JP4136418B2 - Method for producing DNA microarray - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a DNA microarray that can be used in a method for electrochemically detecting DNA or RNA.
[0002]
[Prior art]
Conventionally, there is a DNA microarray (also referred to as a DNA chip) as an analysis unit for efficiently analyzing a large amount of gene structure.
[0003]
As a DNA microarray, there is a method in which probe DNA is applied to an electrode type sensor (see JP-A-9-288080). That is, an electrode having an output terminal, which is a hybrid formed by the probe DNA and the sample DNA by reacting the electrode on which the probe DNA is immobilized with the sample DNA and measuring the current of the electrode after the reaction. This is a method for detecting the presence of DNA or measuring the amount of hybrid DNA. That is, by introducing an electrode bound to a probe DNA modified with a conductive substance into a sample solution containing the sample DNA, the sample DNA having a sequence complementary to the probe DNA is hybridized to form a hybrid DNA. The This hybridization is performed in the presence of an intercalator. Intercalators penetrate between hybrid DNA layers to form a kind of charge transfer complex. If no hybrid is formed, that is, if it remains single-stranded, no intercalation occurs. The amount of current flowing through the electrode changes due to the intercalation. This current is due to the oxidation and reduction of the intercalator intercalated into the double-stranded DNA formed by hybridization. The degree of double-stranded DNA formation can be detected as the degree of intercalation. Therefore, the presence of hybrid DNA is detected by detecting and / or measuring the amount of current flowing through the electrode, or the amount of hybrid DNA is measured. A DNA microarray used for such an electrochemical detection method is also called an ECA (Electronic Chemical Array) chip.
[0004]
As an electrode of a DNA microarray used for an electrochemical detection method, there are electrodes in which different types of synthetic oligo DNAs are covalently bonded to a large number of fine gold electrodes arranged in an array formed by vapor deposition. (See JP 2000-50931). As means for supplying different types of synthetic oligo DNA to each gold electrode, there are a method using a photolithography method and a method of dropping a reactive substance with a dispenser.
[0005]
[Problems to be solved by the invention]
However, hundreds to tens of thousands of electrodes are often formed on the substrate of the DNA microarray. In such a case, there is a problem that a large number of mask processes are required in order to fix the nucleic acid by using the photolithography method. In addition, in order to immobilize nucleic acids using the dispenser method, problems such as non-uniformity in the concentration of nucleic acids due to washing of the pen tip, and high-precision alignment with fine electrodes formed on the substrate There is a problem that is necessary.
[0006]
Accordingly, an object of the present invention is to provide a method for producing a DNA microarray that can solve the above problems and can be produced without using a photolithography method or a dispenser method.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, the method for producing a DNA microarray of the present invention is configured as follows.
[0008]
In other words, the DNA microarray of the present invention continuously prints on the insulating substrate using the conductive ink so that the lead portion and the detection portion are in series, and then detects the conductive fine powder as undried. Then, the lead part and the detection part were dried, and then the nucleic acid was electrically immobilized on the conductive fine powder.
[0009]
In addition, on the insulating substrate, the lead portion is printed with conductive ink and dried, then the detection portion is printed with conductive ink so as to be in series with the lead portion, and then the conductive fine portion is printed. The powder may be attached to an undried detection unit, then the detection unit may be dried, and then the nucleic acid may be electrically immobilized on the conductive fine powder.
[0011]
In the above invention, the conductive fine powder may be composed of metal or graphite.
[0012]
In the above invention, a plurality of rows of lead portions and detection portions may be formed on the substrate.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described in detail with reference to the drawings.
[0014]
FIG. 1 is a cross-sectional view showing an embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention. 2 to 4 are cross-sectional views showing one step of the method for producing the DNA microarray of the present invention. FIG. 5 is a schematic diagram showing one step of the method for producing the DNA microarray of the present invention. 6 to 9 are cross-sectional views showing one step of the method for producing the DNA microarray of the present invention. FIG. 10 is a cross-sectional view showing an embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention. FIG. 11 is a plan view showing one step in the method for producing the DNA microarray of the present invention. FIG. 12 is a cross-sectional view showing an embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention. FIG. 13 is a plan view showing one embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention. In the figure, 1 is a DNA microarray, 2 is a substrate, 3 is a lead part, 4 is a detection part, 5 is a conductive fine powder, 6 is a nucleic acid, and 7 is a masking.
[0015]
In the method of manufacturing the DNA microarray 1 of the present invention, a conductive fine powder is printed on a substrate 2 having an insulating property using a conductive ink so that the lead portion 3 and the detection portion 4 are in series. In this method, 5 is attached to the undried detection part 4, then the lead part 3 and the detection part 4 are dried, and then the nucleic acid 6 is electrically immobilized on the conductive fine powder 5. The DNA microarray 1 referred to in the present invention includes not only DNA detection but also RNA detection.
[0016]
First, it prints continuously using a conductive ink so that the lead part 3 and the detection part 4 may become in series on the board | substrate 2 which has insulation (refer FIG. 2).
[0017]
The substrate 2 has only to be insulating and can be printed with conductive ink. For example, paper, polyethylene resin, ethylene resin, polypropylene resin, polyisobutylene resin, polyethylene terephthalate resin, unsaturated polyester resin. , Fluorine-containing resin, polyvinyl chloride resin, polyvinylidene chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyvinyl acetal resin, acrylic resin, polyacrylonitrile resin, polystyrene resin, acetal resin, polycarbonate resin, polyamide resin, phenol resin, Existence of urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide resin, polysulfone resin, etc. Materials and glasses, and inorganic materials such as alumina can be used. In the DNA microarray 1, in the case of a multi-layer configuration in which a plurality of substrates 2 on which the lead portion 3 and the detection portion 4 are formed are stacked, if each substrate 2 is made as thin as several tens of μm, the DNA microarray 1 can be further reduced in size, and the amount of sample to be brought into contact with the DNA microarray 1 is reduced, which is preferable.
[0018]
The conductive ink is composed of at least a conductive filler, a binder, and a solvent. As the conductive filler, copper powder, gold powder, graphite, carbon black, carbon fiber, nickel powder and the like, which are substances having excellent conductivity, can be used. Since silver powder may migrate due to elution in the sample solution, it is preferable to use other substances as fillers. As the binder, epoxy resin, alkyd resin, acrylic resin, polyurethane resin, melamine resin, phenol resin, vinyl chloride-vinyl acetate copolymer resin and the like can be used. As the solvent, a solvent that dissolves a resin used as a binder may be used. Moreover, you may add additives, such as a dispersing agent, a lubricant, and a coupling agent, as needed.
[0019]
Properties required for the conductive ink include conductivity, printability, and drying properties. If the conductivity is small, the detection signal becomes small and the influence of noise becomes large, so that it is difficult to obtain a correct detection result. In the present invention, the conductivity may be within a range that can be detected appropriately with accuracy according to the length of the lead portion 3 and the like, and is preferably 1500 Ω / cm ² or less in order to extract a stable detection signal with little variation.
[0020]
As a printing method of the conductive ink, for example, a normal printing method such as a screen printing method, a gravure printing method, an offset printing method, and an ink jet printing method can be used. What is necessary is just to select the printing method suitably according to the kind of conductive ink, the pattern shape of the lead part 3 and the detection part 4, etc. FIG.
[0021]
The lead part 3 is formed by printing with conductive ink. The lead part 3 is formed so that the terminal connected to the device for measuring the amount of current and the detection part 4 are connected by a circuit having a desired resistance value.
[0022]
The detection unit 4 is formed by printing conductive ink. The lead part 3 and the detection part 4 may be formed by printing in a continuous printing process in a single printing process so that the lead part 3 and the detection part 4 are arranged in series by printing conductive ink. it can. The detection unit 4 has a desired resistance value, and the conductive fine powder 5 needs to be firmly fixed. That is, in order to ensure that the conductive fine powder 5 can be fixed to the surface of the detection unit 4, the conductive ink film thickness of the printed detection unit 4 and the particle size of the conductive fine powder 5 to be attached are set as follows: It is good to select suitably. Further, when a plurality of types of nucleic acids 6 contained in a sample are detected at the same time, a plurality of detection units 4 are formed on the same substrate 2, and therefore the degree of integration can be achieved by forming the detection units 4 finely. Can be increased.
[0023]
Next, the conductive fine powder 5 is adhered to the undried detection unit 4 (see FIG. 3).
[0024]
As the conductive fine powder 5, one having conductivity and capable of immobilizing the nucleic acid 6 is used. For example, fine powder made of gold, glassy carbon, carbon or the like can be used. The shape and size of the conductive fine powder 5 may be any shape as long as it is formed on the entire surface of the conductive ink printed from the viewpoint of detection capability and reproducibility. For example, spherical, plate-like, needle-like, etc. Various shapes can be used. In particular, a spherical shape is preferable because it can be uniformly attached to the detection unit 4.
[0025]
The conductive fine powder 5 is adhered on the detection unit 4 until the conductive ink printed as the detection unit 4 on the substrate 2 is dried.
[0026]
As a method for attaching the conductive fine powder 5, any method can be used as long as the conductive fine powder 5 can be laminated on the surface of the detection unit 4 which is a printed conductive ink. For example, a method of naturally spraying the conductive fine powder 5 from above the printed conductive ink, a method of spraying the conductive fine powder 5, charging both the substrate 2 and the conductive fine powder 5, and utilizing static electricity There are a method of adsorbing, a method of passing the substrate 2 through a large amount of the conductive fine powder 5, and the like. After such a process, the excess conductive fine powder 5 can be removed with an air gun or the like, and the conductive fine powder 5 can be attached only to the surface of the detection unit 4 (see FIG. 4). Further, the lead 3 may be covered with a masking jig or the like in order to prevent the conductive fine powder 5 from adhering to the lead part 3 when the conductive fine powder 5 is dispersed.
[0027]
Next, the lead part 3 and the detection part 4 are dried.
[0028]
The conductive ink constituting the lead part 3 and the detection part 4 is dried by means such as heating, so that the conductive fine powder 5 is completely fixed to the detection part 4 so as not to peel off. By drying the conductive ink, the lead portion 3 and the detection portion 4 can be set to practical resistance values, and the adhesion with the conductive fine powder 5 can be further strengthened.
[0029]
Next, the nucleic acid 6 is electrically immobilized on the conductive fine powder 5. In the present invention, the nucleic acid 6 refers to a DNA fragment or an RNA fragment.
[0030]
There are the following methods for electrically immobilizing the nucleic acid 6 (see FIG. 5). That is, first, the detection unit 4 is inserted into an acetate buffer (pH 5), and a potential is applied at +1.5 V (vs Ag / AgCl) for 1 minute to activate the surface of the conductive fine powder 5 attached to the detection unit 4. Process. Next, the detection unit 4 is inserted into 0.2 M phosphate buffer pH 7 containing 630 ppm of nucleic acid, and the potential is maintained at +0.5 V for 3 minutes to immobilize the nucleic acid 6 on the conductive fine powder. Next, the detection unit 4 is inserted into 0.2 M phosphate buffer pH 7, rinsed, and then dried in an oven at 55 ° C. for 30 minutes.
[0031]
The DNA microarray 1 can be obtained as described above (see FIG. 1).
[0032]
Further, the DNA microarray 1 may be obtained by the following method.
[0033]
First, the lead portion 3 is printed on the insulating substrate 2 using conductive ink and dried (see FIG. 6). After the lead part 3 is printed, the conductive ink of the lead part 3 is dried to prepare for printing of the detection part 4.
[0034]
Next, the detection unit 4 is printed in series with the lead unit 3 using conductive ink (see FIG. 7). By forming the pattern of the detection unit 4 so as to partially overlap the lead unit 3, conduction between the detection unit 4 and the lead unit 3 can be achieved.
[0035]
Next, the conductive fine powder 5 is adhered to the undried detection unit 4 (see FIG. 8). At this time, since the conductive ink constituting the lead portion 3 is already dried, the conductive fine powder 5 adheres only to the detection portion 4 without performing a process such as masking.
[0036]
Next, the detection unit 4 is dried, and excess conductive fine powder 5 is removed with an air gun or the like (see FIG. 9).
[0037]
Next, the DNA microarray 1 can be obtained (see FIG. 10) by electrically immobilizing the nucleic acid 6 on the conductive fine powder 5 (see FIG. 5).
[0038]
Further, the DNA microarray 1 may be obtained by the following method.
[0039]
First, it prints continuously using a conductive ink so that the lead part 3 and the detection part 4 may become in series on the board | substrate 2 which has insulation (refer FIG. 2).
[0040]
Next, the lead part 3 and the detection part 4 are dried.
[0041]
Next, the lead portion 3 is masked 7 so that the detection portion 4 is exposed (see FIG. 11).
[0042]
Next, the DNA microarray 1 can be obtained (see FIG. 12) by electrically immobilizing the nucleic acid 6 on the detection unit 4 (see FIG. 5).
[0043]
Alternatively, the DNA microarray 1 may have a plurality of rows of lead portions 3 and detection portions 4, and different types of nucleic acids 6 may be attached to the detection portions 4 (see FIG. 13). By individually applying a potential to each detection unit 4, different types of nucleic acids 6 can be immobilized on each detection unit 4.
[0044]
In addition, the DNA microarray 1 of the present invention can have a multilayer structure in which a plurality of substrates 2 on which lead portions 3 and detection portions 4 are formed are stacked. With this configuration, the integration degree of the detection unit 4 can be increased, and detection can be performed with a small amount of sample.
[0045]
The DNA microarray 1 of the present invention can be used for a so-called electrochemical detection method. The electrochemical detection method can be determined immediately without the need for a large-scale detection device, and is much more sensitive than the fluorescence detection method.
[0046]
Examples of the intercalator for electrochemically detecting hybridization as in the present invention include ferrocene compounds, catecholamine compounds, metal bipyridine complexes, metal phenanthrine complexes, viologen compounds, and the like. In the detection of hybridization, since the intercalator itself may adhere to the electrode surface other than the hybrid part, it is necessary to measure the amount of intercalator adhering only to the electrode surface without hybridization in advance as a blank value. Next, hybridization is performed, and a current-potential curve is measured in the same manner as in the blank. If the blank is subtracted from the obtained current-potential curve for correction, the amount of intercalator used in the hybridizing part can be calculated.
[0047]
The DNA microarray 1 of the present invention can be detected by immersing the detection unit 4 in the sample solution together with the counter electrode. Moreover, it can detect by arranging with a counter electrode and dripping a sample solution to the detection part 4. FIG.
[0048]
【Example】
(Example 1) A polyethylene terephthalate film having a thickness of 250 μm and a size of 10 mm × 40 mm is used as a substrate, and conductive ink (carbon paste ink CH-1 manufactured by Jujo Chemical Co., Ltd.) is screen-printed thereon to detect a 5 mm square. The detection part was immersed in a conductive fine powder (manufactured by Naito Shoten Co., Ltd.) that was printed so that the lead part and 5 mm × 30 mm lead part were in series, and then sufficiently ground in a mortar so that the particle diameters were uniform.
[0049]
Next, the substrate was dried in an oven at 70 ° C. for 2 hours, and excess conductive fine powder was removed with an air gun.
[0050]
Next, the substrate was inserted so as to sufficiently immerse the detection part of the substrate in an acetate buffer (pH 5), and a potential of +1.5 V (vs Ag / AgCl) was applied for 1 minute. Next, the detection unit of the substrate prepared above was inserted into a solution prepared by adjusting potassium polyguanylate (POLYGUANLYLICACID potassium salt manufactured by Sigma-Aldrich) to 30 ppm with 0.2 M phosphate buffer solution (pH 7.0), It was kept at +0.5 V (vs Ag / AgCl) and applied for 3 minutes to immobilize nucleic acids on the conductive fine powder to obtain a DNA microarray.
[0051]
The DNA microarray thus obtained was inserted into a 0.2 M phosphate buffer solution (pH 7.0), the potential was scanned from +0.1 V to +1.3 V, and the redox potential was measured. A guanine peak was observed in the vicinity.
[0052]
(Example 2) On a substrate similar to that in Example 1, conductive lead (carbon paste ink CH-1 manufactured by Jujo Chemical Co., Ltd.) was printed on a lead portion of 5 mm × 30 mm by screen printing, and then 70 ° C. in an oven. Dried for 2 hours.
[0053]
Next, similarly, a detection unit having a size of 5 mm square was printed in the same manner as in Example 1, and then the same conductive fine powder as in Example 1 was sprayed from above the substrate.
[0054]
Next, excess conductive fine powder was removed with an air gun, followed by drying in an oven at 70 ° C. for 2 hours.
[0055]
Next, nucleic acids were immobilized on conductive fine powder on the substrate in the same manner as in Example 1 to obtain a DNA microarray.
[0056]
The DNA microarray thus obtained was measured for redox potential in the same manner as in Example 1. As a result, a guanine peak was confirmed.
[0057]
(Example 3) On the board | substrate similar to Example 1, it printed so that a detection part and a lead part may each be in series like Example 1, and this board | substrate dried at 70 degreeC for 2 hours. Went.
[0058]
Next, the lead part is masked by printing with a size of 7 mm × 15 mm by screen printing so that only the detection part and the end part of the lead part are exposed using acrylic resin ink (manufactured by Jujo Chemical Co., Ltd.). It was. Next, in the same manner as in Example 1, the nucleic acid was immobilized on the detection part, and a DNA microarray was obtained.
[0059]
The DNA microarray thus obtained was measured for redox potential in the same manner as in Example 1. As a result, a guanine peak was confirmed.
[0060]
【The invention's effect】
Since this invention consists of an above-described structure, it has the following effects.
[0061]
In the method for producing a DNA microarray of the present invention, a conductive ink is continuously printed on an insulating substrate so that the lead portion and the detection portion are in series, and then the conductive fine powder is undried. Since it is configured to adhere to the detection part, then dry the lead part and the detection part, and then electrically immobilize the nucleic acid on the conductive fine powder, the DNA microarray can be used without using a photolithography method or a dispenser method. Can be manufactured.
[Brief description of the drawings]
FIG. 1 is a cross-sectional view showing an embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention.
FIG. 2 is a cross-sectional view showing one step of a method for producing a DNA microarray of the present invention.
FIG. 3 is a cross-sectional view showing one step of a method for producing a DNA microarray of the present invention.
FIG. 4 is a cross-sectional view showing one step of a method for producing a DNA microarray of the present invention.
FIG. 5 is a schematic view showing one step of the method for producing a DNA microarray of the present invention.
FIG. 6 is a cross-sectional view showing a step of the method for producing a DNA microarray of the present invention.
FIG. 7 is a cross-sectional view showing a step of the method for producing a DNA microarray of the present invention.
FIG. 8 is a cross-sectional view showing one step of a method for producing a DNA microarray of the present invention.
FIG. 9 is a cross-sectional view showing a step of the method for producing a DNA microarray of the present invention.
FIG. 10 is a cross-sectional view showing one embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention.
FIG. 11 is a plan view showing one step in a method for producing a DNA microarray of the present invention.
FIG. 12 is a cross-sectional view showing an embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention.
FIG. 13 is a plan view showing one embodiment of a DNA microarray that can be obtained by the method for producing a DNA microarray of the present invention.
[Explanation of symbols]
1 DNA microarray 2 Substrate 3 Lead part 4 Detection part 5 Conductive fine powder 6 Nucleic acid 7 Masking

Claims (4)

  On the insulating substrate, printing is performed continuously using conductive ink so that the lead portion and the detection portion are in series, and then the conductive fine powder is attached to the undried detection portion, and then the lead portion and A method for producing a DNA microarray, comprising drying a detection part and then electrically immobilizing a nucleic acid on a conductive fine powder.   On the insulating substrate, the lead portion is printed with conductive ink and dried, then the detection portion is printed with the conductive ink so as to be in series with the lead portion, and then the conductive fine powder is applied. A method for producing a DNA microarray, comprising attaching to an undried detection portion, then drying the detection portion, and then electrically immobilizing nucleic acid on the conductive fine powder.   The method for producing a DNA microarray according to claim 1, wherein the conductive fine powder is made of metal or graphite. The method for producing a DNA microarray according to any one of claims 1 to 3 , wherein a plurality of rows of lead portions and detection portions are formed on a substrate.

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