JP6931181B2 - Bio-related molecule immobilization carrier - Google Patents

Description

The present invention relates to a carrier for immobilizing a bio-related molecule and a method for producing the same.

With the demand for safety and soundness of the environment and food, technological development for controlling microbial contamination in environmental samples, food raw materials or products is in progress. As a means for this, a method of detecting a bio-related molecule such as a nucleic acid derived from a microorganism is advantageous in terms of detection sensitivity and specificity, and the bio-related molecule is placed on a surface-treated substrate such as a microarray or a DNA chip. Various immobilized carriers have been developed. With these carriers, a large number of solutions containing different biorelated molecules are spotted individually on the substrate in small spots by using a precision spotting device.

Examples of the carrier for immobilizing the bio-related molecules as described above include a carrier having a polyvalent amine layer on a substrate made of various materials and further having an active ester group on the substrate. Many types and methods of surface treatment are known (Patent Documents 1 and 2). However, the spotting and detection performance of bio-related molecules may be significantly affected by the specific types and application conditions of various substrates and surface treatments, and therefore further technological development is required.

Japanese Unexamined Patent Publication No. 2003-161731 Japanese Patent No. 4764901

As described above, many types and methods of surface treatment of the substrate for immobilizing bio-related molecules are known, and aminoalkylsilane is widely known as one of the representative amino group-containing compounds. However, in a substrate that has been subjected to aminoalkylsilane treatment and then adsorbed with a polyvalent carboxylic acid such as polyacrylic acid, the spot diameter becomes large or the spot diameter becomes large when a bio-related molecule-containing solution is spotted on the surface thereof. There was a problem that the shape became defective. In particular, in general, in order to perform a sufficient surface treatment, it is common to increase the efficiency of forming an aminoalkylsilane layer by performing a hydrophilic treatment on the substrate, and the carrier thus prepared is a spot of a nucleic acid solution. Since the shape becomes poor, there is a need to develop a technique capable of keeping the spot diameter small so that the nucleic acid solution does not spread.

In the case of producing a carrier in which an aminoalkylsilane layer and a polyvalent carboxylic acid layer are sequentially laminated on a resin substrate, the inventors of the present application use a water-repellent resin substrate and surface-treat the substrate to prepare an aminoalkylsilane. We have found that the above problems can be solved by controlling the amount of aminoalkylsilane introduced so that the resin substrate is exposed when the layer is formed, and have completed the present invention.

That is, the present invention is for immobilization of a bio-related molecule having a water-repellent resin substrate, an aminoalkylsilane layer formed on the resin substrate, and a polyvalent carboxylic acid layer formed on the aminoalkylsilane layer. A carrier for immobilizing bio-related molecules, wherein the carboxyl group of the polyvalent carboxylic acid layer is activated esterified and the resin substrate is exposed after the formation of the aminoalkylsilane layer. I will provide a.

Further, the present invention relates to a step of forming an aminoalkylsilane layer on a water-repellent resin substrate, a step of forming a polyvalent carboxylic acid layer on the aminoalkylsilane layer, and a step of forming the polyvalent carboxylic acid layer. A method for producing a bio-related molecule-immobilizing carrier, which comprises a step of active esterifying a carboxyl group, wherein the step of forming the aminoalkylsilane layer is a condition in which the resin substrate is exposed after the formation of the aminoalkylsilane layer. Provides a manufacturing method performed in.

In the present invention, as described above, a water-repellent resin substrate is used, and the amount of aminoalkylsilane introduced is controlled so that the resin substrate is exposed when the substrate is surface-treated to form an aminoalkylsilane layer. Thereby, a carrier for immobilizing a bio-related molecule having a good spot shape can be obtained.

For Experimental Examples 1, 10 and 16, the C1s spectrum chart obtained by XPS analysis of the substrate surface after adsorption of polyacrylic acid is shown. The actual spot observation images of Experimental Examples 1, 10 and 16 are shown. The actual spot observation images of Experimental Examples 11 to 15 are shown.

The present invention is a bio-related molecule-immobilizing carrier having a water-repellent resin substrate, an aminoalkylsilane layer formed on the resin substrate, and a polyvalent carboxylic acid layer formed on the aminoalkylsilane layer. Provided is a carrier for immobilizing a bio-related molecule, which is characterized in that the carboxyl group of the polyvalent carboxylic acid layer is activated esterified and the resin substrate is exposed after the formation of the aminoalkylsilane layer. do.

A water-repellent resin substrate is used as the substrate of the carrier of the present invention. In the present invention, the water-repellent resin means a resin that has not been hydrophilized, or a resin that has been hydrophilized but has a low degree of hydrophilicity. Any kind of resin that has not been hydrophilized can be used in the present invention. As the water-repellent resin of the present invention, it is preferable to use a resin that has not been hydrophilized, but the degree of hydrophilization even in a resin substrate that has been hydrophilized such as corona treatment or vacuum UV treatment. It can also be used in the present invention as long as it has a low value. The water-repellent resin of the present invention has a surface water contact angle of, for example, 66 ° or more, preferably 70 ° or more, more preferably 75 ° or more, and particularly preferably 80 ° or more, regardless of the presence or absence of the hydrophilization treatment.

In order to efficiently form an aminoalkylsilane layer on a resin substrate, it is common to perform a hydrophilic treatment on the resin substrate, but in the present invention, the above-mentioned hydrophilic treatment is not performed or the above-mentioned hydrophilic treatment is not performed. By using a resin substrate having such a low water repellency and using the resin substrate as an exposed carrier even after the formation of the aminoalkylsilane layer as described later, a carrier for immobilizing bio-related molecules having a good spot shape can be obtained. be able to.

The type of resin is not particularly limited as long as the above conditions are satisfied, but since detection of bio-related molecules such as nucleic acids is often performed based on a fluorescent substance bound to the bio-related molecules, a material having as low autofluorescence as possible. Is preferably used. Specific types of resins include, for example, polyethylene, polypropylene, cyclic polyolefin, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluororesin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, and the like. Organic materials such as acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, polyphenylene oxide and polysulfone. And the like, and these two or more kinds of mixed resins may be used. Further, other substances that can improve the performance according to a desired purpose such as improvement of the detection sensitivity may be added as appropriate, and a black pigment such as carbon black may be mixed.

In the present invention, it is preferable to use polycarbonate as a material for the resin substrate, and more preferably polycarbonate containing carbon black as a black pigment is used. The amount of carbon black can be appropriately determined by those skilled in the art, and is, for example, 0.1% by weight to 2% by weight, preferably 0.2% by weight to 1% by weight, and more preferably 0.3% by weight to%. A material containing 0.8% by weight of carbon black in the resin is used.

In the carrier for immobilizing bio-related molecules of the present invention, an aminoalkylsilane layer is formed on the above resin substrate. As the aminoalkylsilane, for example, an alkyl group having 1 to 10 carbon atoms, preferably 2 to 5 carbon atoms is used, and specifically, the alkyl group is a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group or the like. obtain. Of the above, the propyl group is particularly preferable in the present invention. Further, the silane of the aminoalkylsilane may be substituted with one or more substituents, for example, an alkoxy group having 1 to 5 carbon atoms, preferably 2 to 4 groups (methoxy group, ethoxy group, propoxy group, butoxy group). Etc.) can be used, and it is particularly preferable that the ethoxy group is trisubstituted. Specific examples of the aminoalkylsilane include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3-aminopropyldimethoxyethylsilane, and the like, which are used in the present invention. 3-Aminopropyltriethoxysilane is particularly preferred.

The aminoalkylsilane layer in the present invention is merely expressed as a "layer" for convenience based on the relative positional relationship of being formed on the resin substrate, and the aminoalkylsilane layer is at least a part of the substrate. It suffices if it is formed on the surface, and does not mean that it completely covers the entire surface of the substrate. That is, the carrier of the present invention is characterized in that the resin substrate is exposed after the formation of the aminoalkylsilane layer. In the present invention, since the water-repellent resin substrate is exposed after the formation of the aminoalkylsilane layer as described above, a carrier for immobilizing bio-related molecules having a good spot shape can be finally obtained.

As described above, the fact that the resin substrate is exposed in the present invention means that the resin under the aminoalkylsilane layer is exposed on a part of the carrier surface after the formation of the aminoalkylsilane layer. As one of the methods for detecting the exposure of the resin, there is a method of detecting the presence of a substance component on the surface by performing surface analysis of the substrate after the formation of the aminoalkylsilane layer. That is, as one aspect of the present invention, when a peak derived from the resin is detected in the surface analysis, it can be determined that the resin is exposed.

The above-mentioned surface analysis method can be appropriately selected by those skilled in the art, but a method having a detection depth in a small range of about 1 to 10 nm is preferable, for example, X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy. Examples include the method (AES) and the time-of-flight secondary ion mass spectrometry (TOF-SIMS). Of the above, X-ray photoelectron spectroscopy (XPS) is preferred in the present invention. For example, when the surface is measured by X-ray photoelectron spectroscopy after forming an aminoalkylsilane layer using a polycarbonate resin substrate, it appears in the C1s spectrum in the range of 291.5 to 293.5 eV, for example, around 292.5 eV. When the π-π Shake-up peak is confirmed, it can be determined that the polycarbonate is exposed on the surface of the substrate after the formation of the aminoalkylsilane layer. If the presence of a polycarbonate-derived peak is confirmed in the C1s spectrum even slightly, it can be determined that the peak is present.

Further, in the present invention, the aminoalkylsilane is based on the fact that the abundance ratio of silicon obtained by measuring the substrate surface after the formation of the aminoalkylsilane layer by X-ray photoelectron spectroscopy is controlled within a predetermined range. It can also be determined that the resin substrate is exposed without the layer completely covering the resin substrate. In the present invention, if the abundance ratio of silicon is controlled in the range of at least 0.5 to 5.0 atomic%, it can be determined that the resin substrate is exposed. The abundance ratio of silicon is more preferably 0.8 to 3.0 atomic%, still more preferably 1.0 to 2.0 atomic%. In general, the higher the hydrophilicity (wetting property) of the substrate surface, the easier it is for the aminoalkylsilane layer to be formed, and the higher the abundance ratio of silicon. Therefore, particularly when a water-repellent resin substrate (for example, a water contact angle of 66 ° or more) is used, the amount of aminoalkylsilane introduced is suppressed as compared with the hydrophilic resin substrate, and the above-mentioned silicon abundance ratio can be easily obtained. As a result, a carrier with an exposed resin substrate can be obtained.

The method for forming the aminoalkylsilane layer is not particularly limited, but for example, the aminoalkylsilane layer can be formed by immersing the substrate in a solution in which aminoalkylsilane is dissolved in various solvents. Water or various organic solvents such as alcohols such as methanol and ethanol can be used as the type of solvent, but in order to reduce the surface roughness of the substrate and suppress autofluorescence, water is used as the solvent. , It is preferable to immerse the substrate in an aqueous solution in which aminoalkylsilane is sufficiently hydrolyzed.

The concentration and immersion time of the aminoalkylsilane solution may be appropriately set by those skilled in the art so as to obtain the specific type of compound used and the peak or silicon abundance ratio of the surface analysis prescribed in the present application as described above. It is possible, for example, to use a solution of 0.1% to 10% by weight, preferably 1% to 8% by weight, with a soaking time of 15 to 180 minutes, preferably 30 to 150 minutes. Can be done.

In the nucleic acid immobilization carrier of the present invention, a polyvalent carboxylic acid layer is further formed on the aminoalkylsilane layer described above. By forming the polyvalent carboxylic acid layer in this way, a carboxyl group is introduced on the surface side of the carrier. The type of polyvalent carboxylic acid used in the present invention is not particularly limited, but is a homopolymer of a monomer having a carboxyl group such as polyacrylic acid, polymethacrylic acid, polymaleic acid, polyitaconic acid, and acrylic acid-methacrylic acid copolymer. Alternatively, a copolymer can be used. By amide-bonding the carboxyl group of the polyvalent carboxylic acid with the amino group of the aminoalkylsilane layer, the polyvalent carboxylic acid layer can be firmly bonded on the aminoalkylsilane layer. The polyvalent carboxylic acid layer may be formed on the surface of at least a part of the substrate and / or the aminoalkylsilane layer under it, and covers the entire surface of the substrate and / or the aminoalkylsilane layer. No need. As described above, since the polyvalent carboxylic acid layer is formed on the aminoalkylsilane layer by the amide bond between the carboxyl group and the amino group, the resin substrate exposed after the formation of the aminoalkylsilane layer is a polyvalent carboxylic acid. It is assumed that it is still exposed even after the formation of the acid layer.

The method for forming the polyvalent carboxylic acid layer is not particularly limited, and examples thereof include a method of immersing the substrate on which the aminoalkylsilane layer is formed in a solution of the polyvalent carboxylic acid. The solvent used for the solution of the polyvalent carboxylic acid can be appropriately selected by those skilled in the art, and water and various organic solvents such as alcohols such as methanol and ethanol can be used, but in the present invention, the aqueous solution is used. It is preferable to use.

The molecular weight, concentration and immersion time of the polyvalent carboxylic acid layer solution can be appropriately set by those skilled in the art, and for example, the molecular weight is 25,000 to 1,000,000, preferably 50,000 to 500,000, particularly preferably. 100,000 to 200,000 is selected, and the concentration is selected from 0.1% by weight to 10% by weight, preferably 0.5% by weight to 10% by weight, and particularly preferably 1.0% by weight to 5.0% by weight. The immersion time is selected, for example, 1 minute to 60 minutes, preferably 5 minutes to 30 minutes, and particularly preferably 10 minutes to 20 minutes.

In the nucleic acid immobilization carrier of the present invention, the carboxyl group of the polyvalent carboxylic acid layer formed as described above is activated esterified. By forming the active ester group by activating the carboxyl group, it becomes possible to stably immobilize the nucleic acid when the nucleic acid solution is finally spotted as a carrier for immobilizing the nucleic acid. The type of active ester group and the means for forming the active ester group can be appropriately selected by those skilled in the art as appropriate for use as a carrier for immobilizing nucleic acid, and are not particularly limited, but for example, a nitrophenyl group and an N-hydroxysuccinimide group. , N-Hydroxynorbornene-2,3-dicarboxyimide group, succinimide group, phthalateimide group and the like, and N-hydroxysuccinimide group is preferable in the present invention. As a method for forming the active ester group, for example, a dehydration condensing agent such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and various electrophilic groups corresponding to the active ester group as described above are introduced. The carboxyl group of the polyvalent carboxylic acid layer can be activated esterified by immersing the agent (such as N-hydroxysuccinimide) in a solution dissolved in a buffer solution.

The carrier for immobilizing bio-related molecules of the present invention obtained as described above can immobilize bio-related molecules on the surface thereof. The biorelated molecule in the present invention is preferably a nucleic acid. A carrier on which nucleic acid is immobilized can be obtained by instilling a nucleic acid-containing solution onto a carrier for immobilizing a bio-related molecule and washing the unreacted nucleic acid solution that has not been bound on the carrier. Methods for spotting the nucleic acid-containing solution on the carrier include a method in which a pin holding the nucleic acid-containing solution is brought into contact with the carrier and spotted, or a method in which the nucleic acid-containing solution is sprayed onto the carrier by inkjet, but the method is particularly limited. However, it can be carried out by using any device and method known to those skilled in the art.

The nucleic acid-immobilized carrier prepared as described above can be used for detecting the presence of the target nucleic acid in the test sample. For example, in the case of using DNA as a nucleic acid, DNA is extracted from a test sample, amplified, and hybridized with a nucleic acid on a nucleic acid-immobilized carrier (such as a DNA chip or microarray) for detection. , It is possible to confirm the presence or absence of specific microbial contamination in the test sample. Examples of the method for extracting DNA include a phenol extraction method, a phenol / chloroform extraction method, an alkali dissolution method, and a boiling method. Further, a method of extracting DNA using a commercially available DNA extraction reagent or an automatic nucleic acid extraction device can be mentioned.

The target region of the extracted DNA is amplified by a nucleic acid amplification method, if necessary. The target region is a region of chromosomal DNA that can be amplified by a nucleic acid amplification method, and is not particularly limited as long as it can detect a microorganism to be detected, and can be appropriately set according to the purpose. For example, when the test sample contains cells of a type different from that of the microorganism to be detected, the target region preferably has a sequence specific to the microorganism to be detected. Further, depending on the purpose, it may have a sequence common to a plurality of types of microorganisms. Nucleic acid amplification methods include PCR method (polymerase chain reaction), SDA method (strand displacement amplification), LCR method (ligase chain reaction), LAMP method (loop-mediated isothermal amplification), and ICAN method (isothermal and chimeric primer-initiated amplification). of nucleic acids) and the like, and among them, it is preferable to use the PCR method. For example, the length of the target region amplified by the PCR method is usually 80 to 1000 bases, preferably 100 to 500 bases.

The amplified DNA is detected using the nucleic acid-immobilized carrier of the present invention. Nucleic acid (probe) immobilized on a carrier is a target living body when various bio-related molecules such as specific genes and proteins are mixed and it is difficult to distinguish them from others and directly select them. It is a detector that makes it detectable by binding only to related molecules. For example, when detecting a nucleic acid of a specific microorganism as a bio-related molecule, a DNA fragment having a sequence complementary to the base sequence of the nucleic acid of this microorganism is used as a probe, and hybridization with the nucleic acid is performed. .. DNA of 1 to 200 bases, preferably 10 to 150 bases is usually immobilized on the probe. Both single-stranded and double-stranded DNA can be immobilized. Further, by labeling the target bio-related molecule with a fluorescent substance or the like in advance, the bio-related molecule bound to the probe can be detected. The solution used for the binding reaction between the bio-related molecule and the probe contains, for example, a buffer solution obtained by adding SDS (sodium dodecyl sulfate) to citric acid-physiological saline in addition to the bio-related molecule.
Hereinafter, aspects of the present application will be described in more detail based on examples.

[Experimental Example 1]
A polycarbonate substrate containing carbon black (hereinafter, black PC substrate manufactured by Shiraishi Kogyo Co., Ltd.) was prepared, and the water contact angle was measured using a contact angle meter (DropMaster700 manufactured by Kyowa Interface Co., Ltd.). Next, the black PC substrate was immersed in a 5 wt% 3-aminopropyltriethoxysilane aqueous solution for 2 hours, taken out, washed with pure water, and dried at 70 ° C. for 2 hours. This substrate was measured with an X-ray photoelectron spectroscopy analyzer (K-Alpha XPS system manufactured by Thermo Fisher Scientific Co., Ltd.), and the abundance ratio of Si was confirmed. Next, the portion for which the ESCA measurement was not performed was immersed in a 1% aqueous solution of polyacrylic acid for 10 minutes, taken out, washed with pure water, and dried at 80 ° C. for 2 hours. This substrate was measured with an X-ray photoelectron spectroscopy analyzer to obtain a C1s spectrum. The chart after peak separation is shown in FIG.
Then, for the portion where the ESCA measurement was not performed, 200 ml of 0.1 M phosphate buffer (pH 6.8) was mixed with 0.1 M 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride. It was activated by immersing it in an activating solution in which 0.1 M of N-hydroxysuccinimide was dissolved for 10 minutes. A 30% PEG300 solution containing oligo DNA (10 μM) was spotted on the obtained solid support, heated at 80 ° C. for 1 hour, and then the spot shape was photographed with a stereomicroscope (Leica EZ4D, Leica Microsystems, Inc.). Further, the diameter of each spot was measured on the image processing software based on the spot image taken by the stereomicroscope built in the digital camera, and the spot diameter was calculated by averaging the diameters in the X-axis direction and the Y-axis direction.

[Experimental Example 2]
A substrate was prepared in the same manner as in Experimental Example 1 except that the immersion time in the 3-aminopropyltriethoxysilane aqueous solution was changed to 30 minutes.

[Experimental Example 3]
A substrate was prepared in the same manner as in Experimental Example 1 except that the concentration of the 3-aminopropyltriethoxysilane aqueous solution was changed to 1% and the immersion time was changed to 30 minutes.

[Experimental Example 4]
A substrate was prepared in the same manner as in Experimental Example 1 except that the concentration of the 3-aminopropyltriethoxysilane aqueous solution was changed to 0.1% and the immersion time was changed to 30 minutes.

[Experimental Example 5]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with a carbon black-free PC.

[Experimental Example 6]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with polybutylene terephthalate (PBT).

[Experimental Example 7]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with polymethyl methacrylate (PMMA).

[Experimental Example 8]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with polypropylene (PP).

[Experimental Example 9]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with polymethylpentene (PMP).

[Experimental Example 10]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with a black PC in which a hydrophilic group was introduced on the surface by corona irradiation.

[Experimental Example 11]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with a carbon black-free PC in which a hydrophilic group was introduced on the surface by corona irradiation.

[Experimental Example 12]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with PBT having a hydrophilic group introduced on the surface by corona irradiation.

[Experimental Example 13]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with PMMA in which a hydrophilic group was introduced on the surface by corona irradiation.

[Experimental Example 14]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with PP in which a hydrophilic group was introduced on the surface by corona irradiation.

[Experimental Example 15]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with PMP in which a hydrophilic group was introduced on the surface by corona irradiation.

[Experimental Example 16]
A substrate was prepared in the same manner as in Experimental Example 1 except that the black PC was replaced with a black PC in which a hydrophilic group was introduced on the surface by vacuum UV irradiation.

Regarding Experimental Examples 1 to 13, the resin substrate used, the hydrophilization treatment performed, the water contact angle before the surface treatment, the abundance ratio of Si after the formation of the aminoalkylsilane layer, the presence or absence of resin peak detection by X-ray photoelectron spectroscopy, Table 1 shows the spot shape and spot diameter.

＊「−」：測定せず。
Table 1

* "-": Not measured.

As shown in Experimental Examples 1, 10 and 16 in Table 1, the contact angle was decreased by the hydrophilic treatment, and the amount of the aminoalkylsilane layer introduced (the abundance ratio of Si) was increased accordingly. In Experimental Example 1, good spots were obtained, whereas in Experimental Example 10, spots were bleeding, and in Experimental Example 16, spot morphology was observed in which the treated film was damaged.
Further, as shown in the C1s spectrum chart of FIG. 1, in the X-ray photoelectron spectroscopy analysis after the formation of the aminoalkylsilane layer, in Experimental Example 1 and Experimental Example 10, π-π derived from PC appearing in the vicinity of the binding energy of 292.5 eV. The Shake-up peak was confirmed. From this, it is inferred that in the substrate prepared under these experimental conditions, the aminoalkylsilane layer is thinly formed and the PC surface is exposed. On the other hand, in Experimental Example 16, the π-π Shake-up peak could not be confirmed. Therefore, in Experimental Example 16, it is presumed that the surface of the substrate is covered with a thick aminoalkylsilane layer and the PC surface is not exposed.

From these facts, it can be seen that in Experimental Example 1, the aminoalkylsilane layer is thinly formed so that the water-repellent substrate is exposed, and in this state, a good spot shape can be obtained. Further, in Experimental Example 10, the hydrophilic substrate is exposed, and it can be seen that in such a state, although good spots can be obtained, some bleeding occurs. Further, in Experimental Example 16, the substrate is covered with an aminoalkylsilane layer and is not exposed, and it can be seen that the spot shape is poor in such a state.
From the above, it can be seen that the spot shape can be stabilized by controlling the exposed state of the substrate after the formation of the aminoalkylsilane layer and the wettability of the exposed resin substrate surface.

Further, as shown in Experimental Examples 2 to 4 in Table 1, by changing the concentration and the immersion time of the 3-aminopropyltriethoxysilane aqueous solution, a substrate in which the aminoalkylsilane layer is formed thinner and the resin is exposed is prepared. However, it can be seen that the spot shape is good in each case, and the spot shape can be stabilized as in Experimental Example 1 even in the case of a substrate having a Si presence ratio of about 0.64 atomic%.

Further, as shown in Experimental Examples 5 to 9 and 11 to 15 in Table 1, the water contact angle of the sample not subjected to the hydrophilization treatment was 72.8 to 106.6 °, and the spot shape was good. On the other hand, in the sample subjected to the hydrophilization treatment, the water contact angle decreased to 46.6 to 72.8 °, and the Si abundance ratio was 1.42 to 3.11 atomic%. If the Si abundance ratio is within this range, it is presumed that a thin aminoalkylsilane layer is formed and the surface of the resin substrate is exposed. The spot shape is good when a substrate having a water contact angle of 66.1 ° or more is used (Experimental Examples 14 and 15), and when a substrate having a water contact angle lower than that is used (Experimental Examples 11 to 13). ) Has bleeding.
From the above, the wettability of the exposed resin substrate surface greatly affects the spot shape, and in the substrate where the aminoalkylsilane layer is thinly formed and the resin surface is presumed to be exposed, the exposed resin surface It can be seen that when the water contact angle is 66.1 ° or more, a good spot can be obtained without bleeding.

Claims (7)

A bio-related molecule-immobilizing carrier having a water-repellent resin substrate, an aminoalkylsilane layer formed on the resin substrate, and a polyvalent carboxylic acid layer formed on the aminoalkylsilane layer. A carrier for immobilizing bio-related molecules, wherein the carboxyl group of the polyvalent carboxylic acid layer is activated esterified and the resin substrate is exposed after the formation of the aminoalkylsilane layer. The carrier for immobilizing a bio-related molecule according to claim 1, wherein the water contact angle on the surface of the resin substrate is 66 ° or more. The carrier for immobilizing a bio-related molecule according to claim 1 or 2, wherein a peak derived from the resin is detected in the surface analysis of the substrate after the formation of the aminoalkylsilane layer. The carrier for bio-related molecule immobilization according to any one of claims 1 to 3, wherein the abundance ratio of silicon on the substrate surface after the formation of the aminoalkylsilane layer is 0.5 to 5.0 atomic%. Any one of claims 1 to 4, wherein the resin substrate is polycarbonate, and a π-π Shake-up peak is detected in the C1s spectrum of the X-ray photoelectron spectroscopic analysis of the substrate surface after the formation of the aminoalkylsilane layer. The bio-related molecule-immobilized carrier according to. The carrier for immobilizing a bio-related molecule according to any one of claims 1 to 5, wherein the resin substrate is polycarbonate containing a black pigment. A step of forming an aminoalkylsilane layer on a water-repellent resin substrate, a step of forming a polyvalent carboxylic acid layer on the aminoalkylsilane layer, and an active esterification of the carboxyl group of the polyvalent carboxylic acid layer. A method for producing a bio-related molecule-immobilizing carrier, which comprises a step of forming the aminoalkylsilane layer, which is performed under conditions where the resin substrate is exposed after the formation of the aminoalkylsilane layer. ..

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