KR20100049880A - Microarray with micro-well pattern and manufacturing method of the same - Google Patents

Abstract

PURPOSE: A micro array substrate and manufacturing method thereof is provided to quantitatively achieve a detection result from the information of an amount of target DNA which is combined with a fixed amount of a complementary DNA. CONSTITUTION: A micro array substrate comprises: a base substrate(200) which is composed of glass, silicon, and silicone oxide; a self-assembled thin film(210) which is composed of one among thiol group, silanol group, phosphine, L-lysine, amine, aldehyde, epoxy and polylysine and coated on the base substrate; and a polymer substrate(100) which is composed of one among poly- dimethyl siloxane, poly- methyl methacrylate, polycarbonate, polyethylene, polypropylene and polystyrene and wherein a microwell(110) is formed.

Description

Microarray substrate and its manufacturing method {Microarray with micro-well pattern and Manufacturing method of the same}

The present invention relates to a micro array substrate and a method of manufacturing the same, and specifically, to fabricating a micro array substrate having a predetermined micro well pattern by combining a perforated polymer substrate to a base substrate during the manufacture of the micro array substrate. The present invention relates to providing a microarray substrate capable of loading complementary DNA (cDNA) into a well.

Biochip is a chip that attaches high density biomolecules probe such as DNA and protein to be analyzed on the substrate, and can analyze gene expression patterns, gene defects, protein distribution, reaction patterns, etc. in the sample. have. Biochips may be divided into microarray chips attached to a solid substrate and lab-on-a-chips attached to microchannels according to the attachment form of the probe. In other words, the biochip is a biological material such as nucleic acid is fixed on the substrate (substrate), a well-known biochip is a DNA chip, which can be said that the DNA is fixed on the substrate, the protein chip is a protein fixed to the substrate It can be said that there is.

DNA microarrays can be used to create hundreds or hundreds of baseline oligonucleotide probes with known base sequences on solid surfaces such as silicon, surface modified glass, polypropylene, and activated polyacrylamides. It is commonly referred to as microarray by attaching to hundreds of thousands of fixed positions.

When the target DNA fragment to be analyzed is bound to the DNA microarray, the hybridization state is different depending on the complementary degree of the nucleotide sequences on the target DNA fragment and the probes attached to the DNA microarray. The sequencing of the target DNA can be analyzed by observing and interpreting the optical method or radiochemical method (sequencing by hybridization; SBH). DNA chips manufactured using DNA microarrays can be genetically analyzed with only a small amount of sample by miniaturizing the DNA analysis system, and can simultaneously identify several base sequences on the target DNA. Information can be provided.

In addition, DNA chips manufactured using DNA microarrays can not only simultaneously analyze a large amount of genetic information in a short time, but also define correlations between genes. It is expected to be applied to a wide range of fields such as pathogen detection, gene expression analysis and drug development. In addition, by detecting genes for detoxifying substances and applying genetic recombination technology by using them as detectors for microorganisms or environmental pollution, they can be applied to mass production of detoxifying substances, medicinal crops and low-fat meats. It can be used in the biological industry, leading to revolutionary development.

DNA microarrays are divided into oligo chips and cDNA chips according to the type of probes used, and photolithography chips, spotting chips, and inkjet spotting depending on the fabricated method. Sometimes classified as a chip.

Specifically, when divided into methods for manufacturing this field, two methods of synthesizing oligonucleotides on a substrate and a method of fixing a pre-prepared DNA probe on a substrate are generally used. . The former method mainly uses photo lithography to extend and stretch DNA probes on a semiconductor substrate. The latter method processes the substrate surface with a positive charge to fix the prepared DNA probe on the substrate. And a method of surface treatment of a reactive group are utilized.

 Each method has advantages and disadvantages. While the method of synthesizing on a substrate is excellent in reproducibility, the latter method has the advantage that it can be manufactured at a relatively low cost. The most important aspect of microarray fabrication is the high density and stable immobilization of DNA probes on the substrate.

 However, in general, methods of using inkjet, dispenser, or surface processed slide glass, which is a method of spotting probe DNA on a slide glass or a substrate, have a spot shape when the solution is dropped, or are mixed with neighboring probe DNA. Discarding problems may occur.

Various methods have been tried to solve this problem.

 For example, attempts have been made to improve detection sensitivity by forming an uneven surface on a micro array substrate to form a nucleic acid probe, thereby increasing the amount of solid state of probe DNA per spot.

In addition, as shown in FIG. 1A, in the micro array 2, a hydrophilic region 3 to which a probe biopolymer is immobilized and a hydrophobic region 4 to which the probe biopolymer is not immobilized are formed using a photocatalytic technique. Separately, attempts have been made to improve the number of spots of DNA and to ensure that the probes are specifically bound to the desired location. Of course, the detection material used a dropping method using the spotter device (5).

In addition, as shown in FIG. 1B, in addition to the method of increasing the precision in manufacturing the substrate in this manner, the pattern of the positive control 1c surrounded by the negative control 1d in the several blocks 1b. In order to improve the quantitative and precision in the detection determination process with respect to the DNA microarray which consists of (pattern) arrangement | positioning, the method of providing the reference pattern which consists of several heterogeneous position marks 1c on the board | substrate has been proposed.

However, these conventional methods are difficult to position the DNA probe exactly the desired amount in the selected area, and it is difficult to improve the accuracy of image analysis when positioning the detection area and correcting the image when only the surface treatment is performed upon input to the scanner device. There is a problem.

The present invention has been made to solve the above-mentioned problems, an object of the present invention is to combine the perforated polymer substrate with the base substrate in the manufacture of the micro array substrate, by producing a micro array substrate having a constant micro well pattern, respectively The present invention provides a microarray substrate capable of loading complementary DNA (cDNA) into a microwell.

In addition, the present invention can improve the reproducibility and precision in remanufacturing a micro-substrate having a microwell having a certain shape and size, to enable high integration, to improve the sensitivity, and to provide a multi-array inspection microarray substrate. The specific purpose is to provide.

The present invention is to provide a micro-array substrate, characterized in that the polymer substrate to form a micro-well on the base substrate is combined with the configuration for solving the above problems. Since the micro array according to the present invention is clearly distinguished between each spot, it is possible to eliminate the error rate generated during manufacturing, and the amount of reaction with the target DNA is constant, so that the detection sensitivity can be increased, and the pattern position is always constant, so freeze it. It can be used as an key to improve the clarity of the analysis. In addition, the detection result can be quantitatively grasped through the information on the target DNA amount bound to the constant complementary DNA amount dropped.

In the above-described configuration according to the present invention, the base substrate is characterized in that a self-assembled monolayer (SAM) is coated.

In addition, the base substrate may be made of glass, silicon, silicon oxide (slicon oxide).

In addition, the self-assembled thin film of the microarray according to the present invention includes a functional group selected from thiol group, silanol group, phosphine group, L-lysine, amine, aldehyde, epoxy, and poly-L-lysine. It may be formed of a material.

In addition, the polymer substrate constituting the micro array of the present invention, poly-dimethylsiloxane (PDMS), poly-methyl methacrylate (poly methylmethacrylate), polycarbonate, polyethylene (polyethylene), poly It may be formed of any one selected from propylene (polypropylene), polystyrene (polystyrene).

In order to improve the bonding strength of the base substrate and the polymer substrate and the production efficiency in the present invention, it is preferable that the polymer substrate is hydrophobically treated to modify the surface.

The microarray manufacturing process according to the present invention having the above-described structure may be manufactured by bonding a polymer substrate having a micro-well to a base substrate coated with a self-assembled thin film.

In this manufacturing process, the microwells may be formed by punching the polymer substrate through punching, or may be formed by molding the polymer substrate.

In particular, the process of molding and forming the microwells described above,

1) coating a photosensitive material on the silicon wafer and forming a molding pattern through exposure and development;

2) After forming the pattern, trifluoropropyl trimethoxy silane (tirchloro (3,3,3-trifluoropropyl) -silane), 3-aminopropylmethyldiethoxysilane (APDES), 3-aminopropyltriethoxysilane (APTES), 3 in a vacuum silanizing a silicon wafer surface using -methacryloxypropyltrimethoxysilane (MPDMS), 3-methacryloxypropyltrimethoxysilane, or the like;

3) forming a polymer substrate in which a micro well is formed by injecting a liquid polymer material into the silanized pattern.

In addition, in this process, the present invention may further include a step of performing hydrophobic surface treatment on the polymer substrate after step 3), so that the bonding force with the substrate and the production efficiency can be achieved.

In addition, the manufacturing process according to the present invention may further include the step of performing a plasma treatment on the polymer substrate, and bonding to the base substrate after the step 3).

In addition, in the above-described manufacturing process, the photosensitive material of step 1) may be formed of a negative photosensitive agent.

In addition, the polymer material is poly-dimethylsiloxane (PDMS), poly-methyl methacrylate (poly methylmethacrylate), polycarbonate (polycarbonate), polyethylene (polyethylene), polypropylene (polypropylene), polystyrene ( polystyrene).

According to the present invention, when a microarray substrate is manufactured, a perforated polymer substrate is bonded to a base substrate to prepare a microarray substrate having a predetermined microwell pattern, and thus, complementary DNA (cDNA) can be loaded into each microwell. Provided is a microarray substrate.

 Since the microarray substrate according to the present invention can be clearly distinguished from each spot, it is possible to eliminate the error rate generated during manufacturing, and because the amount of reaction with the target DNA is constant, the detection sensitivity can be increased, and the pattern position is always constant. This can be used as an align key to improve the clarity of the analysis. In addition, the detection result can be quantitatively grasped through the information on the target DNA amount bound to the constant complementary DNA amount dropped.

The perforated polymer substrate may be combined with the glass substrate after changing the surface properties, and furthermore, the surface treatment of the polymer substrate may be hydrophobic to maximize the efficiency of spot fabrication.

In particular, the micro array substrate according to the present invention has an advantage that an array substrate capable of multi-item inspection can also be easily manufactured by configuring a predetermined pattern for each zone.

The present invention can be implemented in addition to the advantages of improving the reproducibility and precision in the manufacture of a micro-substrate having a micro well having a certain shape and size, and to improve the sensitivity by enabling high integration.

In particular, compared to the case of making a pattern by photolithography, a perforated polymer substrate is produced by molding or punching, and thus has a low cost. After combining glass and perforated polymer substrate, amine, aldehyde, epoxy, By surface treatment using self assembled monolayers such as poly-L-lysine, various types of microarray substrates can be economically manufactured.

Hereinafter, with reference to the accompanying drawings, it will be described in detail the configuration and manufacturing process of the micro array substrate according to the present invention.

Referring to Figures 2 and 3, shown is a conceptual diagram of a manufacturing process of a micro array substrate according to the present invention.

The micro array substrate according to the present invention is basically formed by bonding the polymer substrate 100 and the base substrate 200 on which the microwells 110 are formed.

Specifically, a polymer substrate having micro wells is prepared (S1). In this case, the micro well 110 basically means a fine perforation pattern formed on the polymer substrate 100. After that, the glass substrate is bonded to the base substrate 200 (S2). The surface treatment may be performed by coating a self-assembled thin film 210 such as a thiol group, silanol group, phosphine group, L-lysine, amine, aldehyde, epoxy, poly-L-lysine, or the like on a substrate. By forming the micro array substrate can be completed (S3).

As the base substrate 200 used in such a manufacturing process, a glass substrate may be used. In addition, the base substrate may be used, such as silicon, silicon oxide (slicon oxide), and not necessarily limited to the type of substrate.

The polymer material forming the polymer substrate 100 may be poly-dimethylsiloxane (PDMS), poly-methylmethacrylate, polycarbonate, polyethylene, polypropylene, or polypropylene. ), Polystyrene (polystyrene) can be made of any one, and particularly preferably PDMS can be used. PDMS is itself a non-toxic, bio-friendly material that has a hydrophobicity in the liquid phase, and has an excellent advantage in terms of efficiency.

In addition, the self-assembled thin film 210 may be formed by including any functional group selected from thiol group, silanol group, phosphine group, L-lysine, amine, aldehyde, epoxy, poly-L-lysine. have.

In addition, the above-described polymer substrate on which the micro well is formed is more preferable to improve the performance of the micro array substrate by hydrophobic treatment of the surface.

Hereinafter, an example as a method of forming a micro well in the above-described polymer substrate will be described. The micro well may be manufactured in various ways using a punching method or a molding method.

1. Punching method

First, a liquid polymer material (for example, PDMS, etc.) of a flat structure is poured slowly, bubbles are removed, and dried at 60 ° C. for 3 hours to form a thin polymer plate. Subsequently, a microwell may be formed by punching a PDMS plate removed from the Petri dish using a punch.

Subsequently, after the plasma treatment of the high-level substrate, the glass substrate is attached to the oven and then bonded.

2. Molding method

First, a negative photosensitive agent (eg, SU-8, etc.) is coated on a silicon wafer, exposed with a mask having a pattern having a desired size, and then developed with a developer to prepare a mold having a pattern for molding.

After forming the negative photosensitive material pattern, trifluoropropyl trimethoxy silane (tirchloro (3,3,3-trifluoropropyl) -silane), 3-aminopropylmethyldiethoxysilane (APDES), and 3-aminopropyltriethoxysilane (APTES) in a vacuum state ), Silanization is performed using a silanization coupling agent such as 3-methacryloxypropyltrimethoxysilane (MPDMS) and 3-methacryloxypropyltrimethoxysilane.

Thereafter, the polymer material (eg, PDMS, etc.) is poured slowly, bubbles are removed, and dried at 60 ° C. for 3 hours to form a polymer substrate on which thin microwells are formed.

Of course, after the plasma treatment to the polymer substrate, it can be bonded to the base substrate and then put in an oven.

As described above, the micro array in which the micro well is implemented has an advantage of being inexpensive because it manufactures a perforated polymer substrate using molding or punching, compared to the case of making a pattern by photolithography, and has a glass and a perforated polymer. After substrate bonding, various types of microarray substrates can be economically produced by surface treatment using self assembled monolayers such as amine, aldehyde, epoxy, poly-L-lysine, etc., depending on the application.

An example of detection using the microarray manufactured by the above method will be described with reference to FIG. 4.

Referring to FIG. 4, the complementary DNA 140 is arrayed using a pin spotting or inkjet device 130 in a manufactured micro array. Thereafter, the target DNA (T) to which the fluorescent material (P) is bound is combined with the complementary DNA (120; C).

Then, wet the target DNA (T) (which is bound to the fluorophore) in solution and wash the unbound after the reaction. Through it, you can check the detection result of specific DNA.

The microarray according to the present invention combines a perforated polymer substrate with a base substrate, and manufactures a microarray substrate having a constant microwell pattern, thereby allowing complementary DNA (cDNA) to be loaded into each microwell.

Such a structure improves reproducibility and precision in manufacturing a micro substrate having a micro well having a certain shape and size, and enables high integration to improve sensitivity. In particular, since each spot is clearly distinguished, it is possible to eliminate the error rate generated during manufacturing, and because the amount of reaction with the target DNA is constant, the detection sensitivity can be increased, and since the pattern position is always constant, it is used as an align key. This has the advantage of improving the clarity of the analysis. In addition, it is possible to grasp the detection result quantitatively through information on the target DNA amount bound to the constant complementary DNA amount loaded. In addition, by configuring a predetermined pattern for each zone, an array substrate capable of multi-item inspection can also be easily manufactured.

In the foregoing detailed description of the present invention, specific examples have been described. However, various modifications are possible within the scope of the present invention. The technical idea of the present invention should not be limited to the embodiments of the present invention but should be determined by the equivalents of the claims and the claims.

1A and 1B illustrate an example of a method for improving precision for manufacturing a substrate in a method of manufacturing a micro array according to the related art.

2 to 3 is a schematic view showing the configuration and manufacturing method of a micro array substrate according to the present invention.

4 is a view showing a method of using a micro array substrate according to the present invention.

Claims (14)

A micro array substrate, characterized in that the polymer substrate is bonded to form a micro-well on the base substrate. The method according to claim 1, The base substrate is a micro-array substrate, characterized in that the self-assembled monolayer (SAM) is coated. The method according to claim 2, The base substrate is a micro array substrate, characterized in that made of glass, silicon, silicon oxide (slicon oxide). The method according to claim 2, The self-assembled thin film is formed of a material containing any one of functional groups selected from thiol group, silanol group, phosphine group, L-lysine, amine, aldehyde, epoxy, poly-L-lysine Micro array substrate. The method according to claim 2, The polymer substrate may be selected from poly-dimethylsiloxane (PDMS), polymethylmethacrylate, polycarbonate, polyethylene, polypropylene, and polystyrene. Micro array substrate, characterized in that made of any one. The method according to any one of claims 1 to 5, The polymer substrate is a micro array substrate, characterized in that the surface is hydrophobic treatment. A method of manufacturing a micro array substrate, comprising bonding a polymer substrate having a micro-well to a base substrate coated with a self-assembled thin film. The method of claim 7, The microwell is a method of manufacturing a micro-array substrate, characterized in that formed by punching the polymer substrate through punching. The method of claim 7, The microwell is formed by molding the polymer substrate. The method according to claim 9, The process of forming the microwell by molding, 1) coating a photosensitive material on the silicon wafer and forming a molding pattern through exposure and development; 2) after forming the pattern, silanizing the silicon wafer surface in vacuum; 3) forming a polymer substrate on which microwells are formed by injecting a liquid polymer material into the silanized wafer and the pattern; Method of producing a micro array substrate comprising a. The method of claim 10, wherein step 2) Trifluoropropyl trimethoxy silane (tirchloro (3,3,3-trifluoropropyl) -silane), 3-aminopropylmethyldiethoxysilane (APDES), 3-aminopropyltriethoxysilane (APTES), 3-methacryloxypropyltrimethoxysilane (MPDMS), 3-methacryloxypropyltrimethoxysilane Method for producing a micro array substrate, characterized in that the silanization is carried out using any one. The method according to claim 10, After step 3) above, The method of manufacturing a micro array substrate further comprising the step of performing a hydrophobic surface treatment on the polymer substrate. The method according to claim 10, After the step 3), Plasma treatment on the polymer substrate, and further comprising the step of bonding to the base substrate. The method according to claim 10, The polymer material is selected from poly-dimethylsiloxane (PDMS), poly-methylmethacrylate, polycarbonate, polycarbonate, polyethylene, polypropylene, and polystyrene. Method for producing a micro array substrate, characterized in that made of any one.

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