Patent Application
20210072179
The present disclosure relates to a film material for protecting a probe constituting a biosensor. More specifically, provided is a polymer material for a protection film that can prevent an enzyme or a mediator constituting a biosensor probe inserted in the body from flowing out.
A biosensor is a system for measuring a substance by utilizing or imitating a molecular recognition ability of a living body, a measurement device that, using among combinations of, for example, an enzyme and a substrate, an antigen and an antibody, a hormone and a receptor, one as an analyte (target substance to be measured), and the other one as a receptor, converts a chemical change generated by a molecular recognition reaction between the analyte and the receptor into electric signals with a transducer, and measures an amount of the analyte according to the intensity of the obtained electric signals.
Besides the above-described living molecules, living molecules used for a biosensor include genes, sugar chains, lipids, peptides, cells, and tissues. Among these, development of a biosensor using an enzyme is mostly advanced, and a representative example thereof is a glucose sensor using glucose oxidase (GOx).
In an electrochemical glucose sensor used for self monitoring of blood glucose, generally, a cover is disposed on an insulating substrate in which an electrode is formed on the surface, with a spacer interposed between the cover and the insulating substrate. A reagent containing an analyte-responsive enzyme, a redox mediator (electron carrier), or the like is disposed on the electrode, and this part serves as an analysis part. The analysis part communicates with one end of a flow path for introducing blood, and the other end of the flow path, which becomes a blood supply port, opens toward the outside. The measurement of the blood glucose level using such a sensor is, for example, carried out as follows. That is, the sensor is first set to a dedicated measurement device (meter). Then, a fingertip or the like is cut with a lancet to cause bleeding, and blood is brought into contact with the blood supply port of the sensor. Blood is absorbed in the flow path of the sensor by capillary phenomenon, introduced into the analysis part through the flow path, and brought into contact with the reagent. Then, an analyte-responsive enzyme E (for example, GOx, and GDH) specifically reacts with glucose in the blood to oxidize glucose. A redox mediator M receives electrons generated due to oxidization. The redox mediator M, which has been reduced by accepting electrons, is electrochemically oxidized in the electrode. The glucose concentration in the blood, that is, the blood glucose level is simply detected from, for example, a magnitude of the current value, or a quantity of electric charge obtained by oxidization of a reduced form of the redox mediator M.
Such an electrochemical blood glucose sensor plays an important role in blood glucose level management for diabetes treatment and allows appropriate insulin administration as well as dietary restriction for diabetic patients based on the blood glucose level. However, such an electrochemical blood glucose sensor requires several times of blood glucose level measurements a day, and patients are forced to endure pain due to blood collection every time. Thus, it has been difficult for the patients to maintain the quality of life (QOL).
An embedded-type amperometric glucose sensor has already been developed. A main body 10 of such an embedded-type amperometric glucose sensor 1 is attached to a living body 2, a probe part 11 is then inserted in the living body, and the blood glucose level is continuously measured (
The Ministry of Health, Labor and Welfare issued “telemedicine notification in 1997 (notification No. 1075, dated on Dec. 24, 1997, issued from the director general of the Health Policy Bureau, Ministry of Health, Labor and Welfare)” and indicated the basic concept of telemedicine as well as points of attention regarding Article 20 of the Medical Practitioners' Act, or the like. Thereafter, the Ministry of Health, Labor and Welfare made an announcement regarding medical examination using information communication devices, that is, telemedicine, to the prefectural governors on Aug. 10, 2015, considering the states of development and spread of the information communication devices. The ban on telemedicine was practically lifted by the notification in 2015 and, in 2016, telemedicine tools, which perform wireless data communication with a biosensor 1 using an information communication device 3 (smartphone) and a dedicated application, have been distributed over the world (
Patent Literature 1 discloses an electrochemical sensor-controlling device using a wireless transmitter, the device being attached to the patient and inserted in the skin. Patent Literature 1 also describes a technique of transmitting data on an amount of analyte collected to a display unit using a wireless transmitter. Patent Literature 1 also discloses a film including a heterocyclic nitrogen group such as vinylpyridine, which is attached to such an electrochemical sensor. These films restrict diffusion of the analyte in a working electrode of the electrochemical sensor. In a glucose sensor having no film, the amount of glucose flowing into a detection layer is linearly increased along with the glucose concentration. Output signals measured are linearly proportional to the flow amount of glucose until all the glucose that reaches the detection layer is consumed. However, in a case where consumption of glucose is restricted in the detection layer, measured signals are not linearly proportional to the flow amount or concentration of glucose, and saturation occurs. Thus, Patent Literature 1 employs a technique of preventing saturation in the sensor, by forming a diffusion restricting film including a heterocyclic nitrogen group such as polyvinylpyridine on the detection layer to decrease the amount of glucose flowing into the detection layer.
Patent Literature 2 discloses a diffusion barrier including a single block copolymer having at least one hydrophilic block and at least one hydrophobic block. Similarly to Patent Literature 1, this diffusion barrier controls diffusion of an analyte from an exterior of an electrode system to enzyme molecules. In addition, such enzyme molecules are immobilized on an electrode to form an enzyme layer. In a production of such an enzyme layer, for example, as disclosed in Patent Literature 3, enzyme molecules can be more firmly immobilized by immobilizing an enzyme on a working electrode by adsorption or capture, and then performing crosslinking with glutaraldehyde or the like.
In an embedded-type sensor, a probe of the sensor is inserted in a body for a long time, and therefore the case is increased where an analyte-responsive enzyme or a redox mediator, which is a constituent element of the sensor, flows out. When the analyte-responsive enzyme or the redox mediator flows out to the outside of the sensor, not only sensitivity of the sensor is deteriorated, but also flowing out causes damage to the living body. Furthermore, when the analyte-responsive enzyme or the redox mediator flows out to the outside, durability of the sensor is lowered. It is therefore very important to take measures to prevent flowing out of the analyte-responsive enzyme or the redox mediator.
When both the analyte-responsive enzyme and the redox mediator constituting the probe of an embedded-type biosensor are immobilized in order to prevent flowing out, a combination of the enzyme and the mediator polymer that can be used, or a structure of the sensor is restricted. Therefore, development of a film that is formed on a detection layer including the enzyme and/or the redox mediator and that can prevent the enzyme and the redox mediator from flowing out to the outside is desired. Moreover, such a film must not inhibit an analyte such as glucose from entering inside. Therefore, an object of the present disclosure is to provide a protection film that does not inhibit an analyte from entering inside and prevents an enzyme and a redox mediator from flowing out to the outside in order to be applied to a probe of an embedded-type biosensor.
The present disclosure provides, as a film structure useful for a probe of a biosensor, a film structure including:
a detection layer including at least an analyte-responsive enzyme and a redox mediator; and
a protection film formed on the detection layer,
in which
the protection film includes alone poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) represented by Formula (1):
where m represents an integer of 1 to 20; x, y, and z each represent mole fractions of three types of monomer units of styrene, 4-vinylpyridine, and propyleneglycol methacrylate, and satisfy a relationship x+y+z=100; and w represents a number average molecular weight; or
in which
the protection film includes a copolymer mixture including:
poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) represented by Formula (1): and
poly (4-vinylpyridine)-block-poly(C1-15 alkyl methacrylate) represented by Formula (2):
where R represents an alkyl group having 1 to 15 carbon atoms; p and q each represent repeating units of two types of monomer units of 4-vinylpyridine and C1-15 alkyl methacrylate; and w represents a number average molecular weight, or
poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) represented by Formula (3):
where s and t each represent mole fractions of two types of monomer units of 4-vinylpyridine and 2-hydroxyethyl methacrylate, and satisfy a relationship s+t=100; and w represents a number average molecular weight. Note that, the oblique lines between monomers in the formula indicate that the three types of monomer units are not bonded in the order described in the formula, but are randomly bonded to each other when excluding deviation caused by reactivity between monomer units.
The polymer represented by Formula (1) above is a random copolymer including styrene, 4-vinylpyridine, and propyleneglycol methacrylate as a monomer unit. The mole fraction x of styrene is 5 to 30, preferably 10 to 30, and more preferably 15 to 25. The mole fraction y of 4-vinylpyridine is 45 to 65, and preferably 50 to 60. The mole fraction z of propyleneglycol methacrylate is 15 to 30, and preferably 20 to 25. Further, x, y, and z need not to be an integer as long as the sum of x, y, and x is 100. Furthermore, the number average molecular weight of the polymer is 40 to 80×103, and preferably 45 to 65×103.
The polymer represented by Formula (2) above is a block copolymer including poly(4-vinylpyridine) and poly(C1-15 alkyl methacrylate). A repeating unit p of 4-vinylpyridine constituting poly(4-vinylpyridine) and a repeating unit q of alkyl methacrylate constituting poly(C1-15 alkyl methacrylate) may each be set so that the number average molecular weight of each block constituting the polymer is 50 to 200×103, and preferably 60 to 100×103.
In the polymer represented by Formula (2) above, C1-15 alkyl of poly(C1-15 alkyl methacrylate) represents an alkyl group having 1 to 15 carbon atoms. Examples thereof include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, a dodecyl group, a tridecyl group, a tetradecyl group, a pentadecyl group, and isomers thereof, and a C3-6 alkyl group is preferred.
The polymer represented by Formula (3) above is advantageous for enhancing biocompatibility, and is a random copolymer including 4-vinylpyridine and 2-hydroxyethyl methacrylate as a monomer unit. The mole fraction s of 4-vinylpyridine is 40 to 80, and preferably 60 to 70. The mole fraction t of 2-hydroxyethyl methacrylate is 20 to 60, and preferably 30 to 40. Further, s and t need not to be an integer as long as the sum of s and t is 100. Furthermore, the number average molecular weight of the polymer is 20 to 500×103, and preferably 60 to 300×103.
The above-described polymer may be crosslinked with a crosslinking agent such as polyethylene glycol diglycidyl ether (PEGDGE).
The protection film may further contain, for example, poly(2-methoxyethylacrylate) as an additive. This enhances biocompatibility of the protection film.
When a protection film including the copolymer or the copolymer mixture of the present disclosure is formed on a detection layer including an enzyme and a mediator constituting a probe of an embedded-type biosensor, it is possible to avoid inhibiting an analyte such as glucose from entering inside and to prevent the enzyme or the mediator included in the detection layer from flowing out to the outside.
1. Production Method of Probe of Embedded-Type Biosensor
A production method of a probe 11 of an embedded-type biosensor 1, to which the film structure of the present disclosure is applied, will be described as a specific example. The structure and production method described below are one of specific examples of the present disclosure, and are not limited to the features described below as long as it can be used as a probe.
(1) Preparation of Insulating Substrate
The embedded-type biosensor 1 includes a main body 10 and a probe 11. The probe 11, having a key shape, generally includes a sensing part to be inserted in a living body, and a terminal part to be electrically connected with an internal circuit of the biosensor main body 10. The sensing part is formed thin so as to be inserted in the body, and the terminal part has a certain size so as to be inserted in the biosensor main body 10 and establish electrical connection. Therefore, an insulating substrate 111 having a key shape is prepared firstly (
(2) Formation of Conductive Thin Film
A conductive thin film 112 is formed by depositing carbon or a conductive metal material selected from the group consisting of metals such as gold, silver, platinum, and palladium on both sides of the insulating substrate 111 by sputtering, deposition, ion plating, or the like (
(3) Formation of Electrode Lead
A groove 113 is formed with a depth that reaches the surface of the insulating substrate 111 by performing laser drawing on the conductive thin film 112 formed on the front side of the insulating substrate 111. The groove 113 separates a working electrode lead 112a and a reference electrode lead 112b and thus electrically insulates them from each other (
(4) Formation of Insulating Resist Film
On the front side of the insulating substrate 111, an insulating resist film 116a having an opening is formed in a part excluding regions used for a working electrode 114 and a reference electrode 115 as well as a working electrode terminal 114a and a reference electrode terminal 115a to be electrically connected with the main body 10, by sputtering, screen printing, or the like. On the back side of the insulating substrate 111, an insulating resist film 116b having an opening is formed in a part excluding regions used for a counter electrode 117 and a counter electrode terminal 117a to be electrically connected with the main body 10, by sputtering, screen printing, or the like (
(5) Formation of Reference Electrode
The reference electrode 115 is formed by depositing Ag/AgCl in the opening for the reference electrode of the resist film 116a formed on the front side of the insulating substrate 111 by screen printing or an inkjet method (
(6) Formation of Detection Layer
A detection layer 118 including conductive particles, an analyte-responsive enzyme, and a redox mediator is formed by applying a suspension of conductive particles such as carbon particles, an aqueous solution of the analyte-responsive enzyme, and an aqueous solution of the redox mediator on the working electrode 114 and dried them (
(7) Formation of Protection Film
A protection film 119 is formed on the both surfaces, side surfaces, and tip of the sensing part by immersing the sensing part in a solution including a polymer for a protection film (
2. Internal Structure of Probe of Embedded-Type Biosensor
The internal structure of the probe of an embedded-type biosensor, to which the film structure of the present disclosure is applied, will be further described.
<Production of Probe>
(1) Preparation of Insulating Substrate
As shown in
(2) Formation of Conductive Thin Film
As shown in
(3) Formation of Electrode Lead
As shown in
(4) Formation of Insulating Resist Film
As shown in
(5) Formation of Reference Electrode
As shown in
(6) Formation of Detection Layer
As shown in
(7) Formation of Protection Film
As shown in
More specifically, the probe produced as described above was immersed 6 times at 10 minutes intervals in a solution in which 600 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.8, m=2) [copolymer (4)] represented by Formula (4):
and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe A was obtained. The formation conditions of the above-described protection film were summarized in Table 1.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The probe A was attached to an embedded-type amperometric glucose sensor, and then the probe was placed in a phosphate-buffered saline solution (PBS, pH 7) at 37° C. To the PBS solution, 50, 100, 200, 300, 400, and 500 mg/dL of glucose was added every 500 seconds in the order of amounts described, and the current response value (nA) was continuously measured.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
The probe A was stored in a phosphate-buffered saline solution (PBS, pH 7) at 37° C. The probe A was attached to an embedded-type amperometric glucose sensor before storage (day 0) and 7 days after storage, and the probe was placed in a phosphate-buffered saline solution (PBS, pH 7) at 37° C. To the PBS solution, 50, 100, 200, 300, 400, and 500 mg/dL of glucose was added every 500 seconds in the order of amounts described, and the current response value (nA) was continuously measured. The response ratio (%) at each concentration was calculated as the current response value at a glucose concentration of 500 mg/dL on day 0 being 100%.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
<Production of Probe>
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film.
More specifically, the probe produced in Example 1 was immersed 6 times at 10 minutes intervals in a solution in which 600 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.6, m=about 16) [copolymer (5)] represented by Formula (5):
and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent. Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe B was obtained. The formation conditions of the above-described protection film were summarized in Table 1.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe B was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe B was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film.
More specifically, the probe produced in Example 1 was immersed 6 times at 10 minutes intervals in a solution in which 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.8, m=2) [copolymer (4)] represented by Formula (4), 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.6; m=about 16) [copolymer (5)] represented by Formula (5), and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent. Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe C was obtained. The formation conditions of the above-described protection film were summarized in Table 1.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe C was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe C was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
<Production of Probe>
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film.
More specifically, the probe produced as described above was immersed 8 times at 10 minutes intervals in a solution in which 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.8, m=2) [random copolymer (4)] represented by Formula (4), 300 mg of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) (Mw/Mn=1.15; w′=77.0−g−80.0×103) [block copolymer (6)] represented by Formula (6),
and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe D was obtained. The formation conditions of the above-described protection film were summarized in Table 3.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe D was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe D was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
<Production of Probe>
In the formation of the protection film, a probe was produced in the same manner as in Example 4, except for changing polymers for a protection film.
More specifically, a protection film was formed in the same manner as in Example 4 except for using poly(4-vinylpyridine)-block-poly(n-butyl methacrylate) (Mw/Mn=1.3; w″=6.0−g−4.5×103) [block copolymer (7)] represented by Formula (7):
in place of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) [block copolymer (6)] represented by Formula (6) in Example 4, and thus a probe E was obtained. The formation conditions of the above-described protection film were summarized in Table 3.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe E was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe E was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 4, except for changing polymers for a protection film.
More specifically, a protection film was formed in the same manner as in Example 4 except for using poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) (Mw/Mn=1.2) [random copolymer (8)] represented by Formula (8):
in place of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) [block copolymer (6)] represented by Formula (6) in Example 4, and thus a probe F was obtained. The formation conditions of the above-described protection film were summarized in Table 3.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe F was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe F was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 4, except for changing polymers for a protection film.
More specifically, a protection film was formed in the same manner as in Example 4 except for using poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) (Mw/Mn=1.6) [random copolymer (9)] represented by Formula (9):
in place of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) [block copolymer (6)] represented by Formula (6) in Example 4, and thus a probe G was obtained. The formation conditions of the above-described protection film were summarized in Table 3.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe G was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe G was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film.
More specifically, the probe produced as described above was immersed 8 times at 10 minutes intervals in a solution in which 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.6, m=about 16) [random copolymer (5)] represented by Formula (5), 300 mg of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) (Mw/Mn=1.15; w′=77.0−g−80.0×103) [block copolymer (6)] represented by Formula (6), and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe H was obtained. The formation conditions of the above-described protection film were summarized in Table 5.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe H was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe H was measured in the same manner as in Example 1.
Response characteristics 7 days after storage showed high linearity at a glucose concentration of 0 to 500 mg/dL and showed almost no change compared to day 0, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 8, except for changing polymers for a protection film.
More specifically, a protection film was formed in the same manner as in Example 8 except for using poly(4-vinylpyridine)-block-poly(n-butyl methacrylate) (Mw/Mn=1.3; w″=6.0−g−4.5×103) [block copolymer (7)] represented by Formula (7) in place of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) [block copolymer (6)] represented by Formula (6) in Example 8, and thus a probe I was obtained. The formation conditions of the above-described protection film were summarized in Table 5.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe I was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe I was measured in the same manner as in Example 1.
Response characteristics 7 days after storage maintained linearity at a glucose concentration of 0 to 500 mg/dL, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 8, except for changing polymers for a protection film.
More specifically, a protection film was formed in the same manner as in Example 8 except for using poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) [random copolymer (9)] represented by Formula (9) in place of poly(4-vinylpyridine)-block-poly(2,2-dimethylethyl methacrylate) [block copolymer (6)] represented by Formula (6) in Example 8, and thus a probe J was obtained. The formation conditions of the above-described protection film were summarized in Table 5.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe J was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe K was measured in the same manner as in Example 1.
Response characteristics 7 days after storage maintained linearity at a glucose concentration of 0 to 500 mg/dL, and durability was favorable. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film and increasing the number of immersions in the crosslinking agent solution.
More specifically, the probe produced as described above was immersed 9 times at 10 minutes intervals in a solution in which 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.8, m=2) [random copolymer (4)] represented by Formula (4), 300 mg of poly(4-vinylpyridine) (Mw=160,000) [P4VP], and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe K was obtained. The formation conditions of the above-described protection film were summarized in Table 7.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe K was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe K was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 48% at a glucose concentration of greater than 100 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film and increasing the number of immersions in the crosslinking agent solution.
More specifically, the probe produced as described above was immersed 9 times at 10 minutes intervals in a solution in which 300 mg of poly(styrene-ran-4-vinylpyridine-ran-propyleneglycol methacrylate) (Mw/Mn=1.6, m=about 16) [random copolymer (5)] represented by Formula (5), 300 mg of poly(4-vinylpyridine) (Mw=160,000) [P4VP], and 47 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe L was obtained. The formation conditions of the above-described protection film were summarized in Table 7.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe L was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe L was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 36% at a glucose concentration of greater than 100 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film, increasing an amount of the crosslinking agent, and increasing the number of immersions in the crosslinking agent solution.
More specifically, the probe produced as described above was immersed 9 times at 10 minutes intervals in a solution in which 800 mg of poly(4-vinylpyridine) (Mw=160,000) [P4VP] and 62 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe M was obtained. Formation conditions of the above-described protection film were summarized in Table 7.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe M was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe M was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 22% at a glucose concentration of greater than 100 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film, increasing the amount of the crosslinking agent, and increasing the number of immersions in the crosslinking agent solution.
More specifically, the probe produced as described above was immersed 12 times at 10 minutes intervals in a solution in which 800 mg of poly(4-vinylpyridine)-block-poly(n-butyl methacrylate) (Mw/Mn=1.3; w″=6.0−g−4.5×103) [block copolymer (7)] represented by Formula (7), and 62 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe N was obtained. The formation conditions of the above-described protection film were summarized in Table 9.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe N was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe N was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 46% at a glucose concentration of greater than 200 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film and increasing the amount of the crosslinking agent.
More specifically, the probe produced as described above was immersed 8 times at 10 minutes intervals in a solution in which 800 mg of poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) [random copolymer (8)] represented by Formula (8) and 62 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe O was obtained. The formation conditions of the above-described protection film were summarized in Table 9.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe O was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe O was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 25% at a glucose concentration of greater than 200 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film and increasing the amount of the crosslinking agent.
More specifically, the probe produced as described above was immersed 8 times at 10 minutes intervals in a solution in which 800 mg of poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) [random copolymer (9)] represented by Formula (9) and 62 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe P was obtained. The formation conditions of the above-described protection film were summarized in Table 9.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe P was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe P was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 32% at a glucose concentration of greater than 200 mg/dL, and durability was poor. The result is shown in
In the formation of the protection film, a probe was produced in the same manner as in Example 1, except for changing polymers for a protection film and increasing the amount of the crosslinking agent.
More specifically, the probe produced as described above was immersed 8 times at 10 minutes intervals in a solution in which 400 mg of poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) [random copolymer (8)] represented by Formula (8), 400 mg of poly(4-vinylpyridine-ran-2-hydroxyethyl methacrylate) [random copolymer (9)] represented by Formula (9), and 62 mg of polyethylene glycol diglycidyl ether (PEGDGE) (Mn=1000) as a crosslinking agent were dissolved in 1 mL of a solvent (ethanol 95%, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer solution (10 mM, pH 8) 5%). Thereafter, the probe was dried over 48 hours at room temperature to form a crosslinked protection film, and thereby a probe Q was obtained. The formation conditions of the above-described protection film were summarized in Table 9.
<Measurement of Probe Characteristics>
[Glucose Response Characteristics]
The glucose response characteristics of the probe Q was measured in the same manner as in Example 1.
High linearity was shown at a glucose concentration of 0 to 500 mg/dL, and glucose response was favorable. The result is shown in
[Durability]
The durability of the probe Q was measured in the same manner as in Example 1.
Response characteristics 7 days after storage decreased to 30% at a glucose concentration of greater than 200 mg/dL, and durability was poor. The result is shown in
It was confirmed that using a protection film including the polymer mixture of the present disclosure for a sensing part of a probe yielded favorable glucose response and enhanced durability compared to the conventional polymers.
The film structure of the present disclosure, including a detection layer including at least an analyte-responsive enzyme and a redox mediator, and a protection film formed on the detection layer, is useful for a probe of an embedded-type biosensor.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-012801 | Jan 2018 | JP | national |
| 2018-045800 | Mar 2018 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2019/002796 | 1/28/2019 | WO | 00 |
