JP2020156911A - Biosensor probe and method for producing the same - Google Patents

Abstract

To improve durability of a biosensor probe by improving an enzyme and a redox mediator from flowing to the outside.SOLUTION: A shape of a longitudinal section of an insulation substrate 111 to be a base constituting a sensing portion of a biosensor probe is a trapezoid with the top shorter than the base.SELECTED DRAWING: Figure 8

Description

The present disclosure relates to, for example, a probe constituting a biosensor for measuring the concentration of a specific substance contained in a subject of a liquid sample, and a method for producing the same. More specifically, the present disclosure improves the durability of biosensor probes by preventing leakage or alteration of enzymes and mediators contained in the detection layer formed on the biosensor probe inserted into the body. The manufacturing method is provided.

A biosensor is a system that measures substances by utilizing or imitating the molecular recognition ability of a living body. For example, one of a combination of enzyme-substrate, antigen-antibody, hormone-receptor, etc. is used as a sample (substance to be measured). ), And the other is a receptor, and the chemical change generated by the molecular recognition reaction between the sample and its receptor is converted into an electrical signal by a transducer, and the sample's strength depends on the strength of the obtained electrical signal. It is a measuring device that measures a quantity.

In addition to the above, biomolecules used in biosensors also include genes, sugar chains, lipids, peptides, cells, tissues and the like. Among them, biosensors using enzymes are being actively developed, and typical examples are glucose oxidase (GOx) and glucose dehydrogenase (GDH) used for self-glycemic measurement. It is an electrochemical glucose sensor using the above.

The electrochemical glucose sensor used for self-blood glucose measurement is generally configured such that a cover is arranged via a spacer on an insulating substrate having electrodes formed on its surface. A reagent containing a sample-responsive enzyme, a redox mediator (electron carrier), and the like is arranged on the electrode, and this portion serves as an analysis unit. One end of a flow path for introducing blood communicates with this analysis unit, and the other end of the flow path is open to the outside, and this is a blood supply port. The blood glucose level is measured using such a sensor, for example, as follows. That is, first, the sensor is set in a dedicated measuring device (meter). Then, the fingertip or the like is injured with a lancet to cause bleeding, and the blood supply port of the sensor is brought into contact with the bleeding. Blood is sucked into the flow path of the sensor by capillarity and introduced into the analyzer through which it comes into contact with the reagent. Then, the sample-responsive enzyme E (for example, GOx, GDH) oxidizes glucose by reacting specifically with glucose in blood. Redox Mediator M accepts electrons generated by oxidation. The redox mediator M, which has received and reduced electrons, is electrochemically oxidized at the electrode. The glucose concentration in blood, that is, the blood glucose level is easily detected from the magnitudes such as the current value and the amount of electric charge obtained by the oxidation of the redox mediator M.

Such an electrochemical blood glucose sensor plays an important role in blood glucose control in the treatment of diabetes, and a diabetic patient can perform appropriate insulin administration and dietary restriction based on the blood glucose level. However, the quality of life (QOL) should be maintained because the blood glucose level must be measured many times a day and blood sampling each time causes pain to the patient. Was difficult.

Implantable electrochemical glucose sensors have already been developed. The main body 10 of such an implantable electrochemical glucose sensor 1 is attached to the living body 2 and the probe 11 is inserted into the living body to continuously measure the blood glucose level (FIGS. 1 and 2). As a result, the blood glucose level can be measured for a long period of time without collecting blood each time.

The Ministry of Health, Labor and Welfare issued the "Notice of Remote Medical Care in 1997 (Notice of Director of Health Policy Bureau, Ministry of Health, Labor and Welfare No. 1075 issued on December 24, 1997)" with the basic concept of remote medical care and Article 20 of the Medical Practitioners Act. The matters to be noted from the relationship of. After that, based on the development and popularization of information and telecommunications equipment, on August 10, 2015, administrative communication was made to each prefectural governor regarding medical treatment using information and communication equipment (so-called "remote medical treatment"). With the 2015 notification, the ban on remote medical care was virtually lifted, and in 2016, remote medical care tools that wirelessly communicate with biosensor 1 using information communication device 3 (smartphone) and a dedicated application began to appear in the world (Fig.) 3). Furthermore, on July 14, 2017, a notification was issued (Medical Administration No. 0714 No. 4) to remind and clarify the handling of remote medical care. According to the 2015 notification, remote medical care will also be direct for remote medical care that combines information and communication devices such as videophones, e-mail, and social networking services, as long as it can be confirmed that the parties are the doctor and the patient. If useful information on the physical and mental condition of the patient can be obtained that can be replaced with face-to-face medical care, it does not immediately violate Article 20 of the Medical Practitioners Act. With this 2017 notification, remote medical care using information and communication equipment will be further developed. Therefore, the demand for embedded sensors is expected to increase more and more.

As a configuration of a conventional in-biosensor (embedded type) biosensor, a base substrate having a main surface, an electrode layer formed on the main surface of the base substrate and having an working electrode and a counter electrode, and a subject. It was provided with a reagent layer that reacts and a biocompatible membrane that covers the space in which the subject and the reagent react, including the reagent layer (Patent Document 1). Further, in an in-vitro type biosensor, at least a measurement electrode and a counter electrode are formed on the surface of an insulating substrate, and a reagent layer is formed on the measurement electrode so as to cover at least a part thereof, and the thickness of the reagent layer is formed. There is also a biosensor in which an insulating portion for adjusting the temperature is provided and a recess is formed in the insulating portion for forming a reagent layer (Patent Document 2).

U.S. Pat. No. 8326393 Japanese Unexamined Patent Publication No. 2000-22156

Since the conventional in vivo implantable sensor inserts the probe into the body for a long time, the chances of the component sample-responsive enzyme and redox mediator leaking out increase. If the sample-responsive enzyme or redox mediator leaks out of the sensor, not only the sensor sensitivity deteriorates, but also the living body is harmed. In addition, if the sample-responsive enzyme or redox mediator leaks to the outside, the durability of the sensor also decreases. Therefore, it is very important to take measures to prevent the outflow of sample-responsive enzymes and redox mediators.

In a conventional in-biosensor embedded biosensor, an electrode layer is formed on a base insulating substrate, a detection layer is formed on the electrode layer, and the reagent layer is covered with a biocompatible protective film. Although it prevents reagents such as sample-responsive enzymes and redox mediators from flowing out to the outside, the protective film becomes thinner than the detection layer formed on the insulating substrate, causing the reagents to become thinner. It may leak out of the membrane, resulting in reduced durability of biosensor measurements.

Therefore, in the present disclosure, in order to improve the measurement durability of the biosensor, the biosensor probe and the manufacturing method thereof do not have a portion where the film thickness of the protective film becomes thin with respect to the detection layer formed on the base substrate. The challenge is to provide.

According to the present disclosure, when the shape of the sensing portion of the probe is substantially trapezoidal in which the length of the upper base is shorter than the length of the lower base as compared with the case where the shape in the vertical cross section is substantially rectangular, the upper part on the detection layer side It was confirmed for the first time that the thickness of the protective film increased.
Therefore, in order to solve the above problems, in the present disclosure, in the biosensor, the vertical cross section of the insulating substrate which is the base constituting the sensing portion of the biosensor probe is formed into a trapezoidal shape in which the upper bottom is shorter than the lower bottom. , A detection layer is formed on the upper bottom thereof.

More specifically, the present disclosure is a probe for a biosensor, which comprises a sensing portion to be inserted into a living body and a terminal portion for electrically connecting to an internal circuit of the biosensor main body in a plan view. The sensing part is
An insulating substrate having a trapezoidal shape in which the upper base is shorter than the lower base in a vertical cross section with the front surface on the upper side and the back surface on the lower side.
Conductive thin films formed on the front and back surfaces of the insulating substrate,
A working electrode and a reference electrode formed on the conductive thin film on the front side of the insulating substrate,
A counter electrode formed on a conductive thin film on the back side of the insulating substrate,
A detection layer formed on a conductive thin film formed on the front surface of the insulating substrate, and a biocompatible protective film covering the working electrode, the reference electrode, the counter electrode, and the detection layer. Provide a probe.

In the embodiment of the present disclosure, the probe for a biosensor uses an insulating substrate having a trapezoidal shape in which the upper bottom is shorter than the lower bottom in a vertical cross section in which the front side surface is the upper side and the back side surface is the lower side.

In the longitudinal section of the sensing portion of the probe formed from the insulating substrate, the length of the upper base is 75 to 90%, preferably 80 to 85% of the length of the lower base, and the height is the lower base. It has a substantially isomorphic pedestal shape, which is 50 to 60% of the length, preferably 53 to 57%. The angle formed by the tangent of the leg and the upper sole at the angle formed by the upper sole and the leg is 105 to 130 °, preferably 110 to 125 °. For example, in an isosceles trapezoid having a lower base length of 360 μm and a height of 200 μm, the upper base length is 270 to 324 μm, preferably 288 to 306 μm. The length of the lower base is not limited depending on the design of the probe, but can be appropriately determined in the range of, for example, 200 to 400 μm.

The method for manufacturing a probe for a biosensor according to the present disclosure is described.
The insulating substrate that is the base of the probe is cut, and the probe is composed of a sensing portion to be inserted into the living body and a terminal portion for electrically connecting to the internal circuit of the biosensor main body in a plan view, and is described above. A process in which the shape of the sensing portion in a vertical cross section with the front surface on the upper side and the back surface on the lower side is a trapezoid in which the upper base is shorter than the lower base.
A step of forming a conductive thin film on the front surface and the back surface of the insulating substrate,
Conductive thin film formed on the front surface of the insulating substrate A step of forming a detection layer on a conductive thin film;
The step of coating the working electrode, the reference electrode, the counter electrode and the detection layer with a biocompatible protective film is included.

In the step of cutting the insulating substrate to form a probe, any method such as laser cutting or die cutting using a pinnacle die can be used as long as the insulating substrate can be cut.

In the step of coating with the biocompatible protective film, the sensing portion is immersed one or more times in the solution containing the resin for forming the biocompatible protective film contained in the container with the axis of the sensing portion of the probe facing downward. .. When the immersion is completed an appropriate number of times, the probe is dried with the back surface facing down.

According to the present disclosure, when the shape of the sensing portion of the probe in the vertical cross section is substantially trapezoidal in which the length of the upper base is shorter than the length of the lower base, the upper part on the detection layer side is protected as compared with the case where the shape is substantially rectangular. The thickness of the membrane is increased by 10-20%.

According to the present disclosure, the sensing portion of the probe formed from the insulating substrate has a trapezoidal shape in which the upper bottom is shorter than the lower bottom in a vertical cross section with the front surface on the upper side and the back surface on the lower side. Therefore, it is possible to form a sufficiently thick protective film on the detection layer, and as a result, it is possible to prevent the reagent from leaking to the outside. This made it possible to improve the measurement durability of the biosensor.

The schematic diagram which shows the state which the embedded biosensor is attached to the living body (human body). A cross-sectional view showing an implantable biosensor attached to a living body (human body). Schematic diagram of an embedded biosensor that wirelessly communicates measurement data with a smartphone. The manufacturing process of the probe of the implantable biosensor of one specific example of the present disclosure. The manufacturing process of the probe of the implantable biosensor of one specific example of the present disclosure. The manufacturing process of the probe of the implantable biosensor of one specific example of the present disclosure. The schematic diagram which shows the formation procedure of the protective film in the manufacturing process of the probe of the implantable biosensor of one specific example of this disclosure. The schematic diagram which shows the comparison of the cross-sectional shape of the protective film by the difference in the shape in the vertical cross section of the sensing part of a probe. Top view of the probe front side of the implantable biosensor of one specific example of the present disclosure. FIG. 9 is a cross-sectional view taken along the line AA'in FIG. FIG. 10 is a cross-sectional view taken along the line B-B'in FIG. FIG. 10 is a cross-sectional view taken along the line C-C'in FIG. A microscopic image of a cross section of a sensing portion of a probe A including a sensing portion having a substantially trapezoidal cross-sectional shape and a sensing portion including a sensing portion having a substantially rectangular cross-sectional shape. The graph which shows the glucose response characteristic of the probe A including the sensing portion which has a substantially trapezoidal cross-sectional shape, and the probe B including the sensing portion which has a substantially rectangular cross-sectional shape.

1. 1. Method for Manufacturing Probe for Implantable Biosensor The method for manufacturing probe 11 for implantable biosensor 1 according to one embodiment of the present disclosure will be described. The structure and manufacturing method shown below are one specific example of the present disclosure, and are limited to the following configurations as long as a sensing portion having a substantially isosceles trapezoidal cross-sectional shape, which is a feature of the present disclosure, is formed. is not.

(1) Preparation of Insulating Substrate The embedded biosensor 1 includes the main body 10 and the probe 11, and the probe 11 is generally used for electrically connecting the sensing portion to be inserted into the living body and the internal circuit of the biosensor main body 10. It is a key shape consisting of terminal parts. The sensing portion is formed thin so that it can be inserted into the body, and the terminal portion has a certain size so as to be inserted into the biosensor main body 10 to form an electrical connection. Therefore, first, the key-shaped insulating substrate 111 is prepared (FIG. 4a). The upper row shows the top view seen from the front side, and the lower row shows the top view seen from the back side (hereinafter, the same applies). The insulating substrate 111 is not particularly limited as long as it has a material and a thickness that can be used as a probe to be inserted into a living body, and for example, polyethylene terephthalate (PET) having a thickness of about 200 μm can be used.
The key-shaped insulating substrate 111 is prepared by cutting the material, but the cutting method is not particularly limited, and the key-shaped insulating substrate 111 can be cut by a method known in the field such as laser cutting and die cutting using a pinnacle die. ..

(2) Formation of Conductive Thin Film Conductive metal materials selected from the group consisting of carbon, gold, silver, platinum, palladium, etc. are sputtered, vapor-deposited, ion-plated, etc. on both sides of the insulating substrate 111. To form a conductive thin film 112 by depositing with (Fig. 4b). The preferred thickness of the conductive thin film is 10 nm to several hundred nm.

(3) Formation of Electrode Leads A groove 113 having a depth reaching the surface of the insulating substrate 111 is formed by drawing a laser on the conductive thin film 112 formed on the front side of the insulating substrate 111, and is referred to as an working electrode lead 112a. Separated into electrode leads 112b for electrical insulation (Fig. 5c).

(4) Formation of Insulating Resist Film The area used as the working electrode terminal 114a and the reference electrode terminal 115a for electrical connection with the working electrode 114 and the reference electrode 115 and the main body 10 is excluded from the front side of the insulating substrate 111. An insulating resist film 116a having an opening is formed in the portion by a sputtering method, a screen printing method, or the like, and an area used as a counter electrode terminal 117a for electrical connection with the counter electrode 117 and the main body 10 on the back side of the insulating substrate 111. An insulating resist film 116b having an opening is formed in the portion excluding the above by a sputtering method, a screen printing method, or the like (FIG. 5d). The preferred thickness of the insulating resist film is 5-40 μm.

(5) Formation of Reference Electrode Ag / AgCl is deposited in the opening for the reference electrode of the resist film 116a formed on the front side of the insulating substrate 111 by a screen printing method or an inkjet method to form the reference electrode 115 (FIG. 5e). ). The preferred thickness of the reference electrode is 5-40 μm.

(6) Formation of detection layer A mixed aqueous solution of conductive particles such as carbon particles, sample-responsive enzyme and redox mediator is applied and dried on the working electrode 114 to obtain the conductive particles, sample-responsive enzyme and redox mediator. A detection layer 118 including the detection layer 118 is formed (FIG. 6f). In order to improve the conductivity of the detection layer, conductive particles such as carbon particles can be appropriately used. In the disclosure, "specimen-responsive enzyme" means a biochemical substance capable of specifically catalyzing the oxidation or reduction of a sample. Any biochemical substance may be used as long as it can be used for the purpose of detecting a biosensor. For example, when glucose is used as a sample, suitable sample-responsive enzymes include glucose oxidase (GOx) and glucose dehydrogenase (GDH). The “redox mediator” means a redox substance that mediates electron transfer, and is responsible for the transfer of electrons generated by the redox reaction of a sample (analyte) in a biosensor. For example, a phenazine derivative and the like are included, but the present invention is not limited to this, and any redox substance may be used as long as it can be used for the detection purpose of a biosensor. The preferred thickness of the detection layer is 5-80 μm.

In the present disclosure, the detection layer may be a multilayer film containing at least a redox mediator and a sample-responsive enzyme, and composed of a mediator layer containing the redox mediator and an enzyme layer containing the sample-responsive enzyme.

(7) Formation of Protective Film The sensing portion is immersed in a solution containing a biocompatible resin for protecting the sensor to form a protective film 119 on both sides, side surfaces and the tip of the sensing portion (FIG. 6 g). The protective film 119 covers at least the working electrode 114, the reference electrode 115, the counter electrode 117 and the detection layer 118 without covering the working electrode terminal 114a, the reference electrode terminal 115a and the counter electrode terminal 117a, and in vivo. Form longer than the length to be inserted. The preferred thickness of the protective film is 5 to 200 μm.

Poly (4-vinylpyridine) can be used as the biocompatible resin for sensor protection, and poly (4-vinylpyridine) is crosslinked with a cross-linking agent such as polyethylene glycol diglycidyl ether (PEGDGE). For example, poly (tert-butyl methacrylate) -b-poly (4-vinylpyridine), polystyrene-co-4-vinylpyridine-co-oligo [propylene glycol methyl ether] methacrylate and the like can be mentioned.

The procedure for forming the protective film will be described in detail with reference to FIG. 7. The key-type probe is immersed in a resin solution for forming a biocompatible protective film contained in a container with the axis of the sensing portion facing downward. The immersion is repeated one or more times, depending on the manufacturing conditions of the probe. After a suitable number of dips, the probe is allowed to stand with the back surface (ie, the trapezoidal bottom in the longitudinal section) facing down. In this state, it is dried at room temperature (18 to 25 ° C.) for 3 to 60 hours.

Poly (4-vinylpyridine) may be crosslinked with a cross-linking agent such as polyethylene glycol diglycidyl ether (PEGDGE).

The protective film may further contain, for example, poly (2-methoxyethyl acrylate) as an additive. This improves the biocompatibility of the protective film.

When the shape of the sensing portion of the probe in the vertical cross section is substantially trapezoidal in which the length of the upper base is shorter than the length of the lower base according to the present disclosure (FIG. 8a), and when the shape in the vertical cross section is made substantially rectangular according to a conventional method. Compared with FIG. 8b, the thickness of the protective film on the upper part on the detection layer side is increased.
The resin solution attached to the probe tends to become spherical due to surface tension, but the shape of the protective film is determined depending on gravity and the shape of the probe inside. When the vertical cross-sectional shape is substantially rectangular, the angle formed by the upper side and the vertical side (the side when the sensing part is viewed three-dimensionally) obstructs the flow of the resin solution, so that it does not easily become spherical and the sensing part. The amount of resin solution present on the front surface of the surface is reduced. On the other hand, if the length of the upper base is shorter than the length of the lower base according to the present disclosure, the resin solution existing above the upper base and the resin solution existing on the sides of both legs are integrated and tend to be spherical. As a result, it is assumed that the amount of the resin solution existing on the front surface of the sensing portion increases.

2. 2. Internal Structure of Probe for Implantable Biosensor The internal structure of probe 11 for implantable biosensor 1 according to one embodiment of the present disclosure will be further described.

FIG. 9 shows a top view of the probe 11 in a state where a protective film is formed on the sensing portion as seen from the front side. A cross-sectional view taken along the AA'cutting line of FIG. 9 is shown in FIG. Conductive thin films 112 are formed on both sides of the insulating substrate 111. In the conductive thin film 112 on the front side, the groove 113 separates the working electrode lead 112a and the reference electrode lead 112b into two leads, which are electrically insulated. A part of the working electrode lead 112a functions as the working electrode 114, and the detection layer 118 is formed on the working electrode 114. Although the detection layer 118 is shown as a single layer, the mediator layer 118a can be formed on the working electrode and the redox mediator layer 118b can be formed on the mediator layer 118a to form a two-layer structure, depending on the embodiment. , Other configurations are also acceptable. Further, the reference electrode 115 is formed in the opening portion of the insulating resist film 116a and is electrically connected to the reference electrode lead 112b. The conductive thin film 112 on the back side serves as a counter electrode lead 112c, and a part of the conductive thin film 112 functions as a counter electrode 117.

A cross-sectional view taken along the BB'cut line of FIG. 10 is shown in FIG. A working electrode 115 is formed on the front side of the insulating substrate 111, a detection layer 118 is formed on the working electrode 115, and a counter electrode 117 is formed on the back side. Furthermore, it can be seen that the entire periphery of the sensing portion is covered with the protective film 119 of the present disclosure.
In FIG. 11, for the purpose of explaining the basic structure inside the probe, the cross-sectional shape of the insulating substrate is simply depicted as a rectangle.

A cross-sectional view of the C-C'cut surface of FIG. 10 is shown in FIG. On the front side of the insulating substrate 111, a working electrode lead 112a and a reference electrode lead 112b electrically separated by a groove 113 are formed, and an insulating resist film 116a is formed on the working electrode lead 112a. A reference electrode 115 is formed in the opening of the insulating resist film 116a. A counter electrode lead 112c is formed on the back side of the substrate 111, and an insulating resist film 116b is formed on the counter electrode lead 112c. Furthermore, it can be seen that the entire periphery of the sensing portion is covered with the protective film 119 of the present disclosure.
In FIG. 12, for the purpose of explaining the basic structure inside the probe, the cross-sectional shape of the insulating substrate is simply depicted as a rectangle.

[Example 1]
<Making a probe>
(1) Preparation of Insulating Substrate As shown in Fig. 4a, a polyethylene terephthalate (PET) sheet (Lumirror R E20 # 188; 189 μm thickness manufactured by Toray Co., Ltd.) was cut to prepare a key-shaped insulating substrate. .. In this example, the PET sheet was cut by a die cut using a pinnacle die (hereinafter, referred to as "pinnacle cut"). The shape of the vertical cross section of the insulating substrate was substantially trapezoidal (shown later).

(2) Formation of Conductive Thin Film As shown in FIG. 4b, a conductive thin film (thickness 30 nm) was formed by depositing gold on both surfaces of the insulating substrate by a sputtering method.

(3) Formation of Electrode Lead As shown in FIG. 5c, a groove having a depth reaching the surface of the insulating substrate is formed by laser drawing on the conductive thin film formed on the front side of the insulating substrate, and the working electrode lead is formed. And the reference electrode lead was separated and electrically separated.

(5) Formation of Insulating Resist Film As shown in FIG. 5d, the working electrode and the reference electrode, and the working electrode terminal and the reference electrode for electrical connection with the main body of the embedded biosensor are provided on the front side of the insulating substrate. An insulating resist film having an opening is formed by a screen printing method in a portion excluding a region used as a terminal, and a portion excluding a counter electrode and a region used as a counter electrode terminal for electrical connection with the main body on the back side of the insulating substrate. An insulating resist film (thickness 10-15 μm) having an opening was formed by a screen printing method.

(6) Formation of Reference Electrode As shown in FIG. 5e, Ag / AgCl was deposited by a screen printing method in the opening for the reference electrode of the resist film formed on the front side of the insulating substrate, and the reference electrode (thickness 10-) was deposited. 15 μm) was formed.

In the figure, various configurations are shown for the substrate cut into a key shape so that the positional relationship of each configuration on the substrate is clarified. However, in the actual probe fabrication, the steps up to this point are shown. After that, the processed substrate was cut into a key shape.

(7) Formation of Detection Layer As shown in FIG. 6f, a conductive thin film exposed from the opening of the insulating resist film formed on the front side of the insulating substrate is used as an acting electrode, and a phenazine derivative is used as a redox mediator on the conductive thin film. Approximately 0.3 μl of the aqueous solution containing it is applied and dried to form a mediator layer, and an aqueous solution containing glucose oxidase (GOD) and sodium polyacrylate as a sample-responsive enzyme is applied onto the mediator layer. By drying to form an enzyme layer, a detection layer (thickness 15 μm) having a two-layer structure was formed.

(8) Formation of Protective Film As shown in FIG. 6g, the sensing portion is immersed in an ethanol solution containing a cross-linking agent and a polymer for a protective film, and protective films (thickness 5-) are formed on both sides, side surfaces and end faces of the sensing portion. 40 μm) was formed.
More specifically, 600 mg of poly (4-vinylpyridine) and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn = 1000) as a cross-linking agent are used as a solvent (95% ethanol, 4- (2-hydroxyethyl)-. The probe prepared above was immersed in a solution of 1-piperazine ethanesulfonic acid (HEPES) buffer (10 mM, pH 8) 5%) 6 times at 10-minute intervals. Then, it was dried at room temperature for 48 hours to form a crosslinked protective film to obtain probe A.

FIG. 13 is a microscope image obtained by vertically cutting the sensing portion (a portion of about 1 mm from the tip) of the probe A and observing the cut surface corresponding to the cross-sectional view taken along the B-B'cutting line shown in FIG. 10 with an optical microscope. Shown in (a).
The insulating substrate had a substantially isosceles trapezoidal shape with an upper base of about 300 μm, a lower base of about 360 μm, and a height of about 200 μm. The thickness of the protective film formed below the lower bottom is about 80 μm, the thickness of the protective film formed above the upper bottom is about 115 μm, and the thickness of the protective film formed on the sides of the left and right legs. The size was 60 to 70 μm. Further, the angle formed by the tangent line of the leg and the upper sole at the angle formed by the upper sole and the leg was within the range of about 115 to 120 °.
The measurement procedure of the dimensions and the angle is shown in FIG. 13 (c) by taking the cut surface of the probe A as an example.

<Measurement of probe characteristics>
[durability]
Probe A was stored in phosphate buffered saline (PBS, pH 7) at 37 ° C. Before storage (day 0) and on days 3, 7 and 15 after storage, probe A was attached to an implantable amperometric glucose sensor and placed in phosphate buffered saline (PBS, pH 7) at 37 ° C. A probe was installed. Glucose was added to this PBS solution at 700 mg / dL, and the current response value (nA) was measured. The response rate (%) at each concentration was calculated with the current response value on day 0 as 100%.
The response characteristics on the 15th day after storage remained 20% or more of that before storage, and the durability was good. The results are summarized in Table 1 and shown in FIG.

[Comparative Example 1]
<Making a probe>
In Example 1, probe B was obtained under the same conditions as in Example 1 except that the PET sheet was cut by laser cutting to make the shape of the vertical cross section of the insulating substrate substantially rectangular (shown later). It was.

FIG. 13 is a microscope image obtained by vertically cutting the sensing portion (a portion of about 1 mm from the tip) of the probe A and observing the cut surface corresponding to the cross-sectional view taken along the B-B'cutting line shown in FIG. 10 with an optical microscope. Shown in (b).
The insulating substrate had a substantially rectangular shape with an upper side of about 360 μm, a lower side of about 360 μm, and a height of about 200 μm. Further, the thickness of the protective film formed below the lower side is about 80 μm, the thickness of the protective film formed above the upper side is about 100 μm, and the thickness of the protective film formed on the left and right sides is about 100 μm. , 60-70 μm. Further, the angle formed by the tangent line of the leg and the upper sole at the angle formed by the upper sole and the leg was about 95 °.
The measurement procedure of the dimensions and the angle is shown in FIG. 13 (c) by taking the cut surface of the probe A as an example.

<Measurement of probe characteristics>
[durability]
The durability of probe B was measured in the same manner as in Example 1.
The response characteristics decreased to about 50% of that before storage on the 3rd day after storage, and fell below 10% on the 15th day, and the durability was poor. The results are summarized in Table 2 and shown in FIG.

In Example 1, since the cross-sectional shape of the sensing portion of the probe is a substantially isosceles trapezoid in which the length of the upper base is shorter than the length of the lower base, the cross-sectional shape of the sensing portion obtained by the conventional method in Comparative Example 1 is substantially the same. The thickness of the protective film above the detection layer was increased compared to the rectangular probe, resulting in improved durability.

By a simple method, the cross-sectional shape of the sensing part of the probe can be made into a substantially isosceles trapezoid whose upper base length is shorter than the lower base length, so that an embedded biosensor with improved durability can be manufactured with high efficiency. It became possible to do.

1 Implantable biosensor
10 body
11 probe
111 Insulating board
112 Conductive thin film
112a working electrode lead
112b Reference electrode lead
112c opposite pole lead
113 groove
114 Working electrode
115 Reference electrode
116 Insulating resist
117 opposite pole
118 Detection layer
119 Protective film
2 Living body
3 Information and communication equipment

Claims (7)

A probe for a biosensor, which is composed of a sensing portion to be inserted into a living body and a terminal portion for electrically connecting to an internal circuit of the biosensor main body in a plan view.
An insulating substrate having a trapezoidal shape in which the upper base is shorter than the lower base in a vertical cross section with the front surface on the upper side and the back surface on the lower side.
Conductive thin films formed on the front and back surfaces of the insulating substrate,
A working electrode and a reference electrode formed on the conductive thin film on the front side of the insulating substrate,
A counter electrode formed on a conductive thin film on the back side of the insulating substrate,
A detection layer formed on a conductive thin film formed on the front surface of the insulating substrate, and a biocompatible protective film covering the working electrode, the reference electrode, the counter electrode, and the detection layer. probe. The probe according to claim 1, wherein the trapezoid is a substantially isosceles trapezoid, and the angle formed by the tangent line of the leg and the upper base at the angle formed by the upper base and the leg is 105 to 130 °. The probe according to claim 1 or 2, wherein the detection layer comprises at least a redox mediator and a sample-responsive enzyme. The probe according to any one of claims 1 to 3, wherein the detection layer is a multilayer film composed of a mediator layer containing a redox mediator and an enzyme layer containing a sample-responsive enzyme. A method for manufacturing a probe for a biosensor.
The insulating substrate that is the base of the probe is cut, and the probe is composed of a sensing portion to be inserted into the living body and a terminal portion for electrically connecting to the internal circuit of the biosensor main body in a plan view, and is described above. A process in which the shape of the sensing portion in a vertical cross section with the front surface on the upper side and the back surface on the lower side is a trapezoid in which the upper base is shorter than the lower base.
A step of forming a conductive thin film on the front surface and the back surface of the insulating substrate,
Conductive thin film formed on the front surface of the insulating substrate A step of forming a detection layer on a conductive thin film;
A method for producing a probe, which comprises a step of coating the working electrode, the reference electrode, the counter electrode and the detection layer with a biocompatible protective film. The method according to claim 5, wherein the trapezoid is a substantially isosceles trapezoid, and the angle formed by the tangent line of the leg and the upper base at the angle formed by the upper base and the leg is 105 to 130 °. The method according to claim 5 or 6, wherein the step of cutting the insulating substrate is formed by a pinnacle cutting method using a pinnacle die.