce a large number of the same array or chips. Various biochemical quantitative analyzes are performed on each array or chip based on, for example, enzymatic action, immunological action, nucleic acid hybridization and small molecular binding under conditions where fluorescence, absorbance or chemiluminescence signals can be obtained, and this signal is obtained. The present invention relates to a method for obtaining images of the above and electronically processing and comparing them to obtain clinically and experimentally useful data.

【0011】

In one aspect, the present invention relates to a long filament or tube comprising the subject substance, coated with the subject substance, or containing the subject substance, and a method for producing the same. The present invention also relates to a method for constructing fibers as a bundle so that the position of each fiber with respect to all other fibers does not change throughout its longitudinal direction.

【0012】

The present invention further relates to means and methods of mounting or adhering all fibers to each other over their entire length. In a related aspect, the present invention is the preparation of a microarray, the fabrication of a microarray, thus the fabrication of a microarray by binding elongated filaments or tubes together and cutting them many times in a direction across them at short intervals to obtain cross-sectional slices thereof. Regarding microarrays.

【0013】

Another aspect of the invention is to include a label either integrated with a tube or fiber or contained in a medium contained within the hollow fiber so that the fiber can be identified throughout its longitudinal direction. Is to be. Further, the aspect of the present invention also includes means for illuminating each fiber at one end of the bundle and identifying the other end by photoelectric means to confirm the maintainability of the fiber configuration.

【0014】

In another aspect, the invention relates to a means of forming a fiber containing a subject material or immobilizing one or one type of subject material to the fiber. In yet another embodiment, the invention is a means of embedding or mounting a whole or fragment of a biological cell, tissue or infectious agent in a fiber or capillary so that the organism is exposed on the cutting end of each fiber or capillary. Regarding.

【0015】

In another aspect of the invention, the array consists of a capillary containing a gel or other polymeric material that adheres to the capillary wall. In another aspect of the invention, the subject matter is attached to a polymerization medium or suspension medium contained in the lumen of a thin tube. In yet another aspect of the invention, the subject matter is attached to the particles suspended in the polymerization medium. The tubules are filled with this suspension and the tubules are used to make array bundles and arrays.

【0016】

The present invention further presents the sequencing of nucleic acids, the analysis of complex mixtures of ribonucleic acids (RNA) and deoxyribonucleic acids (DNA), the detection of other analytes such as proteins, polysaccharides, organic polymers and low molecular weight analysts, and nucleic acids. The present invention relates to a method for mass-producing the same flat two-dimensional array of immobilized nucleic acid-based substances used for quantification of the above-mentioned materials by slicing a long bundle of fibers or tubes containing the above-mentioned materials.

【0017】

In a related embodiment, the present invention relates to the use of microarrays for mass screening of a large number of samples from one type to many types of target substances. In another aspect of the invention, a quality control assay can be performed on each of the manufactured fibers and the fiber bundle can consist only of fully functional fibers. In another related embodiment, the invention relates to the development of test sets on different chips or microarrays that are selectively branched in a procedure, thereby costing, delaying and inconvenience in diagnosing human disease. It is possible to provide complex data obtained by a series of conventional tests, which is usually time consuming, while alleviating the problem.

【0018】

In yet another embodiment, the invention allows the same array to be mounted and compared on a slide and / or test strip for quality control purposes by producing the same array at a sufficiently low cost. Regarding doing so. In yet another embodiment, the present invention incorporates a non-fluorescent dye or other light absorbing material into the material of the array to control the depth at which the light that excites fluorescence penetrates the array. It relates to adjusting the detectable depth to prevent detection of fluorescent analytes that diffuse too deeply into the cell content and do not diffuse light.

【0019】

In another aspect, the invention relates to a method of identifying a capillary tube that is completely filled with a support medium and does not form pores or bubbles. In another aspect, the invention relates to methods and devices that utilize hydrostatic or centrifugal force to completely fill a thin tube with a support medium. In another aspect, the present invention relates to the remanufacturable production of biochips or microarrays for bioanalysis.

【0020】

In another aspect, the invention relates to the design and fabrication of arrays specifically designed to detect and diagnose a particular disease. In yet another aspect, the present invention relates to a multiwell plate and a method for producing the same. In yet another embodiment, the present invention provides means for continuously measuring fluorescence or absorbance over time and specifying the rate of change in fluorescence or absorbance in each element of the array over time. On expanding the dynamic range of parallel assays.

【0021】

Another aspect of the invention is an inexpensive, well-standardized biochip that allows the use of one or more pieces for performing each analysis and comparative and reference tests in parallel on a daily basis. To manufacture. Sections from separate parts or ends of the bundle may be used to further ensure quality. One method of slicing into separate parts of the bundle is to cut or fold the bundle in half and align the two to form one thick bundle, which produces a section in which each fiber appears twice. ..

【0022】

In another embodiment, the present invention presents a chip in which the array elements or cells (sectors) in the tube, support medium, composition in the immobilized surface are different from each other, or the type of the target substance is different if the cells (sectors) are different. Regarding the production of. In another aspect, the invention relates to the fabrication of chips capable of performing different types of reactions, such as immune, enzymatic or hybridization reactions, on the surface of each cell (sector) of the array.

【0023】

Yet another aspect of the present invention relates to the fabrication of fiber or thin tube sub-arrays that adhere to each other to form a single-sided ribbon array that can be stored separately. Quality control analysis may be performed on this "ribbon" before combining it into a two-dimensional array. Different one-dimensional arrays can be used to combine different arrays to provide the option of producing custom-made arrays that meet specific research and clinical requirements.

【0024】

The present invention further relates to a method based on a plurality of parallel chips, which comprises continuously raising the temperature so that a temperature sensitive reaction occurs at a physiological temperature, and then raising the temperature so that a hybridization reaction occurs. Regarding development. In yet another aspect, the invention relates to preparing a library of compounds in which each fiber contains one of them. All of the compounds can be screened simultaneously using this array for a particular chemical or biological effect.

【0025】

The terms "binding component", "target molecule", "target substance", "ligand" or "receptor" may be any of a number of heterologous molecules, biological cells or aggregates thereof, and these terms are interchanged as synonymous. Can be used. Each binding component is immobilized on a cell, sector, site or element of the array and bound to the analyte to be detected. Therefore, the position of the element or cell containing the unique binding component determines the analyte to be bound. Produced by microorganisms such as proteins, polypeptides, peptides, nucleic acids (nucleotides, oligonucleotides and polynucleotides), antibodies, ligands, sugars, polysaccharides, bacteria, fungi and viruses, receptors, antibiotics, test compounds (especially in combination chemistry) Any of these, plant and animal cells, organelles, or their fractions, and other biological components can be binding components if immobilized on the chip. Once they bind to the binding components on the chip, they may in turn be considered as analytes.

【0026】

When the target molecule has a high molecular weight, this is called a "polymer". In the case of biopolymers, high molecular weight means exceeding the length of 100 amino acids, nucleotides or sugar molecules. The term "bonding" includes any physical or chemical adsorption or close association, whether permanent or temporary. In general, hydrogen bond interactions, hydrophobic forces, van der Waals forces, covalent bonds, ionic bonds, and the like allow physical adsorption between the molecule of interest and the object being measured. "Binding" interactions can be short, such as when binding causes a chemical change. This is common when the binding component is an enzyme and the analyte is an enzyme substrate. The reaction that occurs when the binding substance and the analyte come into contact with each other is also within the definition of binding in the present invention.

【0027】

The terms "cell", "sector", "part" or "element" used in the present application generally refer to a unit structure of an array identified by a unique address whose contents and positions are different from those of other cells, sectors, parts or elements. Refers to an element. Living cells are commonly referred to as types such as microorganisms, animals and plant cells. The term "fiber" includes both filaments and hollow capillaries. The filament or rod may be a solid filament in the form of a monosilic, porous or composite material, or an aggregate thereof. A "fiber bundle" is formed by joining a plurality of, usually a large number of fibers, as ribbons or bundles adjacent to each other. The "fiber bundle" may form part of the actual bundle used, such as a ribbon. The cross-sectional shape of this fiber may be any of a circle, a triangle, a quadrangle, a rectangle, a polygon, and the like.

【0028】

The term "particle" includes a large number of insoluble materials, the shape of which may be spherical, needle-like, brush-like or many irregular shapes. The particles are often porous with regular or random internal channels. Examples include silica, cellulose, Sepharose beads, polystyrene (solid, porous and derivative) beads, controlled porous glass, gel beads, sol, living cells, intracellular particles, microorganisms (protozoa, bacteria, yeast). , Virus, etc.) Micelle, liposome, cyclodextrin, two-phase system (agarose beads in wax, etc.) and other structures that enclose or enclose the material. Recombinant hosts and viruses that express the protein of interest are particularly suitable. Certain high molecular weight materials such as polymers and complexes may also act as immobilized structures that make up the "particles".

【0029】

The term "sintering" refers to bonding the surface of a fiber without actually melting the entire fiber. This may be done either chemically or thermally, and an activating autoadhesive component may be used. The terms "array" and "microarray" are used interchangeably to some extent, except that they differ in overall dimensions. The present invention relates to the same method for producing and using both of them. Each array usually contains a large number of cells (usually 100-1,000,000 or more), and each cell is in a well-known location and contains a target-specific component. Therefore, each array contains a large number of heterogeneous components of interest.

【0030】

In the present invention, nucleic acid fragments, nucleotides, antigens, antibodies, proteins, peptides, carbohydrates, ligands, receptors, drug targets, biological cells or subfractions thereof (crushed cells, organelles, solvent extracts, etc.), pathogens or Microarrays, "chips" or by slicing bundles of small plastic rods, fibers, tubes, or tubules containing immobilized binding components containing biomolecules and components such as their subfractions, drugs, toxins or natural products. Make a "biochip". The embedding medium in the present invention can be polymerized or solidified in a small tube, or can be molded into a rod or sheet.

【0031】

The tube can be made of materials such as glass, metal, ceramic or plastic. Immobilized binding components such as nucleic acids, proteins, cells, etc. are coated inside or outside the microtube, contained in a gel inside the microtube, adsorbed on small particles or beads, or in it. It can be embedded and used to fill the tube. The particles or beads may be components of the gelling material or separated components such as latex beads made from various synthetic plastics (such as polystyrene). If each fiber is a solid rod or filament, the material of interest is incorporated onto or inside the plastic before the filament is molded, extruded or drawn from the mold. Each section cut constitutes a microarray used for various binding assays.

【0032】

An important aspect of the invention is that it provides economic advantages, i.e., fibers or tubules are prepared using only methods that provide stable functionality for long-term storage. Unlike other methods that require a protein-containing solution that must be freshly prepared each time, fixed proteins in a relatively dry form are stable over a very long period of time and often do not require freezing.

【0033】

Since each component of the produced microarray can be prepared separately in the fiber / on the surface, each component can be quantified and the functionality or reactivity of each component can be evaluated before being incorporated into the array. Neither spotting techniques nor in situ synthesis techniques can be tested prior to array fabrication, and quality control inspections can only sample a small portion of a microarray. This is different from the present invention in which each fiber can be tested.

【0034】

Various aspects of the present invention are illustrated in FIGS. 1 to 7. The general principle is shown in Fig. 1. The target substance is incorporated in the rod or tube 1. The rods or tubes can be joined as flat, parallel arrays 2 and then a plurality of flat arrays can be joined as a plurality of parallel bundles 3. Alternatively, the bundle 3 may be made from a series of rods 1 in one step. Cutting or slicing the ends of the bundle 3 gives the final array 4 containing a thin section 5 of each rod or tube contained throughout the bundle. By making a long bundle 3 and cutting an ultrathin section 4, the same array or chip can be made in large quantities. For example, if the bundle 3 has a length of 1 m and a region thickness of 10 microns, it is possible to make 100,000 identical chips.

【0035】

In the case of hollow glass fiber such as a channel plate, the hollow fiber is filled with a gel or particles containing an immobilized reactant and the entire bundle is cut into an array. The rods or tubules that make up the bundles that are sliced in this way can be classified into at least eight types, each of which can be further subdivided.

【0036】

The first type is a solid rod or filament in which the immobilized binding component is part of the composition of the rod or filament. The subject matter in the present invention may include a very wide range of chemicals, complexes, tissues, biological cells or fractions thereof. Nucleic acids, sugars and proteins that can be modified or coated with surfactants to increase solubility in organic solvents and a wide range of organic compounds can be incorporated into polymerization mixtures used in the production of plastics and the like. Since the oligonucleotide and nucleic acid dissolve in methylene chloride, they can be contained in an acrylic resin, for example, during polymerization.

【0037】

Numerous polymerization implants have been developed for histological and histochemical studies. Some examples are listed in Table 1 with data on composition, curing temperature, solvent used and viscosity.

【0038】

[table 1]

<img file="JP2003014751A_D0001.tif" />Another example of a method of saturating a solid fiber is to use superpower to assemble the target material by a matrix of solid fibers. The second type of fiber is not homogeneous and the polymerization or gelling material can contain solid structural elements such as filaments, branching elements to further strengthen the gel and provide adsorption sites for the target material. Therefore, the added component acts to strengthen the gel and can provide an adsorption site for inclusions such as dendrimer branched polynucleic acid, branched or crosslinked polymer material, metal or glass fiber. Structural elements in the form of threads, yarns and brushes may be formed into fibers to increase their strength and make the fibers easier to handle or dry. This structural element may act as an immobilization component in the fiber for the desired binding component.

【0039】

Therefore, it is technically feasible to utilize the extrusion processing techniques currently available in the present invention to produce long fibers made of acrylic or other plastics, each containing a different substance. Further, the cut end portion of the fiber can be treated with a diluting solvent for a short time to expose the active group. A third type of fiber includes extruded or molded plastics, which includes a second phase. This second phase is inorganic, for example, hydrocarbons, aqueous or fluorocarbon microdroplets, particles of sugar or other aqueous material, or calcium carbonate particles that can be dissolved in dilute acids to allow active groups to emerge. It may be in the form of particles. Short-term exposure of the cutting surface of the chip to a solvent can dissolve some of this inclusion and increase the surface area of the supporting plastic containing the material of interest.

【0040】

Solid plastics incorporating polystyrene latex or other plastic particles that adsorb proteins or nucleic acids can also be prepared. The supporting plastic can be eroded to a depth of a few microns to reveal the surface of the active subparticles, but the supporting plastic latex beads can be conditioned so that they do not dissolve. For example, proteins derived with fluorinated groups adhere strongly to Teflon® microparticles. For example, such induced Teflon® particles contained in an acrylic resin plastic or other suitable embedding medium are partially exposed to the plastic surface with a dilute acrylic solvent such as methylene chloride and ethyl alcohol. be able to. Alternatively, the particles may be embedded in a porous matrix.

【0041】

The beads that adsorb the target substance may be porous gel beads such as Sephadex, Biogels, etc. used for chromatography, or solid beads such as examples used for chromatography. Various types of methods for inducing support structures and adsorbing polypeptides, proteins, nucleic acids, polynucleotides, sugars, polysaccharides and small molecules to them have been developed so far, and these are well known to those skilled in the art. .. In the structure of the tubules shown in FIG. 2, the tubule 6 consists of a tube 7 containing a gel 8 that supports the particles 9. As shown in the end face view 10 and the enlarged view 11 of this thin tube, the particles 12 are exposed at the cut end portion. In 14 which is a further enlargement of region 13, it is shown that there is a reactant 15 immobilized on the surface of the exposed particles 12.

【0042】

It should be noted that by placing a string or thread in the center of all of the listed rods and molding them together, the strength of the rod can be increased and its handling can be facilitated. A fourth type of fiber is made by sintering glass or plastic beads to form a porous material with a high surface ratio to mass. These materials have traditionally been made from glass, polytetrafluoroethylene (PTFE) (Teflon®), Teflon® AF, polyethylene, polypropylene, from polystyrene, and from a variety of other plastics. It is possible. Plastics can be sintered by exposure to heat, pressure or solvent vapor. The material thus sintered can be guided to the sheet or cutting rod. Polystyrene is preferable from the viewpoint of binding the target substance there. Regarding the induction of polystyrene, a method of adsorbing a protein by its amino group, carboxyl group or sulfhydryl group has been described so far. A solution of metallic sodium in an organic solvent that produces a group to bond other plastics can be used to activate Teflon®, which can be induced. Activation of polyethylene and polystyrene can be performed by corona plasma discharge or electron beam radiation. Producing polystyrene and polyethylene sintered composites is an advantageous technique. It is also possible to sinter and induce nylon beads. There are other sintered materials that are already well known or are currently under development, and it is considered that many of them can be applied to the present invention.

【0043】

The target molecule may be attached to the solid material either before or after sintering. Regarding the attachment of the ligand, the rod is sucked into a tube containing the substance to be attached, or the rod is a general-shaped hollow ball centrifuge rotor equipped with a band-shaped rotor (Anderson, NG, Natl. Cancer Inst. , Monograph No. 21) It can be wound inside, but in this case it may be drained by centrifugal force. Therefore, the solution of the adhering substance is first centrifuged in the sintered body, then taken out from here and washed if necessary. Sintered rods can be dried, coated with a suitable adhesive and combined into bundles for slicing.

【0044】

Alternatively, beads adsorbing the substance of interest and the object may be extruded under pressure to form a rod, which may be sintered together. The combined tubes can be held together in a variety of cements or polymerizable plastics. The outside of the tube may be modified or treated to allow cement or polymerizable plastic to adhere.

【0045】

A fifth type of fiber consists of hollow opaque tubes usually formed from plastics such as, but not limited to, polyethylene, polypropylene, Teflon® or polyvinyl chloride, which are gels or gels to which the substance of interest is directly attached. It is completely filled with other polymerization materials. The outer surface of the tube can be chemically or physically modified to accept the adhesive that binds the tube together. Its inner surface can also be modified such that the gel or polymerization mixture introduced into the tube adheres preferably by covalent bond. The acrylamide derivative can be crosslinked to the wall portion to adhere the acrylamide gel, and the gelatin, agar or agarose derivative can be adsorbed and crosslinked to each gel in the same manner. Methods for cross-linking target substances such as proteins and nucleic acids to linear acrylamide, gelatin and agarose are well known and the inducing molecules can be incorporated into the gel used for molding. Acrylamide can be gelled chemically or by photoactivation at room temperature, but Sepharose, which gels at low temperatures, is also available. Gelatin formation occurs slowly at temperatures below room temperature. The polymer used to fill the tube is usually homogeneous, but may also contain a substance of interest, which adheres to the polymerization medium. An example is a covalent bond of a protein to a short acrylamide chain, which is incorporated into an acrylamide gel to covalently bind the protein to gelatin. Therefore, gels containing variable biomolecules can be obtained or produced without exposure to denaturation temperature. The structure of such a tube is illustrated in FIG. The tube 16 is filled with a crosslinked gel 17, to which the target substance 18 is attached. Side view 19 and end view 20 of the sliced tube show that the immobilized material 21 is available.

【0046】

For arrays made with hollow fibers, the interior of the fibers can be coated with biomolecules by covalent bonds or suitable polymer coatings or in gels prior to array combination. Isocyanate polymers such as oxyethylene diols or polyols, in which most, if not all, hydroxyl groups carry polyisocyanate groups, are suitable. Some of these polymers include polyurea / urethane polymers. This polymer hydrates well and can be classified as a hydrogel. Examples of suitable starting materials include triols such as glycerol, trimethylpropane and triethanolamine, tetraol and polyethylene glycol. Examples of suitable polyicyanates include diisocyanate. The polyisocyanate may be aromatic, aliphatic or alicyclic (US Pat. No. 5,169,720 granted to Braatz et al. And Braatz, J., Biomaterials Applications. 9: 71-96 (1994)). Alternatively, the bundled array can be arranged such that each hollow fiber is filled with a biopolymer in a gel solution before being sliced.

【0047】

The sixth type of fiber or tube includes an empty, impermeable tube with the molecule of interest attached to the inner surface, or otherwise empty or empty. As illustrated in FIG. 4, the sliced tip 22 includes a sliced plastic tube 23 embedded within the supporting plastic 24. The target substance 25 is adsorbed on the inner wall of this tube, and the central portion 26 is in an open state. The result 27, as can be seen from the cross-section, includes a sliced plastic tube 23, an immobilized material 25, and an opening hole 26 formed by them, all of which support each other. Retained by material 24. Considering this chip as an ultratrace element metering plate, flow-through analysis based on immobilized affinity ligand technology, etc. (Hermanson et al., Immobilized Affinity Ligand Techniques, Academic Press, 1992, p. 407), for amplification by replication chain reaction (PCR) of immobilized oligonucleotides, or as described, for example, in US Pat. No. 5,843,767, etc. It can be used for the detection reaction of. When the tube is made of Teflon®, which has been treated to make the inner or outer surface hydrophilic, its cut ends remain hydrophobic. Even when the hydrophilicity test solution is spread over the entire surface of the chip, the solution tends to flow into the holes due to the automatic control volume, so if the total amount of fluid is adjusted appropriately, the adjacent cells Is less likely to affect. The top and bottom surfaces are then sealed with a suitable adhesive tape and the entire surface is then allowed to react, for example, towards DNA replication chain reaction amplification. Alternatively, in the chip 33 sandwiching structure 32 shown in FIG. 4, two pieces of material 34, such as glass or quartz, can be used to seal the end of the tube to form the microchamber 35. Through this transparent end window, changes in fluorescence or optical absorption 36 can be detected for each annular component. The reaction is measured with a colorimeter or a fluorometer.

【0048】

Various reactions other than the above can occur inside the microarray or inside the hollow fiber used for making the microarray. For example, a library of small molecules in combination such as synthesizing polypeptides, polysaccharides or polynucleotides in situ and / or esters, amides, carboxylates, etc. can be prepared. The same reaction, such as PCR, may be carried out on any of the other types of fibers, such as solid fibers, as long as the fibers are sufficiently permeable to the reactants.

【0049】

In the case of a hollow microarray, the target substance does not have to be immobilized inside or on the surface thereof. In such cases, a very small multi-well plate, which is itself a commercial product, is prepared. By placing living cells in "empty" hollow fibers with or without immobilization, this microarray can be used to identify cell responses to specific substances. Substrates or reagents may be immobilized at the same time as the biological cell to stimulate the production of detectable products upon contact or interaction with the specific analyte.

【0050】

Although it is a usual method to put molecules or biological components into the fiber before cutting for forming a microarray, in one embodiment of the present invention, the molecules or biological cells are inserted into a hollow fiber after forming the microarray. Put it inside. One example is the use of microarrays such as replicating biological cells, viruses or other particles by adding dilute suspensions to microarrays. It may take some time to add each of the various target substances, but this is acceptable. To take advantage of the amount of spills and inflows into adjacent array cells, one or more rows of cells may be left empty between each array cell "filled" with the material of interest.

【0051】

The inner surface of the small tubes described above can be chemically modified to attach polynucleotides, polypeptides, polysaccharides or other molecules either directly or via a linker. By attaching molecules, the number of reaction sites inside the tube can be increased. DNA and RNA have traditionally been synthesized on small polystyrene beads, and the most direct method for obtaining nucleic acids is to synthesize oligonucleotides on small polystyrene beads using different groups of beads with different sequences attached to them, and then to make them smaller. A polyethylene, polypropylene, polystyrene or other plastic, metal or ceramic tube is filled with beads and then solidified to completely fill the tube. The beads can be held in place and carefully heated and sintered. Alternatively, residual latex is added to the tube and air is injected into the tube to dry it in place.

【0052】

A seventh type of tube or fiber includes a capillary with a permeable wall. Methods and procedures for making hollow, selectively permeable fibers have been developed for use in renal dialysis machines and for molecular weight fractionation (US Pat. Nos. 4,289,623 and 3,976,576) and are now available. Widely used. A method of embedding this fiber in a solid plastic that can be sliced has also been developed and is used to attach the fiber to a thin tube at the end of a dialyzer.

【0053】

In the present invention, the permeable hollow fiber can be used in two ways. As a first method, the fiber is filled with a reactant-supporting gel that is already embedded in plastic. By carefully spreading the fiber into the molded part, each tube can be selectively filled as described above. This technique has the advantage of being able to quickly form a small array, the step of forming a separate hollow fiber, the step of filling it with a reactant, the step of arranging it in an array, and the step of infiltrating it with supporting plastic. The advantage is that new assays can be developed without performing all the necessary steps.

【0054】

The second use requires filling the hollow fiber before embedding it in the plastic. Techniques for controlling the wall permeability of the permeable tube have been developed so far. Thereby, the inflow and outflow amounts of the monomer and the gelling agent at the time of gelation can be adjusted, and the permeable substance can be removed after gelation. For example, acrylamide gel can be produced by cross-linking acrylamide and bisacrylamide with ultraviolet light in the presence of riboflavin. This technique is suitable when certain binding components are heat sensitive or susceptible to other chemicals. Catalysts that may interfere with subsequent fluorescence measurements can be removed by dialysis through the walls of the tubules after polymerization.

【0055】

Isocyanate-containing prepolymers are another gelling material, which polymerizes on contact with water, producing only carbon dioxide as a by-product of the polymerization. This binding component is first incorporated onto the solid phase (s) or otherwise placed on the fiber and then polymerized and / or dried to be used for gel hydration. Incorporate.

【0056】

The use of permeable support tubules allows the gel in the tube to be impregnated with a substance that can make the reactants more stable, increase the physical strength of the gel and allow slicing. For example, sugars such as lactose, trehalose, glycerol, fructose and other polyhydric alcohols can be added to stabilize the protein and solids can be added to the gel to facilitate slicing. This additive may be partially removed from the exposed surface of the chip when using the chip so that the embedded reactive groups are available. Additives that diffuse into the gel may also be used to increase strength and increase the amount of gel after drying.

【0057】

Further, when particles containing a ligand or a receptor are embedded in the fiber, the embedding medium may be made soluble or meltable so that the particles can be removed after the formation of the microarray. Removal of the embedding medium allows more active sites to be exposed on the particles for binding. Such applications are suitable when the particles are actually microfilament microfibers or microbrushes containing immobilized ligands or receptors similar to the crosslinked polymer (17) shown in FIG.

【0058】

After filling the tube with gel and reagent, the outside of the tube may be cleaned and treated with reagent to enhance the adhesion of the permeable support plastic. Subsequently, this is bundled to prepare a product for slicing. Eighth type tubes or fibers include those synthesized from large blocks, preferably by cleavage from a disc. First, a fiber material containing a target molecule is formed as a disk, and then the disk is rotated to peel off a long fiber from its circumference. This technique is essentially the same as a smaller version of the technique for making veneers from spinning logs. This technique has several specific spatial and handling advantages over long and thin fibers. Discs can be stored more easily, especially when it is necessary to maintain the active ingredient under certain conditions such as freezing, immersion in buffers, or dark places.

【0059】

There are many techniques for mounting arrays or parallel fibers together. One preferred method is thin-film deposition sintering. Steam, often a hot solvent, is allowed to interact with the array for a specific period of time and then evacuated and removed. In heat sintering, the sides of the array are placed in a compressive manner to heat the array to the softening point of its plastic. Another option is to use a metal with a low melting point, such as gallium. Low melting point refers to the temperature at or around the physiological temperature of the binding component.

【0060】

Various histological embedding media that maintain biomolecules in reaction form have been developed so far. For example, Durcupan, Nanoplast and Quetrol 651 can be cured by very slow heating, JB-4 and Immunobed can be polymerized at room temperature, and aqueous acrylic polymers, London Resin Gold and Lowicryl, are UV below freezing point. It can be polymerized by light (all available from Polysciences Inc.). Since conventional embedding media utilize solvents and waxes, this wax must be removed at least partially before analysis.

【0061】

Thus, embedding and slicing methods are available that identify and localize specific biomolecules . In the case of nucleic acids, specific nucleic acid targets can be detected, for example, by in situ hybridization and amplification of specific sequences by replication chain reaction (PCR) and other nucleic acid amplification techniques (LCR, RCA, SDA, etc.). it can.

【0062】

The embedding method shall maintain the desired properties or plurality of properties of the binding component in the living cell. Therefore, when the antibody is immobilized in the cell and the antigen binding specificity to the antibody is desired, the immobilization method retains the antigen binding performance of the antibody. The methods and means by which the fibers are attached to form an array also retain the antigen-binding performance of this antibody.

【0063】

Similarly, if the cell contains a candidate binding molecule for the hormone receptor, its immobilization and adsorption methods and means maintain the shape of the candidate molecule so that the hormone receptor can be identified and bound. And. In addition, many protein or carbohydrate antigens can be detected using immune reagents. This detection is generally done by incorporating the fluorescent dye into the second layer of the analyte or sandwich assay, or by incorporating an enzyme that produces an insoluble dye that can become fluorescent in the second or sandwich assay. This is done by binding to a third layer.

【0064】

Some solid-phase surfaces can be used directly for immobilization of the reactants, but other surfaces must be modified to be ready for addition. Antibodies adhere to clean polystyrene surfaces, and many proteins are the same (Van Oss, CJ and Singer, JM, "The binding of immune globuins and other proteins by polystyrene latex particles," J. Reticuloendothelial Society. 3: 29040, 1966). So far, polystyrene in the form of microtiter plates or beads has been modified to allow biomolecules such as polynucleotides, polypeptides and polysaccharides to bind. Perfluorocarbons containing polytetrafluoroethylene (PTFE) (such as fluorocarbon polymers known as Teflon®), polyvinylidene, polyvinylidene chloride The surface is bound to proteins or other biomolecules (US patent). No. 5,270,193). Such a surface can be produced by containing a fluorinated surfactant that changes the surface into a hydrophilic or positively or negatively charged state. Glass, such as controlled porous glass, can be modified to allow covalent binding of antibodies, antigens, polysaccharides, polynucleotides, nucleic acids, and the like. The plastic surface can be non-specifically modified by corona plasma discharge or electron beam radiation, and various coatings or adhesives can be coated on the plastic surface to attach the polymer. By performing various modifications, more specific covalent bonds of biomolecules become possible, and reactive groups adhere to the polystyrene or acrylic surface. The group then binds to the biopolymer under mild conditions with or without an extended linker.

【0065】

So far, various chromatographic media have been adapted to support an immobilized bioreactor. Examples of such media include soft gel beads composed primarily of acrylamide, agarose, Sepharose and chemically crosslinkable, and low compressible beads designed for high pressure chromatography. A useful natural product as an immobilized support is cellulose, which is readily available as a powder. The support can be chemically modified to covalently bond the bioreactor or purchased as a modified form in a bondable state.

【0066】

Long DNA or RNA molecules can be polymerized and immobilized in the gel and retained purely by physical entanglement. One example is the retention of DNA in agar or acrylamide gels. Furthermore, in other biomolecules such as polypeptides, proteins, polysaccharides or nucleic acids, they are linked to long polymers and do not diffuse when embedded in the gel, so that these biomolecules can be combined with soluble reactants. It can be kept available for reaction. Examples include binding of proteins or nucleic acids to polyethylene glycol (so-called PEGylation) or linear acrylamide chains.

【0067】

In addition to the method of immobilizing the receptor or molecule of interest and using it for binding the reactants, there are general methods for the immobilized components of certain types of reactants. For example, after immobilizing the A or G protein, it can be used for binding specific immunoglobulins. This then binds the unique analyte. A more common approach involves strong and specific reactions between the receptor and other ligands such as avidin and biotin. Avidin can be immobilized on a solid support or attached to a gel and used to bind biotin-linked antibodies or other reactants. This allows the formation of surfaces to which various reactants can easily and quickly adhere (see Savage et al., "Avidin-Biotin Chemistry: A Handbook.", Pierce Chemical Company, 1992). ..

【0068】

So far, a wide variety of methods have been developed to detect reactions between immobilized target molecules and soluble reactants. The main differences in these methods are the mechanism used to generate the signal and the number of different reagents that must be directly or indirectly interleaved with each other for signal generation. Examples include fluorescence with a fluorescent label covalently attached to the analyte (including delayed fluorescence), soluble dyes that bind to the analyte, and fluorescence that includes dyes that significantly increase the amount of fluorescence after binding to the analyte. Can be mentioned. The latter can be used to detect nucleic acids. In more complex systems, such as the so-called sandwich assay, the enzyme is immobilized in the detection complex and combined with a soluble substrate to produce a dye that can be fluorescent and preferably insoluble. Alternatively, the detection complex attached to the bound analyte can contain dendritic molecules such as branched DNA to which many fluorescent dye molecules are attached.

【0069】

A reaction product can be adsorbed by applying a method for producing dental floss in which a short horizontal fiber is attached and a brush-like shape is provided. A pattern that encodes and identifies information on a strand or fiber can be used in the form of dots arranged in small lines. In the development of multi-fiber endoscope arrays, methods for inspecting the arrays have been developed. In this method, a light beam or raster image is injected into one end of a fiber bundle so that the light continuously irradiates each fiber, and an emission pattern emitted from the other end is specified. If the patterns are the same, there are no fibers that have changed positions.

【0070】

Techniques for detecting air bubbles or voids in the liquid filling the tubules are well known and may depend on changes in refractive index, absorbance or fluorescence as measured along each tube. The technique of filling a viscous medium using centrifugal force in the case of short tubes is obvious to those skilled in the art.

【0071】

Tissue blocks may contain samples ranging from soft tissue to bone and are often embedded within blocks of embedding material (such as wax). Microtomes that slice these tissue blocks are commercially available, with sections mounted on glass or plastic slides that are automatically processed to remove some or all of the embedding medium and systematically series the slides. Various techniques and equipment for exposure to the reagents are also available.

【0072】

Microtomes and other slicing or cutting machines are well known that can cut bundles of combined tubes into thin sections and maintain the orientation of the constituent tubes after slicing. Cutting with a blade may reduce the deterioration of coupling components between cells of the microarray. The thickness of the microarray may be arbitrary depending on the expected usage requirements. The stiffness of the fiber bundle may be another specific factor. Section thickness is often less than 1 cm. Section thickness is often less than 50 mm. As illustrated in more detail below, section thickness can be in the order of microns.

【0073】

The sections (as microarray chips) may be mounted directly on the adhesive surface of the flexible film, or on a solid surface such as a glass slide. It is also possible to place multiple sections (in this specification, "sections" are used instead of "chips") with other sections in between and to fit along the filmstrip at intervals. Therefore, it is also possible to place a dozen or more sets of different types of sections along the film in a repeating order and cut the film to obtain one set. For sequencing studies, the inserted DNA can be amplified and labeled and its hybridization pattern examined for large sets of sections.

【0074】

By using a non-deformable fiber bundle, it is possible to form a large number of the same plates that are good if they can be cut or cut across the bundle and realigned. As a result, a highly uniform and reproducible array can be obtained. Realignment can be facilitated by using different types of easily detectable materials for one or more fibers as the microarray alignment means.

【0075】

Most immunochemical or competitive assays rely on signals from reagents rather than analytes. However, methods of fluorescently labeling antigens such as proteins containing aliphatic amino groups in the complex mixture have also been developed, and these are replicable and quantifiable. For example, CyDyes is commercially available from Amersham Life Sciences, with Cy2, Cy3 and Cy5 being particularly useful. When a component of such a labeled mixture reacts with an array of immobilized antibodies, each specific antibody binds to one of the fluorescently labeled analytes, so that each specifically bound labeled analyte fluoresces. This can be detected. To further improve this method, the bound antibody array is exposed to a solution containing a subsaturated amount of each known analytical protein in non-fluorescent form, the array is washed and the array is made into a test mixture of labeled proteins. Exposure produces multiple competitive assays.

【0076】

Any conventional binding assay format using an immobilized binding partner can be used in combination with the microarray system of the present invention. Simply put, a microarray can contain a plurality of ligands or a plurality of receptors, and the analysis product can be a plurality of ligands or a plurality of receptors. Competitive components that bind to either the analyte or the microarray cell may be added. This sample can be labeled and / or competing components can be labeled and / or microarray cells can be labeled. The labels can interact with each other to form a detectable signal or product, or to quench the signal or product. The number of such diverse combinations amounts to dozens, all of which are available to the present invention as well as the various combinations for different cells of the microarray assay.

【0077】

Several types of clinical trials are often required to make a diagnosis of a particular disease. Often, the trials are conducted in succession, with one test or element of the series suggesting the next test to be performed, followed by a third test or test group. Some of these tests most often have to be outsourced. Therefore, a series of branched tests can be performed simultaneously using a method that produces accurate numerical results in a machine-readable form, stable over time, and the clinical analysis currently in use. There is a demand for an inexpensive chip that can be read by a device that is smaller and cheaper than a system.

【0078】

Many biochemical analyzes require a wide dynamic range of the analytical process. Therefore, enzyme and immunochemical assays are often performed by identifying the reaction process over time or by performing multiple analyzes on a series of dilutions. Such analysis can be performed by "reading" the microarray at intervals of time while exposed to the analysis mixture of expression reagents. In addition, parallel analysis using standard type and blank (comparison) is required and includes these. Inexpensive, mass-produced and standardized biochips need to meet these requirements. The biochip can incorporate, for example, different types of reactants capable of detecting and measuring antigens, drugs, nucleic acids or other analytes.

【0079】

Arrays have many uses other than identifying bioactive properties. Chemical interactions and reactions can be tested as well. In such an assay, for example, different reaction chemicals can be tested simultaneously on one type of test material or material to identify corrosion, electrochemical changes or other interactions. This is particularly useful for chemical formulations of multiple substances such as cosmetics, paints, examples in lubricants. Alternatively, the desired interaction between the analyte in the array and all of the molecules of interest may be analyzed.

【0080】

A common problem that arises when using gels to immobilize reactants is that it takes a long time for the reactants, which may adhere to the substance of interest immobilized on the gel, to diffuse into and out of the gel. When the detection is performed by fluorescence, the dye that absorbs the excitation light is taken into the gel for the detection, so that the detection is limited to the region close to the surface. This problem can be solved by incorporating an ultraviolet light absorbing monomer, 4-methacryloxy-2-hydroxybenzophenone (manufactured by Polysciences, Inc.) into an acrylic embedding medium. It may also be useful to add quenching molecules such as DABSYL or DABCYL before fluorescence emission to accept vibrationally excited molecules.

【0081】

If you want to strengthen the bond between the analyte and the binding partner on the surface area of the particles within the microarray fibers, you can etch the embedding matrix of each fiber to increase the surface area of the particles contained on each fiber of the microarray. May be good. When performing a binding assay, the analyte is better diffused into the cells of the microarray to enhance ligand / receptor binding (sensitivity), allowing the microarray to replicate more quantitatively and allowing light to pass through the microarray. May be desired to improve spectrophotometric detection. The ligand may be pushed into the gel material of the microarray to facilitate diffusion into the microarray. This can be done by placing the microarray on a porous membrane and passing the ligand or ligand solution through the microarray using hydrodynamics, electrophoresis or mechanical means. For example, the pressure difference on both sides of the membrane can be used to flow a fluid through a microarray. It is also possible to draw the fluid through the microarray by simply placing a plurality of paper towels on the back side of the membrane and sucking out the fluid. Electrophoretic means apply an electric potential over the entire microarray or using one-point electrodes located on either side of a cell or cell group of the microarray. By providing a pressure difference using pumps of various shapes as a mechanical means, the fluid can be mechanically extruded or drawn through the microarray.

【0082】

The use of the porous membrane also has the specific advantage that the background can be reduced when cleaning the microarray. When embedding porous particles or filamentous components in a fiber, slicing the porous particles or filamentous components in a crossing direction further increases the porosity of the obtained structure, and increases the contact area and cleaning area of the reagent. There is a possibility that it can be expanded. Etching the embedding medium or capillaries can also increase the porosity and increase the exposed area to the target immobilized molecule.

【0083】

Slicing the porous particles, preferably twice, can form thicker channels and provide passages that can contain more fluid. Fibers containing the cut particles can be mounted on the permeable membrane support or on the holes in the solid base support. By doing so, the fluid can pass through the cells of the microarray.

【0084】

By using the present invention, it is possible to avoid the difficulty of spotting each cell on the solid phase or forming a compound in each cell. The former method always requires human involvement and equipment and must be able to quantitatively measure small amounts of liquid. In the latter technique, the types of compounds that can be synthesized on the solid phase are limited. Both of these prior arts are costly and require complex, automated equipment or time-consuming work to create each array individually. In contrast, the present invention, in which a "batch" is a microarray of thousands to millions, is technically simple and rapid. The only individual task required for each microarray is the cutting step.

【0085】

Microarrays prepared from stored multiple sets of reagents or by synthesizing different reaction sequences or compounds on the base chip have difficult quality control problems. In large arrays, the reagents in the final form cannot be analyzed separately prior to use. Moreover, the accuracy of the in-situ synthesized sequence cannot be ascertained until the set of arrays has been manufactured. If errors or non-standard components are found in the batch of arrays, the entire array must be disposed of. These problems are restrictions on the use of "biochips" in everyday clinical research.

【0086】

It is well known that immobilized proteins and nucleic acids are more stable, especially in the dry state, than in solution. The target material in the present invention can include a very wide range of chemical substances, complexes, biological cells or fractions thereof. Nucleic acids, many proteins, and proteins modified or coated with surfactants such as sodium and sodium dodecyl sulfate dissolve in organic solvents and a wide range of organic compounds, so that they are contained in polymerization mixtures such as those used in the production of plastics. Can be incorporated. Therefore, in carrying out the present invention, it is technically feasible to produce long fibers made of acrylic or other plastics containing different target substances by using the extrusion processing technology currently used.

【0087】

By fixing to fibers, bundling, slicing and forming microarrays, a large number of heterogeneous and potentially novel active compounds can be screened simultaneously. The peak fraction after separation, such as a plant extract, can be collected at the same time and used for forming a microarray. By utilizing this microarray in a large number of assay systems at the same time, it is possible to screen all compounds contained for any purpose, while dramatically reducing time and effort.

【0088】

It is particularly preferred to produce a large number of proteins or peptides by mass production techniques. The various fractions obtained by separation techniques from natural sources result in sources containing many heterologous proteins and peptides. Numerous fractionation treatments for separating mixtures of various compounds are well known. A single fiber can be formed using different types of fractions or specific compositions. Two-dimensional electrophoresis gels from serum and other tissues and natural sources produce thousands of heterologous proteins on the gel. Each protein can be individually taken out of the gel (by cleavage, elution, etc.) and used as a target molecule to form a single fiber. In such a method, since the original samples are different in different bundles, it is possible to easily compare the difference in protein between different kinds of samples.

【0089】

When the immobilized macromolecule is an antibody, this microarray can be used to diagnose various protein-based abnormalities. A second antibody labeled against the protein of interest may be used to further highlight the cell. In addition, arrays can be used to immobilize pathogens that are either pre-stained or immobilized and then stained. In this way, microorganisms from biological samples such as serum or crystals can be purified, stained with fluorescent nucleic acid stains such as TOTO-1 or YOPRO-1, and then the combined antibody can be found on the array. The binding analyte can then be detected by scanning the fluorescence and identified from its location.

【0090】

By immobilizing a microorganism or other target molecule, localizing an antibody from a fluid obtained from an individual using this immobilization reagent, and discovering its position with a fluorescent anti-human antibody, the antibody was originally produced. Diagnosing the induced disease is also part of the present invention. So far, arrays have been prepared using phage displays containing inserts from specific genes, synthetic oligonucleotides, or display antigens or antibodies to some extent. In the present application, when each display phage is used for producing one fiber, a population of peptide or antibody display phage may be used. In such a structure, due to the large size of the phage, some of the surface molecules remain embedded in the gel or plastic, leaving the rest exposed. The molecule of interest may be bound to the fiber itself and encapsulated inside the matrix, or may be bound to solid phase particles or microstructures inside or on the surface of the fiber. Phage, recombinant bacteria or other complex biostructures can also be immobilized and, if desired, the contained proteins are crosslinked with glutaraldehyde or similar immobilization material.

【0091】

Each fiber can contain a mixture of target molecules. For example, many isomers are prepared during chemical synthesis. In some cases, it may be convenient to leave this isomer unseparated prior to fiber formation. Similarly, when dividing the mixture, whole and complete isolation is difficult and time consuming, so fibers containing the mixture of receptors may be formed.

【0092】

When using an aggregate of fibers, or otherwise, for example, in embodiments where particles are embedded in the matrix forming the fiber, it is desirable to use material filling that maintains the relative position of the fibers in the longitudinal direction of the bundle. There is. Various glues and adhesives are well known in the art. For example, a filling composition containing a fat component is an aliphatic hydrocarbon having a relatively high molecular weight of 600 or more, and an inorganic component and a block copolymer thicken the material but reduce its viscosity. Antioxidants and anti-aging agents can also be contained. See, for example, US Pat. No. 5,187,763.

【0093】

Select a filling material that maintains the fiber aligned, is machinable, and does not interfere with subsequent processing on the microarray. For example, other examples that can be used include polymerizable materials such as polyacrylamide. The embedding matrix for the fiber may be black or opaque in color, or it may be any other as long as it can absorb the emission signal of the label and reduce crosstalk between the cell and the chip. In addition, the adhesive between the fibers may contain the same absorbent material to reduce the background between the cells of the microarray. Optionally, a specific layer of this material may be placed between the fibers before forming a bundle. If a hollow fiber is used, an opaque material may be incorporated within the hollow fiber shell itself.

【0094】

The array can be equipped with an entire set of antigens / antibodies, etc. in various cells with comparative examples to screen blood samples for common blood-borne diseases prior to transfusion of blood donations. Similarly, certain symptoms have a number of common causes, and arrays may be used to screen them simultaneously. Urethral infections, for example, are common and are caused by numerous types of bacteria that have varying susceptibility to different antibiotics. Being able to test for multiple factors at the same time saves considerable time and money.

【0095】

In the process of using the chip according to the present invention, various well-known techniques and materials are used to reduce non-specific reactions. Thus, as is well known in the art, in the case of protein assays, blocking non-specific sites on the chip by essentially everything except fiber or filament material, embedding material and target binding component, such as albumin or milk. In response to the agent, the blocking agent is bound to a region that does not contain a binding component that can react with all that specifically bind to the ligand, assay, reporter molecule or binding component.

【0096】

The array may have two or more identical cells made of different fibers, but these cells shall contain the same binder. This allows the array to have its own quality assurance checks. In addition, some cells may preferably have different concentrations of binding component for quantitative measurement of the analyte. This allows the microarray to set its own standards for quality detection and quantity detection. For example, a series of cells can contain antibiotics of different concentrations. When sample microorganisms are contacted with this cell and incubated, no growth is seen in one cell, and growth in another gives an approximate minimum inhibitory concentration. The minimum bactericidal concentration can be specified by dyeing with an active dye such as trypan blue or fluorescein acetate and doing the same. Since a microarray can have thousands of cells, it is possible to identify antibiotic susceptibility to various antibiotics at the same time. It is also possible to mass-identify other biological activities with ligands or receptors immobilized within the gel.

【0097】

Essentially the same fiber can be used multiple times in the same microarray. This allows for unique quality control checks and makes the results of the binding assay more reliable. Further quantitative measurements can be made by performing a binding assay to increase accuracy. Empty fibers and fibers without bound molecules of interest are useful negative controls and must be used on any microarray.

【0098】

Long filaments, capillaries or coaxial bipartite filaments are placed in parallel and joined by sintering or adhesive to form a bundle. The bundle is preferably resistant to deformation and each strand or capillary is continuous from one end to the other. The positional configuration of the fibers or capillaries must be invariant throughout the bundle. Filaments made of two types of materials may be used for coaxial formation. This core material is formed of one soluble material and the coating material is resistant to the same dissolution conditions. For example, it is a material that dissolves a specific type of glass with a strong alkali, but does not dissolve other types of glass. Depending on the material contained, this dissolution step can be performed before, or preferably after, the slicing step.

【0099】

Alternatively, the dressing may be soluble and the tolerant core portion may be isolated as an "island" on a microarray mounted on a lining sheet. In either case, the space formed in the melting step can be left empty or filled with a different material. It is also within the scope of the present invention to dissolve a part to obtain a porous material. Porous materials are particularly desirable for coupled arrays because they increase the surface area.

【0100】

Particles, especially porous beads, may be "chemically sintered" to form filaments, sheets, or capillaries. This technique may be used to bond different fibers together. One such method first binds the molecule of interest to the particle. A blocking agent may be added to block other reaction sites or adsorption regions on the particles. If not already filled, the beads are packed in a tube or hollow fiber. Next, when a blocking agent and / or a chemical reaction compound that crosslinks or links with a non-reactive site on the target molecule and / or the bead is added, chemical adhesion occurs at the portion in contact with the bead. The tube or hollow fiber may be removed as is or removed. Since the target molecules in the pores inside the beads do not come into contact with each other, they do not change significantly. Alternatively, the bead pores may be filled with a hydrophilic solution and retained by capillary action while the space between the bead beds is filled with a hydrophobic adhesive or curable liquid.

【0101】

A typical example of chemical sintering is to adsorb G protein on porous beads and then add a gelatin blocking agent. The obtained beads are filled in a 1 mm plastic tube, and a protein cross-linking agent such as carbodiimide is added. After the reaction is complete, the non-reaction reagents are washed and washed away, to which a suitable target antibody is added and bound to the G protein to cleave and cleave a fiber suitable for producing microarrays. Form.

【0102】

Alternatively, the particle surface can be first biotinylated and avidin can be used as the cross-linking agent. An avidin-labeled antibody may be used instead of adsorbing the G protein on the beads. Another method is to use relatively large porous beads and an adhesive or embedding medium that fills the space between the beads. When slicing this fiber, the beads are made large enough to be cleaved and the inside of the beads is opened to expose the bound molecules of interest. Since the target molecule encapsulated therein is exposed when the beads are cleaved, hollow beads or microballoons may be used instead of the porous beads. This concept is similar to slicing tissue or embedded cells to expose and make the intracellular structure visible.

【0103】

In addition, two different sets of beads may be used. The first set is porous and comprises binding receptor / receptor binding material, the second set is coated with a highly reactive material, or the first set of beads or a coating thereof. It has been modified with a reactive group that binds to. The tube is first filled with both beads in a dry form, the tube is shaken, and then a fluid is injected and reacted to form a solid fiber of beads. Alternatively, if the beads in the first set are extremely large, the beads are first filled and then the second set (with or without fluid) is added to allow the beads to pass through the space between the large beads. , React accordingly. The reaction between the beads may be due to a specific binding moiety or a non-specific binding reaction that crosslinks the beads to form a solid that can be sliced. The second bead may be blackened to reduce stray light during fluorescence detection.

【0104】

After the bundled fibers are melted or otherwise glued together to a fixed pattern, the bundle is cut across or at an angle into a number of thin discs, some of which are optionally melted. To do. When a hollow capillary is used, the resulting disc can be used as a channel plate for amplification of optical images and optical waveguides. This thin disc can also be used as a filter because of the uniform hole size, whether rods or fibers are used.

【0105】

Each fiber portion of the sliced two-dimensional array will contain a relatively large amount of binding components such as DNA, RNA, or protein molecules. The first step in using the final resulting array is to wash the plastic surface of the array with a solution that can erode very slowly. This is done at a constant rate to remove any free biopoly molecules. Continue this cleaning to change the plastic into a non-eroding solution. The array may then be dried and stored until use, or it may be used immediately. The surface of the particles is dissolved to form a solution to expose the molecules so that the target reactant is easily exposed in the plastic.

【0106】

Since each fiber contains the target molecule in the same form as that contained in the microarray, quality control inspection can be performed on the fiber itself without using the entire microarray. This is especially important when using microarrays for diagnostic purposes. Quality control inspections can be performed by sampling microarrays from batches, but this does not mean that microarrays on the market are actually inspected. In contrast, the present invention uses a thin section of fiber itself. Analyzing the fiber itself means that each microarray containing a section of the fiber is actually being tested as a microarray cell.

【0107】

On the other hand, when the target molecule is directly synthesized in the solid phase in situ in each cell of the microarray, the actual composition used containing the target molecule is not actually tested after the synthesis. Rather, spot inspections are the basis of quality assurance. In the production of microarrays using droplet spots on the solid phase, quality control inspection can be performed by testing the liquid. However, the droplet sample does not represent the quality of the molecules to be dried that are immobilized on the slide. Therefore, the result of this quality control inspection is not the same as the actual product on sale. Again, this does not provide any quality assurance for the actual composition contained in the cells of the microarrays on the market.

【0108】

In quality control in the present invention, each fiber can be analyzed and this analysis can be performed as a ribbon or small group, or as part of the entire bundle before slicing. Furthermore, by testing one final microarray, the entire microarray can be efficiently tested because the composition of the fiber is identical to that part of the final product.

【0109】

Clinical trials require regulatory approval for the trials and systems and their manufacturing methods. When chips are manufactured using techniques derived from photolithography and other electronic chip manufacturing, the cost of each chip is extraordinarily high, and the error rate when manufacturing chips individually is very high. Since chips are individually manufactured and used only once, quality control is difficult and there is no way to bring every given chip to a satisfactory level. The best possible method is to randomly take large batches of parts to be tested. In the present invention, since a large number of sections can be produced from one composite assembly, adjacent sections and sections separated from each other can be compared with each other. The rate of possible errors can be statistically analyzed and predicted. However, more importantly, because the sections can be produced in large quantities and at a fairly low cost, the same analysis can be performed on clinical samples in duplicate, and confirmation analysis is performed when important diagnostic results are obtained. What you can do. Therefore, the present invention can be widely and routinely applied to gene analysis in medical practice.

【0110】

Important target material components of the fiber are immobilized and retained in the fiber. Immobilization can be performed by a number of techniques that are well known in their own right, such as in-matrix encapsulation and chemical bonding, often by crosslinked moieties via amino, hydroxy, sulfhydryl or carboxyl moieties. Even if this chemical substance is chemically adsorbed on a monomer or polymerized by using it as a monomer, such a component can be effectively incorporated. Binding can also be performed by a number of affinity techniques, such as ligand / receptor pairs such as A or G proteins for antibody adsorption, biotin-avidin, HIV-CD4, sugar-lectin, or digoxygenin-anti. For example, via a ligand having a receptor such as digoxygenin. On the other hand, if gel or non-gel, gel matrix such as wax, silicone polymer and silicone emulsion can be used, no specific bond is required. A solid fiber is formed by simply mixing a liquid wax or gelling agent with its key component, cooling it and molding or extruding it.

【0111】

There is no need to combine arrays in one step. First prepare a flat array containing a set of tubes arranged in parallel, then slice the ends of this array for testing. The flat arrays are attached to each other with a suitable adhesive to form a three-dimensional bundle. If you include a step to provide a flat array in the middle, you can make and store it in this state, and by selecting and mounting different types of 1D arrays, you can make a 2D array according to your order. You will be able to do it. This step-by-step assembly operation allows inspection at each step, minimizing errors due to errors or low coupling effectiveness in a single rod or capillary, resulting in a new pattern of reaction. You also get the flexibility to combine things.

【0112】

In general clinical use, it is important to have an identifying material on the slide holding the chip, which may be integrated with the chip. FIG. 5 illustrates a chip 40 including an array element 41. Bar code 42 is printed along one edge for identification and orientation. In addition, a small amount of concentrate of the dye, which is usually non-fluorescent, is incorporated into the polymer selected for the manufacture of the tube to form a pattern from 43 to 44 containing one or more numbers or one or more letters. May be good. It is also useful to incorporate a fluorescent dye into several cells or elements and use it as a reference for fluorescence measurement. Dyes can also be placed within the components of the selected tube to make the tube even easier to identify. It should be understood that diagonals 43-44 also indicate that the horizontal rows of tubes assembling the array are neatly aligned. If the tubes in an array are not aligned and one tube or rod is missing from the row, the entire pattern will collapse and can be easily identified.

【0113】

The embedding material or adhesive used to hold the tubing as a bundle may be opaque, but the tubing and preferably its contents shall transmit light over its entire length. As a final confirmation of the orientation of the array elements, one element may be illuminated at one time at one end of the bundle, as shown in FIG. By doing so, light is detected at the other end, as shown in FIG. 6, indicating the array position. In FIG. 6, when the bundle 50 containing the fiber 51 is illuminated by a cathode ray tube (CRT) 52, the raster 53 formed is focused by the lens 54 on the end of the bundle. The light transmitted from there is recorded by the CCD camera 55. Each spot 56 becomes a signal 57 to be detected.

【0114】

The configuration for detection using epifluorescence is schematically shown in FIG. The chip 60 is illuminated by a beam 61 emitted by the lamp 62, which passes through the filter 63 to separate light of the optimum wavelength for fluorescence excitation. The split beam prism 64 directs its excitation light to the chip 60. When the light emitted from the chip returns to this split beam prism and passes through it, the emitted wavelength is separated by the filter 65, which is detected by the CCD camera 66. Other systems are well known to those of skill in the art for detecting fluorescence patterns.

【0115】

As another fiber forming method before bundling, the fiber may be formed by cleavage from a large material. In FIG. 8, a sheet of absorbent material 70 is saturated with one type of ligand or receptor. This can be done by dissolving the compound in a solution and then sucking it into a sheet of absorbent paper (such as filter paper). A cross-linking agent may be added to adsorb this receptor to a paper cellulose base or other support. Alternatively, the pulp can be crosslinked to the receptor to form a sheet of paper or felt. This technique provides even more uniform distribution, but requires more receptors. The sheet (70) is manufactured by either method. Many different sheets are prepared and each sheet contains a different receptor.

【0116】

The sheets are then stacked (like a book) with an adhesive and optionally a composite active sheet (not impregnated, preferably black) as a spacer between the sheets. In this way, the book (71) is formed. The book is then taken to a paper cutting tool or similar slicing device to cut a very thin strip (72) similar to the "ribbon" object (2) in Figure 1. Subsequent methods are the same as those shown in FIG. Multiple strips (72) from different books are stacked to form a bundle (73), which is then cut across to form a microarray (74). Adhesives are preferably added to the ribbon to bond them together. Alternatively, prior to the slicing step, an adhesive may be applied to the solid phase or the end of the bundle to bond the solid phase to the end of the bundle.

【0117】

Other films, such as nylon films, that absorb the protein may be used. Inactive films such as polyolefins activated with photoactivated cross-linking reagents with two different reactive functional groups or simple polyurethane films such as Thermedics products may be used. Different proteins may be used on both sides of the sheet or film, and the sheet may be separated with an inert sheet to separate cells (sectors) and signals in the final microarray.

【0118】

The fiber material is preferably glass, metal, plastic or other polymeric material. In the case of coaxial composite fiber, the soluble component can be produced from a significantly wider range of materials. Each material may be a composite material of two or more components. This fiber can act as an optical waveguide or total internal reflection fiber optics to convey information about position alignment and chemical and biological reactions occurring on the surface. It is preferred that the fiber material be selected to support adsorption with target molecules such as cells, oligonucleotides, peptides and polysaccharides. Hollow fibers can be used to store cells in a raw, frozen or dry state. When the fiber material is made of glass or other transparent or translucent material, the light and electrons emitted directly or indirectly from the reaction or component inside the fiber, especially the hollow fiber such as a capillary, are amplified to make it easier to detect. You may. The fiber material contains a component in the fiber or a component that reacts with light, electrons or other chemical components emitted by a reaction occurring in the fiber, a component that detects these, or a component that converts them into other forms. Can be done. Detecting chemiluminescent reactions inside and on the fiber is a suitable method.

【0119】

The gelling material used in the present invention can be selected from various well-known materials. Polymers such as agarose, gelatin, collagen, xanthene, carrageenan, alginate, or thermosetting, thermoplastic, chemically curable or UV polymerizable polymers can be used. Non-polymeric gelling materials, including waxes and clays, can also be used. Hydrogels are particularly suitable when an aqueous environment is required for the reaction between the target substance and the calling additive. After molding the fiber, a polymerization agent or curing agent may be added by submerging the molded product in its solution or by passing the agent along the outside of the fiber molded product.

【0120】

Hydrogels have various gel porosities, can bind proteins during or after polymerization, have low non-specific binding properties, are transparent, are harmless by-products of polymerization, and release time for polymerization. Has a number of desirable features, such as being adjustable and being able to be used with a variety of solvents. Isocyanate polyurethane liquid prepolymers are suitable.

【0121】

These may be modified with a combination of tackifiers, rubbers, hardeners and crosslinkers, plasticizers and various gelling materials. In general, the gelling material should be sufficiently inert so that it does not interfere with the interaction between the binding component and the analyte. In the present invention, the target substance is extracted into an organic solvent. The solvent is miscible with thermosetting plastic mixtures or with mixtures that polymerize chemically or with UV or ionizing radiation. This extraction can be carried out by coating the target substance with a substance containing a surfactant or other reagents to increase the solubility under selective conditions.

【0122】

The mixture is then extruded into long fibers or molded into fibers. The fibers are identified by tabs at the ends of the fibers or tags on the rolls that carry the fibers and / or by incorporating different dyes. The barcode may be printed directly near the end of the fiber. Thermoplastic polymers may be used as long as the embedded product is sufficiently thermostable. Different colors may be colored depending on the fiber to facilitate the location of a particular ligand within the array, or the array itself may be identified.

【0123】

The solvent can be miscible, extractable, or volatile in the gelling material to make the final product porous. Porous products are particularly suitable for self-supporting solid filament fibers. It is also possible to include a dye or other optical absorber in the fiber or its gelling material to view only the analyte / binding component on the surface of each cell. These improvements can reduce the effect of diffusivity due to the gel or porous material, which may change with temperature, time, type of carrier solution, and the like. The use of UV or dyes that absorb luminescent fluorescence can reduce the amount of fluorescence from the non-surface analyte / binding component reaction.

【0124】

Different dyes (fluorescent or non-fluorescent) may be incorporated within each fiber. This makes it possible to confirm the position of each fiber in the two-dimensional array. Solid filaments or capillaries, including fibers, can be glued together using a variety of techniques. Fibers may be sintered against each other as long as the components are sufficiently thermally stable. Alternatively, many adhesives are well known, such as cyanoacrylate adhesives. The space between the fibers can be completely filled with an adhesive or monomer and polymerized. Adhesives can also be constructed from thermoplastics and gelling materials to hold large numbers of fibers together and in blocks. Even with an inert material such as a Teflon® tube, the surface can be made reactive with metallic sodium and the surface can be etched in a hydrocarbon solvent. Non-chemical means may be used, such as passing an electric current through the fiber to fuse the fiber.

【0125】

Sealing the open end of a capillary by pressing a deformable material against its surface to vaporize a plastic (such as parylene) on the surface, or with a chemical such as a thermoplastic or thermosetting plastic material. May be sealed with a flat plate. There are two basic methods for manufacturing a two-dimensional array from such fibers. The first is to make and appraise the ribbon and then form a set of ribbons as a long rectangular bar, and the second is to make the bar first and then make all the fibers in one step. It is meant to be put together. The former option may be advantageous because the ribbons can be individually identified before forming a complete array. If a bar of a two-dimensional array can be formed, it can be sliced using a conventional microtome to form a very large number of sections. It can be attached to, for example, glass, metal or plastic. Alternatively, the solid phase material may first be attached to the ends of the bundle before slicing the bundle. This can be done by first coating the ends of the fiber bundle or the solid phase with an adhesive such as an cyanoacrylate adhesive, if necessary, or by slicing in advance or sintering after slicing.

【0126】

The stained fibers can be seen in this array for identification and orientation confirmation. In addition, the fibers can be stained to form a visible pattern if the array is manufactured correctly, and the pattern may be named or numbered. The advantage of this system is that it can cut a very large number of arrays and a specific percentage can be used as a standard. For example, if a bar with a length of 100 cm is formed and the bar is cut at intervals of 100 microns, 10,000 arrays are produced. If the intercept thickness is 10 microns, the number of arrays is 100,000.

【0127】

If the maximum distance between two points of each fiber is 100 microns, then one ribbon contains 100 fibers, and one fiber bar contains 10,000 fibers, the cross-sectional area of which is 1cm²Will be. If there are 330 fibers per ribbon, the total number of fibers will be 108,900, which is about the same as the number of expressed genes assumed to be contained in the human genome.

【0128】

The present invention is a first array that includes such a large number of different cells per unit area on a microarray without covalently binding the binder to the chip. In the present invention, the array 1 cm²It is preferable to include at least 100 cells per cell, and more preferably to include 250, 500, 1,000, 5,000, 10,000, 100,000 or 1 million or more cells. This is a much higher density than the disposable cells formed by microfluidic in commercially available microarrays.

【0129】

1 cm more than this big number²In order to significantly increase the number of cells per hit, a large fiber bundle of relatively thick fibers can be prepared and the bundle can be stretched or pulled. This makes each fiber thinner, but does not change its basic configuration or orientation and cross-sectional shape with respect to each other. This method has two advantages: the production of more microarrays and the production of smaller microarrays. By using conventional 5 micron porous particles (as in the examples below) and plastic embedding media such as low melting point wax, deformable or ductile fibers can be obtained, which are very less than 20 microns in diameter. It can be stretched to thin fibers. The field of drawing thermoplastic materials is well known in itself. Even when the mold cannot be stretched, the plastic material can be pulled out or extruded between the rollers to lengthen the fiber and reduce its diameter. When heated arbitrarily and gently, the fiber can be similarly elongated and the cross-sectional area can be reduced by simply pulling the end of the fiber bundle. If it contains small, porous particles, the fiber is stretched to a finer size, 1 cm of the microarray.²At least about 1 billion cells can be provided per hit.

【0130】

In the field of fiber optics, a bundle of optical fibers is heated and stretched into an extremely thin optical fiber while maintaining consistency inside the bundle. Similarly, candy canes and candy with a cross-section design are manufactured by stretching large blocks. Even glass beads, which have been used for hundreds of years, have been manufactured in the same way.

【0131】

Until now, high-density cells (sectors) of microarrays have been realized by using photolithography in which target molecules are synthesized on microarray cells. However, the compounds that can be produced by photochemistry are limited. In addition, chemically bound compounds interact differently than the same compounds as when freely suspended. In biological systems, the active moiety may not be freely available for binding. On the other hand, the binding material according to the present invention is only encapsulated in a matrix and can completely retain all chemical and biological activities.

【0132】

When using porous particles and immobilizing the target molecule inside the porous particles, it may be desirable to retain a suitable fluid inside the pores and use an immiscible embedding medium. In such structures, the embedding medium may be of interest, incompatible, or incompatible for use in binding assays, but can be used. For example, aqueous solutions can be used to protect low melting point waxes used for embedding proteins and porous fluids.

【0133】

Well-known photochemical treatment by Fodor et al., Nature 364: 555-6 (1993); Hacia et al., Molecular Psychiatry 3: 483-92 (1998) and Fodor et al., Science 251: 767-773 (1991). Now, prepare short-chain peptides and oligonucleotides covalently attached to the support chip. The method of synthesizing amino acids or nucleotides originally constrains the actual length of the oligomeric polymer to which it binds. It is not feasible to synthesize a protein or an entire gene on a chip. In addition, the secondary, tertiary and quaternary structures of proteins may be important. On the other hand, these are also possible in the present invention.

【0134】

Ultimately, a number of different arrays will be required, and some, especially those developed for pathogen identification, may need to be changed frequently. In addition, new disease-related alleles will need to be incorporated into new arrays. In order to meet these requirements and allow array changes and additions, it is important that individual stable fiber rolls are available and that these rolls are clearly distinguishable. Each roll may be distinguished by applying microstrip along its longitudinal direction at short intervals. Further, the color may be changed for each tube, a non-fluorescent dye may be incorporated into the gel and used as an identification material, or a barcode may be printed on each fiber.

【0135】

The chip according to the present invention can be used not only for identification of pathogens by identifying characteristic nucleic acid sequences, but also for example, using an array of immobilized specific antibodies, intact bacteria, mycoplasma, yeast, nanobacteria. And the virus can be identified. The present invention can be used to identify viruses or other infectious particles isolated by microbanding tubes. The diameter of this micro-banding tube is gradually narrowed from the open end to the closed end, and the desired low-concentration biological component can be concentrated in a small amount according to a suitable centrifugation method. A unique centrifuge tube. See, for example, International Patent Application WO 99/46047. In this way, microorganisms can be concentrated from biological samples such as serum or plasma, stained by fluorescent nucleic acid staining such as TOTO-1 or YOPRO-1, and combined antibodies can be found on the array. The fluorescence can then be scanned and identified by location. Microorganisms or other target molecules are immobilized on the above chip, and after localizing the antibody from the biological fluid using this chip, the position is discovered using the fluorescent anti-human antibody and the antibody production is induced. Diagnosing the disease that was present is also part of the present invention.

【0136】

Maintained as a bundle, additional fibers or ribbons can be added to the bundle as needed before slicing the next array. This allows the detection and measurement of newly discovered emerging diseases, new proteins, genes or compounds without completely reforming another bundle. The present invention can also be applied in another way: the bundle is stored under the user and the array is sliced as needed. This is useful for research purposes where the same array is needed for a long period of time but the number required each time is small.

【0137】

Another method, apart from slicing the bundle and using the section as a separate microarray, is to perform the assay directly at the end of the bundle. The first sample can be applied to the cut section for an assay, washed and then image processed by a detector. The bundle may then be mounted within the microtome device if the assay has not yet been mounted within the microtome device prior to the assay. The blade then removes the used surface of the bundle to expose a new surface for use in the next assay. Repeat this same step. In this way, a series of assays up to 100,000 times or more can be sequentially performed while using the bundle in one machine. Such techniques have certain advantages because optical or electrical detection can be performed through the bundle itself, which includes optical or conductive fibers. The detection system can be continuously attached to the bundle while applying more general light or electrical energy to the ends and using it for testing. It should be noted in Figure 6 that this test technique has been adapted to the detection system.

【0138】

The present invention can also use different immobilization techniques, different types of target fixation materials, different types of analytes, and different types of detection procedures on the same chip. Since the channels can be replicated between the plates, the location of each channel or cell can be accurately located by mechanical means. Adding a reference symbol to the polished edge or other suitable location makes it easier to identify each cell in the array. Visualize each cell, test each cell individually, add material to each cell, or extract from each cell using a sufficiently accurate 2D computer-driven 2D device currently on the market. be able to.

【0139】

The cut surface of each plate may be polished so that the combined plates can face each other with little possibility of cross-leakage. Finally, if the surface treatment is performed with a material that is repellent to the fluid located inside each cell, the cross leak can be further reduced. For example, since a fluorinating agent (Teflon (registered trademark) processing) or a silane agent is water repellent, sufficient surface tension is provided to reduce cross leakage of cells filled with an aqueous solution.

【0140】

After cutting a section from the bundle, the section is generally joined to a solid backing material to serve as a structural support and improve handleability. This solid lining is usually a sheet of plastic or metal, but other materials can be used. Adhesion generally utilizes permanent adhesives or fused deposition. One or several channels at a time by scanning the entire array or parts of it (cells in one or several) with a charge-coupled device (CCD), or by using a condenser lens or objective lens. By illuminating the cell, each cell in the array can be detected or viewed. In this way, the amount of light absorption and the amount of light emission can be detected. Fiber optic bundles that are matched and aligned with the microarray can be used to optically detect differences between cells on the microarray.

【0141】

Detection is radioactive, enzyme, luminescent, optically absorbent dye, magnetic, spin-labeled, oxidant or reducing agent, chemical luminescence, or indirect labeling that interacts with a detectable component that interacts with the substance of interest in the microarray. It can be done on the basis of a number of detectable labels such as. A detectable labeling system based on fluorescence, usually epifluorescence, is suitable. This requires labeling the calling sample with one or more fluorescent dyes. Since the dye is applied to the array as a thin diluted film, the amount of test material required is very small. Nucleic acid hybridization is performed under carefully and strictly controlled conditions.

【0142】

To identify the selected channel, the selected channel can be sealed and / or filled with a substance that is easy to detect. Inks, dyes and coloring materials of different colors are particularly suitable, as well as detectable components similar to the detectable component (s) detected in other cells, or vice versa. There is. Dry inks or plastics, sublimation, solvents containing inks, or printing methods involving inkjet printing may be utilized. The features thus formed either provide good alignment during use of the array or make the markings easier to find, resulting in easier optical alignment.

【0143】

When a ligand binds to a receptor using a microarray in a binding assay, further identification of the ligand may be useful in some cases. In some circumstances, the uniquely bound receptor may not reveal the entire structure of the ligand. For example, if the ligand is a cell, a macromolecular complex, or an inducing molecule containing an inducing moiety that acts as a ligand, further analysis may be desirable. In this case, the ligand can be eluted from the microarray and the ligand can be collected for further analysis. For antibody / antigen binding, the ligand must be stripped in a pH 2-3 environment or other conditions. In nucleic acid hybridization, the ligand must be stripped while increasing the temperature. Various other chemical, physical and electrical techniques for separating this bond are well known in their own right.

【0144】

In order to enhance the peculiarity of the elution method, the substrate can be configured to maintain the electric charge, and the uptake rate of the target biological substance in a specific cell (sector) can be increased. For example, if the target substance is nucleic acid, each cell is configured to carry a positive charge. The opposite electrode carries the opposite charge. Next, if necessary, a singular medium is placed in the cell to reverse the charge in the electrode and release a ligand such as nucleic acid at that position. This counter electrode can be used as part of a micropipette, or a micropipette can be attached to the counter electrode to collect components released from the cell. See U.S. Pat. No. 5,434,049. It is preferable to use a porous membrane and apply currents to both sides of the membrane.

【0145】

The method used for eluent analysis can be capillary electrophoresis, mass spectrometry or a second binding essay. For convenience in mass spectrometry, the microarray itself can be introduced into a laser matrix desorption system incorporated within the mass spectrometry system, where bound molecules can be desorbed and analyzed. Once the analyte is stripped from the microarray, the microarray can be reused. As described above, the reuse process has an advantage that it can be standardized by performing a plurality of comparisons over time.

【0146】

In addition, if the receptor is adsorbed on a microarray matrix by a cleaving linker, the linker can be cleaved to isolate the analyte. Depending on the cell of the microarray, it may contain different linkers or the same linker, so it may need to be purified before the next analysis. Traditional protein chip preparation procedures require the 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 it is frozen, it may have to be frozen and thawed repeatedly in order to be used repeatedly, which also denatures some proteins. In contrast, immobilized proteins have been found to be stable over time.

【0147】

The term "substrate" as used in the present invention refers to a glass capillary array including a "main surface" that refers to the open end of a channel plate, and a "binding reagent" is a DNA, protein or antibody (polymer as a set), cell. / Microorganism / Cell line or other target substance.

【0148】

BEST MODE FOR CARRYING OUT THE INVENTION

Examples are described below for the purpose of exemplifying specific aspects of the present invention. These examples should not be construed as intended for restriction. Example 1: Microarray formation and analysis Antibodies were prepared by affinity purification in which they were reversibly bound to each immobilized antigen on an Integral 100Q biochromatography working terminal and immobilized on a fine particle support (Poros G manufactured by PE Biosystems).

【0149】

The antibody was captured on a Poros G column (a commercially available Poros particle precoated with G protein, a bacterial protein capable of binding more immunoglobulins to the Fc moiety), and the antibody and G protein were crosslinked to dimethylpimelimide. Each antibody support was prepared by immobilizing the antibody on Poros particles by covalent bonding (according to PE Biosystems). This antibody column is extremely stable because it can be reused 100 times or more in subtraction mode (with acidic elution of bound antigen). Each antibody support was characterized to show specificity for one antigen.

【0150】

Antibodies to human serum albumin (HSA), transferrin (Tf) and haptoglobin (Hp) were used. A mixture of these three supports was produced and used for serum subtraction. In the study, a total of three supports were used in combination with 1) rabbit anti-HSA, 2) rabbit anti-human Tf and rabbit anti-human Hp, and 3) mixed anti-HSA, Tf and Hp. Unmodified BA Poros (commercially available streptavidin-coated Poros) was used as a non-antibody comparative example. Therefore, a total of four types of supports were used.

【0151】

Poros particles are nearly spherical with a diameter of approximately 5 microns and are highly reticulated (with numerous interstices). The protein is dispersed and attached to the particles not only on the outer surface but also on the entire inner surface. Embedding the particles in a suitable medium formed a sliceable solid matrix in which the antibody was immobilized and dispersed fairly evenly. Taking advantage of the cubic nature of this support, the volume of sections containing the particles (for antibody and associated antigen binding) is greater than in the simple planes used in current microarrays.

【0152】

Each of the four antibody-carrying particles was mixed with approximately the same amount of 0.75% agarose-fused phosphate buffered saline (PBS). This agarose for rabbit anti-HSA beads contained a green food colorant. Similarly, anti-Tf and Hp agarose were colored blue, anti-HSA, Tf and Hp agarose were colored yellow, and agarose-containing Poros BA was whitened (not colored). Each molten agarose / bead combination was fitted with a 1 ml syringe and placed in a 1 mm diameter, 10 cm long plastic tube immersed in cold water. After a few minutes, the agarose solidified into a jelly-like rod containing approximately 50% by volume of beads. The four rods thus obtained (each containing one of the four types of beads described above and having a different protein coating) were placed in an aluminum channel containing a larger amount of molten agarose and had a square cross section. An array was formed by embedding two parallel rods in the vertical and horizontal directions in the agarose bar.

【0153】

After the bar solidified, the gel was removed from the aluminum channel mold, a thin section perpendicular to the axis of the bar (and filament) was cut to make a section across it, which was mounted on a glass slide. .. Upon microscopic examination, this section showed four circular region patterns (and filaments) containing a particulate material (carrying an immobilized protein) surrounded by a clear embedding matrix of agarose. The circular area of the embedded beads was more stable and did not crack.

【0154】

To test specific protein binding to beads in the four circular regions of the sections forming the microarray, commercially available HSA and Tf proteins were placed on Cellite (Sigma) with fluorescein isothiocyanate (FITC). Signed. Dissolve this protein in approximately 4 ml of 0.4 M sodium bicarbonate buffer (-pH 8.3) and add 4.5 mg or less of HSA to 30 mg of FITC on Celite and 2.8 mg or less of HSA on dry FITC on Celite, respectively. Tf was added to 18 mg FITC on Celite, and 4.5 mg or less of serum protein (20 Ll) was added to 10 mg FITC on Celite.

【0155】

Then, the reaction was carried out at room temperature for 30 minutes. Celite was removed by centrifugation, the supernatant protein and the unreacted dye were placed in a protein centrifuge, and the protein was repeatedly diluted and reconcentrated in buffer for washing. The fluid was centrifuged to remove Celite and supernatant protein, and centrifugation was repeated with 4 ml of sodium bicarbonate buffer until clear.

【0156】

Sections of the four filament arrays were laid flat on glass microscope slides and exposed to a solution of fluorescently labeled HSA. During exposure to the section, the protein was thought to interact specifically with the antibody contained on the two filaments (the circular region of the section). The two filaments carried a carrier for HSA and a mixed carrier for anti-HSA, Tf and Hp. The labeled HSA could not interact with filaments carrying only antibodies to Tf or filaments carrying only streptavidin.

【0157】

These sections were examined with an epifluorescence microscope equipped with a 500 nm low bandpass filter for fluorescein fluorescence detection, a 510 nm high bandpass filter and a 35 nm camera. Vivid fluorescence was seen in all four circular Poros regions before multiple washes, but there was no distinguishable difference. Since the fluorescence in the Poros region was stronger than that in the agarose matrix surrounding the filament, it can be seen that the labeled HSA could be freely diffused in the section in the particle-containing region because the pores of the Poros particles remained unobstructed.

【0158】

The sections were repeatedly washed in PBS and examined again under a fluorescence microscope. When the obtained image was made into a 35 mm color slide, it was found that after washing, the labeled albumin was specifically bound to two filaments containing the HSA antibody and migrated from the other two. From this, it can be seen that the intercept has the ability to specifically detect individual proteins. The positions of the two specifically labeled filaments are diagonally opposite to each other in the 2x2 array, which is the diagonally opposed anti-HSA filaments and the mixed anti-HSA, Tf and Hp agarose filaments. It matched the position. Example 2: Manufacture and use of diagnostic arrays to detect autoantibodies against mitochondrial or lysosomal proteins Suspended substances of fully isolated rat and mouse liver mitochondria, lysosomes and expressed proteins are suspended or dissolved in aqueous buffer at a concentration of 10 mg / ml and optionally fixed with glutaraldehyde (1%). 1 ml of each preparation is mixed with 20 ml of catalyst infiltration resin JB-4 (Polysciences) prepared by mixing 20 ml of monomer A containing 0.17 g of catalyst according to the instructions of the kit. After complete mixing, add 40 ml of monomer B containing 0.17 g of catalyst and stir. After this is completely dissolved, 0.8 g of the accelerator is added, and this mixture is put into a syringe and injected into a Teflon capillary tube having an inner diameter of 0.0625 inch in an oxygen-free environment.

【0159】

Polymerization occurs after approximately 50 minutes at room temperature. The end of the tube is heat-sealed and refrigerated for use, or immediately extruded for use in the production of fiber bundles. Bundles are made by arranging 10 or more fibers in parallel in an elongated Teflon box to form a one-layer array. Add protein-free JB-4 resin and evacuate this box for a short time to remove air bubbles and cure the resin. Several flat arrays can be stacked in parallel to form a three-dimensional structure, and the entire structure can be further evacuated to form a three-dimensional bundle. After polymerization, the bundle is cut with a steel or glass microtome knife to make sections 5-20 microns thick and the sections are placed on glass slides. This section is mounted on a plastic mount (Plastic Mount®) or dried and mounted on Poly-Mount X (Plastic Mount®) (available from Polysciences).

【0160】

Test for autoantibodies by placing 0.25 mL of a 1:10 dilution of human serum on each chip and incubating this array at 25 ° C for 20 minutes. The array is rinsed 4 times with phosphate buffered saline and then impregnated with a goat-derived anti-human globulin solution conjugate with horseradish peroxidase. After incubating for 20 minutes, the array was washed 4 times with buffer and placed in a 3,3', 5,5'-tetramethylbenzidine solution in an organic base, which was subjected to citrate hydrogen peroxide. Add hydrogen peroxide solution (0.02%). The presence of blue insolubles indicates the presence of autoantibodies. Example 3: Manufacture and use of a diagnostic array with a histologically embedded support An array incorporating immobilized infectious particles used to detect convalescent antibodies appearing later in the inflammatory course is made. This is important for pursuing the sentinel population and identifying the inflammation that is occurring.

【0161】

Immuno-Bed GMA water-miscible embedding medium (Polysciences Inc.) was generated as instructed, and a small amount thereof was immobilized with a heterologous suspension of immobilized selective virus (mean titer 109 virus / ml) or immobilized. Mix with the virus cells (107 particles / ml on average). This suspension is placed in a syringe and extruded under pressure into a Teflon® tubule with an inner diameter of 1/16 inch and polymerized at room temperature. The tubules are pretreated with metallic sodium in an organic medium to form a surface that adheres to the epoxy resin. The polymerized fiber is refrigerated in a coiled Teflon® capillary.

【0162】

A jig is used to hold the fibers in a parallel array, the arrays are combined into a bundle, and then the epoxy resin is infiltrated into the array. The bundle including the Teflon® thin tube portion thus obtained is sliced, and the section thereof is mounted on a glass slide with an epoxy resin mounting medium. The sections are washed and rehydrated before exposure to convalescent antisera. The chips are repeatedly washed and exposed to goat-derived anti-human IgG containing the optical brightener fluorescein conjugated. Fluorescence is detected and measured with a CCD camera to identify convalescent antibodies. Example 4: Manufacture of a diagnostic array using sintered strips A 1/16 inch thick sintered polystyrene sheet is cut into multiple strips with square cross sections, each of which is rhinovirus, simple blisters virus, influenza virus type A, respiratory fusion cell virus, blisters zoster virus. 1 against a range of microorganisms including viruses such as (vesicles), tuberculosis, giant cell virus, Epsteiner virus, hepatitis B virus (surface antigen and another core antigen), poliovirus (types 1, 2, 3) and others It was exposed to a dilute solution of monoclonal antibody. After rinsing and drying these strips, they are glued together with an acrylonitrile adhesive to form a three-dimensional array. This is sliced to make an array with a thickness of 5 to 100 microns. Nucleic acid-specific dye YOYO-1 (Molecular) is used to prepare biological samples containing infectious viruses from individuals with viral diseases. After fluorescence staining with Probes) and isolation, the infectious particles are concentrated by centrifugal microbanding to obtain a microliter amount. See International Patent Application WO 099/46047 above. The concentrated virus is applied to the array and mechanically agitated for 1 hour to spread the virus particles throughout the array. The array is then washed, aspirated to remove excess fluid, and irradiated with UV light at 490 nm. The image is captured by an Apogee CCD camera with a 520nm filter. The image is processed by the PMIS image analysis program to obtain quantitative data. Example 5: Manufacture and use of diagnostic arrays containing immobilized oligonucleotides Polystyrene beads (10 to 50 microns in diameter) from a solid-phase oligonucleotide synthesis containing an oligonucleotide covalently bonded are suspended in a buffer and filled in a hollow glass fiber having an inner diameter of 500 microns. The pressure at this time is initially hydrostatic pressure, but then the support liquid is discharged at an atmospheric pressure of 500 psi or less. The fiber is then heated for a short time under conditions adjusted to sinter a portion of its contents. An array of fibers is prepared according to the method described in the above examples, and a vacuum is intermittently formed to remove air bubbles while embedding the resin in a low-viscosity epoxy resin, and then the resin is cured. Slice this bundle using a diamond saw. This array is used in a flow throw arrangement in which the material on it can be manipulated in a manner similar to that performed on large multiwell microtiter plates as described in US Pat. No. 5,843,767 above. Example 6: Manufacture of a multi-well plate The shape of a commercially available glass capillary array (GCA) (Galileo) is a thin disc measuring 2.5 cm x 2.5 cm x 0.5 mm thick. Approximately 50% of the area of this GCA consists of 50 μ holes or approximately 156,000 holes with a total volume of approximately 0.1 ml. The bottom surface of this GCA is glued to a Teflon® sheet with cyanoacrylate adhesive (SUPER GLUE). Example 7: Cloning and replication plate culture in a glass capillary array Streptococcus pyogenes Group A colonies and Group B colonies are selected from plates and mixed in agar to form a suspension of microbial cells (other microbial, animal or plant cells are similarly applicable). did. This is diluted to a concentration of approximately 20,000 cells per ml of medium. Apply about 0.1 ml of suspension to the surface of GCA. This ensures that only one cell is cloned at a rate of about 1 cell per 100 holes. Place this GCA in a sterile Petri dish, cover and incubate overnight at 37 ° C.

【0163】

Two more sterile GCAs without a Teflon® sheet on the bottom are filled with 0.1 ml of heated liquid culture fluid supplemented with 1% agarose. It is then cooled to near condensation and stacked directly on top of the GCA containing the cloned bacterial cells so that the holes in each GCA are aligned. Teflon® top sheets are tightly compressed and the laminates are fastened together. The entire laminate is turned upside down and incubated at room temperature for 5 minutes. The entire laminate is turned sideways and incubated overnight at 37 ° C. Next, the laminated body is made vertical to release the tightened state, and each GCA is separated. Keep the original GCA for continued use.

【0164】

Each of the two added GCAs was placed in a glass flask, placed in a lyophilizer and vacuum dried for 1 hour. Remove the GCA, add 0.1 ml of FITC conjugated binding antibody (manufactured by DIFCO) against Streptococcus pyogenes group A to each GCA, and incubate at room temperature for 10 minutes. Next, each GCA is blotted on absorbent tissue paper (manufactured by KIMWIPE) to remove the fluid. The microarray is immersed in PBS, washed, re-blotted and dried. A CCD scanner with 12.5 μpixels and a resolution of 25 μm required to detect holes containing cell clones detects fluorescent holes contained in GCA and bacteria-containing holes contained in the original GCA.

【0165】

The scanner first scans the amount of fluorescence and then the absorbance to detect the presence or absence of bacterial clones. Absorbance is used to indicate the presence or absence of bacteria and align the holes in the two GCAs. Fluorescence can be detected in some, but not all, holes containing bacterial clones of the original GCA, which corresponds to the presence or absence of Group S bacteria. Example 8: Selection of monoclonal antibody The monoclonal antibody-secreting hybridoma in suspension was diluted in a culture of approximately 20,000 cells per ml of RPMI1640 and 5% fetal bovine serum, 0.1 ml was added to the GCA of Example 6, and the method of Example 7 was repeated. It was. However, CO for 2 days₂Incubated in an incubator at 37 ° C. and GCA was pretreated with 10% fetal bovine serum for 30 minutes. Another GCA was filled with protein-free saline and stacked and tightened. The laminate was incubated at room temperature for 15 minutes without any rotation. After releasing this tightening as described above, it was vacuum dried. Approximately 0.1 ml of FITC covalent goat-derived anti-mouse immunoglobulin was added to this additional GCA, incubated as described above, removed, washed and scanned for fluorescence. The antibody-secreting hybridoma is estimated from the fluorescence position of GCA. Example 9: Protein screening library for biological performance According to Baekkeskov et al., Diabetes 38 (9): pp. 133-41 (1989), human serum proteins are separated by two-dimensional electrophoresis. Cut out 200 spots on the gel and dialyze each protein into 1 ml of PBS. 1 ml of protein solution is mixed with 40 mg of acrylamide monomer containing a catalyst and injected into a polypropylene tube having an inner diameter of 1 mm and a length of 1 m. Heat seal the ends of the tubes and tag each tube. Numerous comparative tubes are made containing various dyes so that the correct orientation of the microarray can be easily confirmed during formation. Polymerize acrylamide overnight. Align the tubes within the bracket and glue between the rows as described above. Under solidification conditions, the bundle is sliced into 10 micrometer-thick sections by a microtome and the microarray is immediately fixed on a plastic sheet.

【0166】

Mouse monoclonal antibodies (Vector Labs) against the following antigens were contacted on separate microarrays, incubated, washed, dried, contacted and scanned with FITC covalent goat-derived anti-mouse immunoglobulins as in Example 8 above. It was. Common antigens to be tested include insulin, calcitonin, glucagon, epidermal growth factor, interferon, CEA, prostatic acid phosphatase and human IgG. Both hormone levels and tumor antigen levels can be identified by semi-quantitative methods. Example 10: Rapid antibiotic susceptibility test A microarray is prepared according to Example 2. However, each tube is filled with an agar medium mixed with various antibiotics in the configuration described below. Five 2-fold dilutions of erythromycin, penicillin V, tetracycline, ampicillin, trimethoprim / sulfamethizole, cefaclor, ofloxacin and nitrofurantoin, and each available as an antibiotic at useful concentrations in the effective spectrum. Use 10 2-fold diluted solutions of 34 new compounds that are candidates.

【0167】

Colonies of unknown E. coli samples grown from patient urine were suspended in 1 ml of culture medium supplemented with fluorescein acetate or trypan blue, placed on each of two microarrays and incubated at 37 ° C. The fluorescence and absorbance of this microarray were scanned at the start of the incubation and 30 minutes later. Microarray cells with a detectable increase in fluorescence (scanned fluorescence-fluorescence from initial scan) were considered to have proliferated, and were alive in microarrays with increased trypan blue absorbance 30 minutes after initiation. Considered that there is a cell. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were thus identified. Similarly, the expected efficacy of the new candidate compound was estimated.

【0168】

1 ml of saline containing another colony in which an unknown sample of E. coli was suspended was plate-cultured on a conventional Mueller-Hinton plate equipped with an antibiotic disc and incubated overnight. The MIC was identified the next day based on the diameter of the stunted area. The MIC of this microarray is comparable to standard growth inhibition measurements. For example, nitrofurantoin is susceptible to infection if its area diameter in millimeters on a 300 mcg disc is> 17 mm, medium if it is 15-16 mm, and resistant if it is <14 mm, respectively. Corresponds to <32, 64 and> 128 of the MIC in units of / ml. The 2-fold diluted nitrofurantoin in this microarray is 16, 32, 64, 128 and 256 mcg / ml.

【0169】

This method is repeated with well-known E. coli strains with different levels of antibiotic resistance and many types of common microorganisms with different levels of antibiotic resistance. The results obtained indicate which of the 34 candidate compounds should be further tested as a potential antibiotic. Example 11: Anticancer properties and drug screening Alkalilytic and protease K degradation suspension of new cells from leukemia patients, several leukemia cell lines (HTB, ATCC, etc.), normal peripheral leukocyte cells and normal bone marrow cells according to the method described in Example 2. Make a microarray containing. This microarray is heat denatured and applied to it with digoxigenin-labeled DNA probes for N-myc, C-myc, K-ras, p53, HER-2 / neu and candidate DNA probes for diagnostic purposes. Texas Red labeled anti-digoxigenin antibody is added to identify binding patterns and amounts. Example 12: Hepatitis test For best treatment of patients, it is desirable to know the type of viral hepatitis and the extent of its infection. A microarray is prepared as described in Example 2. However, use 10 2-fold dilutions of mouse monoclonal antibodies against HAV, HBsAg, HBcAg, HCV, HDV and HEV and 2-fold dilutions for the same antigen. Prepare three tubes for each and use them in the microarray with the pattern of the comparative example.

【0170】

Approximately 3 drops of serum sample are contacted on this microarray, incubated in a 37 ° C water bath for 10 minutes, and then washed 4 times with PBS. Approximately 1 ml of fluorescein-labeled monoclonal antibody reagent against non-overlapping epitopes for each antigen, fluorescein-labeled mouse-derived anti-human IgG and rhodamine-labeled mouse-derived anti-human IgM was added to the microarray and then incubated in a water bath at 37 ° C for 10 minutes. Wash 4 times with PBS. The microarray is scanned for fluorescence at both wavelengths of fluorescein and rhodamine emission, and the results identify the cell that was emitting fluorescence in the microarray, the wavelength of the light, and its level.

【0171】

This microarray is intended for both initial diagnosis and surveillance and remission by detecting antigens and antibodies in convalescent sera. Quantitative results can be obtained by measuring the amount of fluorescence in each cell using a 2-fold diluted solution. Example 13: Screening of active compound candidates Microarrays are made according to Example 2, except that 380 new candidate compounds are introduced into the fiber. After adding 3 drops of the solution containing glutamate receptor 2 to the microarray, incubate at 37 ° C for 10 minutes. As mentioned above, the microarray is washed and dried. After adding a 1:10 dilution of the mouse-derived monoclonal antibody against glutamate receptor 2 (Vector Labs), incubate, wash and dry as described above. FITC conjugated goat anti-mouse IgG is added and this microarray is scanned.

【0172】

The fluorescent cell corresponds to the compound that binds to the receptor. Because this receptor is involved in learning, memory, seizures and other neuropathological conditions, binding to the neurotransmitter glutamate makes both agonists and antagonists pharmacologically targeted. Example 14: Fluorescence Microarray Formation and Analysis As a comparison, a microarray was used as a comparison between a) a columnar polymethacrylate fiber containing microbeads on which an antibody against rat-derived IgG was immobilized, and b) a columnar polymethacrylate fiber containing microbeads on which an antibody against human IgG was immobilized, and c). It is made from a columnar polymethacrylate fiber that does not contain microbeads. These fibers were aligned longitudinally to form an array, sliced with a microtome, and then the sections were transferred to glass slides. These slides were tested on a fluorescent immunoassay and demonstrated specific protein binding to the beads as follows:

【0173】

Two disposable columns, each containing approximately 0.5 ml of UltraLink Immobilized Streptavidin Plus beads (50-80 microns in diameter, 10 mg biotin-BSA volume per ml bead, Pierce Chemical Co., Rockford, Illinois), each containing 0.05% sodium azide. pH 7.2 Washed with phosphate buffered physiological saline. These slides were treated 5 times in a row with 1 ml of solution containing 0.5 g of biotin-labeled goat anti-human IgG in one column and 0.5 g of biotin-labeled goat anti-mouse IgG in the other column. These columns were treated with excess biotin and washed with PBS.

【0174】

The embedding material used was ImmunoBed (Polysciences, Inc., Warrington, PA) prepared according to the manufacturer's instructions. The drying catalyst (225 mg) was dissolved in 25 g of ImmunoBed Solution A. To this solution was added 1 ml of ImmunoBed Solution B. The mixture was introduced into a 4-foot long Teflon tubule (1/32 inch inner diameter) using a syringe attached to the tubule while keeping the temperature of the mixture low. This thin tube filled with ImmunoBed resin was left overnight at room temperature. By cutting the periphery of the end of the thin tube with a single edge razor to expose the fiber and gently pulling the fiber out of the thin tube, the polymerized fiber could be pulled out from the Teflon thin tube.

【0175】

UltraLink beads containing antibodies to human IgG and rat IgG were prepared as described above. Centrifugation at 2000 rpm for 10 minutes collected about 0.5 ml each and mixed these with 5 ml cold ImmunoBed solution (Solution A + catalyst + Solution B) prepared as described above. The beads were then centrifuged at 2000 rpm for 10 minutes at 5 ° C. This was repeated 3 times. The spherical beads were resuspended in 1 ml of ImmunoBed solution and introduced into a Teflon capillary having an inner diameter of 1/32 inch. The tubes were folded into bundles, placed in a centrifuge bucket, and centrifuged at 2500 rpm for 10 minutes. The bucket was removed and left overnight at room temperature to polymerize ImmunoBed. Sections of this bundle were made by cutting the bent apex and the strands were extruded.

【0176】

Two comparative fibers and two experimental fibers were cut to a length of 1.5 cm each. These fibers were aligned longitudinally and placed in a groove within the Teflon block. A glass slide was placed on top of this fiber and pressed in place to expose each fiber of approximately 1 mm. ImmunoBed solution (Solution A + catalyst + Solution B) was poured onto the exposed tip of the fiber and allowed to flow under a glass slide to fill the gap around the fiber and between the fibers. The structure was allowed to stand overnight at room temperature for complete polymerization. The array was removed from the mold and sliced with a Leica Moddel RM-2155 Microtome. The sections (10 microns) were transferred to a glass slide containing 20 μl droplets of water and the water was evaporated at room temperature. By doing so, the sections were brought into close contact with the glass slides. Background fluorescence is stronger in 50 micron thick sections.

【0177】

The 10 micron thick sections prepared above and mounted on glass slides were treated at room temperature for 60 minutes with 100 μl of normal rat serum (containing IgG) diluted 1:50 with PBS containing 1 mg / ml BSA. The solution was drained from the slide, rinsed with 100 μl PBS / BSA for 1 hour, and then washed 3 times with 100 μl PBS / BSA for 5 minutes before draining. After the final wash, 100 μl of R-phycoerythrin-labeled affinity purified goat antibody against rat IgG (H + L) diluted 1: 100 with PBS / BSA was added and left at room temperature for 60 minutes. The solution was then drained and washed 4 times as described above. After fluorescent immunostaining, the sections are Olympus It was viewed with a model BX-40 fluorescence microscope (Olympus America, Inc., Melville, NY) using a green filter (excitation filter 510 to 550 nm, circuit breaker filter 590 nm). The four circular slices containing the 10 micron thick slice also contained two comparative slices, one containing beads with anti-human IgH and the other containing beads with anti-rat IgG. It was included. The fluorescence of the circular slice containing the antibody against rat IgG was stronger than that of the slice containing the anti-human IgH, so that the two comparative slices demonstrated the specificity of the reaction.

【0178】

[Table 2]

<img file="JP2003014751A_D0002.tif" />It should be understood that various modifications can be made to the embodiments disclosed herein. Therefore, the above description is not construed as restrictive and merely illustrates preferred embodiments. It will be apparent to those skilled in the art that other modifications can be made without departing from the scope and intent of the claims attached herein.

【0179】

The entire contents of all the patents and documents cited in this specification shall be cited in this specification. References book Hermanson, Greg T. Bioconjugate Techniques. Academic Press, New York. 1995, 785 pp. Hermanson, GT, Mallia, AK &amp; Smith, PK Immobilized Affinity LigandTechniques. Academic Press, 1992, 454 pp Publications Ogura, M., Agata, Y., Watanabe, K., McCormick, RM Hamaguchi, Y., Aso, Y., and Mitsuhashi, M. RNA chips: Quality assessment of RNA by microchannel linear gel electrophoresis in injection-molded plastic chips. Clin. Chem. 44: 224955, 1998. Johnston, M., Gene chips: Array of hope for understanding gene regulation. Cur. Biol. 8: R171-4, 1998. Jordan, BR, Large-scale expression mesurement by hybridization methods: from high-density membranes to "DNA chips". J. Biochem. (Tokyo) 124: 251-8, 1998. Pevzner, PA, Lysov, Yu.P., Khrapko, KR, Belyavsky, AV, Florentiev, VL and Mirzabekov, ADJ Biol. Struct. Dyn. 9: 399-410, 1991. Hacia, JG, Brody, LC, Collins, FS Applications of DNA chips for genomic analysis. Mol. Psychiatry 3: 483-92, 1998. Ramsay, G., DNA chips: State of the art. Nat. Biotechnol. 16: 40-4. 1998 Kozal, M., Chee, M., Shah, N. Yang, R., Gingeras, T. Development of DNAchips for the rapid sequence analysis and the development of drug resistant mutations for the HIV protease and reverse transcriptase genes. Natl. Conf . Hum. Retroviruses Relat. Infect. (2nd) 1995: 93. Fodor, SP, Rava, RP, Huang, XC, Pease, AC, Holmes, CP, Adams, CL Multiplexed biochemical assays with biological chips. Nature 364: 555-6, 1993. Fodor, SPA, Read, LJ, Pirrung, MC, Stryer, L., Lu, AM, and Solas, D. Light-directed spatially addressable parallel chemical synthesis. Science 251: 767-773, 1991. Cheng, J., Shoffner, MA, Hvichia, GE, Kricka, LJ, and Wilding, P. Chip PCR II. Investigation of different PCR amplification systems in microfabricated silicon-glass chips. Nucleic Acids Research 24: 380-5, 1996. Woolley, AT, and Mathies, RA Ultra-high-speed DNA fragment separations using microfabricated capillary array electrophoresis chips. PNAS USA91: 11348-52, 1994 Southern, EM, DNA chips: Analysing sequence by hybridization to oligonucleotides on a large scale. Trends in Genetics. 12: 110-5, 1996. Birnbaum, S., Uden, C., Magnusson, GM, and Nilsson, S. Latex-based thin layer immunoaffinity chromatography of quantitation of protein assays. Analytical Biochemistry. 206: 168-171, 1992. Bellara, SR, Cui, Z., MacDonald, SL, and Pepper, DS Virus removalfrom bioproducts using ultafiltration membranes modified with latex particle pretreatement. Bioseparations 7: 79-88, 1998. Van Oss, CJ, and Singer, JM The binding of immune globulins and other proteins by polystryene latex particles. J. Reticuloendothelial Society 3: 29040, 1966. Arlinghaus, HF, Kwoka, MN, and K. Bruce Jacoson Analysis of biosensor chips for identification of nucleic acids. Anal. Chem. 69: 3747-3753, 1997. Wang, J., Cai, X., Rivas, G., Shiraishi, H., and Dontha, N. Nucleic-acid immobilization recognition and detection at chronopotentiometric DNA chips. Biosensors &amp; Bioelectronics 12: 587-599, 1997. Livache, T., Bazin, H., Caillat, P., and Roget, A. Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips. Biosensors and Bioelectronics 13: 629-634, 1998. Syvanen, AC. From gels to chips: "Minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms. HumanMutation 13: 1-10, 1999. Shevalier, A., Mikhailov, M., and Nikolaeva, I. New fast low-cost method of HIV dignostics based on carbon-conjugated antigens. Abstr. PB0420Itent International Conferences on AIDS. Abstr. Book Volume 1. Internationatl Conference on STD. Yokohama Japan, 7-12, August, 1994. Inomata, Y., Wada, T., Handa, H., Fujimoto, K., and Kawaguchi, Preparation of DNA-carrying affinity latex and purification of trascription factors with the latex. J. Biomaterial Sci., Polymer Edn. 5: 293-301, 1994. Balhorn, R., Allen, M., Tensch, B., Marzrimaz, JA, Balooch, M., Siekhaus, W., Imaging of DNA molecules deposited on Graphite, in "DOE / NIH Human Genome Contractors. Grantee Workshop", Santa Fe, 34 (1989). Sundarababu, G., Gao, H, and Sigrist, H. Photochemical linkage of antibodies to silicon chips. Photochemistry and Photobiology 61: 540-544, 1995. Regnier, FE, He, B., Lin, S., and Busse, J. Chromatography and electrophoresis on chips: Critical elements of future integrated, microfluidicanalytical systems for life scinece. Trends in Biotechnology 17: 101-106. 1999 Patent US 5,843,767 Microfabricated, flow-through porous apparatus for discrete detection of binding reactions. US 4,289,623 Hollow fiber dialysis US 3,976,576 Dialyzer cartridge --Also, use of dialyzer cartridge by filling hollow fibers and embed protein in fibers as they are formed before the cartridges are cut.

[Simple explanation of drawings]

[Figure 1]

It is a schematic diagram which shows the product of the intermediate stage in the process of making a microarray.

[Figure 2]

It is a schematic diagram which shows each capillary containing the immobilization ligand embedded in a gel.

[Fig. 3]

It is a schematic diagram which shows each capillary containing the gel which adsorbed the ligand.

[Fig. 4]

FIG. 6 is a schematic representation of an array comprising means of adsorbing and containing a ligand on the inner wall of a cell and occluding one surface to form a set of microwells.

[Fig. 5]

FIG. 6 is a schematic showing a means for ensuring that all fibers are maintained in their correct pattern before slicing the bundle.

[Fig. 6]

It is a schematic diagram which shows the means for identifying an array.

[Fig. 7]

It is a schematic diagram which shows the scanning method of an array.

[Fig. 8]

Another method of forming a fiber bundle is shown.