JP5770366B2 - Biochip manufacturing sol-gel kit and biochip manufacturing method using the same - Google Patents

Description

  The present invention uses a sol composition prepared by sequentially mixing a specific solution, or a method for easily manufacturing a biochip by directly dispensing such a specific solution directly onto a substrate without a pretreatment process. The present invention relates to a sol-gel kit for producing a biochip.

  A biochip is a representative example of a technology in which new nanotechnology (NT), biotechnology (BT), and information technology (IT) are fused. A biochip is a high-density microarray in which various biological materials are formed on the surface of a solid support. Depending on the biological material attached to the surface, various biochips such as DNA chips, protein chips, cell chips, and neuron chips can be used. Divided into chips. Biochips have also been developed as LOC (lab-on-a-chip) along with microfluidics technology. The biochip detail technology includes biological material immobilization technology, solid support technology for biomaterial affinity, biomaterial micro-array technology, and assay technology for performing various biological reactions on the produced chip. , Technology for detecting reaction results, protein engineering for producing biological material to be immobilized, and gene recombination technology.

  The protein chip is a kind of biochip, in which various proteins are highly integrated microarray on the surface of a solid support having a unit area. Recently, efforts have been made to manufacture protein chips by introducing the manufacturing principles and manufacturing technology elements of DNA chips that have already been developed and commercialized. In general, many of the commercially available DNA chips are manufactured by immobilizing DNA on a glass plate whose surface has been pretreated with a coating substance. When a protein chip is manufactured by a method similar to the method used to manufacture a DNA chip, that is, when a protein chip is manufactured by immobilizing a protein on a glass plate whose surface has been pretreated with a coating substance, the target to be immobilized Various problems occur due to differences in physicochemical properties of proteins.

The initial protein chip is a simple binding assay after the protein is directly deposited on the surface-treated glass plate. However, depending on whether the immobilized protein is active or not, the protein chip may actually be operated. Depending on the situation, it was difficult to achieve the original purpose (MacBeath and Schreiber, Science 289: 1760, 2000). Such problems are caused by protein denaturation or inactivation, degradation, etc., which are caused by differences in the intrinsic physicochemical properties of proteins as described above. In order to overcome such problems, research has been conducted on a protein adhesion surface treatment technique and a protein immobilizing material suitable for the characteristics of a protein different from DNA. Research on this has focused on providing a way to maintain the activity of the protein while at the same time causing an immobilization reaction on the surface of the protein chip, such as Packard Bioscience recently acquired by PerkinElmer. (Packard Bioscience) Hydrogel ^™ coated slides, Prolinx Versalynx chips, Zyomyx Biochip PDC chips, and the like.

  On the other hand, the sol-gel process is a technique that has been used to create a fine structure through microfabrication. In particular, instead of chemically attaching biomolecules to inorganic substances, a bonding network is formed through a gentle process. It is a technique that has been widely used to form and immobilize biomolecules in this binding network by methods other than covalent bonding (Gill I. and Ballesteros A., Trends Biotechnol., 18: 282, 2000) .

  In addition, many biomolecules including enzymes are immobilized in a massive sol-gel matrix and used for the production of biocatalysts and biosensors (Reetz, Adv. Mater., 9: 943, 1997) It is also used for optical color detection due to its transparent optical properties (Edminston, et al., J. Coll. Interf. Sci., 163: 395, 1994). Biomolecules are known not only to be chemically stabilized but also to be highly thermally stabilized when immobilized by a sol-gel reaction (Dave, et al., Anal. Chem., 66: 1120, 1994).

  In the case of a biosensor, the sol-gel reaction is used not only by simple fixation but also by a method of patterning by forming a fine structure on a solid support. At this time, the patterning method uses hydrodynamics to form a gel with a mold in a sol state that is a liquid phase, and then forms a pattern by removing the mold. For example, micro-moduling in-capillaries (MIMIC) (Kim, J. Ferment. Bioeng., 82: 239, 1995; Marzolin, Adv. Mater., 10: 571, 1998; Schuller, et al , Appl. Optics., 38: 5799, 1999) is a technique for patterning mesoscopic silica. The technique can be used for basic patterning of microfluidics.

  However, since the protein activity is affected by various factors such as pH, it is important to set conditions for maintaining the activity by adding the protein from the sol state in the sol-gel process. For this reason, a technique (Kim, et al, Biotehnol. Bioeng., 73: 331, 2001) was published that pre-mixed protein and sol together using various mild conditions such as neutral pH. However, at a neutral pH, the sol-gel process progresses rapidly, and there are problems such as the formation of cracks and opaqueness depending on the choice of additive. Further, since a pretreatment process in which the protein and the sol are mixed together in advance is required, there is a problem that the spot density tends to be non-uniform.

  In addition, as a prior patent related to the sol-gel process, a sol mixture containing a biological material is subjected to a gelation reaction on a chip substrate, whereby the biological material is supported in a fore formed by the gel matrix and formed by the gel matrix. There is a sol-gel biochip that improves the reactivity of a biological material in a state of being encapsulated by the formed spot, or in the production of a biochip using such a sol-gel process, a fixed biological material There is a patent on a sol-gel biochip sol composition screening method that prevents the deformation of the sol-gel and increases the sensitivity, but the manufacturing method is complicated, and there are problems such as decreased activity and destruction of biological substances in the manufacturing process of the sol composition A point has arisen.

  Therefore, as a result of diligent efforts to prevent a decrease in the activity and destruction of biological materials during the production of the sol composition, the present inventors sequentially mixed specific silicate monomers and additives in a specific order and distributed them to the substrate. In the case of pouring or sequentially dispensing these directly onto the substrate, the gelation time can be delayed compared to the conventional production method to produce a more uniform biochip. The present invention has been completed by confirming that it is possible to manufacture a biochip with extremely excellent sensitivity by preventing the decrease in activity and destruction of substances.

The main object of the present invention is to make a sol-gel material as a kit so that anyone can easily make and analyze a biochip without any special equipment or technology.
Another object of the present invention is to establish a manufacturing method such as sequentially mixing a specific sol composition and dispensing it onto a substrate, or dispensing them directly onto a substrate, so that a uniform process can be performed without a pretreatment process. A method for producing a biochip is provided.
Still another object of the present invention is to provide a method for analyzing a target substance using the biochip.

  In order to achieve the above object, the present invention is, in one embodiment, (a) sol-gel kit (SolB kit), which sequentially detects a liquid composed of SolB1, SolB2, SolB3, SolBH, and SolBS. (B) a sol composition composed of SolB1, SolB2, and SolB3 on a substrate for manufacturing a biochip without a pre-treatment process. A step of dispensing; a step of dispensing SolBH; and a step of dispensing a solution containing SolBS, a detection protein and distilled water onto a substrate and then gelling the biochip using gelation of a sol composition A manufacturing method is provided.

At this time, as SolB1, SolB2, and SolB3 in the SolB kit for biochip production used,
(I) SolB1, methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate (Sodium Silicate), tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (tetraethyl) one or more first silicate monomers selected from the group consisting of orthosilicate (TEOS) and tetramethoxysilicate (TMS);
(Ii) SolB2, 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglycerylsilicate (polyGS), polyvinyl Acetate (polyvinylacetate), polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N, N'-disuccinimidyl carbonate (DSC), 1, 3,5-trimethylbenzene (1,3,5-trimethylbenzene), cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (trie 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutylamide, SIT8189 .5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, pluronic L121 and tetramethylammonium hydroxide One or more selected second silicate monomers; and (iii) SolB3, aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N -Triethoxysilylpropyl-O-polyethylene Oxide urethane (N-triethoxysilylpropyl-O-polyethylene oxide urethane, PEOU), glycerol, characterized in that it comprises a PEG 200, PEG 400, PEG 600, 1 or more additives selected from the group consisting of PEG1350 and PEG 8000.

Specifically, the present invention is a method for producing a biochip using gelation of a sol composition, comprising the following steps:
(A) mixing SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH into a sol composition containing SolB1, SolB2 and SolB3;
(B) a step of mixing SolBS, which is a buffer solution, and distilled water with the solution mixed in step (a) and then stabilizing at −20 to 4 ° C .; and (c) interacting with a target biological substance. A step of mixing a solution containing a biological substance with the solution stabilized in step (b) and dispensing the solution on a substrate, followed by gelling;
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L And one or more second silicate monomers selected from the group consisting of 21 and tetramethylammonium hydroxide; and (iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltri One or more additives selected from the group consisting of methoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000. A featured manufacturing method is provided.

The present invention is also a method for producing a biochip using gelation of a sol composition, comprising the following steps:
(A) After dispensing a sol composition composed of SolB1, SolB2 and SolB3 onto a substrate, SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH is added. A step of dispensing; and (b) a solution II containing SolBS, which is a buffer solution, a biological material that interacts with the target biological material, and distilled water, is gelled after being dispensed on a substrate,
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L And one or more second silicate monomers selected from the group consisting of 21 and tetramethylammonium hydroxide; and (iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltri One or more additives selected from the group consisting of methoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000. A featured manufacturing method is provided.

The present invention also relates to (i) methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetra A first container containing SolB1, which is one or more first silicate monomers selected from the group consisting of methoxysilicate (TMS); (ii) 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane ( DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5- bird Tylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propylsuccinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- ( A second container comprising SolB2, which is one or more second silicate monomers selected from the group consisting of 50% triethoxysilylpropyl) gluconamide (SIT8189.0), Pluronic L121 and tetramethylammonium hydroxide; iii) aminopropyltriethoxylane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG2 0, PEG400, PEG600, PEG1350 and the third container containing SolB3 is one or more additives selected from the group consisting of _{PEG8000; (iv) HCl, H} 2 SO 4, the group consisting of HNO ₃ and _CH 3 COOH A fourth container containing SolBH selected from the above; and (v) a fifth container containing SolBS, which is a buffer solution, and a mixture of SolB1, SolB2, and SolB3 to the sol composition, HCl, H ₂ SO ₄ , The sol mixture is gelated by sequentially mixing SolBH selected from the group consisting of HNO ₃ and CH ₃ COOH, SolBS as a buffer solution, distilled water, and a biological substance for detection. A kit for producing a biochip is provided.

  The present invention also relates to a biochip manufactured by the method and the sol composition and a method of analyzing a target substance that is a biological material using the biochip, and the target biological substance is added to the biochip manufactured by the method. Provided is a method for analyzing a target biological material comprising the step of adding a sample containing a target biological material capable of interacting with an interacting biological material.

The biochip manufactured by dispensing the sol mixed solution manufactured with the sol composition (S-Sol) of this invention, the solution I, and the solution II by an arrayer is shown. Responses to sera from HIV patients are shown with spots containing 5 HIV1 antigens. 1, 2, 3, 4, and 5 are antigen markers for HIV1 antibody diagnosis, and sequentially show p24, p31, gp41, gp120, and gp160. Shows response to HIV patient serum serially diluted with spots containing serially diluted antigen. FIG. 5 is a graph showing the quantification of the response to HIV standard serum in spots containing each of the five HIV1 antigens. 1, 2, 3, 4, and 5 are antigen markers for HIV1 antibody diagnosis, and indicate p24, p31, gp41, gp120, and gp160 in this order. It is the table | surface which compared the detection result using the biochip of this invention and the existing diagnostic kit with respect to the serum of the HIV1 patient extract | collected for every day after infection. It is an Axon GenePix scanner scan photograph (A) and a camera image photograph (B) of a chip manufactured using sciFLEXARRYER S11. The encapsulated structure according to the present invention includes a detection protein and schematically illustrates the shape of a biochip having a fine channel on its surface. It is the photograph which compared the homogeneity of the biochip which concerns on this invention (right side), and the biochip by the conventional method (left side). It is the photograph which compared the homogeneity of the sol mixture (B) when the solution was mixed with the sol mixture (A) according to the present invention in an arbitrary order and the gelation speed. It is the result of having analyzed the specific protein using the protein chip concerning the present invention. It is the result of having analyzed the specific antigen using the protein chip concerning the present invention. It is the result of having analyzed the antibody with respect to a specific disease using the protein chip which concerns on this invention. It is the result of having confirmed the coupling | bonding of a specific compound (bisphenol A) and a DNA aptamer using the sol-gel chip concerning this invention. It is the image of the product which commercialized the sol-gel biochip concerning this invention.

Hereinafter, the present invention will be described in detail.
In one aspect, the present invention is a method for producing a biochip using gelation of a sol composition, comprising the following steps:
(A) mixing SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH into a sol composition containing SolB1, SolB2 and SolB3;
(B) a step of mixing SolBS, which is a buffer solution, and distilled water with the solution mixed in step (a) and then stabilizing at −20 to 4 ° C .; and (c) interacting with a target biological substance. A step of mixing a solution containing a biological substance with the solution stabilized in step (b) and dispensing the solution on a substrate, followed by gelling;
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L And one or more second silicate monomers selected from the group consisting of 21 and tetramethylammonium hydroxide; and (iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltri One or more additives selected from the group consisting of methoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000. The present invention relates to a characteristic manufacturing method.

  In the present invention, the concentration of the SolBH is 1 mM to 100 mM.

In the present invention, the SolBS may be one or more solutions selected from the group consisting of NaH ₂ PO ₄ , Na ₂ HPO ₄ , and Na ₃ PO _{4 in} a concentration range of 1 mM to 100 mM.

  In a conventional method of manufacturing a biochip using a sol-gel method, a sol composition, a buffer solution, a biological substance that interacts with a target biological substance, and the like are arbitrarily mixed in an arbitrary order. First, a method in which a sol mixed solution is first made and then gelled through post-treatment such as vacuum treatment has been used. In addition, the method of mixing the sol composition with the buffer solution and the detection protein includes a method using vortex or ultrasonic vibration, and a method for forming a sol-gel shape on the substrate well. A method of forming a spot after forming a spot, coating the surface with a sol-gel, pouring it into a frame, and the like has been used.

  In such a method, the concentration of the biological substance that interacts with the target biological substance contained in each spot formed with the sol composition mixed solution may not be constant, and the concentration by the practitioner is also different. Differences occur and the possibility of contamination when mixing is large, and the viscosity of the sol-gel mixture varies depending on the sample, so there may be a difference in the degree, shape, and size of spot formation. Therefore, there is a need for a method capable of manufacturing a homogeneous biochip that can solve such problems.

  According to the biochip manufacturing method of the present invention, the biological substance that interacts with the target biological substance has a uniform concentration for each spot, though simple, using the sol-kel method. Biological substances can be detected with higher accuracy and higher efficiency.

The specific description of one embodiment of the biochip manufacturing method of the present invention is as follows.
First, SolB1 which is the first silicate monomer, SolB2 which is the second silicate monomer, and SolB3 which is the additive are sequentially mixed to produce a sol composition.

  SolB1 is selected from the group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more kinds of substances may be used. In one embodiment of the present invention, TEOS was selected and used as a component of the first silicate monomer.

  The SolB2 is composed of 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl. Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-Triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic One or more substances selected from 121 the group consisting of tetramethylammonium hydroxide may be used. In one embodiment of the present invention, DGS was selected and used as a component of the second silicate monomer.

  The SolB3 contains, as additives, aminopropyltriethoxylane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, One or more substances selected from the group consisting of PEG200, PEG400, PEG600, PEG1350, and PEG8000 may be used. In one embodiment of the present invention, PEG was selected and used as the additive.

  The first silicate, the second silicate monomer and the additive may be appropriately selected and used according to the nature of the monomer internal biological material and the shape of the sol-gel chip to be produced.

  In the sol composition for producing a biochip of the present invention, the first silicate monomer is about 2 to 30% by volume relative to the total volume of the sol composition, and the second silicate monomer is about 2 to 8% by volume. , And the additive is preferably included at about 0-5% by volume. At this time, since the sol composition is harmful when inhaled, it is manufactured in a well-ventilated place.

  The sol composition for producing the biochip of the present invention is characterized by forming a fine channel due to pores when gelled. That is, such a channel is a means that can interact with the target substance to be analyzed. In particular, the (iii) component additive is a component utilized to control the size of the channels in the gel.

  In the present invention, the method may be performed by spotting by hand without an arrayer or using a non-contact type arrayer.

  In the present invention, the substrate used in the step (c) is optimized at a temperature higher than the dew point before being used. Here, the temperature higher than the dew point means a temperature higher than a temperature at which dew point starts to be formed on the substrate. Such a dew point changes depending on the humidity condition. For example, when the relative humidity is 50% or more and the atmospheric temperature is 20 ° C., the dew point is 8.6 ° C., and usually the humidity is 70 to 80% and 14 to 14%. 17 ° C.

  In the present invention, the substrate may be polymethylmethacrylate (PMMA), plastic, silicon, glass, or the like.

  In the present invention, the biological substance that interacts with the target biological substance may be any one selected from the group consisting of nucleic acids, proteins, peptides, low molecular weight substances, and cells.

  In the present invention, the solution and the sol mixture mixed in the step (a) can be allowed to stabilize for 30 minutes or more at a temperature of -20 to 4 ° C. A temperature higher than the dew point, usually 14 to 17 ° C. (more than the dew point temperature when the humidity is 70 to 80%), and the dispensing environment can be optimized for the sol-gel by combining the conditions of the humidity 70 to 80% and the atmospheric temperature 20 ° C. it can.

  Through this stabilization and optimization process, the gel formation rate of the sol composition is slowed to facilitate spot formation, and after the gelation, the cracks of the spot are prevented and fine channels in the chip are well formed. So that

  In the present invention, the sol composition and the mixed solution of SolBH and SolBS, distilled water, and the biological substance that interacts with the target biological substance are between 3: 1: 4 and 1: 2: 8, respectively. The concentration of SolBH and SolBS may be 1 mM to 100 mM.

  In the present invention, the volume ratio of the SolBS: distilled water: biological material that interacts with the target biological material ranges between 1: 2: 1 and 2: 5: 1.

  In the present invention, the buffer may be sodium phosphate having a pH in the range of 3-8.

  In the present invention, the substrate is preliminarily plasma-treated, etched, or treated with PDMS, a silicate monomer, or a polymer substance.

In another aspect, the present invention is a method for producing a biochip using gelation of a sol composition, comprising the following steps:
(A) After dispensing a sol composition composed of SolB1, SolB2 and SolB3 onto a substrate, SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH is added. A step of dispensing; and (b) a solution II containing SolBS, which is a buffer solution, a biological material that interacts with the target biological material, and distilled water, is gelled after being dispensed on a substrate,
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L Be one or more second silicate monomers selected from 21 the group consisting of tetramethylammonium hydroxide;
(Iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, One or more additives selected from the group consisting of PEG600, PEG1350 and PEG8000;
(Iv) The SolBH solution is one or more solutions selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH in a concentration range of 1 mM to 100 mM; and (v) the SolBS solution is The present invention relates to a production method characterized by being one or more solutions selected from the group consisting of NaH ₂ PO ₄ , Na ₂ HPO ₄ , and Na ₃ PO _{4 in} a concentration range of 1 mM to 100 mM.

  In the present invention, the substrate used in the step (c) is optimized at a temperature higher than the dew point in advance before being used. Here, the temperature higher than the dew point means a temperature higher than a temperature at which dew starts to form on the substrate. However, such a dew point changes depending on the humidity condition. For example, when the relative humidity is 50% or more and the atmospheric temperature is 20 ° C., it is 8.6 ° C., and usually the humidity is 70 to 80% and 14 to 17 ° C. It is.

  Through such an optimization process, when each solution is sequentially dispensed, the gelation rate of the sol composition is slowed to facilitate spot formation. The fine channel is formed well.

According to the method of the present invention, after dispensing the sol composition, SolBH of solution I selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH is dispensed thereon. Solution I serves to create a pH environment for inducing gelation of the sol composition. The concentration of HCl, H ₂ SO ₄ , HNO ₃ , or CH ₃ COOH is preferably 5 to 30 mM. Adjust to pH 1-3.

  Finally, SolBS, which is a buffer solution, a biological substance that interacts with the target biological substance, and a solution II containing distilled water are dispensed on the substrate to be gelled.

  In the present invention, the SolBS may be sodium phosphate in the range of pH 3-8.

  The buffer solution and the secondary distilled water have a function of preventing destruction of biological substances (for example, proteins). Biological substances are likely to have decreased activity or be destroyed at a pH outside the proper range, and the gelation of the sol proceeds more slowly as the pH is higher, and faster as the pH is lowered. It is important to adjust the pH so that gelation can be performed for an appropriate time while preventing the above. In general, the biological material is stably present in the range of pH 5 to 8, and thus the buffer solution is used particularly in order to prevent destruction of the biological material in the pH environment due to the solution I. The buffer used is not particularly limited, and is appropriately selected by those skilled in the art for the biological material to be added. In the present invention, a sodium phosphate buffer having a pH in the range of 3 to 8 was used. The “biological substance that interacts with the target biological substance” or “biological substance” contained in the solution II is an in vivo body that can interact with the target substance (eg, target protein) to be detected. For example, it can be a nucleic acid, a protein, a peptide, a low molecular weight substance, or a cell. In order to include such a detection protein or biological substance in the solution II, an appropriate buffer solution is used. That is, a biological material for implementation is placed in a buffer solution to make a sample solution for detection. For example, when the biological material is a protein, PBS buffer (phosphate-buffered saline) is mixed and used, and when there is an enzyme reaction, HEPES, NaCl, EDTA, etc. are appropriately concentrated as necessary. Are mixed and used. In one embodiment of the present invention, an HIV1 (Human Immunodeficiency virus 1) patient antibody is used as the target substance (target protein), and five antigen markers that can bind to the HIV antibody using the detection protein are mixed in a PBS buffer. Used.

  At this time, the volume ratio of SolBS: distilled water: biological substance that interacts with the target biological substance in the solution II is 1: 2: 1 to 2: 5: 1. Is preferred, and most preferred is 1: 2: 1. For example, the buffer is about 20-30% by volume, the distilled water is about 40-60% by volume, and the biological material interacting with the target biological material is 20-30% by volume based on the total weight of Solution II. May be. In one embodiment of the present invention, 10 μL of a buffer solution, 20 μL of distilled water, and 10 μL of a solution containing a detection protein (a biological substance that interacts with a target protein) were used.

  The present invention is characterized in that a homogeneous biochip is manufactured by sequentially dispensing the sol composition, the solution I and the solution II in an accurate amount.

  The ratio of the sol composition: solution I: solution II dispensing amount dispensed in the present invention is between 3: 1: 4 and 1: 2: 8, preferably 3: 1: 4. For example, the amount of the sol composition dispensed is preferably 25 to 35 μL, and most preferably about 30 μL. The dispensing amount of the solution I is preferably 5 to 15 μL, and most preferably about 10 μL. The dispensing amount of the solution II is preferably 35 to 45 μL, and most preferably about 40 μL.

  When the sol composition, the solution I and the solution II are mixed in advance in the biochip manufacturing method according to the present invention and then dispensed to the substrate, a non-contact arrayer is used, or a pipette or other It is preferable to spot directly by hand using a tool.

  In the present invention, the manufacturing method is characterized in that there is no pretreatment process, and the pretreatment process includes (i) SolB1, SolB2, SolB3, SolBH, SolBS, or a biological substance that interacts with a target biological substance. A process of mixing a target substance; (ii) a process of mixing and then vortexing; and (iii) a process of stabilizing the mixed solution. And

v In the present invention, the SolB1, SolB2, SolB3, SolBH, SolBS and the biological substance that interacts with the target biological substance are put into a container before being dispensed and sucked up using a nozzle. You may dispense.

  In addition, in the present invention, a large-capacity cartridge to which a pre-dispensing nozzle is connected before dispensing biological materials that interact with SolB1, SolB2, SolB3, SolBH, SolBS and target biological materials. In addition, since the amount dispensed at a time is 100 times or more compared to the dispensing in the form of sucking up using a nozzle, mass production becomes possible.

  When the chip is manufactured by sequentially dispensing the sol composition, the solution I, and the solution II directly onto the substrate without a pretreatment process, an arrayer that dispenses an accurate amount may be used. . At this time, as the arrayer used for dispensing the sol composition, the solution I, and the solution II in an accurate volume, a non-contact type arrayer is preferable.

  Depending on the method of arraying the detection substances on the biochip, it can be divided into “contact type” and “non-contact type”, but the contact type arrayer uses a pin etc. with a very thin space inside. The detector protein is arranged on the chip surface. Such a method has a merit that a variety of detection proteins can be arranged in a short time by a method in which a solution containing the detection protein is gradually discharged outside the pins and arranged in direct contact with the surface. However, since the volume of the solution cannot be adjusted accurately, the uniformity can be lowered. In contrast, a non-contact type arrayer puts a solution containing a detection protein in a thin tube, positions it directly above the chip surface, and then applies a constant pressure to the surface without direct contact. It is the method of arranging on. This method has the advantage that the volume of the solution to be dispensed can be accurately determined. Therefore, when a chip is manufactured by sequentially dispensing the sol composition, the solution I, and the solution II without a pretreatment process, the dispensed volume of each solution can be adjusted to match the ratio. It is preferable to use such a non-contact type arrayer.

Said sol composition; solution I selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH; and biological substances, buffers and distilled water that interact with said target biological substance Solution II of the mixed solution can be sequentially dispensed onto the substrate well using a non-contact type arrayer that dispenses an accurate volume. When another solution is dispensed into a small spot dispensed on the surface, due to the surface tension of the solution, the whole solution will not spread but will form a spot, but will occur when the solution falls The surface energy is converted into vibration, which causes a material flow (convection) within the spot, making it easier for the two solutions to mix. Using this principle, the present invention has devised an automated method for producing a sol-gel chip through spotting directly on the surface without pretreatment.

  For example, Science Array AG's microarrayer can be used. In particular, the use of Science AG's dew point control technology prevents the uncertainty of concentration caused by moisture condensation on the plate surface as much as possible, enabling more accurate volume and size spots. Can be produced. In one embodiment of the present invention, scIFLEXARRAYER S11 (Scienion AG, Germany) was used as the arrayer.

  That is, according to the present invention, when the biochip is manufactured, the biochip can be more easily produced simply by dispensing each solution in an accurate volume using the non-contact type arrayer. Thus, since a pretreatment process such as premixing of a sol-gel monomer, a buffer solution, a detection protein sample or the like is not required, a more homogeneous biochip can be manufactured.

  On the other hand, since the substrate used in the present invention utilizes the property of becoming transparent when the sol composition is gelled, the substrate well or slide is also a material that can maintain good transparency. What was made is good. For example, a plastic made of the same plastic, silicon, or glass component as the polymethyl methacrylate (PMMA) component having very good transparency is used.

  Further, the substrate used in the present invention must be surface-treated so that the sol mixed solution is fixed to the substrate while being gelled. One of the important conditions of the biochip of the present invention is that when the sol mixed solution is gelled and firmly fixed to the substrate and reacted with the solution containing the target substance, the spot should not drop. . Therefore, in the target substance analysis using the biochip, a strong washing process is required after reacting with the target substance. Therefore, in order to overcome such physical force, strong fixation of the spot is indispensable. Plastic substrates that have not been surface-treated for this purpose, plastic substrates that have been surface-treated with plasma, glass substrates that have not been surface-treated, glass substrates that have been surface-treated (eg, etched glass substrates), or It is preferable to use a silicon chip having a porous structure.

In the present invention, the substrate may be previously plasma surface treated, etched, or treated with PDMS, a silicate monomer, or a polymer substance.
In the biochip manufacturing method of the present invention, the following points should be noted when using an arrayer.

First, unlike a DNA chip, the biochip has a property of gelling with the passage of time using a special material called a sol, so that the sol gels during dispensing using an arrayer. It is very important to have it dispensed as soon as possible.
Second is humidity and temperature. The initial humidity and temperature are very important because the spot is spotted on the substrate and the speed at which the spot is gelled and the activity of the spot depend on the amount and temperature of the surrounding moisture. Therefore, when manufacturing a biochip using the sol-gel at all times, it is very important to adjust the humidity and temperature around the arrayer in advance.

  The preferred humidity in the present invention is about 50% or more, or more specifically 70 to 80%. A preferable temperature is about 25 ° C. or less, more specifically, a range of 10 to 25 ° C. which can be said to be a range of normal temperature. In particular, since the first high humidity is an important factor in spot gelation, it is necessary to prepare the humidity before arraying the spots to about 80%, and when the temperature is 25 ° C. or higher. Therefore, it is better to experiment at a temperature as low as possible because the gelation of the sol tends to proceed quickly.

Thus, after preparing the temperature and humidity in advance and a program that can be accumulated earlier, each solution is dispensed for each process.
In the present invention, the biological substance that interacts with the target biological substance may be a nucleic acid, a protein, a peptide, a low molecular weight substance, or a cell.

In another aspect, the present invention provides (i) methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxy. A first container containing SolB1, which is one or more first silicate monomers selected from the group consisting of silicate (TMS); (ii) 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS) ), Methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethyl Benzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propylsuccinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- ( A second container comprising SolB2, which is one or more second silicate monomers selected from the group consisting of 50% triethoxysilylpropyl) gluconamide (SIT8189.0), Pluronic L121 and tetramethylammonium hydroxide; iii) aminopropyltriethoxylane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200 PEG 400, PEG 600, third vessel containing PEG1350 and one or more additives selected from the group consisting of PEG8000 SolB3; _{(iv) HCl,} selected from the group consisting of _H 2 SO 4, HNO ₃ and _CH 3 COOH A fourth container containing the prepared SolBH; and (v) a fifth container containing SolBS as a buffer solution, and the sol composition obtained by mixing SolB1, SolB2 and SolB3 with HCl, H ₂ SO ₄ , HNO _3. And Sol BH selected from the group consisting of CH ₃ COOH, SolBS as a buffer solution, distilled water, and biological material for detection are mixed in order to form a sol mixture into a gel. The present invention relates to a chip manufacturing kit.

  The container is not limited in its material, and such a kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc. These may be partly or wholly formed from plastic, glass, paper, wheels, waxes and the like. The container may be fitted with a fully or partially separable plug that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means. The kit may include an external package, and the external package may include instructions for use of the component.

  In another aspect, the present invention relates to a method for analyzing a target biological substance using the biochip manufactured by the above method.

  Specifically, target biology including the step of adding a sample containing a target biological substance capable of interacting with a biological substance that interacts with the target biological substance to the biochip manufactured by the method The present invention relates to a method for analyzing chemical substances. Here, the target biological substance is any one selected from the group consisting of nucleic acids, proteins, peptides, low molecular substances, and cells, and the target biological substance can be detected. A step of additionally reacting with a biological substance such as a protein or aptamer labeled with a radioisotope, a fluorescent dye, or another type of labeling substance may be included.

  As described above, after the biochip to be reacted with the target biological material is prepared, the biochip is actually reacted with the solution containing the target biological material. In the case of a 96-well type reaction solution, an amount of 50 to 100 μL is appropriate, and the reaction time is 1 hour. The target biological substance that interacts with the biological substance that interacts with the target biological substance may also be a nucleic acid, protein, peptide, low molecular weight substance, or cell as an in vivo substance.

  The reaction solution containing the target biological substance penetrates into the spot through the microporous structure in the spot, contacts the biological substance immobilized in the capsule structure, and binds through the interaction (1st incubation). After the reaction, in order to analyze the target biological substance bound to the biological substance that interacts with the target biological substance in the spot, the labeling protein of the labeling factor is detected so that the target biological substance can be detected And may be reacted. In one embodiment of the present invention, an antibody against a target protein to which a fluorescent dye (Cy3) was attached was used (2nd incubation). At this time, the reaction time is 30 minutes and the amount of the reaction solution is 50 to 100 μL. Both the 1st and 2nd incubation steps are performed at room temperature. When the reaction solution containing the target biological substance is a mixture of various substances, nonspecific binding to the biological substance interacting with the target biological substance contained in the biochip is prevented. To prevent this, a blocking process may be performed before the 1st incubation process. As a blocking solution necessary for this process, skim milk, BSA (bovine serum albumin), or IgG may be used.

  After the 1st and 2nd incubations are completed, a washing process is performed. However, as a component of the washing solution, normal components may be used. In one embodiment of the present invention, 0.2% A PBS buffer containing Tween-20 was used. As the washer, an ELISA washer was used. In the 1st cleaning process, cleaning is performed 4 times, and in the 2nd cleaning process, cleaning is performed 4 times. After the washing step, the well is dried until all the solution is removed.

After the drying process is over, scan the wells that caused the reaction through an image scanner that can detect fluorescent dyes to see if the reaction was correct, and then measure the density of the image using software. This shows how much reaction has occurred. That is, the method for analyzing a target biological substance of the present invention comprises a biological substance such as an aptamer, a protein labeled with a radioisotope, a fluorescent dye, a luminescent substance, a dye, or another type of labeling substance. An additional reaction step. At this time, an aptamer refers to a small single-stranded oligonucleic acid that can specifically recognize a biological substance that interacts with a target biological substance with high affinity.
Furthermore, the present invention includes a detection kit including a biochip manufactured by the manufacturing method.

  Detection biological material detection kits may be in the form of bottles, tubes, sachets, envelopes, tubes, ampoules, etc., which are partially or wholly plastic, glass, paper, wheels, waxes Formed from. The container can be attached to a fully or partially separable plug that is initially part of the container or can be attached to the container by mechanical, adhesive, or other means. The kit may have an external package, which may include instructions for use of the component.

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that this example is merely illustrative of the invention and that the scope of the invention is not to be construed as limited by this example.
<< Example 1: Production of each constituent solution for biochip production >>
SolB1 20 μL, SolB2 6 μL, and SolB3 4 μL, each of which was selected from among the components exemplified in Table 1 below, were mixed to prepare a sol composition. As solution I, 10 μL of SolBH was prepared.

Next, 10 μL of one or more SolBS selected from the group consisting of NaH ₂ PO ₄ , Na ₂ HPO ₄ , and Na ₃ PO ₄ and 20 μL of double distilled water (DDW) are mixed, while the HIV1 antibody and each other are mixed. About 10 to 200 ng of 5 kinds of detection proteins (p24, p31, gp41, gp120, and gp160) that can act are mixed with PBS buffer to make a sample solution of 10 μL, added to the mixture, and then vortexed for 5 seconds. Spin-down to prepare solution II.

<< Example 2: Production of biochip >>
(1) Preparation of substrate wells A PMMA 96 well plate that has been subjected to plasma surface treatment and is commercially available was purchased from SPL (Korea) and prepared.
(2) Production of biochip (protein chip) Before producing spotted protein chips, set an arrayer at a temperature of 16 ° C and a humidity of 80%, and put the sol mixed solution obtained in Example 1 above. As the source well, a general 384 well was prepared, and as the target well, the PMMA 96 well plate of the above (1) was prepared. And the scIFLEXARRYER S11 (Scienion, Germany) arrayer which dispenses an exact volume as set was prepared.
Next, sol composition 30 μL, SolBH 10 μL (solution) prepared by mixing 20 μL of SolB1 in Example 1 above, 6 μL of SolB2 and 4 μL of SolB3 in the source plate of sciFLEXARRYER S11 (Scienion, Germany) I) and 40 μL of the solution II were put on the source plate of the arrayer.
On the PMMA 96 well plate prepared in advance, the sol composition, the solution I, and the solution II were dispensed in a predetermined volume. The amount to be dispensed was 450 pl or less per spot, and at this time, nozzle PDC90 (Scienion AG, Germany) was used. The spotting frequency was set to 500 Hz. The size of the formed spot was about 300 μm (8 drops per spot).

  FIG. 6 shows a photograph A scanned with an Axon GenePix scanner (Axon) at 532 nm and an image photograph B by sciFLEXARRAYER with a camera attached. The mutual spot pitch (dot pitch) on the protein chip was 600 um.

<< Comparative Example 1: Comparison of homogeneity with conventional protein chips >>
It was confirmed whether or not the protein chip according to the method of the present invention has a remarkably excellent homogeneity as compared with the protein chip already used.
First, a protein chip was manufactured by an existing method known as a control group. PMMA 96-well prepared by sequentially mixing silicate monomer, HCl, DW, SP, and sample solution, and then dispensed into a source plate using a pin arrayer Spotted on plate. As a pin array, OnmiGrid Agent Arrayer (Genomic Solutions, USA) was used.

In addition, the camera image photograph (B of FIG. 6) of the protein chip manufactured by the method of the present invention manufactured by the method of Examples 1 and 2 and an image of the protein chip manufactured by the above method observed with a microscope. I compared it with a digital camera.
As a result, as shown in FIG. 8, in the case of a protein chip manufactured by using the specific sol composition of the present invention and by sequentially dispensing without a pretreatment step of mixing with the solution I and the solution II, Although the spot shape and size are arranged in a constant manner, the spot shape and size are not constant in the conventional method. That is, it could be confirmed that the protein chip according to the present invention is remarkably superior in uniformity to the existing protein chip.

<< Comparative Example 2: Comparison when mixing in different order >>
The difference in the sol mixture when the solutions were mixed in the order different from that when the solutions were mixed in the mixing order according to the method of the present invention was compared.
According to the mixing order of the present invention, a sol composition consisting of SolB1, SolB2, and SolB3 was prepared in order by mixing SolBH and SolBS, distilled water and buffer solution, and the same solution was used for comparison. SolBH was mixed at the end to prepare a mixture, and the difference between the two mixtures was compared with a digital camera.
As a result, when the mixture was prepared in accordance with the mixing order of the present invention as shown in FIG. 9, the solutions were easily dissolved in each other, and the gelation did not proceed for a long time (A in FIG. 9). When the mixture was prepared in a different order, it was confirmed that the solution was difficult to dissolve and gelation proceeded (B in FIG. 9).

<< Example 3: HIV analysis and diagnosis using protein chip >>
After blocking the protein chip prepared in Example 2 using a 10% skim milk solution, 50 μL of diluted HIV patient serum was added to each well, followed by primary culture at room temperature for 1 hour. After the primary culture was completed, the serum was removed, a washing solution containing 0.2% Tween-20 was added using an ELISA washing machine, and the process of vortexing for 5 minutes was repeated four times ( Primary wash). After the primary washing, the ∂-Human antibody (∂-Human-Cy3, Jackson ImmunoResearch), which is labeled with Cy3 fluorescent substance and recognizes human antibodies, is diluted and put into 50 μL, and then secondary for 1 hour at room temperature. Cultured. After the secondary culture, ∂-Human-Cy3 was removed, and a washing solution was added using an ELISA washer and vortexed for 5 minutes (secondary washing).

  After the secondary washing, the well in which the reaction was completed was allowed to stand at room temperature for 10 minutes or more and dried, and a spot where the reaction occurred was scanned with a laser scanner FUJI FLA-9000 image scanner. Further, the intensity of the fluorescence signal at each spot where the reaction occurred was measured and quantified using ImageQuant TL, which is an image analysis program, and the degree of the reaction was analyzed.

  As shown in FIG. 2, 5 markers (p24, p31, gp41, gp120 and gp160 (obtained from Abcam and Fitzerald)) each reacted to the serum and no antigen marker was entered. The negative control group chip showed no response.

  FIG. 3 shows that four antigens (p24, p31, gp41, gp120) that caused the most reaction among five antigens and one O-type antigen of HIV1 were serially diluted and spotted on a well. The result of reacting with standard diluted serial serum was shown, and it was confirmed that the quantification was quantitatively performed as expected. Based on the results, it can be seen that the antigen-antibody reaction occurs specifically on the protein chip produced in the present invention.

  FIG. 4 shows the results of quantifying the response to HIV1 standard serum at spots each containing 5 antigens. The X-axis of the drawing shows the titer measured when HIV was diagnosed with standard ELISA using an existing ELISA diagnostic kit. The analysis result of the protein chip of the present invention and the analysis result of the existing diagnostic chip are Show that there is a correlation. X-axis PRB204-00 is described in Boston Biomedica, Inc. Serum standard sample of a patient purchased from Japan, the product name is Anti-HIV1 mixed titer performance panel, and the product number is PRB204 (M). The titer value is an s / co value measured with an existing diagnostic kit, indicates signal to cut-off (positive and negative reference value) ratio, and is positive when it is 1 or more. The Y-axis of the drawing is the value obtained by dividing the intensity (“signal”) value of the fluorescent signal of the spot by the intensity (“control”) value of the fluorescent signal of the negative control group spot.

FIG. 5 is a table comparing the responses to seroconversion panels collected from HIV-infected patients on a daily basis with existing diagnostic kits. Patient sera were obtained from Boston Biomedica, Inc. Detection results using existing diagnostic kits were also provided with the standard samples. The product name of this sample is Anti-HIV1 seroconversion panel V, and the product number is PRB922.
It is understood that HIV infection could not be detected with the existing antibody diagnostic ELISA diagnostic kit shortly after the infection, but the protein chip of the present invention can be detected as positive from the early stage of infection like the antigen detection kit.
Therefore, it can be seen that the biochip has a much higher sensitivity than the existing antibody diagnostic ELISA.

«Example 4: Alternative to Western blot method using protein chip»
Western blotting and immunostaining are techniques for searching for a specific protein from a mixture of various proteins, and revealing the presence or absence of a specific protein by inducing an antigen-antibody reaction using an antibody against the protein to be searched. Is the method.
In general, the process of searching for a specific protein by Western blot is performed by separating the protein mixture by size on an SDS-polyacrylamide gel and then transferring it to a nitrocellulose or nylon membrane. The transferred membrane is searched for an antigen against a specific antibody using an antigen-antibody reaction. The antibody used at this time is labeled with a radioisotope, a specific enzyme (such as horseradish peroxidase), or a fluorescent dye. You can visualize the proteins that are bound and you are looking for.

As described above, the protein mixture prepared by using the protein chip prepared in Example 2 is used instead of the Western blot, which requires a complicated process. We were able to easily locate specific proteins within the protein mixture by assaying with the antibodies present.
In general, when a protein is electrophoresed in a reduced state, it binds to an antibody in a denatured state of the protein, but the specific antibody is the native protein. If only the form is recognized, there is a problem that a general Western blot cannot find the protein, but the sol-gel protein chip according to the present invention immobilizes the protein in its original form. It can be made more useful.

The following experiment was performed using the sol-gel chip manufactured in Example 2.
(1) Binding comparison experiment concerning native form and denaturation form of p24 protein (i) First, experiment using an antibody that binds only to native form but not to denatured form. did. As a result, no band was observed in the Western blot, and the sol-gel protein chip alone was positive.
(Ii) Although it was an antibody against the same antigen, it was shown to be positive as a result of experiments on Western blots and sol-gel protein chips using an antibody that binds to a denatured form.
Therefore, it was revealed that the sol-gel chip according to the present invention can be confirmed both in a denatured and native form.
(2) E. coli expressing p24 protein. Experiment using crude extract of E. coli (i) E. coli expressing p24 protein After the E. coli crude extract was immobilized on the sol-gel protein chip for each concentration (lysate 1, 2, 3), assayed with an antibody against the expressed protein, the sol-gel protein chip showed positive (FIG. 10). ).
(Ii) E. a specific protein was not expressed After the E. coli crude extract (N) was immobilized on a sol-gel protein chip and assayed with the antibody, as shown in FIG. 10, the sol-gel protein chip showed negative (FIG. 10).

  In FIG. 10, N is a negative control group and the specific protein was not expressed. E. coli crude extract was immobilized, and lysate 1 was an E. coli strain in which a specific protein was expressed at a concentration of 0.09 ug / ul. The crude extract of E. coli, lysate 2, is expressed in E. coli in which the specific protein is expressed at a concentration of 0.18 ug / ul. A crude extract of E. coli, lysate 3, is expressed in E. coli in which the specific protein is expressed at a concentration of 0.27 ug / ul. E. coli crude extract is fixed. P is a positive control group in which a Cy3 fluorescent substance is immobilized.

(3) (i) An antibody against p24 protein is immobilized on a sol-gel protein chip for each concentration and then sandwiched by a sandwich assay method. As a result of assaying with a crude E. coli extract and rebinding the antibody, the result was positive in the sol-gel chip (FIG. 11).
(Ii) The antibody against the antigen to be searched was immobilized on the sol-gel protein chip for each concentration, and then the specific antigen was not expressed using the sandwich assay method. As a result of assaying with a crude E. coli extract and rebinding the antibody, it was shown negative in the sol-gel chip (FIG. 11).
In FIG. 11, N is a negative control group, which is a chip on which no antibody is immobilized, and Ab1 and Ab2 are obtained by immobilizing an antibody for each concentration (0.063 ug / ul, 0.125 ug / ul). P is a positive control group in which a Cy3 fluorescent substance is immobilized.
(4) (i) Specific antibodies such as patient serum are included after antigens (p24, p31, gp41, gp120 and gp160) against the antibody of the disease to be searched (AIDS) are immobilized on a sol-gel protein chip As a result of confirming by assaying with the substance (positive serum), it was shown to be positive in the sol-gel chip (FIG. 12).
(Ii) After immobilizing an antigen against the antibody to be searched for on a sol-gel chip and confirming it by assaying with a substance that does not contain a specific antibody such as serum (negative serum), the result is negative on the sol-gel chip. (FIG. 12).

Example 5: Search for a protein or specific substance that binds to a compound
As shown in FIG. 13, the specific compound (bisphenol A) was immobilized on the sol-gel chip prepared in Example 2, and only the buffer solution used to dissolve the compound as a negative control group was immobilized on the chip. And it was labeled with a fluorescent substance (cy3) and analyzed using a single-stranded DNA aptamer (purchased from PSI) that can bind to bisphenol A.

  Protein-protein binding can be confirmed by the yeast two hybrid method or immunoprecipitation (IP), but there are not many methods for easily confirming the binding between the compound and the protein, or the binding between the compound and the DNA. As a result of the experiment, the protein chip manufactured in Example 2 can immobilize various substances ranging from low molecular substances such as compounds and DNA to proteins and antibodies, and easily bind various substances. Can be confirmed.

  When a biochip is manufactured using the gelation of the sol composition according to the present invention, a sol composition composed of SolB1, SolB2, and SolB3, SolBH, SolBS, DW, and a buffer solution are sequentially mixed. By stabilizing at a low temperature, the gelation time of the sol composition is delayed, leading to a stabilized gelation, so that the sol solution can be easily dispensed and the activity of the spot can be maintained. . Further, it is useful because it has an advantageous effect that it is possible to easily produce a homogeneous biochip by using only surface spotting using an arrayer without a pre-processing step of premixing.

  Although specific portions of the subject matter of the present invention have been described in detail above, such specific notation is merely a preferred embodiment for those having ordinary skill in the art, thereby Obviously, the scope of the present invention is not limited. Accordingly, the substantial scope of the present invention is defined by the appended claims and equivalents thereof.

Claims (16)

A method for producing a biochip using gelation of a sol composition, comprising performing the following steps (a) and (b) in that order :
(A) After dispensing a sol composition composed of SolB1, SolB2 and SolB3 onto a substrate, SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH is added. A step of dispensing; and (b) a solution II containing SolBS, which is a buffer solution, a biological material that interacts with the target biological material, and distilled water, is gelled after being dispensed on a substrate,
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L And one or more second silicate monomers selected from the group consisting of 21 and tetramethylammonium hydroxide; and (iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltri One or more additives selected from the group consisting of methoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000. As a feature ,
(I) a process of mixing SolB1, SolB2, SolB3, SolBH, SolBS, or a biological substance that interacts with a target biological substance; (ii) a process of vortexing the mixture of the process (i); iii) The production method described above, which does not include any one pretreatment process selected from the group consisting of processes of stabilizing the mixed solution of the process (i) or (ii) . The manufacturing method according to claim 1 , wherein the method is performed by spotting by hand without an arrayer, or by using a non-contact type arrayer. The SolB1, SolB2, SolB3, SolBH, SolBS, and biological material that interacts with the target biological material are placed in a container before being dispensed, and dispensed using a nozzle. The manufacturing method according to claim 1 . Before dispensing the biological material that interacts with SolB1, SolB2, SolB3, SolBH, SolBS and the target biological material, place it in a large-capacity cartridge with a pre-dispensing nozzle connected to it at once. since the amount for dispensing, compared with the dispensing in the form of sucking by using the nozzle is 100 times or more, the production method according to claim 1, characterized in that it enables mass production. The manufacturing method according to claim 1 , wherein the substrate is selected from the group consisting of polymethyl methacrylate (PMMA), plastic, silicon, and glass. Biological substance interacting with said target biological material, nucleic acids, proteins, peptides, according to claim 1, characterized in that the one selected from the group consisting of low molecular substances and cells Manufacturing method. The manufacturing method according to claim 1 , wherein the substrate is optimized at a temperature higher than a dew point and dispensed under a condition higher than a humidity of 50%. The method according to claim 1 , wherein a ratio of the sol composition: solution I: solution II dispensing amount is in a range between 3: 1: 4 and 1: 2: 8. The manufacturing method according to claim 1 , wherein the concentration of SolBH and SolBS is 1 mM to 100 mM. The solution II is characterized in that the volume ratio of SolBS: distilled water: biological material interacting with the target biological material is in the range between 1: 2: 1 and 2: 5: 1. The manufacturing method according to claim 1 . The SolBS The manufacturing method according to claim 1, characterized in that sodium phosphate in the range of pH 3-8. The manufacturing method according to claim 1 , wherein the substrate is subjected to plasma surface treatment, etching, or treatment with PDMS, a silicate monomer, or a polymer substance. A method for producing a biochip using gelation of a sol composition , comprising performing the following steps (a) to (c) in that order :
(A) mixing SolBH (solution I) selected from the group consisting of HCl, H ₂ SO ₄ , HNO ₃ and CH ₃ COOH into a sol composition containing SolB1, SolB2 and SolB3;
(B) a step of mixing SolBS, which is a buffer solution, and distilled water with the solution mixed in step (a) and then stabilizing at −20 to 4 ° C .; and (c) interacting with a target biological substance. A step of mixing a solution containing a biological substance with the solution stabilized in step (b) and dispensing the solution on a substrate, followed by gelling;
here,
(I) SolB1 is a group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS). One or more first silicate monomers selected from:
(Ii) SolB2 is 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglyceryl silicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl Acrylate, N, N-disuccinimidyl carbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3- (triethoxysilyl) propyl succinic anhydride, N- (3-triethoxysilylpropyl) -4-hydroxybutyramide (SIT8189.5), N- (triethoxysilylpropyl) gluconamide (SIT8189.0) 50%, Pluronic L And one or more second silicate monomers selected from the group consisting of 21 and tetramethylammonium hydroxide; and (iii) SolB3 is aminopropyltriethoxylane (APTES), 3-glycidoxypropyltri One or more additives selected from the group consisting of methoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000. A featured manufacturing method. Adding a sample containing a target biological substance capable of interacting with a biological substance that interacts with the target biological substance to the biochip manufactured by the method according to claim 1 or 13 ; Analysis method of target biological material. Biological substance interacting with said target biological material, nucleic acids, proteins, peptides, according to claim 14, characterized in that the one selected from the group consisting of low molecular substances and cells method towards. 15. The method according to claim 14 , further comprising a step of additionally reacting with a protein, antibody or aptamer labeled with a radioisotope capable of detecting the target biological substance, a fluorescent dye, a dye, or a luminescent substance . mETHODS.