JP2012047691A - Method for manufacturing biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing a biosensor by which a fixed position on an electrode is certainly covered by a reaction part and whose manufacturing costs do not increase.SOLUTION: In the method for manufacturing the biosensor including steps of forming the electrode on a substrate 12 consisting of an insulator, and forming a reaction part 18 which contacts the electrode on the substrate 12, UV (ultraviolet rays) irradiation processing (UV irradiation processing) is performed on the substrate 12 before forming the reaction part 18 after forming the electrode on the substrate 12.

Description

  The present invention relates to a biosensor manufacturing method for detecting a specific component in a sample solution.

  Conventionally, a biosensor has been devised for measuring a blood glucose level of a specimen. As an example of this biosensor, a biosensor 1 is shown in FIG. The biosensor 1 includes a substrate 2 made of an insulator, an electrode 3 provided on the substrate 2, a reaction unit 4 provided on the substrate 2 so as to be in contact with the electrode 3, and a specimen to the reaction unit 4. And a supply port 5 to be introduced. The reaction unit 4 reacts with a specific component in the sample introduced from the supply port 5 so that the specific component can be quantitatively analyzed.

  The reaction part 4 of the biosensor 1 is formed by dropping an aqueous solution that forms the reaction part 4 on the electrode 3. However, even if the same amount of aqueous solution is dropped, the reaction part 4 does not always have a constant area, and thus the measurement accuracy of the biosensor 1 may vary. Further, the position where the aqueous solution is dropped with respect to the substrate 2 provided with the electrode 3 is shifted, and the position of the reaction unit 4 with respect to the electrode 3 is shifted, and the measured value of the biosensor 1 may vary. For this reason, the biosensor which can form a reaction part in a fixed position is also devised (for example, refer patent document 1). In this biosensor, a second slit serving as a breakwater for the spreading reagent is provided in the electrically conductive layer in order to form the reaction part at a certain position.

  However, when the reagent does not reach the slit, the position where the reagent adheres is not always constant. On the other hand, it is conceivable that the reagent that spreads out cannot be stopped sufficiently by only a slit having a very small depth, and the reagent flows out of the electrode. For this reason, it is considered that it is impossible to completely prevent the position where the aqueous solution is dropped from the substrate 2 provided with the electrode 3. Furthermore, it is necessary to perform laser processing on the electrically conductive layer in order to form the slit, which may increase the manufacturing cost.

Japanese Patent No. 4197085

  Therefore, the inventor of the present invention has come to the present invention as a result of intensive studies to investigate the cause of such a problem and solve such a problem.

  The present invention prevents the position where the aqueous solution forming the reaction part is dropped from the substrate provided with the electrode from being shifted, can reliably form the reaction part at a certain position on the substrate, and increases the manufacturing cost. It is an object to provide a method for manufacturing no biosensor.

  The biosensor manufacturing method of the present invention includes a step of forming an electrode on a substrate made of an insulator, and a step of forming a reaction portion in contact with the electrode on the substrate. Before forming the reaction part, UV irradiation treatment is performed on the substrate.

  In the biosensor manufacturing method of the present invention, in the biosensor manufacturing method, the step of forming the electrodes includes a pair of electrodes spaced apart from each other, and a distance from the pair of electrodes. A surrounding electrode surrounding the pair of electrodes.

  The biosensor manufacturing method of the present invention is characterized in that, in the biosensor manufacturing method, the UV irradiation treatment is performed after the electrodes are formed.

  Further, in the biosensor manufacturing method of the present invention, in the biosensor manufacturing method, the step of forming the electrode includes a step of performing sputtering and a step of performing etching, and before performing the sputtering, Glow processing (glow discharge processing) is performed on the substrate.

  The biosensor manufacturing method of the present invention is characterized in that, in the biosensor manufacturing method, the glow process is a glow process using argon or oxygen.

Further, in the biosensor manufacturing method of the present invention, in the biosensor manufacturing method, the step of forming the electrode includes a step of performing sputtering and a step of performing etching, and before performing the sputtering, Silicon or silicon dioxide on the substrate The base sputtering is performed.

  According to the biosensor manufacturing method and biosensor of the present invention, the UV irradiation treatment is performed on the substrate before the reaction portion is formed. Therefore, the hydrophilicity of the surface of the substrate is increased to wet the aqueous solution forming the reaction portion. It is easy to spread, and the reaction part can be reliably formed at a certain position on the substrate. Further, when the surrounding electrode is provided, it is possible to prevent the position where the aqueous solution forming the reaction portion is dropped from the substrate provided with the electrode, and it is not necessary to provide a slit for stopping the dropped aqueous solution. Therefore, it is possible to prevent the manufacturing cost from increasing. Further, by performing glow treatment or base sputtering on the substrate before sputtering, the hydrophilicity of the exposed portion of the substrate can be made higher than the hydrophilicity of the surrounding electrode. For this reason, the dropped aqueous solution spreads smoothly and stays inside the surrounding electrode without exceeding the surrounding electrode. Therefore, the reaction part can be reliably formed at a fixed position inside the surrounding electrode.

It is a figure which shows the biosensor of this invention, the same figure (a) is a top view, The same figure (b) is an AA line | wire cut part sectional drawing. It is a figure for demonstrating the manufacturing method of the biosensor of FIG. 1, The same figure (a) is AA cut | disconnection sectional drawing of the state in which the needle was made to correspond with the center part of an electrode, FIG. ) Is a cross-sectional view taken along line AA in a state where the needle is displaced from the center of the electrode. FIG. 3A is a cross-sectional view of a biosensor in which UV irradiation treatment is not performed on the surface side of the substrate, and FIG. 3B shows that the distance between the pair of electrodes and the inner peripheral side surface of the surrounding electrode is It is sectional drawing of a biosensor less than 10% with respect to the substantially circular diameter of a surrounding side surface. FIG. 4A is a plan view showing another embodiment of the present invention, and FIG. 4B is a plan view showing another embodiment of the present invention. It is AA line cutting | disconnection part sectional drawing which shows the Example of the biosensor of this invention. FIG. 6 is a graph for evaluating the performance of the biosensor of the present invention. FIG. 6A shows the relationship between the glucose concentration and the current integration value, and FIG. 6B shows the UV irradiation time and the current integration. Indicates the relationship with the value. FIG. 7A is a table showing the relationship between the UV irradiation time and the contact angle, and FIG. 7B is a graph showing the relationship between the UV irradiation time and the contact angle. It is a graph which shows the relationship between the process before sputtering, and a contact angle. It is sectional drawing for demonstrating the definition of hydrophilicity. It is a perspective view which shows the conventional biosensor.

  Next, the biosensor manufacturing method according to the present invention will be described in detail with reference to the drawings. In FIG. 1, reference numeral 10 denotes a biosensor according to the present invention.

The biosensor manufacturing method according to the present invention includes a step of forming an electrode on a substrate 12 made of an insulator, and a step of forming a reaction portion 18 in contact with the electrode on the substrate 12. In this method, after the electrodes are formed on the substrate 12 and before the reaction portion 18 is formed, a UV (ultraviolet rays) irradiation process (UV irradiation process) is performed on the substrate 12. In the biosensor manufacturing method according to the present invention described with reference to FIG. 1, the step of forming the electrodes is performed on the pair of electrodes 14 and 16 spaced apart from each other and the pair of electrodes 14 and 16. In this case, the surrounding electrodes 20, 22, and 24 surrounding the pair of electrodes 14 and 16 are formed at intervals. Further, in the biosensor manufacturing method according to the present invention, the step of forming an electrode includes a step of performing sputtering and a step of performing etching, and before performing the sputtering, This is a method of manufacturing a biosensor that performs base sputtering of Si or SiO ₂ .

  The shape of the outer peripheral side surface 26 formed by the electrodes 14 and 16 is substantially circular, and the shape of the inner peripheral side surface 28 formed by the surrounding electrodes 20, 22 and 24 is also substantially circular. is there. The electrodes 14 and 16 are for measuring blood glucose levels and the like. The surrounding electrodes 20, 22, and 24 are not particularly limited in use such as blood introduction confirmation, blood volume detection, or biosensor 10 attachment confirmation on a measurement display. As a result of the test, the interval W between the outer peripheral side surface 26 of the electrodes 14 and 16 and the inner peripheral side surface 28 of the surrounding electrodes 20, 22 and 24 is 10% with respect to the substantially circular diameter D of the inner peripheral side surface 28. It is preferable that it is 40% or less.

When the biosensor 10 is manufactured, first, a metal thin film such as nickel or an alloy is formed on the insulating substrate 12 made of polyethylene terephthalate or the like by sputtering. As a process before sputtering, a glow process is performed with argon, nitrogen, or oxygen gas. Alternatively, before sputtering of the metal, the Si or SiO ₂ layer is sputtered using silicon as a target to form the Si or SiO ₂ layer. Next, the metal thin film is etched by photolithography to form a pattern of the pair of electrodes 14 and 16 and the surrounding electrodes 20, 22 and 24. Further, the surface 13 side of the substrate 12 on which these electrodes are formed is subjected to a UV irradiation treatment as a hydrophilic treatment for improving the spread of a drug attached later.

  Next, as shown in FIG. 2A, the drug 32 is dropped on the electrodes 14 and 16 using the metal needle 30. The outer diameter φ of the metal needle 30 is smaller than the inner diameter D of the surrounding electrodes 20, 22, and 24, and is large enough to cover the electrodes 14 and 16 with the dropped drug 32. The greater the difference between the outer diameter φ and the inner diameter D, the greater the tolerance for misalignment when attaching the drug. When the biosensor 10 is a blood glucose level sensor, an aqueous solution composed of enzymes such as glucose oxidase and glucose dehydrogenase is used as the adhering drug 32. Further, potassium ferricyanide as an electron acceptor may be dissolved and attached simultaneously with the enzyme. In addition, a solution obtained by separately dispersing fine particles of potassium ferricyanide in an organic solvent may be further adhered onto the enzyme layer.

  After the reaction portion 18 is formed on the electrodes 14 and 16 in this way, the biosensor 10 is manufactured by covering the electrodes 14, 16, 20, 22, and 24 on the substrate 12 with a cover via a spacer. Is done. The biosensor 10 performs the above-described glow treatment or the above-described base sputtering before forming a metal thin film such as nickel or an alloy by sputtering, and reacts after forming a metal thin film such as nickel or an alloy by sputtering. Before the portion 18 is formed, it is manufactured by performing UV irradiation treatment.

According to such a biosensor 10, the surface 13 side of the substrate 12 including the electrodes 14 and 16 is subjected to UV irradiation treatment to increase the hydrophilicity. Therefore, an aqueous solution (medicine) 32 containing an enzyme is applied to the electrodes 14 and 16. When dropped on the top, the aqueous solution 32 smoothly spreads on the electrodes 14 and 16 as shown in FIG. 2A without stagnation on the electrodes 14 and 16 as shown in FIG. In particular, by performing glow treatment or sputtering of Si or SiO ₂ before sputtering, the hydrophilicity on the insulating substrate 12 is improved, and the aqueous solution 32 spreads around the central electrodes 14 and 16. However, when the UV irradiation treatment is not performed before coating, the dropped aqueous solution is difficult to spread from the water droplets. The hydrophilicity can be quantitatively expressed by the contact angle θ shown in FIG. 9, that is, the angle of the end of the aqueous solution 32 with respect to the plane 33 on which the aqueous solution 32 is dropped. The smaller the contact angle θ, the higher the hydrophilicity. . In the manufacturing method of the biosensor 10, the surface 13 of the substrate 12 corresponds to the flat surface 33. The effect of the present invention using the contact angle θ will be described later in (Example).

Furthermore, in order for the dropped aqueous solution 32 to cover all the electrodes 14 and 16 and stop wetting and spreading at the boundary with the surrounding electrodes 20 and 22, the hydrophilicity of the substrate 12 is more than the hydrophilicity of the surrounding electrodes 20, 22 and 24. Higher is desirable. On the contrary, when the surrounding electrodes 22 and 24 are much more hydrophilic than the hydrophilicity of the substrate 12, the surrounding electrodes 20 and 22 are wet and spread, or conversely, only the electrodes 14 and 16 remain. In the biosensor manufacturing method of the present invention, since the UV irradiation treatment is performed on the substrate 12 after forming the electrode on the substrate 12 and before forming the reaction portion 18, the substrate exposed portion 19 on which no electrode is formed and The hydrophilicity of the entire surface 13 of the substrate 12 before the formation of the reaction part 18 including the electrodes 14, 16, 20, 22 and 24 can be increased. On the other hand, before the sputtering and before the formation of the electrodes 14, 16, 20, 22, and 24, the substrate 12 is subjected to the glow treatment or the Si or SiO ₂ base sputtering. Can be made higher than the hydrophilicity of the surrounding electrodes 20, 22 and 24. For this reason, the aqueous solution 32 dropped on the substrate 12 after the formation of the electrodes 14, 16, 20, 22 and 24 spreads smoothly and does not exceed the surrounding electrodes 20, 22 and 24. Stop inside. Therefore, the reaction part 18 can be reliably formed at a certain position on the electrode 12.

  On the other hand, since the biosensor 10 includes the surrounding electrodes 20, 22, and 24, as shown in FIG. 2A, the inner circumferences of the surrounding electrodes 20, 22, and 24 do not exceed the surrounding electrodes 20, 22, and 24. Stop at the side. Further, even when the position of the central axis C1 of the metal needle 30 is deviated from the intermediate point C2 of the electrodes 14 and 16, the aqueous solution 32 does not exceed the surrounding electrodes 20, 22, and 24 as shown in FIG. It stops at the inner peripheral side surface of the surrounding electrodes 20, 22 and 24. For this reason, the aqueous solution 32 can be adhered in a certain area inside the surrounding electrodes 20, 22 and 24. Furthermore, in the case of the present invention, the distance W between the outer peripheral side surface 26 of the electrodes 14 and 16 and the inner peripheral side surface 28 of the surrounding electrodes 20, 22 and 24 is 10 with respect to the substantially circular diameter D of the inner peripheral side surface 28. Therefore, the aqueous solution 32 does not extend between the outer peripheral side surface 26 and the inner peripheral side surface 28 and spread on the surrounding electrodes 20, 22 and 24 as shown in FIG. It can be deposited with a constant area inside the electrodes 20, 22 and 24. In the case where an aqueous solution containing an oxidoreductase is attached at a certain area inside the surrounding electrodes 20, 22 and 24 and then attached using a liquid containing an organic solvent, the inside of the surrounding electrodes 20, 22 and 24 Although it is difficult to adhere in a certain area, if the adherent liquid containing the oxidoreductase is adhered in a certain area, the influence on the variation in the measured value is small.

  As mentioned above, although one Embodiment of this invention was described, this invention can be implemented also in another aspect. For example, the shape of the electrodes 14 and 16 is not particularly limited as long as the shape of the inner peripheral side surfaces of the surrounding electrodes 20, 22 and 24 is substantially circular. For example, it may be a rectangle as shown in FIG. 4A or a comb shape as shown in FIG.

  In addition, the present invention can be implemented in a mode not shown. For example, the method for forming the reaction part is not particularly limited as long as it includes an oxidoreductase and an electron acceptor. In addition, the technical scope of the present invention includes embodiments in which various improvements, modifications, and variations are added based on the knowledge of those skilled in the art without departing from the spirit of the present invention. Moreover, you may implement with the form which substituted any invention specific matter to the other technique within the range which the same effect | action or effect produces.

(Example)
Before the formation of the electrodes 14 and 16, a PET film having a thickness of 188 μm was subjected to a glow treatment in a vacuum chamber. The glow treatment was performed using Ar gas, with an electrode applied voltage of 1850 V, a gas flow rate of 30 cc / min, an operating pressure of 4.2 × 10 ⁻³ Torr, and a film speed of 0.5 m / min. After the glow treatment, sputtering was performed at an input power of 0.7 kW, an operating pressure of 2.0 × 10 ⁻³ Torr and a film speed of 1.0 m / min, and a Ni thin film having a thickness of 0.1 μm was formed on the entire surface. Thereafter, the electrode was patterned on the Ni thin film by photolithography. Next, the surface 13 of the substrate 12 on which the electrodes 14 and 16 were formed was subjected to UV irradiation treatment with irradiation energy of 15 mW / cm ² at a wavelength of 254 nm and varying the irradiation time.

  As a method for forming the reaction layer, 0.15 μl of an aqueous solution containing 3.0% GOD (glucose oxidase) and 0.8% CMC (carboxymethylcellulose) was dropped on the electrodes 14 and 16 and dried at 40 ° C. for 6 minutes. By doing so, as shown in FIG. 5, the first layer 18 (1) was formed. When 0.15 μl of the aqueous solution was dropped using a needle with an outer diameter of 0.7 mm, the irradiation time could not be completely wet and spread over 3 minutes, but over the outer electrodes 20 and 22 over 4 minutes. And could be applied to cover all the inside. In addition, the viscosity of the aqueous solution in which the enzyme is dissolved may be appropriately adjusted by changing the CMC concentration. Further, 0.20 μl of a solution obtained by dispersing finely divided potassium ferricyanide (median diameter 3.9 μm) in an ethyl cellosolve solution of 1.13% HPC (hydroxypropyl cellulose) to a concentration of 8.3% It was dropped on the layer 18 (1) to form the second layer 18 (2) as shown in FIG. The ethyl cellosolve solution was applied so as to cover the entire electrode by dropping 0.20 μl using a needle having an outer diameter of 1.5 mm. In this way, the reaction portion 18 composed of the first layer 18 (1) and the second layer 18 (2) was formed.

  The biosensor 10 thus formed was first evaluated for performance. As a performance evaluation method, an aqueous glucose solution was used to obtain the relationship between the glucose concentration and the current integral value. Here, the current integrated value means that the potential between the electrodes is changed from 0 V to -0.2 V, 0 V, +0.2 V at a rate of 200 mV / sec after inhaling the measurement sample, and from -0.1 V to +0.2 V. It is a value obtained by integrating the results of A / D conversion 60 times every 0.025 sec by converting the current flowing between the electrodes during voltage scanning. As shown in FIG. 6 (a), the glucose concentration and the current integrated value are proportional to each other at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, and 7 minutes of the UV irradiation time. It has been found that it can be used as a sensor. The evaluation method is not limited to this, and a constant voltage may be applied between the electrodes, and a current value after a lapse of a predetermined time or an integrated value of the current may be used.

  Next, the output of the biosensor 10 created using the electrodes with different UV irradiation treatment times was measured using an aqueous glucose solution. As a result, as shown in FIG. 6 (b), when the glucose concentration was 100 mg / dl, 300 mg / dl, 500 mg / dl, and 900 mg / dl, the output increased as the irradiation time became longer. Saturated. Therefore, it was found that if the UV irradiation is 3 minutes or more, a constant current integral value can be obtained.

  Next, in order to evaluate the UV irradiation treatment time dependency of the contact angle of the substrate surface, the irradiation time was changed under the same UV irradiation conditions as described above, and an aqueous solution in which the enzyme for forming the first layer was dissolved was dropped onto the treatment surface. The contact angle was evaluated. First, when the contact angle was measured using a normal PET film that was not subjected to sputtering treatment, as shown in FIG. 7A, what was 61 ° before irradiation was up to 30-40 ° after treatment. It is lowered and hydrophilicity is improved. In contrast, on the PET surface on which sputtering and etching were performed after glow treatment with Ar gas, the contact angle decreased to around 20 ° with the irradiation time. Similarly, on the Ni electrode, the contact angle is reduced to about 25 °, but the contact angle is larger than that of the PET surface. For this reason, when the aqueous solution which melt | dissolved the enzyme for 1st layer 18 (1) formation was dripped, it turned out that aqueous solution does not get wet to the outer side of the surrounding electrodes 20, 22, and 24.

Next, the contact angle after 7 minutes of UV irradiation was compared for the pre-sputtering treatment with the type of gas used in the glow treatment, the base sputtering before electrode sputtering, and no treatment. Argon, oxygen, and nitrogen were used for the glow treatment. The electrode application voltage was 1960 to 2000 V, the gas flow rate was 29 cc / min, the operating pressure was 2.0 to 4.8 × 10 ⁻³ Torr, and the film speed was 1.0 m / min. In the base sputtering before electrode sputtering, a Si or SiO ₂ film was formed. In the case of Si, Si was used as a sputtering target, and a layer of about 2 nm was formed at an input power of 0.55 kW, an operating pressure of 2.0 × 10 ⁻³ Torr, and a film speed of 1.4 m / min. In the case of SiO ₂ , Si was used as a sputtering target, and a layer of about 6 nm was formed at an input power of 3.1 kW, an operating pressure of 2.0 × 10 ⁻³ Torr and a film speed of 2.0 m / min. In the case of SiO ₂ sputtering, oxygen is added to argon as a gas introduced during sputtering. The flow rate ratio between argon and oxygen was Ar: O ₂ = 5: 9. Nickel was sputtered onto each pretreated PET film to form an electrode, and then patterned by etching. An aqueous solution in which an enzyme for forming the first layer 18 (1) was dissolved on the PET film exposed after the etching was dropped onto the treated surface, and the contact angle was evaluated.

With no treatment and with nitrogen glow, the contact angle after etching drops to about 25 ° after UV irradiation treatment, but is similar to the contact angle on the Ni electrode surface. On the other hand, the argon and oxygen glows are reduced to about 20 °, indicating that they are more effective. Furthermore, it can be seen that when Si or SiO ₂ is used as the underlayer, the hydrophilicity is further improved by 20 ° or less. When 0.15 μl of an aqueous solution in which the enzyme for forming the first layer 18 (2) is dissolved is dropped onto each of the electrodes using a needle having an outer diameter of 0.7 mm, the nitrogen glow treatment and the untreated case are performed. In some cases, the enveloping electrodes 20, 22, and 24 wet and spread. Further, as described above, since the contact angle after the UV irradiation treatment in the nitrogen glow with no treatment is almost the same as the contact angle of the Ni electrode surface, the electrodes 14 and 16 may be more hydrophilic than the substrate exposed portion 19. As a result, a portion where the aqueous solution was difficult to spread out was generated.

According to the biosensor and the manufacturing method thereof of the present invention, it is possible to improve the measurement accuracy by reliably covering a certain position on the electrode with the reaction part, and the manufacturing cost does not increase. For this reason, it can be widely used for blood glucose level measurement and the like.

10: Biosensor 12: Substrate 13: Surface 14, 16: Electrode 18: Reaction part 20, 22, 24: Surrounding electrode 26: Outer peripheral side 28: Inner peripheral side 30: Metal needle 32: Aqueous solution D: Diameter W: Distance

Claims (6)

In a method for producing a biosensor, comprising: forming an electrode on a substrate made of an insulator; and forming a reaction part in contact with the electrode on the substrate.
A biosensor manufacturing method in which UV irradiation treatment is performed on the substrate before forming the reaction part. 2. The step of forming the electrodes is a step of forming a pair of electrodes spaced apart from each other and an enclosing electrode surrounding the pair of electrodes spaced apart from the pair of electrodes. A method for manufacturing the biosensor described. The biosensor manufacturing method according to claim 1, wherein the UV irradiation treatment is performed after the electrode is formed. Forming the electrodes includes performing sputtering and etching;
The biosensor manufacturing method according to claim 3, wherein a glow process is performed on the substrate before the sputtering. The biosensor manufacturing method according to claim 4, wherein the glow treatment is a glow treatment using argon or oxygen. Forming the electrodes includes performing sputtering and etching;
The biosensor manufacturing method according to claim 3, wherein a base sputtering of silicon or silicon dioxide is performed on the substrate before the sputtering.

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