JP2011095152A - Method and device for electrochemical analysis of body fluid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a device for electrochemical analysis of body fluid capable of selectively inactivating ascorbic acid around a work electrode without requiring complicated pre-treatments. <P>SOLUTION: The method for electrochemical analysis of a body fluid includes: a step of contacting the body fluid to metallic coppers 2, 8, applying a positive voltage, eluting divalent copper ions from the metallic coppers 2, 8, and oxidizing ascorbic acid in the body fluid by the copper ions; and a step of contacting the body fluid to the work electrode 2 and a counter electrode 3, applying a positive voltage and measuring a current value generated between the work electrode 2 and the counter electrode 3. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method and apparatus for electrochemically measuring the concentration of a target substance in a body fluid in which ascorbic acid coexists as an interfering substance.

  Gout is a disease caused by hyperuricemia and causing arthritis and is characterized by severe pain. Gout is a lifestyle-related disease, and it is possible to prevent the onset and progression of disease states by managing the blood uric acid concentration.

  For this reason, it is important to accurately grasp the blood concentration of uric acid. However, ascorbic acid coexists in the blood, and ascorbic acid and uric acid are electrochemically oxidized at a close potential, so when measuring uric acid concentration electrochemically, the current caused by ascorbic acid The value is also measured at the same time.

  Patent Document 1 describes a method for electrochemically measuring uric acid. In this method, ascorbic acid is removed from a measurement sample using an ion exchanger having a divalent copper ion.

JP-A-61-210938

  However, in order to detect uric acid using the method described in Patent Document 1, a pretreatment in which a measurement sample is passed through an ion exchanger in advance is required.

  Therefore, the present invention is intended to provide a method and an apparatus for electrochemical analysis of body fluid that do not require complicated pretreatment and can selectively inactivate ascorbic acid around the working electrode.

  That is, the method for electrochemical analysis of body fluid according to the present invention is a method for electrochemically measuring the concentration of a target substance in a body fluid containing ascorbic acid. And the step of eluting divalent copper ions from the metal copper, oxidizing the ascorbic acid in the body fluid with the copper ions, bringing the body fluid into contact with the working electrode and the counter electrode, Applying a positive voltage and measuring a current value generated between the working electrode and the counter electrode under the voltage.

  In the present invention, examples of the body fluid include blood (whole blood, plasma, serum), urine, and the like. Examples of the target substance include uric acid, hypoxanthine, xanthine, theophylline and the like.

  Moreover, it is preferable that the positive voltage applied to metallic copper in order to elute a bivalent copper ion is 0.5V or less.

  In such a case, a positive voltage is applied to metallic copper provided in the vicinity of the working electrode, and divalent copper ions are eluted from the metallic copper, thereby locally 2 around the working electrode. The subsequent electrochemical reaction can be performed by eluting the valent copper ions to selectively oxidize ascorbic acid in the body fluid around the working electrode and to inactivate it electrochemically (dehydroascorbic acid). In the measurement, only the target substance can be selectively detected. For this reason, pretreatment using an ion exchanger or the like is not necessary, and all the series of steps from inactivation of ascorbic acid to measurement of the concentration of the target substance can be electrochemically processed. In addition, even when a salt containing divalent copper ions is added to the body fluid as the sample solution, the copper ions diffuse into the body fluid and selectively inactivate ascorbic acid around the working electrode. However, according to the present invention, ascorbic acid around the working electrode can be reliably oxidized and inactivated.

  Examples of the electrochemical analyzer for carrying out the method according to the present invention include the following. That is, this apparatus is an apparatus for electrochemically measuring the concentration of a target substance in a body fluid containing ascorbic acid, and is provided in the proximity of the working electrode and the working electrode. Metal copper which is a source of divalent copper ions for oxidizing ascorbic acid, a counter electrode, and a cell containing the working electrode, the metal copper and the counter electrode. And If it is such, since the column etc. which were filled with the ion exchanger are unnecessary, it can be set as a simple apparatus structure. Such an electrochemical analyzer is also one aspect of the present invention.

  The electrochemical analyzer according to the present invention further applies a positive voltage to the metallic copper in order to elute divalent copper ions from the metallic copper, and then applies a positive voltage to the working electrode. Voltage applying means for applying a current, current value measuring means for measuring a current value generated between the working electrode and the counter electrode under the voltage, and calculating the concentration of the target substance from the obtained current value And an information processing apparatus. However, these means and devices may be used by being connected to the electrochemical analyzer according to the present invention as an external device without being included in the equipment constituting the electrochemical analyzer according to the present invention.

  The metallic copper is not particularly limited as long as it is provided close to the working electrode. For example, copper plating applied to the surface of the working electrode or copper on the surface of a substrate made of a carbon paste. The copper dispersed in the surface of the working electrode obtained by press-fitting the powder and then cured, and a copper electrode provided independently of the working electrode, etc. are mentioned, among these, for example, An embodiment in which the working electrode contains the metallic copper, such as copper plating on the surface of the working electrode, is preferable. If it is such, since manufacture is easy and the number of electrodes to be used is small, the structure of the apparatus can be simplified and miniaturized. For example, the electrochemical analyzer according to the present invention It is also possible to make it into a chip and make it disposable.

  As the working electrode, a carbon electrode is preferably used because it is excellent in measurement sensitivity of a target substance such as uric acid. As the working electrode, a conductive diamond electrode can be used instead of the carbon electrode.

  Examples of the conductive diamond electrode include those doped with boron, nitrogen, phosphorus, or the like. Among these, boron-doped diamond electrodes doped with boron at a high concentration have a wide potential window (wide oxidation potential and reduction potential), a low background current compared to other electrode materials, and chemical reactions. It has advantages such as excellent resistance, durability, electrical conductivity, and corrosion resistance.

  In the present invention described above, copper is used to oxidize and inactivate ascorbic acid electrochemically, but instead of copper, an iron complex (ferrocene such as potassium ferricyanide), a cobalt complex Even when silver or the like is used (such as a phenanthroline cobalt complex), ascorbic acid can be similarly oxidized and electrochemically inactivated. Embodiments using these copper substitutes are also one aspect of the present invention.

  As described above, according to the present invention, divalent copper ions are locally eluted around the working electrode, and ascorbic acid in the body fluid around the working electrode is selectively oxidized to be electrochemically inactivated. can do. For this reason, pretreatment using an ion exchanger or the like is not necessary, and all the series of steps from inactivation of ascorbic acid to measurement of the concentration of the target substance can be electrochemically processed. Moreover, even if divalent copper ions are directly added to the body fluid as a measurement sample, the copper ions diffuse into the body fluid, and it is difficult to selectively inactivate ascorbic acid around the working electrode. However, according to the present invention, ascorbic acid around the working electrode can be reliably oxidized and inactivated.

It is a schematic diagram showing the equipment composition of the electrochemical analyzer concerning one embodiment of the present invention. It is a voltammogram which shows the result of the LSV measurement which uses the phosphate buffer containing uric acid and ascorbic acid as a sample solution. It is a perspective view which shows the specific example of the electrochemical analyzer which concerns on the same embodiment. It is a perspective view which shows the specific example of the electrochemical analyzer which concerns on other embodiment. It is a perspective view which shows the specific example of the electrochemical analyzer which concerns on other embodiment.

  An embodiment of the present invention will be described below with reference to the drawings.

  As shown in FIG. 1, the electrochemical analyzer 1 according to the present embodiment includes a working electrode 2, a counter electrode 3, a reference electrode 4, and a measurement cell 5 in which these three electrodes are incorporated. The working electrode 2, the counter electrode 3 and the reference electrode 4 are connected to a potentio galvanostat 6 (corresponding to a voltage applying means and a current value measuring means), and the potentio galvanostat 6 is further connected to an information processing device 7. Is connected.

  Each part will be described below. The working electrode 2 is obtained by performing copper plating on the surface of an electrode body made of a conductive diamond electrode or a carbon electrode. Such copper plating is performed by electrolytically reducing the copper ions by applying a negative voltage to the electrode body in a state where the electrode body is immersed in a solution containing divalent copper ions. By depositing copper on the surface of the body.

The counter electrode 3 compensates for the electrolysis current. For example, an electrode made of carbon, platinum, carbon, stainless steel, gold, SnO ₂ or the like can be used.

  A known electrode can be used as the reference electrode 4, and for example, a silver / silver chloride electrode, a calomel electrode, a standard hydrogen electrode, a hydrogen palladium electrode, or the like can be used.

  The measurement cell 5 is configured to store a sample solution S therein so that the sample solution S can come into contact with the working electrode 2, the counter electrode 3, and the reference electrode 4.

  Examples of the sample solution S include body fluids such as blood (whole blood, plasma, serum) and urine. Examples of the target substance include those having electrochemical properties similar to those of ascorbic acid such as uric acid, hypoxanthine, xanthine, and theophylline and having ascorbic acid as an interfering substance in electrochemical measurement.

  The potentiogalvanostat 6 has functions of a potentiostat (potential control), a galvanostat (current control), and an electrometer (potential measurement), and the current generated by controlling the potential of the working electrode 2 The detection signal is transmitted to the information processing device 7. In addition to the function of keeping the potential constant, the potentiogalvanostat 6 also has a function of scanning the potential at a constant speed and stepping to a specified potential at regular intervals. These functions do not need to be mounted on one unit, and for example, a potential holding function and a potential scanning function may be provided separately.

  The information processing device 7 is a general purpose or dedicated device including a CPU, memory, input / output channels, input means such as a keyboard, output means such as a display, A / D converter, D / A converter, etc. The CPU and its peripheral devices cooperate with each other according to a program stored in a predetermined area of the memory, whereby a signal detected by the potentio galvanostat 6 is analyzed, and a target substance is detected and a concentration is measured. The information processing apparatus 7 does not need to be physically integrated, and may be divided into a plurality of devices by wire or wireless.

  Next, a method for measuring the uric acid concentration in the sample solution S containing uric acid and ascorbic acid using the electrochemical analyzer 1 configured as described above will be described.

  The sample solution S is injected into the measurement cell 5, a positive voltage is applied to the working electrode 2, and the copper plating is electrolytically oxidized to elute divalent copper ions. Thereby, bivalent copper ions are locally eluted in the vicinity of the working electrode 2, and ascorbic acid in the sample solution S around the working electrode 2 is oxidized by the copper ions. In addition, it is preferable that the positive voltage applied to the working electrode 2 for electrolytically oxidizing the copper plating to elute divalent copper ions is 0.5 V or less.

  Immediately after elution of copper ions from the working electrode 2, uric acid is oxidized on the working electrode 2 by applying a positive voltage to the working electrode 2, and this oxidation occurs between the working electrode 2 and the counter electrode 3. An electric current accompanying the reaction is generated. This current value (electric signal) is transmitted to the potentio galvanostat 6 to control / detect signals at each electrode. The signal detected by the potentiogalvanostat 6 is transmitted to the information processing device 7, and a calibration curve between the concentration of uric acid and the current value prepared in advance is compared with the obtained current value, so that The uric acid concentration is calculated.

  FIG. 2 shows a case where a phosphate buffer solution containing uric acid (UA) and ascorbic acid (AA) is used as a sample solution S, a carbon electrode plated with copper is used as the working electrode 2, and a voltage of 500 mV is applied to the working electrode 2. Is a voltammogram showing the result of performing linear sweep voltammetry (LSV) measurement after applying divalent copper for 5 seconds to elute divalent copper ions from the copper plating. Each condition is as follows.

Working electrode: Carbon electrode reference electrode obtained by applying a voltage of −300 mV to a carbon sheet (5 mm × 5 mm) for 1 minute to subject the surface to copper plating; Ag / AgCl (silver / silver chloride)
Counter electrode; Pt (platinum)
Sweep condition: Sweep from 0 mV to 900 mV at 50 mV / sec

  As shown in FIG. 2, a current value dependent on the uric acid concentration was obtained even in the presence of ascorbic acid. Moreover, even if the ascorbic acid concentration was changed with the uric acid concentration kept constant, the current value did not change.

  As a specific example of the electrochemical analyzer 1 according to the present embodiment, as a disposable chip-shaped device using blood (plasma) as the sample solution S, for example, the one shown in FIG. That is, the electrochemical analyzer 1 is composed of a base portion 11 and a lid portion 12 each made of polymethyl methacrylate (PMMA). A long channel 13 having a longitudinal section of 1 to 4 μm (width) × 1 μm or more (height) is formed in the base portion 11, and the inner surface of the channel 13 is subjected to a hydrophilic treatment by oxygen plasma. ing. Further, the surface of the lid portion 12 facing the flow path 13 is also subjected to a hydrophilic treatment, whereby the flow path 13 sucks only plasma from the blood dropped on the sample dropping section 14 by capillary action. be able to. In the present electrochemical analyzer 1, the flow path 13 functions as the measurement cell 5. A working electrode 2, a counter electrode 3, and a reference electrode 4 are embedded in the lid portion 12 so as to be in contact with plasma that has flowed into the flow path 13. Carbon electrodes are used as the working electrode 2 and the counter electrode 3, and copper plating is applied to the surface of the working electrode 2. On the other hand, a silver / silver chloride electrode is used as the reference electrode 4. Each electrode is connected to a potentiogalvanostat 6 (not shown). In the present electrochemical analysis device 1, the potentiogalvanostat 6 and the information processing device 7 are used as external devices.

  In order to measure the uric acid concentration in blood using this electrochemical analyzer 1, first, one drop of blood is dropped on the sample dropping unit 14. Then, only plasma is sucked into the flow path 13. And as above-mentioned, the elution of a bivalent copper ion and subsequent electrochemical measurement can measure the uric acid concentration in blood.

  Therefore, according to the electrochemical analyzer and method according to the present embodiment configured as described above, a positive voltage is applied to the working electrode 2 and divalent copper ions are eluted from the copper plating applied to the electrode surface. Thus, divalent copper ions are locally eluted around the working electrode 2 to selectively oxidize ascorbic acid in the body fluid around the working electrode 2 and to inactivate it electrochemically (dehydroascorbic acid In the subsequent electrochemical measurement, only the target substance can be selectively detected. For this reason, pretreatment using an ion exchanger or the like is not necessary, and all the series of steps from inactivation of ascorbic acid to measurement of the concentration of the target substance can be electrochemically processed.

  Further, even if divalent copper ions are directly added to the sample solution S, the copper ions diffuse into the sample solution S, and the ascorbic acid around the working electrode 2 is selectively inactivated. Although difficult, according to this embodiment, ascorbic acid around the working electrode 2 can be reliably oxidized and inactivated.

  Also, by providing metallic copper as a copper plating formed on the surface of the working electrode 2, there is no need to provide a separate copper electrode, so the number of electrodes can be reduced, the device configuration can be simplified, and electrochemical It is also possible to make the analytical device 1 into a chip and make it disposable.

  The present invention is not limited to the above embodiment.

  The working electrode in the present invention is not limited to the electrode surface plated with copper. For example, after pressing copper powder onto the surface of a substrate made of carbon paste, the working electrode is cured to form the working electrode. Also good. Moreover, you may provide a copper electrode separately in proximity to a working electrode, without including copper in a working electrode.

  The electrochemical analysis apparatus 1 according to the embodiment performs measurement by the three-electrode method provided with the working electrode 2, the counter electrode 3, and the reference electrode 4, but the electrochemical analysis apparatus according to the present invention. 1 may be a two-electrode method including only the working electrode 2 and the counter electrode 3. In the three-electrode method, the absolute value of the potential applied between the working electrode 2 and the counter electrode 3 can be controlled, so that measurement with higher accuracy and sensitivity can be performed. According to the above, since two electrodes, that is, the working electrode 2 and the counter electrode 3 are used, the structure of the electrochemical analyzer 1 can be further simplified and miniaturized.

  In the electrochemical analysis apparatus 1 in the form of a chip as shown in FIG. 3, when metallic copper is provided independently of the working electrode 2, for example, as shown in FIG. Although the copper electrode 8 may be provided in parallel with the plasma electrode 8, as shown in FIG. 5, the copper electrode 8 may be provided upstream of the working electrode 2 with respect to the plasma flow.

  In addition, it is needless to say that some or all of the above-described embodiments and modified embodiments may be appropriately combined, and various modifications can be made without departing from the scope of the invention.

DESCRIPTION OF SYMBOLS 1 ... Electrochemical analyzer 2 ... Working electrode 3 ... Counter electrode 4 ... Reference electrode 5 ... Measurement cell 6 ... Potentiogalvanostat 7 ... Information processing apparatus S ...・ Sample solution

Claims (5)

A method for electrochemically measuring the concentration of a target substance in a body fluid containing ascorbic acid,
Contacting the body fluid with metallic copper, applying a positive voltage, eluting divalent copper ions from the metallic copper, and oxidizing the ascorbic acid in the body fluid with the copper ions;
A step of bringing the body fluid into contact with the working electrode and the counter electrode, applying a positive voltage, and measuring a current value generated between the working electrode and the counter electrode under the voltage. A method for electrochemical analysis of body fluids. An apparatus for electrochemically measuring the concentration of a target substance in a body fluid containing ascorbic acid,
A working electrode;
Metallic copper, which is provided close to the working electrode and is a source of divalent copper ions for oxidizing ascorbic acid in the body fluid;
A counter electrode;
A body fluid electrochemical analyzer comprising: the working electrode, the metal copper, and a cell containing the counter electrode. Voltage application means for applying a positive voltage to the metallic copper in order to elute divalent copper ions from the metallic copper, and then applying a voltage in the positive direction to the working electrode;
Current value measuring means for measuring a current value generated between the working electrode and the counter electrode under the voltage;
The body fluid electrochemical analyzer according to claim 2, further comprising: an information processing device that calculates the concentration of the target substance from the obtained current value.   The body fluid electrochemical analyzer according to claim 2 or 3, wherein the working electrode contains the metallic copper.   The body fluid electrochemical analyzer according to claim 2, 3 or 4, wherein the working electrode is a carbon electrode.