JP2003202314A - Manufacturing method of biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem that entire products cannot be warranted since the quality of a population is warranted by sampling using a statistic method to warrant the performance of a group of biosensors to be mass-produced, and further intermediate materials during a manufacturing process may include the risk of mass abandonment. <P>SOLUTION: During a biosensor-manufacturing process, incident light having a specific wavelength is applied to a biosensor, and the area value of a contact section 14 coming into contact with a liquid sample on an operation electrode 1 formed on the biosensor is measured according to the luminance information of a reflected light that is reflected from a operation electrode section, thus judging the performance of the biosensor. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a biosensor for carrying out rapid and highly accurate quantification of a measurement component in a liquid sample, and in particular, evaluating the performance of the biosensor by an indirect method. It has the characteristic of

2. Description of the Related Art As a quantitative analysis method of saccharides such as sucrose and glucose, a polarimetric method, a colorimetric method, a reduction titration method, a method using various chromatographies and the like have been developed.
However, none of these methods had high specificity for saccharides, and thus the accuracy was poor. According to the polarimetric method, the operation is simple, but it is greatly affected by the temperature at the time of operation, so it is not suitable as a method for easily quantifying sugars by ordinary people at home. By the way, in recent years, various types of biosensors that utilize the specific catalytic action of enzymes have been developed. The glucose quantification method will be described below as an example of the substrate quantification method in the sample solution. As an electrochemical quantification method of glucose, a method of using glucose oxidase (EC 1.1.3.4: hereinafter abbreviated as GOD) and an enzyme electrode or a hydrogen peroxide electrode is generally known. . (For example, Shuichi Suzuki's "Biosensor" Kodansha). GOD uses oxygen as an electron acceptor to selectively oxidize the substrate β-D-glucose into D-glucono-δ-lactone. Oxygen is reduced to hydrogen peroxide in the redox process by GOD in the presence of oxygen. The amount of decrease in oxygen is measured, or the amount of increase in hydrogen peroxide is measured by a hydrogen peroxide electrode. Since the decrease amount of oxygen and the increase amount of hydrogen peroxide are proportional to the glucose content in the sample, glucose can be quantified from the decrease amount of oxygen or the increase amount of hydrogen peroxide.
As can be inferred from the reaction process, the above method has a drawback that the measurement result is greatly affected by the oxygen concentration contained in the sample solution, and further, if oxygen is not present in the sample solution, measurement becomes impossible. . Therefore, without using oxygen as an electron acceptor, potassium ferricyanide, a ferrocene derivative,
A new type of glucose sensor using an organic compound such as a quinone derivative or a metal complex as an electron acceptor has been developed. In this type of sensor, the reduced form of the electron acceptor generated as a result of the enzymatic reaction is oxidized on the electrode, and the concentration of glucose contained in the sample solution is obtained from the oxidation current. By using such an organic compound or a metal complex as an electron acceptor instead of oxygen, a known amount of GOD and those electron acceptors can be accurately and stably supported on an electrode to form a reaction layer. Is possible. In this case, since the reaction layer can be integrated with the electrode system in a state close to a dry state, a disposable glucose sensor based on this technique has recently received much attention. FIG. 7 is an exploded perspective view showing a conventional biosensor for measuring blood glucose level as described above, and FIG. 8 is a sectional view thereof. On an insulating substrate 5 such as polyethylene terephthalate, a working electrode 1 serving as an electrode, a counter electrode 2, and a resist layer 6 for insulating and protecting both electrodes are formed by printing. Glucose oxidase and an electron acceptor are formed on these electrodes. And a surfactant layer 12 made of egg yolk lecithin or the like is formed on the reagent layer 11. Then, in order to form a cavity 13 for detecting a current value generated by a reaction between the collected blood and the reagent layer 11 with a certain amount of blood, a spacer in which a portion above the electrode and the reagent is cut out is elongated. 8 and the cover 7 in which the air escape hole 10 is formed are attached to the insulating substrate 5.
In the biosensor having such a configuration, blood is introduced from the suction port 9 into the cavity 13 and guided to a position where the electrode and the reagent are present. The current value generated by the reaction between the blood and the reagent on the electrode is read by connecting to an external measuring device (not shown) through the leads 3 and 4. Conventionally, in order to mass-produce biosensors having the same performance, biosensors having such a configuration use a means such as printing on an insulating substrate 5 that is processed into a sheet having a certain size, and a plurality of biosensors can be manufactured by one operation. The device is designed so that individual biosensors can be produced.

The biosensor thus manufactured cannot be repeatedly used once because of its constitution. Therefore, it is mass-produced in order to guarantee performance or quality. The performance is guaranteed by a sampling method using a statistical method such as AQL with the biosensor group as a population. Specifically, an example of a quality inspection method of a glucose sensor for measuring a blood glucose level in blood will be shown below. Blood or glucose concentration standard solution having a known blood glucose level is sucked into a prescribed amount of blood glucose sensor from a large amount of produced glucose group population, and the measured value from the measuring device is read. Next, the performance of all biosensor groups in the population is guaranteed from the absolute value and coefficient of variation of the measured values of the specified amount obtained. Therefore, it is not possible to manage or guarantee the total number of biosensor groups by the above method, and some biosensor groups may include biosensors that show a measurement value different from the true value. There is a problem such as. Furthermore, the performance of the biosensor group may be guaranteed only by the sampling method from the completed products, and if the measurement results deviate from the quality assurance range, all the products in process during the manufacturing process are discarded. There is no choice but to take a huge risk in the manufacturing sector.

In order to solve the above problems, a method of manufacturing a biosensor according to the present invention uses a working electrode and a counter electrode formed on an insulative substrate to form a liquid sample and a reagent. In the biosensor for analyzing the components in the liquid sample by detecting the reaction with, by measuring the contact area between the working electrode and the liquid sample during the manufacturing process, the performance of the biosensor group is It is characterized by making a judgment.

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing a biosensor according to claim 1 of the present invention uses a working electrode and a counter electrode formed on an insulating substrate to react a liquid sample with a reagent. In a biosensor for analyzing components in the liquid sample by detecting, the performance of the biosensor is evaluated by measuring a contact area between the working electrode and the liquid sample. . According to this
Since it is not necessary to take a method of sucking a liquid sample to obtain a measurement value in order to directly evaluate the performance of the biosensor,
Since it is possible to perform 100% inspection or 100% guarantee of the sensor group population, which has not been done so far, further it is possible to confirm the performance by simply measuring the contact area value of the working electrode during the biosensor manufacturing process. It is possible to provide a biosensor manufacturing method in which the risk of discarding products in process in the manufacturing process is greatly reduced. Next, the method for manufacturing a biosensor according to claim 2 of the present invention comprises the steps of irradiating the working electrode with light having a specific wavelength, detecting reflected light reflected from the working electrode portion, and detecting the light. The method for manufacturing a biosensor according to claim 1, further comprising the step of calculating a contact area value of the working electrode from the reflected light thus obtained, and an effect similar to that of claim 1 can be expected. Next, the method for manufacturing a biosensor according to claim 3 of the present invention is characterized in that a contact area value is calculated from luminance information of reflected light.
The method for manufacturing a biosensor described above can determine the bleeding or foreign matter in the electrode opening, or even the pinhole in the electrode, so that the effective area that actually affects the performance can be easily determined. Next, in the biosensor manufacturing method according to claim 4 of the present invention, the contact area is measured while shielding the light so as to control the amount of reflected light reflected from the working electrode. This is a method of manufacturing a biosensor, and in particular, by installing a light-shielding function near the focal point of a camera or the like, a large contrast ratio level can be obtained and the working electrode interface can be regulated in more detail even with a small amount of light, and the effective area can be easily determined It can be realized. Next, in the method for producing the biosensor according to claim 5 of the present invention, the working electrode is irradiated with light having a peak in a wavelength range of 450 nm to 510 nm. This is a method for determining the effective area that affects the performance more accurately because the brightness information of the reflected light can be accurately obtained even when the color tone or the solid step around the contact portion of the working electrode changes. Can be easily realized. (Embodiment 1) Hereinafter, claim 1 and claim 2 of the present invention
An embodiment of the invention described in 1. will be described with reference to FIGS. It should be noted that the configuration numbers in FIGS. 1 and 2 are the same as those in FIGS. In FIG. 1, reference numeral 5 is an insulating substrate made of, for example, polyethylene terephthalate, which is supplied in the form of a sheet having a constant size. On the insulating substrate 5, a working electrode 1 serving as an electrode, a counter electrode 2, and a resist layer 6 for insulating and protecting both electrodes are formed by means of screen printing or the like. On these electrodes, a reagent layer 11 containing glucose oxidase and an electron acceptor, and a surface active material layer 12 made of egg yolk lecithin or the like are formed. Further, in FIG. 2, reference numeral 14 is a contact portion with the opened liquid sample on the working electrode 1 formed on the biosensor, and the contact area is regulated by the resist layer 6. 1
Reference numeral 5 is a camera, 16 is a light shielding plate, and 17 is an illumination for irradiating light of a specific wavelength. Here, in the biosensor manufacturing method of the present invention, after the electrodes 1 and 2 are formed on the insulating substrate 5 and then the resist layer 6 is formed, the optical system shown in FIG. The performance of the biosensor is determined by measuring the area of the contact portion 14 with the liquid sample opened on the working electrode 1 by using its brightness. In an actual biosensor manufacturing process, n × m biosensors are two-dimensionally arranged in one sheet (not shown) so as to be suitable for mass production. Then, each biosensor is formed by punching from this sheet with a press or the like. In the manufacturing process until the sheet is punched out, the sheet is processed at a preset work speed (tact) in each work process and the process proceeds to the next process. In addition, since the takt time is determined by the processing capacity of computer devices such as personal computers and workstations in measurement to arithmetic processing, at present, n cameras are arranged in the same row in the advancing direction (provisionally n direction) in the process of insulating substrate, The tact is balanced. The irradiation range of the illumination 17 is set so as to include the counter electrode on the biosensor, but what is important here is to reduce the amount of reflected light reflected from the biosensor as much as possible to further increase the dynamic range width in contrast. Is to spread. That is, in the camera optical system, by installing the light shielding plate 16 near the in-focus position, the minimum amount of light necessary for area determination or optimum information can be obtained. FIG. 3 is a diagram showing a correlation with the glucose value measured using a glucose standard solution after intentionally changing the area of the resist layer that regulates the area of the contact portion on the working electrode. In the figure, when three types of contact portion areas were intentionally set, glucose measurement values also showed a similar distribution, and their correlation coefficient was 0.9683, which was very high. Since the sensitivity of the glucose sensor is mathematically corrected by the variation of the measured value with respect to the standard solution, the sensitivity of the glucose sensor can be indirectly and nondestructively determined by measuring the contact area. (Embodiment 2) Claims 3 and 4 of the present invention
2, FIG. 4, regarding the embodiment of the invention described in FIG.
This will be described with reference to FIGS. FIG. 4 shows the luminance distribution of the illumination 17 in FIG. 2, and has a luminance peak near about 500 nm. As a result, in the image obtained by the camera, the contrast is enhanced in the boundary area, and the boundary area of the necessary portion can be visually determined. FIG. 5 shows an input image photograph by a fluorescent lamp, and FIG. 6 shows an input image photograph by a single wavelength illumination using an LED according to the present invention. Since the input image itself has a high contrast ratio, the processing time to actually calculate the contact area from the image data is very fast and accurate, and the processing time required for one image was 80 ms, but it was as high as 26 ms. It was possible to process it approximately 3 times as much. As described above, according to the present invention, it becomes possible to perform non-destructive and high-speed image processing, measurement, and determination of the biosensor itself during the manufacturing process of the biosensor, and all sensors can be determined inline.

As described above, according to the method for manufacturing a biosensor of the present invention, it is possible to perform 100% inspection or 100% guarantee of a sensor group population, which has not been possible by the conventional methods. Since it becomes possible to confirm the performance only by measuring the contact area value of the working electrode on the way, it is possible to provide a method for manufacturing a biosensor in which the risk of discarding a product in process in the manufacturing process is significantly reduced. Furthermore, by calculating the contact area value of the working electrode from the brightness information of the reflected light, it is possible to analyze the contact area value due to the bleeding of the electrode opening, determine the presence of foreign matter, or even pinholes in the electrode, so that the actual performance It is possible to easily realize the determination of the effective area having an influence. Further, by having a light-shielding device that controls the amount of light input to the camera, the amount of reflected light reflected from the biosensor can be reduced as much as possible, and the dynamic range width in contrast can be further widened. That is, in the camera optical system, by installing the light shielding plate 16 near the in-focus position, the minimum amount of light necessary for area determination or optimum information can be obtained. Further, by irradiating light having a peak in the wavelength range of 450 nm to 510 nm, it is possible to accurately obtain the luminance information of the reflected light even when the color tone or the three-dimensional step in the peripheral region of the contact portion of the working electrode changes. Therefore, it is possible to easily and more accurately determine the effective area that affects the performance.

[Brief description of drawings]

FIG. 1 is a sectional view of a biosensor according to a first embodiment of the present invention.

FIG. 2 is a schematic diagram of contact area measurement of the biosensor according to the first embodiment of the present invention.

FIG. 3 is a correlation diagram of contact area and sensitivity according to the first embodiment of the present invention.

FIG. 4 is a wavelength characteristic diagram of a light source according to the second embodiment of the present invention.

FIG. 5 is an acquired image comparison diagram according to the second embodiment of the present invention.

FIG. 6 is an acquired image comparison diagram according to the second embodiment of the present invention.

FIG. 7 is an exploded perspective view showing a conventional biosensor for measuring blood glucose level.

FIG. 8 is a cross-sectional view showing a conventional biosensor for measuring blood glucose level.

[Explanation of symbols]

1 Working electrode 2 counter electrodes 3, 4 leads 5 insulating substrate 6 Resist layer 7 cover 8 spacers 9 Suction port 10 Air escape holes 11 Reagent layer 12 Surfactant layer 13 cavities 14 Working electrode opening 15 camera 16 light shield 17 Lighting

Continued front page    (72) Inventor Konobu Sawada             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. (72) Inventor Takashi Nishioka             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. (72) Inventor Fukuyama Teruaki             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. (72) Inventor Katsuaki Kawamoto             1 Juden Matsushita, 1-8 Koshinmachi, Takamatsu City, Kagawa Prefecture             Child Industry Co., Ltd. F term (reference) 2F065 AA58 BB00 CC00 FF04 FF44                       GG03 GG07 GG17 GG22 HH13                       JJ03 JJ05 JJ09 QQ31                 2G051 AA61 AB07 BA04 CA04 CC07                       EA11 EA12 EA16

Claims (5)

[Claims] 1. A biosensor for analyzing a component in a liquid sample by detecting a reaction between a liquid sample and a reagent using a working electrode and a counter electrode formed on an insulating substrate. A method for manufacturing a biosensor, characterized in that the performance of the biosensor is evaluated by measuring a contact area between the working electrode and a liquid sample. 2. A step of irradiating the working electrode with light of a specific wavelength, a step of detecting reflected light reflected from the working electrode portion, and a step of calculating a contact area value of the working electrode from the detected reflected light. The method of manufacturing a biosensor according to claim 1, further comprising: 3. The method for manufacturing a biosensor according to claim 2, wherein the contact area value is calculated from the brightness information of the reflected light. 4. The method for manufacturing a biosensor according to claim 2, wherein the contact area is measured while shielding the light so as to control the amount of reflected light reflected from the working electrode. 5. The method for producing a biosensor according to claim 2, wherein the working electrode is irradiated with light having a peak in a wavelength range of 450 nm to 510 nm.