JP5061342B2 - Carbon nanotube electrode and sensor using the electrode - Google Patents

Description

  The present invention relates to an electrode for a sensor for detecting a biomolecule and the like and a sensor using the electrode, and more particularly to a carbon nanotube electrode and a sensor using the electrode.

  Conventionally, the proposed electrochemical biosensor forms a thin film on the electrode with a reactive group that selectively reacts with a specific molecule, and changes in potential when the thin film adsorbs the specific molecule, or The current that flows through the electrode when the molecule undergoes a reaction involving the exchange of electrons with the electrode is measured. Specifically, this is a method of detecting the amount of glucose by forming a thin film having glucose oxidase on an electrode and measuring a change in current value accompanying an oxidation reaction with glucose. Regarding this type of biosensor, for example, the following Patent Document 1 can be cited.

By the way, carbon nanotube is a substance in which a graphite sheet is rounded on a tube, and its electrical characteristics are predicted to be metallic and semiconducting depending on the structure. Carbon nanotubes have a diameter of 1 to several tens of nanometers and a length of several tens of micrometers, and are therefore expected to exhibit typical one-dimensional electrical conduction. Such a tube-shaped substance has a one-dimensional electric conduction characteristic and is expected to exhibit ballistic conduction. For this reason, attempts to apply tubular inorganic materials to various devices have been active recently.
JP 10-260156 A (publication date: September 29, 1998)

  However, since the above-described biosensor in the prior art is a method for directly detecting a current value associated with a chemical reaction as described above, the biosensor is low in sensitivity and difficult to detect a low concentration of glucose. It had the disadvantage that the high selectivity of the sensor could not be fully demonstrated.

  In addition, biosensors that directly detect currents by electrochemically oxidizing and reducing biomolecules using electrodes such as Au and Pt have been studied. However, when a sensor is manufactured using the above-described electrodes, there are a drawback that sufficient sensitivity cannot be obtained, and a further disadvantage that integration is difficult.

  Therefore, there is a strong development of biosensors that detect direct currents by electrochemically oxidizing and reducing biomolecules using electrodes and that have sufficient sensitivity and can be integrated. It was desired.

  The present invention has been made in view of the above problems, and an object thereof is to provide an electrode that can be used for a highly sensitive sensor and the like and a sensor using the electrode.

  As a result of intensive investigations in view of the above problems, the present inventors have significantly increased the surface area of an electrode when carbon nanotubes are formed on a metal surface. It has become possible to detect molecules and the like with high sensitivity, and furthermore, since the underlying electrode area can be reduced, it has been found that integration is possible, and the present invention has been completed. That is, the invention according to the present invention includes the following inventions.

  (1) An electrode having carbon nanotubes on the metal surface.

  (2) The electrode according to (1), wherein the carbon nanotube is a single-walled carbon nanotube.

  (3) The carbon nanotube is obtained by immobilizing one kind of substance selected from the group consisting of an antibody, a protein, a sugar, an enzyme, a peptide nucleic acid, and an aptamer nucleic acid on the surface (1) or (2) The electrode as described.

  (4) The electrode according to any one of (1) to (3), wherein the carbon nanotube is in good electrical or mechanical contact with the metal surface by directly growing on the metal surface.

  (5) The electrode according to any one of (1) to (4), wherein the carbon nanotube is grown directly on a fine metal having a diameter of several microns.

  (6) When the carbon nanotube electrode is formed, the catalyst is patterned on the metal electrode with a photoresist, and the carbon nanotube is formed by a thermal chemical vapor deposition method (1) to (5) electrode.

  (7) When forming the carbon nanotube electrode, the catalyst is patterned on the metal electrode with a photoresist, and the carbon nanotube is formed by plasma enhanced chemical vapor deposition (1) to (5).

  In the case of (7) above, the catalyst is preferably iron, nickel, or cobalt.

  (8) An electrode group formed by integrally forming a plurality of electrodes according to any one of (1) to (7) on a substrate.

  (9) A sensor comprising the electrode according to any one of (1) to (7) or the electrode group according to (8).

  (10) The sensor according to (9), wherein the measurement target substance is measured in a label-free manner by an electric current generated by oxidation or reduction of an amino acid or a base contained in the measurement target substance or a substance immobilized in advance on the electrode surface. .

  (11) The sensor according to (9) or (10), which is a biosensor.

  (12) In any one of (9) to (11), in the plurality of carbon nanotube electrodes, a different substance is immobilized on each carbon nanotube electrode, and a large number of items can be inspected simultaneously by measuring a single sample. The sensor according to Crab.

  (13) The sensor according to any one of (9) to (12), wherein a potentiostat is incorporated on a silicon substrate of the sensor using an integrated circuit technique.

  (14) The sensor according to (13), wherein the potentiostat and the sensor part are removable and can be disposable.

  Since the electrode according to the present invention has the above-described unique configuration, when used in a sensor, the electrode can produce a highly sensitive sensor. Furthermore, sensors using this electrode can be integrated.

  Hereinafter, one embodiment of the present invention will be described, but it is added again that the present invention is not limited to the following embodiment.

  The electrode according to the present invention is not particularly limited as long as it has carbon nanotubes on the metal surface, and other specific configurations. Such an electrode can be said to be an electrode in which carbon nanotubes are formed on a metal surface, for example. The “carbon nanotube” as used in the present invention may be a conventionally known carbon nanotube, and other specific configurations are not particularly limited. For example, it is preferably a one-dimensional nanomaterial made of only carbon atoms, having a diameter of 0.4 to 50 nm (1 nanometer: one billionth of a meter) and a length of approximately 1 to several hundreds of μm.

  As said metal, the conventionally well-known metal used for an electrochemical electrode can be utilized, Although it does not specifically limit about a specific structure, For example, Pt, Au, etc. can be used.

  In addition, a technique for forming carbon nanotubes on the metal surface can be performed by a conventionally known method, and is not particularly limited. For example, as shown in Examples, it can be easily formed by chemical vapor deposition.

  In the electrode, the carbon nanotubes are preferably single-walled carbon nanotubes (SWNTs). “Single-walled carbon nanotubes” have a chemical structure of carbon nanotubes expressed by rolling and joining graphite layers, and the number of graphite layers is only one. It is known that the electronic structure becomes metallic or semiconducting depending on how the graphite layer is wound (helicality). According to the said structure, since a diameter is small, it can grow at higher density, As a result, a surface area can increase markedly. Further, since the crystallinity is good, there is an advantage that the substance can be easily fixed on the surface.

  In addition, the carbon nanotube is preferably obtained by immobilizing one substance selected from the group consisting of antibodies, proteins, sugars, enzymes, and nucleic acids such as peptide nucleic acids and aptamer nucleic acids on the surface. A technique for immobilizing an antibody or the like on the carbon nanotube can be suitably used by a conventionally known technique, and is not particularly limited. For example, as shown in Examples described later, a technique for immobilization via a linker substance can be used.

  Further, it is desirable that the carbon nanotubes are directly grown from the metal surface using a thermal chemical vapor deposition method or the like, as shown in Examples described later. As a result, the metal surface can be satisfactorily contacted electrically and mechanically, and the performance and stability of the electrode can be greatly improved.

  Moreover, since the carbon nanotube electrode can be formed on a metal surface of several microns using a lithography method as in the embodiments described later, a plurality of carbon nanotube electrodes can be formed on one substrate at the same time. This facilitates simultaneous multiple item measurement.

  The present invention also includes a sensor including the above electrode. In particular, a biosensor for detecting a biomolecule is preferable.

  In addition, when measuring a molecule having an amino acid or base in its internal structure according to the present invention, or when using a probe molecule having an amino acid or base in its internal structure, the amino acid or base is oxidized and reduced to the electrode. It is preferable to adopt a method of detecting the molecule to be measured in a label-free manner based on the change of the current flow. By this method, measurement can be performed more easily, and there is no need to hold and act on a dangerous labeled molecule, and the safety is also high. In addition, many biomolecules can be measured in the same manner on the same principle without labeling, which is advantageous for simultaneous measurement of a plurality of items.

  Carbon nanotubes are ideal as electrochemical electrodes because they are chemically stable, have a very wide potential window, and have a very large specific surface area. Therefore, when used as a sensor electrode, high sensitivity can be expected, and further integration is possible.

  As shown in the examples described later, it has been clarified that when amino acids are detected using a carbon nanotube electrode, the sensitivity is increased by several orders of magnitude as compared with the conventional electrode. Therefore, it was shown that a highly sensitive biosensor (for example, antigen sensor, amino acid sensor, protein sensor, nucleic acid sensor, enzyme sensor, etc.) can be developed by using the electrode.

  Furthermore, since the sensor using the carbon nanotube electrode has high sensitivity characteristics, it can be applied not only to biomolecules but also to sensors of various ions.

  Further, the present invention includes the following embodiments. That is, when the carbon nanotube electrode is formed, the catalyst is patterned on the metal electrode with a photoresist, and the carbon nanotube is formed by a thermal chemical vapor deposition method.

  The carbon nanotube electrode may be formed by patterning a catalyst (iron, nickel, cobalt) with a photoresist on a metal electrode to form a carbon nanotube by plasma chemical vapor deposition.

  A sensor characterized in that, in the plurality of carbon nanotube electrodes, a different substance is immobilized on each carbon nanotube electrode, and a plurality of items can be inspected simultaneously by measuring a single sample.

  A sensor comprising a potentiostat incorporated on the silicon substrate of the sensor using an integrated circuit technology.

  Furthermore, the above-mentioned potentiostat and the sensor part are removable and can be disposable.

  Hereinafter, examples will be shown, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

As shown in FIG. 1, the present inventors made a highly sensitive and label-free current measurement biosensor using microelectrodes formed using single-walled carbon nanotubes (hereinafter referred to as SWNTs). . SWNTs are known to have a high ability to promote electron transfer reactions in electrochemical measurements. This is because SWNTs have a high aspect ratio that dramatically increases the total surface area of the electrode in the same region. In this example, K ₃ [Fe (CN) ₆ ] and an electron-activated amino acid (for example, tyrosine) are subjected to cyclic voltammetry (CV) or differential pulse voltammetry (DPV) as shown in FIG. The electrochemical identification of the device was investigated. Furthermore, after immobilizing a PSA antibody on an electrode on which SWNTs are arranged (hereinafter referred to as SWNT array electrodes), the electrochemistry of prostate specific antigen (PSA), which is a specific marker for prostate cancer, using DPV Label-free detection was performed.

  As shown in FIG. 1, the SWNT array electrode was formed by directly forming on the platinum by thermal chemical vapor deposition (CVD). This electrode was placed in various regions of the device to investigate the region dependence of electrochemical current intensity.

FIG. 3 is a diagram showing an electrochemical signal of K ₃ [Fe (CN) ₆ ] at the SWNT electrode or the platinum electrode (where the area of the base of the SWNT electrode is the same as the area of the platinum electrode) by CV. is there. A strong peak from redox can be clearly observed. The peak separation was about 0.1 V, which was almost the same as the theoretical value. Furthermore, it was found that the peak intensity at the SWNT electrode was higher than that at the platinum electrode. FIG. 4 is a diagram showing the area dependence of the base of the SWNT electrode with respect to the electrochemical signal. The areas of the bases of the SWNT electrodes in FIGS. 4A to 4D are 40,000, 20,000, 10,000, and 2500 μm ² , respectively. The peak current increases linearly with the area of the base of the SWNT electrode. These results indicate that the SWNT array electrode works effectively as a working electrode.

  FIG. 5 is a diagram showing an electrochemical signal of tyrosine at a SWNT electrode or a platinum electrode (where the area of the base of the SWNT electrode is the same as the area of the platinum electrode) by DPV. The peak intensity from the SWNT electrode was more than 100 times higher than that from the platinum exposed electrode. After detection of tyrosine by DPV by SEM, adsorption of tyrosine oxide was observed on the SWNT array electrode. These results indicate that the present device having the SWNT array electrodes is very sensitive to biomolecules because each SWNT acts as an electrode.

  Next, as shown in FIG. 6, electrochemical detection of PSA which is a cancer marker was performed by DPV. Specifically, first, a linker was formed on the SWNT electrode. Next, the PSA antibody was immobilized on the SWNT electrode. Next, PSA was introduced onto the electrode. Thereafter, the sample was rinsed with a buffer. Finally, an electrochemical signal was measured by the DPV method.

  FIG. 7 is a diagram showing the electrochemical signal of the protein at the SWNT array electrode by DPV. As shown in FIG. 7 (a), the electrochemical signal was detected only from the PSA antibody. On the other hand, when 1 ng / mL PSA was introduced onto the SWNT electrode having the PSA antibody, the electrochemical signal increased significantly as shown in FIG. This is due to the formation of an antigen-antibody complex. On the other hand, when other non-specific protein (BSA (bovine serum albumin)) is introduced on the SWNT electrode equipped with the PSA antibody, as shown in FIG. It did not increase. This indicates that no antigen-antibody reaction has occurred. Therefore, it was found that this device has high selectivity.

  Furthermore, as shown in FIG. 8, the PSA concentration dependency was clearly observed. Therefore, according to this apparatus, even PSA with a concentration of 0.5 ng / mL can be detected effectively. The concentration of 0.5 ng / mL is much smaller than the conventional reference value (4 ng / mL). Therefore, this device (biosensor) is also very useful as a clinical diagnosis method for prostate cancer.

  In conclusion, the present device having SWNT array microelectrodes developed by the present inventors is very useful as an amperometric biosensor for detecting biomolecules with high sensitivity in real time without labeling.

  As described above, the present invention can be used for sensor technology such as a highly sensitive biosensor. Therefore, there are industrial applicability in a wide range of fields such as diagnosis in the medical field and detection of trace substances in the environmental field as well as in the academic and research fields.

It is a figure which shows typically the structure of the sensor and electrode in a present Example. It is a figure which shows typically the structure of three electrode systems in the biosensor of a present Example. According to CV, it is a diagram illustrating an electrochemical signal of K ₃ [Fe (CN) _6] in SWNT electrodes or platinum electrodes. It is a figure which shows the area dependence of the foundation | substrate of a SWNT electrode about an electrochemical signal. It is a figure which shows the electrochemical signal of tyrosine in a SWNT electrode or a platinum electrode (here, the area of the foundation | substrate of a SWNT electrode and the area of a platinum electrode is the same) by DPV. It is a figure which shows typically the method of electrochemically detecting PSA which is a cancer marker by DPV. It is a figure which shows the electrochemical signal of the protein in a SWNT arrangement | sequence electrode by DPV. It is a figure which shows the result of having investigated PSA density | concentration dependence.

Claims (12)

It has carbon nanotubes on the metal surface,
The carbon nanotube is provided with a longitudinal direction perpendicular to the metal surface ,
When the carbon nanotube electrode is formed, a catalyst is patterned on the metal electrode with a photoresist, and the carbon nanotube is formed by a thermal chemical vapor deposition method (provided that the carbon nanotube is an insulator). Except those covered by). It has carbon nanotubes on the metal surface,
The carbon nanotube is provided with a longitudinal direction perpendicular to the metal surface,
When the carbon nanotube electrode is formed, a catalyst is patterned on the metal electrode with a photoresist, and the carbon nanotube is formed by plasma enhanced chemical vapor deposition (wherein the carbon nanotube is an insulator) Except those covered by). The carbon nanotubes, electrodes according to claim 1 or 2, characterized in that a single-walled carbon nanotubes. The carbon nanotubes are antibodies, proteins, sugars, enzymes, and claims, characterized in that is obtained by immobilizing a peptide nucleic acid, one of the substances selected from the group consisting of aptamers nucleic acid on the surface 1-3 The electrode according to any one of the above. The electrode according to any one of claims 1 to 4 , wherein the carbon nanotube is in electrical or mechanical contact with the metal surface by directly growing on the metal surface. An electrode group comprising a plurality of the electrodes according to any one of claims 1 to 5 integrally formed on a substrate. A sensor comprising the electrode according to any one of claims 1 to 5 or the electrode group according to claim 6 . Advance the measurement substance or the electrode surface contained in the immobilized material, the current due to oxidation or reduction of an amino acid or a base, according to claim 7, characterized in that the analyte is to measure the label-free Sensor. The sensor according to claim 7 or 8 , wherein the sensor is a biosensor. In the plurality of carbon nanotubes electrodes, different materials to each of the carbon nanotube electrode is fixed, the measurement of a single sample, more of claims 7-9, characterized in that as it can inspect the large number of items simultaneously The sensor according to claim 1. The sensor according to any one of claims 7 to 10 , wherein a potentiostat is incorporated on a silicon substrate of the sensor by using an integrated circuit technique. The sensor according to claim 11 , wherein the potentiostat and the sensor part are removable and disposable.