JP4404433B2 - Disposable BUN sensor and manufacturing method thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a disposable BUN sensor for measuring urea nitrogen in biological fluids such as whole blood, plasma, serum, and urine, and a method for producing the same.
[0002]
[Prior art]
In recent years, small-sized and disposable enzyme sensors that do not require calibration of calibration curves or electrode cleaning 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 counter electrode) that forms an electric circuit, and is a substance resulting from an enzyme reaction at the working electrode. The change is taken out as a change in electric signal by these electrodes, and the concentration of the substrate that specifically acts on the enzyme is measured from the change. Among them, enzyme sensors that are often used include those that use oxidoreductases. For example, an amperometric enzyme sensor that uses hydrogen peroxide produced or oxygen consumed as a detection substance (for example, a glucose enzyme sensor disclosed in Japanese Patent Laid-Open No. 2005-151867). There is also an amperometric enzyme sensor that detects an electric signal via an electron mediator (called a mediator) that mediates redox of the enzyme. Furthermore, the concentration of the substrate can be quantified by using a hydrolase and potentiometrically measuring the change in electrode signal by the hydrolysis product of the substrate that selectively reacts with it.
[0003]
For example, urea, especially urea nitrogen (BUN) in the blood, is an important clinical test item that reflects the liver's intermediary metabolic function and kidney function, and therefore, many developments of enzyme sensors for its measurement have been reported. . In general, urease, which is a hydrolase, is used for measuring urea. The reaction formula of the hydrolysis reaction is as follows:
CO (NH ₂ ) ₂ + H ₂ O → CO ₂ + 2NH ₃
Since the reaction produces carbon dioxide (CO ₂ ) and ammonia (NH ₃ ), the prior art uses a PCO ₂ electrode, ie a pH electrode with a CO ₂ gas permeable membrane (eg, GGGuilbault et al., Anal. Chem., 44, 2161 (1972)), or an NH ₃ electrode, that is, a pH electrode having an NH ₃ gas permeable membrane (see, for example, US Pat. No. 3,926,734). Alternatively, NH _{3 in} an aqueous solution can be an ammonia ion (NH ₄ ⁺ ) selective electrode (see, eg, US Pat. No. 4,476,005), a change in electrical conductivity (eg, US Pat. No. 5,698, 083) or changes in pH (eg RMIanniello et al., Anal. Chim. Acta, 146, 249 (1983)) have also been studied.
[0004]
Thus, the BUN sensor is a kind of biosensor, and this type of sensor is generally like an ammonium ion or bicarbonate ion generated by an enzymatic reaction between urea nitrogen in the blood to be measured and an enzyme called urease. The product is configured to detect a change in pH associated with the production of such a product or such ions, and the detected value is converted into the concentration of urea nitrogen.
[0005]
By the way, such a BUN sensor is discarded after use from the viewpoint of the safety of the operator in view of its use environment, that is, a disposable sensor has become common. Therefore, it is required to keep the cost of the sensor itself as low as possible while maintaining high measurement reliability. However, conventional manufacturing of this type of sensor usually uses electronic circuit manufacturing technology, particularly thin film formation technology such as etching and ion plating, and requires expensive and large-scale manufacturing equipment. The actual situation is that the process is complicated and the manufacturing cost cannot be sufficiently reduced.
In addition, this type of sensor is desired to have a sensor calibration and measurement time as short as possible.
[0006]
[Problems to be solved by the invention]
Problems in the conventional urea measuring sensor will be described.
In urea measurement using a PCO ₂ electrode and an NH ₃ electrode, since a gas selective permeable membrane is used, it is necessary to hold a supporting electrolyte solution between the pH electrode and the gas permeable membrane. This is a manufacturing disadvantage for disposable sensors.
Further, if the solution is stored in the solution for a long time, the performance of the pH electrode is deteriorated and there is a problem of evaporation of the solution, so that the storage life of the sensor is limited.
In addition, the measurement method using an ammonia ion selective electrode is not practical because the selectivity of the electrode itself is poor (it reacts with other ions). A conventional urea sensor based on pH measurement is a combination of a glass pH electrode and a urease enzyme, and thus requires a supporting electrolyte, and is used as a disposable sensor in terms of miniaturization and ease of manufacture. It is difficult. In the measurement by electric conductivity, since the ionic strength of the specimen sample is high, the amount of change in ionic strength due to the enzyme reaction is small, and there are many problems in practical use.
Accordingly, it is expected to provide a dry-type disposable potentiometric enzyme sensor that can be manufactured at low cost, has a simple structure, and does not require a supporting electrolyte solution.
[0007]
Accordingly, the present invention solves the problems associated with the prior art as described above, and is a disposable BUN sensor that detects a pH change with a pH electrode at a low cost, with a short measurement time and high measurement accuracy, and a method for manufacturing the same. The purpose is to provide.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, the disposable BUN sensor provided by the first invention of the present invention sequentially forms an electrode layer and an insulating layer surrounding the electrode layer on the insulating film by screen printing. PH electrode for detecting pH change formed by printing and dripping and drying organic synthetic substance on carbon layer, urease, water-swellable polymer material, surfactant and buffer substance on said pH electrode An enzyme layer formed by dripping and drying an aqueous solution of the mixture of the above, and covering the surface of the enzyme layer, preventing the enzyme from flowing out of the enzyme layer, limiting the diffusion of urea, and contaminants in the measurement solution (for example, And a filter layer formed by dropping and drying a solution of a polymer substance that functions to remove blood cells and proteins in whole blood .
pH electrode may preferably consist of a material that reacts to the hydrogen ion such as tridodecylamine.
In the enzyme layer, the measurement solution penetrates quickly during the measurement and the enzyme dissolves and the reaction is likely to occur, and the surfactant in the enzyme layer helps to absorb moisture and generate bubbles that are likely to cause an error in the measurement. It works to suppress.
[0009]
The disposable BUN sensor provided by the first invention of the present invention employs a coated wire type pH liquid film electrode that uses screen printing and has a low cost, good storage stability and a simple structure. This pH liquid film electrode has a certain sensitivity, and has a shelf life of more than half a year when stored at room temperature. In addition, by providing an enzyme reaction layer directly on the surface of the pH liquid membrane electrode, when injecting the 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 this invention, the structure of the supporting electrolyte solution for a pH electrode to work is unnecessary. In addition, since a coated wire type pH liquid film electrode having a certain sensitivity is used, troublesome procedures such as calibration of a calibration curve of a conventional potentiometric enzyme sensor can be omitted. Accordingly, it is possible to provide a potentiometric enzyme sensor that can be stored in a dry state and is inexpensive, stable, and disposable.
[0010]
The method for manufacturing a disposable BUN sensor according to the second invention of the present invention includes a step of sequentially printing an electrode layer and an insulating layer surrounding the electrode layer on an insulating film by a screen printing method, and a carbon layer. A step of forming a pH electrode for detecting pH change by dripping and drying an organic synthetic substance on the aqueous solution, and an aqueous solution of a mixture of urease, a water-swellable polymer material, a surfactant and a buffer substance on the pH electrode The process of forming the enzyme layer by dripping and drying, covering the surface of the enzyme layer, preventing the flow of urease from the enzyme layer, limiting the diffusion of urea, and removing contaminants in the measurement solution And a step of forming a filter layer by dripping and drying a solution of the polymer substance to be formed.
[0011]
Preferably, the enzyme layer can be formed by dripping and drying a predetermined amount of an aqueous solution comprising an enzyme (urease ), a water-swellable polymer material 0.01 to 2 mg, a surfactant 0 to 1 μl, and a buffer substance 50 μl. The organic synthetic material forming the pH electrode may be a pH sensitive material based on polyvinyl chloride (PVC).
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described 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 constituting a substrate, on which a silver paste layer 2, a carbon layer 3, and an insulator layer 4 are provided, and these layers are formed by a screen printing method. The region defined by the insulator layer 4 on the carbon layer 3 is provided with a pH electrode 5 for detecting a pH change. The pH electrode 5 is an organic synthetic substance that reacts with hydrogen ions such as tridodecylamine . It consists of a PVC film. On the pH electrode 5, an enzyme layer 6 made of a mixture of an enzyme, a water-swellable polymer material, a surfactant and a buffer substance is provided. The enzyme layer 6 has a diameter as shown in the figure. It is formed smaller. The water-swellable polymer material in the enzyme layer 6 functions to absorb and retain moisture, and Na2HPO4-NaH2PO4, CH3COONa-HCl, Tris-HCl, etc. can be used as a buffer substance. On the enzyme layer 6, the upper surface and the side peripheral surface of the enzyme layer 6 are covered to prevent the loss of the enzyme in the enzyme layer 6, the diffusion of urea and nitrogen is restricted, and the contaminants (blood cells) in the measurement solution A filter layer 7 is provided to remove water. As a material for the filter layer, a polymer substance such as Nafion or polyvinyl butyral can be used.
[0014]
FIG. 2 shows a preferred embodiment of the method for producing the disposable BUN sensor of the present invention.
In the 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 film layer 2 of the silver paste.
In the second step shown in FIG. 2B, the carbon layer 3 is printed by screen printing so as to cover the outer surface of the thin film layer 2 of silver paste.
In the third step shown in FIG. 2C, a resist layer 4 made of an insulator is formed by screen printing so as to surround the carbon layer 3, and this resist layer 4 is formed on the carbon layer 3 with a pH electrode 5 as shown in the drawing. A forming region is defined.
[0015]
In the fourth step shown in FIG. 2D, an aqueous solution of a pH sensitive substance such as tridodecylamine based on polyvinyl chloride is dropped into the recesses on the carbon layer 3 defined by the resist layer 4, And dried. Thus, the pH electrode 5 is formed on the carbon layer 3. In the fifth step shown in FIG. 2E, the pH electrode 5 is composed of an enzyme (urease ), a water-swellable polymer material 0.01-2 mg, a surfactant 0-1 μl, and a buffer substance 50 μl. The aqueous solution is added dropwise and dried at the expected drying rate. Thus, the enzyme layer 6 is formed on the pH electrode 5, and the peripheral edge of the enzyme layer 6 extends on the resist layer 4 as shown.
[0016]
In the last step, an aqueous solution of a polymer material such as polyvinyl butyral is dropped on the enzyme layer 6 and dried at a predetermined drying speed. Thus, a filter layer 7 is formed so as to cover the enzyme layer 6 (see FIG. 1). In the illustrated embodiment, the enzyme layer 6 is configured to maintain the initial pH value at 7.4 in order to measure the pH change caused by the enzyme reaction.
[0017]
【Example】
Next, specific examples of the urea sensor of the present invention will be described.
Example 1
A urea sensor for measuring urea in a biological sample was prepared as follows.
(1) Production of pH electrode substrate.
First, as shown in FIG. 3, the silver indicator electrode 11 and the base electrode of the reference electrode 12 are provided on the insulating substrate 10 in an inner circle shape and an outer ring shape. Next, a conductive carbon paste was printed and dried to form a carbon layer 13 so that the silver was not exposed on the inner circle silver and was not connected to the outer ring silver. Further, a resist is printed on the substrate so that the exposed diameter of the inner carbon layer 13 is 1.0 mmΦ and not covered with the reference electrode 12 and dried to form a resist layer 14 to produce an electrode substrate. .
[0018]
(2) Configuration of coated wire type pH liquid film.
The silver reference electrode of the above electrode substrate was subjected to silver chloride formation treatment in 0.7 M potassium chloride at a constant current of 1 mA for 10 seconds, washed and dried to complete the reference electrode 12. Further, a fixed volume of pH liquid film solution prepared with a prescribed composition was dropped on the indicator electrode carbon layer 13 and dried at room temperature to prepare a coated wire pH liquid film electrode 15.
[0019]
(3) Configuration of the enzyme reaction layer.
First, an enzyme-polymer solution was prepared as follows, urease (Toyobo Co., Ltd., 120 IU / mg) 10 mg, PVA (Wako Pure Chemical Industries, Ltd., polyvinyl alcohol, n = 500) 1.0 g Dissolved in 100 ml (0.02 M) of an acid buffer and mixed uniformly.
Next, 0.5 ul of the above solution was dropped onto the above pH liquid membrane indicator electrode 15 and dried at room temperature to form the enzyme reaction layer 16. 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. For example, 0.5 ul of Nafion solution (5%, Wt% in water: n-propanol = 1: 1) was dropped on the enzyme reaction layer and dried at room temperature.
[0020]
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 a test example.
(1) Preparation of urea standard measurement solution First, a urea standard aqueous solution is prepared as follows. Urea dried at 80 ° C. (manufactured by Wako Pure Chemical Industries, Ltd., biochemical supplies) was dissolved in 50 mM phosphate buffer (pH 7.38) to prepare a final urea concentration of 40 mM.
Next, the 40 mM urea solution was diluted with 50 mM phosphate buffer (pH 7.38) to prepare a final urea concentration of about 15 steps from 0 mM to 40 mM.
[0021]
(2) Urea Response by Potentiometric Method 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 Digital Electrometer (manufactured by Advantest). 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 transferred to the computer via the GPIB connector for 120 seconds. Next, the sensor was taken out from the above solution, immersed in a solution with a urea concentration of 2 mM in the urea standard measurement solution, and the potential response was measured for 60 seconds. Further, a new sensor was replaced, and a new concentration of urea solution was repeated with the same measurement. All measurements were made in about 15 steps from 0 to 40 mM urea concentration. The measurement results are shown in FIGS.
[0022]
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 between urea and urease, the output potential of the pH liquid film electrode is lowered. This potential change (absolute value) tends to increase as the urea concentration increases.
FIG. 5 shows a logarithmic relationship between the urea concentration and the sensor potential change based on 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 addition, in the range of 1 to 20 mM, there was a linear relationship and a sufficient dynamic range was shown.
[0023]
(3) pH dependence of urea response In the present invention, since the enzyme reaction layer placed on the pH liquid membrane electrode does not block the diffusion of hydrogen ions in the measurement liquid, the hydrogen ions in the measurement liquid are It diffuses through the reaction layer to the pH electrode and directly affects the response of the pH electrode. Here, the influence on the urea measurement from the pH of the sample was confirmed as follows.
First, several types of phosphate buffers (50 mM) having a pH of 6.2 to 7.8 were prepared. A certain amount of urea was dissolved therein to prepare a final concentration of 8 mM. Next, this 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. In other words. In the confirmed pH range (6.2 to 7.8), the potential change (absolute value) of the sensor also increased as the pH increased.
In order to solve the pH dependence as shown in FIG. 6, it is necessary to simultaneously measure the pH of the sample at the time of measurement, and to correct the measured value of urea based on the measured value.
[0024]
The present invention can be used as long as the concentration of hydrogen ions in the solution is changed by a biological sample, for example, creatine in blood, urine and the like, and other enzyme reactions. For example, since glucose ions are generated from a reaction with glucose using glucose dehydrogenase (Glucose Dehydrogenase) and the coenzyme NAD (P) +, glucose can be measured according to the present invention.
[0025]
【The invention's effect】
As described above, in the disposable BUN sensor according to the present invention , the electrode layer and the insulating layer surrounding the electrode layer are sequentially printed on the insulating film by the screen printing method, and the organic system is formed on the carbon layer. A pH electrode that detects a pH change formed by dripping and drying a synthetic substance, and an aqueous solution of a mixture of urease, a water-swellable polymer material, a surfactant and a buffer substance is dripped and dried on the pH electrode. Covers the surface of the enzyme layer and the surface of the enzyme layer, prevents the loss of enzyme in the enzyme layer, restricts the diffusion of urea, and contaminants in the measurement solution (for example, blood cells and proteins in whole blood) since a filter layer formed by dropping drying a solution of a polymer substance which serves to remove, it can be significantly reduced important manufacturing cost as a disposable sensor, Goods with the less expensive, calibration fluid penetrates the or quickly the filter layer during measurement, it is likely state reaction melts the enzyme layer, moreover bubble to the enzyme layer by the action of the buffer substance contained in the enzyme layer Therefore, the concentrations of urea and nitrogen can be measured with high accuracy.
[0026]
In addition, 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 a pH electrode, an enzyme layer, and a filter layer are formed in a dropping drying step, respectively. Since the BUN sensor is manufactured, the manufacturing cost can be greatly reduced, and an inexpensive product can be provided as a disposable sensor.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view showing a disposable BUN sensor according to an embodiment of the present invention.
FIG. 2 is a schematic cross-sectional view showing each process state of the manufacturing method of the BUN sensor shown in FIG.
FIG. 3 is a schematic view of a urea enzyme sensor according to an embodiment of the present invention.
FIG. 4 is a potential response curve diagram of a urea enzyme sensor measured by potentiometry with respect to a urea standard solution, and the urea concentration of the measured urea solution is 0, 2, 5, 10, 20, and 30 mM.
FIG. 5 is a diagram showing a logarithmic relationship between urea concentration and the potential response of a sensor.
FIG. 6 is a graph showing the pH dependence of the urea enzyme sensor of the present invention.
[Explanation of symbols]
1: Polyethylene film 2: Silver paste thin film layer (electrode layer)
3: Carbon layer 4: Resist layer 5: PH electrode 6: Enzyme layer 7: Filter layer 10: Insulating substrate 11: Silver indicator electrode 12: Reference electrode 13: Carbon layer 14: Resist layer 15: pH liquid film electrode 16 : Enzyme reaction layer

Claims (2)

On the insulating film, an electrode layer and an insulating layer surrounding the electrode layer are sequentially printed by a screen printing method, and a pH change formed by dropping and drying an organic synthetic material on the carbon layer is detected. a pH electrode;
On the pH electrode, an enzyme layer formed by dripping and drying an aqueous solution of a mixture of urease, water-swellable polymer material, surfactant and buffer substance,
It is formed by dripping and drying a solution of a polymer substance that covers the surface of the enzyme layer, prevents the loss of enzyme in the enzyme layer, restricts the diffusion of urea, and removes contaminants in the measurement solution. Filter layer and
Disposable BUN sensor, characterized in that it comprises a. 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;
Forming a pH electrode for detecting a pH change by dripping and drying an organic synthetic material on the carbon layer;
On the pH electrode, a step of dripping and drying an aqueous solution of a mixture of urease, a water-swellable polymer material, a surfactant and a buffer substance to form an enzyme layer;
Cover the surface of the enzyme layer, prevent the urease from flowing out of the enzyme layer, limit the diffusion of urea, and drop and dry a solution of a polymer substance that functions to remove contaminants in the measurement solution. A process for producing a disposable BUN sensor characterized by comprising the steps of:

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