JP4557699B2 - Patterned microchip and manufacturing method thereof - Google Patents

Description

  The present invention relates to a patterned microchip and a method for manufacturing the same.

  Analysis using various nucleic acid probe molecules such as DNA chip, RNA chip, etc. provides important indicators for diagnosis, prognosis prediction, treatment policy determination, etc. of cancer, genetic diseases, lifestyle-related diseases, infectious diseases, etc. Expected. Conventionally, in an analysis system using a DNA chip, a DNA chip on which DNA of a known structure is fixed is used, and the unknown DNA is dropped onto the DNA chip to react with the known DNA (hybridization). The DNA was washed, and the DNAs that were bonded and those that were not bonded were selected, and the reaction was analyzed by color development or the like.

  These DNA chips have a high density by applying several thousand to tens of thousands of points of DNA whose structure has already been analyzed on a flat glass or silicon substrate using a coating device called a spotter. A DNA molecule is fixed. Further, by analyzing whether or not the DNA molecule used for the DNA chip and the test DNA are bound in this reaction (hybridization), it is possible to check whether the target DNA exists in the sample. For example, when a part of DNA is damaged due to a disease or the like, it is possible to detect that the complementarity between DNAs is lost.

  A DNA chip on which a known DNA is fixed is currently very expensive. As a method for manufacturing a DNA chip, several methods are known. For example, a method of sequentially synthesizing DNA probes directly on a substrate using photolithography (Patent Documents 1 and 2), and supplying pre-synthesized DNA or cDNA (complementary DNA) on the substrate. The binding method (Patent Documents 3 to 5 and Non-Patent Document 1) is a typical method for producing a DNA chip. In general, a nucleic acid chip is produced by any of the above methods, but when the produced nucleic acid chip is to be used for the above-mentioned purpose, the reliability of analysis, that is, the quantitativeness and reproducibility are guaranteed. For this purpose, it is important to know the amount of nucleic acid used for the probe, that is, the nucleic acid used for the probe, that is, the density. It is also important from the viewpoint of securing quantitativeness and reproducibility to know what matrix shape (shape, size, state) actually exists (imaging).

Moreover, the following methods (nonpatent literature 2) which pattern avidin are proposed. First, a mercapto group is formed on a substrate surface using 3-mercaptopropyl-trimethoxysilane on a silicon substrate having an oxide film. Thereafter, 1- [4-azido-alisilamide] -6- [biotintinamide] -hexane, which is a photoactive biotin, is adjusted with anhydrous methanol so as to be 2 μg / ml, and is placed on the substrate having the mercapto group. Apply. Next, by exposing with UV light (365 nm, 5 min) through a photomask having a desired pattern, the photoactive biotin and the mercapto group on the substrate surface are covalently bonded. The biotin pattern is then completed by rinsing with dimethyl sulfoxide for 15 minutes, followed by a brief rinse with H ₂ O. By binding avidin to patterned biotin, an avidin pattern can also be obtained. However, in this method, since there is an exposure step using UV light, it is necessary to store the substrate in an environment where there is no exposure by light from the surroundings until exposure and washing with anhydrous methanol. That is, compared with the present invention, the method of the prior art has a problem that the number of steps is increased because development is required, and an environmental facility that blocks the influence of light from the surroundings is required.

On the other hand, cell adhesion control is characterized in that cell adhesion is controlled by irradiating an ion beam to a biopolymer material coated on a substrate, and the irradiation site is a cell non-adhesive surface. Materials have been reported (Patent Document 6). Here, it is described that gelatin, collagen, or the like is used as the biopolymer material, and the ion beam is irradiated at an acceleration energy of 50 to 150 keV and an irradiation amount of 1 × 10 ¹⁵ pieces / cm ² or more. Further, it is described that by selecting conditions such as ionic species, a selective adhesive surface is formed in which the irradiation site is a cancer cell non-adhesive surface or a normal cell adhesive surface. In addition, a technique for controlling cell adhesion by irradiating an ion beam to a culture substrate coated with an extracellular matrix has been reported (Non-Patent Documents 3 and 4).

Japanese National Patent Publication No. 4-505863 US Pat. No. 5,405,783, etc. Japanese National Patent Publication No. 10-503841 US Pat. No. 5,601,980 JP 11-187900 A JP-A-7-108060 Science Vol.270,467,1995 Chandran R. Sabanayagam, Cassandra L. Smith, Charles R. Cantor, “Oligonucleotide immobilization on micropatterned Streptabidin surfaces”, Nucleic Acids Research, 2000, Vol.28, No.8 Cell adhesion control by irradiation of ion beam to culture matrix coated with extracellular matrix, Yoshiaki Suzuki et al., Artificial organ, 23 (3), (1994) pp 700-707 Cell Adhesion Control by Ion Implantation into Extracellular Matrix, Y. Suzuki, et al., Nucl. Instr. And Meth., B91, (1994) pp 588-592

  An object of the present invention is to provide a substrate on which a target substance to be patterned such as protein is micropatterned. In particular, the present invention has an object to be solved by providing a substrate which is excellent in quantification and reproducibility, and which is formed by arbitrarily patterning an arbitrary target substance to form a microstructured surface.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have radiated high-energy charged particles (ion beams) onto target substances such as proteins physically or chemically adsorbed on the substrate surface. The present inventors have found that a surface in which a substance is patterned can be created by using the function of the remaining surface (surface not irradiated with the ion beam) by disappearing the material, and the present invention has been completed.

  That is, according to the present invention, the target substance is patterned on the surface, which is produced by performing ion implantation on the patterned region of the substrate on which the target substance to be patterned is physically or chemically adsorbed. A substrate is provided.

  According to another aspect of the present invention, the target substance is a surface, wherein ion implantation is performed on a patterned region of the substrate on which the target substance to be patterned is physically or chemically adsorbed. A method of manufacturing a substrate that has been patterned is provided.

Preferably, ion implantation is performed on the patterned region of the substrate by performing ion implantation after mounting the patterned mask on the substrate surface.
Preferably, the target substance is an affinity substance, protein, peptide or nucleic acid.
Preferably, the affinity substance is an avidin.
Preferably, a dose of phi is the ion implantation within an amount of ^{1 × 10 12 ≦ φ <1} × 10 16 pieces / cm ^2.

  According to the present invention, an arbitrary protein function or functional group population can be arbitrarily patterned to form a microstructured surface.

Hereinafter, embodiments of the present invention will be described in detail.
The present invention relates to a substrate on which a target substance is patterned on a surface, which is produced by performing ion implantation on a patterned region of a substrate on which a target substance to be patterned is physically or chemically adsorbed. Manufacturing a substrate on which a target substance is patterned on the surface, wherein ion implantation is performed on a patterned region of the substrate on which the target substance to be patterned is physically or chemically adsorbed It is about the method.

  Surface / surface layer processing technology has been developed as a method for forming surface layers with new superior functions and composite functions. This technique includes a method of modifying only the surface layer of the base material without changing the properties of the base material and a method of forming a new layer on the surface. The ion implantation method (ion beam irradiation technique) used in the present invention is the former, and is an established technique as an impurity addition method to silicon in the example for the purpose of the addition.

The ion implantation method is a method in which particles to be added are ionized in a high vacuum (for example, 10 ⁻⁴ Pa), accelerated from several tens of kV to several MV, and added to a solid substrate. Since the ion beam produced by this is mass-separated, the ion purity is very good, the controllability such as acceleration energy and irradiation amount is high, and processing with good reproducibility and uniformity is possible. In addition, there is an advantage that the ion beam irradiation effect on the structural change of the irradiation material can be easily analyzed.

  The surface layer modification method using an ion beam is roughly divided into two. One is a doping effect due to the addition of ions, and the other is an irradiation damage effect such as bond breakage caused by the passage of ions. When a target substance (for example, an affinity substance, protein, peptide, or nucleic acid) is irradiated with an ion beam, the primary structure or higher-order structure of the target substance is destroyed, and the target substance loses its function. Further, when the functional group in the target substance is irradiated with an ion beam, the functional group may be destroyed and the reactivity may be lost.

  The ion beam in the ion implantation method has straightness. When an ion beam is irradiated after a patterned mask made of metal or the like is mounted on the sample surface using this property, an irradiation surface according to the created pattern can be formed. The shape of the pattern is arbitrary such as a linear shape or a circular shape, and the size thereof can be arbitrarily selected whether it is submicron or several mm. In the present invention, a patterned functionalized surface can be formed by using this feature to eliminate the function of the target substance in the irradiated portion (FIG. 1).

  On the other hand, in order to produce DNA microchips and microarrays, it is necessary to fix probe DNA on a substrate. In order to immobilize probe DNA on a substrate, for example, a method of immobilizing probe DNA modified with a thiol (SH) group at its end to the Au surface or the substrate surface on which the SH group is formed by silane coupling reaction by thiol bond Alternatively, probe DNA modified with amino groups can be fixed to the surface of the substrate on which carboxyl groups are formed by silane coupling reaction, etc. by peptide bonds, or probe DNA can be incorporated into a gel or polymer layer formed on the substrate. There are various methods such as fixing. Above all, the method of immobilizing avidin on the substrate surface and immobilizing it with a biotinylated probe DNA has a fixed position of the biotinylated probe DNA due to the tetramer structure of avidin. Since it is determined, it has an advantage that the probe fixing density can be easily controlled.

  Examples of the target substance to be patterned used in the present invention include affinity substances, proteins, peptides, and nucleic acids.

  Examples of the affinity substance include avidins. Examples of the avidins mentioned here include avidin, streptavidin, or modified compounds capable of forming a stable complex with biotin.

  The type of the protein or peptide is not particularly limited, and may be an arbitrary protein having physiological activity or a fragment peptide thereof, an antigen, a receptor, an antibody or antibody fragment, a nucleic acid recognition protein, or the like. Examples of nucleic acid recognition proteins include double-stranded DNA recognition substances such as transcription factors.

  The type of nucleic acid is not particularly limited, and examples thereof include oligonucleotides, polynucleotides, and peptide nucleic acids. The nucleic acid may be a naturally occurring nucleic acid (DNA, RNA or a fragment thereof), a chemically synthesized nucleic acid, or a recombinant nucleic acid produced by a gene recombination technique. For example, when examining the expression of a gene, cDNA, a part of cDNA, or EST can be used as DNA. Further, when analyzing gene mutations and polymorphisms, various oligonucleotides corresponding to the mutations and polymorphisms can be chemically synthesized and used based on known base sequences.

  In the present invention, a substrate on which the target substance is physically or chemically adsorbed is used. The substrate is not particularly limited as long as the target substance can be fixed, and may be a substrate having a smooth surface or a substrate having irregularities on the surface. The material of the substrate is not particularly limited, and examples thereof include silicon, porous silicon, glass, porous glass, ceramics, porous ceramics, polyethylene terephthalate, cellulose acetate, polystyrene, and polymethyl methacrylate. When the substrate is porous, the pore size of the porous material is preferably about 1 to 1000 nm. The substrate may have a coating layer on the surface. For example, a silicon substrate having a thermal oxide film or a glass substrate coated with an organic coating layer may be used.

  In the present invention, ion implantation is performed on the patterned region of the substrate on which the target substance is adsorbed. Specifically, for example, ion implantation can be performed on a patterned region of the substrate by performing ion implantation after mounting a patterned mask on the substrate surface.

Examples of ion species to be implanted include H ⁺ , He ⁺ , C ⁺ , N ⁺ , Ne ⁺ , Na ⁺ , N ⁺ , O ⁺ , N ₂ ⁺ , O ₂ ⁺ , Ar ⁺ and Kr ⁺ . It is not limited to these as long as the structure and / or function of the target substance can be lost. The ionic species is particularly preferably He ⁺ , Ne ⁺ , Ar ⁺ or Kr ⁺ .

The dose amount φ at the time of ion implantation is preferably in the range of 1 × 10 ¹² ≦ φ <1 × 10 ¹⁶ pieces / cm ² . If the dose amount φ is lower than 1 × 10 ¹² pieces / cm ² , the structure of the target substance is not destroyed, and the improvement effect becomes small, which is not preferable.

It is desirable to set the beam current density within a range not exceeding about 3 μA / cm ² . This is because when the beam current density is excessive, the temperature of the target material is excessively increased, and the material itself other than the patterned portion is deteriorated and the gas permeability may be decreased.

  Regarding the ion acceleration energy, it is considered that the energy transfer mechanism varies depending on the height, but in practice, it can be set within a range of several MeV or less, preferably 2 MeV or less.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

Example 1: Preparation of a sample (Avidin fixing step)
A sample was prepared by forming an amino group on a silicon substrate having a thermal oxide film of about 50 nm by a silane coupling reaction, followed by carboxylation and active esterification steps, and fixing avidin on the substrate by a covalent bond. Specifically, a sample was prepared as follows.

The surface of a silicon substrate having an oxide film of 5 mm × 10 mm is cleaned by plasma ashing (200 W, 5 min), and then immersed in hydrogen peroxide for 10 minutes to form —OH groups on the substrate surface. After rinsing with pure water, it is dried in an oven (120 ° C, 5 minutes). Next, the substrate is immersed in an anhydrous toluene solution of 2% 3-aminopropyltriethoxysilane, and —NH ₂ groups are formed on the surface of the substrate while stirring at 50 ° C. for 30 minutes. After rinsing with toluene, drying is performed at 120 ° C. for 1 hour to fix the —NH ₂ group. Next, the substrate is immersed in anhydrous dimethylformamide in which 10 mg / ml succinic anhydride and 0.05 M dimethylaminopyridine are dissolved, and the reaction is carried out at 50 ° C. for 2 hours to form a carboxyl group. After rinsing with dimethylformamide, 100 mg each of 1-ethyl-3- (3-dimethylamino) propylcarbodiimid Hydrochloride (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysiccinimide (N-hydroxysuccinimide) Carboxyl group is activated ester by immersing in water solution at room temperature for 30 min.After rinsing with 100 mM HEPES Buffer, the surface is immersed in 0.1 mg / ml avidin or streptavidin aqueous solution at room temperature for 1 hour. Is fixed and rinsed with 100 mM HEPES Buffer to remove excess avidin, thereby completing the avidin-fixed substrate.

Example 2: Ion beam irradiation In an ion implanter RIKEN 200 kV Low Current Implanter, He ⁺ , Ne ⁺ , Ar ⁺ , or Kr ⁺ ions are accelerated at 150 keV, and irradiation dose is 1 × 10 ¹³ , 1 × 10 ¹⁴ , 1 × 10 ¹⁵ ions / cm ² And ion beam irradiation was performed. The ion beam current was 0.1 μA / cm ² or less. Patterning was performed by attaching a patterned stainless steel mask to the sample surface and irradiating with an ion beam. Since the ion beam irradiated to the metal portion cannot reach the sample surface, it is patterned.

Example 3: Evaluation 1 of ion beam irradiation array
The concentration of a solution containing poly-T 30mer modified with biotin at one end and Cy3 modified at the other end was adjusted to 1 μM, dropped onto a sample (substrate), and left at room temperature for 30 min. Thereafter, rocking washing was performed at room temperature for 5 min using Phosphate Buffer Solution (PBS) containing 0.5% Tween (surfactant) to remove excess Cy3-modified poly-T. Then, Cy3 fluorescence observation was performed with a fluorescence microscope under the following imaging conditions.

(Shooting conditions)
・ Exposure time: 1500 ms
・ Threshold: Low 800, High 3000

  2 and 3 show the results of Biotin-Cy3 labeling of an avidin-immobilized sample irradiated with an ion beam. It was observed that avidin was destroyed and inactivated by ion beam irradiation, and the fluorescence intensity decreased. From these results, it was demonstrated that a new biochip and microarray can be formed by an ion beam.

Example 4: Evaluation of ion beam irradiation array 2
In addition, poly-T 30mer with one end biotinylated is immobilized on an avidin-immobilized substrate irradiated with an ion beam by the same operation as in Example 3, and then poly-A 30mer labeled with Cy3 at one end is included. Hybridization was performed using 1 μM solution while shaking at 55 ° C. for 3 hours. Using a Phosphate Buffer Solution (PBS) containing 0.5% Tween (surfactant), washing was performed at room temperature for 5 minutes to remove excess poly-A. As a result, it was confirmed that the same pattern as that obtained when the probe was fixed was obtained.

FIG. 1 shows an overview of the formation of a patterned functionalized surface according to the present invention. FIG. 2 shows the result (He ⁺ , or Ne ⁺ ) obtained by labeling an avidin-immobilized sample irradiated with an ion beam with Biotin-Cy3. FIG. 3 shows the result (Ar ⁺ , or Kr ⁺ ) obtained by labeling an avidin-immobilized sample irradiated with an ion beam with Biotin-Cy3.

Claims (8)

A substrate is patterned by mounting a patterned mask on the surface of a substrate on which a target substance selected from affinity substances, proteins, peptides or nucleic acids is physically or chemically adsorbed, and then performing ion implantation . A microarray in which a target substance is patterned on the surface, which is produced by performing ion implantation on a formed region. The microarray according to claim 1 , wherein the affinity substance is avidin. The microarray according to claim 1 or 2 , wherein ion implantation is performed in a range where the dose amount φ is 1 × 10 ¹² ≦ φ <1 × 10 ¹⁶ pieces / cm ² . The microarray according to any one of claims 1 to 3 , wherein the ion species is He ⁺ , Ne ⁺ , Ar ⁺ or Kr ⁺ . By performing ion implantation after mounting a patterned mask on the surface of a substrate on which a target substance to be patterned selected from affinity substances, proteins, peptides or nucleic acids is physically or chemically adsorbed, A method for producing a microarray in which a target substance is patterned on the surface, wherein ion implantation is performed on the patterned region of The method according to claim 5 , wherein the affinity substance is an avidin. 7. The method according to claim 5 , wherein the ion implantation is performed in a range where the dose amount φ is 1 × 10 ¹² ≦ φ <1 × 10 ¹⁶ pieces / cm ² . The method according to claim 5, wherein the ionic species is He ⁺ , Ne ⁺ , Ar ⁺ or Kr ⁺ .