JP2005330329A - Silicon resin surface modification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface modification method of a silicon resin capable of producing a biochip excellent in cost performance while maintaining excellent transparency even after activation of a substrate surface.
A method for modifying a surface of a silicon resin, comprising performing an ozone contacting step of bringing ozone into contact with a silicon resin surface and then performing a reducing agent contacting step of bringing a reducing agent into contact with the silicon resin surface.
[Selection] Figure 1

Description

  The present invention relates to a method for modifying the surface of a silicon resin, and more particularly to a method for modifying the surface of a silicon resin that is a constituent material of a biochip.

  In performing recent genome analysis or proteome analysis, biochips have been widely introduced to comprehensively analyze large amounts of DNA or proteins, determination of nucleic acid sequence information, gene expression / mutation / diversity, etc. Analysis and functional analysis such as protein purification / identification, protein expression / interaction / post-translational modification. As such biochips, DNA chips and protein chips manufactured by microarray technology are known, and for example, microfluidic devices in which complicated flow paths and reaction chambers are formed are known as DNA chips. ing.

  Microarray technology has made tremendous technological progress in recent years. A DNA chip is produced, for example, by aligning and fixing DNA on a substrate such as glass at high density. Then, the target nucleic acid labeled with a fluorescent molecule or the like is hybridized with the immobilized DNA, and the label bonded on the substrate is imaged with a fluorescence scanner or the like and analyzed.

In addition, microfluidic devices are manufactured by applying microfabrication technology (MEMS) such as semiconductors. For example, a minute capillary fluid flow path or a structure such as a reaction chamber, an electrophoresis column, a membrane separation mechanism, etc. as a reaction region connected to the flow path is formed in the member. Such microfluidic devices are mainly used for measuring biological samples such as DNA analysis devices, microelectrophoresis devices, microchromatography devices, microsensors, and the like.
For example, Patent Document 1 includes a plurality of independent reaction chambers formed by anisotropic etching on the surface of a silicon substrate, and a flat plate (heat-resistant glass) that is anodically bonded to the surface of the silicon substrate and seals the reaction chamber. A microfluidic device is disclosed. If this microfluidic device is integrated, a large number of biochemical reactions can be performed simultaneously in parallel.

As described above, glass is used as a constituent material of the biochip. While glass has the advantages of being inexpensively available and having excellent chemical resistance, it is difficult to process, and because it absorbs ultraviolet rays (UV), it is not suitable for applications that use UV in the detection system. There is.
On the other hand, in addition to glass, silicon resin is used as a constituent material for biochips. Among these silicon resins, polydimethylsiloxane (hereinafter referred to as PDMS) is particularly preferred from the viewpoint of ease of molding and optical properties. Has attracted attention.

PDMS has extremely high transparency, excellent optical characteristics, and low absorption in a wide wavelength region. In particular, the absorption in the visible light region is extremely small and hardly affects the fluorescence detection. For this reason, S / N value can be made low by using PDMS as a substrate of a microfluidic device.
Furthermore, it has a high follow-up property to the mold, can be easily processed in nano to micron order, and can be easily formed into an arbitrary fine structure. In this respect, there is an advantage that microfabrication can be performed into an optimum shape for various optical instruments, and therefore, cost performance for device fabrication is excellent.
In addition, PDMS itself has a good adhesion property with glass, acrylic resin, etc., and adhesion is achieved by bringing a flat glass / acrylic resin member into contact with a finely processed PDMS substrate. It is possible to form a fluid flow path, a chamber, or the like without using an adhesion means such as adhesion with an agent.

  Here, since the main component of PDMS is dimethylsiloxane and has only an inactive functional group having poor affinity with biological molecules such as DNA and protein, the surface of the PDMS substrate becomes hydrophobic and inactive. Has surface properties. Therefore, it is difficult to immobilize biological related molecules, various chemical substances involved in biochemical reactions, etc. on the surface of the PDMS substrate.

  On the other hand, many biologically relevant molecules are generally hydrophilic and have high affinity with functional groups such as a hydroxyl group, an amino group, and a carboxyl group. Therefore, if the surface of the PDMS substrate has these functional groups, for example, has hydrophilic and high surface activity characteristics, it becomes easy to immobilize biological molecules on the substrate surface, and biochemical reactions on the device are performed. It is thought that it can be done more reliably.

  That is, when the microfluidic device is used as a reactor for performing a biochemical reaction, it is important to bind biologically relevant molecules and various chemical substances directly to the substrate surface. Therefore, when PDMS is adopted as the constituent material of the microfluidic device, for example, the surface of the chamber in which the reaction is performed is hydrophilized so as to improve affinity with biological molecules and various chemical substances. It is conceivable to perform a modification that improves the above.

  As such surface property modification, for example, Non-Patent Document 1 discloses that a PDMS substrate surface is subjected to ultraviolet (UV) irradiation treatment, or a PDMS substrate surface is subjected to ultraviolet-ozone (UVO) treatment. Discloses a technique for performing photo-ozone oxidation. By these treatments, the PDMS substrate surface can be modified so as to have a hydroxyl group or the like, so that the PDMS substrate surface can be made hydrophilic.

  In addition, a hydroxyl group is generated on the surface of the PDMS substrate by performing a strong alkali treatment on the surface of the PDMS substrate or by performing an oxygen radical irradiation treatment on the surface of the PDMS substrate using a plasma irradiation apparatus or the like. The surface of the PDMS substrate can be hydrophilized.

Japanese Patent Application Laid-Open No. 10-337173 (see claims) K. K. Efimenko, William E. Wallace, J. J. Genzer, “Surface Modification of Sylgard-184 Poly (dimethyl siloxane) Networks by Ultraviolet and Ultraviolet / Ozone Treatment”, Journal of Colloid and Interface Science 254, 306-315 (2002)

In the above-described property modification of the PDMS substrate surface, when the UV irradiation treatment or UVO treatment is performed on the PDMS substrate surface (Patent Document 2), or when a strong alkali treatment is performed, There was a problem that cracks occurred and it became cloudy and transparency was significantly impaired. This is considered to be caused by the molecular chain of PDMS being destroyed by exposure to ultraviolet light on the surface of the PDMS substrate.
When a microfluidic device is used as a DNA analysis device, a dye or fluorescently labeled DNA may be detected after a biochemical reaction on the device. At this time, if the transparency of the device is impaired, it becomes difficult to detect fluorescence with a scanner, and accurate DNA analysis cannot be performed.

  In addition, when oxygen radical irradiation treatment is performed on the surface of the PDMS substrate, the generated hydroxyl group is not stable, and there is a problem that it is only a temporary surface modification. In addition, since a very expensive plasma irradiation apparatus or the like is used, there is a problem that the manufacturing cost increases. High production costs hinder the widespread use of PDMS as a constituent material for microfluidic devices.

  Accordingly, an object of the present invention is to provide a method for modifying the surface of a silicon resin that can produce a biochip having excellent cost performance while maintaining excellent transparency even after activation of the substrate surface.

  In order to achieve the above object, the first feature of the surface modification method for a silicon resin according to the present invention is that after performing an ozone contact step in which ozone is brought into contact with the silicon resin surface, a reducing agent is brought into contact with the silicon resin surface. It is in the point which performs the reducing agent contact process.

According to the first characteristic configuration, when ozone is brought into contact with the surface of the silicon resin, the ozonide is formed without breaking the molecular chain due to the ozone particularly acting on the surface of the silicon resin having an unsaturated bond. Since the molecular chain is not cleaved, cracks are not generated on the surface of the silicon resin and white turbidity occurs, so that the transparency of the resin is maintained.
Then, a reducing agent is brought into contact with the silicon resin surface on which the ozonide is formed in this way. Thereby, the unstable ozonide can be efficiently converted into a stable hydroxyl group or carboxyl group, and as a result, modification can be made to make the surface of the silicon resin hydrophilic. In this way, the surface of the silicon resin is hydrophilized, and then the affinity between the silicon resin and biological molecules and various chemical substances is achieved by, for example, coupling a coupling agent such as aminosilane or poly-L-lysine. It becomes possible to increase the property. Thus, if the modification | reformation which hydrophilizes a silicon resin is attained, the modification | reformation to a silicon resin with high surface activity can be performed easily.
Therefore, it becomes easy to fix bio-related molecules and various chemical substances on the surface of the silicon resin, and the biochemical reaction on the silicon resin can be performed more reliably.
Moreover, since it is not necessary to use an expensive plasma irradiation apparatus etc. with this structure, the biochip excellent in cost performance can be provided.

  The second characteristic configuration of the method for modifying the surface of a silicon resin according to the present invention is that after the reducing agent contacting step, a surface function modifying agent contacting step of bringing a surface function modifying agent into contact with the silicon resin surface is performed. is there.

  According to the second characteristic configuration, when a hydroxyl group or a carboxyl group generated on the surface of the silicon resin is reacted with, for example, an amino group-containing surface function modifier, various functional groups such as an amino group are introduced on the surface of the silicon resin. can do. Therefore, by using various surface function modifiers, it is possible to produce silicon resins having affinity for many kinds of biologically relevant molecules and various chemical substances. Therefore, a constituent material for a biochip having a wide range of application can be provided.

  The third characteristic configuration of the method for modifying the surface of a silicon resin according to the present invention is that the silicon resin contains polydimethylsiloxane (PDMS) as a main component.

  According to the third characteristic configuration, PDMS having physically excellent characteristics such as transparency, optical characteristics, followability to a mold, ease of processing, and adhesion can be suitably used as the silicon resin. . PDMS is easy to obtain at low cost and can save labor required for processing. For this reason, a biochip or the like having excellent cost performance can be manufactured, and it becomes easy to construct a disposable system, and problems relating to contamination hardly occur.

  A fourth characteristic configuration of the method for modifying a surface of a silicon resin according to the present invention is that the reducing agent is hydrogen peroxide.

  According to the fourth characteristic configuration, hydrogen peroxide can be used as a reducing agent that is easy to obtain and handle, so that the surface of the silicon resin can be easily made hydrophilic.

  A fifth characteristic configuration of the method for modifying a surface of a silicon resin according to the present invention is that the surface function modifier is a silane coupling agent.

  According to the fifth characteristic configuration, since the silane coupling agent can be used as a surface function modifier that can be easily obtained and handled, the silicon resin surface has an affinity for biologically related molecules such as amino groups and phenyl groups. High functional group can be easily added.

  The 6th characteristic structure of the surface modification method of the silicon resin which concerns on this invention exists in the point which treats ozone for 1 to 3 hours at 0.8 g / h or more with respect to the said resin surface in the said ozone contact process.

  As shown in the examples described later, the result that the silicon resin (PDMS) maintains transparency even after the surface activation treatment is obtained by performing the ozone contact step under the conditions of this configuration. Therefore, according to the sixth characteristic configuration, since the ozone contact step can be performed in a short time, the silicon resin surface modification method is excellent in terms of cost performance and efficiency.

Embodiments of the present invention will be described below with reference to the drawings.
The present invention is a surface modification method for a silicon resin that is a constituent material of a biochip, in particular. As shown in FIG. 1, after performing an ozone contact step in which ozone is brought into contact with the silicon resin surface, A reducing agent contact step of bringing a reducing agent into contact is performed.

Silicone resin is an organosilicon polymer generally called silicon rubber, and there are several types depending on the side chain bonded to the main chain composed of siloxane bonds. The most common silicone resin is tridimethylsiloxy-terminated polydimethylsiloxane (PDMS). The properties of polydimethylsiloxane can be variously changed by replacing the methyl group bonded to the silicon atom with hydrogen, alkyl, phenyl, or an organic functional group.
This PDMS is available at low cost and has physical properties such as transparency, optical properties, followability to molds, ease of processing, adhesion, etc. Suitable as a material. Silicon resin is not limited to PDMS.

Although the main component of PDMS is dimethylsiloxane, polymerization alone does not occur, and cross-linking occurs in some places by entering a siloxane compound having a vinyl group or the like in the side chain to form a network structure. For example, when polymerized with a polymerization agent containing a vinyl group, an unsaturated bond is formed in the molecular chain. As described later, the molecular chain is cut by ozone acting on the surface of the PDMS substrate. This makes it easier to form ozonides.
In the present specification, a resin base material containing PDMS as a main component is referred to as a PDMS base material. In this specification, the “main component” refers to a component contained in the resin component by 50% or more, preferably 70% or more, and may contain other substances as appropriate. Therefore, it is possible to add various additives in addition to the polymerization agent as long as PDMS is a main component.

  The silicon resin surface usually has poor affinity with biological molecules. For this reason, surface characteristics are modified so that biochemical reactions can be suitably performed on the microfluidic device.

  First, an ozone contact process is performed in which ozone is brought into contact with the silicon resin surface. At this time, the ozonide is formed without breaking the molecular chain due to ozone acting on the surface of the silicon resin having an unsaturated bond. Since the molecular chain is not cleaved, cracks are not generated on the surface of the silicon resin and white turbidity occurs, so that the transparency of the resin is maintained. And when the reducing agent contact process which makes a reducing agent contact the silicon resin surface in which the ozonide is formed, unstable ozonide is efficiently converted into a stable hydroxyl group or carboxyl group, and the silicon resin surface is hydrophilized. Can be modified. In this way, by modifying the surface of the silicon resin to be hydrophilic, it can be modified to a silicon resin having high surface activity. Therefore, it becomes easy to fix bio-related molecules and various chemical substances on the surface of the silicon resin.

  The reducing agent is preferably exemplified by hydrogen peroxide, but is not limited thereto. As another reducing agent, for example, sodium sulfide can be used.

After performing a reducing agent contact process, the surface function modifier contact process which makes a surface function modifier contact a silicon resin surface is performed.
In this process, hydroxyl groups and carboxyl groups generated on the surface of the silicon resin are reacted with surface function modifiers containing amino groups and phenyl groups, and various functional groups such as amino groups and phenyl groups are formed on the silicon resin surface. Can be introduced. Therefore, it becomes possible to produce a silicone resin having affinity with many kinds of biologically relevant molecules and various chemical substances.

  As the surface function modifier, various silane coupling agents are preferably exemplified, but not limited thereto. The silane coupling agent uses, for example, aminosilane such as 3-aminopropyltriethoxysilane when introducing an amino group, and phenylsilane such as phenyltriethoxysilane (PTES) when introducing a phenyl group, respectively. it can. As the surface function modifier other than the silane coupling agent, for example, poly-L-lysine can be suitably used.

Embodiments of the present invention will be described below in detail with reference to the drawings.
<Biochip>
Various forms can be applied to a biochip for immobilizing DNA, which is an example of a biological molecule.
For example, PDMS (Sylgard (registered trademark) 184 on the substrate (first member) 38,
There is a DNA chip 31 provided with a PDMS base material (second member) 39 formed mainly from Dow Corning (see FIG. 2). The PDMS base material 39 includes a polymerization agent and an additive, which will be described later, as components in addition to the main component PDMS.
In the PDMS substrate 39, grooves for forming minute capillary fluid flow paths 36, 37, recesses for forming reaction chambers 34a, 34b as reaction regions connected to these flow paths, and injection holes for injecting reaction liquid 33, the discharge hole 35 for discharging the reaction liquid (reacted liquid) is finely processed.
That is, the space between the substrate 38 and the PDMS base material 39 is surrounded by two members, thereby allowing the sample to flow therethrough (fluid flow channels 36 and 37), and the space serving as a reaction region that can carry the sample. (Reaction chamber 34) is formed.
The substrate 38 can be a solid phase carrier such as a glass plate. Other preferable examples of the solid support include a quartz plate and a silicon wafer.

  Then, the finely processed PDMS base material 39 and the substrate 38 are closely bonded. The bonding method can be performed by applying an adhesive, but PDMS itself has the property of adhering to a flat surface such as glass or acrylic resin, so PDMS is used for one member and the other member is used. For example, when flat glass is used, it is possible to bond without using an adhesive by removing organic substances and dust with an alkaline detergent for glass.

In addition, the biochip is not limited to the above-described configuration, and it is also possible to form a DNA chip having reaction spaces arranged in parallel in a barcode by performing fine processing on a PDMS substrate.
In this case, both the first member and the second member are configured as flat plate-like bodies, and the second member has a plurality of grooves extending in one direction formed in parallel in a barcode shape on the contact surface with the first member. The configuration is as follows.

In the case where PDMS is used as a base material constituting a member to be subjected to a fine structure, a method of forming using a mold is particularly preferable because PDMS has high followability to the mold. Once the mold is produced, a large amount of chips can be produced easily and inexpensively, so that the manufacturing cost can be reduced. The mold is formed by appropriately using a known fine processing technique such as a combination of a photolithography technique and an etching technique.
The mold having the uneven spatial structure produced in this way is fixed in the mold, uncrosslinked PDMS is mixed with an appropriate polymerizing agent and additives, poured into the mold, and heated to be cured. Then, the template structure is transferred to PDMS. At this time, the curing time is appropriately set according to the curing temperature. After curing, the PDMS substrate having a shape pattern with a desired structure can be produced by peeling from the mold.
In this embodiment, methylhydrogen siloxane is used as the polymerization agent. At this time, it is preferable to mix PDMS: polymerizing agent = 10: 1.
Moreover, as an additive, the adhesive containing methoxy groups, such as 3-acryloxypropyl trimethoxysilane (KBM-5103), is mixed so that it may become 0.1 to 2%. Thereby, it can be expected that the efficiency of fixing DNA to the PDMS substrate is improved.

<PDMS substrate surface modification>
An experiment for activating the surface of the PDMS substrate 39 was performed.
For example, in the case of the DNA chip 31 shown in FIG. 2, the surface activation of the PDMS base material 39 faces the substrate 38 at least when the reaction chamber 34 surface, that is, the substrate 38 and the PDMS base material 39 are bonded. It is applied to the concave surface of the PDMS substrate 39. The surface of the fluid flow paths 36 and 37 can be appropriately activated, but when the fluid flow paths 36 and 37 are regarded as mere fluid (reagent) passages, bio-related molecules and various chemical substances are used. Therefore, it is not always necessary to perform surface activation.

  The “biologically relevant molecules” are preferably exemplified by DNA fragments such as cDNA and PCR products, nucleic acids such as oligonucleotides, polynucleotides and peptide nucleic acids, proteins, peptides, sugars, cells, microorganisms, etc. It is not limited to.

  Below, the process which activates the PDMS base material surface is explained in full detail. The PDMS base material used in the following experiments was a flat plate member without a groove or a recess for forming a fluid flow path or a reaction chamber.

This PDMS substrate is brought into contact with ozone (ozone contact step). The contact of ozone with the PDMS substrate is performed by an ozonizer (ED-OG-R4: manufactured by Ecodesign) at a discharge tube voltage of 75 V and an ozone concentration of 0.8 g / h (flow rate 2 L / min) for 1 to 3 hours. I went there.
After the ozone contact step, the PDMS substrate was immersed in 30% hydrogen peroxide solution for 10 minutes or 24 hours (reducing agent contact step).

  Thereafter, the PDMS substrate was immersed in a 50% ethanol solution containing 3-aminopropyltriethoxysilane as a silane coupling agent for 10 minutes or 24 hours (surface function modifier contact step). A silane coupling agent having a pH adjusted to 7.0 or 4.5 was used. The silane coupling agent was heated to room temperature or 75 ° C.

  Thereafter, the PDMS substrate surface was immersed in 100% ethanol and the process of stirring for 2 minutes was repeated twice to perform a cleaning step of cleaning the PDMS substrate surface. After washing, a drying process at 40 ° C. for 15 minutes was performed.

Thus, the substrate samples 1-7 processed by changing various processing time, temperature conditions, etc. in the ozone contact process / reducing agent contact process and the surface function modifier contact process were produced.
Table 1 shows the experimental conditions when Samples 1 to 7 were produced.

  At this time, as surface findings of Samples 1 to 7, it was recognized that transparency was maintained in all samples. Therefore, it is considered that the ozone contact process is preferably performed for about 1 to 3 hours from the viewpoint of cost, efficiency, and the like.

<Affinity between surface-modified PDMS and biological molecules>
DNA was immobilized on the surface of the PDMS base material 39 subjected to the surface modification treatment, and the strength of affinity with DNA, which is a biologically relevant molecule, was examined.
The DNA was immobilized on the PDMS substrate surface-modified under the conditions of Sample 1 as follows.
A 1 pmol / μL oligo DNA solution fluorescently labeled with fluorescein isothiocyanate (FITC) is applied (spot) to the surface of the PDMS substrate by a conventional method, and UV irradiation is performed at 120 mJ to apply the fluorescently labeled DNA to the surface of the surface modified PDMS substrate. Fixed to. Thereafter, the DNA not fixed was removed by washing with pure water, followed by drying at 40 ° C. for 20 minutes.

  The PDMS substrate on which the fluorescence-labeled DNA was thus immobilized was subjected to image processing by measuring the fluorescence intensity at an absorption wavelength of 550 nm and an excitation wavelength of 570 nm with a scanner dedicated to DNA microarrays. The result of image processing is shown in FIG. The image in FIG. 3 (a) is comparison data 1 in which the ozone treatment process of the present invention is not performed, and the image in FIG. 3 (b) is the ozone treatment of the present invention on the PDMS substrate mixed with the additive. It is the comparison data 2 which does not perform a process. In order to obtain an average value, 8 DNA spots (spots 1 to 8) are prepared. And if the spot after image processing becomes dark, it has shown that DNA has been fix | immobilized efficiently.

The measurement results of the DNA concentration immobilized on the surface of the PDMS substrate (Table 2) and the evaluation of each sample based on the surface findings and the like are as follows.
The concentration of DNA was calculated from a standard curve with fluorescence intensity (reference: DNA concentration = (fluorescence intensity + 3451.9) / 134647). The fluorescence intensity is a value obtained by subtracting the background from the average value of the eight data. Spots 1 to 8 in Table 2 are spots 1 to 4 in order from the upper left to the lower left, and spots 5 to 8 in order from the upper right to the lower right in each image data in FIG.

  In comparative data 1 (FIG. 3 (a)) in which no additive is mixed into the PDMS substrate and no ozone treatment is performed, it is clear that DNA can hardly be fixed. In comparative data 2 (FIG. 3 (b)) in which an additive is mixed with a PDMS substrate, a slight DNA binding ability is observed. On the other hand, in the data (Fig. 3 (c)) in which the additive of the present invention was mixed and the ozone treatment was applied (Fig. 3 (c)), it was recognized that DNA was immobilized with high efficiency because a dark spot was detected. It was. In either case, the transparency of the PDMS substrate is not impaired.

  As described above, since the PDMS base material has been modified to improve the affinity with DNA and the DNA fixing ability by performing the ozone treatment process of the present invention, the substrate material of the biochip that can be suitably analyzed It was recognized that we could provide

[Another embodiment]
In the above-described embodiments, the silicon resin containing PDMS as a main component is exemplified, but the present invention is not limited to this. For example, the present invention can also be carried out with an acrylic resin such as polymethyl methacrylate (PMMA, polymethyl methacrylate).

The figure which showed the process of the surface modification method of the silicon resin of this invention Diagram showing an example of a biochip The figure which showed the result of fixing fluorescence label DNA on the surface of PDMS substrate and measuring fluorescence intensity

Claims (6)

  A method for modifying a surface of a silicon resin, comprising performing a reducing agent contact step of bringing a reducing agent into contact with the silicon resin surface after performing an ozone contacting step in which ozone is brought into contact with the silicon resin surface.   The method for modifying the surface of a silicon resin according to claim 1, wherein after performing the reducing agent contact step, a surface function modifier contact step of bringing a surface function modifier into contact with the surface of the silicon resin is performed.   The method for modifying a surface of a silicon resin according to claim 2, wherein the silicon resin contains polydimethylsiloxane (PDMS) as a main component.   The method for modifying the surface of a silicon resin according to claim 1, wherein the reducing agent is hydrogen peroxide.   The method for modifying the surface of a silicon resin according to claim 2 or 3, wherein the surface function modifier is a silane coupling agent.   The method for modifying the surface of a silicon resin according to any one of claims 1 to 5, wherein, in the ozone contact step, ozone is applied to the resin surface at a rate of 0.8 g / h or more for 1 to 3 hours.