JP5002993B2 - Method for producing selective binding substance-immobilized carrier - Google Patents

Description

  The present invention relates to a method for producing a selective binding substance-immobilized carrier, and more specifically, high-quality selective binding substance immobilization capable of sufficiently achieving spotter pin cleaning and reducing carryover. The present invention relates to a method for producing a carrier.

  The DNA microarray method (DNA chip method) expresses a large number of genes using a glass flat substrate piece in which a large number of DNA fragments, proteins, and sugar chains are aligned and fixed at high density (microarray or DNA chip). It is an analysis method for analyzing a single nucleotide polymorphism (SNP) of various organisms and a genetic information analysis of various organisms. In addition, development of a chip in which a nucleic acid or protein other than DNA is bound by applying the above-described DNA chip is also progressing.

  As a technique for immobilizing a selective binding substance such as nucleic acid on a substrate, a spotting device called a spotter is used by coating poly-L-lysine, aminosilane, etc. on a flat substrate such as a slide glass. A method for immobilizing each nucleic acid has been developed (see, for example, Patent Document 1). In this method, a solution containing a desired DNA is brought into contact with the tip of a spotter pin, and then the pin is brought into contact with a predetermined position of the carrier and spotted, and this operation is repeated to apply a predetermined type of DNA to the carrier. To spot.

  In the case of the above method using a spotter, it is desirable to wash the pins before bringing the pins into contact with the solution containing the DNA so that other DNA is prevented from being mixed and the predetermined DNA is spotted at a predetermined position. . In particular, when a spotter is used repeatedly, it is necessary to perform cleaning with every spotting in order to prevent mixing. Conventionally, there has been a spotter in which an aqueous solution of SDS (sodium dodecyl sulfate), pure water, or ethanol (a stationary tank) is used as a pin cleaning liquid.

Japanese National Patent Publication No. 10-503841

However, in the conventional spotter pin cleaning liquid described above, the cleaning is often not performed sufficiently, and there are cases where the cleaning is insufficient even when three kinds of SDS aqueous solution, pure water and ethanol are combined. In particular, in the case of a highly sensitive chip, if the cleaning is insufficient, a significant carry-over is observed, which may cause a problem in detection using the chip.
In view of the above problems, the present invention can sufficiently achieve pin washing and reduce carry-over in producing a selective binding substance-immobilized carrier using a spotter. It aims at providing the means which can do.

The present invention provides the following inventions.
[1] In producing a selective binding substance-immobilized carrier using a spotter,
After immersing the spotter pin in a solution containing the selective binding substance, the step of bringing the pin into contact with the carrier and spotting the selective binding substance on the carrier, and the pin into the solution containing the aprotic polar solvent A method for producing a selective binding substance- immobilized carrier, comprising a step of dipping.
[2] The method according to [1], further comprising a step of immersing the pin in water or an aqueous solution not containing a selective binding substance.
[3] The manufacturing method according to [1] or [2], wherein a fluid solution is used for the immersion of the pin.
[4] The production method according to any one of [1] to [3], wherein the selective binding substance is DNA, RNA, protein, peptide, sugar, sugar chain, or lipid.
[5] The aprotic polar solvent is acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, N-methyl-2-pyrrolidone, or γ-butyllactone, The manufacturing method as described in any one of said [1]-[4].
[6] Any one of the above [1] to [5], wherein the carrier is a carrier having a concavo-convex portion on a surface thereof, and a selective binding substance is spotted on the top surface of the concavo-convex portion. The manufacturing method according to one item.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of the selective binding substance fixed support | carrier which can fully achieve the washing | cleaning of the pin of a spotter and reduced carryover is provided.

The method for producing a selective binding substance- immobilized carrier of the present invention relates to a method for producing the carrier using a spotter, and after the pins are immersed in a solution containing the selective binding substance, the pins are brought into contact with the carrier to form the carrier. It includes a step of spotting a selective binding substance (step (1)) and a step of immersing the pin in a solution containing an aprotic polar solvent (step (2)).

First, step (1) will be described. In the step (1), after spotter pins are immersed in a solution containing a selective binding substance, the pins are brought into contact with the support to spot the selective binding substance on the support.
A pin is a structure that can hold a solution containing a selective binding substance and can be spotted by bringing the solution into contact with a carrier, and can have various shapes depending on the amount of solution retained and the efficiency at the time of spotting. Things can be used. The shape of the tip is not particularly limited, but the shape of the tip end face (symbol X in FIG. 1) is a flat type (for example, FIG. 1), a single-character groove type (for example, FIG. 2), or a cross-shaped groove type (for example, for example). 3) and the like, and the tip diameter is generally about 0.01 to 0.5 mm. Moreover, not only the end face of the tip but also a pin having a groove formed on the side face of the tip (reference numeral Y in FIG. 1) can be used. The material may be various resins in addition to a metal such as stainless steel, but is preferably a metal such as stainless steel.

When a pin having a groove on the end surface of the pin, such as a single-character groove type (for example, FIG. 2) or a cross-shaped groove type (for example, FIG. 3), is used as such a pin, for example, as follows A selective binding substance can be spotted on the carrier by being immersed in a solution containing When the tip of the pin is immersed in a solution containing a selective binding substance, the groove is filled with the solution by capillary action, and the liquid level is located in the groove on the end face of the tip. After immersing the pin in the solution containing the selective binding substance as described above, for example, the pin is lifted from the solution and moved until the tip of the pin comes just above the position to be spotted on the surface of the carrier. When the pin is lowered vertically, and the tip of the pin is lightly struck against the surface by instantaneous contact with the surface of the carrier, the solution filled in the groove on the end surface of the pin tip is drawn to the surface of the carrier and spotted on the surface of the carrier .
A specific example of a spotter having such a pin is disclosed in Japanese Patent Laid-Open No. 10-503841.

  The carrier means a substance that can immobilize the selective binding substance. Examples of the shape of the carrier include a flat plate shape and a spherical shape, and a flat plate shape is preferable. As the material of the carrier, for example, inorganic materials such as glass, ceramic and silicon, polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate and silicone rubber can be used. Among these, polymethyl methacrylate, polystyrene, polydimethylsiloxane (PDMS) elastomer, glass, and silicon can be particularly preferably used. The carrier can be produced by an injection molding method or hot embossing method in the case of plastic, a sand blasting method in the case of glass or ceramic, or a known semiconductor process in the case of silicon.

  In the production method of the present invention, any carrier can be applied, but among them, a carrier with high sensitivity is preferable. A representative carrier having a high sensitivity is a carrier having an uneven portion on the surface. The carrier having such a structure can bind the selective binding substance to the convex part of the concavo-convex part in a stable spot shape, and thus has excellent quantitative properties and adsorbs nonspecifically during optical detection. Since the specimen is not detected, the noise is small, and as a result, a result with better S / N can be obtained. Furthermore, fine particles and the like can be stored between the convex portions of the concave and convex portions, and by moving these by external force, the sample solution is stirred without damaging the selective binding substance bound to the upper end surface of the columnar structure. Therefore, the inspection time can be shortened, and as a result, ultra-sensitive and highly accurate inspection is possible.

  Regarding the height of the convex portions of the concavo-convex portions, it is preferable that the height of the upper surface of each convex portion is substantially the same. Specifically, the difference in height is usually 50 μm or less and 30 μm or less. It is particularly preferable that the heights are the same. Moreover, it is preferable that the upper surface of a convex part is substantially flat. Here, the fact that the top surface of the convex portion is substantially flat means that there is no unevenness of 20 μm or more. Further, it is preferable that a flat portion is further provided on the surface of the carrier having the uneven portion. The height of the upper surface of the convex part of the concave and convex part and the height of the flat part are preferably substantially the same, preferably 50 μm or less, and particularly preferably.

  A specific example showing an outline of a carrier having an uneven portion on the surface is shown in FIG. In the example shown in FIG. 4, the surface of the selective binding substance-immobilized base material a is composed of a plurality of convex portions 2 and a concavo-convex portion 4 including a concave portion 3, and a flat portion 1 is provided therearound. Yes. A selective binding substance can be bonded to the convex upper surface 2A.

  The selective binding substance means a substance that specifically interacts with a specific substance included in the substance group. Specific interaction is direct or indirect binding to a specific substance, and can be referred to as binding having selectivity. Specific examples of the selective binding substance include nucleic acids such as DNA, RNA and PNA, peptides, amino acids and other proteins, saccharides, lipids, and other antigenic compounds. Among them, DNA, RNA, Proteins, peptides, sugars, sugar chains or lipids are preferable, and DNA and RNA are particularly preferable. In the case of a nucleic acid, the base length is preferably 10 to 100 bases, and most preferably 20 to 100 bases, particularly 40 to 100 bases. Those in this base length range can be easily artificially synthesized with a synthesizer, and can be easily immobilized on the substrate surface because the amino group and mercapto group can be easily modified at the end of the nucleic acid. Therefore, hybridization can be performed stably.

  The solution containing a selective binding substance means a solution in which the above selective binding substance is dissolved in a solvent. The solvent is not particularly limited as long as the selective binding substance is soluble. Moreover, the density | concentration of the selective binding substance in a solution can be set suitably as needed, and the density | concentration of 0.1-100 micromol can be used preferably. The solution containing the selective binding substance can be stored in a 384 plate or the like that can be stored separately for each selective binding substance spotted at each position of the carrier.

Next, at a process (2), the above-mentioned pin is immersed in the solution containing an aprotic polar solvent. The solution containing the aprotic polar solvent is a solution for washing different from the solution containing the selective binding substance in the above step (1), and is a solution not containing the selective binding substance.
An aprotic polar solvent means a solvent composed of polar molecules that does not dissociate under normal conditions to generate protons. Specifically, acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, N-methyl-2-pyrrolidone, or γ-butyllactone is preferable, and acetonitrile, N, N-dimethyl are particularly preferable. Formamide and N-methyl-2-pyrrolidone are more preferable. The solution containing the aprotic polar solvent used in the step (2) may be a solution containing these aprotic polar solvents in another solvent. For example, an aqueous solution containing the aprotic polar solvent is preferably used. be able to.

In the step (2), conditions such as the immersion time can be appropriately set. In particular, it is preferable to swing the pin while the pin is immersed in a solvent because the cleaning efficiency can be further improved. The immersion method can be performed by providing a stationary tank containing a solution containing an aprotic polar solvent and sequentially immersing the pins, but in a fluidized state, that is, in a state where each solution is flowed at an appropriate speed. The contact with the pin is preferable because the cleaning efficiency can be further increased. Specifically, the immersion time can be in the range of 1 to 60 seconds, and the flow rate can be in the range of 0.02 to 10 L / min. Moreover, it can also be set as an ultrasonic cleaning system by emitting an ultrasonic wave directly or indirectly to a solvent.
Step (2) can be performed before and after step (1). Further, the selective binding immobilization carrier can be obtained by repeating steps (1) and (2) so that a desired spot can be realized.

In addition to the above steps (1) and (2), the production method of the present invention preferably further includes a step of immersing the pin in water or an aqueous solution not containing a selective binding substance (step (3)). Thereby, the cleaning efficiency can be further increased.
The water or aqueous solution not containing the selective binding substance is not particularly limited as long as it does not contain the selective binding substance.
The order of this step (3) can be between step (1) and step (2) and between step (2) and step (1). In particular, it is preferably performed after the step (2), particularly preferably every time after the step (2).

  In the manufacturing method of this invention, the drying process (4) for drying a pin can be added between each process. Examples of the drying method in the drying step (4) include vacuum drying, vacuum drying, hot air drying, and the like, but vacuum drying is preferable from the viewpoint of drying efficiency. By performing the drying step, it is possible to avoid bringing in the aprotic solvent in the step (2) and the water or aqueous solution in the step (3) the selective binding substance attached to the pin. The cleaning efficiency can be further increased.

  In the production method of the present invention, a cleaning step with a surfactant such as SDS and a cleaning step with an alcohol such as ethanol can be performed between the steps as necessary. Also in this case, the above-described drying step (4) can be inserted after washing.

  Conditions such as the immersion time in the step (3) can be appropriately set. The dipping method can be carried out by providing a stationary tank containing the solution used in each step and dipping the pins in order, but it is flow type, that is, the pins are contacted with each solution flowing at an appropriate speed. This is preferable because the cleaning efficiency can be further increased. Specifically, the immersion time can be in the range of 1 to 60 seconds, and the flow rate can be in the range of 0.02 to 10 L / min. Moreover, it can also be set as an ultrasonic cleaning system by emitting an ultrasonic wave directly or indirectly to a solvent.

  The production method of the present invention comprises a solution tank filled with a solution containing a selective binding substance corresponding to step (1), a washing tank filled with a solution containing an aprotic polar solvent corresponding to step (2), optional A spotter is attached with a cleaning tank filled with water or an aqueous solution corresponding to the step (3) performed in the above, and a device having a moving means capable of moving the pin between the tanks and adjusting the height between the upper and lower sides. Thus, when cleaning, the spotter can be temporarily stopped and the pins can be sequentially moved to the respective tanks. Furthermore, when performing immersion in a fluid solution, ultrasonic cleaning, etc. in each process, a flow means such as a pump, an ultrasonic transmitter, etc. can be attached to each tank, and a drying process is provided. The can also be provided with drying means.

  Such a selective binding substance-immobilized carrier produced by the production method of the present invention uses a substance that selectively interacts (for example, binds and hybridizes) to the selective binding substance as a test substance. By applying a sample (sample solution) that may contain a test substance, it can be used for analysis of the test substance in the sample.

  The invention is illustrated in more detail by the following examples. However, the present invention is not limited to the following examples.

Reference example 1
(Preparation of DNA immobilization carrier)
A mold for injection molding was produced by using a known LIGA (Lithographie Galvanoformung Abformung) process, and a carrier made of polymethyl methacrylate (PMMA) having a shape as described later was obtained by the injection molding method. The average molecular weight of PMMA used in this example is 50,000, carbon black (Mitsubishi Chemical # 3050B) is contained in PMMA at a ratio of 1% by weight, and the carrier is black. When the spectral reflectance and spectral transmittance of this black carrier were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength of 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Note that the spectral reflectance is the spectrum when specularly reflected light from a carrier is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light receiving optical system that conforms to condition C of JIS Z 8722. The reflectance was measured.

  The shape of the carrier was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central part of the carrier. In the center of the carrier, a recessed portion having a diameter of 10 mm and a depth of 0.2 mm is provided. In this recess, 64 (8 × 8) convex portions having a diameter of 0.2 mm and a height of 0.2 mm are provided. Provided. When the height difference between the height of the upper surface of the convex portion of the concavo-convex portion (average value of the height of the 64 convex portions) and the flat portion was measured, it was 3 μm or less. Further, the height variation of the 64 convex upper surfaces (the difference in height between the highest convex upper surface and the lowest convex upper surface), and further, the average height of the convex upper surface and the flat portion The difference in height of the upper surface was measured and found to be 3 μm or less. Furthermore, the pitch (the distance from the central portion of the convex portion to the central portion of the adjacent convex portion) of the concave and convex portion was 0.6 mm.

  The PMMA carrier was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed with pure water, 0.1N HCl aqueous solution, and pure water in this order to generate carboxyl groups on the surface of the carrier.

(Spotting on carrier)
A probe DNA of SEQ ID NO: 1 (60 bases, 5 ′ terminal amination) was synthesized. The DNA of SEQ ID NO: 1 is aminated at the 5 ′ end.

This DNA was dissolved in pure water at a concentration of 0.3 nmol / μl to obtain a stock solution. When spotting on a carrier, PBS (NaCl 8 g, Na ₂ HPO ₄ · 12H ₂ O 2.9 g, KCl 0.2 g, KH ₂ PO ₄ 0.2 g in pure water was made up to 1 l. To adjust the final concentration of probe DNA to 0.03 nmol / μl by adding hydrochloric acid for adjusting pH to pH 5.5) and condensing the carboxylic acid on the carrier surface with the amino group at the end of the probe DNA 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to obtain a probe solution having a final concentration of 50 mg / ml. In addition, samples obtained by removing the probe DNA from the probe solution (spot solutions 1 and 2) were separately prepared. Dispense the probe solution and spot solutions 1 and 2 onto a 384 plate and spot the spot solution 1, wash the pins, and spot the probe solution using a special spotter (GTMAS Stamp II: manufactured by Nippon Laser Electronics Co., Ltd.). Spotting was performed in the order of pin washing, spot solution 2 spotting. The pin used is a single-character groove type with a tip diameter of 0.15 mm. The pins were washed by dipping on the substrate, then immersing the pure water and the cleaning liquid A in the order of acetonitrile and pure water for 10 seconds each, followed by vacuum drying for 15 seconds.

(Incubation of substrate)
Next, the carrier was placed in a sealed plastic container, incubated at 37 ° C. and 100% humidity for about 20 hours, and washed with pure water. This reaction scheme is shown in FIG.

(Preparation of sample DNA)
As the sample DNA, a DNA having a base sequence represented by SEQ ID NO: 4 having a base sequence capable of hybridizing with the probe DNA immobilized on the DNA immobilization carrier (968 bases, hereinafter referred to as DNA of SEQ ID NO: 4) Used). The preparation method is shown below.

  DNA having the base sequence represented by SEQ ID NO: 2 (hereinafter sometimes referred to as DNA of SEQ ID NO: 2) and DNA having the base sequence represented by SEQ ID NO: 3 (hereinafter referred to as DNA of SEQ ID NO: 3) Was synthesized). This was dissolved in pure water to a concentration of 100 μM. Next, pKF3 plasmid DNA (Takara Bio Inc. product number; 3100) (DNA having the base sequence represented by SEQ ID NO: 5: 2264 bases, hereinafter sometimes referred to as DNA of SEQ ID NO: 5) is prepared. Then, amplification was performed by PCR reaction (Polymerase Chain Reaction) using this as a template and DNAs of SEQ ID NO: 2 and SEQ ID NO: 3 as primers.

  The conditions for PCR are as follows. That is, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (the above is attached to product number RR001A manufactured by Takara Bio Inc.), DNA solution of SEQ ID NO: 2 is 2 μl, DNA solution of SEQ ID NO: 3 is 2 μl, template (sequence 0.2 μl of DNA No. 5) was added, and the volume was made up to 400 μl with pure water. These mixed solutions were divided into four microtubes, and PCR reaction was performed using a thermal cycler. This was purified by ethanol precipitation and dissolved in 40 μl of pure water. When a part of the solution after the PCR reaction was taken and confirmed by electrophoresis, it was confirmed that the base length of the amplified DNA was about 960 bases and the DNA of SEQ ID NO: 4 was amplified.

  Subsequently, a 9-base random primer (manufactured by Takara Bio Inc .; product number 3802) was dissolved at a concentration of 6 mg / ml, and 2 μl was added to the DNA solution purified after the PCR reaction. This solution was heated to 100 ° C. and then rapidly cooled on ice. To this, 5 μl of the buffer attached to Klenow Fragment (Takara Bio Inc .; product number 2140AK) and 2.5 μl of dNTP mixture (dATP, dTTP and dGTP concentrations of 2.5 mM and dCTP concentration of 400 μM, respectively) were added. . Furthermore, 2 μl of Cy3-dCTP (manufactured by Amersham Pharmacia Biotech; product number PA53021) was added. To this solution, 10 U of Klenow Fragment was added and incubated at 37 ° C. for 20 hours to obtain sample DNA labeled with Cy3. Since the random primer is used for labeling, the length of the sample DNA varies. The longest sample DNA is the DNA of SEQ ID NO: 4. When the sample DNA solution was taken out and confirmed by electrophoresis, the strongest band appeared in the vicinity corresponding to 960 bases, and the region corresponding to a shorter base length was thinly smeared. This was purified by ethanol precipitation and dried.

  This labeled sample DNA is obtained by diluting 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC is 20 × SSC (manufactured by Sigma) 4 times with pure water. 20 × SSC diluted twice with pure water is expressed as 10 × SSC, 20 × SSC 2 × diluted solution is expressed as 10 × SSC, and 100 × diluted solution is expressed as 0.2 × SSC). A 0.1 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA solution (each concentration is a final concentration) and 400 μl were dissolved to obtain a stock solution for hybridization.

  In the following examples and comparative examples, the sample solutions used for hybridization were 1 wt% BSA, 5 × SSC, 0.01 wt% salmon sperm DNA, unless otherwise specified. What was diluted 200 times with a 0.1 wt% SDS solution (each concentration is a final concentration) was used. The sample DNA concentration in this solution was measured and found to be 1.5 ng / μL.

(Modification of glass beads)
After 10 g of glass beads having a diameter of 150 μm were immersed in a 10N NaOH solution, the glass beads were washed with pure water. Next, after APS (3-aminopropyltriethoxysilane; manufactured by Shin-Etsu Chemical Co., Ltd.) was dissolved in pure water at a ratio of 2% by weight, the glass beads were immersed for 1 hour and taken out from this solution. Dried at 110 ° C. for 10 minutes. In this way, amino groups were introduced on the surface of the glass beads.

  Subsequently, 5.5 g of succinic anhydride was dissolved in 335 ml of 1-methyl-2-pyrrolidone. 1M 50 ml sodium borate (3.09 g boric acid and sodium hydroxide for pH adjustment was added to make up to 50 ml with pure water. PH 8.0) was added to the succinic acid solution. The glass beads were immersed in this mixed solution for 20 minutes. After soaking, it was washed with pure water and dried. In this way, amino groups on the surface of the glass beads were reacted with succinic anhydride to introduce carboxyl groups on the surface of the glass beads.

(Adhesion of cover member)
A cover member b having four through-holes 5 shown in FIG. 6 is manufactured by cutting from a PMMA plate having a thickness of 1 mm, and this is covered with a probe DNA immobilization region using a double-sided tape (thickness 100 μm). And glued. At this time, 2 mg of the glass beads modified by the above procedure were put in the concave portion of the carrier, and then the cover member was adhered.

(Hybridization)
The sample DNA was hybridized to the carrier on which the probe DNA obtained above was immobilized. Specifically, 50 μl of hybridization solution is injected from the through hole using a micropipette. Then, the through hole was closed with Kapton tape (As One 5-5018-01), and this was fixed in a plastic container provided on the rotating surface of a microtube rotator (manufactured by AS One, product number: 1-4096-01). Incubated at 0 ° C. for 16 hours. At that time, the rotation speed of the rotator was 3 rpm, and the rotation surface of the rotator was set to be perpendicular to the horizontal plane. Furthermore, the probe DNA immobilization surface of the carrier was set to be perpendicular to the rotation surface of the rotator. By doing so, the glass beads continue to move in the direction of gravity, and hybridization can be performed while stirring the sample solution. After the incubation, the cover member and the double-sided tape were detached from the carrier, and the carrier was washed and dried.

(Measurement)
The carrier after the above treatment was set on a DNA chip scanner (Axon Instruments GenePix 4000B), and measurement was performed with the laser output 33% and the photomultiplier voltage set to 500. The results are shown in Table 1. Here, the fluorescence intensity is an average value of the fluorescence intensity in each spot. The convex portion spotted with the probe solution, the convex portion spotted with the spot solutions 1 and 2, and the background noise are immobilized probe DNA. It is the fluorescence intensity of the convex part which is not.

In this experiment, the carryover of the selective binding substance can be evaluated by comparing the fluorescence intensities of the spot solutions 1 and 2. That is, when the spot solution 2 has a higher fluorescence intensity than the spot solution 1, it is evaluated that there is a carry-over. As shown in Table 1, there was no carryover of the selective binding substance in Reference Example 1 in which acetonitrile was used for washing the pins.

Comparative Example 1
A similar experiment was performed using pure water instead of acetonitrile as the cleaning liquid A of Reference Example 1. That is, immersion in pure water was performed 3 times (10 seconds / 1 time). The results are shown in Table 1.
It is considered that the fluorescence intensity of the spot solution 2 is three times that of the spot solution 1 and the result is that the probe solution that was previously spotted was brought in. Therefore, carry-over of the selective binding substance was observed with pure water, and it was found that the cleaning effect was higher when acetonitrile of Reference Example 1 was used.

Examples 2-6
Instead of using acetonitrile washing solution A of Example 1, N, N- dimethylformamide, N- methyl-2-pyrrolidone, dimethyl sulfoxide, using hexamethylphosphoric triamide, .gamma.-butyrolactone, respectively, Example 1 The same experiment was conducted. The results are shown in Table 1.

  Both the N, N-dimethylformamide and N-methyl-2-pyrrolidone solvents show the same fluorescence intensity in the spot solutions 1 and 2, and there is no carryover of the selective binding substance, with the same cleaning effect as acetonitrile. I found out. It was also found that any solvent of dimethyl sulfoxide, hexamethylphosphoric triamide, and γ-butyl lactone has a higher cleaning effect than the pure water of Comparative Example 1.

Reference Example 7
In the pin cleaning method of the experiment of Reference Example 1, an experiment was conducted in which the immersion time of the pin was 5 seconds each. The results are shown in Table 1.
It was found that although the immersion time was shorter than the case of washing with pure water of Comparative Example 1, the washing effect was sufficient.

Reference Example 8
The step of attaching the pump to the spotter used in Reference Example 7 and washing the pins in the order of pure water, acetonitrile, and pure water was carried out by using the pump to flow each solution (1 L / min), and the immersion time was 5 for each. The second experiment was conducted. The results are shown in Table 1.
Even when the immersion time was 5 seconds, the spot solutions 1 and 2 showed the same fluorescence intensity, and it was found that this method enables sufficient pin cleaning equivalent to that of Reference Example 1. That is, it was found that the pins can be efficiently cleaned in a short time by immersing the pins in the fluid type solution.

FIG. 1 is a diagram illustrating an example of a flat type spotter pin tip. FIG. 2 is a diagram illustrating an example of a one-character groove type spotter pin tip. FIG. 3 is a diagram illustrating an example of a tip portion of a cross groove type spotter pin. FIG. 4 is a perspective view schematically showing an example of a carrier provided with uneven portions. FIG. 5 is a schematic diagram showing the reaction scheme of the examples. 6 is a perspective view schematically showing an example of the combination of the carrier and the cover member shown in FIG.

Explanation of symbols

X Pin tip end face Y Pin tip side face 1 Flat part 2 Convex part 2A Convex part top face 3 Concave part 4 Concave part 5 Through hole a Selective binding substance immobilization carrier b Cover member

Claims (6)

In producing a selective binding substance-immobilized carrier using a spotter,
A step of immersing a spotter pin in a solution containing a selective binding substance, then bringing the pin into contact with the carrier and spotting the selective binding substance on the carrier, and the pin being aprotic polar solvent (provided that acetonitrile A method for producing a selective binding substance- immobilized carrier, comprising a step of immersing in a solution containing   The manufacturing method according to claim 1, further comprising a step of immersing the pin in water or an aqueous solution not containing a selective binding substance.   The manufacturing method according to claim 1, wherein a fluid type solution is used for the immersion of the pin.   The method according to any one of claims 1 to 3, wherein the selective binding substance is DNA, RNA, protein, peptide, sugar, sugar chain, or lipid. Wherein the aprotic polar solvent is, N, N- dimethylformamide, dimethyl sulfoxide, hexamethylphosphoric triamide, characterized in that it is a N- methyl-2-pyrrolidone, or γ- butyrolactone, claims 1 to 4 The manufacturing method as described in any one of these.   The said support | carrier is a support | carrier with an uneven | corrugated | grooved part on the surface, Comprising: The selective binding substance is spotted on the convex-part upper surface of an uneven | corrugated | grooved part, The manufacture as described in any one of Claims 1-5 Method.

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