JP2009281907A - Method, device and instrument for measuring creatinine, and method, device and instrument for measuring amount of salt in urine using those means - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method, device and instrument capable of performing the precise quantification of creatinine contained in a sample. <P>SOLUTION: The creatine measuring method includes step (A) of bringing the sample containing creatinine into contact with a creatine measuring reagent containing a quinone compound to directly react creatinine contained in the sample with the quinone compound to thereby produce the reduced substance of the quinone compound, step (B) of optically measuring the reaction in the step A, and step (C) of calculating the concentration or amount of creatinine contained in the sample on the basis of the measured value in the step B. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a measurement method, a measurement device, and a measurement apparatus for quantifying creatinine and salt contained in a sample.

  Measurement of creatinine contained in a sample is important in the fields of clinical chemistry and analytical chemistry. Since creatinine is a product of the endogenous metabolism of muscle, it is known that the amount of creatinine in urine reflects the total muscle mass. Therefore, it is said that the amount of creatinine in urine excreted by humans in a day is generally constant for each individual and does not vary from day to day. Therefore, the amount of creatinine in urine is sometimes used as a measure of the density of excreted urine. Moreover, since the amount of creatinine in urine and blood increases or decreases due to uremia or a decrease in renal function, the presence or absence of uremia or a decrease in kidney function can be known by measuring the amount of creatinine in urine or blood.

  As a method for measuring creatinine, a method based on a Jaffe reaction using an alkaline picric acid solution is known. In this method, an orange-red product obtained by reacting picric acid with creatinine is measured spectroscopically (see, for example, Patent Document 1).

  As another creatinine measurement method, a method using an enzyme that specifically reacts with creatinine is known. As a method using an enzyme, for example, there is a method in which ammonia is obtained by decomposing creatinine using creatinine deiminase, and the concentration of creatinine is obtained by measuring the amount of ammonia from a pH change (for example, patents). Reference 2).

  As another method using an enzyme, there is a method of measuring creatinine by the following reactions (Chemical Formula 1) to (Chemical Formula 3).

  As the enzymes that catalyze the reactions of (Chemical Formula 1) to (Chemical Formula 3), creatinine amide hydrolase (creatininase), creatine amidinohydrolase (creatinase), and sarcosine oxidase or sarcosine dehydrogenase are used, respectively. Here, as a method for quantifying creatinine, for example, a method in which a leuco dye or a Trinder reagent is used together with peroxidase and the hydrogen peroxide generated in (Chemical Formula 3) is colored and spectroscopically quantified is used. (For example, refer to Patent Document 3). As another creatinine quantification method, a method of electrochemically oxidizing the hydrogen peroxide generated in (Chemical Formula 3) with an electrode and quantifying creatinine from a flowing current is used (see, for example, Patent Documents 4 and 5). ).

  In addition to the reactions of (Chemical Formula 1) and (Chemical Formula 2), in addition to the reactions of (Chemical Formula 3), as another method using an enzyme, the reaction of sarcosine and an electron carrier is used. Has been proposed (see, for example, Patent Documents 6 and 7). Patent Document 6 discloses a reagent solution in which creatininase, creatinase, sarcosine oxidase and an electron carrier are dissolved in a pH 7 to 8.5 buffer in a sensor having at least a pair of working electrodes and counter electrodes on a substrate. A creatinine biosensor is disclosed which is immobilized by drying on an electrode or a substrate around the electrode. Specifically, it is disclosed that potassium ferricyanide is used as an electron carrier, and a phosphate buffer having a pH of 6.86 is used as a buffer. Further, it is disclosed that when the pH of the buffer solution is pH 7 or lower or pH 8.5 or higher, the enzyme activity decreases, which is not preferable. Patent Document 7 discloses that creatinine is measured by a colorimetric method or an electrochemical detection method using a mediator encapsulated by cyclodextrin together with sarcosine oxidase. Specifically, α-naphthoquinone (1,4-naphthoquinone) is mentioned as an example of a mediator incorporated by cyclodextrin. However, in Patent Document 7, when mediators such as α-naphthoquinone and 1-methoxy-5-methylphenazinium methyl sulfate (M-PMS) are not included in cyclodextrin, creatinine is measured using an enzyme. It is disclosed that it is inappropriate.

  In addition to the reactions of (Chemical Formula 1) and (Chemical Formula 2), in addition to the reactions of (Chemical Formula 3), as another method using an enzyme, a reaction between sarcosine and a tetrazolium indicator is used for spectroscopy. In particular, a method for measuring creatinine has been proposed (see, for example, Patent Document 8). Patent Document 8 discloses that a mixed reagent consisting of creatinine hydrolase, creatine amidinohydrolase, sarcosine dehydrogenase, tetrazolium indicator thiazolyl blue and potassium phosphate pH 7.5 is used as a composition for measuring creatinine. .

  In addition, as another method using an enzyme, creatinine amide hydrolase, creatine amidinohydrolase and sarcosine dehydrogenase are used to change creatinine into glycine and formaldehyde. Has been proposed (see, for example, Patent Document 9). In Patent Document 9, in addition to creatinine amide hydrolase, creatine amidinohydrolase, sarcosine dehydrogenase and a colorant, a pH 7.5 phosphate buffer and potassium ferricyanide as a reaction accelerator for promoting the formation of formaldehyde are used. It is disclosed.

  Further, as another method using an enzyme, a method of measuring creatinine using a polymer that catalyzes hydrolysis of creatinine, an electrode on which a sarcosine oxidase and a mediator are immobilized has been proposed (for example, Patent Document 10). reference). Patent Document 10 discloses that potassium ferricyanide, ferrocene, osmium derivatives, phenazine methosulfate (PMS), and the like can be used as mediators.

As another creatinine measurement method, a method using potassium 1,4-naphthoquinone-2-sulfonate has been proposed (see, for example, Patent Document 11 and Non-Patent Documents 1 to 3).
US Pat. No. 3,705,003 JP-T-2001-512692 JP-A-62-257400 Special table 2003-533679 gazette US Pat. No. 5,466,575 JP 2006-349412 A JP 2005-1118014 A Japanese Patent Laid-Open No. 55-023998 JP 54-151095 A JP 2003-326172 A JP-A-63-033661 Sullivan, 1 other, “Highly specific test of creatinine”, Journal of Biological Chemistry, 1958, Vol. 233, No. 2, p. 530-534 (Sullivan, “A Highly Specific Test for Creatine”, 1958, Vol. 233, No. 2, p. 530-534) Narayanan, 1 other, “Creatinine: Review”, Clinical Chemistry, 1980, Vol. 26, No. 8, p. 1119-1126 (Narayanan, “Creatinine: A Review”, Clinical Chemistry, 1980, Vol. 26, No. 8, p. 1119-1126) Cooper, et al., “Evaluation of four methods for measuring urinary creatinine”, Clinical Chemistry, 1961, Vol. 7, No. 6, p. 665-673 (Cooper, “An Evaluation of Four Methods of Measuring Primary Creatine”, 1961, Vol. 7, No. 6, P. 665-673)

  However, the conventional method has the following problems.

  In the method described in Patent Document 1, amino acids such as glycine, histidine, glutamine, and serine and sugars such as glucose react with picric acid, and therefore samples containing amino acids and sugars, for example, biological samples such as urine and blood It is difficult to accurately quantify creatinine in it.

  Further, in the method described in Patent Document 2, since changes in pH and potential are unstable, it is difficult to accurately quantify creatinine.

  In addition, in the methods described in Patent Documents 2 to 10, the presence of ionic species such as salinity and urea in the sample causes enzyme activity to be reduced due to denaturation of the enzyme. The reaction rate varies depending on the concentration of. Therefore, when creatinine is quantified in a sample containing ionic species and urea, for example, a biological sample such as urine and blood, an error occurs in the measurement result depending on the concentration of the ionic species and urea contained in the sample.

  In addition, regarding the method using potassium 1,4-naphthoquinone-2-sulfonate described in Patent Document 11 and Non-Patent Document 1, in Non-Patent Documents 2 and 3, the reproducibility of measurement results is very low. A problem has been reported.

  Therefore, in view of the above-described conventional problems, the present invention has a first object to provide a creatinine measurement method, a creatinine measurement device, and a creatinine measurement apparatus that can accurately quantify creatinine contained in a sample. To do.

  It is a second object of the present invention to provide a urinary salt content measuring method, a urinary salt content measuring device, and a urinary salt content measuring apparatus capable of accurately quantifying the salt content in urine. And

In order to solve the above-mentioned conventional problems, the creatinine measurement method of the present invention comprises (A) a sample containing creatinine and a creatinine measurement reagent containing a quinone compound, thereby bringing the creatinine contained in the sample into contact. And a step of directly reacting the quinone compound to produce a reduced product of the quinone compound,
(B) electrochemically measuring the reaction in the step A, and (C) determining the concentration or amount of the creatinine contained in the sample based on the measurement value in the step B.

Moreover, the urinary salt content measuring method of the present invention uses urine as a sample, and in addition to steps A and B of the creatinine measuring method,
(F) a step of measuring electrical characteristics of the urine before the urine and the reagent for measuring creatinine come into contact; and (G) a measured value in the process B and the electrical characteristics measured in the process F. And a step of obtaining a value reflecting the amount of salt excreted in the urine based on the above.

  Further, the creatinine measuring device of the present invention is a creatinine measuring device used in the above creatinine measuring method, and is connected to the sample storing chamber for storing a sample containing creatinine, the sample storing chamber, and the sample storing chamber. A sample introduction port for introducing the sample into the chamber; at least two electrodes disposed in the sample storage chamber; and a creatinine measurement reagent including a quinone compound disposed in the sample storage chamber.

  The urinary salt content measuring device of the present invention is a urinary salt content measuring device used in the above urinary salt content measuring method, and includes a first sample storage chamber for storing urine as a sample. A first sample inlet for introducing the urine into the first sample storage chamber, and at least two electrodes disposed in the first sample storage chamber. And a reagent for measuring creatinine containing a quinone compound, a second sample storage chamber for storing the urine, and the second sample storage chamber, disposed in the first sample storage chamber, A second sample introduction port for introducing the urine into the second sample storage chamber; and at least two electrodes disposed in the second sample storage chamber.

  Further, the creatinine measuring apparatus of the present invention includes a measuring device mounting portion for mounting the creatinine measuring device, a creatinine in the sample storage chamber of the creatinine measuring device mounted on the measuring device mounting portion, and the creatinine measuring reagent. A measurement unit that electrochemically measures the reaction with the calculation unit, and a calculation unit that obtains the concentration or amount of the creatinine contained in the sample based on the measurement value in the measurement unit.

  Further, the urinary salt content measuring apparatus of the present invention is a measuring device mounting portion for mounting the urinary salt content measuring device, and the first of the urinary salt content measuring device mounted on the measuring device mounting portion. A first measurement unit for electrochemically measuring a reaction between creatinine and the creatinine measurement reagent in the sample storage chamber, and the second sample of the urinary salt content measurement device attached to the measurement device attachment unit Based on the second measurement unit for measuring the electrical characteristics of the urine in the storage chamber, and the measured values in the first measurement unit and the electrical characteristics measured by the second measurement unit, A calculation unit for obtaining a value reflecting the amount of salt excretion is provided.

  According to the creatinine measurement method of the present invention, creatinine contained in a sample can be accurately quantified.

  The inventor of the present invention newly found that creatinine and a quinone compound react directly. In this reaction, even in the absence of an enzyme that acts on creatinine, creatinine and a quinone compound react with each other to reduce the amount of creatinine and quinone compound, and creatinine oxide and quinone compound reduced product. And generate.

  The present invention has been made based on the above-mentioned new findings, and is characterized by using a quinone compound as an active ingredient of a reagent composition for measuring creatinine.

The method for measuring creatinine of the present invention comprises (A) a sample containing creatinine and a reagent for measuring creatinine containing a quinone compound, whereby the creatinine contained in the sample reacts directly with the quinone compound. A step of producing a reduced product of the quinone compound,
(B) electrochemically measuring the reaction in the step A, and (C) determining the concentration or amount of the creatinine contained in the sample based on the measurement value in the step B.

  According to this method, unlike conventional measurement methods, creatinine reacts directly with quinone compounds in the absence of an enzyme or picric acid that acts on creatinine, thus interfering with ionic species such as salinity, urea, amino acids, sugars, etc. The reaction proceeds without being affected by the components. Therefore, even when a biological sample such as urine or blood is used, creatinine contained in the sample can be quantified with higher accuracy than conventional measurement methods.

  As the quinone compound used in the present invention, a quinone compound having an oxidation-reduction potential at pH 7 and 25 ° C. which is more positive than −180 mV with respect to a silver-silver chloride electrode (hereinafter abbreviated as “vs Ag / AgCl”) is used. It is preferable. A preferable upper limit value of the oxidation-reduction potential at pH 7 and 25 ° C. is 0 mV (vs Ag / AgCl). A more preferable range of the oxidation-reduction potential of the quinone compound at pH 7 and 25 ° C. is −100 mV to 0 mV (vs Ag / AgCl).

As the quinone compound, 1,2-naphthoquinone (chemical formula: C ₁₀ H ₆ O ₂ ) represented by the structural formula of the following (Chemical Formula 4), 5-hydroxy-1 represented by the structural formula of the following (Chemical Formula 5) , 4-naphthoquinone (chemical formula: C ₁₀ H ₆ O ₃ ) or the like can be used. The oxidation-reduction potential at pH 7 and 25 ° C. is -60 mV (vs Ag / AgCl) for 1,2-naphthoquinone and -180 mV (vs Ag / AgCl) for 5-hydroxy-1,4-naphthoquinone.

  In the step A, a buffer may be brought into contact with the sample.

  Examples of the buffer used in the present invention include phosphate buffers such as dipotassium hydrogen phosphate and potassium dihydrogen phosphate, citric acid buffers, phthalic acid buffers, acetic acid buffers, MES buffers, and the like. Is mentioned.

  In the creatinine measuring method of the present invention, in the step A, it is preferable that the pH of the sample is adjusted to 4 to 8 by bringing the phosphate buffer into contact with the sample. More preferably, the pH of the sample is adjusted to 4-6. More preferably, the pH of the sample is adjusted to 4-5.

  If it does in this way, since the reaction rate of the direct reaction of creatinine and a quinone compound becomes quick, measurement time can be shortened.

  In the creatinine measurement method of the present invention, the phosphate buffer is preferably composed of dipotassium hydrogen phosphate and potassium dihydrogen phosphate. If it does in this way, when these phosphates melt | dissolve in a sample, the pH of the sample after a phosphate buffer will melt | dissolve can be adjusted in the range of 4-8.

  In the creatinine measurement method of the present invention, in step A, the concentration of the phosphate buffer in the sample after the phosphate buffer is dissolved is preferably 5 to 1000 mM. The present inventor has found that the rate of reaction between creatinine and a quinone compound increases as the concentration of phosphate ions increases. If the concentration of the phosphate buffer is 5 mM or more, a sufficient reaction rate can be obtained. 1000 mM is the upper limit of the solubility of the phosphate buffer having a buffering ability to adjust the pH of the sample to 4-8.

In the step B, at least two electrodes are brought into contact with the sample,
Step B is
(D) applying a voltage between the two electrodes, and (E) detecting a value of electric current or a charge amount flowing between the two electrodes,
In step C,
The concentration or amount of the creatinine contained in the sample may be obtained based on the current value or the charge amount detected in the step E.

  In this way, the concentration or amount of creatinine contained in the sample can be easily determined electrochemically.

  Reducing substances such as uric acid and ascorbic acid are contained in blood and urine. By using a quinone compound having a low oxidation-reduction potential as a reagent for measuring creatinine, when the reduced form of the quinone compound produced by the reaction with creatinine is oxidized in step E, reducing substances such as uric acid and ascorbic acid are oxidized. Therefore, the reduced form of the quinone compound can be oxidized, so that the influence of the reducing substance contained in the sample can be eliminated and the creatinine concentration can be quantified with high accuracy.

The urinary salt content measuring method of the present invention uses urine as a sample, and in addition to steps A and B of the above creatinine measuring method,
(F) a step of measuring electrical characteristics of the urine before the urine and the reagent for measuring creatinine come into contact; and (G) a measured value in the process B and the electrical characteristics measured in the process F. And a step of obtaining a value reflecting the amount of salt excreted in the urine based on the above.

  The electrical characteristics of urine before the urine and the creatinine measurement reagent come into contact with each other and the creatinine measurement reagent dissolves in the urine reflect the concentration of the electrolyte contained in the urine. The concentration of the electrolyte contained in urine has a correlation with the concentration of salt contained in urine. Ingredients such as salt are affected by water intake, sweating, etc., and concentrated or diluted to be excreted in urine. For this reason, the concentration of urine components such as salt contained in the urine collected at any time, which is urine collected at any time during the day and at night, varies under the influence of urine concentration / dilution.

  On the other hand, as described above, since creatinine is produced depending on muscle mass, it is known that the amount of creatinine excreted in urine per unit time is constant. Therefore, even when urine is used at any time, for example, the ratio of the measured urinary component concentration to the creatinine concentration (urine component / creatinine ratio) is determined to correct the influence of urine concentration / dilution. be able to.

  According to the urinary salt content measuring method of the present invention, by using the measured value in Step B that accurately reflects the creatinine concentration and the electrical characteristics measured in Step F that reflect the salinity concentration. Since the influence of urine concentration / dilution is accurately corrected, a value that appropriately reflects the amount of salt excreted in urine can be obtained.

  Here, as electrical characteristics of urine, resistance, conductivity, impedance, voltage (or current) signal output with respect to the input current (or voltage) signal, and phase of the input AC signal are output. For example, a phase difference from the phase of the AC signal.

  Examples of values that reflect the amount of urinary salt excretion determined in step G include the amount of salt per unit amount of creatinine, the amount of urinary salt excretion per unit time (for example, one day), and the unit time (for example, one day). The amount of salt intake per hit is listed.

  A creatinine measuring device of the present invention is a creatinine measuring device used in the above creatinine measuring method, and is in communication with the sample storage chamber for storing a sample containing creatinine, and in the sample storage chamber. A sample introduction port for introducing the sample; at least two electrodes disposed in the sample storage chamber; and a creatinine measurement reagent including a quinone compound disposed in the sample storage chamber.

  According to this device, unlike conventional measurement devices, creatinine and quinone compounds react directly even in the absence of an enzyme that acts on creatinine or picric acid in the sample storage chamber. The reaction proceeds without being affected by interfering components such as amino acids and sugars. Therefore, even when a biological sample such as urine or blood is used, creatinine contained in the sample can be quantified with higher accuracy than conventional measurement devices.

  The creatinine measuring device of the present invention may further include a phosphate buffer disposed in the sample storage chamber.

  The urinary salt content measuring device of the present invention is a urinary salt content measuring device used in the above urinary salt content measuring method, the first sample storage chamber for storing urine as a sample, A first sample introduction port for communicating with the first sample storage chamber and for introducing the urine into the first sample storage chamber; and at least two electrodes disposed in the first sample storage chamber; A reagent for measuring creatinine containing a quinone compound, a second sample storage chamber for storing the urine, and a second sample storage chamber disposed in the first sample storage chamber, A second sample introduction port for introducing the urine into the two sample storage chambers, and at least two electrodes disposed in the second sample storage chamber.

  According to this device, it is possible to accurately measure the amount of reaction between creatinine contained in urine and the reagent for measuring creatinine, which accurately reflects creatinine concentration, and electrical characteristics of urine, which reflects salt concentration. it can. By using the reaction amount and the electrical characteristics, the influence of urine concentration / dilution is corrected with high accuracy, so that a value that appropriately reflects the amount of salt excreted in urine can be obtained.

  The creatinine measuring apparatus of the present invention includes a measuring device mounting portion for mounting the creatinine measuring device, a creatinine in the sample storage chamber of the creatinine measuring device mounted on the measuring device mounting portion, and the creatinine measuring reagent. A measurement unit that electrochemically measures the reaction, and a calculation unit that obtains the concentration or amount of the creatinine contained in the sample based on the measurement value in the measurement unit are provided.

  According to this measuring apparatus, the concentration or amount of creatinine contained in the sample can be electrochemically measured using the creatinine measuring device mounted on the measuring device mounting portion. Unlike the conventional measurement apparatus, the creatinine measurement apparatus of the present invention reacts directly with creatinine and a quinone compound even in the absence of an enzyme that acts on creatinine or picric acid in a sample storage chamber. The reaction proceeds without being affected by interfering components such as seeds, urea, amino acids and sugars. Therefore, even when a biological sample such as urine or blood is used, creatinine contained in the sample can be quantified with higher accuracy than conventional measurement devices.

  The creatinine measuring apparatus of the present invention is such that the creatinine measuring device further includes at least two electrodes arranged in the sample storage chamber, and the measuring unit applies a voltage between the two electrodes. And a detection unit that detects a value of electric current or a charge amount flowing between the two electrodes, and the calculation unit is included in the sample based on the current value or the charge amount detected by the detection unit. The concentration or amount of the creatinine contained may be determined.

  According to this configuration, the concentration or amount of creatinine contained in the sample can be easily determined electrochemically using the creatinine measurement device attached to the measurement device attachment portion.

  A urinary salt content measuring apparatus according to the present invention includes a measurement device attachment portion for attaching the urinary salt content measurement device, and the first sample of the urinary salt content measurement device attached to the measurement device attachment portion. A first measurement unit for electrochemically measuring a reaction between creatinine and the reagent for measuring creatinine in the storage chamber, and the second sample storage chamber of the urinary salt content measurement device attached to the measurement device mounting portion A second measuring unit for measuring the electrical characteristics of the urine in the blood, and based on the measured values in the first measuring unit and the electrical characteristics measured by the second measuring unit, the salinity in the urine A calculation unit for obtaining a value reflecting the excretion amount is provided.

  According to this measuring device, the concentration of urine can be corrected with respect to the electrical characteristics of the measured urine using the urine creatinine concentration quantified with high accuracy. Can be determined.

  According to the urinary salt content measurement method of the present invention, the amount of reaction between creatinine contained in urine and the reagent for measuring creatinine, which accurately reflects the creatinine concentration, and the urine electricity that reflects the salt concentration. By using the characteristics, the influence of concentration / dilution of urine is corrected with high accuracy, so that a value that appropriately reflects the amount of salt excreted in urine can be obtained.

  Examples of the sample include body fluids such as blood, serum, plasma, urine, interstitial fluid, lymph fluid, and saliva in addition to an aqueous solution. In particular, urine is a very effective sample for non-invasive daily health management at home. Since the concentrations of ionic species and urea in these body fluids are relatively high, the effects of the present invention can be obtained very high.

  The material of at least two electrodes in the present invention is preferably one containing at least one of gold, platinum, palladium, an alloy or mixture thereof, and carbon. These materials are chemically and electrochemically stable, and a stable measurement can be realized. As the third electrode, a stable electrode, for example, a reference electrode such as an Ag / AgCl or saturated calomel electrode may be used in combination with the above two electrodes. It is preferable to regulate the potential of one of the two electrodes with respect to the third electrode because the potential for measurement becomes stable. Further, for example, an Ag / AgCl or saturated calomel electrode may be used as the other of the two electrodes.

  In the measurement device of the present invention, it is preferable that the creatinine measurement reagent is provided in a dry state and is disposed so as to dissolve in the sample when the sample is introduced into the sample storage chamber.

  For example, after impregnating a porous carrier composed of glass fiber, filter paper, or the like with a solution containing a creatinine measurement reagent, the creatinine measurement reagent is supported on the carrier by drying, and the carrier contacts the sample. What is necessary is just to provide in a part. Alternatively, the reagent for measuring creatinine may be arranged by directly applying a solution containing the reagent for measuring creatinine on the wall surface of the part in contact with the sample in the measuring device and then drying.

  The measurement device is preferably attached to a measurement device attachment portion of the measurement apparatus in a detachable state. In particular, when a biological fluid such as urine or blood is used, the measuring device is preferably disposable from a hygienic viewpoint.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1)
A creatinine measuring device according to Embodiment 1 of the present invention will be described with reference to FIG. FIG. 1 is an exploded perspective view showing a configuration of a creatinine measuring device according to the present embodiment.

  The creatinine measuring device 100 according to the present embodiment is used in a creatinine measuring method for electrochemically quantifying the concentration of creatinine contained in a sample. The creatinine measuring device 100 according to the present embodiment has a structure in which an insulating first substrate 102 and a second substrate 104 having air holes 108 are combined with a spacer 106 having a slit 110 interposed therebetween. Yes.

  The first substrate 102 is electrically connected to the first electrode 112, the second electrode 114, the first lead 122 electrically connected to the first electrode 112, and the second electrode 114. A second lead 124 is disposed. A reagent layer 130 containing a reagent for measuring creatinine is disposed on the first electrode 112 and the second electrode 114. The dimensions of the first substrate 102 may be set as appropriate. For example, the width is about 7 mm, the length is about 30 mm, and the thickness is about 0.7 mm.

  Next, a method for manufacturing the creatinine measuring device 100 will be described. In the present embodiment, 1,2-naphthoquinone, which is a quinone compound, is used as an active ingredient of a reagent for measuring creatinine.

  First, palladium is sputtered on a first substrate 102 made of polyethylene terephthalate with a resin electrode pattern mask placed thereon, whereby a first electrode 112, a second electrode 114, a first lead 122, And a second lead 124 is formed. The first electrode 112 and the second electrode 114 are electrically connected to terminals of a creatinine measuring device described later by a first lead 122 and a second lead 124, respectively.

Next, an aqueous solution in which 1,2-naphthoquinone, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate are dissolved over the first electrode 112 and the second electrode 114 provided over the first substrate 102 is used. After a predetermined amount is dropped using a microsyringe or the like, the first substrate 102 is left in an environment of about room temperature to 30 ° C. and dried to form the reagent layer 130. The concentration and amount of the aqueous solution containing the reagent to be applied may be selected according to the characteristics and size of the required device. For example, the concentration of 1,2-naphthoquinone in the aqueous solution containing the reagent is about 0.5 mM. Yes, the dripping amount is about 0.7 mL. Also, the area of the region for forming the reagent layer 130 may be appropriately selected in view of such as solubility of the reagent to the sample. For example, for the area ² about 3 mm.

  Next, the first substrate 102 on which the electrode and reagent layer 130 is formed, the polyethylene terephthalate spacer 106 having the slit 110, and the second substrate 104 having the air holes 108 are combined. An adhesive is applied to each joint portion of the first substrate 102, the spacer 106, and the second substrate 104, and these are bonded to each other, and then pressed to stand and adhere. Instead of this method, after combining without applying an adhesive, the joining portion may be welded by heat or ultrasonic waves using a commercially available welding machine.

  When the first substrate 102, the spacer 106, and the second substrate 104 are combined, the space formed by the slits 110 and the second substrate 104 provided in the first substrate 102, the spacer 106 is stored in the sample. Functions as a chamber. In addition, the opening of the slit 110 functions as the sample inlet 132.

  Next, a creatinine measuring apparatus according to the present embodiment and a creatinine measuring method using the same will be described with reference to FIGS. FIG. 2 is a perspective view showing the appearance of the creatinine measuring apparatus 200 according to the present embodiment, and FIG. 3 is a block diagram showing the configuration of the creatinine measuring apparatus 200.

  First, the configuration of the creatinine measuring apparatus 200 will be described with reference to FIG.

  The casing 202 of the creatinine measuring apparatus 200 has a measurement device mounting portion 208 for mounting the creatinine measuring device 100 on the creatinine measuring apparatus 200, a display 204 on which the measurement results are displayed, and the measurement of creatinine by the creatinine measuring apparatus 200. A measurement start button 206 for starting is provided. In addition, the measurement device attachment portion 208 includes a first terminal electrically connected to the first lead 122 and the second lead 124 of the creatinine measurement device 100 attached to the measurement device attachment portion 208, respectively. A second terminal is provided.

  Next, the configuration inside the housing 202 of the creatinine measuring apparatus 200 will be described with reference to FIG.

  The creatinine measuring apparatus 200 includes a voltage application unit 302, an electric signal detection unit 304, a control unit 306, a timing unit 308, and a storage unit 310 inside the housing 202.

  The voltage application unit 302 is connected to the first lead 122 and the second lead 124 of the creatinine measurement device 100 attached to the measurement device attachment unit 208 via a first terminal and a second terminal, respectively. Thus, the voltage or potential is applied to the first electrode 112 and the second electrode 114 of the creatinine measuring device 100.

  The electric signal detection unit 304 has a function of detecting and measuring the electric signals from the first electrode 112 and the second electrode 114 via the first terminal and the second terminal. The electric signal detection unit 304 corresponds to the detection unit in the present invention.

  The storage unit 310 stores correlation data corresponding to a calibration curve representing the correlation between the concentration of creatinine and the electrical signal detected by the electrical signal detection unit 304. As the storage unit 310, for example, a memory such as a RAM or a ROM can be used.

  The control unit 306 has a function of converting the electrical signal detected and determined by the electrical signal detection measurement unit 304 into a creatinine concentration with reference to the correlation data. The control unit 306 corresponds to the calculation unit in the present invention. As the control unit 306, for example, a microcomputer such as a CPU (Central Processing Unit) can be used.

  Next, the creatinine measuring method according to the present embodiment using the creatinine measuring device 100 and the creatinine measuring apparatus 200 will be described.

  First, the user inserts the lead side of the creatinine measuring device 100 into the measuring device mounting portion 208 of the creatinine measuring apparatus 200. As a result, the first lead 122 and the second lead 124 of the creatinine measuring device 100 come into contact with the first terminal and the second terminal provided inside the measuring device mounting portion 208, respectively. Conducted to.

  When the creatinine measurement device 100 is inserted into the measurement device attachment unit 208, a measurement device insertion detection switch including a micro switch provided in the measurement device attachment unit 208 is activated and outputs a signal to the control unit 306. When the control unit 306 detects the insertion of the creatinine measurement device 100 based on the output signal from the measurement device insertion detection switch, the control unit 306 controls the voltage application unit 302, via the first terminal and the second terminal, A voltage (for example, 0.2 V) is applied between the first electrode 112 and the second electrode 114.

  Next, the user brings the sample into contact with the sample inlet 132 of the creatinine measuring device 100. By this contact, the sample is sucked into the sample storage chamber of the creatinine measuring device 100 through the sample introduction port 132 by capillary action, and the sample storage chamber is filled with the sample. When the sample comes into contact with the first electrode 112 and the second electrode 114, current flows between the first electrode 112 and the second electrode 114 through the sample. The electrical signal detection unit 304 detects it.

  When the control unit 306 detects that the sample has been introduced into the sample storage chamber based on the output signal from the electrical signal detection unit 304, the control unit 306 controls the voltage application unit 302 to apply the voltage applied by the voltage application unit 302. Switch to a different voltage (eg 0V or open circuit). In addition, along with the detection of sample introduction, the control unit 306 starts timing by the timer unit 308 that is a timer.

  When the reagent layer 130 exposed in the sample storage chamber comes into contact with the sample, 1,2-naphthoquinone in the creatinine measurement reagent contained in the reagent layer 130 is dissolved in the sample. The 1,2-naphthoquinone dissolved in the sample reacts directly with the creatinine contained in the sample, whereby creatinine oxide and reduced 1,2-naphthoquinone are generated.

  When the control unit 306 determines that a predetermined time (for example, 60 seconds) has elapsed based on a signal from the time measuring unit 308, the control unit 306 controls the voltage application unit 302 to control the first electrode 112 and the second electrode. A different voltage is applied between the first electrode 112 and the second electrode 114 (for example, a voltage at which the first electrode 112 becomes +0.6 V compared to the second electrode 114). In this way, an electrical signal such as a current flowing between the first electrode 112 and the second electrode 114 is measured by the electrical signal detection unit 304 after a certain time (for example, 5 seconds) has elapsed since the voltage was applied. At this time, the reduced 1,2-naphthoquinone is oxidized at the first electrode 112. The electrical signal measured by the electrical signal detection unit 304 depends on the concentration of creatinine contained in the sample.

  The control unit 306 reads the correlation data representing the correlation between the electrical signal stored in the storage unit 310 and the creatinine concentration, and refers to it to convert the electrical signal detected by the electrical signal detection unit 304 into the creatinine concentration in the sample. Convert to.

  The obtained creatinine concentration is displayed on the display 204. By displaying the creatinine concentration on the display 204, the user knows that the creatinine concentration measurement has been completed. The obtained creatinine concentration is preferably stored in the storage unit 310 together with the time measured by the time measuring unit 308.

  According to the creatinine measuring device 100 according to the present embodiment, unlike the conventional measuring device, creatinine and 1,2-naphthoquinone are present in the sample storage chamber even if there is no enzyme or picric acid acting on creatinine. Since it reacts directly, the reaction proceeds without being affected by interfering components such as ionic species such as salt, urea, amino acids, and sugars. Therefore, even when a biological sample such as urine or blood is used, creatinine contained in the sample can be quantified with higher accuracy than conventional measurement devices.

  In the present embodiment, an example is shown in which 1,2-naphthoquinone is used as an active ingredient contained in the reagent for measuring creatinine, but 5-hydroxy-1,4-naphthoquinone may be used instead. Even when 5-hydroxy-1,4-naphthoquinone is used as an active ingredient contained in a reagent for measuring creatinine, conventional measurement is not affected by interference components such as ionic species such as salt, urea, amino acids, and sugars. Creatinine contained in the sample can be quantified with higher accuracy than the device.

  Moreover, in this Embodiment, it is not limited to this which showed the example in which a creatinine measuring device is provided with one reagent layer. The creatinine measuring device may include two reagent layers, for example, a first reagent layer containing a creatinine measuring reagent and a second reagent layer containing a phosphate buffer.

  In the present embodiment, an example in which the control unit detects that a sample has been introduced into the sample storage chamber and the voltage applied by the voltage application unit is switched to a different voltage has been described, but the present invention is not limited thereto. As long as a current depending on the concentration of creatinine is obtained, the voltage value necessary for measurement (here, as described above, for example, the voltage at which the first electrode becomes +0.6 V compared to the second electrode) is used as the creatinine. If the voltage is applied from the time of detecting the insertion of the measuring device, the voltage value may be continuously applied after the sample is detected.

  Further, although an example in which the potential applied to the first electrode for obtaining an electric signal is 600 mV with respect to the second electrode is described in this embodiment mode, the present invention is not limited to this. The voltage between the first electrode and the second electrode may be a voltage at which 1,2-naphthoquinone reduced by the oxidation-reduction reaction with creatinine is oxidized.

  In the present embodiment, an example in which the time (reaction time) until detection of an electrical signal after detection of a sample is set to 60 seconds is shown, but the value is not necessarily required. As long as a difference in current value with respect to a difference in creatinine concentration can be detected significantly, a reaction time smaller than the above can be used. On the other hand, when the reaction time is increased, the possibility that the reaction between creatinine and 1,2-naphthoquinone reaches a complete state or a steady state increases, so the amount of creatinine is reduced without being affected by environmental conditions such as temperature. It becomes easy to quantify more accurately.

  Moreover, although the example which detects an electrical signal 5 seconds after applying an electric potential to an electrode was shown, it is not limited to this time. This time should just be the time which can detect the difference of an electrical signal with respect to the difference in creatinine concentration significantly.

  In this embodiment, an example of the electrode system and the lead / terminal is shown, but the shape, number, arrangement, etc. thereof are not limited to these.

  Further, in order to more smoothly introduce the sample into the sample storage chamber of the creatinine measuring device, a solution obtained by dissolving lecithin in toluene or other organic solvent is applied to the inner wall of the second substrate and dried. A lecithin layer may be formed. By adopting such a structure, the amount of the sample can be made constant with higher reproducibility, so that creatinine contained in the sample can be quantified with higher accuracy.

  The creatinine measuring apparatus may further include a recording unit for recording the creatinine concentration obtained as a measurement result on a storage medium such as an SD card. By storing in a removable storage medium, the measurement result can be easily taken out from the creatinine measurement device, so that it becomes easy to request an analysis related company to analyze the measurement result.

  Moreover, the creatinine measuring device may further include a transmission unit for transmitting the creatinine concentration obtained as a measurement result to the outside of the creatinine measuring device. As a result, measurement results can be sent to analysis-related departments or analysis-related contractors in hospitals and analyzed at analysis-related departments or analysis-related contractors, so the time from measurement to analysis can be reduced. .

  The creatinine measuring apparatus may further include a receiving unit for receiving the result of analysis in an analysis related department or an analysis related business. As a result, the analysis result can be quickly fed back to the user.

(Embodiment 2)
Next, a urinary salt content measuring device according to Embodiment 2 of the present invention will be described with reference to FIGS. FIG. 4 is an exploded perspective view showing a configuration of the urinary salt content measuring device according to the present embodiment as viewed from the first surface side of the first substrate, and FIG. 5 is a urinary salt content according to the present embodiment. It is a disassembled perspective view which shows the structure which looked at the measuring device from the 2nd surface side of the 1st board | substrate.

  The urinary salt content measuring device 700 according to the present embodiment electrochemically measures creatinine contained in urine as a sample and measures the electrical characteristics of urine, and uses these measurement results for one day. It is used in a method for estimating the amount of salt contained in urine excreted in the urine.

  In the urinary salt content measuring device 700 according to the present embodiment, the first substrate 102 having the insulating property and the second substrate 104 having the air holes 108 are connected to the first surface 702 of the first substrate 102 and the first substrate 702. The third substrate 704 having the first spacer 102 having the slit 110 and the insulating first substrate 102 and the air hole 708 is combined with the first spacer 106 so as to be in contact with the first spacer 106. 102 has a structure in which a second spacer 706 having a slit 710 is sandwiched so that the second surface 802 of 102 and the second spacer 706 are in contact with each other.

  Similar to the creatinine measurement device 100 according to Embodiment 1, the first electrode 112, the second electrode 114, and the first electrode 112 are electrically connected to the first surface 702 of the first substrate 102. A first lead 122 connected and a second lead 124 electrically connected to the second electrode 114 are disposed. A reagent layer 130 containing a reagent for measuring creatinine is disposed on the first electrode 112 and the second electrode 114.

  On the other hand, on the second surface 802 of the first substrate 102, the third electrode 712, the fourth electrode 714, the fifth electrode 716, the sixth electrode 718, and the third electrode 712 are electrically connected. The third lead 722 connected, the fourth lead 724 electrically connected to the fourth electrode 714, the fifth lead 726 electrically connected to the fifth electrode 716, and the sixth electrode A sixth lead 728 electrically connected to 718 is disposed. The dimensions of the first substrate 102 may be set as appropriate. For example, the width is about 7 mm, the length is about 30 mm, and the thickness is about 0.7 mm.

  Next, a method for manufacturing the urinary salt content measuring device 700 will be described.

  First, palladium is sputtered on the first surface 702 of the first substrate 102 made of polyethylene terephthalate with a resin electrode pattern mask placed thereon, whereby the first electrode 112 and the second electrode 114 are sputtered. First lead 122 and second lead 124 are formed. The first electrode 112 and the second electrode 114 are electrically connected to a terminal of a urinary salt content measuring device described later by a first lead 122 and a second lead 124, respectively.

  Next, palladium is sputtered on the second surface 802 of the first substrate 102 with an electrode pattern mask having a pattern different from the above electrode pattern mask, whereby the third electrode 712, The fourth electrode 714, the fifth electrode 716, the sixth electrode 718, the third lead 722, the fourth lead 724, the fifth lead 726, and the sixth lead 728 are formed. The third electrode 712, the fourth electrode 714, the fifth electrode 716, and the sixth electrode 718 are respectively a third lead 722, a fourth lead 724, a fifth lead 726, and a sixth lead. 728 is electrically connected to a terminal of a urinary salt content measuring device described later.

Next, on the first electrode 112 and the second electrode 114 provided on the first surface 702 of the first substrate 102, 1,2-naphthoquinone which is a reagent for measuring creatinine, potassium dihydrogen phosphate And a predetermined amount of an aqueous solution in which dipotassium hydrogen phosphate is dissolved using a microsyringe or the like, and then the first substrate 102 is left to stand in an environment of about room temperature to 30 ° C. and dried to thereby form the reagent layer 130. Form. The concentration and amount of the aqueous solution containing the reagent to be applied may be selected according to the characteristics and size of the required device. For example, the concentration of 1,2-naphthoquinone in the aqueous solution containing the reagent is about 0.5 mM. Yes, the dripping amount is about 0.7 mL. In addition, the area of the region in which the reagent layer 130 is formed may be appropriately selected in view of the solubility of the reagent with respect to the sample, but the area is, for example, about 3 mm ² .

  Next, the air holes 108 are formed so that the first surface 702 of the first substrate 102 is in contact with the first spacer 106 and the second surface 802 of the first substrate 102 is in contact with the second spacer 706. A second substrate 104 having a slit, a first spacer 106 made of polyethylene terephthalate having a slit 110, a first substrate 102, a second spacer 706 made of polyethylene terephthalate having a slit 710, and a third having an air hole 708. The substrate 704 is combined. An adhesive is applied to each joint portion of each member, and after pasting them together, they are pressed and allowed to stand and adhere. Instead of this method, after combining without applying an adhesive, the joining portion may be welded by heat or ultrasonic waves using a commercially available welding machine.

  When the first substrate 102, the first spacer 106, and the second substrate 104 are combined, the first substrate 102, the slit 110 provided in the first spacer 106, and the second substrate 104 are formed. The space functions as a first sample storage chamber for creatinine measurement. Further, the opening of the slit 110 functions as a first sample introduction port 132 for measuring creatinine.

  On the other hand, when the first substrate 102, the second spacer 706, and the third substrate 704 are combined, the first substrate 102, the slit 710 provided in the second spacer 706, and the third substrate 704 are formed. The space portion to be functioned as a second sample storage chamber for electrical characteristics of urine. Further, the opening of the slit 710 functions as a second sample introduction port 732 for electrical characteristics of urine.

  Next, a urinary salt content measuring apparatus and a urinary salt content measuring method using the same according to the present embodiment will be described with reference to FIGS. FIG. 6 is a perspective view showing the external appearance of the urinary salt content measuring apparatus 900 according to the present embodiment, and FIG. 7 is a block diagram showing the configuration of the urinary salt content measuring apparatus 900.

  First, the configuration of the urinary salt content measuring apparatus 900 will be described with reference to FIG.

  The housing 202 of the urinary salt content measuring apparatus 900 has a measurement device mounting unit 208 for attaching the urinary salt content measuring device 700 to the urinary salt content measuring apparatus 900, a display 204 on which measurement results and the like are displayed, and A measurement start button 206 for starting measurement of the electrical characteristics of creatinine and urine by the urinary salt content measuring apparatus 900 is provided. In addition, the measurement device attachment portion 208 includes a first lead 122, a second lead 124, a third lead 722, and a fourth lead of the urinary salt content measurement device 700 attached to the measurement device attachment portion 208. A first terminal, a second terminal, a third terminal, a fourth terminal, a fifth terminal, and a fifth terminal electrically connected to the lead 724, the fifth lead 726, and the sixth lead 728, respectively. Six terminals are provided.

  Next, the internal structure of the housing 202 of the urinary salt content measuring apparatus 900 will be described with reference to FIG.

  The urinary salt content measuring apparatus 900 includes a voltage application unit 302, an electric signal detection unit 304, a constant current AC power source 902, a voltage detector 904, a control unit 306, a timing unit 308, and a storage unit 310 inside the housing 202. I have.

  The voltage application unit 302 includes a first terminal and a second terminal that are electrically connected to the first lead 122 and the second lead 124 of the urinary salt content measurement device 700 attached to the measurement device attachment unit 208, respectively. It has a function of applying a voltage or a potential to the first electrode 112 and the second electrode 114 of the urinary salt content measuring device 700 via the terminal.

  The electric signal detection unit 304 has a function of detecting and measuring the electric signals from the first electrode 112 and the second electrode 114 via the first terminal and the second terminal. The electric signal detection unit 304 corresponds to the detection unit in the present invention.

  The constant current AC power source 902 includes a third terminal and a sixth terminal electrically connected to the third lead 722 and the sixth lead 728 of the urinary salt content measuring device 700 attached to the measuring device attachment unit 208, respectively. The terminal has a function of applying a constant alternating current between the third electrode 712 and the sixth electrode 718 of the urinary salt content measuring device 700 through the terminal. The alternating current to be applied has, for example, a frequency of about 1 kHz and a current value of about 0.1 mA.

  The voltage detector 904 has a function of detecting a voltage (effective value of an alternating voltage) between the fourth electrode 714 and the fifth electrode 716 through the fourth terminal and the fifth terminal.

  The storage unit 310 detects the first correlation data corresponding to the first calibration curve representing the correlation between the concentration of creatinine and the electrical signal detected by the electrical signal detection unit 304, and the salt concentration and voltage detector 904 detects the correlation. Second correlation data corresponding to a second calibration curve representing the correlation with the applied voltage, and a third calibration curve representing the correlation between the daily urinary salt excretion and the salt concentration corrected by the creatinine concentration The third correlation data corresponding to is stored. As the storage unit 310, for example, a memory such as a RAM or a ROM can be used.

  The control unit 306 refers to the first correlation data, functions to convert the electrical signal detected by the electrical signal detection measurement unit 304 into a creatinine concentration, and refers to the second correlation data by the voltage detector 904. The function of converting the detected voltage into a salinity concentration, the function of correcting the salinity concentration using the obtained creatinine concentration, and the third correlation data, the corrected salinity concentration per day in the urine salt Has a function to convert to excretion. The control unit 306 corresponds to the calculation unit in the present invention. As the control unit 306, for example, a microcomputer such as a CPU (Central Processing Unit) can be used.

  Next, the urinary salt content measuring method according to the present embodiment using the urinary salt content measuring device 700 and the urinary salt content measuring apparatus 900 will be described.

  First, the user inserts the lead side of the urinary salt content measurement device 700 into the measurement device mounting portion 208 of the urinary salt content measurement device 900. Accordingly, the first lead 122, the second lead 124, the third lead 722, the fourth lead 724, the fifth lead 726, and the sixth lead 728 of the urinary salt content measuring device 700 are measured. The first terminal, the second terminal, the third terminal, the fourth terminal, the fifth terminal, and the sixth terminal provided in the device mounting portion 208 are electrically connected to each other. Conduct.

  When the urinary salt content measurement device 700 is inserted into the measurement device attachment unit 208, a measurement device insertion detection switch including a micro switch provided in the measurement device attachment unit 208 is activated to output a signal to the control unit 306. To do. When the control unit 306 detects the insertion of the urinary salt content measurement device 700 based on the output signal from the measurement device insertion detection switch, the control unit 306 controls the voltage application unit 302 to switch the first terminal and the second terminal. Thus, a voltage (for example, 0.2 V) is applied between the first electrode 112 and the second electrode 114.

  Next, the user brings the sample into contact with the first sample inlet 132 and the second sample inlet 732 of the urinary salt content measuring device 700. By this contact, the sample is sucked into the two sample storage chambers of the urinary salt content measuring device 700 through the first sample introduction port 132 and the second sample introduction port 732 by capillary action, and thus the two sample storage chambers. Is filled with the sample. When the sample comes into contact with the first electrode 112 and the second electrode 114, current flows between the first electrode 112 and the second electrode 114 through the sample. The electrical signal detection unit 304 detects it.

  When the control unit 306 detects that the sample has been introduced into the sample storage chamber based on the output signal from the electrical signal detection unit 304, the control unit 306 controls the voltage application unit 302 to apply the voltage applied by the voltage application unit 302. Switch to a different voltage (eg 0V or open circuit). In addition, along with the detection of sample introduction, the control unit 306 starts timing by the timer unit 308 that is a timer.

  Along with the detection of the sample introduction, the control unit 306 controls the constant current AC power source 902 to be constant between the third electrode 712 and the sixth electrode 718 via the third terminal and the sixth terminal. AC current (for example, frequency 1 kHz, current value 0.1 mA) is applied. The voltage detector 904 measures the voltage (effective value of the alternating current) between the fourth electrode 714 and the fifth electrode 716 after a predetermined time has elapsed from the application of the alternating current (for example, after 5 seconds).

  The control unit 306 reads the second correlation data representing the correlation between the salinity concentration stored in the storage unit 310 and the voltage detected by the voltage detector 904, and the voltage detector 904 detects the correlation data by referring to it. The applied voltage is converted to the salinity concentration in the sample. The obtained salinity concentration is displayed on the display 204.

  When the reagent layer 130 exposed in the first sample storage chamber for creatinine measurement comes into contact with the sample, 1,2-naphthoquinone in the creatinine measurement reagent contained in the reagent layer 130 is dissolved in the sample. The 1,2-naphthoquinone dissolved in the sample reacts directly with the creatinine contained in the sample, whereby creatinine oxide and reduced 1,2-naphthoquinone are generated.

  When the control unit 306 determines that a predetermined time (for example, 60 seconds) has elapsed based on a signal from the time measuring unit 308, the control unit 306 controls the voltage application unit 302 to control the first electrode 112 and the second electrode. A different voltage is applied between the first electrode 112 and the second electrode 114 (for example, a voltage at which the first electrode 112 becomes +0.6 V compared to the second electrode 114). In this way, an electrical signal such as a current flowing between the first electrode 112 and the second electrode 114 is measured by the electrical signal detection unit 304 after a certain time (for example, 5 seconds) has elapsed since the voltage was applied. At this time, the reduced 1,2-naphthoquinone is oxidized at the first electrode 112. The electrical signal measured by the electrical signal detection unit 304 depends on the concentration of creatinine contained in the sample.

  The control unit 306 reads the first correlation data representing the correlation between the electrical signal stored in the storage unit 310 and the creatinine concentration, and refers to the first correlation data to detect the electrical signal detected by the electrical signal detection unit 304 in the sample. Convert to creatinine concentration.

  Next, the control unit 306 corrects the salinity concentration using the obtained creatinine concentration. Subsequently, the control unit 306 stores third correlation data corresponding to a third calibration curve representing the correlation between the daily urinary salt excretion amount stored in the storage unit 310 and the salt concentration corrected by the creatinine concentration. Is read, and the corrected salt concentration is converted into the amount of urinary salt excretion per day.

  The obtained creatinine concentration and the daily urinary salt excretion amount are displayed on the display 204. By displaying the creatinine concentration and the daily urinary salt excretion amount on the display 204, the user knows that the measurement has been completed. The obtained creatinine concentration and daily urinary salt excretion amount are preferably stored in the storage unit 310 together with the time measured by the time measuring unit 308.

  According to the urinary salt content measuring device 700 according to the present embodiment, unlike the conventional measuring device, creatinine and 1,2-in the sample storage chamber even if there is no enzyme or picric acid acting on creatinine. Since naphthoquinone reacts directly, the reaction proceeds without being affected by interfering components such as ionic species such as salt, urea, amino acids, and sugars. Therefore, even when a biological sample such as urine or blood is used, creatinine contained in the sample can be quantified with higher accuracy than conventional measurement devices.

  Moreover, according to the urinary salt amount measuring apparatus 900 according to the present embodiment, the urinary salt excretion amount per day is calculated based on the salt concentration corrected by the creatinine concentration measured with high accuracy. Therefore, the amount of urinary salt excretion per day can be obtained with high accuracy.

  In the present embodiment, an example is shown in which 1,2-naphthoquinone is used as an active ingredient in the reagent for measuring creatinine, but 5-hydroxy-1,4-naphthoquinone may be used instead. Even when 5-hydroxy-1,4-naphthoquinone is used as an active ingredient in a reagent for measuring creatinine, a conventional measuring device is not affected by interfering components such as ionic species such as salt, urea, amino acids, and sugars. The creatinine contained in the sample can be quantified with higher accuracy.

  Moreover, in this Embodiment, it is not limited to this which showed the example in which the urinary salt content measuring device is provided with one reagent layer. The urinary salt content measuring device may include two reagent layers, for example, a first reagent layer containing a reagent for measuring creatinine and a second reagent layer containing a phosphate buffer.

  In the present embodiment, an example in which the control unit detects that a sample has been introduced into the sample storage chamber and the voltage applied by the voltage application unit is switched to a different voltage has been described, but the present invention is not limited thereto. As long as a current depending on the concentration of creatinine is obtained, the voltage value necessary for measurement (here, as described above, for example, the voltage at which the first electrode becomes +0.6 V compared to the second electrode) is used as the creatinine. If the voltage is applied from the time of detecting the insertion of the measuring device, the voltage value may be continuously applied after the sample is detected.

  Further, although an example in which the potential applied to the first electrode for obtaining an electric signal is 600 mV with respect to the second electrode is described in this embodiment mode, the present invention is not limited to this. The voltage between the first electrode and the second electrode may be a voltage at which 1,2-naphthoquinone reduced by the oxidation-reduction reaction with creatinine is oxidized.

  In the present embodiment, an example in which the time (reaction time) until detection of an electrical signal after detection of a sample is set to 60 seconds is shown, but the value is not necessarily required. As long as a difference in current value with respect to a difference in creatinine concentration can be detected significantly, a reaction time smaller than the above can be used. On the other hand, when the reaction time is increased, the possibility that the reaction between creatinine and 1,2-naphthoquinone reaches a complete state or a steady state increases, so the amount of creatinine is reduced without being affected by environmental conditions such as temperature. It becomes easy to quantify more accurately.

  Moreover, although the example which detects an electric signal 5 seconds after applying a voltage between a 1st electrode and a 2nd electrode was shown, it is not limited to this time. This time should just be the time which can detect the difference of an electrical signal with respect to the difference in creatinine concentration significantly.

  Further, in the present embodiment, the storage unit stores the first correlation data corresponding to the first calibration curve representing the correlation between the concentration of creatinine and the electrical signal detected by the electrical signal detection unit 304, the concentration of salinity, and the like. The second correlation data corresponding to the second calibration curve representing the correlation with the voltage detected by the voltage detector 904, and the correlation between the daily urinary salt excretion amount and the salt concentration corrected by the creatinine concentration Although the example in which the third correlation data corresponding to the third calibration curve to be expressed is stored is shown, the present invention is not limited to this. Instead, correlation data indicating the correlation between the electrical signal detected by the electrical signal detection unit, the voltage detected by the voltage detector, and the urinary salt excretion per unit time (for example, 1 day) is stored in the storage unit. It may be stored. In this case, the urinary salt excretion per unit time can be directly calculated using the electric signal detected by the electric signal detector and the voltage detected by the voltage detector without obtaining the creatinine concentration and the salt concentration. Can be sought.

  In this embodiment, an example of the electrode system and the lead / terminal is shown, but the shape, number, arrangement, etc. thereof are not limited to these.

  In order to more smoothly introduce the sample into the sample storage chamber of the urinary salt content measuring device, a solution obtained by dissolving lecithin in toluene or another organic solvent is used as the inner wall of the second substrate and the third substrate. You may form a lecithin layer by apply | coating to and drying. By adopting such a structure, the amount of the sample can be made constant with higher reproducibility, so that creatinine and salt contained in the sample can be quantified with higher accuracy.

  The urinary salt content measuring device may further include a recording unit for recording the creatinine concentration, the salinity concentration, and the daily urinary salt excretion obtained as a measurement result in a storage medium such as an SD card. Good. By storing in the removable storage medium, the measurement result can be easily taken out from the urinary salt content measuring device, so that it becomes easy to request the analysis related company to analyze the measurement result.

  The urinary salt content measuring device further includes a transmission unit for transmitting the creatinine concentration, the salinity concentration and the daily urinary salt excretion obtained as a measurement result to the outside of the urinary salt content measuring device. Also good. As a result, measurement results can be sent to analysis-related departments or analysis-related contractors in hospitals and analyzed at analysis-related departments or analysis-related contractors, so the time from measurement to analysis can be reduced. .

  In addition, the urinary salt content measuring device may further include a receiving unit for receiving the result of analysis in an analysis related department or an analysis related business. As a result, the analysis result can be quickly fed back to the user.

Example 1
In order to confirm the effect of the creatinine measuring method according to the present invention, the following experiment was conducted. In this example, 1,2-naphthoquinone, which is a quinone compound, was used as an active ingredient in the reagent for measuring creatinine.

  First, a 100 mM dipotassium hydrogen phosphate aqueous solution and a 100 mM potassium dihydrogen phosphate aqueous solution were prepared. The two aqueous solutions were alternately mixed while monitoring with a pH meter, thereby adjusting the pH of the resulting mixed aqueous solution to 7. 1,2-naphthoquinone was dissolved in the thus obtained 100 mM phosphate buffer (pH = 7) to a concentration of 0.5 mM.

The obtained aqueous solution is put into a glass cell container, a gold electrode (electrode area 2 mm ² ) is used as a first electrode, a platinum wire 5 cm in length is wound as a second electrode, and a third electrode As a reference electrode using Ag / AgCl (saturated KCl aqueous solution), each was immersed in the aqueous solution in the cell container. All of these electrodes were commercially available from BAS Co., Ltd. The connection terminals of the first electrode, the second electrode, and the third electrode were sequentially connected to the connection terminal of the working electrode, the counter electrode, and the reference electrode of the ALS-660A electrochemical analyzer.

  Next, a small amount of a 500 mM creatinine aqueous solution was added to the aqueous solution in the cell container. The addition amount of the creatinine aqueous solution was adjusted for each measurement so that the creatinine concentration contained in the aqueous solution in the cell container became a predetermined value.

  Timing was started with the addition of creatinine, 10 minutes after the addition of creatinine, a potential of 0.6 V was applied to the first electrode with respect to the third electrode, and the current value was measured 5 seconds after the potential application. The above experiment was performed at room temperature (about 25 ° C.).

  When the creatinine concentration contained in the aqueous solution in the cell container was 0, 9, and 27 mM, the above measurements were performed.

  FIG. 8 is a graph in which measured current values are plotted against creatinine concentration. In FIG. 8, the horizontal axis represents the creatinine concentration (mM) contained in the aqueous solution in the cell container, and the vertical axis represents the measured current value (μA). As apparent from FIG. 8, the obtained current value increased linearly with the increase in the concentration of creatinine contained in the aqueous solution in the cell container, and the current value and the creatinine concentration showed a high correlation. Therefore, it can be seen that creatinine can be quantified based on the current value obtained by the creatinine measurement method according to the present invention.

  From the above results, the reaction in the creatinine measurement method according to the present invention can be explained as follows.

  In the sample, 1,2-naphthoquinone is reduced by reacting with creatinine in the presence of a phosphate buffer. That is, creatinine is oxidized by donating electrons to 1,2-naphthoquinone by this reaction. Since a potential that accepts electrons from the reduced 1,2-naphthoquinone is applied to the first electrode, the reduced 1,2-naphthoquinone is electrochemically oxidized at the first electrode. The Along with this, a current flows through the first electrode. The concentration of 1,2-naphthoquinone that is reduced over a period of time depends on the concentration of creatinine. Further, since the oxidation current of reduced 1,2-naphthoquinone depends on the concentration of reduced 1,2-naphthoquinone present in the sample, the obtained current value depends on the concentration of creatinine.

(Example 2)
Next, the following experiment was conducted in order to investigate a preferable pH range in the creatinine measurement method according to the present invention. Since the sample adjustment method and apparatus configuration used in the experiment and the procedure of the experiment are the same as those in Example 1, the description thereof is omitted. However, in this example, the creatinine concentration in the sample is 27 mM, and the pH of the phosphate buffer added to the sample is 3, 4, 5, 6, 7, 8 using a 400 mM buffer. , 9, 10, 11, and 12, the same measurement as in Example 1 was performed.

  FIG. 9 shows the measurement result of the current value. The current value is remarkably large in the pH range of 4-8. From this result, it was found that the determination of creatinine can be measured particularly sensitively in the pH range. The pH range of 4 to 8 is imparted by hydrogen phosphate ions and dihydrogen phosphate ions. Therefore, it is considered preferable to use a combination of, for example, dipotassium hydrogen phosphate or disodium hydrogen phosphate and potassium dihydrogen phosphate or sodium dihydrogen phosphate as the phosphate buffer.

  Further, from the results of FIG. 9, the current value is remarkably large and the current value is stable in the pH range of 4-6. From this result, it can be seen that the determination of creatinine in the pH range can be measured with particularly high sensitivity and high reproducibility.

  The present invention is useful for quantifying creatinine contained in a sample, particularly a biological sample such as urine.

The disassembled perspective view which shows the structure of the creatinine measuring device in one embodiment of this invention The perspective view which shows the external appearance of the creatinine measuring apparatus in the embodiment Block diagram showing the configuration of the creatinine measuring apparatus The disassembled perspective view which shows the structure which looked at the urinary salt content measuring device in other embodiment of this invention from the 1st surface side of the 1st board | substrate. The exploded perspective view which shows the structure which looked at the salt content measuring device in the urine from the 2nd surface side of the 1st board | substrate. The perspective view which shows the external appearance of the urinary salt content measuring apparatus in the embodiment Block diagram showing the configuration of the urinary salt content measuring device The graph which shows the relationship between the creatinine density | concentration in the sample in one Example of this invention, and the measured electric current value The graph which shows the relationship between pH in a sample and the measured electric current value in the other Example of this invention.

Explanation of symbols

100 Creatinine Measurement Device 102 First Substrate 104 Second Substrate 106 Spacer (First Spacer)
108,708 Air hole 110,710 Slit 112 First electrode 114 Second electrode 122 First lead 124 Second lead 130 Reagent layer 132 Sample inlet (first sample inlet)
200 creatinine measuring apparatus 202 casing 204 display 206 measurement start button 208 measuring device mounting unit 302 voltage applying unit 304 electrical signal detecting unit 306 control unit 308 timing unit 310 storage unit 700 urinary salt content measuring device 702 first surface 704 first 3rd substrate 706 2nd spacer 712 3rd electrode 714 4th electrode 716 5th electrode 718 6th electrode 722 3rd lead 724 4th lead 726 5th lead 728 6th lead 732 6th lead 2 Sample introduction port 802 Second surface 900 Urine salt content measuring device 902 Constant current AC power supply 904 Voltage detector

Claims (14)

(A) By bringing a sample containing creatinine into contact with a reagent for measuring creatinine containing a quinone compound, the creatinine contained in the sample reacts directly with the quinone compound, thereby reducing the quinone compound. The process of generating
(B) a step of electrochemically measuring the reaction in the step A; and (C) a creatinine measurement method including a step of determining the concentration or amount of the creatinine contained in the sample based on the measurement value in the step B. . The method for measuring creatinine according to claim 1, wherein the oxidation-reduction potential of the quinone compound at pH 7 and 25 ° C is more positive than -180 mV with respect to the silver-silver chloride electrode. The method for measuring creatinine according to claim 1 or 2, wherein the quinone compound is 1,2-naphthoquinone. In step A,
The creatinine measuring method according to any one of claims 1 to 3, wherein the pH of the sample is adjusted to 4 to 8 by bringing a phosphate buffer into contact with the sample. In the step B, at least two electrodes are brought into contact with the sample,
Step B is
(D) applying a voltage between the two electrodes, and (E) detecting a value of electric current or a charge amount flowing between the two electrodes,
In step C,
The method for measuring creatinine according to any one of claims 1 to 4, wherein the concentration or amount of the creatinine contained in the sample is determined based on the current value or the amount of charge detected in the step E. Using urine as a sample,
In addition to steps A and B of the method for measuring creatinine according to claim 1,
(F) a step of measuring electrical characteristics of the urine before the urine and the reagent for measuring creatinine come into contact; and (G) a measured value in the process B and the electrical characteristics measured in the process F. A method for measuring urinary salt content, further comprising a step of obtaining a value reflecting the amount of salt excreted in urine based on the above. A creatinine measuring device used in the creatinine measuring method according to claim 1,
A sample storage chamber for storing a sample containing creatinine;
A sample inlet for communicating with the sample storage chamber and for introducing the sample into the sample storage chamber;
At least two electrodes disposed in the sample storage chamber;
A creatinine measurement device comprising a creatinine measurement reagent containing a quinone compound, which is disposed in the sample storage chamber. The creatinine measuring device according to claim 7, wherein the oxidation-reduction potential of the quinone compound at pH 7 and 25 ° C is more positive than -180 mV with respect to the silver-silver chloride electrode. The creatinine measuring device according to claim 7 or 8, wherein the quinone compound is 1,2-naphthoquinone. The creatinine measuring device according to any one of claims 7 to 9, further comprising a phosphate buffer disposed in the sample storage chamber. A urinary salt content measuring device used in the urinary salt content measuring method according to claim 6,
A first sample storage chamber for storing urine as a sample;
A first sample inlet for communicating with the first sample storage chamber and for introducing the urine into the first sample storage chamber;
At least two electrodes disposed in the first sample storage chamber;
A creatinine measurement reagent containing a quinone compound, disposed in the first sample storage chamber;
A second sample storage chamber for storing the urine;
A second sample inlet for communicating with the second sample storage chamber and for introducing the urine into the second sample storage chamber;
A urinary salt content measuring device comprising: at least two electrodes arranged in the second sample storage chamber. A measuring device mounting portion for mounting the creatinine measuring device according to claim 7,
A measurement unit that electrochemically measures a reaction between the creatinine measurement device attached to the measurement device attachment unit and the creatinine measurement reagent in the sample storage chamber, and the sample based on the measurement value in the measurement unit A creatinine measuring apparatus comprising a calculation unit for obtaining the concentration or amount of the creatinine contained therein. The creatinine measuring device further comprises at least two electrodes disposed in the sample storage chamber;
The measurement unit is
A voltage application unit that applies a voltage between the two electrodes, and a detection unit that detects a value of electric current or a charge amount flowing between the two electrodes,
The computing unit is
The creatinine measuring apparatus according to claim 12, wherein the concentration or amount of the creatinine contained in the sample is obtained based on the current value or the charge amount detected by the detection unit. A measuring device mounting portion for mounting the urinary salt content measuring device according to claim 11,
A first measurement unit for electrochemically measuring a reaction between creatinine and the creatinine measurement reagent in the first sample storage chamber of the urinary salt content measurement device attached to the measurement device attachment unit;
A second measurement unit for measuring electrical characteristics of the urine in the second sample storage chamber of the urinary salt content measurement device attached to the measurement device attachment unit; and a measurement value in the first measurement unit; A urinary salt content measuring device including a calculation unit that obtains a value reflecting the amount of salt excreted in the urine based on the electrical characteristics measured by the second measuring unit.

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