KR20050078268A - Method for producing a biomolecular probe layer and a biomolecular probe layer produced by using the same and method for producing a microarray using the same - Google Patents

Abstract

The present invention comprises the steps of mixing the biomolecular solution and the liquid polymer; Molding the biomolecule-polymer mixture to form a biomolecule-polymer film; Cutting the biomolecule-polymer film to form a fiber; And laminating the biomolecule-polymer fibers and cutting the bundle of biomolecule-polymer fibers inclined with respect to the longitudinal direction to form a biomolecule layer, the method of producing a biomolecule probe layer for a biomolecule microarray. To provide.

Description

Method for producing a biomolecular probe layer and a biomolecular probe layer produced by using the same and method for producing a microarray using the same }

The present invention relates to a method for producing a biomolecular probe layer, a biomolecular probe layer and a microarray manufacturing method produced thereby.

Generally, a "microarray" is a group of biomolecules such as polynucleotides or proteins immobilized at a high density on a solid substrate, and the polymer molecules are immobilized in a constant region, respectively. Such microarrays are well known in the art. Microarrays are disclosed, for example, in US Pat. Nos. 5,445,934 and 5,744,305. Examples of such microarrays include protein and polynucleotide arrays. In addition, "prov" generally refers to a molecule that is immobilized on the microarray, and specifically binds to a target molecule.

Various methods have conventionally been used to immobilize biomolecules on microarray substrates. Methods for transferring the reaction liquid used for the immobilization reaction onto the substrate include, for example, spotting, ink-jet, and electrical methods. The spotting method delivers the reaction liquid using a mechanical field, and the ink-jet method uses a mechanical force derived from a thermal force (bubble jet) or an electrical force (piezoelectric effect) to eject liquid from the nozzle. Discharge and transfer to the substrate. In addition, the method of immobilizing the biomolecule on the substrate includes, for example, in situ synthesis method by photolithography, covalent bonding method and adsorption method. In situ synthesis is a method of synthesizing a nucleic acid by extending one or more nucleotide units on a substrate, and covalent bonding and adsorption are immobilization of the already synthesized nucleic acid on a substrate by covalent bonding or adsorption. This method uses a solution containing a high concentration of salt, and since such a solution is used in a small amount at each spot, a problem arises in that the reaction solution evaporates. In other words, the nozzle, which is the part that transfers the reaction solution and comes into contact with the substrate, especially the string part of the nozzle, needs to have a small diameter because it handles a small amount of the reaction solution. As the diameter decreases, the ratio of the surface area to the volume of the reaction liquid discharged therefrom increases, so that the water evaporates on the droplet surface is high. As a result, the salt inside the droplets crystallizes and becomes a solid state, and the solidified salt blocks the nozzle. In addition, since most of the reaction solution distributed on the solid substrate does not participate in the reaction, the loss of the reaction solution is large. In particular, when the spotting method is used, it is known that the loss of the reaction solution is 90% or more.

In the production of conventional microarrays, a method of using fibers in producing the probe layer has been known. US Patent No. 6,534,270 discloses a fiber wound on a solid support in a solution containing biomolecules to immobilize biomolecules on each fiber, and arrange the fibers so that they are spaced apart from each other by a set distance from each other. Immersion of the arrayed fibers in the frozen aqueous material by immersing in an aqueous solution and freezing, cutting the impregnated fibers in a direction perpendicular to the longitudinal direction of the fibers to obtain chips, which are placed on a substrate and the frozen aqueous material A method of manufacturing a biochip is disclosed, wherein the fibers having immobilized biomolecules remain immobilized on the substrate by removing them. European Patent Publication No. EP 1162262 also discloses fibers in which nucleic acids are immobilized. However, these methods have a problem in that the biomolecules are not uniformly adsorbed by adsorbing the biomolecules to the fiber prepared already. In addition, there is a disadvantage that it can not be confirmed whether the uniformly adsorbed until the final microarray is manufactured.

 The present inventors, while studying to solve the problems of the prior art as described above, by using a method of homogeneously mixing the mixture of biomolecule and polymer, it is possible to avoid the problem caused by evaporation while reducing the loss of the reaction solution. And the fact that biomolecules can be concentrated at high density, the present invention has been completed.

It is an object of the present invention to provide a method for producing a biomolecule probe layer for a microarray.

It is also an object of the present invention to provide a biomolecular probe layer prepared by the above method.

It is also an object of the present invention to provide a method for producing a microarray using a biomolecular probe layer prepared by the above method.

The present invention comprises the steps of mixing the biomolecular solution and the liquid polymer;

Molding the biomolecule-polymer mixture to form a biomolecule-polymer film;

Cutting the biomolecule-polymer film to form a fiber; And

Stacking the biomolecule-polymer fibers and cutting the bundle of biomolecule-polymer fibers inclined with respect to the longitudinal direction to form a biomolecule layer, the method of producing a biomolecule probe layer for a biomolecule microarray. to provide.

In the process of the invention, the biomolecular solution is first mixed with at least one monomer which is the polymer or the basic unit of the polymer. The polymer or monomer of the present invention may be liquid or solid. When the polymer or monomer is in solid phase, it may be present as a solution or suspension dissolved in water or a solvent miscible with water. As used herein, the term "biomolecule" refers to a molecule of natural, synthetic or semisynthetic origin, including polynucleotides or polypeptides. Preferably a polynucleotide or polypeptide. In the present invention, the polymer is not limited to a specific polymer as long as it can be mixed with the biomolecule solution. Preferably, the polymer is a water soluble, water affinity or water dispersible polymer. Examples of water-soluble or water-miscible polymers include polyethylene oxide, NiPaAm (N-isopropyl acrylamide), polyacrylamide, agarose gel, PPO (polypropylene oxide), chitin, chitosan, poly ( Vinylalcohol) and combinations thereof. In addition, although the nature of the polymer itself is not high in water affinity, materials that can be dispersed at a microlevel in water using methods such as grafting, copolymerization, ionic bonding, hydrogen bonding, and micelle formation Included. Examples of such water dispersible materials may include poly (lactic acid) / poly (ethylene oxide) system, poly (glycolic acid) / poly (ethylene oxide) system, PEDOT dispersed in PSS, that is, PEDOT / PSS and the like. Mixing of the biomolecular solution with the polymer or monomers thereof can be accomplished by conventional methods and is thoroughly mixed to obtain a homogeneous mixture.

Optionally, the biomolecule-polymer or mixture of monomers thereof obtained in the present invention may be subjected to physical or chemical treatment to enhance processability in subsequent film formation or fiber formation processes. For example, a crosslinking agent or catalyst can be added to the mixture. By doing so, heat or light may be applied to the mixture of biomolecule-polymers or monomers thereof to carry out a reaction such as crosslinking to improve the mechanical properties or processability of the mixture.

The method of the present invention comprises molding the biomolecule-polymer or mixture of monomers thereof to form a biomolecule-polymer film. As used herein, the term "film" means a very thin film, and preferably a thin film having a thickness of several to several hundred micrometers. Processes for forming such thin films are known in many industries, including semiconductor manufacturing processes. For example, such films can be made using spin coating, dip coating, and doctor-blade.

The present invention also includes cutting the biomolecule-polymer film to form a fiber. As used herein, the term "fiber" refers to a cut body that is cut out from the film, and has a length that is long compared to the width and has a thread-like shape regardless of the shape of the cross section. When the film is cut, the fiber may be manufactured by using a device such as a general microtome or by using a spinning machine that emits the fiber itself without generating a film. In this case, the fiber is a state in which the biomolecule is uniformly contained therein.

In another embodiment of the method of the present invention, the step of directly molding the biomolecule-polymer or a mixture of monomers thereof comprises forming a biomolecule-polymer fiber. Methods of forming fibers from polymers or mixtures of monomers thereof are well known. The formation of the fibers may for example form the fibers of the biomolecule-polymer by a wet or dry spinning process.

The method also includes laminating the biomolecule-polymer fibers and cutting the obtained bundle of biomolecule-polymer fibers obliquely with respect to the longitudinal direction to form a biomolecule layer. The biomolecule-polymer fibers may be laminated to have a cross section suitable for the microarray in consideration of the arrangement of the probes. Such lamination may, for example, but not be limited to laminating the polymer fibers in a predetermined container. The polymer fiber bundles thus laminated can be cut obliquely, preferably vertically, with respect to the longitudinal direction of the fibers using suitable cutting means, for example a microtome. By doing so, it is possible to form a biomolecule probe layer in which biomolecules such as DNA or RNA are homogeneously dispersed in the polymer. The biomolecule pro layer may be immobilized on a microarray substrate to be useful for preparing a microarray.

The present invention also provides a biomolecule probe layer for a biomolecule microarray produced by the method of the present invention. The biomolecule probe layer of the present invention has a high density of biomolecule-polymer fibers in which biomolecules such as DNA or RNA are evenly dispersed in the polymer. The biomolecule probe layer may be used in the manufacture of a microarray by immobilizing on a microarray substrate.

The present invention also provides a method of manufacturing a microarray comprising immobilizing a biomolecular probe layer prepared by the method of the present invention on a microarray substrate. When the biomolecular probe layer is immobilized, covalent / ion / hydrogen bonds between the substrate and the polymer and physical / chemical adsorption may be utilized by external energy such as heat. In addition, the biomolecule / polymer probe layer may be fixed to the substrate using a method of inserting a chemical for adhesion between the film and the substrate.

Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

According to the probe layer manufacturing method of the present invention, there is no problem caused by evaporation of the biomolecule solution or clogging of the array fin by the biomolecule solution. In addition, since the biomolecules are directly mixed and processed by the polymer, the loss of the reaction sample can be reduced. Whether the biomolecules are uniformly immobilized on the microarrays can be confirmed by checking whether the mixture of the biomolecules and the polymer is homogeneous, rather than checking the microarrays in a fixed state, thereby facilitating the quality assurance of the microarrays. In addition, since the biomolecule is mixed in the polymer and the solution state, the density of the biomolecule immobilized in the microarray spot can be increased.

According to the probe layer for the microarray of the present invention, a high quality microarray can be easily manufactured.

According to the microarray manufacturing method of the present invention, high-quality biomolecule microarray can be efficiently produced at low cost.

Claims (9)

Mixing the biomolecule solution with the liquid polymer; Molding the biomolecule-polymer mixture to form a biomolecule-polymer film; Cutting the biomolecule-polymer film to form a fiber; And Stacking the biomolecule-polymer fibers and cutting the obtained biomolecule-polymer fiber bundles obliquely with respect to the longitudinal direction to form a biomolecule layer, the method of producing a biomolecule probe layer for a biomolecule microarray. Mixing the biomolecule solution with the liquid polymer; Molding the biomolecule-polymer mixture to form a biomolecule-polymer fiber; And Stacking the biomolecule-polymer fibers and cutting the bundle of biomolecule-polymer fibers obliquely with respect to the longitudinal direction to form a biomolecule layer, the method of producing a biomolecule probe layer for a biomolecule microarray. The method of claim 1 or 2, wherein the biomolecule is a polynucleotide or a polypeptide. The method of claim 1 or 2, wherein the polymer is a water soluble, water affinity, or water dispersible polymer. The method of claim 1 or 2, wherein the polymer is selected from the group consisting of polyethylene oxide, NiPaAm, polyacrylamide, agarose gel, PPO, chitin, chitosan, poly (vinyl alcohol) and PEDOT / PSS. How to. The method of claim 1, wherein the film is made using spin coating, dip coating, and doctor-blade. A method according to claim 1 or 2, wherein the cut is cut perpendicular to the longitudinal direction of the fiber bundle. A biomolecule probe layer for a biomolecule microarray prepared by the method of claim 1. A method of manufacturing a microarray comprising immobilizing a biomolecular probe layer prepared by the method of claim 1 or 2 on a microarray substrate.