JP2008002974A - Covering body and using method of covering body - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a covering body capable of improving developability of sample solution, and reducing adsorption of a physiologically active substance, when the sample solution is brought into contact onto a microarray. <P>SOLUTION: This covering body to be installed on a substrate when retaining liquid on the solid phase substrate has a characteristic wherein at least a surface on the contact side with the liquid of the covering body is coated with a material having a phospholipid. In the covering body, the material having the phospholipid is preferably a polymer material having a phosphorylcholine group, and the polymer material having the phosphorylcholine group is a copolymer including 2-methacryloyloxyethyl phosphorylcholine as a monomer unit. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

    The present invention relates to a covering used for the purpose of holding a solution on a solid substrate when the sample solution is brought into contact with the substrate, and a method of using the same.

    The microarray 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 microarray, the nucleic acid is fixed on a solid phase substrate such as plastic or glass. The nucleic acid microarray is used in such a manner that a solution containing nucleic acid extracted and amplified from a specimen such as a cell is developed on the microarray and reacted for a predetermined time. The nucleic acid immobilized on the microarray and the nucleic acid in the sample solution undergo a hybridization reaction corresponding to the homology of the base sequence, and this reaction rate is quantified by using a label such as a fluorescent dye or a radioisotope. Information on the sequence of the nucleic acid contained in the sample solution can be obtained.

    Since the reaction of physiologically active substances is generally a liquid phase reaction, it is necessary to bring the sample into contact with the microarray surface in a solution state and hold it for a certain period of time in order to cause the sample to react with the physiologically active substance immobilized on the microarray. There is. In this operation, it is important that the sample solution and the microarray surface are uniformly contacted 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 microarray is uneven or a part of the sample solution evaporates, the reaction efficiency varies depending on the position on the microarray, and the reliability of the obtained data is lowered.

    As the covering, a cover glass for preparing a preparation is often used. A cover glass provided with a spacer for keeping the thickness of the liquid layer constant is also used. However, since the cover glass is thin, it is very easy to break, and handling is relatively complicated, and therefore, a plastic covering that is hard to break is attracting attention (see, for example, Patent Document 1). However, in general, the plastic surface is often hydrophobic, and the wettability with the sample solution is low, so that uniform development of the solution is difficult. That is, when the cover is placed on the microarray and the sample solution is poured into the gap, the sample solution cannot smoothly wet the surface of the cover.

Further, in the reaction of the physiologically active substance on the microarray, nonspecific adsorption of the sample physiologically active substance to the cover glass used for the covering or the cover glass provided with the spacer is also a problem. For example, in the nucleic acid microarray, when the sample nucleic acid is adsorbed to the coating of the sample nucleic acid, the absolute amount of the nucleic acid in the sample solution is decreased, thereby affecting the hybridization with the nucleic acid immobilized on the substrate.
If non-specific adsorption to the covering is intense, the concentration in the sample nucleic acid changes, resulting in data that is not reproducible and lacks reliability.
For this reason, the covering which can solve the above-mentioned problems, that is, the wettability with the sample solution is high, the spreadability of the sample solution on the microarray is improved, and the adsorption of the physiologically active substance is small. A coating was sought.

JP 2004-177345 A

  The present invention provides a covering used for bringing a sample solution into contact with a solid phase substrate, improving the developability of the sample solution, and reducing the adsorption of a physiologically active substance in the sample solution. With the goal.

The present invention
(1) A covering that is placed on a substrate when the liquid is held on the solid phase substrate, and at least a surface of the covering that is in contact with the liquid is coated with a substance having phospholipid. A covering,
(2) The covering according to (1), wherein the substance having phospholipid is a polymer substance having a phosphorylcholine group,
(3) The covering according to (2), wherein the polymer substance having a phosphorylcholine group is a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit,
(4) The covering according to any one of (1) to (3), wherein a spacer is provided as a means for keeping the thickness of the liquid layer constant when holding the liquid on the solid phase substrate,
(5) The covering according to any one of (1) to (4), wherein the surface of the covering that is in contact with the liquid is a rough surface having a center line average roughness (Ra value) of 0.001 to 10 μm.
(6) Any one of (1) to (5), wherein a fine groove-like structure having a groove width and depth of 0.1 to 500 μm is provided on a surface of the covering body that contacts the liquid. Coverings,
(7) The covering according to any one of (1) to (6), wherein the material of the covering is plastic.
(8) The covering according to (7), wherein the plastic is polystyrene,
(9) The covering according to any one of (1) to (6), wherein the material of the covering is glass,
(10) When the liquid containing the second physiologically active substance is brought into contact with the substrate surface on which the first physiologically active substance is immobilized, the covering according to any one of (1) to (9) is used. A method of using a covering, characterized by holding a liquid on the substrate;
(11) After covering the surface of the substrate on which the first physiologically active substance is immobilized with the cover according to any one of (1) to (9), the gap between the substrate and the cover A method of using a covering, wherein a liquid containing a second physiologically active substance is allowed to flow into
(12) The physiologically active substance is at least one selected from nucleic acids, peptide nucleic acids, aptamers, oligopeptides, proteins, enzymes, sugar chains, and derivatives thereof, or at least one of these A method for using the covering according to (11) or (12), which is a composite comprising:
It is.

    According to the present invention, when a sample solution is brought into contact with a solid phase substrate, a coating body that improves the developability of the sample solution and reduces the adsorption of a physiologically active substance in the sample solution can be obtained, particularly using a microarray. It has become possible to improve the reliability of the evaluation.

Hereinafter, the present invention will be described in detail.
The uniformity of the hybridization reaction can be cited as an element that influences reliability in evaluations such as gene analysis and gene identification using a nucleic acid microarray. In order to perform the hybridization reaction uniformly, it is important that the contact between the microarray surface and the sample solution is uniform over the entire surface of the array. That is, it is important that the thickness of the sample solution is constant, that the sample solution is not dried during the reaction, and that no bubbles are mixed into the sample solution layer.

      The present invention relates to a covering that is placed on a substrate when a liquid is held on a solid-phase substrate, and at least the surface of the covering that comes into contact with the liquid is coated with a substance having a phospholipid. It is.

    The substance having phospholipid is a general term for a combination of phosphoric acid and lipid, and is mainly on the surface of a cell and plays a role of constituting a cell membrane. In the present invention, glycerol derivatives such as phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and serine derivatives such as sphingoemilin can be preferably used.

    In the present invention, a polymer substance containing a phosphorylcholine group is preferably used as the substance having a phospholipid. As a polymer substance containing a phosphorylcholine group, a copolymer (MPC polymer) having 2-methacryloyloxyethyl phosphorylcholine as a monomer unit can be suitably used. MPC polymers have a structure similar to that of phospholipid bilayer membranes, and are known to have the effect of suppressing the adsorption of bioactive substances and to stabilize bioactive molecules (Ishihara K, Tsuji T, Kurosaki T, Nakabayashi N, Journal of Biomedical Materials Research, 28 (2), pp.225-232, (1994)).

    As a method for forming the MPC polymer layer on the surface of the covering, a dip coating method can be used. That is, after the substrate is immersed in the MPC polymer solution, the MPC polymer layer is formed on the surface by volatilizing the solvent. The concentration of the MPC polymer is preferably 0.01 to 10% by weight, more preferably 0.01 to 5% by weight, and still more preferably 0.1 to 5% by weight.

(Cover body material)
Examples of the covering material that can be used in the present invention include glass and plastic. The plastic material is preferably one that does not deform at the temperature during the hybridization reaction, is resistant to solvents and buffers, and has low water absorption. Further, in order to keep the thickness of the sample solution constant, it is preferable that the material has rigidity enough to prevent deformation due to its own weight. Furthermore, it is preferable that the state of the solution can be visually confirmed when the sample solution is developed on the microarray, and the covering material is preferably transparent or translucent. As materials satisfying these conditions, polystyrene, cyclic polyolefin, polycarbonate, polypropylene, polymethyl methacrylate, styrene-methyl methacrylate copolymer, styrene-acrylonitrile copolymer, poly-4-methylpentene-1 and the like are used. However, it is preferably polystyrene, cyclic polyolefin, polycarbonate, or polypropylene.

(Hydrophilic coating)
In addition, when covering the microarray with the covering and pouring the sample solution into the gap, means for improving the wettability made of plastic can be taken in order to smoothly wet the sample solution onto the surface of the covering. As means for improving the wettability of the plastic surface, an oxidation treatment is preferable. As the oxidation treatment, methods such as plasma treatment, corona discharge treatment, ultraviolet irradiation, flame treatment, and chemical treatment are preferably used, and plasma treatment is most preferred.

(Surface roughening)
By roughening the plastic surface, the wettability with the liquid can be controlled. The method of roughening the surface is roughly divided into a dry method and a wet method. As the dry method, mechanical surface roughening, plasma contact treatment, ion beam treatment, excimer laser treatment, or the like can be used. Mechanical roughening is a method of imparting microscopic irregularities to the surface by applying a physical force. Specifically, mold shape transfer, polishing with abrasive grains, scratching with sandpaper or cloth, and spraying of particles can be used. When it is necessary to highly control the surface roughness, plasma contact treatment, ion beam treatment, excimer laser treatment, or the like can be used. Examples of the wet method include chemical treatment and solvent treatment. Specifically, roughening can be realized by dissolving a part of the surface by treatment with a good solvent of acid, alkali, or material. Of the above roughening methods, mechanical roughening is preferred from the viewpoint of ease of production. Among them, a method by transferring a mold shape is more preferable. As means for transferring the shape of the mold, injection molding, compression molding, extrusion molding, embossing or the like can be used.

    As for the surface roughness of the roughened covering, the center line average roughness (Ra value) is preferably 0.001 to 10 μm, more preferably 0.005 to 1 μm, and more preferably 0.05 to More preferably, it is 0.1 μm. It is also possible to perform a hydrophilization treatment such as oxidation treatment, graft polymerization, coating, etc., on the surface of the coated body which has been roughened by the above means.

(Surface fine processing)
By providing fine grooves on the plastic surface, wettability with the sample solution can be enhanced. As a method for providing a fine groove, a method such as injection molding, compression molding, extrusion molding, embossing, or the like can be used, such as mold shape transfer, cutting, laser processing, or lithography. From the viewpoint of ease of production, transfer of the mold shape is preferred.

The width and depth of the groove are preferably 0.1 to 500 μm, more preferably 1 to 200 μm, and still more preferably 10 to 200 μm.
It is also possible to subject the surface of the coated body provided with fine grooves to a hydrophilic treatment such as oxidation treatment, graft polymerization, and coating.

(Shape of covering)
When the microarray substrate is flat, the shape of the covering is preferably flat in order to keep the thickness of the sample solution constant. Further, in order to keep the gap between the surface of the covering and the surface of the microarray constant, it is preferable to provide a spacer portion (thickness portion) at the peripheral portion of the covering or a part thereof as shown in FIGS.

    The spacer thickness (gap between the covering and the microarray) is preferably 5 to 100 μm, more preferably 5 to 50 μm, and most preferably 10 to 30 μm.

  The present invention is specifically described by the following examples, but the present invention is not limited to the scope of the examples.

(Example 1)
Using polystyrene as a material, a covering was produced by an injection molding method. The shape of the covering is a rectangular smooth plate (60 mm × 25 mm × 0.7 mm), and a clogged tooth-like spacer (60 mm × 1 mm × 20 μm) is provided on the edge of the long side as shown in FIG. It was a thing. The obtained injection-molded article was added to a 0.5 wt% ethanol solution of a phospholipid / polymer composite (MPC (2-methacryloylphosphorylcholine) / BMA (butyl methacrylate) molar ratio = 3/7 copolymer) at room temperature. After being immersed for a minute, it was pulled up and dried overnight at room temperature to obtain a coated body having a surface coated with a phospholipid / polymer complex.

(Example 2)
Using polystyrene as a material, a covering was produced by an injection molding method. The shape of the cover is a rectangular smooth plate (60 mm x 25 mm x 0.7 mm), and a clogged tooth-like spacer (60 mm x 1 mm x 20 µm) is provided on the long side edge as shown in Fig. 1. It was. The obtained injection-molded product is subjected to plasma treatment to make the surface of the coated body hydrophilic, and then a phospholipid / polymer composite (MPC (2-methacryloylphosphorylcholine) / BMA (butyl methacrylate) molar ratio = 3/7 co-weight). After being soaked in a 0.5 wt% ethanol solution of coalescence) at room temperature for 10 minutes, it was pulled up and dried overnight at room temperature to obtain a coated body having a surface coated with a phospholipid / polymer complex.

(Example 3)
A glass product having the same shape as in Example 1 was prepared. A covering was produced by the same coating method as in Example 1.

(Comparative Example 1)
A coated body having the same shape as in Example 1 was produced by an injection molding method and used as it was without coating.

(Comparative Example 2)
A commercially available glass coating was obtained and used as it was. The shape of the glass covering was such that spacers were installed at the four corners of the flat glass.

(Evaluation 1)
The contact angle with the water of the coating body surface of Examples 1-3 and Comparative Examples 1-2 was measured. The results are shown in Table 1.

(Evaluation 2)
The coatings of Examples and Comparative Examples were placed on the surface of a microarray substrate (75 mm × 25 mm × 1.0 mm) whose surface was treated with aldehyde, and a hybridization reaction was performed using a pipette in the gap between the substrate and the coating. Buffer solution (0.3 M sodium chloride, 0.03 M trisodium citrate, 0.2% aqueous sodium dodecyl sulfate) was poured. The developability of the buffer solution and the presence or absence of air bubbles between the coating and the substrate were visually evaluated. The results are shown in Table 1.

(Evaluation 3: Non-specific adsorption of the coating)
The coated bodies of Examples and Comparative Examples were immersed in a phosphate buffer (pH 7.4) obtained by diluting a stock solution of Streptavidin-Cy3 (Amersham Biosciences Corp. PA43001) 1000 times and immersed at 37 ° C. for 30 minutes. . After washing with phosphate buffer, it was dried.
Using the microarray scanner “ScanArray Lite” (manufactured by Packard Biochip Technology), the fluorescence of each substrate was detected from the covering obtained by the above operation. The measurement conditions at this time were laser output 90%, PMT sensitivity 60%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 50 μm.
Quantification of fluorescence intensity was performed using analysis software “QuantArray” attached to the scanner. The results are shown in Table 2.

(Evaluation 4: Hybridization using a covering)
An aqueous solution (10 μM) of an oligonucleotide (atagaagttt gtccatttgt aaactcccgg attgcgctcc ctcccgcctt (SEQ ID NO: 1)) having an amino group introduced at the end thereof on the surface of an aldehyde-treated microarray substrate (75 mm × 25 mm × 1.0 mm) ). After heat treatment at 80 ° C. for 1 hour, it was washed with pure water and subjected to blocking treatment. Subsequently, it was washed with pure water and centrifuged to remove water droplets.
The substrates of Examples and Comparative Examples were placed on the substrate, and the hybridization solution was injected from the injection port (gap between the substrate and the cover). The hybridization solution was a solution in which a 50 bp long oligo DNA (AAGGCGGGAGGGAGCGCAATCCGGGAGTTTACAAATGGACAAACTTCTAT (SEQ ID NO: 2)) having Cy3 introduced at the 5 ′ end was dissolved in 5 × SSC, 0.3% SDS buffer. The hybridization reaction was performed at 65 ° C. for 3 hours. After completion of hybridization, the cells were immersed in 2 × SSC and 0.1% SDS for 10 minutes. Thereafter, washing was performed in the order of 0.2 × SSC and 0.02 × SSC. The substrate was then dried by centrifuging.
Using the microarray scanner “ScanArray Lite” (manufactured by Packard Biochip Technology), the fluorescence of the spot on each substrate was detected from the substrate obtained by the above operation. The measurement conditions at this time were laser output 90%, PMT sensitivity 60%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 50 μm.
Quantification of the fluorescence intensity of the spot was performed using analysis software “QuantArray” attached to the scanner. Table 3 shows the fluorescence values and CV values obtained in Examples and Comparative Examples. The CV value is a value obtained by dividing the average value of each fluorescence value of 96 spots by the standard deviation of each fluorescence value of 96 spots, and represents a variation in signal.

In the results of Evaluations 1 and 2, it was shown that the coating method in the present invention is effective for improving the wettability.
In the result of Evaluation 3, in Examples 1 to 3, the fluorescence signal value was significantly reduced as compared with Comparative Examples 1 and 2. This was a result supporting that the coating body coated with the substance having a phospholipid in Examples 1 to 3 suppressed nonspecific adsorption.
In Evaluation 4, in Examples 1 to 3, the fluorescence signal value was higher than in Comparative Examples 1 and 2. This is considered to be a result of the increase in the nucleic acid concentration in the hybridization solution due to the suppression of the nucleic acid adsorbed on the coating coated with the phospholipid-containing substance in Examples 1 to 3, This supported the effect of the present invention.

Schematic which shows an example of the shape of the coating body of this invention. Schematic which shows another example of the shape of the coating body of this invention. Schematic which shows another example of the shape of the coating body of this invention.

Explanation of symbols

1: Plane part 2: Thick part (spacer), indicated by diagonal lines

Claims (12)

A coating that is placed on a substrate when a liquid is held on a solid-phase substrate, and at least a surface of the coating that comes into contact with the liquid is coated with a substance having a phospholipid. body. The covering according to claim 1, wherein the substance having phospholipid is a polymer substance having a phosphorylcholine group. The coated body according to claim 2, wherein the polymer substance having a phosphorylcholine group is a copolymer containing 2-methacryloyloxyethyl phosphorylcholine as a monomer unit. The covering according to any one of claims 1 to 3, wherein a spacer is provided as a means for keeping the thickness of the liquid layer constant when the liquid is held on the solid phase substrate. The coated body according to any one of claims 1 to 4, wherein the surface of the coated body that comes into contact with the liquid is a rough surface having a center line average roughness (Ra value) of 0.001 to 10 µm. 6. The coating according to any one of claims 1 to 5, wherein a fine groove-like structure having a groove width and depth of 0.1 to 500 [mu] m is provided on a surface of the covering body in contact with the liquid. body. The covering according to any one of claims 1 to 6, wherein a material of the covering is plastic. The covering according to claim 7, wherein the plastic is polystyrene. The covering according to any one of claims 1 to 6, wherein a material of the covering is glass. When the liquid containing the second physiologically active substance is brought into contact with the substrate surface on which the first physiologically active substance is immobilized, the liquid is placed on the substrate using the covering according to any one of claims 1 to 9. A method of using a covering, characterized in that the covering is held on the surface. After covering the surface of the substrate on which the first physiologically active substance is immobilized with the cover according to any one of claims 1 to 9, the second physiology is placed in a gap between the substrate and the cover. A method of using a covering, comprising flowing a liquid containing an active substance. The physiologically active substance is at least one selected from nucleic acids, peptide nucleic acids, aptamers, oligopeptides, proteins, enzymes, sugar chains, and derivatives thereof, or a complex containing at least one of these The method of using the covering according to claim 11 or 12.

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