Biorecovery

Abstract

This invention provides an EGFET-type biosensor that offers excellent detection sensitivity, enabling accurate detection of glucose in test solutions even at low concentrations such as saliva or sweat. It can be used continuously for extended periods, exhibits minimal sensitivity degradation even after repeated long-term use, and is suitable for non-invasive applications and wearable devices. [Solution] A biosensor for detecting glucose concentration in a test solution using a field-effect transistor (EGFET) having an extended gate electrode, wherein the biosensor has a detection unit in the test solution in which the extended gate electrode and a reference electrode are facing each other, and the extended gate electrode has an insulating film containing amorphous vanadium oxide, and an enzyme immobilization film formed on the surface of the insulating film on which glycolytic enzymes are immobilized.

Description

[Technical Field]

[0001] This invention relates to a biosensor. More specifically, it relates to an extended-gate field-effect transistor (EGFET) type biosensor used for measuring the concentration of glucose or other substances in a test solution. [Background technology]

[0002] Currently, self-monitoring glucose sensors using enzyme-immobilized electrodes (enzyme electrodes) are the primary method used to measure blood glucose levels required by diabetic patients. In these self-monitoring sensors, electrons generated by an enzymatic oxidation-reduction reaction with glucose in the blood are picked up by a voltage-applied electrode, and the glucose concentration is calculated from the magnitude of the current that flows. However, measurement using self-monitoring glucose sensors with enzyme electrodes is an invasive procedure that requires blood sampling, which places a significant burden on the patient. Furthermore, it is designed for single-use applications and is unsuitable for prolonged continuous use.

[0003] Therefore, there is a need for the development of non-invasive blood glucose sensors that do not require blood sampling. Biosensors such as glucose sensors that can be applied to non-invasive applications and can be used continuously for long periods are highly anticipated, and the development of biosensors that can be applied to non-invasive blood glucose measurement methods is progressing. Known biosensors of this type include electrode-type biosensors (amperometry type) that have an enzyme electrode with a membrane on which an enzyme is immobilized on a conductive electrode, and EGFET-type biosensors (potentiometry type) that use an extended-gate field-effect transistor (EGFET) and immobilize the enzyme on the insulating film of the extended gate electrode to form the sensor.

[0004] For example, Non-Patent Document 1 discloses an EGFET-type biosensor using an electro-effect transistor (FET), in which an enzyme such as glucose oxidase (GOD) is immobilized on the surface of an insulating film (TiO2 / Ti extended electrode) formed by creating an ultrathin titanium oxide (TiO2) thin film on a Ti metal plate, using silk fibroin. Non-Patent Document 2 also discloses an example in this EGFET-type biosensor in which silane coupling treatment using an aminosilane monolayer is performed to immobilize the enzyme on the TiO2 thin film. These biosensors are said to be applicable to non-invasive applications and wearable devices, exhibit excellent detection sensitivity capable of detecting even low concentrations of glucose found in saliva and sweat, can be used continuously for long periods, and can be used repeatedly for long periods. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Journal of the Society of Material Science, Japan), Vol.69, No.9, pp.692-697 [Non-Patent Document 2] Koike et al., "Glucose detection characteristics of extended-gate field-effect transistors with enzyme immobilized using long-chain aminosilanes," Journal of the Institute of Electrical Engineers of Japan, 139 (6) pp. 143-148, 2019. [Overview of the project] [Problems that the invention aims to solve]

[0006] However, while the EGFET-type biosensor disclosed in Non-Patent Document 1 is capable of detecting low concentrations of glucose in saliva and sweat, its detection sensitivity is not sufficient. Therefore, there is a need to improve detection sensitivity in order to perform highly accurate measurements of saliva, sweat, and other substances besides blood. Other types of biosensors, such as amperometric and EGFET-type biosensors, also suffered from the problem of low sensitivity in detecting glucose and other substances.

[0007] The present invention aims to solve the above-mentioned problems of conventional EGFET-type biosensors and provide an EGFET-type biosensor that has excellent detection sensitivity that can accurately detect glucose in a test solution even at low concentrations such as in saliva or sweat, can be used continuously for long periods of time, exhibits little decrease in sensitivity even after repeated use over a long period, and can be applied to non-invasive applications and wearable devices. [Means for solving the problem]

[0008] As a result of extensive research to solve the aforementioned problems, the inventors of the present invention discovered that by using an amorphous vanadium oxide film as the insulating film in the transducer section (extended gate electrode) of an EGFET, the detection sensitivity is dramatically improved compared to conventional glucose sensors, such as glucose sensors where the insulating film is a titanium oxide film, and thus completed the present invention. In particular, when the enzyme immobilization membrane is a membrane made using the silk fibroin encapsulation method, both the detection sensitivity and response voltage are dramatically improved compared to conventional biosensors. In other words, the above problems are solved by the configuration shown below.

[0009] The first aspect of this invention is, A biosensor that detects glucose concentration in a test solution using a field-effect transistor (EGFET) with an extended gate electrode, An extended gate electrode is provided in the detection unit into which the test solution is introduced, facing the reference electrode and connected to the gate unit. The extended gate electrode is a biosensor having an insulating film containing amorphous vanadium oxide, and an enzyme-immobilized film formed on the surface of the insulating film, on which glycolytic enzymes are immobilized. Here, glycolytic enzymes refer to enzymes that selectively react with glucose to produce H+, such as glucose oxidase and hexonase.

[0010] A second aspect of the present invention provides the biosensor according to the first aspect, wherein the enzyme-immobilized membrane is a silk fibroin membrane having glucose oxidase immobilized thereon.

[0011] A third aspect of the present invention provides the biosensor according to the first or second aspect, wherein the insulating film has a thickness of 5 nm or more and 300 nm or less.

[0012] A fourth aspect of the present invention provides the biosensor according to the first aspect, wherein the insulating film containing amorphous vanadium oxide has a structure in which no vanadium oxide peak appears when the diffraction angle (2θ) is from 10° to 70° inclusive in a measurement result obtained by an X-ray diffractometer. In this measurement, diffraction peaks derived from a substrate and an underlying material are also observed, but the peak referred to in the fourth aspect of the present invention means a diffraction peak only for the insulating film excluding diffraction peaks of the substrate and the underlying material. Effects of the Invention

[0013] According to the first aspect of the present invention, there is provided a glucose sensor having dramatically improved detection sensitivity for glucose in a test solution compared to conventional EGFET biosensors. In particular, according to the second aspect of the present invention, both glucose detection sensitivity and response voltage are dramatically improved compared to conventional biosensors. Therefore, even when the test solution is a solution with a low glucose concentration such as saliva or sweat, the glucose concentration can be detected with high accuracy. Furthermore, the biosensors according to the first to fourth aspects of the present invention can be applied to non-invasive uses and wearable devices, are capable of being used continuously for a long time and repeatedly over a long period of time, and are considered to be suitably applicable to medical biosensors, rapid diagnostic kits and the like intended for non-invasive point-of-care. Brief Description of the Drawings

[0014] [Figure 1] FIG. 1 is a schematic cross-sectional view schematically showing the structure of the biosensor of the present invention. [Figure 2]It is a graph showing the relationship between the change in glucose concentration (change in time) and the response voltage (ΔV (mV)) in an example. [Figure 3] It is a graph showing the relationship between glucose concentration and response voltage (ΔV (mV)) in an example. [Figure 4] It is a graph showing the relationship between glucose concentration and response voltage (ΔV (mV)) in a comparative example. [Figure 5] It is a graph showing the XRD measurement results of vanadium oxide insulating layers A, B, and C produced in an example (comparative example). [Figure 6] It is a graph showing the XPS measurement results of vanadium oxide insulating layer A produced in an example (comparative example). [Figure 7] It is a graph showing the XPS measurement results of vanadium oxide insulating layer B produced in an example (comparative example). [Figure 8] It is a graph showing the XPS measurement results of vanadium oxide insulating layer C produced in an example. DETAILED DESCRIPTION OF EMBODIMENTS

[0015] Hereinafter, modes for carrying out the present invention will be described more specifically, but the scope of the present invention is not limited to the following modes.

[0016] FIG. 1 is a schematic cross-sectional view schematically showing the structure of the biosensor of the present invention. In the figure, 1 denotes a field effect transistor, S denotes a source, D denotes a drain, and G denotes a gate. 2 denotes an extended gate electrode connected to the gate G. The extended gate electrode 2 is composed of an insulating film 3 and an enzyme-immobilized film 4 formed on the surface of the insulating film 3.

[0017] In the figure, 7 denotes a detection unit of the biosensor, 6 denotes a liquid to be detected introduced into the detection unit 7, and the extended gate electrode 2 and a reference electrode 5 are provided so as to be immersed in the liquid to be detected 6. The reference electrode 5 is provided so as to face (opposite each other) the enzyme-immobilized film 4 of the extended gate electrode 2.

[0018] In the biosensor of the present invention, the insulating film 3 of the extended gate electrode 2 is characterized by containing amorphous vanadium oxide. Containing amorphous vanadium oxide means that amorphous vanadium oxide VO2 X This refers to a high ratio of [the crystalline component]. Specifically, in the X-ray diffraction of vanadium oxide constituting insulating film 3, the total peak area (peak area of ​​the crystalline component + amorphous component VO2) is high. X Amorphous component VO2 (in relation to the halo pattern area) X This refers to a large ratio of the halo pattern area, which is preferably 75% or more, more preferably 90% or more, and especially preferably 100%, i.e., when no peaks of crystalline components are observed, resulting in improved detection sensitivity. As described above, conventional EGFET-type biosensors are known to use a titanium oxide film as the insulating film of the extended gate electrode (Non-Patent Literature 1). However, by making the insulating film 3 contain amorphous vanadium oxide, the detection sensitivity of glucose in the test solution is dramatically improved compared to conventional EGFET-type biosensors. In addition, crystalline vanadium oxide (amorphous component in X-ray diffraction VO) can be used instead of amorphous vanadium oxide. X This method provides superior glucose detection sensitivity compared to using a device with a smaller ratio of halo pattern area.

[0019] Amorphous vanadium oxide is, as mentioned above, non-crystalline vanadium oxide (VO2). X That is, vanadium oxide that is not crystallized and does not have long-range order, but vanadium oxides include, for example, VO2, V2O3, V2O5, V3O5, V6O 13 Examples include V3O7, V4O9, and mixtures thereof.

[0020] In the biosensor of the present invention, the thickness of the insulating film 3 containing amorphous vanadium oxide is preferably 5 nm or more and 300 nm or less. If the thickness is less than 5 nm, there is a concern that the insulating properties will decrease and the detection sensitivity will decrease. If it exceeds 300 nm, there is a concern that the capacitance of the expanded gate will decrease and the glucose detection sensitivity will decrease. The thickness of the insulating film is more preferably 20 nm or more and 100 nm or less, which will more reliably provide superior detection sensitivity.

[0021] The insulating film 3 containing amorphous vanadium oxide may be formed on an electrode layer such as a thin film of titanium. Other support substrates, such as a glass substrate on which a thin metal film such as titanium has been formed by sputter deposition, can also be used as substrates on which the insulating film 3 is formed. In this case, the extended gate electrode 2 has a three-layer structure consisting of an ion-sensitive film (first layer) made of the insulating film 3 (VOx film) and an enzyme-immobilized film 4 formed on its surface, an electrode layer (second layer) on which the ion-sensitive film (first layer) is formed on its surface, and a support layer on which the electrode layer (second layer) is formed. Although titanium is an example of a material for forming the electrode layer, other metals can also be used. The support layer is formed of an insulating material such as glass or plastic.

[0022] The insulating film 3 containing amorphous vanadium oxide can be obtained, for example, by spin-coating a mixed solution of ammonium metavanadate, polyvinyl alcohol, and water onto an electrode layer such as the titanium thin film mentioned above, followed by drying and firing. The thickness of the insulating film 3 can be adjusted by adjusting the amount of polyvinyl alcohol, the spin-coating conditions, etc.

[0023] In the biosensor of the present invention, the enzyme-immobilized membrane 4 is formed on the surface of the insulating film 3 and has glycolytic enzymes such as glucose oxidase and hexonase immobilized on it. It is preferable that the enzyme-immobilized membrane 4 is made using the silk fibroin encapsulation method, as this further improves both the glucose detection sensitivity and the response voltage.

[0024] The enzyme-immobilized membrane of the silk fibroin inclusion method is an enzyme-immobilized membrane formed by immobilizing glucose oxidase within a silk fibroin membrane. For example, it can be formed by dropping an aqueous solution consisting of silk fibroin, glucose oxidase (enzyme), and water onto the surface of an insulating film 3, forming a film by spin coating or the like, and then immersing it in an ethanol aqueous solution to insolubilize the silk fibroin membrane containing the enzyme. When the enzyme-immobilized membrane is made of silk fibroin, both the detection sensitivity and response voltage of glucose are dramatically improved.

[0025] The glucose oxidase content in the enzyme-immobilized membrane 4 of the silk fibroin-based method is preferably in the range of 4.7 to 35% by mass relative to the mass of the enzyme-immobilized membrane 4. A higher glucose oxidase content improves the sensitivity of glucose detection, but if it exceeds 35% by mass, problems may arise such as the enzyme not being immobilized in the silk fibroin in the correct ratio or the immobilized enzyme leaking out during measurement. On the other hand, if it is less than 4.7% by mass, it may be difficult to detect glucose accurately in samples with low glucose concentrations, such as sweat or saliva.

[0026] The thickness of the enzyme-immobilized membrane 4 can be the same as that of conventional EGFET-type biosensors, and can be selected from a range of, for example, 0.1 to 10 μm. If it exceeds 10 μm, the immobilization treatment may not be sufficiently effective, and the enzyme-immobilized membrane may peel off. Also, if it is less than 0.1 μm and the amount of enzyme included is insufficient, it may be difficult to detect glucose with high accuracy when detecting glucose in low concentrations such as sweat or saliva.

[0027] In the biosensor of the present invention, glucose oxidase (enzyme) in the enzyme-immobilized membrane 4 acts on glucose in the liquid to be detected 6 introduced into the detection unit 7, and the electrons released are picked up by the reference electrode 5. The resulting change in voltage between the drain D and source S of the EGFET is used to detect the amount of glucose in the liquid to be detected 6.

[0028] A FET is a transistor that controls the current between the source and drain electrodes by controlling the density of electrons or holes through an electric field generated in the channel region by applying a voltage to the gate electrode. FETs operate by voltage drive to the gate, which is the control point. An EGFET is a field-effect transistor (FET) that has an extended gate electrode connected to the gate. In the biosensor of the present invention, the extended gate electrode is provided in the detection unit.

[0029] Examples (Example 1 of fabrication of a vanadium oxide insulating layer) A 10 mm square titanium sputtered film was formed by spin-coating a solution of ammonium metavanadate, polyvinyl alcohol, and water in a mass ratio of 1:1:100 at 6000 rpm for 30 seconds. The resulting film was pre-baked at 200°C for 10 minutes, and then fired at 600°C for 120 minutes to form an amorphous vanadium oxide insulating layer on the titanium film. The resulting insulating layer is referred to as insulating layer C.

[0030] (Example 2 of fabrication of a vanadium oxide insulating layer) A vanadium oxide insulating layer was formed on a titanium film in the same manner as in Example 1 of vanadium oxide insulating layer preparation, except that a solution prepared by mixing ammonium metavanadate, polyvinyl alcohol, and water in a mass ratio of 1:1:100 was replaced with a solution prepared by mixing ammonium metavanadate, polyvinyl alcohol, and ammonia water in a mass ratio of 1:1:100. The resulting insulating layer is referred to as insulating layer A.

[0031] (Example 3 of fabrication of a vanadium oxide insulating layer) A vanadium oxide insulating layer was formed on a titanium film in the same manner as in Example 1 of vanadium oxide insulating layer preparation, except that the solution prepared by mixing ammonium metavanadate, polyvinyl alcohol, and water in a mass ratio of 1:1:100 was replaced with a solution prepared by mixing ammonium metavanadate, polyvinyl alcohol, and ammonia water in a mass ratio of 2:1:100. The resulting insulating layer is referred to as insulating layer B.

[0032] X-ray diffraction and X-ray photoelectron spectroscopy were performed on the obtained insulating layer A. As a result, the vanadium oxide insulating layer was an insulating layer formed mainly of amorphous vanadium oxide, and the thickness thereof was 30 nm. From the X-ray diffraction measurement results of the obtained insulating layer A, no crystal peaks other than the peak of titanium as the electrode material and TiO2 generated by the oxidation reaction of titanium were observed, and the amorphous material is VO x was determined to be the main component of the film. Further, the obtained vanadium oxide insulating layer was analyzed by X-ray photoelectron spectroscopy to obtain V 4+ (VO2) and V 5+ it was revealed that the composition was derived from (V2O5). From this, V 4+ (VO2) and V 5+ it was determined to be an amorphous structure having the composition of (V2O5). From these analysis results, it was found that the vanadium oxide insulating layer is an insulating layer formed mainly of amorphous vanadium oxide.

[0033] (Enzyme-immobilized membrane: Preparation of silk fibroin membrane with immobilized glucose oxidase) Silk fibroin (manufactured by Matsuda Sericulture Farm; Nanofibroin (registered trademark) powder), glucose oxidase, and water were mixed at a mass ratio of 60:3:140 and sufficiently stirred. The aqueous solution thus obtained was dropped onto the surface of the insulating layer C obtained in the above (Preparation 1 of amorphous vanadium oxide insulating layer) and spin-coated. Thereafter, the product was immersed in an 80 Vol / % ethanol aqueous solution for 1 hour to insolubilize the silk fibroin enzyme membrane, and an extended gate electrode having an amorphous vanadium oxide insulating layer and an enzyme-immobilized layer (glucose oxidase-immobilized silk fibroin membrane) formed on the surface thereof was obtained.

[0034] (Measurement of response voltage) The obtained extended gate electrode was used as the extended gate electrode 2 of a biosensor having the structure schematically shown in Figure 1. The liquid to be detected 6 was introduced into the detection unit 7 with varying glucose concentrations, and the response voltage was measured at 37°C, which corresponds to the temperature of human body fluids. The results are shown in Figures 2 and 3. Specifically, the graph in Figure 2 shows the relationship between the change in glucose concentration (change over time) and the response voltage (ΔV(mV)). The graph in Figure 3 shows the relationship between glucose concentration and the response voltage (ΔV(mV)).

[0035] A biosensor (Comparative Example 1) was prepared in the same manner as in the example, except that the insulating layer 3 was formed from titanium dioxide instead of amorphous vanadium oxide. The liquid to be detected 6 was introduced into the detection unit 7 in the same manner as in the example, with varying glucose concentrations, and the response voltage was measured. The results are shown in Figure 4. Specifically, the graph in Figure 4 shows the relationship between glucose concentration and response voltage (ΔV(mV)) when using the biosensor of the comparative example.

[0036] As is clear from Figures 2-4, the biosensor in the example using amorphous vanadium oxide (VOx) as the insulating film on the extended gate electrode and the silk fibroin encapsulation method as the enzyme-immobilized membrane shows a dramatic improvement in detection sensitivity (approximately 10 times higher) and response voltage (2.5 times higher) compared to the comparative biosensor (conventional biosensor: insulating film; titanium dioxide). This result indicates that the biosensor of the present invention may enable accurate testing even for samples with low glucose concentrations, such as sweat and saliva. In other words, the present invention enables non-invasive measurement of saliva and other samples, and is expected to contribute to medical and healthcare applications.

[0037] (XRD measurement) For insulating layers A, B, and C obtained in the above-mentioned vanadium oxide insulating layer fabrication examples 1 to 3, the insulating layer (VO) was analyzed using an X-ray diffraction (XRD) apparatus. XStructural analysis of the thin film was performed. The results are shown in Figure 5. As is clear from Figure 5, diffraction peaks originating from crystalline VO2 or V2O5 can be confirmed in insulating layers A and B. This indicates that the obtained vanadium oxide thin film is a thin film containing VO2 or V2O5 crystals. On the other hand, for insulating layer C, no diffraction peaks originating from the crystal were observed within the measurement range (2θ: 10° to 70°). In X-ray diffraction measurements, excluding diffraction peaks of the substrate and underlying material, a structure in which no peaks appear at diffraction angles (2θ) between 10° and 70° is defined as amorphous. Therefore, from the results in Figure 5, it can be said that the vanadium oxide thin film of insulating layer C has an amorphous structure as its main component. It is thought that these diffraction peaks disappear and become invisible when the proportion of amorphous material in the thin film exceeds approximately 75% by mass. Furthermore, when an insulating layer is deposited on a Ti electrode under the fabrication conditions of the above-described examples, only peaks for the underlying Ti and its oxide, TiO2, may be observed. However, since some peaks originating from VO2 or V2O5 crystals are observed at different positions from those of Ti and TiO2, these peaks can be distinguished.

[0038] (XPS analysis) The results of X-ray photoelectron spectroscopy (XPS) analysis of insulating layers A, B, and C are shown in the left figures of Figures 6-8, respectively. The fitting analysis (V2p 3 / 2 The results (with peak magnification) are shown in the left figures of Figures 6-8, respectively. For insulating layers A and B, the obtained vanadium oxide thin film (V2p) was determined from the spectra and fitting analysis shown in the left figures of Figures 6 and 7. 3 / 2 ) contains V 4+ (VO2) and V 5+ It can be seen that the composition of the oxidized state of (V2O5) is included. Furthermore, the main component of insulating layer A (Figure 6) is V derived from VO2. 4+ This is a peak, and for insulating layer B (Figure 7), the V is derived from V2O5. 5+ It can be said that this is the peak.

[0039] Regarding the insulating layer C, the obtained vanadium oxide thin film (V2p) was obtained from the spectrum in the left figure of Figure 8 and its fitting analysis. 3 / 2 ) contains V 4+ (VO2) and V 5+ It can be seen that the composition of the oxidized state of (V2O5) is included. The ratios of these compositions are almost equal. As for the insulating layer C, the results of the XRD measurement above show that the main component of the thin film is an amorphous structure, so V 4+ (VO2) and V 5+ It is presumed that (V2O5) is in an amorphous state where molecules are bound together in a disordered manner. Table 1 shows the composition ratio (molar ratio) of VO2 and V2O5 calculated from the spectra in Figures 6-8.

[0040] [Table 1] [Explanation of Symbols]

[0041] 1. Field-effect transistor 2 Extended Grid Control 3. Insulating film 4. Enzyme-immobilized membrane 5 Reference electrode 6. Liquid to be detected 7 Detection unit

Claims

1. A biosensor that detects glucose concentration in a test solution using a field-effect transistor (EGFET) with an extended gate electrode, An extended gate electrode is provided in the detection unit into which the test solution is introduced, facing the reference electrode and connected to the gate unit. The extended gate electrode comprises an insulating film containing amorphous vanadium oxide, and an enzyme-immobilized film formed on the surface of the insulating film, on which glycolytic enzymes are immobilized.

2. The biosensor according to claim 1, wherein the enzyme-immobilized membrane is a silk fibroin membrane containing glucose oxidase.

3. The biosensor according to claim 1 or claim 2, wherein the thickness of the insulating film is 5 nm or more and 300 nm or less.

4. The biosensor according to claim 1, wherein the insulating film containing amorphous vanadium oxide has a structure in which, in the measurement results by an X-ray diffractometer, the diffraction angle (2θ) is 10° or more and 70° or less, and no vanadium oxide peak appears.

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