KR20100038569A - Microarray with black matrix pattern and manufacturing method of the same - Google Patents

Abstract

PURPOSE: A micro array substrate and a manufacturing method thereof are provided to improve detection sensitivity by constantly maintain a reaction amount with a target DNA. CONSTITUTION: A unit cell pattern(20) is formed on a substrate and is comprised of a black matrix. The unit cell pattern includes a plurality of unit cells. A fixing probe and a target probe are fixed inside the unit cell. The fixing probe is a complementary DNA(30).

Description

Microarray substrate and its manufacturing method {Microarray with Black Matrix Pattern and Manufacturing method of the same}

The present invention relates to a microarray substrate and a method of manufacturing the same, and to a technique of forming a constant black pattern during fabrication of a microarray substrate to achieve high integration, high integration, and improved detection sensitivity. will be. In particular, the present invention relates to a technology for providing a substrate that can be utilized for multi-item inspection by using a pattern as an alignment key.

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.

In particular, with the phenomenal progress of recent human genome projects, there has been a great demand for a way to quickly provide a huge amount of genetic information in the diagnosis, treatment and prevention of genetic diseases. Sanger's method, which has been used as a sequencing method until then, has been steadily developed due to the development and automation of polymerase chain reaction (PCR) method that replicates DNA. Because of the cost, effort, cost, and high level of proficiency, it is not possible to analyze large quantities of genes, and new sequencing systems are constantly being searched for. The needs of the times have made progress in many areas related to the fabrication and use of DNA microarrays over the last few years.

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 accuracy in manufacturing the substrate in this manner, the positive control 1c enclosed 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 arrangement | positioning of a pattern, the method of providing the reference pattern which consists of several heterogeneous position marks 1c on the board | substrate was 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-described problem, an object of the present invention is to form a unit cell pattern on a substrate when manufacturing a micro-array substrate, it is possible to put a fixed material and a target material in the section partitioned by the pattern In order to improve the accuracy of detection and to provide a highly integrated micro array substrate.

In order to solve the above-mentioned problems, the present invention specifically forms a section through a unit cell pattern, preferably a black pattern, having a predetermined standard in manufacturing a micro array substrate, in order to improve detection accuracy and detection sensitivity. Complementary DNA is loaded in each section, so that it can be accurately distinguished between spots. Through this, the error rate generated during the manufacture of the microarray substrate can be removed, and the amount of reaction with the target DNA is constant, thereby increasing the detection sensitivity.

Furthermore, the spot divided by the pattern of the microarray substrate, that is, the surface of the unit cell can be modified to maximize the efficiency of spot fabrication, the darkroom contrast ratio due to the black pattern can be improved, and the position of the pattern is aligned. Can also be used as a key to improve clarity of analysis. In particular, by configuring a predetermined pattern for each zone, it is possible to provide an array substrate capable of multi-item inspection using one substrate.

According to the present invention, a section is formed through a black pattern in the manufacture of a micro array substrate, and complementary DNA is dropped in each section, thereby accurately distinguishing between spots, thereby eliminating an error rate generated during manufacturing. As a result, the amount of reaction with the target DNA is constant, thereby increasing the detection sensitivity.

In addition, the efficiency of spot fabrication can be maximized by modifying the section of the microarray substrate, that is, the surface of the unit cell, and the darkroom contrast ratio due to the black pattern when detecting the target DNA in the unit cell divided by the black pattern. In addition, the clarity of the analysis can be improved by using the position of the pattern as a key for alignment. In particular, when a certain pattern is configured for each zone, there is also an effect that can easily produce an array substrate capable of multi-item inspection.

The present invention provides a micro array substrate in which a unit cell pattern is formed on a substrate in a micro array, by using a standardized pattern to improve the precision of biometric information acquisition, and to enable high integration to increase detection sensitivity. To improve.

In another aspect, the present invention provides a micro-array substrate, characterized in that the unit cell pattern is made of a black matrix (BM), when detecting a target material such as target DNA existing between the unit cell pattern with a fluorescent scanner The contrast ratio of the darkroom can be improved to improve the detection sensitivity.

In another aspect, the present invention provides a micro-array substrate, characterized in that the substrate and the unit cell pattern has opposite properties to each other, so that the fixed probe, i.e. Allow complementary DNA to bind easily.

In addition, the present invention is to provide a micro-array substrate, characterized in that the unit cell pattern is made of a material having water repellency.

In addition, the present invention is to provide a micro-array substrate, characterized in that the unit cell pattern is formed of a structure in which a plurality of unit cells in which the fixed probe and the target probe is fixed. In particular, when the pattern is implemented as a regular standardized pattern, it is possible to implement the clarity of analysis by utilizing the position of the predetermined pattern as an alignment.

The present invention also provides a micro array substrate, characterized in that the fixed probe is complementary DNA (Complementary DNA).

In addition, the present invention provides a micro-array substrate, characterized in that the immobilizing agent is applied on the substrate on which the pattern is implemented, so that the fixing probe, ie, complementary DNA, is easily bound to the substrate region exposed between the patterns after the implementation of the pattern. To help.

In particular, since the unit cell pattern has the same size and pattern shape of each unit cell, the pattern is implemented as a regular standardized pattern, and the target DNA is detected by dropping and fixing the complementary DNA by a certain method. To increase the accuracy. Furthermore, the clarity of the analysis can be realized by utilizing the position of a certain pattern as an alignment, and further, the substrate on which the unit cell pattern is formed is heat-treated to modify the surface to control the degree of hydrophilicity, so that the complementary DNA is patterned (in the unit cell). Make sure to fill evenly between).

In another aspect, the present invention provides a method of manufacturing a micro-array substrate, characterized in that the unit cell pattern is formed by any one method selected from the photolithography method, the inkjet method, the dispenser method. In particular, in addition to the above-described method, any method that can form a pattern may be used to implement a variety of manufacturing methods.

The present invention also provides a method of manufacturing a micro-array substrate, further comprising the step of dropping and fixing complementary DNA to a unit cell formed by the unit cell pattern. The complementary DNA serves as a fixed probe to allow the target DNA to bind, and a certain amount of the complementary DNA is applied inside a predetermined pattern, thereby increasing the accuracy of detection of the target DNA.

In another aspect, the present invention provides a method for producing a micro-array substrate, characterized in that it further comprises the step of treating the immobilizing agent to the substrate exposed in the unit cell, it is possible to implement even filling of the complementary DNA.

In addition, the present invention uses a complementary DNA formed in each section as a fixed probe on a micro array substrate having a black pattern partitioned into a plurality of predetermined sections on the substrate, the target DNA combined with a fluorescent material is complementary It is possible to provide a method of using a micro array substrate, characterized in that the binding to the DNA to detect the detection by a scanner. In particular, the target DNA is bound to the complementary DNA on the substrate, and the washing does not occur (washing) so that the specific detection result can be confirmed through fluorescence expression of the target DNA of the bound portion.

The present invention also provides a method of using a micro array substrate, wherein analyzing the detection by the scanner aligns the position of the pattern by utilizing the coordinates of the edge of the unit cell pattern of the unit cell pattern. to provide. In particular, since the unit cell pattern has a certain standard, it is possible to quantitatively add complementary DNA to the unit cell without displaying an additional alliance, and to analyze the target DNA analysis result expressed by coordinates of the corners of the pattern. The precision can be improved. That is, the biometric information of the location can be made into data from a positionally accurate point of view.

In addition, the present invention provides a method of using a micro-array substrate, comprising a plurality of zones formed through the unit cell pattern, and performs a multi-item inspection for each zone at the same time, coordinates the unit cell pattern Each detection information can be partitioned as a position for each coordinate, so that a plurality of detection items can be classified on one substrate, and this can be divided into specific coordinate areas for inspection of multiple items. Enable usability.

Hereinafter, with reference to the accompanying drawings will be described in detail the configuration and operation of the present invention.

2 is a plan view of a micro array substrate according to the present invention.

The micro array substrate according to the present invention is characterized by implementing a pattern 20 divided into unit cells having a predetermined standard on the upper surface of the substrate 10. The substrate 10 may utilize various materials such as slide glass and plastic, and the unit cell pattern 20 may be formed by any method such as photolithography, inkjet, and dispenser. The material for embodying the unit cell pattern may be used as long as the material has a black color (black), and the lower the light transmittance, the more preferable. However, it is better to use a material that can be surface modified after implementing the pattern. One example of such a preferred material may be a black matrix (BM).

The unit cell pattern may be implemented in various shapes and sizes, and preferably, a section (unit cell; C) partitioned into each pattern is formed to have a predetermined size and size as shown.

Complementary DNA 30 is filled in the exposed substrate region, ie, the unit cell C, between the pattern and the pattern. In particular, before filling the complementary DNA, it is preferable to apply an immobilizing agent (poly-L-lysine, etc.) so that the complementary DNA can easily bind.

The application of the complementary DNA can be carried out by various methods such as an inkjet, dispenser method, and the like, in particular, quantitative dropping is possible in a unit cell partitioned by a predetermined capacity, thereby improving accuracy.

2B is a side view of a micro array substrate in accordance with the present invention. As illustrated, the unit cell pattern 20 is formed on the substrate 10, and the unit cell C partitioned into the unit cell pattern has a structure in which the complementary DNA 30 is filled.

That is, in the manufacture of the microarray substrate according to the present invention, a black matrix pattern, that is, a black pattern 20 is formed on the substrate 10, and the complementary DNA 30 is formed on the substrate by inkjet, dispensing, coating, or transcription. ) Is added to the unit cell. In this case, in order to increase the binding force between the complementary DNA and the substrate, an immobilizing agent may be treated between the patterns after the formation of the pattern.

An embodiment of another method of manufacturing a micro array substrate according to the present invention will be described with reference to FIG. 2C.

Forming the unit cell pattern 20 on the substrate 10 and heat-treating the substrate on which the unit cell pattern is formed are different from the basic manufacturing method of the present invention described above. As a matter of course, the formed pattern may be formed as a black pattern such as a black matrix having low light transmittance as described above. Preferably, the heat treatment is performed at 25 to 400 ° C., and in particular, the heat treatment can control the degree of hydrophilicity of the substrate. In addition, after the substrate is heat treated, the fixative may be additionally treated between the patterns.

Subsequently, complementary DNA is added to the unit cell by inkjet, dispensing, coating, or transcription on the substrate. As shown in FIG. 2C, the surface of the unit cell C having the black pattern 20 formed on the substrate 10 is modified by (b) heat treatment, and the complementary DNA is filled in the unit cell. . In this case, complementary DNA is uniformly distributed in the unit cell to the edge of the unit cell. This shows that, as shown in (c), the water repellency of the black pattern is weakened through the heat treatment, so that the complementary DNA solution is not pushed out, and the distribution in the pattern is perfect. In particular, when the microarray is manufactured, spots (complementary DNA are loaded onto the substrate) can be clearly distinguished from each other, and thus, error rates generated during manufacturing can be eliminated. Through the information on the amount of target DNA binding, there is an advantage that can detect the detection result quantitatively. In particular, as described above, since the surface properties can be changed in a certain black pattern, it is also possible to maximize the efficiency of spot fabrication.

An example of use of a detection method using a micro array substrate according to the present invention will be described with reference to FIG. 3.

As described above, the target DNA 40 to which the fluorescent material 50 is bound on the microarray substrate is coupled with the complementary DNA 30 in accordance with the present invention in which the complementary DNA 30 is filled. This binding basically wets the target DNA (bound with the fluorescent substance) in the solution state on the substrate, and after the reaction occurs, washing the non-binding with the complementary DNA, leaving only the portion where the binding occurred.

After that, when the scanner detects whether it detects the fluorescence is expressed only in the portion where the binding occurs, it is possible to confirm the detection result for a specific DNA. Particularly, when the unit cell pattern is formed as a black pattern (black matrix), there are materials to be detected between the black patterns. When the detection material is analyzed and analyzed by a fluorescent scanner, the dark pattern contrast ratio may be improved. In addition, since the position of the pattern is always constant, it can be used as an alignment key, thereby improving the clarity of analysis. The use of patterns as alignment keys is described below.

4, another use case of the micro array substrate according to the present invention will be described.

Basically, a preferred embodiment according to the present invention in which the unit cell pattern is formed on the substrate as a black pattern will be described as an example. In the microarray substrate according to the present invention, when the black pattern is recognized as the coordinate of the coordinate system, and the coordinate position is given at the corner portion of the pattern, the biometric information on the accurate array substrate can be converted into data for each position. In particular, when the edge of the pattern is coordinated and processed, the analysis accuracy of the detection result can be improved. This solves the process problems of the conventional method of forming a reference pattern for forming a plurality of heterogeneous position marks on a substrate in order to increase the precision in manufacturing a micro substrate at a time, and furthermore, The advantage that the multi-item inspection can be performed by partitioning the location area on one micro array substrate according to the present invention.

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.

2A to 2C are schematic diagrams showing the configuration and manufacturing method of the micro array substrate according to the present invention.

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

4 conceptualizes a method of using the black pattern according to the present invention as an align key.

Claims (14)

In a micro array, The micro array substrate, characterized in that the unit cell pattern is formed on the substrate. The method according to claim 1, The unit cell pattern is a micro array substrate, characterized in that the black matrix (BM). The method according to claim 2, And the substrate and the unit cell pattern have opposite hydrophilic properties. The method according to claim 2, The unit cell pattern is a micro array substrate, characterized in that made of a water-repellent material. The method according to claim 3 or 4, The unit cell pattern may include a structure in which a plurality of unit cells in which a fixed probe and a target probe are fixed therein are formed. The method according to claim 5, The fixed probe is a micro array substrate, characterized in that the complementary DNA (complementary DNA). The method according to claim 5, Microarray substrate, characterized in that the fixing agent is applied on the substrate on which the pattern is implemented. A method of manufacturing a micro array substrate, characterized in that to form a pattern having a unit cell divided into a plurality of sections having a predetermined shape on the substrate. The method according to claim 8, The unit cell pattern is a method of manufacturing a micro array substrate, characterized in that formed in any one method selected from photolithography method, inkjet method, dispenser method. The method according to claim 8, And dropping and fixing the complementary DNA in the unit cell pattern. The method according to claim 10, And treating the immobilizing agent with the substrate exposed in the unit cell. Complementary DNA formed in each section is used as a fixed probe on a micro array substrate having a unit cell pattern divided into a plurality of regular sections on the substrate, A method of using a micro array substrate, characterized in that a target DNA combined with a fluorescent material is bound to the complementary DNA and detected by a scanner. The method according to claim 12, Analyzing whether the scanner detects the position of the pattern using the coordinates of the edges of the unit cell patterns of the substrate, wherein the position of the pattern is aligned. 14. The method of claim 13, A method of using a micro array substrate, comprising: configuring a plurality of zones formed through the unit cell pattern, and performing multi-item inspection for each zone simultaneously.

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