KR100544860B1 - Sample Plates and Manufacturing Method Thereof - Google Patents

Abstract

Disclosed are a sample plate and a method of manufacturing the same, which are applied to analyze a sample for analysis. The sample plate includes patterns having a substrate and an upper surface on which the sample is placed to concentrate a sample formed and provided on the substrate. And a hydrophobic coating layer covering an upper surface of the substrate and having an opening for selectively exposing the central portions of the patterns so that the solvent contained in the sample is evaporated and concentrated in the central portions of the patterns. The pattern of the sample plate having the above-described configuration is formed by a photo-etching process, the solvent contained in the sample can be evaporated faster for analysis, and can be used for one-time without cleaning process, so that the sample used for analysis can be stored. The sample can be reanalyzed after analysis.

Description

Sample plate and method of manufacturing the same

1 is a block diagram showing a Maldi anchor plate that is a conventional sample plate.

2 is a block diagram showing a sample plate according to an embodiment of the present invention.

FIG. 3 is a cross-sectional view of the sample plate illustrated in FIG. 2 taken along the line II ′. FIG.

4 to 10 are cross-sectional views showing a method of manufacturing a sample plate of the present invention.

11 is a graph showing MALDI spectral data of a standard peptide sample using the sample plate of the present invention.

12 is a graph showing MALDI spectral data of a standard peptide sample using a conventional Maldi anchor plate.

Explanation of symbols on the main parts of the drawings

100 substrate 110 first photoresist film

110a: first photoresist pattern 120: pattern

130a: second photoresist pattern 140a: hydrophobic coating film

D: opening

The present invention relates to a sample plate metal and a method for producing the same, and more particularly, to a sample plate and a method for manufacturing the same applied to the analysis of the concentration of the analyte contained in the sample.

Recently, with the rapid development of the semiconductor industry and the bio industry, analytical devices and methods for analyzing the physical and chemical properties of analytes applied to the semiconductor industry and the bio industry more quickly and quantitatively have been proposed.

In particular, among the methods for analyzing the analyte, Maldi (Matrix Assisted laser Desorption Ionization; MALDI) mass spectrometry is a sample plate (Sample) by mixing the analyte to be analyzed with a matrix sensitive to the laser (laser) Spotting is formed on the surface of the plate, and the sample plate is irradiated with a laser to ionize the sample and then analyze the mass of the ionized sample.

In the MALDI analysis method, the analysis sensitivity, resolution, and quality of analytical data are greatly influenced by the prepared analytical sample. This is because when the salt contains a large amount of the analyte, ionization is not well performed, and when an impurity is included, it is difficult to interpret data of the analyte to be analyzed by a peak from the impurity. In addition, in order to perform an effective Maldi analysis method, the analytical sample should be mixed with the matrix and provided as small spots as possible on the surface of the sample plate.

Currently, the Maldi anchor plate 10, which is a sample plate illustrated in FIG. 1, which is applied to perform the Maldi analysis method, is mainly used by chemically stable and less reactive strainless substrate 12. Since the anchor plate 10 needs to be reused after performing a separate cleaning process after one use, if the sample residue is present on the surface of the patterns 14 formed on the surface of the plate due to the operator's carelessness, subsequent analysis is performed. Sometimes failures occur. In addition, despite the need to reanalyze the results of the analyte for post-reconfirmation, there is a problem in that samples that are difficult to prepare for reuse of the sample plate must be removed. In addition, the plate has a thickness of 5mm or more, so that it is difficult to handle in the division apparatus, and has a disadvantage that the evaporation rate of the solvent contained in the sample is remarkably slow.

In addition, the sample plate may be applied to biological sample analysis such as surface enhanced laser desorption ionization (SELDI), protein chip, surface plasma resonance (SPR) as well as MALDI.

For example, in the case of the sensor chip used as the SELDI sensor, the core technology is a protein immobilization technology that binds a protein to the surface of the chip (sample plate), and may be classified into three methods as follows. The first method is to immobilize a specific protein on the surface of the sensor chip. One of the most widely used protein immobilization methods is CM (carboxy methyl) -dextran. Amine bonds are most commonly used, and thiol bonds and avidin-biotin bonds are also used to bind acidic proteins and DNA. The second method is to treat the surface of the sensor chip to combine a group of proteins with the same characteristics. Currently available surfaces are hydrophilic surface and hydrophobic surface. ), Ion exchange surfaces, immobilized metal surfaces, and the like. Finally, polylysine or calix crowns may be used to bind a number of unspecified proteins.

In addition, the protein chip has a variety of applications compared to the DNA chip, the diagnostic chip (Screening), quantification (Kinetic Measurement), mass analysis (Mass Spectrometric Analysis) The application is based on four characteristics. The surface treatment of the protein chip is applied to the case of the sensor chip mentioned above, and the preparation of a protein suitable for the purpose becomes more problematic than the surface treatment of the sample plate used as the protein chip. In addition, in the case of SPR, sample play is used by coating a thin film on the glass substrate for efficient total reflection of the laser used, and then surface treatment to have various surface properties.

However, the sample plates applied to the MALDI, surface enhanced laser desorption ionization (SELDI), protein chips, and surface plasma resonance (SPR) are mostly expensive (protein chips: thousands of dollars ($), SELDI chips: $ 1500/10 each). ($), SPR chip: $ 700/3 each) Not only is there a limit to use, but it is not available for sample storage or disposable.

In addition, the pattern for concentrating the sample is formed on the sample plate only by surface etching without protruding shape. Since the patterns are formed by etching of the substrate using a laser, it is difficult to mass-produce and the manufacturing cost is high. In addition, there is a problem that takes a lot of production time.

Accordingly, an object of the present invention for solving the above problems is to significantly reduce the manufacturing cost and manufacturing time, to provide a sample plate that can be used for disposable or storage, and excellent in the volatility of the solvent.

In addition, another object of the present invention for solving the above problems is to provide a method of manufacturing a sample plate that can significantly reduce the manufacturing cost and manufacturing time.

Therefore, the sample plate according to an embodiment of the present invention for achieving the above object of the present invention is a substrate for improving the volatility of the solvent contained in the sample, and formed on the substrate to concentrate the sample provided It includes patterns having a top surface on which the sample is placed. And a hydrophobic coating layer covering an upper surface of the substrate, the hydrophobic coating layer having an opening that selectively exposes the central portions of the patterns such that the solvent contained in the sample evaporates and is concentrated at the central portions of the patterns.

In addition, the method of manufacturing a sample plate according to an embodiment of the present invention for achieving another object of the present invention is etching the upper portion of the substrate exposed to the first photoresist pattern. Subsequently, the first photoresist pattern is removed to prepare a substrate on which protruding patterns are formed, and then second photoresist patterns are formed at the centers of the patterns. Subsequently, a hydrophobic coating layer is continuously formed on the surface of the substrate on which the second photoresist patterns are formed. Subsequently, the sample plate is completed by removing the second photoresist patterns to form a hydrophobic coating layer having an opening that selectively exposes only the central portions of the patterns.

In the sample plate and its manufacturing method according to the present invention, the sample plate is formed by applying a photolithography process using a photoresist rather than a laser etching technique, so that mass production is possible at a low cost and is used for single use in analytical experiments or sample storage. Can be used for In addition, the sample plate not only can dry the solvent contained in the sample faster than the conventional sample plate, but also has all the characteristics necessary for quantitative analysis and qualitative analysis.

Hereinafter, the sample plate and the method of manufacturing the sample plate of the present invention will be described in detail below.

Sample plate

FIG. 2 is a diagram illustrating a sample plate according to an embodiment of the present invention, and FIG. 3 is a cross-sectional view of the sample plate illustrated in FIG. 2 taken along the line II ′.

2 and 3, the sample plate 150 of the present invention covers a substrate 100, patterns 120 having an upper surface on which a sample is provided, and covers an upper surface of the substrate. It has a configuration including a hydrophobic coating film (140a) having an opening for selectively exposing the upper surface center.

Examples of the substrate 100 include an aluminum substrate, a stainless substrate, a tin plate, a copper plate, and a silicon substrate.

In particular, it is preferable to use an aluminum substrate having excellent thermal conductivity. This is because the thermal conductivity of the aluminum substrate is about 175 kcal / mh ^o C, which is about 4 times higher than that of 45 kcal / mh ^o C, which is the thermal conductivity of a material such as stainless steel. In addition, even in the case of the same aluminum substrate, the thin film is more thermally efficient than the thick substrate, and relatively high thermal conductivity than other metals, as well as the low cost of the material is suitable for mass production. It is also environmentally friendly and can be recycled after collection, which is environmentally friendly compared to environmentally hazardous substances such as plastics.

In addition, in the case of aluminum, a stable oxide film is naturally formed on the surface, and discoloration or discoloration does not occur well even when stored for a long time. In addition, since it has excellent weather resistance, it does not change easily even after long-term exposure to high temperature and high humidity in the distribution process. For example, in the case of a copper plate, an uneven oxide film is formed on the surface and corrosion is generated. In the case of a steel plate, when the moisture is high, it becomes a red iron oxide, which causes the quality of the product and shortens the shelf life. it's difficult.

In addition, the formation of a dense and stable oxide film on the aluminum surface increases the reliability of the surface and prevents further contamination by the substrate when the used substrate is reused. In addition, it does not react with the sample for sample analysis, which is advantageous for long-term storage. In addition, the aluminum substrate has a relatively higher reflectance than other materials, which effectively increases the efficiency of the laser irradiated onto the Maldi plate.

When using an aluminum substrate as the substrate 100, the substrate preferably has a thickness of 0.05mm to 2mm. This causes the user to easily bend or tear when handling the substrate when the thickness is less than 0.05 mm. On the other hand, if the thickness of the substrate exceeds 2mm, the drying time of the selectively dropped solution becomes long, and when mounted on the equipment, a special device to withstand the load is required. Thus, the aluminum substrate has a thickness of 0.05 mm to 2 mm, in particular 0.1 mm to 1 mm thick.

The patterns 120 are formed on the substrate 100 so as to be spaced apart at equal intervals from each other, and have a top surface on which a sample provided is placed. The patterns 120 have patterns having a first height H1 protruding from the surface of the substrate 100 and may have a cylindrical, triangular, square, and polygonal shape.

The height of the pattern 120 should exceed 0.01 μm to determine the exact position and shape, and cannot exceed 50 μm to maintain the correct pattern shape from the erosion of the bottom by etching. The pattern thus has a height of 0.01 to 50 μm or less, in particular a height of 0.01 to 20 μm.

In addition, the top surface has a diameter of 1 to 5 mm, particularly 1 to 3 mm, to include the size of the sample spot that is easily visually confirmed for effective dropping of the reagents and concentrated according to the amount of the sample. Has a diameter.

The hydrophobic coating layer 140a is formed on the substrate on which the patterns are formed, so that the surface of the substrate may be surface-modified in a hydrophobic state to reduce the contact area of the sample on the upper surface of the pattern. The hydrophobic coating layer includes an opening (D) to expose the center of the upper surface of the pattern. The opening D exposes the center of the upper surface of the pattern such that a sample provided to the upper surfaces of the patterns on which the hydrophobic coating layer 140a is formed is focused on the center of the pattern 120.

That is, the hydrophobic coating layer 140a having the opening D may selectively expose a center portion of the upper portions of the patterns 120 to focus the sample provided to the center portion of the pattern 120 and at the same time, the solvent contained in the sample. When evaporated, the sample can be concentrated only at the center.

The hydrophobic coating layer 140a is formed using a material such as Teflon, polytetrafluoroethylene (PTFE), Ethylene Fluoro Ethylene (ETFE), PolyChloro Tri-Fluoroethylene (PCTFE), Cytop, Teflon AF, and the like. It is formed to have a thickness of 10 ⁶ nm. When the hydrophobic coating film 140a is less than 1 nm, the degree of hydrophobicity is lowered, and performance may be degraded when used for a long time, and a weak problem occurs on the surface scratch. If it exceeds 1 × 10 ⁶ nm, it is difficult to form holes of the correct size in the next process. Therefore, the hydrophobic coating film has a thickness of 1 to 1 × 10 ⁶ nm, especially 10 to 0.5 × 10 ⁶ nm or less.

An opening of the hydrophobic coating layer is necessary to expose the top of the pattern. The opening of the coating film can be selected according to the amount of the sample, and it is generally larger than the cross-sectional area of the beam of the laser to be used, and generally has a diameter of 100 μm to 1000 μm.

The sample plate having the above-described configuration is not only significantly lower in manufacturing cost than the conventional Maldi sample plate but also short in manufacturing time, so it can be used for single use in analytical experiments or for storing analytical sample. In addition, the sample plate not only allows the solvent contained in the analytical sample to be dried faster than the conventional sample plate, but also provides excellent sensitivity and resolution of the analyte during the analytical process.

Method of making a sample plate.

4 to 8 are cross-sectional views illustrating a method of manufacturing the sample plate shown in FIG. 3.

Referring to FIG. 4, first, substrates 100 for preparing a sample plate are prepared. The substrates 100 of the present embodiment preferably use aluminum substrates on which a surface treatment process has been performed. The surface treatment process is to remove the organic dirt and fine dust on the surface through ultrasonic cleaning and pickling to ensure homogeneity on the surface of the aluminum substrate and to improve the adhesion of the photoresist.

5 and 6, a first photoresist pattern 110a defining a region in which a pattern is formed is formed on the aluminum substrate 100.

To form the first photoresist pattern 110a, first, a photoresist is applied on the aluminum substrate 100 to have a thickness of about 0.5 to 100 μm by spin coating or film deposition. Subsequently, the photoresist coated substrate is soft baked for about 1 minute to evaporate the solvent included in the photoresist to form the first photoresist layer 110. Next, the first photoresist film 110 is selectively exposed by applying an exposure mask M that defines the shape of the pattern of the sample plate.

Subsequently, the post exposure baking, developing, and cleaning processes are sequentially performed to form the first photoresist patterns 110 illustrated in FIG. 5. The post exposure baking is a process of amplifying the acid generated by the exposure to cause a difference in solubility of the photoresist, and is preferably performed at a temperature of about 100 to 130 ° C. for about 1 minute.

Referring to FIG. 7, the upper surface of the exposed substrate is etched by applying the first photoresist pattern as an etching mask to form a pattern 120 having a first height H1.

Referring to FIG. 8, the first photoresist pattern 110a is removed from the substrate 100 by applying a solvent capable of dissolving the first photoresist pattern 110a. The patterns 120 thus formed are formed on the substrate 100 to be spaced at equal intervals into patterns having a first height H1 of 10 to 1 × 10 ⁴ nm protruding from the surface of the substrate 100. It has an upper surface on which the sample to be placed is placed.

9, a second photoresist pattern 130a defining a central portion of the upper surface of the pattern is formed on the patterns 120 formed on the substrate.

The second photoresist pattern 130a is first formed on the aluminum substrate 100 on which the patterns 120 are formed by coating a photoresist having a thickness of about 0.5 to 100 μm by spin coating or film deposition. In particular, it is applied to have a thickness of about 0.5 to 50㎛.

Subsequently, the photoresist coated substrate is soft baked for about 1 minute to evaporate a solvent included in the photoresist to form a second photoresist film (not shown). Subsequently, an exposure mask defining a size of the center portion of the patterns 120 is applied to selectively expose the second photoresist film. Subsequently, post exposure baking, developing and cleaning processes are sequentially performed to form a second photoresist pattern having a diameter of 100 to 1000 μm at the center of the upper surface of the pattern.

Here, the centers of the patterns 120 are areas exposed by the openings (not shown) of the coating film formed on the upper surface of the substrate in a subsequent process, and are areas where the sample provided on the upper surfaces of the patterns is focused (concentrated).

Referring to FIG. 10, a hydrophobic material is coated on the substrate on which the second photoresist pattern 130a is formed without removing the second photoresist pattern 130a to form a hydrophobic coating layer 140. The material for forming the hydrophobic coating layer 140 is formed using a material such as Teflon (Teflon), polytetrafluoroethylene (PTFE), Ethylene Fluoro Ethylene (ETFE), PolyChloroTri-Fluoroethylene (PCTFE), Cytop, Teflon AF, etc. It is formed to have about 10 to 0.5 × 10 ⁶ nm.

In this case, since the second photoresist patterns 130a are formed at the centers of the upper surfaces of the patterns 120, the hydrophobic coating layer 140 may have a uniform thickness on the surface of the patterns except the center of the patterns and the surface of the substrate. Is formed. A portion of the hydrophobic coating layer remains on the second photoresist pattern 130a.

Subsequently, the second photoresist pattern 130a is removed and a portion of the coating film remaining thereon is removed to form a hydrophobic coating film having an opening that exposes the center of the upper surface of the pattern. Since the hydrophobic coating layer 140a having the opening D is formed, the sample plate shown in FIG. 3 is completed.

Here, the opening D has a diameter of 100 to 1000 μm, and the hydrophobic coating layer 140a having the opening D exposes the central portion of the upper surface of the patterns to focus the sample provided on the upper surface of the pattern. When the solvent contained in the sample is evaporated, the sample may be concentrated only at the center portion.

Since the method of forming the sample plate is a photolithography process using a photoresist pattern, a mass production system is possible, and the sample plate can be manufactured in a short time at a low cost.

Hereinafter, the present invention will be described in detail by evaluating the characteristics of the sample plate.

Solvent Dryness Evaluation of Sample Plate

Three sample plates to which aluminum substrates having a thickness of 0.05 mm, 0.2 mm and 2 mm and stainless steel substrates having a thickness of 2 mm were applied, respectively. The sample plate was formed with patterns having a top surface on which the sample was placed, and then, after spotting 1 ul of acetone on the top surfaces of the plate patterns, the drying time of the acetone was measured.

Kinds Thickness of substrate (mm) Acetone Volume (μl) Acetone drying time (sec) Aluminum substrate 0.05 One 8 ± 1 Aluminum substrate 0.2 One 15 ± 2 Aluminum substrate 2 One 37 ± 3 Stainless steel substrate 2 One 45 ± 3

Table 1 shows a result of measuring the acetone drying time for the type and thickness of metal substrates not considering the formation of a hydrophobic coating film. As a result of drying time measurement, it was found that the aluminum substrate was slightly faster than the stainless steel substrate when the same thickness was obtained. However, it can be seen that the drying time of acetone is remarkably faster as the thickness of the aluminum substrate becomes thinner. Here, the drying time measurement was measured at 25 ^° C temperature, 50% humidity conditions.

Evaluation of Analytical Capability of Sample Plates

An aluminum substrate having a thickness of about 0.2 mm, openings for selectively exposing the center portions of the patterns such that the patterns provided to the aluminum substrate are concentrated and the samples provided as the patterns are concentrated at the center of the pattern. Disposable sample plate and maldi anchor plate (manufacturer: Bruker) of the present invention containing a hydrophobic coating film having a prepared respectively. Subsequently, six standard peptide samples were spotted on each of the disposable sample plate and the Maldi anchor plate, and the standard peptide samples were analyzed by a Maldi analyzer. The results are shown in the graphs of FIGS. 11 and 12.

FIG. 11 is a graph showing MALDI spectral data of a standard peptide sample using a sample plate of the present invention, and FIG. 12 is a graph showing MALDI spectral data of a standard peptide sample using a conventional Maldi anchor plate.

Referring to the graphs shown in FIGS. 11 and 12, all six standard samples were measured with high sensitivity for 20 femto mole standard peptide samples. The performance of the disposable sample plate of the present invention showed a somewhat superior sensitivity in the peptide having a large molecular weight compared to the conventional Maldi anchor plate, it was found that the focusing of the sample is more efficient. In addition, there was no interference action of the aluminum substrate used as the substrate of the sample plate of the present invention. In addition, although not shown a separate graph, the disposable sample plate of the present invention showed similar results as above for the standard protein.

Here, as the standard peptide sample, six standard samples such as Angiotensin I, II, substance P, Bombasin, ACTH (1-17), ACTH (18-39) were used, and the device for analyzing the standard sample Bruker's Ultraflex system was used as a Maldi spectrometer, and carbohydrocinnanic acid (CHCA) was used as a matrix applied to analyze the standard sample, and a nitrogen laser of 337 nm was used. .

The sample plate according to the present invention is not only significantly lower manufacturing cost than the conventional sample plate but also short production time can be used for single use in the assay or storage of the analytical sample.

In addition, the sample plate not only can dry the solvent contained in the analytical sample faster than the conventional sample plate, but also has all the characteristics necessary for quantitative analysis and qualitative analysis.

In addition, the method of manufacturing the sample plate is formed by applying a photolithography process using a photoresist rather than a laser etching technique, it is possible to significantly reduce the manufacturing cost and manufacturing time as well as to mass-produce the sample plate.

In addition, the sample plate is easy to handle and can increase the accuracy of the measurement by using a sample plate for each sample, as well as having a thin film form can be used directly without any special apparatus for existing analysis equipment.                     

Although described above with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and changed within the scope of the invention without departing from the spirit and scope of the invention described in the claims below I can understand that you can.

Claims (13)

Board: Patterns formed on the substrate to have a columnar shape and having a top surface on which the sample is placed to concentrate the sample provided; And And a hydrophobic coating layer covering an upper surface of the substrate, the hydrophobic coating layer having an opening for selectively exposing the center portions of the patterns such that the solvent contained in the sample evaporates to concentrate the sample at the center portions of the patterns. The sample plate of claim 1, wherein the substrate is any one selected from the group consisting of an aluminum substrate, a stainless substrate, a tin plate, a copper plate, and a silicon substrate. The sample plate of claim 1, wherein the substrate is an aluminum substrate having a thickness of 0.05 to 2 mm. The sample plate of claim 1, wherein the patterns have a height of 10 to 1 × 10 ⁴ nm. The sample plate of claim 1, wherein the patterns have a cylindrical, square, or polygonal shape, the upper surface of which has a diameter of 1 to 3 mm. The method of claim 1, wherein the coating film is any one selected from the group consisting of Teflon (Teflon) coating film, PTFE (polytetrafluoroethylene) coating film, ETFE (Ethylene Fluoro Ethylene) coating film, PCTFE (PolyChloro Tri-Fluoroethylene) coating film and Cytop coating film A sample plate. The sample plate of claim 1, wherein the opening has a diameter of 100 to 1000 μm. Etching the upper portion of the substrate exposed to the first photoresist pattern; Removing the first photoresist pattern to provide a substrate on which protruding patterns are formed; Forming second photoresist patterns in the center of the patterns; Continuously forming a hydrophobic coating layer on a surface of the substrate on which the second photoresist patterns are formed; And Removing the second photoresist patterns to form a hydrophobic coating layer having an opening that selectively exposes only the central portions of the patterns. The method of claim 7, wherein the patterns, Forming a first photoresist film on the substrate; Forming the first photoresist film into a first photoresist pattern defining a region in which the patterns are formed; And And applying the first photoresist pattern as an etching mask to etch the top surface of the exposed substrate to form patterns having a height of 10 to 1 × 10 ⁴ nm. The method of claim 8, wherein the second photoresist patterns, Forming a second photoresist film having a uniform thickness on the patterned substrate; And And forming the second photoresist film as a second photoresist pattern covering only the centers of the patterns. The method of claim 8, wherein the hydrophobic coating film, Applying a hydrophobic material on the substrate on which the second photoresist pattern is formed to have a uniform thickness; And And removing the hydrophobic material remaining on top of the photoresist pattern simultaneously with removing the second photoresist pattern. The method of claim 8, wherein the hydrophobic coating layer is formed to have a thickness of 1 to 1 × 10 ⁶ nm. The method of claim 8, wherein the opening is formed to have a diameter of 0.1 to 1.5 × 10 ⁵ μm.

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