JP4170336B2 - Catecholamine sensor - Google Patents

Description

  The present invention relates to a catecholamine sensor, and more particularly to a catecholamine sensor for electrochemically measuring a catecholamine concentration in a biological sample.

  Catecholamine is a type of neurotransmitter that is released from nerve cells or released from the adrenal glands in the brain. Since this catecholamine has high electrochemical activity, an electrochemical measurement method is often used to detect catecholamine.

  Regarding the detection of catecholamines, a system combining liquid chromatography and an electrochemical detector is mainly used in clinical practice. On the other hand, for detection of catecholamines in a living body such as the brain, a minute electrode produced by processing carbon fiber is used. Even in the brain, the electrode surface is covered with a cation exchange material such as Nafion (registered trademark) or polypyrrole peroxide because it is susceptible to measurement interference by anionic molecules such as L-ascorbine and uric acid (Non-patent Document 1). reference). A sensor chip in which an enzyme reactor for removing a measurement interfering substance and an electrochemical detector for the purpose of realizing continuous measurement is also reported (Non-Patent Document 2).

1. K.Puhel, Q.D.Walker, R.M.Wightman. “Overoxidized Polypyrrole-Coated Carbon Fiber Microelectrodes for Dopamine Measurements with Fast-Scan Cyclic Voltammetry” Anal, Chem., 68, 2084-2089, 1996 K. Hayashi, Y. Iwasaki, R. Kurita, K. Sunagawa, O. Niwa, A. Tate. “The Highly sensitive detection of catecholamines using a microfluidic device integrated with an enzyme-modified pre-reactor for interferent elimination and interdigitated array electrode ”J. Electroanal. Chem., 579, 215-222, 2005

  The catecholamine concentration in blood is expected as a heart disease index. However, since it is difficult to continuously measure the catecholamine concentration in blood, biomarkers other than catecholamine (blood gas, pH, electrolyte, etc.) are used in clinical practice as heart disease indicators. The reason why the catecholamine concentration is not used as an index of heart disease is that the concentration of catecholamine is low in blood and urine, and there are many substances having high electrochemical activity such as L-ascorbic acid. This is because it is difficult to measure continuously.

  In order to realize continuous measurement of the catecholamine concentration, it is necessary to remove a measurement interfering substance such as L-ascorbic acid before reaching the detection electrode. In an enzyme reactor, a measurement interfering substance can be selectively removed from the substrate specificity of the enzyme, but the amount of the measurement interfering substance that can be removed by an enzyme reaction is limited. In addition, since there is an electrochemically active substance that cannot be enzymatically decomposed, the enzyme reactor alone cannot remove the electrochemically active substance that cannot be enzymatically decomposed. It has been difficult to achieve improvements, preferably complete removal of measurement interfering substances.

  Thus, with conventional catecholamine sensors, it was possible to perform continuous measurement with the accuracy desired at the time, for example, using an enzyme reactor. It was required to be able to perform continuous measurement with high accuracy. That is, it is desired to improve the removal efficiency of measurement interfering substances regardless of whether or not enzymatic degradation is possible.

  The present invention has been made in view of such problems, and its purpose is to reduce the influence of measurement interfering substances and to continuously monitor the catecholamine concentration in a biological sample such as blood. It is to provide a catecholamine sensor.

In order to achieve the above object, the present invention provides a catecholamine sensor for detecting a catecholamine concentration by flowing a measurement sample from an upstream side to a downstream side in a flow path, A first electrode for oxidizing catecholamine and a measurement interfering substance contained in the measurement sample formed in the flow path, and a catecholamine oxidized in the first electrode formed in the flow path. comprising a second electrode for reducing, than the first electrode and the second electrode in the flow path is formed downstream, and a third electrode for detecting the catecholamine, the first The first electrode and the second electrode are comb-shaped electrodes, and the branch portions of the comb-shaped electrode serving as the first electrode and the branch-shaped portions of the comb-shaped electrode serving as the second electrode are alternately arranged. And alternately The most downstream location has been interdigital electrodes characterized in that it is a branch portion of the comb electrode serving as the second electrode.

According to a second aspect of the present invention, in the first aspect of the invention, the surface of the first electrode is modified with a substance having a functional group having a positive charge.

  According to a third aspect of the present invention, in the second aspect of the present invention, the substance is a thiol.

According to a fourth aspect of the present invention, in the invention according to any one of the first to third aspects, the third electrode is an array-type electrode.

The invention according to claim 5 is the invention according to any one of claims 1 to 4 , further comprising an enzyme reaction means formed on the upstream side of the first electrode in the flow path. To do.

The invention according to claim 6 is the catecholamine sensor according to any one of claims 1 to 5 and a capillary for sampling, connected to the catecholamine sensor, a tube connected to the catecholamine sensor, or the catecholamine Any one of the probes with a microdialysis membrane connected to the sensor is provided.

  According to the present invention, since an electrode for oxidizing the measurement interfering substance and an electrode for reducing catecholamine are provided upstream of the electrode for detecting catecholamine, the influence of the measurement interfering substance is reduced. It becomes possible to continuously monitor the catecholamine concentration in a biological sample such as blood.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.
In one embodiment of the present invention, a flow path through which a measurement sample such as a biological sample flows is formed, and in this flow path, the measurement interfering substance is oxidized in order from the upstream side to the downstream side. Electrode (also referred to herein as “oxidation electrode”), electrode for reducing oxidized catecholamine (also referred to herein as “reduction electrode”), electrode for detecting catecholamine (this specification) (Also referred to as “detection electrode”). With such a configuration, when catecholamine is to be detected from a measurement sample such as a biological sample, the measurement interference substance can be removed even if the measurement sample contains a measurement interference substance.

In the present specification, “catecholamine” includes biogenic amines having an o-dihydroxybenzene skeleton such as dopamine, noradrenaline, and adrenaline.
In the present specification, “measurement interfering substances” include metabolites of catecholamines having similar electrochemical properties such as L-ascorbic acid, uric acid, DOPAC (3,4-dihydroxyphenylacetic acid), and the like.

FIG. 1 is a top view of a catecholamine sensor according to an embodiment of the present invention.
In FIG. 1, a flow path 12 is formed in the substrate 1 by an insulating layer 17, and a measurement sample 16 flows in the flow path 12 in the direction of arrow P (a direction from upstream to downstream). In this specification, “upstream” is upstream of the flow of the measurement sample flowing in the flow path of the catechol sensor, and is the left side of FIG. Further, “downstream” is the downstream of the flow of the measurement sample flowing in the flow path of the catechol sensor, and is the right side of FIG.

  An oxidation electrode 13 is formed in the flow path 12, a reduction electrode 14 is formed on the downstream side of the oxidation electrode 13, and a detection electrode 15 is provided on the downstream side of the reduction electrode 14. Is formed. Thus, the measurement sample 16 flows through the flow path 12 in which the oxidation electrode 13, the reduction electrode 14, and the detection electrode 15 are formed in order from the upstream side to the downstream side.

  In FIG. 1, first, the oxidation electrode 13, the reduction electrode 14, and the detection electrode 15 are formed on the substrate 11, and then the insulating layer 17 is formed so as to secure the flow path 12. By doing so, each electrode is formed at the bottom of the flow path 12.

  When the measurement sample 16 flows in such a flow path 12 and the measurement sample 16 contains a measurement interfering substance, the measurement interfering substance is used for oxidation when an oxidation potential is applied to the oxidation electrode 13. Oxidized by the electrode 13. At the same time, the catecholamine in the measurement sample 16 is also oxidized. Next, the measurement sample 16 containing the measurement interfering substance and catecholamine that has been oxidized by the oxidation electrode 13 flows to the reduction electrode 14, and when the reduction potential is applied to the reduction electrode 14, the measurement that has flowed above. The oxidized catecholamine contained in the sample 16 is reduced. This is because catecholamine has the property of being reversibly oxidized and reduced on the electrode. At this time, the oxidized measurement interfering substance contained in the measurement sample 16 is hardly reduced by the reduction electrode 14.

  Next, the catecholamine reduced by the reduction electrode 14 and the oxidized measurement interfering substance that is hardly reduced by the reduction electrode 14 flow to the detection electrode 15. At this time, by applying a potential to the detection electrode 15, only the catecholamine in the measurement sample 16 is detected, and the oxidized measurement interfering substance flows downstream as it is. In this manner, catecholamine is selectively detected from the measurement sample 16 by the detection electrode 15.

  As described above, catecholamine as a measurement target has a property of being reversibly oxidized and reduced on the electrode as described above, but a measurement interfering substance is basically not reversible for oxidation and reduction. However, if the conditions are met, the measurement interfering substance also exhibits the property of reversibly oxidizing / reducing, but the ratio is very small compared to catecholamine. Therefore, even if the conditions are met and a part of the measurement interfering substance is reduced on the reducing electrode 14, the amount is very small, so that the catecholamine detected by the detecting electrode 15 is hardly affected. . Therefore, the influence of the measurement interfering substance can be reduced.

  That is, in one embodiment of the present invention, the measurement interfering substance is oxidized upstream of the detection electrode 13 in order to reduce the amount of the measurement interfering substance detected when catecholamine is detected by the detection electrode 13. For this purpose, an oxidation electrode 13 is provided. However, at this time, the catecholamine to be detected is also oxidized, but the oxidized catecholamine is reduced by the reducing electrode 14 disposed downstream of the oxidizing electrode 13. At this time, since the oxidized measurement interfering substance is not reduced due to the above-mentioned property or is reduced even if reduced, the influence of the measurement interfering substance on the detection of catecholamine at the detection electrode 15 is substantially eliminated. It will be. That is, the removal efficiency of the measurement interfering substance can be improved as compared with the conventional case. Therefore, the catecholamine concentration can be continuously detected.

  As described above, in order to reduce or eliminate the amount of the measurement interfering substance detected by the detection electrode 15, in one embodiment of the present invention, from the upstream side to the downstream side of the detection electrode. In order, an oxidation electrode and a reduction electrode are arranged. That is, the oxidation electrode 13 has a function of converting a measurement interfering substance into a substance that does not affect the detection of the detection electrode 15, and the reduction electrode 14 uses only the catecholamine to be measured by the detection electrode 15. It has a function of returning to a detectable state, that is, a function of selecting a substance that can be detected by the detection electrode 15 and a substance that cannot be detected, and the function of the oxidation electrode 13 and the reduction electrode 14 allows the measurement sample 16 to Only the measurement interfering substances are selectively removed.

  As described above, in one embodiment of the present invention, the oxidation electrode oxidizes both the catecholamine and the measurement interfering substance, but it is desirable to suppress the oxidation of the catecholamine as much as possible in order to increase the subsequent detection efficiency. That is, in the oxidation electrode, it is desirable to increase the amount of oxidation of the measurement interfering substance and decrease the amount of oxidation of catecholamine.

  Therefore, in one embodiment of the present invention, the surface of the oxidation electrode may be modified with a positive charge in order to suppress catecholamine oxidation. Specifically, a substance having a functional group having a positive charge such as a polymer having a positive charge, such as a thiol, may be modified on the surface of the oxidation electrode. Thiols include single thiols and multiple thiols. The modification of thiols can be performed by a self-assembly method.

  Since the oxidation of the catecholamine on the oxidation electrode is suppressed by modifying the surface of the oxidation electrode with a polymer having a positive charge, the amount of catecholamine detected by the detection electrode 15 Can be suppressed.

In FIG. 1, the electrode for oxidation, the electrode for reduction, and the electrode for detection are planar electrodes. However, in one embodiment of the present invention, the present invention is not limited to this. Other shape electrodes may also be used.
In one embodiment of the present invention, in order to improve the detection sensitivity of catecholamine, it is desirable that each electrode is a comb-type electrode. The comb-shaped electrode means an electrode composed of a trunk part and a plurality of branch parts protruding from the trunk part, and the plurality of branch parts arranged in parallel.

  When the oxidation electrode and the reduction electrode are both comb-type electrodes, it is desirable that they be a pair of comb-type array electrodes. A pair of comb-shaped array electrodes means an electrode pair in which trunk portions of two comb-shaped electrodes are arranged in parallel and branch portions are alternately arranged. In this configuration, except for the end portion of the alternate arrangement region of the branch portions, the branch portions of the oxidation electrode are arranged between two adjacent branch portions of the reduction electrode, and the branches of the reduction electrode are arranged. The section is arranged between two adjacent branches of the oxidation electrode. The reduction electrode is intended to reduce catecholamines oxidized by the oxidation electrode, and therefore, the most downstream of the pair of comb array electrodes is preferably the reduction electrode.

  In one embodiment of the present invention, all of the oxidation electrode, the reduction electrode, and the detection electrode may be array electrodes, or at least one of the electrodes may be an array electrode.

In one embodiment of the present invention, it is desirable that the oxidation electrode, the reduction electrode, and the detection electrode include a material that hardly dissolves even when a potential is applied. Examples of materials for the oxidation electrode and the reduction electrode include carbon, gold, platinum, iridium, and the like. Moreover, carbon, gold | metal | money, platinum, iridium etc. are mentioned as an example of the material for electrodes for a detection. In the above, an example of each electrode is given. However, in the present invention, the material of the electrode is not essential, and any electrode may be used as long as it functions as an electrode.
In one embodiment of the present invention, the substrate is preferably an insulating substrate.

  Furthermore, in one embodiment of the present invention, an enzyme reaction means may be provided on the upstream side of the oxidation electrode in order to further improve the removal efficiency of the measurement interfering substance.

  In this specification, the “enzyme reaction means” is a means for removing a measurement interfering substance from a measurement sample by an enzyme reaction. The enzyme reaction means includes a membrane that performs an enzyme reaction with a measurement interfering substance, an enzyme reactor having a mechanism for removing the measurement interfering substance from the measurement sample by an enzyme reaction, and the like.

  Furthermore, in one embodiment of the present invention, in the flow cell having the above-described catechol sensor, either a tube, a capillary such as a glass capillary, or a probe with a microdialysis membrane may be used for collecting a sample connected to the catechol sensor. good.

  Hereinafter, a catechol sensor according to an embodiment of the present invention will be described in detail based on examples. However, the present invention is not limited to the following examples, and various modifications can be made without departing from the scope of the present invention. Needless to say.

(Example)
The schematic diagram of the catecholamine sensor based on a present Example is shown to Fig.1 (a) and (b). The catechol sensor according to the present embodiment is formed by bonding two glass substrates to the surfaces on which the electrodes of the respective glass substrates are formed.
1A and 1B, a flow path 4 is formed by an insulating layer 10 in a glass substrate 8a as an insulating substrate. In the flow path 4, a first pair of comb array electrodes composed of an oxidation electrode 1 and a reduction electrode 2 and a second pair of combs composed of two detection electrodes 3 in order from the upstream side. A mold array electrode is formed. Each of the oxidation electrode 1, the reduction electrode 2, and the detection electrode 3 is a comb-type electrode. The oxidation electrode 1 and the reduction electrode 2 are gold films, and the detection electrode 3 is a carbon thin film.

  In the present embodiment, first, the oxidation electrode 1, the reduction electrode 2, and the detection electrode 3 are formed on the glass substrate 8a. Next, the insulating layer 10 is formed on the glass substrate 8a on which each electrode is formed so that the flow path 4 is formed. That is, in the flow path 4, the insulating layer 10 is formed so that the glass substrate 8a and the surface of each electrode are exposed.

  In the first pair of comb array electrodes, the trunk portion of the oxidation electrode 1 and the trunk portion of the reduction electrode 2 are arranged substantially in parallel, and the branch portions of the oxidation electrode 1 and the reduction electrode 2 The branch portions are alternately arranged so as to be substantially parallel to each other. Furthermore, the most upstream of the first pair of comb array electrodes is the branch portion of the oxidation electrode 1, and the most downstream is the branch portion of the reduction electrode 2.

  In the second pair of comb-shaped array electrodes, the trunk portions of the two detection electrodes 3 are arranged substantially in parallel, and the branch portions of the two detection electrodes 3 are alternately arranged in parallel to each other. Has been.

  Such an oxidation electrode 1, a reduction electrode 2, and a detection electrode 3 are formed on the same substrate, that is, a glass substrate 8a. Further, an ascorbic acid oxidase film 7 as an enzyme reaction means is formed on the upstream side of the oxidation electrode 1 in the flow path 4.

  The glass substrate 8b as an insulating substrate has a counter electrode 5 at a position facing the region including the first pair of comb array electrodes when the glass substrate 8a and the glass substrate 8b are bonded to each other. A reference electrode 6 is formed at a position facing the formed region including the second pair of comb array electrodes. The counter electrode 5 is a platinum electrode, and the reference electrode is a silver electrode.

  In the present embodiment, the catecholamine sensor is completed by bonding the glass substrate 8a and the glass substrate 8b having such a configuration. In the catecholamine sensor having such a configuration, the catecholamine concentration is continuously monitored by flowing the measurement sample 9 through the flow path 4.

  In this embodiment, the counter electrode 5 and the reference electrode 6 are formed on separate substrates from the oxidation electrode 1, the reduction electrode 2, and the detection electrode 3, but they are formed on the same substrate. May be.

  The oxidation electrode 1 is produced by patterning gold, and a monomolecular film of aminoethanethiol is modified on the gold surface by a self-assembly method. The monomolecular film of thiol used here may be a single type or may be formed using a plurality of thiols at the same time.

  The above electrodes were connected to ALS1000 (CH1, USA) and measured. With respect to the reference electrode 6, a potential of 600 mV was applied to the oxidation electrode 1, a potential of −200 mV was applied to the reduction electrode 2, and a potential of 600 mV and −200 mV were applied to the two electrodes constituting the detection electrode 3. When the measurement sample 9 was introduced, a syringe pump was used, and the flow rate was set to 2 μl / min.

  First, a potential was applied only to the detection electrode 3. When a 100 nM dopamine solution was introduced into the flow path 4 as the measurement sample 9, a current value of 10 nA was obtained. Next, when a potential of 600 mV and −200 mV was applied to the oxidation electrode 1 and the reduction electrode 2 respectively and the same measurement was performed, a current value of 10 nA was obtained. This indicates that dopamine is not consumed when passing through the oxidation electrode 1 and the reduction electrode 2 but reaches the detection electrode 3. Subsequently, when a mixed solution of 50 μM L-ascorbic acid as a measurement interfering substance and 100 nM dopamine was introduced into the flow path 4 as the measurement sample 9, a current value of 10 nA was also obtained. This indicates that L-ascorbic acid is completely removed on the ascorbate oxidase membrane 7 and the oxidation electrode 1.

  As a comparison, when the measurement was performed without using the oxidation electrode 1, a current value of 11 nA was obtained. This indicates that L-ascorbic acid cannot be completely removed by L-ascorbate oxidase. Further, when the self-assembled film was not formed on the oxidation electrode 1, the current value decreased to 8 nA. This indicates that the oxidation of the dopamine in the electrode for oxidation 1 has not been reduced by the reduction electrode 2. Therefore, it is preferable to modify the surface of the oxidation electrode 1 with thiols.

  In this example, ascorbate oxidase was used as the enzyme of the enzyme reaction means, but not limited thereto, and uric acid can be decomposed by fixing uricase or catalase. In addition, although the comb-shaped electrode is used as the detection electrode, an array electrode in which a large number of minute electrodes on the order of micrometers is arranged may be used. When performing measurement while collecting a biological sample, a microdialysis probe or a glass capillary may be connected.

  As described above, by using an electrode modified with a positively charged thiol as an electrode for electrolytic oxidation of a measurement interfering substance, the measurement interfering substance that could not be removed by the enzymatic reaction was selectively removed, and catecholamine was removed. A device that can be selectively detected was constructed.

It is a top view of the catecholamine sensor based on one Embodiment of this invention. (A) is a top view of the catecholamine sensor based on the Example of this invention, (b) is the sectional view on the AA line of (a).

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Substrate 12 Channel 13 Oxidation electrode 14 Reduction electrode 15 Detection electrode 16 Measurement sample 17 Insulating layer

Claims (6)

A catecholamine sensor that detects a catecholamine concentration by flowing a measurement sample from an upstream side to a downstream side in a flow path,
A first electrode for oxidizing catecholamines and measurement interfering substances contained in the measurement sample formed in the flow path;
A second electrode for reducing catecholamines formed in the flow path and oxidized by the first electrode;
A third electrode for detecting the catecholamine formed downstream of the first electrode and the second electrode in the flow path ;
The first electrode and the second electrode are comb-type electrodes,
The branch part of the comb electrode that becomes the first electrode and the branch part of the comb electrode that becomes the second electrode are alternately arranged, and the most downstream of the alternately arranged comb electrode is A catecholamine sensor, wherein the catecholamine sensor is a branch portion of a comb-shaped electrode to be the second electrode . The catecholamine sensor according to claim 1 , wherein the surface of the first electrode is modified with a substance having a functional group having a positive charge.   The catecholamine sensor according to claim 2, wherein the substance is a thiol. The catecholamine sensor according to any one of claims 1 to 3, wherein the third electrode is an array-type electrode. The catecholamine sensor according to any one of claims 1 to 4 , further comprising enzyme reaction means formed upstream of the first electrode in the flow channel. A catecholamine sensor according to any one of claims 1 to 5 ,
One of a capillary for sample collection connected to the catecholamine sensor, a tube connected to the catecholamine sensor, or a probe with a microdialysis membrane connected to the catecholamine sensor is provided. Flow cell.

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