JP5531911B2 - Aqueous composition for immobilizing protein on carrier, and method for immobilizing protein on carrier - Google Patents

Description

  The present invention relates to an aqueous composition used for immobilizing a biologically relevant molecule that specifically binds to the biologically relevant molecule on the surface of a carrier used for detecting biologically relevant molecules and the like. It relates to an aqueous composition for immobilization on top.

Conventionally, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, environment and food inspection, etc., using a carrier such as a microarray or DNA chip on which probes are immobilized, it is related to living organisms such as nucleic acids and proteins. It is widely performed to detect molecules. A probe is a substance for binding to a target biologically relevant molecule so that the target can be detected. For example, biologically relevant molecules (such as physiologically active substances) such as DNA fragments and proteins are used. A large number of such probes are immobilized on a carrier such as a microarray or a DNA chip.
As a method for immobilizing a probe on a carrier, a method is known in which a probe is dissolved in an aqueous composition and the aqueous composition is spotted on the carrier.

Here, in order to accurately detect biologically relevant molecules using the carrier on which the probe is immobilized, it is important that the probe is appropriately and stably spotted on the carrier. That is, it is desirable that there is no variation in the shape and size of the spots formed by spotting, and the amount of probe at each spot is constant.
As a technique for stably spotting the probe on the carrier in this way, a method of immobilizing the polynucleotide described in Patent Document 1 on a solid support can be mentioned. According to this method, when the probe is a nucleic acid, the spotting can be appropriately performed.

In addition, according to the method for producing a microarray for DNA analysis described in Patent Document 2, a carrier having a uniform spot shape and size can be obtained by adjusting the viscosity by adding a thickener to the aqueous composition. It is supposed to be possible.
Furthermore, Patent Documents 3 and 4 describe that the reproducibility of the spot shape and size can be improved by adding polyvinyl alcohol or polyethylene glycol to the aqueous composition.

JP 2004-198406 A Japanese Patent No. 3398366 JP 2004-286728 A JP 2007-163354 A

However, these methods are suitable when the probe is a nucleic acid, and not suitable when the probe is a protein.
In other words, when the probe is a protein, depending on the type and viscosity of the aqueous composition, if the protein is dissolved in the aqueous composition, and this is spotted on a carrier and immobilized, and then washed, The part was adsorbed to a part other than the spot on the carrier surface, and an appropriate spot could not be obtained.
For this reason, when such a carrier is used for detection of biologically relevant molecules, there is a problem that the background becomes high and appropriate detection cannot be performed.

Thus, as a result of intensive studies, the present inventors have found that the adsorption of protein to the carrier can be suppressed by containing a certain monoalkylene glycol in the aqueous composition to be spotted on the carrier, thereby completing the present invention. .
That is, the present invention provides a monoalkylene glycol having 4 to 6 carbon atoms in an aqueous composition used for immobilizing a protein on a carrier surface for detecting a bio-related molecule that specifically binds to the protein. It is an object of the present invention to provide an aqueous composition for immobilizing a protein on a carrier and a method for immobilizing the protein on the carrier, which can suitably perform the spotting of the probe on the carrier by containing .

  In order to achieve the above object, the aqueous composition of the present invention is an aqueous composition used for immobilizing a protein on the surface of a carrier for detecting a bio-related molecule that specifically binds to the protein. And it is set as the structure containing at least any one of C4-C6 monoalkylene glycol.

  The method for immobilizing a protein on a carrier according to the present invention is a method for immobilizing a protein on the surface of a carrier for detecting a biologically relevant molecule that specifically binds to the protein, comprising 4 carbon atoms. A step of dissolving a protein in an aqueous composition containing at least one of -6 monoalkylene glycols, spotting the aqueous composition on the surface of the carrier, a step of immobilizing the protein on the surface of the carrier, and a carrier Cleaning the surface of the substrate.

  According to the present invention, when a protein is immobilized on a carrier surface for detecting a biologically relevant molecule that specifically binds to the protein, the protein can be prevented from adsorbing to a portion other than the spot of the carrier. It becomes possible to detect molecules appropriately.

It is a figure which shows the list of the various glycol type additives in each experiment, and its density | concentration. It is a figure which shows the spotted state of the protein on the support | carrier surface in Experiment 1 and 2. FIG. It is a figure which shows the spotted state of the protein on the support | carrier surface in Experiment 3 and 4. FIG. It is a figure which shows the spotted state of the protein on the support | carrier surface in Experiment 5 and 6. FIG. It is a figure which shows the spotted state of the protein on the carrier surface in Experiment 7 and 8. It is a figure which shows the spotted state of the protein on the support | carrier surface in Experiment 9 and 10. FIG.

  Hereinafter, preferred embodiments of an aqueous composition for immobilizing a protein of the present invention on a carrier and a method for immobilizing a protein on a carrier will be described in detail, but the present invention is limited to the configurations of the following embodiments. It is not something.

(Aqueous composition for immobilizing protein on a carrier)
The aqueous composition for immobilizing the protein of this embodiment on a carrier (hereinafter sometimes referred to as an immobilization solution) contains a specific glycol-based additive in an aqueous solution containing the protein to be immobilized on the carrier. It is added.
The type of protein to be immobilized on the carrier is not particularly limited. For example, it is possible to use a protein that becomes an antigen, various antibody proteins such as immunoglobulin, and various proteins that can bind to other biologically relevant molecules. it can.
Moreover, a general thing can be used for the solvent of the aqueous solution containing protein, For example, a phosphate buffered saline solution (PBS) etc. can be used conveniently.

A monoalkylene glycol having 4 to 6 carbon atoms is used as a glycol-based additive added to the aqueous composition for immobilizing the protein of the present embodiment on a carrier.
That is, 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol can be suitably used as the additive. These additives may be used alone or in combination. In addition, these modified products can be used alone or in combination.
By using these as additives in aqueous compositions to immobilize proteins on a carrier, it is possible to prevent proteins from adsorbing nonspecifically to parts other than spots on the carrier surface and to suppress drying of the spot solution. It becomes possible to do.

The concentration of these monoalkylene glycols in the aqueous composition is preferably 10% by weight to 70% by weight.
If it is less than 10% by weight, the spot liquid tends to dry, and therefore spot unevenness tends to occur. On the other hand, if it exceeds 70% by weight, it is considered that the binding efficiency of the protein to the substrate is lowered due to the change in the viscosity of the solvent of the spot solution and the affinity with the protein.
In particular, when 1,4-butanediol is used as an additive, the concentration is preferably 10% by weight to 70% by weight, and when 1,5-pentanediol is used, the concentration is 10% by weight to 50%. From the results of Examples described later, it is clear that the concentration is preferably 10% by weight to 50% by weight when 1,6-hexanediol is used.

(Carrier)
The carrier used in this embodiment is a carrier such as a microarray or a DNA chip, and a large number of the above-described proteins are immobilized on the surface.
The carrier substrate is, for example, silicon, glass, fiber, wood, paper, ceramics, polyester resin, polyethylene resin, polypropylene resin, ABS (Acrylonitrile Butadiene Styrene) resin, nylon, acrylic resin, fluororesin, polycarbonate resin, Can be made of polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin, or other plastics. It is preferable to use a substrate whose autofluorescence (background) is small.

It is also preferable that the surface of the carrier is subjected to a surface treatment such as vapor deposition of diamond-like carbon so that the protein can be firmly immobilized on the carrier.
The shape and size of the carrier are not particularly limited. For example, a flat plate having a width of 0.1 to 100 mm, a length of 0.1 to 100 mm, and a thickness of 0.01 to 10 mm is preferably used. it can.

In addition, a single layer of Ti, Au, Pt, Nb, Cr, TiC, TiN, or a composite film of these may be formed on the front or back surface of the carrier, and an electrostatic layer may be provided. It is also preferable.
In order to introduce a functional group that can be covalently bonded to a protein to the surface of the carrier to which the electrostatic layer has been applied, chemical modification can be performed. Examples of the functional group include a carboxyl group, an active ester group, a haloformyl group, a hydroxyl group, a sulfate group, a cyano group, a nitro group, and a thiol group.

In particular, the protein is immobilized on the carrier by introducing a carboxyl group into the carrier on which the electrostatic layer has been applied using dicarboxylic acid or polyvalent carboxylic acid, and binding the amino group of the protein to this carboxyl group. Is preferred.
As a method for performing chemical modification, a general method can be used. For example, when a reaction for introducing a functional group capable of covalently binding to a protein is performed in a solution on a carrier on which an electrostatic layer has been applied, It is preferable to introduce a functional group capable of covalently bonding to a protein after immersion in a solution containing a compound having an unsubstituted or monosubstituted amino group. As the solvent of the solution, for example, water, N-methylpyrrolidone, ethanol or the like can be used.

(Method of immobilizing protein on carrier)
(1) Preparation of aqueous composition First, a protein to be immobilized on a carrier is added to a phosphate buffered saline solution. Next, any monoalkylene glycol having 4 to 6 carbon atoms is added to prepare an aqueous composition as an immobilization solution. The order of adding protein and monoalkylene glycol may be reversed.
In addition, the concentration of any monoalkylene glycol having 4 to 6 carbon atoms is preferably 10% by weight to 70% by weight, and 10% by weight to 50% by weight as a final concentration in the immobilization solution as described above. This is more preferable because the stability of the spot can be further improved.

(2) Immobilization reaction Next, the prepared immobilization solution is dispensed into a well microplate, the well microplate and the carrier are set in a spotting machine, and the immobilization solution is spotted on the carrier (spotting). ) And let stand for a fixed time. Thereby, the immobilization reaction of the protein to the carrier is performed.

(3) Washing Next, the carrier on which the protein is immobilized is washed. The washing method is not limited as long as unreacted protein after the immobilization reaction can be removed, and a buffer and a surfactant that do not denature the protein can be used.
Furthermore, it is preferable to perform blocking on the washed carrier surface. This blocking is for suppressing nonspecific adsorption of the protein to be reacted on the substrate in the actual use stage of the protein-immobilized substrate. Generally, the blocking is performed with a reaction such as albumin or skim milk. In many cases, irrelevant proteins are adsorbed on the substrate surface, or the substrate surface is coated with a non-living substance. In the present embodiment, these can be used as appropriate.

(Method for confirming the presence or absence of nonspecific adsorption of protein to the carrier)
Next, a method for confirming whether the protein is non-specifically adsorbed on the surface of the carrier other than the spot by performing a binding reaction between the protein immobilized on the carrier and the biologically relevant molecule will be described. . In general, in order to evaluate nonspecific adsorption on a substrate, a fluorescent substance is directly labeled on the protein to be immobilized, and nonspecific adsorption is often detected with a detector. A method for detecting non-specific adsorption is described below.

(1) Binding reaction between protein and biological molecule First, a biological molecule (hereinafter sometimes referred to as a sample) that specifically binds to a protein immobilized on a carrier is prepared, and this is prepared with a fluorescent substance or the like. Label. The labeling method is not particularly limited, and a commercially available labeling kit or the like can be used. If there is a commercially available product, a labeled sample may be purchased and used.
In addition, as a labeling substance, fluorescent labels such as CyDye such as Cy3 and Cy5, FITC, RITC, rhodamine, Texas red, TET, TAMRA, FAM, HEX, and ROX can be preferably used. A biotin method, a chemiluminescence method, etc. can also be used instead.
The composition of the aqueous solution of the labeled sample is not particularly limited as long as the sample can sufficiently bind to the immobilized protein, and the buffer and interface that do not denature the protein and do not inhibit the binding reaction. An activator or the like can be used as appropriate.

Next, the obtained aqueous solution is spotted on a spot on which the protein on the carrier is immobilized, and a binding reaction between the protein immobilized on the carrier and the labeled sample is performed. For example, when a protein immobilized on a carrier is an antigen, an antibody is used as a sample, and an antigen-antibody reaction is performed between them.
After the binding reaction between the protein and the sample, the carrier is washed with a washing solution to remove the unreacted sample from the surface of the carrier. As the cleaning solution, a mixed solution of a surfactant and a buffer solution can be used.

(2) Detection Next, the fluorescence intensity of the spot on the carrier is measured. At this time, the fluorescence signal of the label of the sample bound to the protein immobilized on the carrier is detected by the fluorescence detector. As the fluorescence detector, for example, a fluorescence scanning device in which a cooled CCD camera and a computer are connected can be used. Thereby, the fluorescence on the carrier can be obtained as image data.

When the protein is appropriately immobilized in the spot area on the carrier and the protein is not non-specifically adsorbed on other areas on the carrier surface by the above method, circular fluorescence is generated in the spot area on the carrier. Is detected, and image data from which fluorescence is not detected from other regions can be obtained.
On the other hand, when protein is non-specifically adsorbed in a region other than the spot on the carrier surface, fluorescence is detected in the adsorbed portion. Such fluorescence increases the background and hinders detection of the sample.
In addition, when the protein is not properly immobilized in the spot region on the carrier, fluorescence having an unstable shape is detected. Such a state hinders detection of a sample and hinders obtaining quantitative data.

  Therefore, in the method for immobilizing a protein on a carrier according to the present embodiment, nonspecific adsorption or non-specific adsorption of the protein onto the surface of the carrier is performed by adding a monoalkylene glycol having 4 to 6 carbon atoms to the immobilization solution. It is possible to prevent formation of a spot having a stable shape.

  Here, it is known that when nucleic acid is immobilized on a carrier, non-specific adsorption of the nucleic acid to the carrier can be suppressed by adding polyethylene glycol (PEG) to the immobilization solution. Thus, when protein is immobilized on a carrier, it was expected that the protein could be prevented from adsorbing non-specifically to the carrier by adding polyethylene glycol to the immobilization solution.

However, as shown in Comparative Examples described later, even when an immobilization solution to which polyethylene glycol was added was used, it was not possible to suppress nonspecific adsorption of proteins to the carrier. This is because the immobilization solution containing polyethylene glycol has a high viscosity, so in the case of a protein having a large molecular weight compared to nucleic acid, the protein that did not bind to the sample during washing after immobilization moves from the carrier surface to the washing solution. It is presumed that the rate of diffusion of the water became very slow and nonspecifically adsorbed on the support surface.
Therefore, the present inventors screened the same glycols with lower molecular weights, but nonspecific adsorption occurred when alkylene glycols having about 4 to 6 carbon atoms were added to the immobilization solution. I found nothing.

On the other hand, nonspecific adsorption was observed in ethylene glycol having 2 carbon atoms and propylene glycol having 3 carbon atoms. When these immobilization solutions were used, the protein was dried on the substrate immediately after spotting. From this, it is presumed that when these immobilization solutions were used, the spot solution was easily dried, and proteins were aggregated by drying and were easily adsorbed on the substrate surface.
Therefore, the optimal immobilization solution for immobilizing the protein on the carrier is considered to be a low-viscosity solution in which substance diffusion is likely to occur, and a solution that does not dry during the spot-immobilization reaction.

  From the above, in this embodiment, the protein is adsorbed to a portion other than the spot of the carrier by containing the monoalkylene glycol having 4 to 6 carbon atoms in the aqueous composition for immobilizing the protein on the carrier. This makes it possible to appropriately detect biologically relevant molecules.

  The following was performed to verify the presence or absence of non-specific adsorption of protein to the carrier when various glycols were added as additives to the aqueous composition for immobilizing the protein on the carrier. Examples, comparative examples and reference examples will be described.

<Experiment 1: 1,4-butanediol>
Example 1
1 × phosphate buffered saline solution (Rabbit IgG, manufactured by Nippon Gene Co., Ltd., pH 7.4, hereinafter sometimes referred to as PBS) containing Rabbit IgG (manufactured by Life Technology Japan Co., Ltd.) as a protein to be immobilized on the carrier. , 4-butanediol (manufactured by Wako Pure Chemical Industries, Ltd.) was added to prepare a Rabbit IgG solution having a final concentration of 100 μg / mL, which was used as an immobilization solution. 1,4-butanediol was added to a final concentration of 10% by weight. In addition, the additive made from Wako Pure Chemical Industries Ltd. was used similarly in the following Examples, Comparative Examples, and Reference Examples. A list of the concentrations of glycols used in each experiment is shown in FIG.

  Next, a 384-well microplate in which 5 μL of the prepared immobilization solution is dispensed and Gene Silicon (manufactured by Toyo Kohan Co., Ltd.) as a spotting machine SPBIOTM2000 (manufactured by Hitachi Software Engineering Co. Ltd.) are used. Set in position and spotted.

The carrier spotted with the immobilization solution was allowed to stand at 4 ° C. overnight, and then washed with 1 × PBS / 0.5% Tween 20 for 15 minutes. Further, 1 × Carbo-Free Blocking Solution (Vector Laboratories) of blocking agent was used. , Inc.) for 3 hours at room temperature.
The carrier was washed with Milli-Q water, centrifuged and vacuum packed with an aluminum pouch. The prepared Rabbit IgG-immobilized carrier was stored at −20 ° C. until use.

  Next, Alexa Fluor 647 goat anti-Rabbit IgG (H + L) (manufactured by Life Technology Japan Co., Ltd.) is used as a bio-related molecule to be bound to the protein immobilized on the carrier, and the final concentration becomes 20 μg / mL. As described above, a reaction solution containing a sample was prepared by diluting in 1 × PBS / 0.05% Tween 20 solution. In addition, this biologically relevant molecule is already fluorescently labeled and is commercially available.

This reaction solution was dropped onto the Rabbit IgG-immobilized carrier prepared above, and the cover plate was put on, followed by standing at room temperature (25 ° C.) for 60 minutes under light shielding to carry out an antigen-antibody reaction.
After the reaction, the carrier was washed by immersing it in 50 mM TTBS buffer (20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 0.05% Tween 20) at room temperature, and a cover glass was placed on the fluorescence detector Bioshot (Toyo Kohan). Fluorescent image data was obtained by measuring the fluorescence intensity of the carrier surface at 632 nm. The result is shown in FIG.

(Examples 2, 3, and 4)
Except that 1,4-butanediol was added as an additive to be contained in the immobilization solution so that the final concentrations in Examples 2, 3, and 4 were 30% by weight, 50% by weight, and 70% by weight, respectively. Experiments were performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.

  When 1,4-butanediol was used as an additive for the immobilization solution, almost no fluorescence was observed in the portions other than the spots in any of the fluorescence images of Examples 1, 2, 3, and 4. The fluorescent shape of the spot is a stable circle. Therefore, in these cases, it can be seen that the protein is hardly adsorbed on the portion other than the spot and is appropriately immobilized on the spot. For this reason, when 1,4-butanediol is used as an additive for the immobilization solution, the concentration in the immobilization solution is preferably 10 wt% to 70 wt%.

<Experiment 2: 1,5-pentanediol>
(Examples 5, 6, and 7)
Except that 1,5-pentanediol was added as an additive to be contained in the immobilization solution so that the final concentrations in Examples 5, 6 and 7 were 10% by weight, 30% by weight and 50% by weight, respectively. Experiments were performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.

(Reference Example 1)
Except that 1,5-pentanediol was added as an additive to be contained in the immobilization solution so that the final concentration was 70% by weight, an experiment was performed in the same manner as in Example 1 to obtain an image of fluorescence on the surface of the carrier. I got the data. The result is shown in FIG.

  When 1,5-pentanediol is used as an additive for the immobilization solution, almost no fluorescence is observed in portions other than the spots in the fluorescent images of any concentration of Examples 5, 6, 7 and Reference Example 1. . On the other hand, the fluorescent shape of the spot is a stable circle in Examples 5, 6, and 7, but is slightly distorted in Reference Example 1. This is considered to be because when 70% by weight of 1,5-pentanediol is contained in the immobilization solution, the reaction of the protein bound to the surface of the carrier is reduced and the amount of protein adsorbed on the spot is reduced. . Therefore, when 1,5-pentanediol is used as an additive for the immobilization solution, its concentration in the immobilization solution is preferably 10 wt% to 50 wt%.

<Experiment 3: 1,6-hexanediol>
(Examples 8, 9, and 10)
Except that 1,6-hexanediol was added as an additive to be contained in the immobilization solution so that the final concentrations in Examples 8, 9, and 10 were 10 wt%, 30 wt%, and 50 wt%, respectively. Experiments were performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.

(Reference Example 2)
Except that 1,6-hexanediol was added as an additive to be contained in the immobilization solution so that the final concentration was 70% by weight, an experiment was performed in the same manner as in Example 1 to obtain an image of fluorescence on the surface of the carrier. I got the data. The result is shown in FIG.

  When 1,6-hexanediol is used as an additive for the immobilization solution, almost no fluorescence is observed in portions other than the spots in any of the fluorescence images of Examples 8, 9, 10 and Reference Example 2. . On the other hand, the fluorescent shape of the spot is a stable circular shape in Examples 8, 9, and 10, but in the reference example 2, it is an irregular shape. This is considered to be because when 70% by weight of 1,6-hexanediol is contained in the immobilization solution, the reaction of the protein bound to the surface of the carrier is reduced, and the amount of protein adsorbed on the spot is reduced. . Therefore, when 1,6-hexanediol is used as an additive for the immobilization solution, the concentration in the immobilization solution is preferably 10% to 50% by weight.

<Experiment 4: Ethylene glycol>
(Comparative Example 1)
Except that ethylene glycol was added as an additive to be contained in the immobilization solution so that the final concentration was 30% by weight, an experiment was performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. . The result is shown in FIG.
In this case, in the fluorescence image of Comparative Example 1, the fluorescence is also seen in portions other than the spot, the shape of the spot is not circular, and is unstable and distorted, and the protein is adsorbed on the portion other than the spot. ing. Therefore, it can be seen that it is difficult to properly immobilize the protein on the carrier even if ethylene glycol is used as an additive.

<Experiment 5: Propylene glycol>
(Comparative Examples 2, 3, and 4)
Example 1 except that propylene glycol was added as an additive to be contained in the immobilization solution so that the final concentrations in Comparative Examples 2, 3, and 4 were 10% by weight, 30% by weight, and 50% by weight, respectively. An experiment was performed in the same manner to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.
In these cases, in any of the fluorescence images of Comparative Examples 2, 3, and 4, the fluorescence is also observed in portions other than the spot, and the shape of the spot is not circular but is unstable and distorted. Is adsorbed to a portion other than the spot. Therefore, it can be seen that it is difficult to properly immobilize the protein on the carrier even if propylene glycol is used as an additive.

<Experiment 6: Diethylene glycol>
(Comparative Examples 5, 6, 7)
As in Example 1, except that diethylene glycol was added as an additive to be contained in the immobilization solution so that the final concentrations in Comparative Examples 5, 6, and 7 were 10%, 30%, and 50%, respectively. Experiments were conducted to obtain image data of fluorescence on the surface of the carrier. The result is shown in FIG.
In these cases, in any of the fluorescence images of Comparative Examples 5, 6, and 7, the fluorescence is also seen in portions other than the spot, and the shape of the spot is not circular but has an unstable and distorted shape. Is adsorbed to a portion other than the spot. Therefore, it can be seen that it is difficult to properly immobilize the protein on the carrier even if diethylene glycol is used as an additive.

<Experiment 7: Glycerol>
(Comparative Example 8)
Experiments were performed in the same manner as in Example 1 except that glycerol was added as an additive to be contained in the immobilization solution so that the final concentration was 30% by weight, and fluorescence image data on the surface of the carrier was obtained. The result is shown in FIG.
In this case, in the fluorescence image of Comparative Example 8, the fluorescence is also seen in the portion other than the spot, the shape of the spot is not circular, and is unstable and distorted, and the protein is adsorbed on the portion other than the spot. ing. Therefore, it can be seen that it is difficult to properly immobilize the protein on the carrier even if glycerol is used as an additive.

<Experiment 8: Polyethylene glycol (molecular weight 200)>
(Comparative Examples 9, 10, 11)
Except that polyethylene glycol (molecular weight 200) was added as an additive to be contained in the immobilization solution so that the final concentrations in Comparative Examples 9, 10, and 11 were 1 wt%, 10 wt%, and 30 wt%, respectively. Experiments were performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.

<Experiment 9: Polyethylene glycol (molecular weight 300)>
(Comparative Examples 12 and 13)
As in Example 1, except that polyethylene glycol (molecular weight 300) was added as an additive to be contained in the immobilization solution so that the final concentrations were 10 wt% and 30 wt% in Comparative Examples 12 and 13, respectively. Experiments were performed to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.

<Experiment 10: Polyethylene glycol (molecular weight 6000)>
(Comparative Examples 14, 15, 16)
Except that polyethylene glycol (molecular weight 6000) was added as an additive to be contained in the immobilization solution so that the final concentrations in Comparative Examples 14, 15, and 16 were 1% by weight, 10% by weight, and 30% by weight, respectively. Experiments were performed in the same manner as in Example 1 to obtain fluorescence image data on the surface of the carrier. The result is shown in FIG.
In any of the fluorescence images of Comparative Examples 9-16, the fluorescence was also observed in portions other than the spot, the shape of the spot was not circular, and was unstable and distorted, and the protein was in a portion other than the spot. Adsorbed. Therefore, it can be seen that it is difficult to properly immobilize the protein on the carrier even if polyethylene glycol is used as an additive.

The present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, it is possible to make appropriate changes such as adding two or more of monoalkylene glycols having 4 to 6 carbon atoms in combination to an aqueous composition for immobilizing a protein on a carrier.

  The present invention can be suitably used for producing a carrier such as a DNA chip or a microarray on which a protein is immobilized as a probe.

Claims (5)

An aqueous composition used to immobilize a protein on the surface of a carrier for detecting a bio-related molecule that specifically binds to the protein, comprising 1,4-butanediol, 1,5-pentane An aqueous composition comprising one or more selected from the group consisting of diol and 1,6-hexanediol as an additive . When the additive is 1,4-butanediol, the concentration of 1,4-butanediol is 10% by weight to 70% by weight,
When the additive is 1,5-pentanediol, the concentration of the 1,5-pentanediol is 10 wt% to 50 wt%,
The aqueous composition according to claim 1, wherein when the additive is 1,6-hexanediol, the concentration of 1,6-hexanediol is 10 wt% to 50 wt% . A method of immobilizing a protein on the surface of a carrier for detecting a bio-related molecule that specifically binds to the protein,
Protein is dissolved in an aqueous composition containing one or more selected from the group consisting of 1,4-butanediol, 1,5-pentanediol, and 1,6-hexanediol. Spotting the surface of the carrier;
Immobilizing the protein on the surface of the carrier;
And a step of washing the surface of the carrier. A method for immobilizing a protein on the carrier.   The method for immobilizing a protein on a carrier according to claim 3, further comprising a step of blocking the surface of the carrier after washing the surface of the carrier. The protein on the carrier according to claim 3 or 4, wherein the protein is immobilized on the surface of the carrier by forming a covalent bond between a carboxyl group on the surface of the carrier and an amino group of the protein. Immobilization method.