JP4352849B2 - Plastic surface treatment method, plastic substrate and plastic biochip - Google Patents

Description

  The present invention relates to plastic surface treatment, and more particularly to surface treatment of a plastic substrate for a biochip.

In recent years, biochips, which are devices in which a physiologically active substance is immobilized on a solid phase substrate, have attracted attention as means for achieving high throughput in drug discovery research and clinical tests. Typical examples of physiologically active substances to be immobilized include nucleic acids, proteins, antibodies, sugar chains, glycoproteins, etc. In particular, nucleic acid microarrays, which are biochips on which nucleic acids are immobilized, already have a variety of products. It is on the market (Non-Patent Document 1). In addition, in order to detect a specific protein, a biochip in which an antibody against the protein is immobilized has attracted widespread attention.
The fluorescence label method is often used for signal detection on a biochip. For example, in the case of a nucleic acid microarray, the nucleic acid of the sample is fluorescently labeled in advance, and the amount of hybridization with the nucleic acid immobilized on the microarray is read with a fluorescent scanner. In fluorescence signal detection, if the fluorescence (background) derived from the substrate is high, the detection accuracy decreases due to a decrease in the S / N ratio. Therefore, the biochip substrate is often made of a low fluorescent material. In addition, chemical modification for efficiently immobilizing nucleic acid is usually performed on the substrate surface, and aldehyde groups or amino groups are often used. Reducing fluorescence resulting from the introduction of these functional groups is important from the viewpoint of improving the accuracy of assays using biochips.

The material of the biochip substrate is glass or plastic. As a method for surface modification of glass or plastic, there is surface coating with a silane coupling agent. This method is frequently used because it is possible to introduce a functional group on the surface by a relatively simple process. For example, when aminoalkylsilane is used as the silane coupling agent, it is possible to introduce amino groups on the surface only by the immersion treatment step and the heat treatment step. Furthermore, an aldehyde group can be introduce | transduced on the substrate surface by making a polyfunctional aldehyde act on the amino group introduce | transduced by said method.
Usually, the surface of the plastic is inactive, and the reactivity with the silane coupling agent is low as it is, so that the surface treatment cannot be performed sufficiently. Therefore, the plastic surface is often activated by oxidation treatment. That is, by introducing an oxygen-containing functional group having a strong polarity such as a hydroxyl group or a carbonyl group by oxidation treatment, the surface is activated and then the silane coupling agent is contacted. However, the plastic surface that has been surface-treated by such a method emits relatively strong fluorescence, which has been a cause of reducing the detection accuracy as a biochip.
"Cell engineering separate volume DNA microarray and latest PCR method" Shujunsha, 2000

  An object of the present invention is to provide a plastic surface treatment method and a plastic substrate, particularly a biochip plastic substrate, in which the amount of fluorescence generated is suppressed to a low level.

The present invention is as follows.
(1) A plastic surface treatment comprising a step of oxidizing a plastic surface and a step of reducing an oxygen-containing functional group generated by the oxidation treatment, wherein the first functional group is introduced into the plastic surface. In the method , the oxidation treatment includes at least one treatment selected from plasma treatment, corona discharge treatment, flame treatment, and ultraviolet irradiation treatment, and the oxygen-containing functional group to be reduced contains a carbonyl group A method for treating the surface of a plastic, wherein the reduction treatment comprises a step of immersing in a solution containing a metal hydride .
( 2 ) The plastic surface treatment method according to ( 1 ), wherein the carbonyl group contains an aldehyde group and / or a ketone group.
( 3 ) The plastic surface treatment method according to (1) or (2) , wherein the oxidation treatment includes plasma treatment under an oxygen atmosphere.
( 4 ) The plastic surface treatment method according to any one of ( 1) to (3), wherein the metal hydride contains sodium borohydride and / or lithium aluminum hydride.
( 5 ) The surface treatment of plastic according to any one of (1) to ( 4 ), further comprising a step of introducing a second functional group via the first functional group introduced to the plastic surface after the reduction treatment step. Method.
( 6 ) The plastic surface treatment method according to ( 5 ), wherein the introduction of the second functional group comprises bonding of an alkylsilane compound and the plastic surface.
( 7 ) The plastic surface treatment method according to ( 6 ), wherein the alkylsilane compound includes an aminoalkylsilane having an amino group, and the second functional group is an amino group.
( 8 ) The plastic surface treatment method according to ( 7 ), wherein the aminoalkylsilane compound contains 3-aminopropyltrimethoxysilane.
( 9 ) A plastic substrate having a second functional group introduced on the surface of the plastic substrate by the plastic surface treatment method according to any one of ( 5 ) to ( 8 ).
( 10 ) The method for treating a plastic surface according to any one of ( 5 ) to ( 8 ), further comprising the step of introducing a third functional group through the second functional group after introducing the second functional group. .
( 11 ) The plastic surface treatment method according to ( 7 ) or ( 8 ), further comprising a step of introducing a third functional group through the amino group after introducing the amino group.
( 12 ) The plastic surface treatment method according to ( 11 ), wherein the step of introducing a third functional group comprises a step of introducing an aldehyde group via an amino group by reacting with a polyfunctional aldehyde.
( 13 ) The plastic surface treatment method according to ( 12 ), wherein the polyfunctional aldehyde is glutaraldehyde.
( 14 ) A plastic substrate having a third functional group introduced on the surface of the plastic substrate by the plastic surface treatment method according to any one of ( 10 ) to ( 13 ).
( 15 ) A plastic biochip in which a physiologically active substance is further immobilized on the surface of the plastic substrate according to ( 9 ) via a second functional group.
( 16 ) A plastic biochip in which a physiologically active substance is further immobilized on the surface of the plastic substrate according to ( 14 ) via a third functional group.
( 17 ) The plastic biochip as set forth in ( 15 ) or ( 16 ), wherein the physiologically active substance has a property of specifically capturing another substance.
( 18 ) The plastic biochip according to ( 17 ), wherein the physiologically active substance is at least one selected from nucleic acids, proteins, antibodies, sugar chains, glycoproteins, and peptide nucleic acids.
( 19 ) The plastic biochip as set forth in any one of ( 15 ) to ( 18 ), wherein the physiologically active substance is detected by a fluorescent signal.

  According to the method of the present invention, a plastic substrate with a small amount of fluorescence generation can be produced. Such a plastic substrate is useful as a biochip substrate for detecting a signal by fluorescence.

The characteristics required when producing a plastic substrate for a biochip are the following two. That is, (1) a functional group for immobilizing a physiologically active substance (nucleic acid, protein, antibody, sugar chain, etc.) is introduced on the surface, and (2) the substrate itself has noise during signal detection. (Background) is not. Since fluorescence is often used for biochip signal detection, the amount of fluorescence generated on the surface of the plastic substrate is required to be low.
Generally, since the surface of plastic is inactive, it is difficult to bind a surface treatment agent such as alkylsilane as it is. For this reason, it is common to perform an oxidation treatment as a pretreatment. The surface treatment of the plastic of the present invention is characterized in that the proportion of hydroxyl groups in the surface functional groups is increased by reducing the oxygen-containing functional groups generated by the oxidation treatment. By introducing the oxygen-containing functional group by oxidation treatment, the reactivity of the plastic surface is improved, and the affinity with the surface treatment agent is increased. When an active functional group such as an aldehyde group is introduced by the oxidation treatment, the surface treatment performed after the oxidation treatment may be hindered. For example, consider the case where an alkylsilane having an amino group is used as the surface treatment agent. When an aldehyde group is introduced by the oxidation treatment, a Schiff base is formed with the amino group of the alkylsilane. The inventor has found that the presence of a Schiff base on the surface increases the amount of fluorescence generated on the plastic surface. Therefore, in order to reduce the amount of fluorescence generated, it is effective to convert carbonyl and other chemically active functional groups generated by the oxidation treatment into more inactive functional groups in advance.

  The plastic surface treatment method of the present invention mainly includes three steps. That is, (1) introduction process of oxygen-containing functional group by oxidation treatment, (2) conversion process of active functional group to more inactive functional group, (3) introduction process of second functional group by surface treatment agent It is. Each step will be described in detail below.

(Oxidation treatment)
In the present invention, the oxidation treatment refers to a treatment for introducing an oxygen-containing functional group into at least a part of the plastic surface. Means suitably used as the oxidation treatment are plasma treatment, corona discharge treatment, flame treatment, ultraviolet irradiation treatment, etc., preferably plasma treatment and corona discharge treatment, more preferably plasma treatment, most preferably oxygen treatment. This is a plasma treatment under an atmosphere. The oxygen-containing functional group is a group of polar functional groups represented by carbonyl group (including aldehyde group, ketone group, etc.), carboxyl group, hydroxyl group, ether group, ester group, peroxide group, epoxy group, etc. However, it is not limited to these. By introducing the oxygen-containing functional group by oxidation treatment, the reactivity of the plastic surface is improved, and the affinity with the surface treatment agent is increased.

(Reduction treatment)
The step of converting an active functional group into a more inactive functional group preferably has a reduction reaction as a main mechanism. This step is preferably an operation in which a reducing agent is allowed to act on the plastic surface, and specifically, for example, an operation of immersing the plastic substrate in a reducing agent solution. As the reducing agent, metal hydrides are preferable, and sodium borohydride, sodium cyanotrihydroborate, lithium aluminum hydride, and the like can be used. Sodium borohydride is preferable from the viewpoint of safety in handling. The reducing agent solution is water, a buffer solution, or a solution in which the reducing agent is dissolved in another solvent, and the most preferable solvent is a buffer solution having a pH of 6 to 8 containing ethanol. Preferably, 5 to 50% by volume, more preferably 10 to 30% by volume of ethanol is added to the buffer solution. By adding ethanol, it is possible to suppress the generation of bubbles when the reducing agent is dissolved in the buffer solution, and a more uniform reduction treatment is possible. The concentration of the reducing agent in the reducing agent solution is preferably 0.001 to 10% by weight, more preferably 0.01 to 1% by weight, still more preferably 0.1 to 0.5% by weight, and most preferably 0. .25% by weight. By the above reducing agent treatment, for example, an aldehyde group is converted into an alcohol group (hydroxyl group) which is a more inactive functional group.

(Reaction of surface treatment agent)
On the surface of the plastic substrate that has undergone oxidation treatment and reduction treatment, a first functional group mainly composed of hydroxyl groups is introduced. Various second functional groups can be introduced by reacting this with a surface treatment agent. Alkylsilane is preferably used as the surface treatment agent. There are a wide variety of functional groups that can be introduced by alkylsilanes, and those useful as biochip substrates include amino groups, carboxyl groups, epoxy groups, hydroxyl groups, acid anhydrides, thiol groups, and the like. Of these, aminoalkylsilanes having an amino group (such as a primary amino group) are particularly useful. Examples of the aminoalkylsilane having a primary amino group include 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-2- (aminoethyl) -3-aminopropyltrimethoxysilane, N-2- ( Common materials such as aminoethyl) -3-aminopropyltriethoxysilane can be used, but 3-aminopropyltrimethoxysilane is most preferred. As an aminoalkylsilane not containing a primary amino group, for example, N, N′-bis [3- (trimethoxysilyl) propyl] ethylenediamine and the like can be suitably used.

For the reaction of the alkylsilane on the plastic substrate, a method of immersing the substrate in an alkylsilane solution is suitable. As a solvent, water, a buffer solution, alcohol, etc. can be used conveniently, and water or alcohol is more preferable. The concentration of the alkylsilane is preferably 0.1 to 10% by weight, more preferably 0.5 to 5% by weight, further preferably 1 to 3% by weight, and most preferably 2% by weight. It is preferable to perform washing and drying following immersion in the alkylsilane solution. The drying is most preferably performed by vacuum drying at 20 to 100 ° C. In addition, for the reaction of the alkylsilane on the substrate surface, a method such as spraying an alkylsilane solution onto the substrate surface or exposing the substrate surface to an alkylsilane vapor can be used as appropriate.
When the above reduction treatment is not performed, an active carbonyl group such as an aldehyde group is present on the plastic surface. When aminoalkylsilane is reacted here, a Schiff base is formed between the aldehyde group and the amino group. Since the Schiff base tends to emit fluorescence, the fluorescence generation amount (background) of the substrate increases. On the other hand, when the reduction treatment is performed according to the present invention, since no active carbonyl group is substantially present on the plastic surface, no Schiff base is formed and the amount of fluorescence generated does not increase.

(Reaction of second surface treatment agent)
The third functional group can be introduced by reacting a substance having reactivity with an amino group or the like on the plastic surface into which the amino group or the like has been introduced by the above-described means. Examples of the third functional group include, but are not limited to, an aldehyde group, a carboxyl group, an epoxy group, a hydroxyl group, an acid anhydride, and a thiol group. Among them, an aldehyde group can be exemplified as a particularly useful functional group. Introduction of an aldehyde group can be achieved, for example, by allowing a compound having two or more aldehyde groups in the molecule (polyfunctional aldehyde) to act. That is, an aldehyde group can be introduced on the surface by reacting an amino group introduced on the plastic surface with at least one aldehyde group in the polyfunctional aldehyde molecule. For example, glutaraldehyde can be suitably used as the polyfunctional aldehyde. The reaction of glutaraldehyde on the substrate surface is achieved, for example, by immersing the substrate in a glutaraldehyde solution. In this case, the concentration of glutaraldehyde is preferably 0.1 to 20% by weight, more preferably 0.1 to 5% by weight, further preferably 0.5 to 5% by weight, and most preferably 0.5 to 2% by weight. %. As the solvent, water, a buffer solution, and other organic solvents can be used, but a buffer solution is particularly preferable. The pH of the buffer solution is preferably 6 to 14, more preferably 6 to 12, and most preferably 6 to 11. It is possible to change the amount of glutaraldehyde introduced by changing the temperature of the glutaraldehyde solution during immersion and the immersion time.

(Substrate material)
As a material for the biochip substrate, glass, plastic, metal or the like is generally used. From the viewpoint of easy surface treatment and mass productivity, plastic is particularly preferable, and thermoplastic resin is particularly preferable. As the thermoplastic resin, those having a small amount of fluorescence generation are preferable, for example, linear polyolefins such as polyethylene and polypropylene, cyclic polyolefins, fluorine-containing resins, etc. are preferably used, heat resistance, chemical resistance, low fluorescence, It is more preferable to use a saturated cyclic polyolefin that is particularly excellent in moldability. Here, the saturated cyclic polyolefin refers to a saturated polymer obtained by hydrogenating a polymer having a cyclic olefin structure or a copolymer of a cyclic olefin and an α-olefin.

(Biochip)
The physiologically active substance can be immobilized via the second or third functional group on the plastic substrate having the second or third functional group introduced on the surface by the above-described means. Examples of the physiologically active substance include, but are not limited to, nucleic acids, proteins, antibodies, sugar chains, glycoproteins, and the like. The physiologically active substance is preferably immobilized by a chemical bond between the second or third functional group on the surface of the plastic substrate and the functional group contained in the physiologically active substance. It may be due to hydrogen bonding or the like. It is preferable that the physiologically active substance to be immobilized substantially maintains the original physiological activity. Here, the original physiological activity is, for example, a hybridization phenomenon in the case of a nucleic acid and an antigen recognition ability in the case of an antibody. Information on the specimen can be obtained by reacting a physiologically active substance immobilized on the substrate with a physiologically active substance (specimen) separately prepared and quantifying the reaction. A method generally used as a means of quantification is a method in which a specimen is fluorescently labeled and the amount of fluorescence is read. Since the plastic substrate of the present invention has a low fluorescence generation amount, it can be suitably used as a biochip substrate using a fluorescent label for quantitative determination. It can also be used as a biochip substrate that does not use a fluorescent label.

Example 1
(Oxidation treatment, reduction treatment)
A saturated cyclic polyolefin resin was molded by an injection molding method to produce a flat substrate having a thickness of 1 mm. The substrate surface was oxidized by plasma treatment in an oxygen atmosphere. The substrate immediately after the plasma treatment was immersed in a reducing agent solution for 15 minutes. Here, the reducing agent solution was a solution in which 0.25 wt% sodium borohydride was dissolved in a phosphate buffer solution (pH 7.4) / ethanol mixed solvent (volume ratio 4: 1). After immersion, the substrate was washed with pure water and dried.
(Introduction of amino group)
3-aminopropyltrimethoxysilane was dissolved in pure water at a concentration of 2% by weight. The above-mentioned substrate was immersed in this solution. Immersion was performed at 25 ° C. for 1 hour. After immersion, the substrate was washed with pure water and dried. This was vacuum dried using a vacuum dryer maintained at 45 ° C.
(Measurement of substrate fluorescence)
The amount of fluorescence generated on the substrate surface after introduction of amino groups was measured using a microarray scanner (ScanArrayLITE, manufactured by Packard BioChip Technologies). The measurement conditions were laser output 90%, PMT sensitivity 70%, scan resolution 30 μm, and measurement wavelengths Cy3 and Cy5 channels. The results are shown in Table 1.
(Evaluation of DNA immobilization / hybridization performance)
DNA (β-actin site, approximately 600 bp) synthesized by PCR was dissolved in 2 × SSC (citrate buffer). This solution was spotted on the surface of the substrate into which amino groups had been introduced, and was left in an oven at 80 ° C. for 1 hour. After washing the substrate surface, the remaining amino groups were blocked by immersing in a buffer solution containing 0.1 wt% BSA (bovine serum albumin) for 1 hour. Fluorescently labeled DNA (β-actin site, approximately 600 bp, Cy5 label) synthesized by PCR was dissolved in 5 × SSC containing 0.5 wt% sodium lauryl sulfate. This solution was dropped onto the surface of the substrate after blocking, covered with a cover glass, and then reacted in an incubator at 50 ° C. for 18 hours. After completion of the reaction, the substrate surface was washed and dried. The amount of binding between the DNA immobilized on the substrate surface and the fluorescently labeled DNA was quantified using a microarray scanner. The measurement conditions were laser output 90%, PMT sensitivity 45%, scan resolution 30 μm, and measurement wavelength Cy5 channel. The results are shown in Table 1.

Comparative Example 1
(Oxidation treatment)
A substrate was molded in the same manner as in Example 1, and the surface was oxidized. No reduction treatment was performed after the oxidation treatment.
(Introduction of amino group)
An amino group was introduced onto the substrate surface in the same manner as in Example 1.
(Measurement of substrate fluorescence)
The amount of fluorescence generated on the substrate surface was measured in the same manner as in Example 1. The results are shown in Table 1.
(Evaluation of DNA immobilization / hybridization performance)
DNA was immobilized in the same manner as in Example 1, and the amount of fluorescently labeled DNA bound was measured. The results are shown in Table 1.

  Comparing the results of the example and the comparative example, it can be seen that the amount of fluorescence generated from the substrate surface is low in the example. In addition, there was no significant difference between Examples and Comparative Examples in DNA immobilization / hybridization evaluation. Furthermore, when the signal-to-noise ratio (S / N ratio) was determined, a significant improvement of 45 in the example was recognized compared to 15 in the comparative example. From the above results, it was shown that the surface treatment method of the present invention is excellent as a treatment method for producing a biochip substrate.

  According to the present invention, it is possible to produce a plastic biochip substrate with reduced fluorescence generation. Reduction in the amount of fluorescence generated on the substrate leads to a reduction in noise (background) when reading the signal on the biochip surface as the amount of fluorescence, and as a result, the signal-to-noise ratio of the biochip is improved. The plastic surface treatment method and plastic substrate of the present invention can be effectively used in the production of biochips such as DNA microarrays (DNA chips), protein arrays, antibody arrays, and sugar chain arrays.

Claims (19)

A step of oxidizing the plastic surface, and has a step of reduction treatment an oxygen-containing functional groups produced by oxidation treatment, met the surface treatment method for a plastic, which comprises introducing a first functional group to the plastic surface The oxidation treatment includes at least one treatment selected from plasma treatment, corona discharge treatment, flame treatment, and ultraviolet irradiation treatment, and the oxygen-containing functional group to be reduced contains a carbonyl group. A plastic surface treatment method, wherein the reduction treatment comprises a step of immersing in a solution containing a metal hydride . The surface treatment method of plastic according to claim 1, wherein the carbonyl group is one that contains an aldehyde group and / or ketone group. The plastic surface treatment method according to claim 1 or 2 , wherein the oxidation treatment includes a plasma treatment in an oxygen atmosphere. The plastic surface treatment method according to any one of claims 1 to 3, wherein the metal hydride contains sodium borohydride and / or lithium aluminum hydride. The method for treating a plastic surface according to any one of claims 1 to 4 , further comprising a step of introducing a second functional group through the first functional group introduced to the plastic surface after the reduction treatment. The plastic surface treatment method according to claim 5 , wherein the introduction of the second functional group is performed by bonding an alkylsilane compound and the plastic surface. The plastic surface treatment method according to claim 6 , wherein the alkylsilane compound includes an aminoalkylsilane having an amino group, and the second functional group is an amino group. The plastic surface treatment method according to claim 7 , wherein the aminoalkylsilane compound contains 3-aminopropyltrimethoxysilane. A plastic substrate in which a second functional group is introduced on the surface of the plastic substrate by the plastic surface treatment method according to any one of claims 5 to 8 . The plastic surface treatment method according to any one of claims 5 to 8 , further comprising a step of introducing a third functional group through the second functional group after introducing the second functional group. The plastic surface treatment method according to claim 7 or 8 , further comprising a step of introducing a third functional group through the amino group after the amino group is introduced. The plastic surface treatment method according to claim 11, wherein the step of introducing the third functional group includes a step of introducing an aldehyde group via an amino group by reacting with a polyfunctional aldehyde. The plastic surface treatment method according to claim 12, wherein the polyfunctional aldehyde is glutaraldehyde. Third functional group introduced plastic substrates to claim 10-13 plastic substrate surface by surface treatment method of plastic according to any one. A plastic biochip in which a physiologically active substance is further immobilized on the surface of the plastic substrate according to claim 9 via a second functional group. A plastic biochip in which a physiologically active substance is further immobilized on the surface of the plastic substrate according to claim 14 via a third functional group. The plastic biochip according to claim 15 or 16 , wherein the physiologically active substance has a property of specifically capturing another substance. The plastic biochip according to claim 17 , wherein the physiologically active substance is at least one selected from nucleic acids, proteins, antibodies, sugar chains, glycoproteins, and peptide nucleic acids. The plastic biochip according to any one of claims 15 to 18, wherein the physiologically active substance is detected by a fluorescence signal.