JP4225888B2 - Analysis chip and preparation method thereof. - Google Patents

Description

  The present invention relates to an analysis chip for causing a predetermined reaction to be performed using a small amount of sample and detecting the reaction qualitatively or quantitatively, and a method for preparing the same.

Analyzing a very small amount of biological sample on an analysis chip has been widely performed. Examples of such an analysis chip include a DNA chip used for analyzing the base sequence of DNA (deoxyribonucleotide) using a hybridization method, a diagnostic chip using an antigen-antibody reaction or an enzyme reaction, and the like. .
Conventionally, such an analysis chip is produced by fixing a large number of samples, that is, functional molecules such as specimens and detection reagents in a spot shape on a chip substrate. Therefore, it is required to increase the number of spots per unit area.

For this reason, Patent Document 1 proposes a method in which a frame body is formed of a photosensitive resin on the surface of a chip substrate, and functional molecules are supplied into the frame for immobilization. However, in the photosensitive resin, since the height of the frame is as low as 1 to 20 μm, the amount of functional molecules per spot is small, so the analysis sensitivity is poor. In addition, patterning, etching, There is a problem that a plurality of steps such as cleaning are necessary and time-consuming.
Therefore, Patent Document 2 proposes a method of forming a frame having a height of 3 to 500 μm on the surface of the chip substrate by laminating a thin film member having a large number of fine through holes on the chip substrate.
JP 11-187900 A JP 2002-27984 A

This method is an excellent method because the formation of the frame is simple and the height of the frame can be arbitrarily set.
However, if it is surrounded by a tall frame, it is difficult to reach the bottom of the frame with the inert treatment solution or cleaning solution, so the deactivation treatment and cleaning after immobilizing functional molecules can be performed sufficiently. However, there is a problem that non-specific reaction occurs and analysis accuracy is lowered. In addition, when analyzing using this analysis chip, it is difficult to reach the specimen or sample to the bottom of the immobilized functional molecule, so that the reaction does not occur sufficiently at the bottom, and the functional molecules per spot There is also a problem that the analytical sensitivity does not increase so much despite the increase in volume.
The present invention seeks to provide a method of enjoying the advantages of a frame when immobilizing functional molecules, and avoiding the adverse effects of the frame when analyzing.

The present inventors have found that the above problem can be solved by using a frame when fixing functional molecules on a substrate and removing the frame when performing analysis, and have reached the present invention.
That is, the gist of the present invention is a thin film member having a through hole and a chip substrate that is detachably stacked on the chip substrate , and can be easily stacked and adhered to the substrate by pressure bonding and easily removed from the substrate. A member made of a resin , and the through-hole and the chip substrate surface cooperate to accommodate a sample that is a protein, peptide, low molecular weight compound, nucleic acid, sugar, sugar chain, or lipid . A method for preparing an analytical chip, comprising: supplying a solution containing a sample to the concave portion of the laminate forming the concave portion to fix the sample on the surface of the chip substrate; and removing the thin film member Exist.

  According to the present invention, similar to the method described in Japanese Patent Application Laid-Open No. 2002-27984, the sample is prepared by cooperating the through hole and the chip substrate by a simple means of simply laminating the thin film member having the through hole on the substrate. Since the concave portions to be accommodated can be formed at a high density, there is an advantage that analysis efficiency can be improved. In addition, since the thin film member can be peeled off, after the functional molecules are immobilized, the thin film member can be removed by peeling or the like, whereby cleaning can be performed more accurately. Further, in the analysis, since the reagent reaches the bottom of the immobilized functional molecule, there is an advantage that the analysis sensitivity is improved.

As the chip substrate, the same material and shape as the conventional one can be used. For example, examples of the material for the substrate include resin materials such as polyolefin, polystyrene, polycarbonate, polyamide, and acrylic resin, and inorganic materials such as glass, alumina, carbon, and metal. A porous chip substrate having an average pore diameter of 100 nm to 100 μm and a porosity of about 30 to 80% is preferable because functional molecules can be fixed in a limited space at a high density and three-dimensionally.
The shape of the chip substrate is not particularly limited, but a flat plate shape is preferable, and the thickness is usually about 0.5 to 5 mm.

When the analysis chip according to the present invention is used for the surface plasmon resonance method, the chip substrate is preferably provided with a diffraction grating for generating an evanescent wave on the surface portion of the substrate. In order to induce surface plasmon waves, the surface of the diffraction grating is preferably coated with metal.
Examples of the diffraction grating include a plurality of grooves each having a depth of about 10 to 100 nm that are engraved in parallel with a distance between the grooves of about 200 to 2000 nm.
The metal is not particularly limited as long as it can induce surface plasmon waves, and examples thereof include gold, silver, copper, aluminum, and alloys containing these. Of these, silver is preferable in terms of sensitivity and inexpensiveness, and gold is preferable in terms of stability. A metal layer is formed by vapor deposition, sputtering, plating, other coatings, etc., and the thickness is 20-300 nm normally, Preferably it is about 30-160 nm.

  The surface of the chip substrate is preferably coated with an organic polymer substance having a functional group capable of binding to a functional molecule immobilized on the substrate. Examples of such a functional group include a carboxyl group, amino group, hydroxyl group, aldehyde group, carbonyl group, epoxy group, vinyl group, thiol group, maleimide group, succinyl group, phosphate group, and imidazole group. Among these organic polymer materials having these functional groups, water-soluble organic polymer materials such as polyacrylic acid, polyethylene glycol, polyvinyl alcohol, polyacrylamide, dextran, carboxymethyl dextran, agarose, carrageenan, alginic acid, starch, and cellulose. Is preferably used because it has high solubility in water and can improve the fixing density of functional molecules. Of these, polyacrylic acid, carboxymethyldextran, and the like that provide a coating layer having a three-dimensional structure are preferable.

  The organic polymer substance coating layer having such a functional group can be formed by directly coating the organic polymer substance. Or, for example, (meth) acrylic acid having a carboxyl group and a salt thereof, 2-hydroxyethyl (meth) acrylate having a hydroxyl group, acrolein having an aldehyde group, glycidyl (meth) acrylate having an epoxy group, N having a succinyl group -After applying a solution containing a monomer such as hydroxy succinimide (meth) acrylate, phosmer having a phosphate group, vinyl imidazole having an imidazole group on the chip substrate, it is irradiated with active energy rays or heated. Can be formed by polymerization.

  When the functional group possessed by the organic polymer substance is a carboxyl group, a functional group covalently bonded to the carboxyl group is introduced in advance on the substrate, and the monomer having the carboxyl group is covalently bonded under acidic conditions in the presence of a condensing agent. It is preferable to form a coating layer of an organic polymer substance by the method.

  Examples of the functional group to be introduced in advance onto the substrate include an amino group, a hydroxyl group, and a thiol group, and an amino group is preferable because the introduction of the functional group is easy and the control of the immobilization reaction of the organic compound is easy. . As a method for introducing a functional group, when an amino group is introduced on a metal surface, a thiol compound having an amino group such as cysteamine hydrochloride is allowed to act on the metal surface to introduce an amino group on the glass surface. A silane coupling agent having an amino group such as aminopropyltriethoxysilane can be easily introduced by acting on the glass surface.

  Examples of the condensing agent include carbodiimide. The carboxyl group is deprotonated in an alkaline solution and protonated in an acidic solution. For this reason, it is considered that the organic polymer substance having a carboxyl group has a chain extending in the alkaline solution and tends to have a relatively compact structure in the acidic solution. By immobilizing the polymer chain having carboxyl groups on the substrate surface with a compact structure, a surface having high density of carboxyl groups can be obtained. For this reason, the reaction is preferably carried out under acidic conditions, pH 5 or less, particularly pH 4 or less, particularly preferably pH 3 or less.

  When the organic polymer substance forming the coating layer is bonded to the functional molecule, the functional group of the organic polymer substance is preferably activated by a known method. In the case of an organic polymer substance having a carboxyl group, it can be activated by bringing the surface of the coating film of the organic polymer substance into contact with an aqueous solution containing an activating agent such as carbodiimide and a salt thereof.

  The thin film member is preferably a thin film member that can be easily laminated, particularly in close contact with the substrate, and can be easily removed from the substrate. For example, if a thin film layer having an adhesive layer having a weak adhesive force is used on the surface, it can be adhered to the substrate by pressure bonding and removed from the substrate by peeling. Preferably, a resin having a self-adsorption force, specifically, (1) when using a reaction solution, a sample solution, etc., it can be in close contact with an object to be adsorbed (substrate) so that gas, liquid, etc. do not leak, (2) When peeling, a thin film member made of a resin that can be easily peeled without damaging the thin film member or the adsorption object (substrate), for example, silicon rubber is used. The thin film member can be laminated on the substrate simply by pressure bonding without using an adhesive, and can be easily peeled off from the substrate. The thin film member can be easily removed from the substrate by peeling. However, if the functional molecule fixed on the substrate is not impaired, it can be removed with an organic solvent.

The thin film member is provided with a through-hole for forming a recess that accommodates a functional molecule in cooperation with the chip substrate in a stacked state on the chip substrate. An example is shown in FIG. The thickness of the thin film member is 1 μm or more, preferably 100 μm or more, more preferably 300 μm or more, and usually 2000 μm or less, preferably 1000 μm or less. If the thin film member is too thin, the amount of functional molecules that can be accommodated in the recesses is reduced, and if it is too thick, it becomes difficult to process the thin film member. The cross-sectional shape of the through hole of the thin film member is arbitrary, but is usually circular or a shape similar thereto. The bottom area of the recess and the opening area of the through hole are preferably 0.5 × 10 ^{−10 to} 7 mm ² .

  The thin film member can be produced by forming a through hole in a resin sheet by laser processing or punching. Moreover, after inject | pouring molten resin into the metal mold | die provided with many convex parts, you may demold and shape | mold. The latter is suitable for mass production and can reduce the manufacturing cost.

  Next, functional molecules are supplied to the concave portion of the laminate formed by laminating the thin film member on the chip substrate and having a large number of concave portions in cooperation with the through-holes of the thin film member and the substrate. The work of immobilizing the sex molecule may be performed by a known method such as the method described in the above-mentioned publication. For example, after a solution containing functional molecules in the recesses is supplied by a method such as an ink jet method, the functional molecules are fixed on the substrate by being left for a certain period of time.

Functional molecules include proteins such as enzymes, antibodies, antigens, lectins, low molecular compounds such as peptides, hormones, drugs, sugar chains such as nucleic acids, sugars, oligosaccharides, and polysaccharides, molecules that constitute lipids, viruses, or cells. And those capable of specific reaction such as allergens. Of these, proteins and DNA are preferably used.
The functional molecule may be supported on particles having a particle diameter of about 1 nm to 100 μm made of a metal such as gold or a polymer such as polystyrene.

After the functional molecule is immobilized on the substrate, the thin film member is peeled off from the chip substrate to obtain an analysis chip in which the functional molecule is immobilized on the chip substrate.
After the thin film member is peeled off, it can be washed by a conventional method to remove contaminants, or to prevent non-specific reactions from occurring in the organic polymer material layer. Inactivate the group. When the functional group is a carboxyl group, the inactivation can be performed by immersing in an aqueous solution containing a blocking agent such as ethanolamine at a ratio of about 1 mol / liter for about 15 minutes. In the present invention, when the immobilized functional molecule is washed or the functional group of the organic polymer substance is deactivated, there is no partition wall between the functional molecules, which may prevent washing and deactivation. Absent.

  The sample is analyzed by bringing a solution containing the sample into contact with the surface of the analysis chip according to the present invention by a conventional method and measuring the interaction between the immobilized functional molecule and the sample.

  The interaction between the functional molecule and the analyte is usually due to the force acting between the molecules resulting from at least one of the covalent bond, the hydrophobic bond, the van der Waals bond, the electrostatic force bond, etc. between the target molecule and the analyte. The action is used. Specific examples of interactions include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and partner molecule, binding and dissociation between enzyme and substrate, Binding and dissociation between enzyme and coenzyme, binding and dissociation between nucleic acid and protein binding to it, binding and dissociation between proteins in information transmission system, binding and dissociation between glycoprotein and protein, sugar Examples include binding and dissociation between chains and proteins.

  Examples of the measurement method of the interaction include a fluorescence method, a chemiluminescence method, an RI method, a surface plasmon resonance method, a mass spectrometry method, a crystal oscillator method, and a detection method using an electrochemical method. Among them, in the fluorescence method, chemiluminescence method, RI method, etc., the interaction is usually measured by measuring the signal of the labeling substance bound to the specimen. You may measure a signal using what couple | bonded the labeling substance with the molecule | numerator. Detection by the surface plasmon resonance method is preferably used because the sample can be analyzed without labeling.

  When the analysis chip according to the present invention is used for the surface plasmon resonance method, the signal is strong and uniform. The reason for this is not clear, but in the analysis chip where functional molecules are spotted without using a conventional frame, the signal is weak because the amount of functional molecules immobilized is small, and the functional molecules are contained in the frame. In the analysis chip formed in this way, when the substrate is treated with a solution such as activation or cleaning, or when the solution containing the sample reacts with the functional molecule during analysis, the entry of the solution is limited. The signal is weakened because the body is obstructed and the reaction cannot be performed sufficiently. However, in the analysis chip obtained by the method of the present invention, it is presumed that a sufficient amount of fixation is obtained due to the presence of the frame, and that the signal becomes strong because various treatments and reactions can be sufficiently performed.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these Examples.

<Example 1 and Comparative Example 1>
(1) Production of a substrate having an amino group on the surface A gold-coated grating chip having a size of 2.5 cm × 2.5 cm (a flat polycarbonate substrate) in a solution obtained by dissolving 34 mg of cysteamine hydrochloride in 60 ml of water On the surface, a concave / convex shape having a groove pitch of about 870 nm and a groove depth of about 40 nm is formed, and a gold-deposited sensor chip having a thickness of about 80 nm is immersed on the surface of the concave / convex shape for 20 minutes at room temperature After holding, the sensor chip was washed with distilled water. In this treatment, an amino group is introduced into a gold-coated substrate surface via a gold-sulfur bond.

(2) Immobilization of polyacrylic acid on substrate surface In 25 ml of water, 1.25 g of polyacrylic acid (molecular weight 50,000) (manufactured by Polyscience) and 24 mg of 1-ethyl-3- (3-dimethyl) Aminopropyl) -carbodiimide hydrochloride (EDC) was dissolved. After adjusting the pH of the solution to 2.2, the aminated substrate obtained in (1) above was immersed in 2.5 ml of this solution and reacted for 1 hour. After completion of the reaction, the chip was washed with distilled water. In this treatment, an amino group on the gold surface and a carboxyl group of polyacrylic acid are condensed by EDC to form an amide bond.

(3) Immobilization of functional molecule The substrate covered with polyacrylic acid obtained in (2) above contains N-hydroxysuccinimide (NHS) at a concentration of 0.1M and EDC at a concentration of 0.4M. After immersing in the solution at room temperature for 10 minutes, the solution was washed with distilled water to activate carboxyl groups.
100 through-holes having a diameter of 0.5 mm were formed in a silicon rubber having a size of 2.5 cm × 2.5 cm and a thickness of 0.5 mm to produce a thin film member. The thin film member was laminated on the half of the substrate surface, and the thin film member was not laminated on the other half.
A solution in which mouse IgG (manufactured by Lumpia Biological Laboratory) was dissolved in an acetate buffer (10 mM, pH 5.0) so as to be 100 μg / ml was used with a spotter (“BioChip Array ^™ ” manufactured by Packard). On the surface of the substrate on which the thin film member was laminated, it was injected into the through hole. The amount of solution per through hole was 70 nl. Example 1
Further, on the surface of the substrate on which the thin film member was not laminated, the spot was directly spotted on the substrate so as to have a diameter of 0.5 mm. The amount of solution per spot was 17.5 nl. (Comparative Example 1)
After reacting for 15 minutes, the thin film member was peeled off, the entire substrate was immersed in a 1 mol / liter ethanolamine aqueous solution, and then dried to produce an analytical chip on which a ligand (mouse IgG) was immobilized. The obtained analysis chip was attached to an SPR measurement device (“FLEX CHIPS ^™ Kinetic Analysis System” manufactured by HTS Biosystems Inc.), and the amount of change in the resonance angle accompanying the immobilization of the ligand (mouse IgG) was measured. In addition, the part which injected the ligand (mouse IgG) and the part which is not injected were measured, and the difference was made into the variation | change_quantity accompanying fixation. The results are shown in Table-1.

(4) Surface plasmon resonance (SPR) measurement during analyte reaction 10 μg of anti-mouse Fab. Obtained by cleaving anti-mouse IgG (manufactured by Immunoprobe) on an analysis chip attached to an SPR measurement device / ml, a solution containing 10 mM HEPES, 10 mM NaCl, and 3 mM EDTA-2Na was allowed to flow at a flow rate of 500 μl / min for 8 minutes and reacted at 37 ° C.
After the injection of this solution, the intensity of the reflected light was measured while performing angle scanning, and the value of the change in resonance angle accompanying protein immobilization was calculated from the difference in resonance angle with respect to the resonance angle of the substrate surface on which the ligand was immobilized. The results are shown in Table-1.

<Reference example>
A chip substrate that forms the bottom surface of a recess, such as cleaning of the surface of the chip substrate, activation reaction or deactivation reaction of a functional group, and interaction reaction between a functional molecule and a specimen, in a laminate having a recess for containing a sample As a model experiment for examining the influence of the frame on the reaction in the substrate, the substrate surface activation treatment was performed before the frame was laminated (Reference Example 1) or after (Reference Example 2) to attach the ligand. We compared how much the activation treatment was hindered by the frame from the amount.

<Reference Example 1>
Gold-coated grating chip having a size of 2.5 cm × 2.5 cm (an uneven shape having a groove pitch of about 870 nm and a groove depth of about 40 nm is formed on the surface of a flat polycarbonate substrate, and the surface of the uneven surface is formed. A sensor chip on which gold is deposited to a thickness of about 80 nm is dipped in a solution of 16-mercaptohexadecanoic acid dissolved in ethanol to a concentration of 5 mM, reacted for about 2 hours, and then distilled water. By washing with C, a C16 film was formed on the substrate to produce an analysis chip.
Next, after activating the carboxyl group in the same manner as in Example 1 (3), a thin film member was laminated in the same manner as in Example 1, and then mouse IgG was injected into the through hole.
The obtained analysis chip was attached to an SPR device, and the amount of change in the resonance angle accompanying the immobilization of the ligand (mouse IgG) was measured. Further, a value (CV) obtained by dividing the standard deviation by the average value was obtained. The results are shown in Table-2.

<Reference Example 2>
After the thin film member was laminated on the analytical chip having the C16 film formed on the substrate formed in Reference Example 1 on the substrate, the carboxyl group was activated in the same manner as in (3) of Example 1. And mouse IgG was injected into the through-hole.
The obtained analysis chip was attached to an SPR device, and the amount of change in the resonance angle accompanying the immobilization of the ligand (mouse IgG) was measured. Further, a value (CV) obtained by dividing the standard deviation by the average value was obtained. The results are shown in Table-2.

From Table 2, it can be seen that Reference Example 2 has a smaller amount of resonance angle change than Reference Example 1. This is presumably because the amount of ligand immobilized was reduced because the activation liquid was difficult to enter the frame and the activation of the substrate surface was insufficient.
Further, CV indicates that the larger the numerical value, the more the amount of attached ligand varies. In Reference Example 2, since the amount of the activation liquid entering the inside of the frame differs for each through-hole, it is considered that as a result, the amount of attached ligand was more varied than that in Reference Example 1.
From this, it is inferred that the frame obstructs various processes.

Plan view of thin film member used in the present invention Sectional drawing for demonstrating the preparation method of the chip | tip for analysis of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chip substrate 2 Thin film member 3 Through-hole 3a Recessed part 4 Coating layer of organic polymer substance

Claims (17)

A chip substrate and a thin film member that is detachably laminated on the substrate and has a through-hole, and a member made of a resin that can be easily laminated and adhered to the substrate by pressure bonding and can be easily removed from the substrate. A laminate in which the through hole and the chip substrate surface cooperate to form a recess for accommodating a sample of protein, peptide, low molecular weight compound, nucleic acid, sugar, sugar chain, or lipid. A method for preparing an analytical chip, comprising: supplying a solution containing a sample to the concave portion of the substrate and immobilizing the sample on the surface of the chip substrate, and then removing the thin film member. The method for preparing an analytical chip according to claim 1, wherein the thin film member is removed by peeling the thin film member from the laminate. 3. The analytical chip adjustment method according to claim 1, wherein the thin film member is made of silicon rubber . Process for the preparation of the analysis chip according to any one of claims 1 to 3, wherein the chip substrate is a porous substrate. Process for the preparation of the analysis chip according to any one of claims 1 to 4 the surface of the chip substrate is characterized in that it is formed on the diffraction grating coated with a metal. Process for the preparation of the analysis chip according to any one of claims 1 to 5, characterized in that the surface of the chip substrate is coated with an organic polymer material having a functional group capable of immobilizing them in combination with the sample . 7. The method for preparing an analytical chip according to claim 1, wherein the thin film member has a thickness of 1 to 2000 [mu] m. The analysis according to any one of claims 1 to 7 , wherein the laminate has a plurality of recesses, and the bottom area of one recess is 0.5 x 10 ^{-10 to} 7 mm ^2. Method of preparing chips. It claims 1 to analysis chip which is adjusted by the method according to any one of 8. A chip substrate and a thin film member that is detachably laminated on the substrate and has a through-hole, and a member made of a resin that can be easily laminated and adhered to the substrate by pressure bonding and can be easily removed from the substrate. A laminate in which the through hole and the chip substrate surface cooperate to form a recess for accommodating a sample of protein, peptide, low molecular weight compound, nucleic acid, sugar, sugar chain, or lipid. An analysis chip substrate comprising: The analysis chip substrate according to claim 10 , wherein the thin film member is made of silicon rubber. The analytical chip substrate according to claim 10 or 11 , wherein the thin film member has a thickness of 1 to 2000 µm. The thin film member has a plurality of through holes, and the opening area of one through hole is 0.5 × 10 ^{−10 to} 7 mm ² . The chip substrate for analysis described in 1. Analysis chip substrate according to any one of claims 10 to 13, wherein the chip substrate is a porous substrate. Analysis chip substrate according to any one of claims 10 to 14 surface portion of the chip substrate is characterized in that it is formed on the diffraction grating coated with a metal. 16. The analytical chip substrate according to claim 10 , wherein the surface of the chip substrate is coated with an organic polymer substance having a functional group capable of binding and immobilizing the sample. A peelable thin film member used for the analysis chip substrate according to any one of claims 10 to 16 .

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