Protective film material for biosensor probe
阅读说明:本技术 生物传感器探针用保护膜材料 (Protective film material for biosensor probe ) 是由 远藤太志 池田纯子 于 2019-01-28 设计创作,主要内容包括:本发明使用一种膜结构体作为在埋入型生物传感器的探针中有用的膜结构体,其具备包含检测体响应性酶的检测层、以及在上述检测层上形成的保护膜,上述保护膜包含交联剂和聚(苯乙烯-无规-4-乙烯基吡啶-无规-甲基丙烯酸丙二醇酯)、以及聚(4-乙烯基吡啶)-嵌段-聚(甲基丙烯酸C<Sub>1-15</Sub>烷基酯)或者聚(4-乙烯基吡啶-无规-甲基丙烯酸2-羟基乙酯)。(The present invention uses a membrane structure comprising a detection layer containing a detection-responsive enzyme, and a protective film formed on the detection layer, the protective film comprising a crosslinking agent, poly (styrene-random-4-vinylpyridine-random-propylene glycol methacrylate), and poly (4-vinylpyridine) -block-poly (methacrylic acid C-methyl acrylate), as a membrane structure useful for a probe of an embedded biosensor 1‑15 Alkyl ester) or poly (4-vinylpyridine-random-2-hydroxyethyl methacrylate).)
1. A membrane structure comprising a detection layer containing at least a detection-responsive enzyme and a redox mediator, and a protective film formed on the detection layer, the protective film comprising:
poly (4-vinylpyridine) -block-poly (methacrylic acid C) represented by formula (1)1-15Alkyl esters); or
Comprising poly (4-vinylpyridine) -block-poly (methacrylic acid C) represented by the formula (1)1-15Alkyl ester) with poly (4-vinylpyridine-random-2-hydroxyethyl methacrylate) of formula (2),
in the formula (1), R represents alkyl with 1-15 carbon atoms, p and q respectively represent 2 monomer units, namely 4-vinylpyridine and methacrylic acid C1-15Alkyl ester repeating units, w represents a number average molecular weight,
in formula (2), s and t represent the mole percentages of 2 monomer units, i.e., 4-vinylpyridine and 2-hydroxyethyl methacrylate, respectively, and s + t is 100, and w represents the number average molecular weight.
2. The membrane structure of claim 1, wherein the protective membrane further comprises poly (2-methoxyethyl acrylate).
3. A probe for a biosensor, comprising an insulating substrate, conductive thin films formed on both surfaces of the insulating substrate, a working electrode and a reference electrode formed on the conductive thin film on the front surface side of the insulating substrate, a counter electrode formed on the conductive thin film on the back surface side of the insulating substrate, a detection layer formed on the working electrode, and a protective film covering the working electrode, the reference electrode, the counter electrode, and the detection layer, wherein the detection layer contains a sample-responsive enzyme and a redox mediator, and the protective film contains:
a poly represented by the formula (1)(4-vinylpyridine) -block-poly (methacrylic acid C)1-15Alkyl esters); or
Comprising poly (4-vinylpyridine) -block-poly (methacrylic acid C) represented by the formula (1)1-15Alkyl ester) with poly (4-vinylpyridine-random-2-hydroxyethyl methacrylate) of formula (2),
in the formula (1), R represents alkyl with 1-15 carbon atoms, p and q respectively represent 2 monomer units, namely 4-vinylpyridine and methacrylic acid C 1-15Alkyl ester repeating units, w represents a number average molecular weight,
in formula (2), s and t represent the mole percentages of 2 monomer units, i.e., 4-vinylpyridine and 2-hydroxyethyl methacrylate, respectively, and s + t is 100, and w represents the number average molecular weight.
Technical Field
The present disclosure relates to a membrane material for protecting a probe constituting a biosensor. More specifically, a polymer material for a protective film capable of preventing the outflow of an enzyme or a mediator constituting a biosensor probe inserted into the body is provided.
Background
A biosensor is a system for measuring a substance by utilizing or simulating the molecular recognition ability of a living body, and is, for example, a measuring apparatus in which one of combinations of an enzyme-substrate, an antigen-antibody, a hormone-receptor, and the like is used as a detection body (a substance to be measured), the other is used as a receptor, a chemical change caused by a molecular recognition reaction between the detection body and the receptor is converted into an electric signal by a transducer, and the amount of the detection body is measured based on the intensity of the obtained electric signal.
The biomolecule used in the biosensor includes genes, sugar chains, lipids, peptides, cells, tissues, and the like in addition to the above. Among them, biosensors using enzymes have been developed most rapidly, and a representative example thereof is a Glucose sensor using Glucose oxidase (GOx).
An electrochemical glucose sensor used for self-blood glucose measurement is generally configured such that a cap is disposed on an insulating substrate having electrodes formed on the surface thereof, with a spacer interposed therebetween. The electrode is provided with a reagent containing a sample-responsive enzyme, a redox mediator (electron conductor), and the like, and this portion serves as an analysis section. One end of a channel for introducing blood communicates with the analysis section, and the other end of the channel opens to the outside, and is referred to as a blood supply port. The blood glucose level using such a sensor is measured, for example, as follows. That is, first, the sensor is mounted on a dedicated measuring device (measuring instrument). Then, a fingertip or the like is pricked with a lancet to bleed, and the blood supply port of the sensor is brought into contact with the fingertip or the fingertip. The blood is drawn into the channel of the sensor by capillary action, and is introduced into the analysis section through the channel, where it comes into contact with the reagent. Then, the sample-responsive enzyme E (e.g., GOx, GDH) oxidizes glucose by a specific reaction with glucose in blood. The redox mediator M accepts electrons generated by oxidation. The redox mediator M that has received electrons and has been reduced is electrochemically oxidized by an electrode. The glucose concentration in blood, i.e., the blood glucose level, can be easily detected based on the magnitude of the current value, the charge amount, and the like obtained by oxidation of the reducing redox mediator M.
Such an electrochemical blood glucose sensor plays an important role in blood glucose management in diabetes treatment, and a diabetic patient can appropriately administer insulin or control diet based on the blood glucose level. However, the blood glucose level must be measured several times a day, and each blood collection can increase the pain of the patient and make it difficult to maintain the Quality of Life (Quality of Life; QOL).
Embedded amperometric glucose sensors have been developed. The
"telemedicine notification in 9 years (healthcare issue No. 1075 health policy bureau of pachyson department, 24 days in 12 months in 9 years)" is performed by the japan university labour, and shows the basic ideas of telemedicine and the matters to be noticed in relation to the japanese medical law,
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 describes a technique of transmitting collected data on the amount of a detected object to a display device using a wireless transmitter. Patent document 1 also discloses a film containing a heterocyclic nitrogen group such as vinylpyridine, which is mounted on such an electrochemical sensor. These membranes limit diffusion of the detection body in the electrochemical sensor to the working electrode. In a glucose sensor having no membrane, the flow rate of glucose to the detection layer increases linearly together with the glucose concentration, and when the glucose consumption is limited in the detection layer, the measurement signal is saturated and does not linearly proportion to the flow rate or concentration of glucose, while the output signal measured linearly proportion to the flow rate of glucose in the course of the consumption of all the glucose that has reached the detection layer. Therefore, patent document 1 adopts a technique of forming a diffusion limiting film containing a heterocyclic nitrogen group such as polyvinylpyridine on the detection layer to reduce the flow rate of glucose to the detection layer, thereby preventing saturation of the sensor.
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 is a member for controlling diffusion of an analyte from the outside of an electrode system to an enzyme molecule, as in patent document 1. In addition, such enzyme molecules are immobilized to an electrode to form an enzyme layer. In the production of such an enzyme layer, as disclosed in patent document 3, for example, the enzyme can be immobilized on the working electrode by adsorption and capture, and further firmly immobilized by crosslinking with glutaraldehyde or the like.
Disclosure of Invention
Problems to be solved by the invention
Since the probe of the embedded sensor is inserted into the body for a long time, the chance of elution of the detection-responsive enzyme and the redox mediator, which are components, increases. If the sample-responsive enzyme or the redox mediator flows out of the sensor, not only the sensitivity of the sensor deteriorates, but also the living body is damaged. In addition, if the sample-responsive enzyme or the redox mediator is eluted to the outside, the durability of the sensor is also reduced. Therefore, measures for preventing the outflow of the sample-responsive enzyme and the redox mediator are very important.
If it is desired to immobilize both a detection-responsive enzyme and a redox mediator constituting a probe of an embedded biosensor to prevent elution, the combination of an enzyme and a mediator polymer that can be used, and the structure of the sensor, are limited. Therefore, it is desired to develop a membrane that is formed on a detection layer containing an enzyme and/or a redox mediator and can prevent the enzyme and the redox mediator from flowing out to the outside. In addition, such a membrane cannot prevent a specimen such as glucose from entering the inside. Accordingly, an object of the present disclosure is to provide a protective film that prevents an enzyme and a redox mediator from flowing out to the outside without inhibiting the entry of a detection object into the inside, in order to be applied to a probe of an embedded biosensor.
Means for solving the problems
The present disclosure provides a membrane structure useful as a probe for a biosensor, comprising a detection layer containing at least a detection-responsive enzyme and a redox mediator, and a protective film formed on the detection layer, the protective film comprising: poly (4-vinylpyridine) -block-poly (methacrylic acid C) represented by formula (1)1-15Alkyl esters); or poly (4-vinylpyridine) -block-poly (methacrylic acid C) represented by the formula (1)1-15Alkyl ester) with poly (4-vinylpyridine) of the formula (2)-random-2-hydroxyethyl methacrylate). In the formula, the diagonal lines between the monomers do not indicate that the 3 kinds of monomer units are in the order described in the formula, but indicate that the monomer units are bonded irregularly except for unevenness due to reactivity between the monomer units.
[ chemical formula 1]
[ wherein R represents an alkyl group having 1 to 15 carbon atoms, and p and q each represent 2 monomer units, i.e., 4-vinylpyridine and methacrylic acid C1-15Alkyl ester repeating units, w represents a number average molecular weight.]
[ chemical formula 2]
In the formula, s and t represent the mole percentages of 2 monomer units, i.e., 4-vinylpyridine and 2-hydroxyethyl methacrylate, respectively, s + t is 100, and w represents the number average molecular weight. ]
The polymer represented by the above formula (1) is a copolymer of poly (4-vinylpyridine) and poly (methacrylic acid C)1-15Alkyl ester), repeating units p of 4-vinylpyridine constituting poly (4-vinylpyridine), and poly (methacrylic acid C)1-15Alkyl ester) of methacrylic acid alkyl ester each having a number average molecular weight of 50 to
Among the polymers represented by the above formula (1), poly (methacrylic acid C)1-15Alkyl ester) C1-15The alkyl group represents an alkyl group having 1 to 15 carbon atoms, and 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 preferably C3-6An alkyl group.
Polymerization represented by the above formula (2)The composition is advantageous for improving biocompatibility, and is a random copolymer comprising 4-vinylpyridine and 2-hydroxyethyl methacrylate as monomer units, wherein the molar percentage s of 4-vinylpyridine is 40 to 80, preferably 60 to 70, and the molar percentage t of 2-hydroxyethyl methacrylate is 20 to 60, preferably 30 to 40, and s and t may not be integers as long as the total of s and t is 100. The number average molecular weight of the polymer is 20 to
The polymer may be crosslinked with a crosslinking agent such as polyethylene glycol diglycidyl ether (PEGDGE).
The protective film may further contain, for example, poly (2-methoxyethyl acrylate) as an additive. Thus, the biocompatibility of the protective film is improved.
Effects of the invention
When the protective film is formed using the polymer for protective film of the present disclosure on the detection layer containing the enzyme and the mediator constituting the probe of the embedded biosensor, the enzyme and the mediator contained in the detection layer can be prevented from flowing out without inhibiting the entry of a detection substance such as glucose into the interior.
Drawings
Fig. 1 is a schematic view showing a state in which an embedded biosensor is attached to a living body (human body).
Fig. 2 is a sectional view showing an embedded biosensor attached to a living body (human body).
Fig. 3 is a schematic diagram of an embedded biosensor that wirelessly communicates measurement data with a smartphone.
FIG. 4 shows a process for producing a probe of an embedded biosensor, which is one specific example of the present disclosure.
FIG. 5 shows a process for producing a probe of an embedded biosensor, which is one specific example of the present disclosure.
FIG. 6 shows a process for producing a probe of an embedded biosensor, which is one specific example of the present disclosure.
Fig. 7 is a plan view of the probe surface side of an embedded biosensor, which is one specific example of the present disclosure.
FIG. 8 is a cross-sectional view at the cut line A-A' of FIG. 7.
Fig. 9 is a sectional view at the B-B' cut line of fig. 8.
FIG. 10 is a cross-sectional view at the cut line C-C' of FIG. 8.
Fig. 11 is a graph showing glucose response characteristics of a probe using the copolymer or copolymer mixture of the present disclosure for a protective film.
Fig. 12 is a graph showing durability of a probe using the copolymer or copolymer mixture of the present disclosure for a protective film.
FIG. 13 is a graph showing glucose response characteristics of a probe using a polymer of a comparative example for a protective film and a probe using a conventional copolymer for a protective film.
Fig. 14 is a graph showing durability of a probe using the polymer of the comparative example for the protective film and a probe using the conventional copolymer for the protective film.
FIG. 15 is a graph showing glucose response characteristics of a probe using the polymer of the reference example as a protective film and a probe using a conventional copolymer as a protective film.
Fig. 16 is a graph showing durability of a probe using the polymer of the reference example for the protective film and a probe using the conventional copolymer for the protective film.
Detailed Description
1. Method for manufacturing probe of embedded biosensor
A method for producing probe 11 of embedded biosensor 1 to which a specific example of the membrane structure of the present disclosure is applied will be described. The structure and the manufacturing method shown below are specific examples of the present disclosure, and are not limited to the following configurations as long as they can be used as probes.
(1) Preparation of insulating substrate
The embedded biosensor 1 includes a
(2) Formation of conductive thin film
A conductive metal material such as carbon or a metal selected from gold, silver, platinum, or palladium is deposited on both surfaces of the insulating
(3) Formation of electrode leads
The conductive thin film 112 formed on the front surface side of the insulating
(4) Formation of insulating resist film
An insulating resist
(5) Formation of reference electrode
The
(6) Formation of detection layer
A detection layer 118 (fig. 6f) containing conductive particles, a sample-responsive enzyme, and a redox mediator is formed by applying and drying a suspension of conductive particles such as carbon particles, an aqueous solution of a sample-responsive enzyme, and an aqueous solution of a redox mediator on the working
(7) Formation of protective film
The sensing part is immersed in a solution containing a polymer for a protective film to form a
2. Internal structure of probe of embedded biosensor
Further, the internal structure of the probe of the embedded biosensor to which the membrane structure of the present disclosure is applied will be described.
The cross-sectional view at the cut line A-A' of FIG. 7 is shown in FIG. 8. Conductive thin films 112 are formed on both sides of the insulating
The cross-sectional view at the B-B' cut line of FIG. 8 is shown in FIG. 9. A working
The cross-sectional view at the C-C' cut plane of fig. 8 is shown in fig. 10. A working
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