Sol-gel kit for preparing biochip and methods for preparing biochip using the same

Abstract

A method of preparing a protein chip by gelation of a sol composition. In the method, a mixture of specific silicate monomers, such as SolB 1, SolB 2 and SolB 3, SolBH, and a mixture of SolBS, distilled water and a detector protein are mixed sequentially, so that the gelation rate of the sol-composition can be delayed, thus inducing the stable gelation of the composition. Also, the biochip can be fabricated in a simple and easy manner by dispensing the sol composition by hand using an arrayer or a tool such as a pipette. In addition, a uniform biochip can be prepared by dispensing the sol composition, solution I (SolBH) and solution II (a mixture of buffer, SolBS, distilled water and a detector protein) sequentially onto a substrate without needing a conventional pretreatment process such as mixing.

1 . A method for preparing a biochip by gelation of a sol composition,, the method comprising the steps of:
 (a) adding to a sol composition comprising SolB1, SolB2 and SolB3 and SolBH solution (solution I) selected from the group consisting of HCl, H 2 SO 4 , HNO 3  and CH 2 COOH;   (b) mixing the solution of step (a) with buffer SolBS and distilled water, and then stabilising the mixed solution at a temperature ranging from −20° C. to 4° C.; and   (c) mixing the stabilized solution of step (b) with a solution containing a biological material which interacts with target biological material, dispensing the mixed solution onto a substrate and gelling the dispensed solution,   wherein (i) said SolB1 is at least one first silicate monomer selected from the group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS);   wherein (ii) said SolB2 is at least one second silicate monomer selected from the group consisting of 3-aminotrimethoxysilane (3-ATMS;), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglycerylsilicate (PGS), polyvinyl acetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N,N-dicusinimidilcarbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3-(triethoxysily)propyl succinic anhydride, N-(3-triethoxysily propyl)-4-hydroxy butylamide (SIT8189.5), N-(triethoxysilylpropyl)gluconamide (SIT8189.0), pluronic L121 and tetramethyl ammonium hydroxide; and   wherein (iii) said SolB3 is at least one additive selected from the group consisting of aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000.   
     
     
         2 . The method of  claim 1 , wherein a hand-spotting technique is performed without using any arrayer or a non-contact arrayer is used. 
     
     
         3 . The method of  claim 1 , wherein the substrate is made of any one selected from the group consisting of polymethylmethacrylate (PMMA), plastic, silicon, and glass. 
     
     
         4 . The method of  claim 1 , wherein the biological material which interacts with target biological material is any one selected from the group consisting of nucleic acid, protein, peptide, low-molecular-weight material, and cell. 
     
     
         5 . The method of  claim 1 , wherein the added solution in step (a) is stabilized by allowing it to be left to stand at a temperature ranging from −20° C. to 4° C. for 30 minutes or more. 
     
     
         6 . The method of  claim 1 , wherein the substrate used in step (c) is optimized at a temperature higher than dew point before its use, and the mixed solution is dispensed onto the substrate at a humidity higher than 50% in step (c). 
     
     
         7 . The method of  claim 1 , wherein the mixed solution of the sol composition, the SolBH and the SolBS, and the distilled water and the biological material which interacts with target biological material are mixed with each other in a ratio between 3:1:4 and 1:2:8. 
     
     
         8 . The method of  claim 1 , wherein the SolBH and the SolBS have a concentration ranging from 1 mM to 100 mM. 
     
     
         9 . The method of  claim 1 , wherein the volume ratio of the SolBS: the distilled water: the biological material which interacts with target biological material is between 1:2:1 and 2:5:1. 
     
     
         10 . The method of  claim 1 , wherein the buffer is sodium phosphate buffer having a pH ranging from 3 to 8. 
     
     
         11 . The method of  claim 1 , wherein the substrate is plasma-surface treated, is etched, or is treated with PDMS, silicate monomer, or polymeric material. 
     
     
         12 . A method for preparing a biochip by gelation of a sol composition, the method comprising the steps of:
 (a) dispensing onto a substrate a sol composition consisting of SolB1, SolB2 and SolB3 and dispensing SolBH (solution I) selected from the group consisting of HCl, H 2 SO 4 , HNO 3  and CH 3 COOH onto the substrate onto which the sol composition has been dispensed; and   (b) dispensing solution II, comprising buffer SolBS, a biological material which interacts with target biological material and distilled water, onto the substrate onto which the solution I has been dispensed, and then gelling the dispensed solutions,   wherein (i) said SolB1 is at least one first silicate monomer selected from the group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS);   wherein (ii) said SolB2 is at least one second silicate monomer selected from the group consisting of 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglycerylsilicate (PGS), polyvinylacetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N,N-dicusinimidilcarbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3-(triethoxysily)propyl succinic anhydride, N-(3-triethoxysilypropyl)-4-hydroxy butylamide (SIT8189.5), N-(triethoxysilylpropyl)gluconamide (SIT8189.0), pluronic L121 and tetramethyl ammonium hydroxide; and   wherein (iii) said SolB3 is at least one additive selected from the group consisting of aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide methane (PEOU), glycerol, PEG200), PEG400, PEG600, PEG1350 and PEG8000.   
     
     
         13 . The method of  claim 12 , wherein a hand-spotting technique is performed without using any arrayer or a non-contact arrayer is used. 
     
     
         14 . The method of  claim 12 , wherein the method does not have a pretreatment process. 
     
     
         15 . The method of  claim 14 , wherein the pretreatment process is one or more selected from the group consisting of (i) mixing process of SolB1, SolB2, SolB3, SolBH, SolBS, or biological material which interacts with target biological material; (ii) vortexing process of the mixed solution of (i); and (iii) stabilizing process of the mixed solution of (i) or (ii). 
     
     
         16 . The method of  claim 12 , wherein the SolB1, SolB2, SolB3, SolBH, SolBS and the biological material which interacts with target biological material are contained in the container before the dispensing, and the dispensing is performed by sucking out through nozzle. 
     
     
         17 . The method of  claim 12 , wherein the SolB1, SolB2, SolB3, SolBH, SolBS and biological material which interacts with target biological material are contained in a mass production cartridge connecting with dispensing nozzle, and a dispensing quantity is more than 100 times compared with that of dispensing by sucking process through nozzle, thus the method is capable of achieving a mass production. 
     
     
         18 . The method of  claim 12 , wherein the substrate is made of any one selected from the group consisting of polymethylmethacrylate (PMMA), plastic, silicon, and glass. 
     
     
         19 . The method of  claim 12 , wherein the biological material which interacts with target biological material is any one selected from the group consisting of nucleic acid, protein, peptide, low-molecular-weight material, and cell. 
     
     
         20 . The method of  claim 12 , wherein the substrate is optimized at a temperature higher than dew point before its use, and the second mixed solution is dispensed onto the substrate at a humidity higher than 50%. 
     
     
         21 . The method of  claim 12 , wherein the ratio of the amounts of the sol composition: the solution I:the solution II that are dispensed is between 3:1:4 and 1:2:8. 
     
     
         22 . The method of  claim 12 , wherein the SolBH and the SolBS have a concentration ranging from 1 mM to 100 mM. 
     
     
         23 . The method of  claim 12 , wherein the volume ratio of the SolBS:the distilled water: biological material which interacts with target biological material in solution II is between 1:2:1 and 2:5:1. 
     
     
         24 . The method of  claim 12 , wherein the buffer SolBS is sodium phosphate buffer having a pH ranging from 3 to 8. 
     
     
         25 . The method of  claim 12 , wherein the substrate is plasma-surface treated, is etched, or is treated with PDMS, silicate monomer, or polymeric material. 
     
     
         26 . A kit for preparation of a biochip, the kit comprising:
 a first container containing at least one first silicate monomer, SolB1, selected from the group consisting of methyltriethoxysilane (MTES), ethyltriethoxysilane (ETrEOS), sodium silicate, tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) and tetramethoxysilicate (TMS);   a second container containing at least one second silicate monomer, SolB2, selected from the group consisting of 3-aminotrimethoxysilane (3-ATMS), diglycerylsilane (DGS), methyltrimethoxysilicate (MTMS), polyglycerylsilicate (PGS), polyvinylacetate, polyvinylpyrrolidone, glyceryl methacrylate, hydroxyethyl acrylate, N,N-dicusinimidilcarbonate (DSC), 1,3,5-trimethylbenzene, cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, 3-(triethoxysily)propyl succinic anhydride, N-(3-triethoxysily propyl)-4-hydroxy butylamide (SIT8189.5), N-(triethoxysilylpropyl)gluconamide (SIT8189.0), pluronic L121 and tetramethyl ammonium hydroxide;   a third container containing at least one additive, SolB3, selected from the group consisting of aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMOS), N-triethoxysilylpropyl-O-polyethylene oxide urethane (PEOU), glycerol, PEG200, PEG400, PEG600, PEG1350 and PEG8000;   a fourth container containing SolBH selected from the group consisting of HCl, H 2 SO 4 , HNO 3  and CH 3 COOH; and   a fifth container containing Buffer SolBS,   wherein SolBH selected from the group consisting of HCl, H 2 SO 4 , HNO 3  and CH 3 COOH, buffer SolBS, distilled water and biological material which interacts with target biological material are added sequentially to a sol composition consisting of SolB1, SolB2 and SolB3 so that the sol composition is gelled.   
     
     
         27 . A method for analyzing a target biological material, the method comprising a step of adding a sample, which contains the target biological material capable of interacting with the biological material, which interacts with target biological material, to the biochip prepared by the method of  claim 1 . 
     
     
         28 . The method of  claim 27 , wherein the target biological material is any one selected from the group consisting of nucleic acids, proteins, peptides, low-molecular-weight materials, and cells. 
     
     
         29 . The method of  claim 27 , further comprising a step of allowing the target biological material to react with a protein, antibody, or aptamer labeled with a radioactive isotope, dye, a fluorescent dye, or luminescent material which can detect the target biological material.