JP2009162658A - Surface modification method of liquid contact part of channel tip made of polymer resin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a surface modification method of a liquid contact part of a channel chip made of polymer resin, which can provide long lasting improved hydrophilicity to a liquid contact part. <P>SOLUTION: The surface modification method of a liquid contact part of a channel chip made of polymer resin comprises: introducing an application solution containing a silane coupling agent, and polyethylene glycol and/or polyvinyl alcohol into the inside of a channel 12 of a polymer resin channel chip 1; forming an applied coating on a liquid contact part 12a; and drying the applied coating under conditions of a humidity of 70% and a temperature of 100°C or lower. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for modifying the surface of a polymer resin flow channel tip wetted portion.

  For example, in the analysis of biomolecules such as nucleic acids and proteins, for example, a flow path chip made of a polymer resin having a pump, a valve, a sensor, a flow path, etc., such as μ-TAS (Micro Total Analysis System) was used. It is widely performed to analyze various types of samples by performing analysis. Such an analysis method has an advantage that a large amount of analysis can be performed at a low cost and in a short time. Further, by automating the work necessary for the analysis, it is possible to easily perform the analysis without requiring troublesome work and skilled techniques. Therefore, it is suitably used particularly in the fields of medical care and environmental measurement.

  In such an analysis, when the sample solution passes through the flow path of the polymer resin flow path chip, the nucleic acid and protein in the sample solution, the fluorescent organic compound used as the labeling compound, etc. are in contact with the flow path. When adsorbed on the inner wall (inner wall surface), the quantitativeness and accuracy of the analysis are lowered and the reliability is lowered. In particular, in the case of μ-TAS that requires a calibration curve, the effect becomes large. As the material of the polymer resin flow channel chip, generally, a hydrophobic polymer resin such as cyclic olefin, polymethyl methacrylate, polyimide, silicon rubber or the like is used. Therefore, it is required to suppress the adsorption of samples such as nucleic acids, proteins, fluorescent organic compounds, etc. on the liquid contact part of the polymer resin flow channel chip.

As a method for suppressing the adsorption of biomolecules such as nucleic acids to the wetted part of the polymer resin flow channel chip, the hydrophilicity of the wetted part is improved, and the wetted part and the living body such as nucleic acid in the sample solution There are known methods for weakening the interaction with molecules.
Specifically, for example, there is a method in which serum albumin, which is known to have a weak interaction with biomolecules, and a phospholipid component constituting a biological membrane are held in the wetted part by adsorption (Patent Document 1).
JP 2006-292472 A

However, in the method of Patent Document 1, depending on the type of polymer resin material, serum albumin and phospholipid components are difficult to adsorb on the wetted part, and the retention efficiency is poor. Therefore, the effect of suppressing the adsorption of biomolecules in the sample is effective. It may not be obtained sufficiently. There is also a problem that serum albumin and phospholipid components adsorbed on the wetted part are desorbed into the sample solution.
Therefore, there is a demand for a surface modification method that can improve the hydrophilicity of the liquid contact portion of the polymer resin flow channel chip and has a high hydrophilic sustainability.

  Accordingly, the present invention is directed to a method for modifying the surface of a polymer resin flow channel tip wetted part, which can improve the hydrophilicity of the wetted part and has a high hydrophilic sustainability.

  The method for modifying the surface of a polymer resin channel chip wetted part according to the present invention includes a coating solution containing polyethylene glycol and / or polyvinyl alcohol and a silane coupling agent in the channel of the polymer resin channel chip. And forming a coating film on the liquid contact portion of the flow path, and drying the coating film under conditions of a humidity of 70% or more and 100 ° C. or less. Thereby, the modified film containing the silane coupling agent and the hydrophilic polymer can be formed in the liquid contact portion of the flow path.

In the method for modifying the surface of the polymer resin flow channel tip wetted part of the present invention, the drying is preferably performed by heat treatment at 50 to 100 ° C. As a result, the solvent in the coating film evaporates quickly, and the modified film is formed in a short time.
Moreover, it is preferable that content of the said hydrophilic polymer in the said coating solution (100 mass%) is 1-20 mass%. By setting the content of the hydrophilic polymer within the above range, the hydrophilicity of the surface of the molded body is improved at a low cost.
The content of the silane coupling agent in the coating solution (100% by mass) is preferably 5 to 30% by mass. By setting the content of the silane coupling agent within the above range, it becomes easy to fix the hydrophilic polymer without increasing the film thickness of the modified film.

  According to the method of the present invention, the hydrophilicity of the liquid contact portion of the polymer resin channel chip can be improved, and the imparted hydrophilicity lasts for a long time.

In the method for modifying the surface of a polymer resin flow channel chip wetted part according to the present invention, a coating solution is introduced into the flow path of the polymer resin flow channel chip, and a coating film is formed on the liquid wetted part. It is a method including the process of drying.
As an embodiment of the surface modification method of the present invention, a method for improving the hydrophilicity of the wetted part of the polymer resin flow channel chip 1 (hereinafter referred to as the flow channel chip 1) illustrated in FIGS. explain.

[Polymer resin channel chip]
As shown in FIG. 3, the flow path chip 1 is formed by joining a long upper substrate 2 and a lower substrate 3 having the same shape as the upper substrate 2. Further, the upper substrate 2 is formed with recesses 2 a (three) along the longitudinal direction of the upper substrate 2, and the upper substrate 2 and the lower substrate 3 are joined together, whereby the flow path 12. Is formed.
Further, as shown in FIGS. 1 and 2, the flow channel chip 1 has an opening 11 for introducing the sample solution 20 and an opening 13 for discharging the sample solution 20. Therefore, the sample solution 20 introduced from the opening 11 passes through the flow path 12 and is discharged from the opening 13, so that various samples can be continuously obtained by arranging a sensor or the like in the middle of the flow path 12. Can be measured.

  Examples of the material of the upper substrate 2 and the lower substrate 3 of the flow path chip 1 include polymer resin materials made of a hydrophobic polymer such as cyclic olefin, polymethyl methacrylate, polyimide, and silicon rubber. The liquid contact portion 12a (the inner wall surface of the flow channel 12) of the flow channel chip 1 made of such a polymer resin material is hydrophobic. The material of the upper substrate 2 and the lower substrate 3 may be the same or different.

[Coating solution]
The coating solution used to form the coating film on the liquid contact portion 12a of the flow path chip 1 contains a hydrophilic polymer and a silane coupling agent in the solvent.
Examples of the solvent include organic solvents such as ethanol, n-hexanol, and 1,4-dioxane, and may be a mixed solvent of the organic solvent and water. Moreover, it is preferable that acids, such as an acetic acid, hydrochloric acid, and a sulfuric acid, are added to these solvents.

(Hydrophilic polymer)
The hydrophilic polymer plays a role of improving the hydrophilicity of the surface by being fixed to the liquid contact part 12a of the flow path chip 1.
Polyvinyl alcohol and / or polyethylene glycol can be used as the hydrophilic polymer.
When polyvinyl sulfate or the like having a low acidity constant (pKa) is used as the hydrophilic polymer, since the polyvinyl sulfate exists in the dissociated state in the flow path 12, the wetted part is caused by electrostatic interaction. Samples such as nucleic acids and proteins are adsorbed to 12a. In the surface modification method of the present invention, adsorption of the sample due to such electrostatic interaction can be avoided by using polyvinyl alcohol or polyethylene glycol.

The average molecular weight of polyethylene glycol is preferably 400 to 8000, more preferably 600 to 6000, and even more preferably 600 to 4000. If the average molecular weight is 600 or more, the film surface density of polyethylene glycol tends to increase. If the average molecular weight is 6000 or less, it is easy to ensure solubility in the coating solution.
The polymerization degree of polyvinyl alcohol is preferably 1000 to 3000, and more preferably 1500 to 2000. If the degree of polymerization is 1500 or more, the film surface density of polyvinyl alcohol is likely to be increased. If the degree of polymerization is 2000 or less, it is easy to ensure solubility in the coating solution.

The content of the hydrophilic polymer in the coating solution (100% by mass) is preferably 1 to 20% by mass, more preferably 3 to 15% by mass, and 5 to 10% by mass. Further preferred.
If the content of the hydrophilic polymer is 1% by mass or more, it is easy to improve the hydrophilicity of the surface of the polymer resin material. Further, when the content of the hydrophilic polymer exceeds 20% by mass, the effect of improving the hydrophilicity hardly changes.
When polyvinyl alcohol and polyethylene glycol are used in combination as the hydrophilic polymer, it is preferable that the total amount thereof satisfies the above range.

(Silane coupling agent)
The silane coupling agent plays a role of forming a modified film on the liquid contact portion 12a by drying a coated film formed by introducing a coating solution, and fixing a hydrophilic polymer to the modified film.
Examples of the silane coupling agent include methyltrimethoxysilane, methyltriethoxysilane, tetraethoxysilane, vinyltrimethoxysilane, 3-aminopropyltriethoxysilane, N- (2-aminomethyl) -3-aminopropyltri Methoxysilane, N- (2-aminomethyl) -3-aminopropylmityldimethyoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-mercaptopropyltrimethoxysilane, hexamethylsilazane, 1,3-bis (3-glycidoxypropyl) -1,1,3,3-tetramethyldisiloxane, phenyltrie G Shishiran, and the like.
A silane coupling agent may be used individually by 1 type, and may use 2 or more types together.

The content of the silane coupling agent in the coating solution (100% by mass) is preferably 5 to 30% by mass, more preferably 10 to 20% by mass, and 12 to 16% by mass. Further preferred.
When the content of the silane coupling agent is 10% by mass or more, it is easy to fix the hydrophilic polymer on the surface of the polymer resin material. Moreover, if content of a silane coupling agent is 20 mass% or less, it will be easy to prevent the film thickness of the modified film after drying becoming too thick.
When using 2 or more types of silane coupling agents together, it is preferable to make the total amount satisfy the above range.

[Surface modification method]
In the surface modification method of the present invention, a coating solution is introduced into the flow channel 12 of the flow channel chip 1 to form a coating film on the liquid contact portion 12a, and the coating film is subjected to conditions of a humidity of 70% or more and 100 ° C. or less. The process of drying with.
The method for forming the coating film on the liquid contact portion 12a is not particularly limited. For example, the coating solution is pushed into the flow path 12 using a syringe or the like and introduced into the flow path 12, and the air is conditioned after a certain period of time. A method of forming a coating film by removing an excessive coating solution by introducing is mentioned. Alternatively, after reducing the pressure from the opening 13 side, the coating solution is drawn into the flow path 12 from the opening 11 to be acclimatized, and then the excess coating solution is removed in the same manner as described above. Further, the coating solution may be introduced into the flow path 12 from the opening 13 side.

In the method of the present invention, in order to improve the bonding strength between the modified film formed by the hydrophilic polymer and the silane coupling agent after drying the coating film and the liquid contact portion 12a, the coating solution is provided in the flow path 12. The surface treatment of the wetted part 12a may be performed before introducing.
For example, when the channel chip 1 is made of a polyimide resin material, alkali hydrolysis is performed by introducing a sodium hydroxide aqueous solution or the like into the channel 12 to expose the carboxy group and amino group in the liquid contact part 12a. There is a need.

The coating film formed on the liquid contact part 12a can sufficiently improve the hydrophilicity of the liquid contact part 12a of the flow path chip 1 by including a step in which the humidity during drying is 70% or more. Further, after drying under conditions of 70% or higher humidity, drying may be performed under conditions of less than 70% humidity. In this case, it is preferable to dry at a humidity of 50 to 80%.
The humidity can be adjusted by introducing air of the humidity into the flow path 12, and it is preferable that the humidity also serves as removal of the coating solution in the flow path 12.

In the method of the present invention, drying is performed at 100 ° C. or lower. Drying is preferably performed by heat treatment at 50 to 100 ° C, and more preferably by heat treatment at 85 to 95 ° C.
When the drying temperature is set to 50 ° C. or higher, the coating film can be dried in a shorter time, so that the manufacturing efficiency of the flow channel chip 1 having the highly hydrophilic liquid contact portion 12a is improved. Further, if the drying temperature is 95 ° C. or lower, it is easy to prevent cracks from occurring in the modified film formed by the rapid evaporation of the solvent during drying.

  Moreover, after drying on the said humidity conditions, when drying on the conditions of 70% or less of humidity, a drying temperature shall be 100 degrees C or less similarly to the said drying temperature. The preferable temperature conditions are also the same.

  The film thickness of the modified film formed on the liquid contact portion 12a is preferably 0.01 to 2 μm. If the film thickness is 0.01 μm or more, it is easy to improve the hydrophilicity of the liquid contact part 12a. Moreover, if the film thickness is 2 μm or less, there is little risk of impeding the passage of the sample solution 20 and reducing the analytical performance.

According to the method of the present invention described above, it is possible to improve the hydrophilicity by modifying the liquid contact portion of the polymer resin channel chip at low cost. In addition, the hydrophilic surface of the obtained polymer resin flow channel tip wetted part is superior in sustainability compared to the conventional method. Therefore, it is possible to prevent the sample such as nucleic acid, protein, and fluorescent organic compound that is a labeling compound in the sample solution 20 from adsorbing to the wetted part, thereby improving the quantitativeness and accuracy of the analysis and improving the reliability. Get higher.
The reason is considered to be that the hydrophilic polymer can be firmly fixed to the wetted part by including the hydrophilic polymer in the film formed of the silane coupling agent.

  In addition, since the present invention includes a step of setting the humidity during drying to 70% or higher, high hydrophilicity can be imparted to the wetted part. The reason for this is that, by drying under high humidity conditions, there is an interaction (hydrogen bond, etc.) between water molecules present in the gas phase at the time of drying and the hydroxy group of the silane coupling agent in the coating film. This is considered to be because the density of hydroxy groups of the silane coupling agent near the surface of the modified film formed after drying is increased.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. However, the present invention is not limited by the following description.
[Example 1]
A polymer resin channel chip 4 (hereinafter, channel chip 4) as shown in FIG. 4 was prepared. The flow path chip 4 is formed by joining a long upper substrate 5 and a lower substrate 6 having the same shape as the upper substrate 5. Further, the upper substrate 5 is formed with a groove 5a (one) along the longitudinal direction of the upper substrate 5, and the upper substrate 5 and the lower substrate 6 are joined together to form the flow path 32. (FIG. 6).
Further, as shown in FIGS. 4 and 5, the flow path chip 4 has an opening 31 for introducing the sample solution and an opening 33 for discharging the sample solution. The upper substrate 5 and the lower substrate 6 are made of a cyclic olefin resin (COC resin).
In addition, to a solvent composed of ethanol (20 mL), n-hexanol (2 mL), acetic acid (0.9 mL), and 0.01N hydrochloric acid (4.3 mL), phenyltriethoxysilane (1 mL), methyltriethoxysilane (1 mL) ) And aminopropyltriethoxysilane (0.5 mL) were sequentially added and mixed well. Next, after standing at room temperature for a certain period, an ethanol-water mixed solution (10 mL) containing polyethylene glycol (2 g) having an average molecular weight of 3000 was added and mixed well to obtain a coating solution.
Next, the coating solution was filled by introducing it into the flow path 32 through the opening 31 using a 1 mL capacity resin syringe. After that, the coating solution is allowed to stand at room temperature (25 ° C.) for 5 minutes, and the coating solution is made to conform to the liquid contact part 32a of the flow path 32. The excess coating solution was removed by introduction, and a coating film was formed on the liquid contact part 32a.
Next, using a heating device with a humidification adjustment function (TEMP. & HUMID. CHAMBER SH-240, manufactured by ESPEC), continuously heated at 80% humidity and a temperature of 95 ° C for 12 hours. It is 80%, and it becomes dry with the passage of time.) After continuously drying at a temperature of 95 ° C. for 12 hours, it is returned to room temperature over a period of about 3 hours, thereby fixing the polyethylene glycol to the liquid contact part 32a. 4A was obtained.
In addition, from the electron micrograph of the cut cross section of the flow path of the flow path chip 4A, it was estimated that the film thickness of the modified film was 0.16 μm.

[Examples 2-3]
Except having changed the drying conditions by the heating apparatus with a humidification adjustment function as shown in Table 1, it carried out similarly to Example 1, and obtained flow-path chip 4B and 4C.

[Example 4]
As the flow path chip, the flow path chip 4 made of polyimide resin is used, and a 1N sodium hydroxide aqueous solution is introduced into the flow path 32 and subjected to alkali hydrolysis for 15 minutes, washed with ion-exchanged water, and brought into contact with the liquid contact portion 32a. The carboxy group and amino group were exposed.
In addition, to a solvent composed of ethanol (20 mL), n-hexanol (2 mL), acetic acid (0.9 mL), and 0.01N hydrochloric acid (4.3 mL), phenyltriethoxysilane (1 mL), methyltriethoxysilane (1 mL) ) And aminopropyltriethoxysilane (0.5 mL) were sequentially added and mixed well. Next, after standing at room temperature for a certain period, an aqueous solution (10 mL) containing polyvinyl alcohol (2 g) having a polymerization degree of 1700 and a saponification degree of 99.5 mol% was added and mixed well to obtain a coating solution.
Formation of a coating film on the liquid contact part 32a of the channel chip 4 and drying of the coating film using the coating solution were performed in the same manner as in Example 1 to obtain a channel chip 4D.

[Comparative Example 1]
A flow channel chip made of a polymer resin not subjected to the surface modification method of the present invention was defined as a flow channel chip 4E.

[Comparative Examples 2-3]
Flow channel chips 4F and 4G were obtained in the same manner as in Example 1 except that the drying conditions were changed as shown in Table 1 after forming the coating film on the liquid contact portion.

The hydrophilicity evaluation of the polymer resin channel chip wetted part obtained in this example was performed by the following contact angle measurement and adsorptivity measurement.
[Measurement of contact angle]
For the channel chips 4A to 4G obtained in the examples and comparative examples, after the channel chip was cut, 100 μL of ion-exchanged water was dropped on the liquid contact part, and the contact angle of the droplets was measured. The contact angle was measured using a contact angle meter CA-X type (manufactured by Kyowa Interface Science Co., Ltd.) at 25 ° C.

[Measurement of adsorptivity]
A sample solution containing a protein in which chicken ovalbumin (2 × 10 ⁻⁷ M) was fluorescently modified with 10-fold molar amount of 4-nitro-7-chlorobenzotriazole was prepared, and channels obtained in Examples and Comparative Examples The sample solution was continuously flowed into the chips 4A to 4G at a flow rate of 50 μL / min for 10 minutes, and the change in protein concentration in the sample solution before and after that was evaluated from the fluorescence spectrum intensity. The fluorescence spectrum measurement was performed using a fluorescence spectrometer 650 (manufactured by Hitachi, Ltd.).
Table 1 shows the measurement results of the contact angle and the adsorptivity of the flow path chips 4A to 4G obtained in the examples and comparative examples. However, the adsorptive measurement result is the fluorescence spectrum intensity of the sample solution after flowing into the channel chip, and is a value based on the fluorescence spectrum intensity of the sample solution before flowing into the channel chip as a reference (100). is there.

In Examples 1 to 3 in which the liquid contact part of the channel chip made of the COC resin was modified by the method of the present invention, the contact angle after drying was a very small value and had high hydrophilicity. Moreover, the contact angle was maintained after 10 days, and the imparted hydrophilic sustainability was high. In addition, in the measurement of the adsorptivity, the fluorescence spectrum intensity of the sample solution flowed in the flow channel chip was not changed at all from that before the passage, indicating an excellent low adsorptivity.
Further, in Example 4 in which the liquid contact part of the channel chip made of polyimide resin was modified by the method of the present invention, the contact angle after drying was a very small value, as in Examples 1 to 3, and high hydrophilicity. Had sex. Moreover, the contact angle was maintained after 10 days, and the imparted hydrophilic sustainability was high. In addition, in the measurement of the adsorptivity, the fluorescence spectrum intensity of the sample solution flowed in the flow channel chip was not changed at all from that before the passage, indicating an excellent low adsorptivity.

On the other hand, in Comparative Example 1 in which the liquid contact part of the channel chip made of COC resin is not modified, the contact angle is 65 degrees and the hydrophilicity is low. In addition, the fluorescence spectrum intensity of the sample solution flowed into the flow channel chip was 75% before liquid passage, and the sample was adsorbed on the liquid contact part of the flow channel chip.
Moreover, in Comparative Example 2 in which the heat treatment of the coating film formed in the liquid contact portion was performed in a dry state, the hydrophilicity was inferior compared to the Examples.
Further, even in Comparative Example 3 in which the heat treatment of the coating film formed on the liquid contact portion was performed at a humidity of 60%, the hydrophilicity was inferior to that of the Example.

  The surface modification method of the present invention can modify the wetted part of the polymer resin flow channel chip, impart high hydrophilicity, and maintain the hydrophilicity for a long period of time. Therefore, adsorption of biological molecules such as nucleic acids and proteins and fluorescent organic compounds that are labeling compounds to the wetted part can be suppressed, and quantitative and accurate analysis can be improved and reliability can be improved. . Therefore, it can be suitably used in fields such as medical care and environmental measurement.

It is the perspective view which showed an example of the polymeric resin-made flow-path chips. It is sectional drawing which cut | disconnected the polymeric resin flow-path chip | tip of FIG. 1 by the A-A 'line. FIG. 2 is a cross-sectional view of the polymer resin flow channel chip of FIG. 1 cut along line B-B ′. It is the perspective view which showed the polymeric resin-made flow-path chip | tip used in the present Example. FIG. 5 is a cross-sectional view of the polymer resin flow channel chip of FIG. 4 cut along line C-C ′. FIG. 5 is a cross-sectional view of the polymer resin flow channel chip of FIG. 4 cut along line D-D ′.

Explanation of symbols

  1 Polymer resin channel chip 12 Channel 12a Wetted part

Claims (4)

  A coating solution containing polyethylene glycol and / or polyvinyl alcohol and a silane coupling agent is introduced into the flow path of the polymer resin flow path chip, and a coating film is formed at the liquid contact portion of the flow path. A method for modifying the surface of a wetted part made of a polymer resin flow channel chip, comprising a step of drying at a humidity of 70% to 100 ° C.   The surface modification method for a polymer resin flow channel chip wetted part according to claim 1, wherein the drying is performed by heat treatment at 50 to 100C.   The surface modification of the polymer resin channel chip wetted part according to claim 1 or 2, wherein the content of the hydrophilic polymer in the coating solution (100% by mass) is 1 to 20% by mass. Method.   Content of the said silane coupling agent in the said application | coating solution (100 mass%) is 5-30 mass% of the polymer resin channel chip wetted part in any one of Claims 1-3 Surface modification method.

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