JP2013019707A - Biochip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide efficient immobilization of a bioactive substance in a well on a base material without drying even when the amount of a used solution is suppressed, with respect to a biochip which immobilizes the bioactive substance in the well on the base material.SOLUTION: A microchip which has one or more wells for measurement of the bioactive substance sectioned by partitions is characterized in having removable coating bodies above bottom surfaces of the wells in the wells. The coating bodies allow suppressing the amount of the used solution and drying and thereby make the biochip efficient.

Description

The present invention relates to a biochip having a well on a substrate and immobilizing a physiologically active substance in the well.

The microchip is a device in which a physiologically active substance is immobilized on the surface of a solid phase substrate. For example, in the case of a nucleic acid microchip, the nucleic acid is fixed on a solid phase substrate such as plastic or glass. The nucleic acid microchip is used in such a manner that a solution containing nucleic acid extracted and amplified from a specimen such as a cell is spread on the microchip and reacted for a predetermined time. The nucleic acid immobilized on the microchip and the nucleic acid in the sample solution cause a hybridization reaction corresponding to the homology of the base sequence, and the reaction rate is quantified with a label such as a fluorescent dye or a radioisotope. Thus, information on the sequence of the nucleic acid contained in the sample solution can be obtained.

In general, for microchips in which many types of nucleic acids are immobilized on a solid phase substrate, a method is used in which a sample is brought into contact with the surface of the microchip in a solution state. Is the method.
In the case of a general single-chip microchip, in order to reduce the amount of sample, a cover glass for preparing a preparation is used as a covering, and the sample is spread over the entire reaction surface. For this reason, there is a problem that one chip is used for one sample, and a plurality of samples cannot be processed at the same time.
In recent years, in order to react a plurality of samples on one microchip, a chip in which a well-like depression is formed on the microchip has been developed (for example, Patent Document 1).

This microchip with well-like depressions is very good in that it can detect multiple types of samples with a single chip, but the shape of the chip itself is special, so it can be used for reaction detection. There is a high possibility that the number of scanners to be limited is limited or correction is necessary.
Therefore, generally, a technique is employed in which a well is formed on a substrate by disposing a partition portion on the upper surface of the substrate (Patent Document 2). In the case of this method, when the partition portion is removed after the reaction is completed, the original substrate is returned to, so that the signal can be easily read by an existing scanner.
Since the reaction of physiologically active substances is generally a liquid phase reaction, in order to react the sample with the physiologically active substance immobilized on the microchip, the sample is brought into contact with the microchip surface in a solution state and held for a certain period of time. There is a need to. In this operation, it is important that the sample solution and the microchip surface come into uniform contact over the entire surface. Uniform contact means that the liquid layer has a constant thickness and does not contain bubbles. It is also important to prevent evaporation of the sample solution during the reaction. If the contact between the sample solution and the microchip is uneven or a part of the sample solution evaporates, the reaction efficiency varies depending on the position on the microchip, and the reliability of the obtained data is lowered. . Therefore, in the case of a technique for preparing a well by attaching a partition, in order to fill the reaction surface with the sample solution, a volume of at least about 100 μL is required for each well, and the microchip can be detected with a small amount of sample. There was a problem that the merit of halved.

In Patent Document 3, a method of preventing drying using a polymerized hydrogel carrying a physiologically active substance on the bottom surface of the well is employed. However, the structure is complicated and the hydrogel is used. It is necessary to pay more attention than necessary, and there is a disadvantage that it is not universal.

Japanese Patent Laid-Open No. 2005-43241 JP 2009-542222 A Special table 2005-506530 gazette

The present invention relates to a biochip having a well on a substrate and immobilizing a physiologically active substance in the well.

Means for Solving the Invention

Such an object is achieved by the present invention described in the following (1) to (19).
(1) A microchip having one or more wells separated by a partition for measuring a physiologically active substance, wherein the well has a removable covering on the bottom of the well. Microchip (2) The area of the bottom surface of the covering is 75 to 95% of the area of the well bottom. (3) The microchip according to (1), wherein the covering is 0.1 to 2 from the well bottom. The biochip according to (1) or (2), which is suspended at a constant height in a range of 0.0 mm.
(4) The covering body has at least one or more legs on the well bottom surface side, or the covering body has at least one knob portion on the well top surface side and is suspended ( The biochip according to any one of 1) to (3).
(5) The microchip according to any one of (1) to (4), wherein a solution is developed between the covering and the bottom of the well. (6) At least the bottom of the covering is hydrophilized. The biochip according to any one of (1) to (5).
(7) The covering according to any one of (1) to (6), wherein two or more of the coverings are connected.
(8) The covering according to any one of (1) to (7), wherein a material of the covering is a plastic resin.
(9) The covering according to (8), wherein the plastic resin is polystyrene or cyclic polyolefin.
(10) A biochip in which the microchip according to (1) has a removable cylindrical or polygonal cylinder partition member on a planar chip.
(11) The biochip as set forth in (10), wherein the removable partition member is made of a plastic resin.
(12) A biochip in which a physiologically active substance is immobilized on the bottom of the well according to (1) (13) A biochip in which the physiologically active substance according to (12) is immobilized in a spot shape.
(14) A biochip in which the physiologically active substance according to (12) or (13) is immobilized via a primary amino group introduced into the bottom of the well. (15) The primary amino group has a primary amino group as a side chain. The biochip according to (13), wherein the polymer having the polymer is introduced into the bottom of the well (16) The polymer is obtained by copolymerizing a monomer having a primary amino group in the side chain and a monomer having a hydrophilic group in the side chain ( The biochip according to 14) or 15).
(17) The biochip as set forth in (16), wherein the hydrophilic group is a polyalkylene glycol residue or a 2-methacryloyloxyethyl phosphorylcholine group.
(18) The biochip according to any one of (13) to (16), wherein the primary amino group is a hydrazide group or an aminooxyl group.
(19) The physiologically active substance is at least one selected from sugar chains, lectins, nucleic acids, peptide nucleic acids, aptamers, oligopeptides, proteins, antibodies, enzymes, and derivatives thereof, or among these The biochip according to any one of (12) to (14), which is a complex comprising at least one of

In a biochip having a well on a substrate, it is possible to efficiently immobilize a physiologically active substance in the well without drying even if the amount of the solution used is suppressed.

Biochip according to the present invention, (a) cylindrical well, (b) polygonal well. A well covering according to the present invention, (a) a well covering with legs, and (b) a well covering in which a knob is a suspension. The figure showing the detection result of the sugar_chain | carbohydrate chip | tip by this invention.

Hereinafter, embodiments of the present invention will be described.

FIG. 1 shows an example of a microchip having a well-like depression described in the present invention. The shape of the macro chip is not particularly limited as long as it has a recess, but a shape in which a substrate is engraved or a shape in which a side surface is raised can be suitably used.
Although the shape of the well is not particularly limited, it may be a circle or a polygon more than a triangle in plan view, and a square is preferable in consideration of efficient use of the substrate area, and is used for optical measurement. It is preferable to have a flat bottom surface.
The number of wells may be one or more.
In addition, it is desirable that the partition portion of FIG. 1 be removable after the reaction is completed so that it can be easily detected by an existing scanner.

The covering of the present invention covers the upper surface of the bottom surface of the well, and its role corresponds to a cover glass for preparing a preparation in the case of a single microchip described in the background art. is there. However, if the cover glass is simply a flat cover glass according to the well size, the tip of the tweezers will be removed when the cover glass is removed after the reaction, especially when the well size is smaller than 5 mm diameter. It is difficult to pick up and remove the cover glass.

As shown in FIG. 2, it is desirable that the bottom surface covering in the present invention has a shape having legs or a shape suspended from the top of the well so as not to stick to the bottom surface of the well of the microchip. However, the covering body which does not have a leg part may be sufficient. Moreover, the covering body which provided the knob part may be sufficient so that it may be easy to pinch with tweezers etc. at the time of removal to the upper part of a covering body.

As described above, having a certain height from the bottom surface of the well makes it possible to hold the solution in the space portion formed between the bottom surface and the covering.
The shape of the covering is not particularly limited, but is desirably the same as the shape of the well bottom surface of the microchip. Also. Since the size of the covering cannot be dropped into the well unless it is smaller than the area of the bottom of the well, it is preferably 75 to 95%, more preferably 80 to 95%, most preferably 85 to 95% of the area of the bottom of the well. 95%.

The height from the bottom of the well of the covering is preferably a constant height of 0.1 to 2.0 mm, more preferably 0.1 to 1.0 mm. This height allows the amount of solution dispensed into the well to be determined.

As the first shape of the covering, FIG. 2 shows a shape having legs. The shape of the leg is not particularly limited, but a conical shape, a polygonal pyramid shape, or a claw-like shape in which the contact point with the well bottom surface is a dot is useful because it does not reduce the effective area of the well bottom surface. is there. Further, for example, a trapezoidal leg portion in which the contact point with the well bottom surface is linear may be used.

The number of legs is preferably 3 or more because the purpose is to maintain a certain height, and in order to secure an effective area of the well bottom surface, when the contact point with the well bottom surface area is dotted, Three is the most effective. Further, when the contact point with the bottom surface of the well is on a line, the number of leg portions may be one or more.
Further, a knob for facilitating removal may be present on the upper surface of the covering. Since the knob portion extends above the end surface portion of the well upper surface, the covering can be easily removed.
Further, by connecting the plurality of coverings with the knob portion, it is possible to remove the plurality of coverings at once, and there is an effect of improving workability.

Next, the shape of the covering that is suspended from the top of the well in a suspended manner will be described.
Since the shape of the covering is the same as that of the covering having the legs, only the knob portion will be described.
The knob part is provided on the upper surface of the cover, so that it can be used not only for easy removal but also for the purpose of hanging on the end surface of the well upper surface, and the cover can be suspended above the bottom of the well. Become.
In this case, the shape of the knob portion is not particularly limited, but a claw-like shape is preferable.
The number of knobs is preferably one or more, and two are sufficient for maintaining the level, but there is no problem with three or more knobs.
By connecting the knobs of the plurality of covering bodies, it is possible to remove the plurality of covering bodies at once, and there is an effect of improving workability.

Since the material constituting the covering needs to have protrusions and knobs, a molded product using a plastic resin is most preferable.
As the plastic resin, polystyrene, cyclic polyolefin, polycarbonate, polypropylene, polymethyl methacrylate, styrene-methyl methacrylate copolymer, styrene-acrylonitrile copolymer, poly-4-methylpentene-1, and the like can be used. Polystyrene, cyclic polyolefin, polycarbonate, polypropylene, or silicone resin composition can be used. Since the solution is developed between the covering and the bottom surface of the well, it is preferably a transparent resin for determining whether bubbles are generated or insufficiently developed in the developing solution, and polystyrene and cyclic polyolefin resin are preferable. Can be used.

Since the covering is for developing the solution, it is preferable that the bottom surface of the covering, that is, the solution developing side of the covering, is subjected to a hydrophilic treatment.
The hydrophilization method can be achieved by hydrophilization by surface oxidation treatment or by application of a hydrophilic polymer compound.
In consideration of mass production, the hydrophilic treatment is simple by the surface participation treatment.
Specifically, corona discharge treatment is given as oxygen, air, and argon plasma treatment.

One microchip body will be described.
Since the microchip of the present invention has a well portion as described above, the microchip is preferably a resin molded product.
The resin to be used is the same as that of the covering, but the microchip has a particularly strong demand for optical characteristics for use in measurement. For example, at the time of measurement using a fluorescent label, a material with less autofluorescence is required, and polystyrene and cyclic polyolefin resin can be suitably used.
The molding method is not particularly limited, but injection molding is preferred in view of mass productivity and structural stability.

The microchip of the present invention is characterized by having one or more wells separated by a partition, but may be integrally molded, or a partition that is removable on a microchip on a single flat substrate. A part may be provided. Since a microchip measuring device dedicated to the measurement of a flat microchip is commercially available, a device provided with the latter removable partition portion is suitable.

The material of the removable partition part is not particularly limited, but is preferably made of plastic resin or metal. Although the shape of a partition part is not specifically limited, A cylindrical thing is preferable and a cylindrical thing and a polygonal cylindrical thing are especially preferable. These partition portions may be a single cylinder, or may be one in which several cylinders or polygonal cylinders are integrated.

The partition portion needs to be detachably provided on the flat substrate, and its method is not particularly limited, but it is necessary to closely contact the flat substrate to the extent that liquid leakage does not occur between the wells. Therefore, a method of bonding using an adhesive is suitable. Further, when the partition portion is made of a silicone resin molded product, it can be stably fixed to the flat substrate due to the adhesiveness of silicone.

It is necessary to immobilize a physiologically active substance in the well portion of the microchip. For this purpose, the surface may be subjected to treatment for immobilizing a physiologically active substance.
The surface treatment method is not particularly limited. As an example, there is a method of introducing a primary amino group into the well portion.
As a method for introducing an amino group into the well portion, there are a method of performing a plasma treatment in the presence of ammonia gas and a method of coating a polymer compound having a primary amino group.

About what was coat | covered with the high molecular compound which has a primary amino group, it is preferable that the said high molecular compound further contains the unit for hold | maintaining hydrophilicity. In the substrate of this form, the unit for maintaining hydrophilicity plays a role of suppressing physical adsorption (non-specific adsorption) of the detection target substance to the substrate.

In addition, the polymer compound may have a unit having a hydrophobic group, which serves to bind the polymer compound to the substrate.

The structure of the polymer compound is not particularly limited as long as it has the unit, but a polymer compound of the following formula 1 can be preferably used.

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. , M, and n are natural numbers.)

Application of the polymer compound to the well can be achieved by making it into a solution state, dispensing the solution into the well, allowing it to stand for a certain period of time and fixing, and then washing.

Examples of the physiologically active substance immobilized on the microchip produced as described above include, for example, the above-mentioned physiologically active substance is a sugar chain, a lectin, a nucleic acid, a peptide nucleic acid, an aptamer, an oligopeptide, a protein, an antibody, an enzyme, and the like. A complex containing at least one selected from these derivatives or including at least one of these can be used.
In particular, it is suitable for immobilizing a sugar chain sample having a reducing end.

Next, a method for using the coated body of the present invention will be described.
The step of using the covering can be used in a step of immobilizing a physiologically active substance on the chip or a step of reacting another physiologically active substance with the immobilized physiologically active substance.
First, a solution containing an appropriate amount of a physiologically active substance is dropped into the well, and the covering is suspended from the solution so as to suppress the droplet.

As another method, a method of dispensing the solution from the gap between the side surface of the well and the covering body after the covering body is first inserted into the well can be used. In consideration of high-throughput treatment, a method of suspending the coated body after the solution dropping is preferable.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

<Example>
(Preparation of sugar chain immobilization substrate)
For the production of the microarray, a glass substrate made of glass slide made of Sumitomo Bakelite and cyclic polyolefin resin was used.
2-methacryloyloxyethyl phosphorylcholine-cyclohexyl methacrylate-N- [2- [2- [2- [t-butoxycarbonylaminooxyacetylamino) ethoxy] ethoxy] ethyl] -methacrylamide copolymer 1.0 wt% ethanol The slide was immersed in the solution to apply the polymer compound.
After coating, the Boc group was deprotected by treatment with 2M HCl at 37 ° C. for 2 hours.

(Fixation of sugar chains)
Subsequently, disaccharide lactose (Wako Pure Chemicals, 124-00092) in 100 mM sodium acetate, 0.01% Triton X-100, 0.01% PVA (degree of polymerization = 1500) solution (
1 mg / mL using pH 5.0). The produced lactose solution was spotted on the produced substrate, and then reacted at 80 degrees for 1 hour to immobilize sugar chains on the substrate. After completion of the reaction, washing with ultrapure water was performed to remove unreacted lactose.

(Well preparation)
A rubber plate with an 8 mm square hole was attached to the prepared sugar chain-fixed substrate, and a well was provided on the substrate. Sixteen sugar chains are spotted in one well.

(Detection of fixed sugar chains)
Biotin-labeled RCA120 lectin (Vector, B-1085) was adjusted to 5 μg / mL with 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20 and wells. 3 μL was dispensed. Subsequently, the cover glass cut into a 7.5 mm square was dropped into the well, and the RCA120 solution was developed in the well. In order to prevent the solution from drying, it was stored in a sealed container together with a Kimwipe soaked in water and allowed to react at room temperature for 2 hours. After the reaction, the rubber plate forming the well was removed, and 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.5% Triton X-100 was used at room temperature. Washing was performed for 2 minutes.

Next, a solution in which Cy3-Streptavidin (GE Healthcare, PA43001) was prepared to ₂ ug / mL with 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20 Was spread over the entire surface of the substrate and reacted at room temperature for 1 hour. After the reaction, 50 mM Tris.HCl (pH 7.
5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.5%
Washing was performed for 2 minutes at room temperature using Triton X-100.

  The amount of fluorescence was measured for the sugar chain-fixed part and the part other than the sugar chain-fixed part (background), and the subtracted value was calculated. The measurement was performed by measuring the fluorescence intensity of Cy3 using an X-100 microarray reader, ScanArrayLite (manufactured by PerkinElmer) (Laser = 90, PMT = 60). The results are shown in FIG. The sugar chains spotted on the substrate could be detected properly.

<Comparative example> When a covering is not used Preparation of a sugar chain immobilization substrate, fixation of sugar chains, and preparation of wells were performed in the same manner as in Example 1.

(Detection of fixed sugar chains)
Biotin-labeled RCA120 lectin (Vector, B-1085) was adjusted to 5 μg / mL with 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20 and wells. Dispensed into In order to fill the whole well bottom with the solution, a solution of 80 μL or more was required. After reacting at room temperature for 2 hours, the rubber plate forming the well was removed, and washing was performed for 2 minutes at room temperature using a Tris buffer containing 0.05 wt% Triton X-100.
Next, a solution in which Cy3-Streptavidin (GE Healthcare, PA43001) was prepared to ₂ ug / mL with 50 mM Tris · HCl (pH 7.5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.05% Tween 20 Was spread over the entire surface of the substrate and reacted at room temperature for 1 hour. After the reaction, 50 mM Tris.HCl (pH 7.
5), 100 mM NaCl, 1 mM CaCl ₂ , MnCl ₂ , MgCl ₂ , 0.5%
Washing was performed for 2 minutes at room temperature using Triton X-100.
The amount of fluorescence was measured for the sugar chain-fixed part and the part other than the sugar chain-fixed part (background), and the subtracted value was calculated. The measurement was performed by measuring the fluorescence intensity of Cy3 using an X-100 microarray reader, ScanArrayLite (manufactured by PerkinElmer) (Laser = 90, PMT = 60). The results are shown in FIG.

A microchip with a structure separated by wells has a removable covering on the bottom of the well, and the covering reduces the amount of solution used and drying, and reduces the amount of effective biological substances used. Can be suppressed. In addition, the present invention provides a microchip that can react various specimens on one biochip and can use a general-purpose device by removing a partition.

Claims (19)

A microchip having one or more wells separated by a partition for measuring a physiologically active substance,
A microchip having a removable covering on the bottom of the well in the well 2. The microchip according to claim 1, wherein the area of the bottom surface of the covering is 75 to 95% of the area of the well bottom. The biochip according to claim 1 or 2, wherein the covering is suspended at a certain height within a range of 0.1 to 2.0 mm from the bottom of the well. The said covering body has at least 1 or more leg part in the well bottom face side, or the covering body has at least 1 or more knob part in the well upper face side, and is suspended. 3. The biochip according to any one of 3 above. The microchip according to claim 1, wherein a solution is developed between the covering and the bottom of the well. The biochip according to any one of claims 1 to 5, wherein at least a bottom surface side of the covering is subjected to a hydrophilic treatment. The coated body according to any one of claims 1 to 6, wherein two or more coated bodies are connected. The covering according to any one of claims 1 to 7, wherein a material of the covering is a plastic resin. The covering according to claim 8, wherein the plastic resin is polystyrene or cyclic polyolefin. A biochip, wherein the microchip according to claim 1 has a removable cylindrical or polygonal partition member on a planar chip. The biochip according to claim 10, wherein the removable partition member is made of a plastic resin. A biochip in which a physiologically active substance is immobilized on the bottom of the well according to claim 1 A biochip in which the physiologically active substance according to claim 12 is immobilized in a spot shape. A biochip on which the physiologically active substance according to claim 12 or 13 is immobilized via a primary amino group introduced into the bottom of the well The biochip according to claim 13, wherein the primary amino group is a polymer having a primary amino group in a side chain introduced into the bottom of the well. The biochip according to claim 14 or 15, wherein the polymer is obtained by copolymerizing a monomer having a primary amino group in a side chain and a monomer having a hydrophilic group in the side chain. The biochip according to claim 16, wherein the hydrophilic group is a polyalkylene glycol residue or a 2-methacryloyloxyethyl phosphorylcholine group. The biochip according to any one of claims 13 to 16, wherein the primary amino group is a hydrazide group or an aminooxyl group. The physiologically active substance is at least one selected from sugar chains, lectins, nucleic acids, peptide nucleic acids, aptamers, oligopeptides, proteins, antibodies, enzymes, and derivatives thereof, or at least one of these. The biochip according to any one of claims 12 to 14, which is a complex containing two.