JP2007187491A - Electrode for electrochemical detection and method of manufacturing same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrode for electrochemical detection constituted so as to fix nucleic acid with good reproducibility and stability. <P>SOLUTION: The electrode for electrochemical detection is used for fixing nucleic acid formed on a substrate 1 and constituted of at least two layers a first Au layer 2 relatively low in crystallinity on the substrate 1 and a second Au layer 3, which is relatively high in crystallinity and comprises an Au (111) plane in the plane for fixing nucleic acid on the first Au layer 2. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an electrode for electrochemical detection used for an electrochemical detection chip for detecting the presence or absence of hybridization of nucleic acids by utilizing an oxidation-reduction reaction at an electrode, and a method for producing the same.

  Genetic information in the living body is stored as a DNA base sequence, and analyzing gene expression is effective for prevention of various diseases, early diagnosis treatment, tailor-made medicine, and the like. In gene analysis in the fields of biology and medicine, a hybridization method is widely used as a method for detecting a nucleic acid fragment having a specific sequence. The hybridization method uses the formation of a double-stranded nucleic acid by hydrogen bonding between base pairs having complementarity when the base sequences of single-stranded nucleic acids are complementary. As a method capable of specifically detecting the target gene, the Southern hybridization method is generally used.

  In Southern hybridization, sample DNA is first fragmented with one or more types of restriction enzymes, subjected to agarose gel electrophoresis or polyacrylamide gel electrophoresis, and separated into fractions according to their molecular weight sizes, and then the separated sample DNA fragments are separated. After denaturation to a single strand, it is immobilized on a nylon filter or nitrocellulose paper. Then, the sample DNA fragment is hybridized with a single-stranded DNA fragment labeled with a radioisotope that is complementary to the sample DNA fragment, and then the washed filter is subjected to autoradiography and developed. A sample DNA fragment having a specific base sequence that hybridizes with complementary DNA can be detected.

  However, since the method including the Southern hybridization method uses a radioisotope as a label, detection takes a long time, the band tends to be unclear, and there is a problem in terms of handling and cost of the radioactive material. .

  A method is also known in which a fluorescent substance is used as a label instead of labeling with a radioisotope. A method using a fluorescent substance as a label is an excellent method in terms of safety and speed. For example, a DNA fragment labeled with a fluorescent substance is hybridized to a DNA fragment that is fixed on a substrate such as a glass slide or silicon by non-covalent bonding (electrostatic bonding) or covalent bonding and aligned at high density. DNA chips for detecting the hybridization have already been used. However, fading due to excitation light occurs, a dedicated fluorescence measurement device is required for measurement, and it is difficult to introduce a certain amount of fluorescent material due to internal quenching and aggregation of fluorescence, etc. There is a problem.

  On the other hand, a method using an electrochemical detection chip that detects the presence or absence of nucleic acid hybridization using an oxidation-reduction reaction at an electrode is also known. In this method, when a probe DNA fragment is fixed to an electrode having an output terminal, and the sample DNA fragment and the electrochemically active threaded intercalator are brought into contact with the electrode, the probe DNA fragment and the sample DNA fixed on the electrode are contacted. A double-stranded DNA is formed between the fragment and an intercalator is bound inside the double-stranded DNA. Therefore, when a potential is applied to the electrode, a current is passed through the intercalator between the counter electrode provided separately. Since it flows, hybridization can be detected by measuring the amount of current.

  However, since the method using an electrochemical chip requires the probe DNA to be fixed to an electrode (generally an Au electrode), the reproducibility regarding the amount of the fixed DNA and the stability to the fixation of the fixed DNA are low. There is a problem that the result (data) obtained by the process is low in reliability.

  As a method for immobilizing DNA on an Au electrode, the immobilization method utilizes a bond between a thiol group modified at the end of the DNA and an Au atom, or via a SAM (self-assembled monomolecular film) formed on the Au electrode. A typical method is DNA immobilization.

  However, these fixing methods bond with good reproducibility on the Au (111) surface, but have a problem that they are difficult to bond with different surface orientations and surfaces with poor crystallinity. Further, the substrate for obtaining the Au (111) surface is only mica (mica), and the substrate is not versatile because the Au (111) surface cannot be obtained with an amorphous material such as glass or a substrate having a different crystal structure. There is a problem of being scarce.

As a method for solving the problem of reproducibility related to fixation, for example, in Patent Document 1, a microchannel is formed by combining a base substrate and an upper plate, and a pair of exposed microchannels is exposed on the base substrate. An electrode is formed and a mixed solution containing a nucleic acid fragment having a known base sequence and a sample nucleic acid fragment is introduced into a microchannel together with an intercalator, and a current flowing between the electrodes is measured by applying a potential between the pair of electrodes. Thus, an electrochemical detection chip for detecting the presence or absence of hybridization in a mixed solution is disclosed.
JP 2004-109049 A

  The electrochemical detection chip described in Patent Document 1 does not fix the probe DNA to the electrode, and thus the reproducibility of the measurement is improved, but problems such as performance and productivity have not been solved. It is not used for general purpose.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an electrode for electrochemical detection and a method for producing the same that can fix nucleic acids with good reproducibility and stability.

  The electrode for electrochemical detection of the present invention is an electrode for immobilizing a nucleic acid formed on a substrate, the electrode comprising a first Au layer having relatively low crystallinity directly above the substrate, On the first Au layer, the surface having relatively high crystallinity and fixing the nucleic acid is composed of at least two layers including a second Au layer composed of an Au (111) surface.

  Preferably, the (111) plane existence ratio of the first Au layer is less than 30%, and the (111) plane existence ratio of the second Au layer is 30% or more.

  The method for manufacturing an electrode for electrochemical detection of the present invention is a method for manufacturing an electrode for immobilizing a nucleic acid formed on a substrate, wherein Au is deposited on the substrate by sputtering to form a first Au layer. Then, the substrate is heated to 300 ° C. or higher and lower than 700 ° C., and Au is deposited by sputtering to form a second Au layer.

  In forming the first Au layer, the substrate is preferably heated at less than 300 ° C., more preferably from 50 ° C. to less than 200 ° C., and further preferably from 50 ° C. to 100 ° C.

  The electrochemical detection chip of the present invention is an electrochemical detection chip for detecting the presence or absence of nucleic acid hybridization using an oxidation-reduction reaction at an electrode, for fixing the nucleic acid formed on the substrate by the electrode. A first Au layer having relatively low crystallinity directly on the substrate, and a surface on which the nucleic acid is immobilized on the first Au layer having relatively high crystallinity from the Au (111) surface. It consists of at least two layers with the second Au layer.

  The electrode for electrochemical detection of the present invention is an electrode for immobilizing a nucleic acid formed on a substrate. The electrode comprises a first Au layer having relatively low crystallinity directly above the substrate, and the first Au layer. Since the surface having relatively high crystallinity and immobilizing nucleic acid on one Au layer is composed of at least two layers including the second Au layer composed of Au (111) surface, the surface immobilizing nucleic acid is Au (111) surface The regular gaps are formed by four Au atoms, and the thiol group (SH group) formed at the end of the nucleic acid enters here, so that the bond is strengthened by the coordination bond of Au-S. It is possible to fix the nucleic acid with high stability.

  In general, Au is easily oxidized and easily affected by contact with other substances, so that the ability to fix a thiol group is likely to deteriorate. However, the electrochemical detection electrode of the present invention fixes nucleic acids. Since the surface is made of Au (111) and Au atoms are regularly arranged, it can be fixed stably, and since Au atoms are regularly arranged, peeling in the C-axis direction is suppressed. Can do.

  In the method for manufacturing an electrode for electrochemical detection according to the present invention, Au is deposited on a substrate by sputtering to form a first Au layer, and then the substrate is heated to 300 ° C. or more and less than 700 ° C., and Au is sputtered. Since the second Au layer is formed by deposition, a relative crystal having an Au (111) plane abundance of less than 30% in the vicinity of the substrate is used to buffer the unevenness of the substrate when the first Au layer is formed. When the second Au layer is formed, the substrate is sputtered in a high energy state by heating the substrate to 300 ° C. or higher and lower than 700 ° C. on the first Au layer buffering the unevenness of the substrate. , Au is self-crystallized, and a second Au layer having a relatively high crystallinity with an abundance of the Au (111) plane of 30% or more can be formed.

  Hereinafter, the electrode for electrochemical detection of the present invention will be described with reference to the drawings. FIG. 1 is a schematic sectional view of an electrochemical detection electrode of the present invention. As shown in FIG. 1, the electrochemical detection electrode 10 of the present invention includes a first Au layer 2 having relatively low crystallinity directly above a substrate 1 and a relatively crystalline structure on the first Au layer 2. It is characterized in that the surface on which the nucleic acid is immobilized is composed of at least two layers including the second Au layer 3 composed of an Au (111) surface.

  Here, the nucleic acid is a base unit consisting of a nucleotide consisting of a base, phosphate and sugar, and this phosphate is polymerized by forming a diester bond between the 3 ′ and 5 ′ carbons of the sugar between each nucleotide. It is a long chain polynucleotide. There are RNA and DNA depending on whether the sugar moiety is ribose or deoxyribose. Since nucleic acids are immobilized on electrodes, thiol groups are formed at the ends.

  The substrate is not particularly limited, and examples include highly versatile amorphous materials such as glass, semiconductor materials such as Si, and insulating materials such as ceramics and alumina.

  The (111) plane abundance of the first Au layer is preferably less than 30%, and the (111) plane abundance of the second Au layer is preferably 30% or more, more desirably (111) of the first Au layer. ) The plane presence rate is preferably less than 50%, and the (111) plane presence rate of the second Au layer is preferably 50% or more. Here, the presence ratio of the (111) plane of the Au layer is determined by taking a TEM image at an arbitrary point, and visually observing a region where the crystal arrangement is aligned as the Au (111) plane, and visually checking the Au (111) ) Determined by measuring the surface.

  In order to manufacture the electrode as described above, Au is deposited on the substrate by sputtering to form a first Au layer, and then the substrate is heated to 300 ° C. or more and less than 700 ° C. to deposit Au by sputtering. A second Au layer is formed.

  Sputtering may be any of direct current sputtering (DC), radio frequency (RF) sputtering, magnetron sputtering, ion beam sputtering, etc., but high frequency sputtering is preferred from the viewpoint of electrode film quality and versatility.

  When the first Au layer is formed, the heating temperature during sputtering is preferably heated to less than 300 ° C., more preferably from 50 ° C. to less than 200 ° C., and even more preferably from 50 ° C. to less than 100 ° C. It is preferable to do. When forming the second Au layer, it is preferable to heat the substrate at 300 ° C. or higher and lower than 700 ° C., more preferably 500 ° C. or higher and 600 ° C. or lower.

  The thicknesses of the first Au layer and the second Au layer cannot be generally specified because of the sputtering method and sputtering conditions, but the first Au layer is preferably 30 to 70 nm, and the second Au layer is preferably 100 nm or more.

  Here, a case where Au is deposited by sputtering to form a first Au layer, and then the substrate is heated to 300 ° C. or higher and lower than 700 ° C. and Au is deposited by sputtering to form a second Au layer is described. However, when forming the Au layer on the substrate, first, sputtering of Au is started at room temperature to less than 200 ° C., and then the heating temperature is gradually increased, and finally at 300 ° C. or more and less than 700 ° C. Sputtering may be performed.

  FIG. 2 is an enlarged schematic view of the uppermost Au (111) surface on which the nucleic acid of the second Au layer 3 is fixed. As shown in FIG. 2, the Au (111) surface of the uppermost layer on which the nucleic acid of the second Au layer 3 is fixed has regular gaps formed by four Au atoms, and the thiol formed at the end of the nucleic acid here Since the group is contained, the bond can be strengthened and the nucleic acid can be immobilized with good stability.

  The electrode for electrochemical detection of the present invention can be used in an electrochemical detection chip for detecting the presence or absence of nucleic acid hybridization using an oxidation-reduction reaction. In this electrochemical detection chip, a single-stranded nucleic acid (for example, a DNA probe) is immobilized on the electrode surface, and in the presence of an intercalator in a solution system, a complementary double-stranded DNA of a test DNA and a probe DNA is formed, Detection of the formed double-stranded DNA is performed by detecting a change in the current of the electrode as an electrochemical signal.

  FIG. 3 shows a conceptual diagram of electrochemical measurement in which an electrochemical detection method is performed using a three-electrode system including a working electrode (WE), a counter electrode (CE), and a reference electrode (RE). When a probe DNA is fixed to a working electrode having an output terminal and a test DNA fragment and an electrochemically active threaded intercalator are brought into contact therewith, a space between the probe DNA fragment fixed on the electrode and the test DNA fragment is obtained. A double-stranded DNA is formed, and an intercalator binds to the inside of the double-stranded DNA. Therefore, when a potential is applied to the electrode, a current flows between the counter electrode provided separately via the intercalator. Hybridization can be detected by measuring the amount of current. Note that the reference electrode is for measuring the potential of the working electrode, and virtually no current flows through the reference electrode.

  Hereinafter, the electrode for electrochemical detection of the present invention and the production method thereof will be described in more detail with reference to examples.

Example 1
High frequency sputtering is performed on a glass substrate that has been subjected to organic cleaning (ultrasonic cleaning with acetone and ultrapure water for 5 minutes / 2 times) under conditions of a film formation rate of 100 nm / min and a power of 200 W. The first Au layer was formed. Next, while heating the glass substrate to 500 ° C., high-frequency sputtering was performed under the conditions of a film formation rate of 100 nm / min and a power of 200 W to form a second Au layer having a film thickness of about 150 nm to manufacture an electrode.

  As a result of taking a TEM image of the obtained electrode surface, it was found that the second Au layer of the electrode of Example 1 had a surface with a relatively high crystallinity with an Au (111) plane existence ratio of 50% or more. On the other hand, when the second Au layer was scraped off, and the surface layer of the first Au layer was scraped off and TEM image was taken again in the vicinity of the substrate, the presence ratio of Au (111) plane was 25%, which was relatively crystalline. The low side was obtained.

(Comparative Example 1)
An electrode was manufactured by depositing Au with a thickness of 200 nm by vapor deposition on a glass substrate prepared in the same manner as in Example 1 in a vacuum of 10 ⁻³ Pa or less.

(Comparative Example 2)
An electrode was manufactured by depositing Au having a thickness of 250 nm at a power of 200 W at a deposition rate of 100 nm / min on a glass substrate that had been pretreated in the same manner as in Example 1.

(Comparative Example 3)
The electrode obtained in Comparative Example 2 was placed in an electric furnace and annealed in air at 530 ° C. for 7 hours to produce an electrode.

  4 to 7 show RHEED (reflection high-energy electron diffraction) patterns of the electrodes obtained in Example 1 and Comparative Examples 1 to 3. FIG. As apparent from the RHEED pattern, the Au (111) surface was obtained only with the electrode manufactured in Example 1.

  Moreover, the result of having investigated the response curve (DPV) which fixed the DNA probe which modified the terminal with the thiol group to Au electrode obtained in Example 1 and Comparative Examples 1-3 is shown in FIGS. In the DPV graph, the response curve when DNA is fixed is indicated by a circular connecting line, and the response curve when nothing is fixed is indicated by a square connecting line. In the case of the electrode of Example 1, it can be seen that the amount of immobilized DNA is greatly improved as compared with the electrodes of Comparative Examples 1 to 3. This is because, in the case of the electrodes manufactured in Comparative Examples 1 to 3, since the Au layer is amorphous and not single-crystallized, a sufficient bond with the thiol group at the end of the DNA is obtained. However, the surface of the electrode manufactured in Example 1 has an Au (111) surface, and it is considered that a thiol group was reliably bonded between four Au atoms on the (111) surface. It is considered that the reproducibility can be improved because the portion to be securely bonded is formed.

  As described above, the electrochemical detection electrode of the present invention has a first Au layer with relatively low crystallinity directly above a substrate, and a nucleic acid is immobilized on the first Au layer with relatively high crystallinity. The surface to be fixed is composed of at least two layers including a second Au layer composed of an Au (111) surface, and the surface on which the nucleic acid is immobilized is on the Au (111) surface, and regular gaps are formed by four Au atoms. Since a thiol group formed at the end of the nucleic acid is contained in the structure, the bond can be strengthened by the coordination bond of Au—S, and the nucleic acid can be immobilized with good stability.

Schematic sectional view of the electrode for electrochemical detection of the present invention An enlarged schematic diagram of the uppermost Au (111) surface on which the nucleic acid is immobilized Conceptual diagram of electrochemical measurement RHEED pattern of the electrode obtained in Example 1 RHEED pattern of the electrode obtained in Comparative Example 1 RHEED pattern of the electrode obtained in Comparative Example 2 RHEED pattern of the electrode obtained in Comparative Example 3 Response curve when DNA was immobilized on the electrode obtained in Example 1 Response curve when DNA is immobilized on the electrode obtained in Comparative Example 1 Response curve when DNA is immobilized on the electrode obtained in Comparative Example 2 Response curve when DNA is immobilized on the electrode obtained in Comparative Example 3

Explanation of symbols

1 Substrate 2 First Au layer 3 Second Au layer 10 Electrode

Claims (5)

  An electrode for immobilizing a nucleic acid formed on a substrate, wherein the electrode has a relatively low crystallinity immediately above the substrate and a relatively low crystallinity on the first Au layer. An electrode for electrochemical detection, characterized in that the surface on which the nucleic acid is immobilized has a high crystallinity and is composed of at least two layers including a second Au layer composed of an Au (111) surface.   2. The electrochemical according to claim 1, wherein the (111) plane existence ratio of the first Au layer is less than 30%, and the (111) plane existence ratio of the second Au layer is 30% or more. Detection electrode.   A method for manufacturing an electrode for immobilizing a nucleic acid formed on a substrate, wherein Au is deposited on the substrate by sputtering to form a first Au layer, and then the substrate is heated to 300 ° C. or higher and lower than 700 ° C. And depositing Au by sputtering to form a second Au layer.   The method for manufacturing an electrode for electrochemical detection according to claim 3, wherein the first Au layer is formed by heating the substrate to less than 300 ° C.   An electrochemical detection chip for detecting the presence or absence of nucleic acid hybridization using an oxidation-reduction reaction at an electrode, wherein the electrode is for fixing a nucleic acid formed on the substrate, and is directly above the substrate. At least two of a first Au layer having relatively low crystallinity and a second Au layer having a relatively high crystallinity on the first Au layer and a surface on which the nucleic acid is immobilized is an Au (111) surface. An electrochemical detection chip comprising a layer.