JP3940027B2 - Method for producing substrate for nucleic acid sensor and method for producing nucleic acid sensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid sensor substrate for immobilizing a nucleic acid on an electrode.
[0002]
[Prior art]
Conventionally, an electrode having a structure in which a nucleic acid serving as a probe is immobilized on a conductor in order to detect the presence of a specific base sequence or a specific gene has been proposed in JP-A-10-146183 (hereinafter referred to as Conventional Example A). ing. As described in the conventional example A, in recent years, the relationship between nucleic acid base sequences and diseases has been clarified. By detecting the presence of a specific base sequence or a specific gene, it is possible to cause disease. Genetic diagnosis that explores sex and the degree of disease progression has become an important part of clinical testing. In general, an electrode for detecting the presence of a specific base sequence or a specific gene has a structure in which a nucleic acid to be a probe is fixed on a conductor, and the presence of a specific base sequence or a specific gene is a nucleic acid to be a probe. The pair (hybrid) formed between the sample and the sample nucleic acid can be detected by applying an electric voltage to the probe nucleic acid and measuring an electrical or optical signal.
[0003]
In Conventional Example A, since the conventional electrode has a structure in which a wire electrode is embedded in a resin, there is a problem that the surface area on which the nucleic acid for the probe can be immobilized tends to vary. A conductor and an insulator having an opening so as to expose a part of the conductor are formed on the upper surface of the substrate, and an electrode is formed by immobilizing nucleic acid on the conductor exposed at the opening.
[0004]
[Problems to be solved by the invention]
However, the electrode of Conventional Example A has a problem that it cannot sufficiently cope with further increase in detection sensitivity, and as a result, reproducibility and quantitativeness are insufficient. That is, in order to further increase the detection sensitivity, it is necessary to increase the amount of nucleic acid immobilized on the electrode by increasing the orientation index of the (111) plane of the crystal structure of the gold layer that is the uppermost layer of the electrode. Although effective, it has not been achieved so far to significantly increase the orientation index of the (111) plane of the crystal structure of the gold layer.
[0005]
Therefore, the present invention has been completed in view of the above-mentioned problems, and its purpose is to increase the orientation of the (111) plane of the crystal structure of the gold layer on the uppermost surface of the electrode. The detection sensitivity is further increased by increasing the amount of the nucleic acid immobilized on the electrode for immobilizing the nucleic acid.
[0006]
[Means for Solving the Problems]
The method for producing a nucleic acid sensor substrate of the present invention comprises a step of preparing an insulating substrate made of silicon, a step of forming a first silicon oxide layer on the insulating substrate by a thermal oxidation method, and a vacuum film forming technique. Forming a second silicon oxide layer having a thickness of 0.005 to 1 μm on the first silicon oxide layer, and forming a conductor layer having a thickness of 0.05 to 1 μm on the second silicon oxide layer. And a step of forming a gold layer on which the nucleic acid is immobilized on the conductor layer.
[0007]
The method for producing a nucleic acid sensor substrate of the present invention is characterized in that the second silicon oxide layer is formed using nitrogen gas.
[0008]
The nucleic acid sensor substrate manufacturing method of the present invention is characterized in that the conductor layer is made of a nickel-chromium alloy.
[0009]
It has the process of fixing the said nucleic acid through the thiol group to the said gold | metal layer of the substrate for nucleic acid sensors manufactured by the manufacturing method in any one of the above-mentioned.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
An example of an embodiment of the substrate for nucleic acid sensors of the present invention is shown in FIG. In FIG. 1, 1 is an insulating substrate made of ceramics such as silicon (Si) and alumina (Al ₂ O ₃ ) ceramics, and 2 is formed on the upper surface of the insulating substrate 1 by thermal oxidation when Si is Si. An oxide layer. 3 is a silicon oxide (SiO ₂ ) layer having a thickness of 1 μm or less formed on the upper surface of the oxide layer 2 by a vacuum film forming technique such as sputtering, 4 is a conductor layer and a gold layer made of a thin film, and 4a is an adhesion metal layer. 4b is a diffusion prevention layer and 4c is a gold layer. Reference numeral 5 denotes a nucleic acid immobilized on the upper surface of the gold layer 4c. The adhesion metal layer 4a and the diffusion prevention layer 4b correspond to conductor layers having a thickness of 1 μm or less.
[0011]
When the insulating substrate 1 is made of Si, the oxide layer 2 is preferably formed on the upper surface thereof. The oxide layer 2 is important for insulating Si, which is a semiconductor, and is generally formed using a thermal oxidation furnace exceeding 1000 ° C. Its thickness is preferably about 0.1 to 3 μm. If the thickness is less than 0.1 μm, the insulation between Si and the conductor layer 4 cannot be sufficiently maintained. If the thickness exceeds 3 μm, peeling from the insulating substrate 1 tends to occur due to the residual stress of the oxide layer 2.
[0012]
Examples of the material for the insulating substrate 1 of the present invention include silicon, glass, quartz glass, alumina ceramics, mullite (3Al ₂ O ₃ .2SiO ₂ ) ceramics, cordierite (2MgO.2Al ₂ O ₃ .5SiO ₂ ) ceramics, stellite (2MgO · SiO ₂₎ ceramics, silicon carbide (SiC) ceramics, silicon nitride (Si ₃ N ₄₎ ceramics, or inorganic materials such as sapphire (single crystal alumina). Polyethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate Organic materials such as polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used.
[0013]
A silicon oxide (SiO ₂ ) layer 3 is formed on the main surface of the insulating substrate 1 or the upper surface of the oxide layer 2 by a vacuum film formation technique such as vacuum deposition, sputtering, or CVD. The thickness is 1 μm or less, and if it exceeds 1 μm, the silicon oxide layer 3 is easily damaged from the inside due to the stress of the conductive layer made of a thin film formed on the upper surface and the gold layer 4. The thickness is preferably 0.1 μm or less, and if it is thinner than this, no breakage occurs. Further, the thickness of the silicon oxide layer 3 is 0.005 μm or more, and if it is less than 0.005 μm, the variation in thickness becomes large during film formation.
[0014]
By forming the silicon oxide layer 3 having a thickness of 0.005 to 1 μm, the orientation of the (111) plane of the crystal structure of the gold layer 4c, which is the uppermost layer of the nucleic acid fixing electrode, is improved. Although the principle is not known in detail, when the orientation of the (111) plane of the crystal is quantitatively confirmed by the X-ray diffraction method for the gold layer 4c, it is affected by the orientation in the absence of the silicon oxide layer 3. The peak intensity ratio of the (111) plane of the gold layer 4c based on the difficult (311) plane peak intensity is about 16000, whereas the peak intensity ratio when the silicon oxide layer 3 is present is about 32000, for example. It was found that the intensity ratio was about twice.
[0015]
In the present invention, the peak intensity of the (111) plane with respect to the peak intensity of the (311) plane by the X-ray diffraction method of the gold layer 4c is preferably 25000 times or more. In this case, 10 ¹¹ nucleic acids are applied to the surface of the gold layer 4c. It can be fixed at about copy / cm ² or more, and the detection sensitivity of nucleic acid is greatly improved. More preferably, the peak intensity of the (111) plane with respect to the peak intensity of the (311) plane by the X-ray diffraction method should be 30000 times or more. In this case, the nucleic acid is fixed to the surface of the gold layer 4c by about 10 ¹³ copies / cm ² or more. The detection sensitivity of nucleic acids is greatly improved. As described above, by adopting the above-described configuration of the present invention, the gold layer 4c with improved orientation of the (111) plane can be formed, and as a result, the nucleic acid detection sensitivity is improved.
[0016]
Further, when nitrogen is added to the atmospheric gas when the silicon oxide film 3 is formed by a vacuum film formation technique, the silicon oxide film 3 becomes a silicon oxynitride film containing nitrogen, and chemical resistance is improved.
[0017]
The conductive layer and the gold layer 4 made of a thin film are formed by a thin film forming method such as a vapor deposition method, a sputtering method, a CVD method, or a plating method, and are processed into a predetermined pattern by a photolithography method, an etching method, a lift-off method, or the like.
[0018]
The conductor layer and the gold layer 4 may have a three-layer structure in which, for example, an adhesion metal layer 4a, a diffusion prevention layer 4b, and a gold layer 4c are sequentially laminated. That is, the adhesion metal layer 4a firmly adheres the nucleic acid immobilization electrode to the insulating substrate 1, and the diffusion prevention layer 4b prevents mutual diffusion between the gold layer 4c and the adhesion metal layer 4a.
[0019]
The adhesion metal layer 4a is preferably composed of at least one of Ti, Cr, Ta, Nb, Ni—Cr alloy, Ta ₂ N, and the like in terms of adhesion with the insulating substrate 1. The thickness of the adhesion metal layer 4a is preferably about 0.01 to 0.2 μm. If it is less than 0.01 μm, it is difficult to firmly adhere, and if it exceeds 0.2 μm, peeling tends to occur due to internal stress during film formation.
[0020]
The diffusion prevention layer 4b is made of at least one of Pt, Pd, Rh, Ni, Ni—Cr alloy, Ti—W alloy and the like in order to prevent mutual diffusion between the adhesion metal layer 4a and the gold layer 4c. good. The thickness of the diffusion preventing layer 4b is preferably about 0.05 to 1 [mu] m, and if it is less than 0.05 [mu] m, defects such as pinholes are generated and the function as the diffusion preventing layer 4b becomes difficult. If it exceeds 1 μm, peeling tends to occur due to internal stress during film formation. When a Ni—Cr alloy is used for the diffusion preventing layer 4b, the adhesion can be secured, so that the adhesion metal layer 4a can be omitted.
[0021]
The total thickness of the adhesion metal layer 4a and the diffusion prevention layer 4b as the conductor layers is 0.05 to 1 μm. If the thickness is less than 0.05 μm, the conductor layer and the silicon oxide layer 3 cannot be firmly adhered, and mutual diffusion between the adhesion metal layer 4a and the gold layer 4c cannot be prevented. If the thickness exceeds 1 μm, the improvement in the orientation of the gold layer 4c due to the provision of the silicon oxide layer 3 becomes difficult to be exhibited.
[0022]
Furthermore, the thickness of the gold (Au) layer 4c is preferably about 0.1 to 5 μm. If it is less than 0.1 μm, the electric resistance tends to increase. If it exceeds 5 μm, peeling tends to occur due to internal stress during film formation. Further, since Au is a noble metal and expensive, it is preferable to form it as thin as possible in terms of cost reduction.
[0023]
When nucleic acid 5 is immobilized on the upper surface of the nucleic acid immobilization electrode, the surface is washed with sulfuric acid, mixed acid, aqua regia, hydrogen chloride, etc., and then a thiol group is introduced at the 5 ′ or 3 ′ end of nucleic acid 5 One end of the nucleic acid 5 is adsorbed on the surface of the gold layer 4c through the group and firmly fixed. Nucleic acid 5 may be single-stranded DNA (Deoxyribonucleic Acid) or single-stranded RNA (ribonucleic acid: ribonucleic acid), and the number of bases is not particularly limited, but 15 to 3000 bases. Any length is acceptable.
[0024]
Nucleic acids detected as a pair (hybrid) with the nucleic acid 5 immobilized on the surface of the nucleic acid immobilizing electrode include nucleic acids such as various viruses, bacteria, parasites and fungi, and nucleic acids of cells having various diseases, Alternatively, it may be a gene of cells of various viruses or various diseases, or a part of those genes.
[0025]
The hybridized nucleic acid 5 can be detected by measuring the light emission intensity using the property that the nucleic acid emits light when a voltage is applied. Thereby, the nucleic acid having the same base sequence as the nucleic acid 5 present in the measurement target can be detected by detecting the amount of the paired (hybridized) nucleic acid 5 on the nucleic acid fixing electrode. Further, the amount of nucleic acid 5 paired on the nucleic acid fixing electrode can be electrically detected. That is, the nucleic acid 5 on the nucleic acid immobilizing electrode is labeled with an electrochemically active substance such as Hoechst 33258, an insertion agent, phylocenes, and a metal complex, and a voltage is applied to the nucleic acid 5 to cause the above substance. An electric signal is detected.
[0026]
The means for applying a voltage to the nucleic acid immobilization electrode is a thin film of a lead wire made of Au or the like formed by being drawn out from the part of the upper surface of the gold layer 4c onto the oxide layer 2 via the side surface of the nucleic acid immobilization electrode. It can be provided by a forming method or by bonding a bonding wire made of Au, Al or the like on the upper surface of the gold layer 4c.
[0027]
【Example】
Examples of the substrate for nucleic acid sensors of the present invention will be described below.
[0028]
The nucleic acid sensor substrate of FIG. 1 was constructed as follows. The insulating substrate 1 made of Si was placed in a thermal oxidation furnace and oxidized at about 1000 ° C. for about 9 hours to form an oxide layer 2 having a thickness of about 0.3 μm on the surface of the insulating substrate 1. A silicon oxide layer 3 having a thickness of 0.02 μm was formed on a part of the upper surface of the oxide layer 2 by sputtering. An adhesion metal layer 4a made of Ti having a thickness of 0.1 μm, a diffusion preventing layer 4b made of Pt having a thickness of 0.2 μm, and a gold layer 4c having a thickness of 0.5 μm are formed on the upper surface of the silicon oxide layer 3 by sputtering, A nucleic acid sensor substrate (sample A) was prepared.
[0029]
Further, as Comparative Example 1, a nucleic acid sensor substrate (sample B) was prepared in the same manner as in the above example except that the silicon oxide layer 3 was not provided.
[0030]
As Comparative Example 2, a nucleic acid sensor substrate (sample C) was prepared in the same manner as in the above example except that the thickness of the silicon oxide layer 3 was 3 μm.
[0031]
As Comparative Example 3, a nucleic acid sensor substrate (sample D) was prepared in the same manner as in the above example except that the total thickness of the adhesion metal layer 4a and the diffusion preventing layer 4b was 2 μm.
[0032]
As Comparative Example 4, a nucleic acid sensor substrate (sample E) was prepared in the same manner as in the above example except that the thickness of the silicon oxide layer 3 was 0.001 μm.
[0033]
As Comparative Example 5, a nucleic acid sensor substrate (sample F) was prepared in the same manner as in the above example except that the total thickness of the adhesion metal layer 4a and the diffusion preventing layer 4b was 0.01 μm.
[0034]
For each of 10 samples A to F, the orientation of the (111) plane of the gold layer 4c was measured by an X-ray diffraction method using an X-ray of Cu element Kα. In the sample A, the peak intensity ratio of the (111) plane of the gold layer 4c on the basis of the peak intensity of the (311) plane which is hardly affected by the orientation was about 32000 on average. In contrast, sample B had an average peak intensity ratio of about 16000, and sample C had an average peak intensity ratio of about 31000. Nine cracks occurred in the silicon oxide layer 3 and were defective. became. Sample D had an average peak intensity ratio of about 22000. Sample E had an average peak intensity ratio of about 17000, and sample F was a defective product because peeling occurred between the silicon oxide layer 3 and the adhesion metal layer 4a in all ten samples.
[0035]
From the above, it can be expected that sample A can immobilize nucleic acid 5 by about 10 ¹⁴ copies / cm ² , whereas sample B has about 10 ⁸ copies / cm ² , sample D has about 10 ¹⁰ copies / cm ² , sample E Was found to be expected only at about 10 ⁸ copies / cm ² . Further, in sample C, cracks are likely to occur in the silicon oxide layer 3, and in sample F, peeling occurs between the silicon oxide layer 3 and the adhesion metal layer 4a, so that the samples C and F have poor yield and the substrate for nucleic acid sensors. Found to be difficult to use as.
[0036]
The present invention is not limited to the above-described embodiments and examples, and various modifications may be made without departing from the scope of the present invention.
[0037]
【The invention's effect】
The nucleic acid sensor substrate of the present invention has a nucleic acid immobilization electrode on which one end of nucleic acid is immobilized on the surface of an insulating substrate. The nucleic acid immobilization electrode is a silicon oxide layer having a thickness of 0.005 to 1 μm. And a 0.05 to 1 μm thick conductor layer and a gold layer are sequentially laminated, and the orientation of the (111) plane of the crystal structure of the gold layer, which is the uppermost layer of the nucleic acid immobilizing electrode, is dramatically increased. As a result, a large number of nucleic acids can be immobilized on the surface of the gold layer, and thus a highly sensitive nucleic acid sensor substrate can be provided. Further, by setting the thickness of the conductor layer to 0.05 to 1 μm, it becomes easy to improve the orientation of the gold layer due to the provision of the silicon oxide layer.
[0038]
In the nucleic acid sensor substrate of the present invention, preferably, the gold layer has a peak intensity of the (111) plane of 25000 times or more of the peak intensity of the (311) plane by X-ray diffractometry of the crystal. Nucleic acids can be immobilized on the surface of about 10 ¹¹ copies / cm ² or more, and the nucleic acid detection sensitivity is greatly improved.
[Brief description of the drawings]
FIG. 1 is a cross-sectional view showing an example of an embodiment of a nucleic acid sensor substrate of the present invention.
[Explanation of symbols]
1: Insulating substrate 2: Oxide layer 3: Silicon oxide layer 4a: Adhesion metal layer 4b: Diffusion prevention layer 4c: Gold layer 5: Nucleic acid

Claims (4)

Preparing an insulating substrate made of silicon;
Forming a first silicon oxide layer on the insulating substrate by a thermal oxidation method;
Forming a second silicon oxide layer having a thickness of 0.005 to 1 μm on the first silicon oxide layer by a vacuum film formation technique;
Forming a conductor layer having a thickness of 0.05 to 1 μm on the second silicon oxide layer;
Forming a gold layer on which the nucleic acid is immobilized on the conductor layer. The method for producing a nucleic acid sensor substrate according to claim 1, wherein the second silicon oxide layer is formed using nitrogen gas. The method for producing a nucleic acid sensor substrate according to claim 1, wherein the conductor layer is made of a nickel-chromium alloy. A nucleic acid sensor production comprising a step of fixing the nucleic acid to the gold layer of the nucleic acid sensor substrate produced by the production method according to claim 1 through a thiol group. Method

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