JP2019170701A - Protection film for preventing outward flow of specimen responsive enzyme and biosensor probe having same formed thereon - Google Patents

Abstract

To provide a protection film which does not inhibit intrusion into a specimen and prevents specimen responsive enzyme from flowing out to the outside in order to be applied to a probe of an embedded biosensor.SOLUTION: As a film structure useful for a probe of a biosensor, the film structure is used which includes an enzyme layer including specimen responsive enzyme, and a protection film formed on the enzyme layer. The protection film includes polyurethane.SELECTED DRAWING: Figure 1

Description

  The present disclosure relates to a film for protecting a probe constituting a biosensor and a biosensor probe in which such a protective film is formed. More specifically, a biosensor probe in which a protective film capable of preventing the outflow of an enzyme or the like constituting the biosensor probe inserted into the body is provided.

  A biosensor is an electrochemical sensor that uses the molecular recognition ability of biomolecules. For example, one of a combination of enzyme-substrate, antigen-antibody, hormone-receptor, etc. is used as a specimen (measurement target substance), and the other A chemical change generated by a molecular recognition reaction between a specimen and its receptor is immobilized as a receptor on the sensing site, and converted into an electrical signal by a transducer, and the specimen is analyzed according to the strength of the obtained electrical signal. It is a measuring device which measures the quantity of.

  In addition to the above, biomolecules used in biosensors include genes, sugar chains, lipids, peptides, cells, tissues, and the like. Among them, biosensors using enzymes have been most developed, and a typical example is a glucose sensor using glucose oxidase (GOx).

The glucose sensor generally has a probe including at least a working electrode and an enzyme layer containing an analyte-responsive enzyme formed thereon. When the sample (glucose) contained in the subcutaneous interstitial fluid to be examined reaches the enzyme layer, an enzyme reaction occurs due to the action of the coenzyme in the enzyme. By this enzymatic reaction, glucose is oxidized to gluconolactone, and at the same time, oxygen is reduced to produce hydrogen peroxide (H ₂ O ₂ ). In order to detect the glucose concentration contained in the test object, the oxygen consumption is measured with an oxygen electrode, or the amount of H ₂ O ₂ produced is measured with a hydrogen peroxide electrode. In the measurement method using a hydrogen peroxide electrode, H ₂ O ₂ generated by GOx in the enzyme layer is expressed on the working electrode by the following formula:

に従って酸化されて、電子が発生する。この発生した電子を電流値として測定する（図１）。

Are oxidized to generate electrons. The generated electrons are measured as a current value (FIG. 1).

  Such a glucose sensor is used for autologous blood glucose measurement by diabetic patients. Control of blood glucose level is important for diabetes treatment, and appropriate insulin administration and dietary restriction based on blood glucose level are necessary. For this reason, blood sugar levels must be measured several times a day, and taking blood each time can be painful for the patient, thus maintaining the quality of life (QOL). It was difficult.

  Implantable amperometric glucose sensors have already been developed. The body 10 of such an implantable amperometric 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. 2 and 3). Thereby, a blood glucose level can be measured over a long time without collecting blood each time.

  The Ministry of Health, Labor and Welfare issued the “Notice of Telemedicine in 1997 (Notice of Health Policy Department No. 1075 from Kensei, December 24, 1997)” The matters to be noted from the relationship are shown. After that, based on the status of development and popularization of information and communication equipment, on August 10, 2015, we contacted the prefectural governors regarding medical care using information communication equipment (so-called “remote medical care”). In 2015, telemedicine was virtually lifted, and in 2016, telemedicine tools for wireless data communication with biosensor 1 using information communication equipment 3 (smartphones) and dedicated applications began to appear in the world (Fig. 4). Furthermore, as of July 14, 2017, a notice (Medical Administration 0714 No. 4) was issued with the aim of re-disseminating and clarifying the handling of telemedicine. According to the 2015 notification, telemedicine can also be used for telemedicine combining videophones, e-mails, social networking services and other information communication devices as long as the parties can be confirmed to be doctors and patients. If useful information about the patient's mental and physical condition that can be substituted for face-to-face medical care is available, it would not immediately violate Article 20 etc. of the Medical Doctor Law. This 2017 notification will further develop telemedicine using information and communication equipment. Therefore, the demand for embedded sensors is expected to increase.

  Patent Document 1 discloses an electrochemical sensor control device that is attached to a patient and inserted into the skin using a wireless transmitter, and transmits data related to the collected specimen amount to a display device using the wireless transmitter. The technology is described. Patent Document 1 also discloses a film containing a heterocyclic nitrogen group such as vinylpyridine attached to such an electrochemical sensor. These membranes limit analyte diffusion to the working electrode in the electrochemical sensor. In a glucose sensor without a membrane, the flow rate of glucose to the enzyme layer increases linearly with the glucose concentration, and while all the glucose that reaches the enzyme layer is consumed, the measured output signal is the glucose flow rate. However, if glucose consumption is limited in the enzyme layer, the measurement signal is not linearly proportional to glucose flow or concentration and saturation occurs. Therefore, in Patent Document 1, a technique for preventing the saturation of the sensor by forming a diffusion limiting film containing a heterocyclic nitrogen group such as polyvinyl pyridine on the enzyme layer and reducing the flow rate of glucose to the enzyme layer. Is adopted.

  Patent document 2 discloses a diffusion barrier comprising a single block copolymer having at least one hydrophilic block and at least one hydrophobic block, which, like patent document 1, is an enzyme from outside the electrode system. It controls the diffusion of analytes into the molecule. Such enzyme molecules are immobilized on the electrode to form an enzyme layer. For example, as disclosed in Patent Document 3, the enzyme layer can be more firmly fixed by immobilizing the enzyme on the working electrode by adsorption or capture and crosslinking with glutaraldehyde or the like. .

Special table 2010-517054 specification Special Table No. 2015-515305 Specification International Publication No. 2007/147475

  Since the implantable sensor inserts the probe into the body for a long time, the chance that the component-responsive enzyme that is a component flows out increases. If the analyte-responsive enzyme flows out of the sensor, not only the sensor sensitivity is deteriorated, but also harms the living body. In addition, if the analyte-responsive enzyme flows out, the durability of the sensor also decreases. Therefore, it is very important to take measures to prevent the sample-responsive enzyme from flowing out.

  In order to prevent outflow of the analyte-responsive enzyme constituting the probe of the implantable biosensor, it is desired to form a film on the enzyme layer containing the analyte-responsive enzyme. Such a membrane should not inhibit the entry of an analyte such as glucose into the interior. Therefore, in the present disclosure, in order to be applied to the probe of the implantable biosensor, a protective film that does not inhibit the entry of the specimen into the interior and prevents the specimen-responsive enzyme from flowing out to the outside, and such protection. It is an object to provide a biosensor probe in which a film is formed.

  The present disclosure includes, as a membrane structure useful for a biosensor probe, an enzyme layer containing at least an analyte-responsive enzyme and a protective film formed on the enzyme layer, wherein the protective film has the general formula (1):

で表されるポリウレタンを含む、膜構造体を提供する。

A membrane structure comprising a polyurethane represented by the formula:

  The polyurethane used in the present invention has the general formula (2):

で表されるジオールと、一般式（３）：

A diol represented by the general formula (3):

で表されるジイソシアネートとの反応により得ることができる。

It can obtain by reaction with the diisocyanate represented by these.

  In this invention, the sample is glucose, and the sample-responsive enzyme is glucose oxidase. Glucose oxidase is immobilized by forming a bovine serum albumin (BSA) cross-linked film or a polyvinyl alcohol cross-linked film. For example, glucose oxidase can be immobilized using BSA and glutaraldehyde.

  If a protective film is formed using the polyurethane of the present disclosure on the enzyme layer containing the enzyme that constitutes the probe of the implantable biosensor, the enzyme such as glucose is not inhibited from entering the inside, and the enzyme The analyte-responsive enzyme contained in the layer can be prevented from flowing out.

Schematic explaining the measurement principle of the specimen (glucose) by enzyme reaction. Schematic which shows the state by which the implantable biosensor was attached to the biological body (human body). Sectional drawing which shows the implantable biosensor of the state attached to the biological body (human body). Schematic of an implantable biosensor that wirelessly communicates measurement data with a smartphone. A manufacturing process of an implantable biosensor probe according to one specific example of the present disclosure. A manufacturing process of an implantable biosensor probe according to one specific example of the present disclosure. A manufacturing process of an implantable biosensor probe according to one specific example of the present disclosure. The top view of the probe front side of the implantable biosensor of one specific example of the present disclosure. Sectional drawing in the A-A 'cutting line in FIG. Sectional drawing in the B-B 'cutting line in FIG. Sectional drawing in the C-C 'cutting line in FIG. The graph which shows the glucose response characteristic of the probe in which the protective film of this indication was formed. The graph which shows durability of the probe in which the protective film of this indication was formed.

1. Method for Producing Implantable Biosensor Probe A method for producing the probe 11 of the implantable biosensor 1 of a specific example to which the membrane structure of the present disclosure is applied will be described. The structure and manufacturing method described below are one specific example of the present disclosure, and are not limited to the configurations described below as long as they can be used as probes.

(1) Preparation of Insulating Substrate The implantable biosensor 1 includes a main body 10 and a probe 11. The probe 11 is generally used for electrical connection with a sensing portion to be inserted into a living body and an internal circuit of the biosensor main body 10. It is a key shape consisting of a terminal part. The sensing portion is thinly formed so as to be inserted into the body, and the terminal portion has a certain size so as to be inserted into the biosensor body 10 to form an electrical connection. Therefore, first, a key-shaped insulating substrate 111 is prepared (FIG. 5a). The top view shows a top view seen from the front side, and the bottom shows a top view seen from the back side (hereinafter the same). The insulating substrate 111 is not particularly limited as long as it is a material and thickness that can be used as a probe inserted into a living body, and for example, polyethylene terephthalate (PET) having a thickness of about 200 μm can be used.
(2) Formation of a conductive thin film A conductive metal material selected from the group consisting of metals such as carbon, gold, silver, platinum or palladium is formed on both surfaces of the insulating substrate 111 by sputtering, vapor deposition, ion plating, etc. A conductive thin film 112 is formed by depositing (FIG. 5b). The preferred thickness of the conductive thin film is 10 nm to several hundred nm.
(3) Formation of Electrode Lead 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 the working electrode lead 112a is referred to. Separated into electrode leads 112b and electrically insulated (FIG. 6c).
(4) Formation of Insulating Resist Film On the front side of the insulating substrate 111, the working electrode 114, the reference electrode 115, and the region used as the working electrode terminal 114a and the reference electrode terminal 115a for electrical connection with the main body 10 are excluded. An insulating resist film 116a having an opening is formed on the portion by sputtering, screen printing, or the like, and a region used as a counter electrode 117a for electrically connecting 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 by a sputtering method, a screen printing method, or the like (FIG. 6d). A preferable thickness of the insulating resist film is 5 to 40 μm.
(5) Formation of reference electrode Ag / AgCl is deposited by the screen printing method or the inkjet method in the opening for reference electrodes of the resist film 116a formed on the front side of the insulating substrate 111 to form the reference electrode 115 (FIG. 6e). ). The preferred thickness of the reference electrode is 5-40 μm.
(6) Formation of enzyme layer An aqueous solution containing a specimen-responsive enzyme is applied and dried on the working electrode 114 to form an enzyme layer 118 containing a specimen-responsive enzyme (FIG. 7f). In the present disclosure, “analyte responsive enzyme” means a biochemical substance that can specifically catalyze the oxidation or reduction of an analyte. Any biochemical substance may be used as long as it can be used for the detection purpose of the biosensor. For example, when glucose is used as a sample, suitable sample-responsive enzymes include glucose oxidase (GOx) and glucose dehydrogenase (GDH). The preferred thickness of the enzyme layer is 5 to 80 μm.
(7) Formation of Protective Film The sensing portion is immersed in a solution containing the protective film polymer to form the protective film 119 on both sides, side surfaces, and the tip of the sensing portion (FIG. 7g). The protective film 119 covers at least the working electrode 114, the reference electrode 115, the counter electrode 117, and the enzyme layer 118 without covering the working electrode terminal 114a, the reference electrode terminal 115a, and the counter electrode terminal 117a. It is formed longer than the length to be inserted. The preferred thickness of the protective film is 5 to 200 μm.

2. Internal structure of probe of implantable biosensor The internal structure of the probe of the implantable biosensor to which the membrane structure of the present disclosure is applied will be further described.

  FIG. 9 shows a cross-sectional view taken along the line A-A ′ of FIG. Conductive thin films 112 are formed on both sides of the insulating substrate 111. The conductive thin film 112 on the front side is separated into two leads, a working electrode lead 112a and a reference electrode lead 112b, by a groove 113, and is electrically insulated. A part of the working electrode lead 112a functions as the working electrode 114, and the enzyme layer 118 is formed on the working electrode 114. A reference electrode 115 is formed in the opening 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 thereof functions as the counter electrode 117.

  FIG. 10 is a cross-sectional view taken along the line B-B ′ of FIG. A working electrode 114 is formed on the front side of the insulating substrate 111, an enzyme layer 118 is formed thereon, 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.

  A cross-sectional view taken along the line C-C 'of FIG. 9 is shown in FIG. A working electrode lead 112a and a reference electrode lead 112b electrically separated by the groove 113 are formed on the front side of the insulating substrate 111, and an insulating resist film 116a is formed thereon. 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 thereon. Furthermore, it can be seen that the entire periphery of the sensing portion is covered with the protective film 119 of the present disclosure.

[Example 1]
<Preparation of probe>
(1) Preparation of Insulating Substrate As shown in FIG. 6a, polyethylene terephthalate (PET) (Lumirror R E20 # 188; 189 μm thickness, manufactured by Toray Industries, Inc.) was cut to prepare a key-shaped insulating substrate.

(2) Formation of conductive thin film As shown in FIG. 6b, a conductive thin film (thickness 30 nm) was formed on both surfaces of the insulating substrate by depositing palladium by sputtering.

(3) Formation of electrode lead As shown in FIG. 6c, a groove having a depth reaching the surface of the insulating substrate is formed by drawing a laser on a conductive thin film formed on the front side of the insulating substrate, thereby forming a working electrode lead. And separated into a reference electrode lead and electrically separated.

(4) Formation of Insulating Resist Film As shown in FIG. 6d, on the front side of the insulating substrate, the working electrode and the reference electrode, and the working electrode terminal and the reference electrode for electrical connection with the body of the embedded biosensor An insulating resist film having an opening is formed by screen printing in a portion excluding a region used as a terminal, and a portion excluding a region used as a counter electrode and a counter electrode terminal for electrical connection to the back side of the insulating substrate An insulating resist film (thickness: 10-15 μm) having an opening was formed by screen printing.

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

(6) Formation of enzyme layer As shown in FIG. 7f, the conductive thin film exposed from the opening of the insulating resist film formed on the front side of the insulating substrate is used as a working electrode, and further, bovine serum albumin (Bovine serum albumin) is formed thereon. albmin (BSA) solution and glucose oxidase (GOD) solution as an analyte-responsive enzyme for glucose are applied, and glutaraldehyde is added dropwise and dried to form an enzyme layer (thickness 15 μm). did.

(7) Formation of Protective Film As shown in FIG. 7g, the sensing part is immersed in a dioxolane solution containing a cross-linking agent and polyurethane, and a protective film (thickness 5-40 μm) is formed on both sides, side surfaces, and the tip of the sensing part. Formed.
More specifically, the probe produced above was immersed nine times at intervals of 10 minutes in a solution obtained by dissolving 60 mg of polyurethane in 1 mL of a solvent (dioxolane). Then, probes (samples 1 and 2) were obtained by drying at room temperature for 12 hours.

<Measurement of probe characteristics>
[Glucose response characteristics]
The obtained two probes were attached to an implantable amperometric glucose sensor, and the probes were placed in 37 ° C. phosphate buffered saline (PBS, pH 7). Glucose was added to this PBS solution every 50 seconds at 50, 100, 200, 300, 400, and 500 mg / dL, and the current response value (nA) was continuously measured.
When the glucose concentration was 0 to 500 mg / dL, high linearity was exhibited and the glucose responsiveness was good. The results are shown in FIG. 12 and summarized in Table 1.

[durability]
The two obtained probes were stored in phosphate buffered saline (PBS, pH 7) at 37 ° C. for 2 weeks. Before storage (Day 1) and after storage, each of the two probes was attached to an implantable amperometric glucose sensor, and the probes were placed in 37 ° C. phosphate buffered saline (PBS, pH 7). 500 mg / dL was added to this PBS solution, and the current response value (nA) was continuously measured. The response rate (%) in each storage period was calculated with the current response value at the glucose concentration of 500 mg / dL on the first day as 100%.
The response characteristics after 2 weeks of storage maintained 80% or more of the response value on the first day, and the durability was good. The results are shown in FIG. 13 and summarized in Table 2.
The response rate after several days is higher than that after one day of storage, but this is presumed to be due to the fact that they were sufficiently familiar with PBS. The response rates after 15 days of storage when the maximum value is 100% are 88.7% and 72.5%, respectively, and are sufficiently high.

  It was confirmed that when the protective film using the polyurethane of the present disclosure was used for the sensing portion of the probe, the glucose responsiveness was good and the durability was improved.

  The membrane structure including an enzyme layer containing at least an analyte-responsive enzyme and a protective film formed on the enzyme layer of the present disclosure is useful as a probe for an implantable biosensor.

1 Implantable biosensor
10 Body
11 Probe
111 Insulating substrate
112 Conductive thin film
112a Working electrode lead
112b Reference electrode lead
112c counter lead
113 groove
114 working electrode
115 Reference electrode
116 Insulating resist
117 Counter electrode
118 Enzyme layer
119 Protective film
2 Living body
3 Information and communication equipment

Claims (4)

An enzyme layer containing at least an analyte-responsive enzyme and a protective film formed on the enzyme layer, wherein the protective film has the general formula (1):

[Wherein, R and R ′ represent an organic group, and x represents an integer. ] The film | membrane structure containing the polyurethane represented by this.   The membrane structure according to claim 1, wherein the analyte-responsive enzyme is glucose oxidase.   The membrane structure according to claim 1, wherein the glucose oxidase is immobilized on the membrane by bovine serum albumin and glutaraldehyde. Insulating substrate, conductive thin film formed on both surfaces of the insulating substrate, working electrode and reference electrode formed on the conductive thin film on the front side of the insulating substrate, on the conductive thin film on the back side of the insulating substrate A probe for a biosensor comprising a counter electrode formed on the working electrode, an enzyme layer formed on the working electrode, and a protective film covering the working electrode, the reference electrode, the counter electrode and the enzyme layer,
The enzyme layer includes at least an analyte-responsive enzyme,
The conductive thin film includes palladium; and
The protective film has the general formula (1):

[Wherein, R and R ′ represent an organic group, and x represents an integer. ] The probe for biosensors containing the polyurethane represented by this.

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