JP2001242133A - Disposable bun sensor and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a disposable 'BUN' sensor and a production method thereof, which enable detection of pH changes, using a pH electrode at lower cost, and moreover along with a shorter measurement time and a higher measuring accuracy. SOLUTION: The disposable BUN sensor is provided with a pH electrode which is formed on an insulating film to detect pH changes, an enzyme layer which is provided on the pH electrode and comprises a mixture of an enzyme, a water-swelling high polymer material, a surfactant, a buffer material and the like and a filter layer, which is provided covering the enzyme layer to prevent the spillage of the enzyme from the enzyme layer and restricts the diffusion of urea and nitrogen, to remove cross substance in a measurement liquid. In the production method of the disposable BUN sensor, an electrode layer is formed by a screen printing method on a polyethylene film and the pH electrode, the enzyme layer and the filter layer are formed separately in a drip drying process.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a disposable BU for measuring urea nitrogen in a biological fluid such as whole blood, plasma, serum, urine and the like.
The present invention relates to an N sensor and a method for manufacturing the same.

[0002]

2. Description of the Related Art In recent years, small and disposable enzyme sensors which do not require calibration of a calibration curve or washing of electrodes have been put to practical use. An enzyme sensor generally has a structure having a working electrode (also referred to as an enzyme electrode or a measuring electrode) for detecting an enzyme reaction and a reference electrode (or a counter electrode) forming an electric circuit. The change is extracted as a change in the electric signal by the electrodes, and the concentration of the substrate that specifically acts on the enzyme is measured from the change. Among them, some of the commonly used enzyme sensors utilize oxidoreductase. For example, it is an amperometric enzyme sensor using hydrogen peroxide generated or consumed oxygen as a detection substance (for example, a glucose enzyme sensor disclosed in JP-A-Hei). There is also an amperometric enzyme sensor that detects an electric signal via an electron mediator (referred to as a mediator) that mediates the oxidation-reduction of an enzyme. Furthermore, it is also possible to quantify the concentration of the substrate by utilizing a hydrolase and potentiometrically measuring the change in the electrode signal with the hydrolysis product of the substrate that selectively reacts with the enzyme.

[0003] For example, urea, especially urea nitrogen (B
UN) is an important clinical test item that reflects liver protein metabolic function and renal function, and thus many enzyme sensors have been developed for its measurement. Urease, which is a hydrolase, is generally used for measuring urea. The reaction equation of the hydrolysis reaction is as follows: CO (NH ₂ ) ₂ + H ₂ O → CO ₂ + 2NH _{3 The} reaction produces carbon dioxide (CO ₂ ) and ammonia (NH ₃ ), so that the conventional technology PCO ₂ electrode, a pH electrode with a CO ₂ gas permeable membrane (eg, GGGuilbault et al., Anal.
Chem., 44, 2161 (1972)), or an NH ₃ electrode, ie, a pH electrode having an NH ₃ gas permeable membrane (see, for example, US Pat.
Measured using 6,734). Or NH ₃ in aqueous solution
Is an ammonium ion (NH ₄ ⁺ ) selective electrode (see, eg, US Pat. No. 4,476,005), a change in electrical conductivity (see, eg, US Pat. No. 5,698,083) or pH. Changes (eg RMIanniello et al., An
al. Chim. Acta, 146, 249 (1983)).

[0004] As described above, the BUN sensor is a kind of biosensor, and this type of sensor generally has ammonium ion or bicarbonate generated by an enzymatic reaction between urea nitrogen in blood to be measured and an enzyme called urease. It is configured to detect a product such as an ion or a change in pH accompanying the generation of such an ion, and converts the detected value into a concentration of urea nitrogen.

[0005] By the way, such a BUN sensor is discarded after use from the viewpoint of the environment of use, from the viewpoint of the safety of the operator, that is, a disposable sensor is becoming common. Therefore, it is necessary to keep the cost of the sensor itself as low as possible while maintaining high measurement reliability. However, the manufacture of this type of conventional sensor typically involves the use of electronic circuit manufacturing techniques, especially thin film forming techniques such as etching and ion plating.
The fact is that an expensive and large-scale manufacturing apparatus is required, the manufacturing process is complicated, and the manufacturing cost cannot be sufficiently reduced. It is also desired that this type of sensor requires as little time as possible to calibrate and measure the sensor.

[0006]

Problems with the conventional urea measurement sensor will be described. PCO ₂ electrode and NH ₃
In the urea measurement using an electrode, p is selected because a gas selective permeable membrane is used.
It is necessary to hold a supporting electrolyte solution between the H electrode and the gas permeable membrane. This is a manufacturing disadvantage for disposable sensors. Also, if you keep it in solution for a long time,
The performance of the pH electrode is degraded and the solution evaporates, which limits the shelf life of the sensor. In addition, the measurement method using an ammonia ion selection electrode is not practical because the electrode itself has poor selectivity (reacts with other ions). In a urea sensor based on conventional pH measurement,
Since it is a combination of a glass pH electrode and a urease enzyme, it also requires a supporting electrolyte and is difficult to use as a disposable sensor in terms of miniaturization and ease of production. In the measurement by the electric conductivity, since the ionic strength of the sample is high, the amount of change in the ionic strength due to the enzymatic reaction is small, and there are many practical problems. Therefore, it is expected to provide a dry disposable potentiometric enzyme sensor that can be manufactured at low cost, has a simple structure, and does not require a supporting electrolyte solution.

Therefore, the present invention solves the above-mentioned problems associated with the prior art, and provides a disposable BUN sensor which is inexpensive, has a short measurement time, has high measurement accuracy, and detects a pH change with a pH electrode. It is intended to provide a method for producing the same.

[0008]

In order to achieve the above object, a disposable B provided by the first invention of the present invention is provided.
The UN sensor is provided on the pH electrode for detecting at least a pH change formed on the insulating film, and a UN electrode comprising a mixture of an enzyme, a water-swellable polymer material, a surfactant and a buffer substance. And an enzyme layer formed over the enzyme layer to prevent the enzyme from flowing out of the enzyme layer, limit the diffusion of urea, and remove contaminants (eg, blood cells and proteins in whole blood) from the measurement solution. And a filter layer. The pH electrode may preferably be composed of a substance that reacts with hydrogen ions, such as triiddecylamine. In the enzyme layer, the measurement solution penetrates quickly during measurement, the enzyme dissolves easily, and the reaction easily occurs.The surfactant in the enzyme layer assists in the absorption of moisture and generates bubbles that easily cause errors in measurement. It works to suppress.

In the disposable BUN sensor provided by the first aspect of the present invention, a coated wire type pH liquid membrane electrode having a simple structure, which is low cost, has good storage stability, and has a simple structure using screen printing is used. This pH liquid membrane electrode has a certain sensitivity and has a storage life of more than half a year at room temperature. In addition, by providing an enzyme reaction layer directly on the surface of the pH liquid membrane electrode, when injecting a specimen sample, moisture and ions in the sample reach the surface of the pH electrode through the enzyme reaction layer so that the pH electrode works. Become. At the same time, the measurement substrate also diffuses into the enzyme layer, reacts with the enzyme (and coenzyme) immobilized therein, and changes in pH due to the reaction are sensed by a pH electrode provided thereunder. Therefore, in the present invention, the structure of the supporting electrolyte solution for the operation of the pH electrode is unnecessary. In addition, since a coated wire type pH liquid membrane electrode having a constant sensitivity is used, complicated procedures such as calibration of a calibration curve of a conventional potentiometric enzyme sensor can be omitted. Therefore, it can be stored in a dry state and is inexpensive, stable,
A disposable potentiometric enzyme sensor can be provided.

[0010] A method of manufacturing a disposable BUN sensor according to a second aspect of the present invention includes a step of sequentially printing an electrode layer and an insulating layer surrounding the electrode layer on the insulating film by screen printing. Forming a pH electrode for detecting a change in pH by dropping and drying an organic synthetic substance on a carbon layer, and forming an enzyme, a water-swellable polymer material, a surfactant and a buffer substance on the pH electrode. Step of forming an enzyme layer by dropping and drying the aqueous solution of the mixture, and covering the surface of the enzyme layer, preventing the enzyme from flowing out of the enzyme layer, limiting diffusion of urea, and removing contaminants in the measurement solution Forming a filter layer by dropping and drying a solution of a polymer substance that functions as a filter layer.

Preferably, the enzyme layer comprises an enzyme (urease) of 0 to 100 V, a water-swellable polymer material of 0.01 to 2 m
g, an aqueous solution consisting of 0 to 1 µl of a surfactant and 50 µl of a buffer substance is dropped and dried in a predetermined amount to form a solution. The organic synthetic material forming the pH electrode is polyvinyl chloride (PV).
It can consist of a pH sensitive substance based on C).

The method of the present invention can be applied not only to the measurement of BUN but also to the change of pH due to the reaction and the measurement of other substances. For example, it can be applied to creatine, creatinine and the like.

[0013]

Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 shows a disposable BUN sensor according to an embodiment of the present invention. In the illustrated BUN sensor, reference numeral 1 denotes a polyethylene film forming a base, on which a silver paste layer 2, a carbon layer 3,
And an insulator layer 4, which are formed by a screen printing method. In a region defined by the insulator layer 4 on the carbon layer 3, a pH electrode 5 for detecting a change in pH is provided. The pH electrode 5 is made of an organic synthetic substance that reacts with hydrogen ions such as triid decylamine. It consists of a PVC film. On the pH electrode 5 is provided an enzyme layer 6 made of a mixture of an enzyme, a water-swellable polymer material, a surfactant, a buffer substance, and the like.
It is formed smaller than the diameter of the electrode 5. The water-swellable polymer material in the enzyme layer 6 functions to absorb and retain moisture.
As the buffer substance, Na ₂ HPO ₄ —NaH ₂ PO
_{4, CH} 3 COONa-HCl, etc. Tris-HCl may be used. On the enzyme layer 6, the upper surface and the side peripheral surface of the enzyme layer 6 are covered to prevent the enzyme from flowing out of the enzyme layer 6,
A filter layer 7 for limiting the diffusion of urea and nitrogen and removing contaminants (blood cells) in the measurement solution is provided. As a material for the filter layer, a polymer material such as Nafion or polyvinyl butyral may be used.

FIG. 2 shows a preferred embodiment of the method for manufacturing a disposable BUN sensor according to the present invention. In a first step shown in FIG. 2A, a silver paste is printed on the upper surface of the polyethylene film 1 by a screen printing method to form a thin layer 2 of the silver paste. In a second step shown in FIG. 2B, the carbon layer 3 is printed by a screen printing method so as to cover the outer surface of the thin film layer 2 of the silver paste. In a third step shown in FIG. 2C, a resist layer 4 made of an insulator is formed around the carbon layer 3 by a screen printing method, and the resist layer 4 is formed on the carbon layer 3 as shown in FIG. Is defined.

In the fourth step shown in FIG. 2D, a recess such as triid decylamine based on polyvinyl chloride is formed in the concave portion on the carbon layer 3 defined by the resist layer 4.
An aqueous solution of the H-sensitive material is added dropwise and dried. Thus, the pH electrode 5 is formed on the carbon layer 3. FIG.
In the fifth step (E), an enzyme (urease) of 0 to 100 V, a water-swellable polymer material of 0.01 to 2 mg, a surfactant of 0 to 1 μl, and a buffer substance of 50 μl are placed on the pH electrode 5. The resulting aqueous solution is added dropwise and dried at a predetermined drying rate. Thus, the enzyme layer 6 is formed on the pH electrode 5, and the peripheral portion of the enzyme layer 6 extends on the resist layer 4 as shown in the figure.

In the last step, an aqueous solution of a high molecular substance such as polyvinyl butyral is dropped on the enzyme layer 6 and dried at a predetermined drying speed. Thus, the filter layer 7 is formed so as to cover the enzyme layer 6 (see FIG. 1). In the illustrated embodiment, the enzyme layer 6 has an initial pH for measuring a pH change caused by an enzyme reaction.
It is configured to keep the value at 7.4.

[0017]

Next, an embodiment of the urea sensor of the present invention will be specifically described. Example 1 A urea sensor for measuring urea in a biological sample was prepared as follows. (1) Preparation of pH electrode substrate. First, as shown in FIG. 3, base electrodes of a silver indicator electrode 11 and a reference electrode 12 are provided on an insulating substrate 10 in an inner circle and an outer ring shape. Next, a carbon layer 13 was formed by printing and drying a conductive carbon paste so that silver was not exposed on the inner circle silver and was not connected to silver on the outer ring.
Further, a resist was printed on the substrate so that the exposed diameter of the inner circular carbon layer 13 became 1.0 mm Φ, and was not covered with the reference electrode 12, and was dried to form a resist layer 14, thereby producing an electrode substrate. .

(2) Structure of coated wire type pH liquid film. Place the silver reference electrode of the above electrode substrate in 0.7 M potassium chloride,
A silver chloride forming treatment was performed at a constant current of 1 mA for 10 seconds, followed by washing and drying to complete the reference electrode 12. In addition, a fixed volume of a pH liquid film solution prepared with a prescribed composition was dropped on the indicator electrode carbon layer 13 and dried at room temperature.
An H liquid film electrode 15 was produced.

(3) Structure of enzyme reaction layer. First, 10 mg of urease (manufactured by Toyobo Co., Ltd., 120 IU / mg) and PVA (manufactured by Wako Pure Chemical Industries, Ltd., polyvinyl alcohol, n = 500) were prepared as follows.
g was dissolved in 100 ml (0.02M) of phosphate buffer and mixed uniformly. Next, 0.5 ul of the above solution is dropped on the pH liquid membrane indicator electrode 15 and dried at room temperature to remove the enzyme reaction layer 16.
Was formed. Thus, a urea enzyme sensor was produced. The surface of the enzyme reaction layer 16 of the urea sensor was further coated with a polymer film. This polymer membrane was completed by, for example, dropping 0.5 ul of a Nafion solution (5%, Wt% in water: n-propanol = 1: 1) onto the enzyme reaction layer and drying at room temperature.

Test Example 1 Next, using the urea sensor described in Example 1 as an example of the present invention, the sensor performance was tested in Test Examples. (1) Preparation of urea standard measurement solution First, a urea standard aqueous solution is prepared as follows. Urea (biochemical products, manufactured by Wako Pure Chemical Industries, Ltd.) dried at 80 ° C. was dissolved in 50 mM phosphate buffer (pH 7.38) and the final concentration of urea was 40
It was adjusted to be mM. Next, the above 40 mM urea solution was diluted with 50 mM phosphate buffer (pH 7.38) to prepare a final concentration of urea of about 15 steps from 0 mM to 40 mM.

(2) Urea response by potentiometry First, the working electrode terminal 17 and the reference electrode terminal 18 of the urea enzyme sensor were connected to a potential difference measuring terminal of a Digital Electrometer (manufactured by Advantest Co., Ltd.). Next, the sensor was immersed in 50 mM phosphate buffer (pH 7.38, hereinafter referred to as blank), and the potential response of the sensor was taken into a computer via a GPIB connector for 120 seconds. Next, the sensor was taken out of the above solution, immersed in a urea standard measurement solution having a urea concentration of 2 mM, and the potential response was measured for 60 seconds. A new sensor was replaced and a new concentration of urea solution was repeated with similar measurements. Urea concentration is 0 to
All measurements were performed in about 15 steps up to 40 mM. The measurement results are shown in FIGS.

FIG. 4 is a response curve of the urea enzyme sensor to blank and urea. Since the solution becomes alkaline due to the enzymatic reaction of urea and urease, the output potential of the pH liquid membrane electrode decreases. This potential change (absolute value) tends to increase as the urea concentration increases. FIG. 5 shows the logarithmic relationship between the urea concentration and the sensor potential change based on the data from the response curve as shown in FIG. As a result, the urea sensor of the present invention had a urea concentration of about 5 mM and a sensitivity of about 55 mV / Decade. In the range of 1 to 20 mM, there was a linear relationship, indicating a sufficient dynamic range.

(3) pH Dependency of Urea Response In the present invention, the enzyme reaction layer placed on the pH liquid membrane electrode is
Because it does not block the diffusion of hydrogen ions in the measurement solution,
Hydrogen ions in the measurement solution diffuse through the enzyme reaction layer to the pH electrode, directly affecting the response of the pH electrode. Here, the influence of the pH of the sample on the urea measurement was confirmed as follows. First, several kinds of phosphate buffers (50 mM) of pH 6.2 to pH 7.8 were prepared. A certain amount of urea was dissolved therein to prepare a final concentration of 8 mM. Next, the urea solution was measured by potentiometry according to the procedure described in (2). The measurement results are shown in FIG. As a result, it was found that the potential response of urea greatly depends on the pH of the measurement solution. I mean. Confirmed pH range (6.2
In 7.8), the change in the potential of the sensor (absolute value) also increased as the pH increased. In order to solve the pH dependence as shown in FIG. 6, at the time of measurement, the pH of the sample is also measured, and it is necessary to correct the measured value of urea based on the measured value.

The present invention can be used as long as the concentration of hydrogen ions in a solution changes due to creatine, creatinine and the like in a biological sample such as blood and urine and other enzyme reactions. For example, glucose dehydrogenase (Glucose Dehydrogenase) and the coenzyme NAD (P) +
Is used to generate hydrogen ions from the reaction with glucose, so that glucose can be measured according to the present invention.

[0025]

As described above, in the disposable BUN sensor according to the present invention, a PH electrode formed on an insulating film to detect a change in PH,
An enzyme layer comprising a mixture of an enzyme, a water-swellable polymer material, a surfactant, a buffer substance, etc., provided on the H electrode, and provided over the enzyme layer to prevent the enzyme from flowing out of the enzyme layer; A filter layer that limits the diffusion of nitrogen and removes contaminants in the measurement solution.The calibration solution quickly penetrates the filter layer during measurement, and the enzyme in the enzyme layer dissolves and reacts easily. In addition, bubbles do not occur in the enzyme layer due to the action of the buffer substance contained in the enzyme layer, so that the concentrations of urea and nitrogen can be accurately measured.

Further, according to the method for manufacturing a disposable BUN sensor according to the present invention, an electrode layer is formed on a polyethylene film by a screen printing method, and P
Since the BUN sensor is manufactured by forming the H electrode, the enzyme layer, and the filter layer, respectively, the manufacturing cost can be significantly reduced, and an inexpensive product as a disposable sensor can be provided.

[Brief description of the drawings]

FIG. 1 shows a disposable BUN according to an embodiment of the present invention.
FIG. 2 is a schematic sectional view showing a sensor.

FIG. 2 is a schematic cross-sectional view showing each step of a method of manufacturing the BUN sensor shown in FIG.

FIG. 3 is a schematic diagram of a urea enzyme sensor of an embodiment according to the present invention.

FIG. 4 is a potential response curve of a urea enzyme sensor measured by potentiometry with respect to a urea standard solution. The measured urea solutions have urea concentrations of 0, 2, 5, 10, 20, and 30 mM.
It is.

FIG. 5 is a diagram showing a logarithmic relationship between a urea concentration and a potential response of a sensor.

FIG. 6 is a diagram showing the pH dependence of the urea enzyme sensor of the present invention.

[Explanation of symbols]

 1: Polyethylene film 2: Thin layer of silver paste (electrode layer) 3: Carbon layer 4: Resist layer 5: PH electrode 6: Enzyme layer 7: Filter layer 10: Insulating substrate 11: Silver indicating electrode 12: Reference electrode 13: carbon layer 14: resist layer 15: pH liquid membrane electrode 16: enzyme reaction layer

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA13 AA15 AA25 CA25 CA26 CB03 DA16 FB01 FB05 GC18 JA01 JA02 JA07

Claims (4)

[Claims] 1. A pH electrode formed on an insulating film for detecting at least a change in pH, and a mixture of an enzyme, a water-swellable polymer material, a surfactant and a buffer substance provided on the pH electrode. And a filter layer provided over the enzyme layer, which prevents the enzyme from flowing out of the enzyme layer, restricts the diffusion of urea, and removes contaminants in the measurement solution. Disposable BUN sensor. 2. A step of sequentially printing an electrode layer and an insulating layer surrounding the electrode layer on the insulating film by a screen printing method, and dropping and drying an organic synthetic substance on the carbon layer to obtain a pH value. A step of forming a pH electrode for detecting a change, and a step of dropping and drying an aqueous solution of a mixture of an enzyme, a water-swellable polymer material, a surfactant and a buffer substance on the pH electrode to form an enzyme layer. , Covering the surface of the enzyme layer,
Forming a filter layer by dropping and drying a solution of a polymer substance that functions to prevent the enzyme from flowing out of the enzyme layer, limit the diffusion of urea, and remove contaminants in the measurement solution. The manufacturing method of a disposable BUN sensor characterized by the above-mentioned. 3. The method according to claim 1, wherein the enzyme layer comprises an enzyme (urease) of 0 to 100.
V. The disposable BUN according to claim 2, wherein an aqueous solution comprising 0.01 to 2 mg of a water-swellable polymer material, 0 to 1 µl of a surfactant and 50 µl of a pH buffer solution is dropped and dried. Sensor manufacturing method. 4. The method for producing a disposable BUN sensor according to claim 2, wherein the organic synthetic substance forming the pH electrode is a pH-sensitive substance based on polyvinyl chloride (PVC).