Enzyme electrode and sensor for glucose detection and application of enzyme electrode and sensor

Abstract

The invention relates to an enzyme electrode for glucose detection, the enzyme electrode comprises an electrode substrate layer and a sensing layer covering the electrode substrate layer, and the sensing layer comprises NAD (P) (H) dependent glucose oxidoreductase and NAD (P) (H) oxidoreductase; the enzyme electrode further comprises a first polymer layer which covers the sensing layer and is used for protecting the sensing layer and / or limiting permeation of glucose and / or interferents; the enzyme electrode further comprises NAD (P) (H) which is positioned on the sensing layer; and / or a derivative of NAD (P) (H), which is a polymer covalently bound to NAD (P) (H), located in the sensing layer and / or the first polymer layer. The enzyme electrode disclosed by the invention can realize stable, accurate and / or anti-interference continuous detection on the blood glucose level.

Description

Technical Field

[0001] The invention relates to the field of electrochemistry, and in particular to an enzyme electrode and a sensor for glucose detection and applications thereof. Background Art

[0002] Enzyme electrodes can be used as biosensors to detect and quantify specific chemicals. For example, enzyme electrodes can be made into glucose sensors to provide rapid and quantitative blood glucose monitoring (BGM) for personal blood glucose control.

[0003] As the incidence of diabetes continues to rise, blood glucose monitoring has become an integral part of daily management for diabetic patients. Traditional blood glucose testing methods, such as fingertip blood sampling, are accurate but invasive and inconvenient, limiting their use in continuous blood glucose monitoring. To address this problem, continuous glucose monitoring (CGM) technology has emerged, which provides continuous, real-time monitoring of blood glucose levels in a minimally invasive or non-invasive manner, thereby helping patients better manage their blood glucose.

[0004] Patent CN116698945A discloses a new type of continuous blood glucose monitoring technology, which uses an enzyme electrode system, whose core components include NAD (P) (H) dependent glucose oxidase or glucose dehydrogenase, cofactor NAD (P) (H) and NAD (P) (H) oxidoreductase, which can achieve direct electron transfer with the electrode. This direct electron transfer mechanism not only improves the response speed and sensitivity of the sensor, but also enhances its stability and anti-interference ability. Compared with the traditional first-generation CGM technology, the enzyme electrode system no longer relies on oxygen as an electron transfer agent, thereby overcoming the sensitivity and accuracy problems caused by oxygen concentration limitations. At the same time, compared with the second-generation technology, it does not need to use artificial electron mediators, improves system stability and eliminates the biological toxicity that may be caused by leakage of electron mediators.

[0005] However, due to the long-term contact and reaction between the electrode and body fluid, this new continuous blood glucose monitoring technology faces problems such as enzyme and cofactor loss, and interference from biochemical substances in the detection solution receptor. The accuracy and stability of continuous blood glucose monitoring technology are challenged.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to achieve stable, accurate, and / or interference-resistant continuous detection of blood sugar levels.

[0008] The purpose of the present invention can be achieved by the following technical solutions:

[0009] In a first aspect, the present invention provides an enzyme electrode for glucose detection, the enzyme electrode comprising an electrode substrate layer and a sensing layer covering the electrode substrate layer, wherein the sensing layer comprises: NAD(P)(H)-dependent glucose oxidoreductase and NAD(P)(H) oxidoreductase;

[0010] The enzyme electrode further comprises a first polymer layer covering the sensing layer, for protecting the sensing layer and / or for limiting the penetration of glucose and / or interfering substances;

[0011] The enzyme electrode further comprises: NAD(P)(H), which is located in the sensing layer; and / or, a NAD(P)(H) derivative, which is a polymer covalently bound to NAD(P)(H), located in the sensing layer and / or the first polymer layer.

[0012] The enzyme electrode of the present invention uses a polymer layer to cover the sensing layer, which plays the following roles: it has a fixing and stabilizing effect on the enzyme and / or cofactor (NAD(P)(H) and / or NAD(P)(H) derivative) on the surface of the sensing layer; and / or, it prevents the sensing layer from directly contacting the detection environment, reduces the mass transfer of glucose, oxygen, etc., and thus reduces the interference of the detection environment on the sensing layer. In some cases, the enzyme electrode of the present invention includes an NAD(P)(H) derivative, which is a covalent polymer of NAD(P)(H), which is covalently bound to the polymer, is more stable than NAD(P)(H), is not easy to lose, provides NAD(P)(H) for the sensing layer, and plays a role of supplementing or replacing NAD(P)(H), thereby solving the problem of unstable detection results caused by the loss of NAD(P)(H); in addition, the polymer structure of the NAD(P)(H) derivative can also play a role of embedding and stabilizing the enzyme and / or NAD(P)(H).

[0013] In some specific embodiments, the enzyme electrode further comprises a second polymer layer covering the first polymer layer for limiting the penetration of glucose and / or interfering substances, and the second polymer layer further comprises or does not comprise the NAD(P)(H) derivative.

[0014] In some specific embodiments, the enzyme electrode further comprises a supporting substrate, and the electrode substrate is located on the supporting substrate.

[0015] In some specific embodiments, the NAD(P)(H) covalently bound polymer includes a NAD(P)(H) cross-linked polymer or a NAD(P)(H) grafted polymer; preferably includes NAD(P)(H) cross-linked PEI, NAD(P)(H) cross-linked CMC or NAD(P)(H) grafted PVP.

[0016] In some specific embodiments, the NAD(P)(H) cross-linked PEI has a chemical structure as shown below:

[0017]

[0018] Herein, n1 represents an integer of 5 to 50, preferably 11; n2 represents an integer of 1 to 200, preferably 6.

[0019] In some specific embodiments, the NAD(P)(H) cross-linked CMC has the following chemical structure:

[0020]

[0021] Here, n represents an integer of 200 to 3000, and is preferably 400.

[0022] In some specific embodiments, the NADP + Grafted PVP has the following chemical structure:

[0023]

[0024] Among them, x=0.05~0.3, y=0.05~0.5, z=0.3~0.8, x+y+z=1; preferably, x=0.7, y=0.1, z=0.2.

[0025] In some specific embodiments, the first polymer layer and / or the second polymer layer are selected from polymer dissolution membranes or in situ synthesized polymer membranes; preferably, the polymer layer is selected from PEI cross-linked membranes, TPU membranes, PU membranes, PVP membranes, chitosan membranes, NAD(P)(H) cross-linked polymer membranes or NAD(P)(H) grafted polymer membranes.

[0026] In some specific embodiments, the sensing layer further comprises a crosslinking agent; preferably, the crosslinking agent is selected from one or more of epoxy crosslinking agents, glutaraldehyde and polyethylene glycol; more preferably, the crosslinking agent is selected from PEG or PEGDGE.

[0027] In some specific embodiments, the NAD(P)(H)-dependent glucose oxidoreductase is NADP + Glucose oxidase or NADP-dependent + Dependent on glucose dehydrogenase, the NADP + The oxidoreductase is diaphorase.

[0028] In some specific embodiments, the NAD(P)(H) is NADH, NADPH, NAD + and NADP +One or more of, wherein the NAD(P)(H) is NADH, NADPH, NAD + and NADP + In some specific embodiments, the NAD(P)(H) is NADP + , the NAD(P)(H) derivative is NADP + derivative.

[0029] In a second aspect, the present invention also provides a method for preparing the enzyme electrode described in any of the above items, the method comprising forming the sensing layer on the surface of the electrode substrate, and forming the first polymer layer on the surface of the sensing layer, and optionally, the method further comprises forming the second polymer layer on the surface of the first polymer layer.

[0030] In some specific embodiments, the sensing layer is formed by pull-up dip coating or drop coating, and the at least first polymer layer and / or the second polymer layer is formed by pull-up dip coating.

[0031] In a third aspect, the present invention further provides a sensor for glucose detection, wherein the sensor comprises the enzyme electrode described in any of the preceding items as a working electrode, and further comprises a counter electrode and a reference electrode.

[0032] In some specific embodiments, the counter electrode is a carbon electrode, and the reference electrode is an Ag / Cl reference electrode.

[0033] In a fourth aspect, the present invention further provides use of any of the aforementioned enzyme electrodes or sensors in the preparation of a continuous detection instrument for glucose.

[0034] In a fifth aspect, the present invention further provides a detection method, which comprises using any of the aforementioned sensors to continuously detect glucose.

[0035] In summary, the present invention has at least one of the following beneficial effects:

[0036] (1) The enzyme electrode includes a first polymer layer and / or a second polymer layer, which has the following functions: fixing and stabilizing the enzyme and / or cofactor (NAD(P)(H) and / or NAD(P)(H) derivatives) on the surface of the sensing layer; and / or preventing the sensing layer from directly contacting the detection environment, reducing the mass transfer of glucose, oxygen, etc., thereby reducing the interference of the detection environment on the sensing layer.

[0037] (2) In some cases, the enzyme electrode described in the present invention includes an NAD(P)(H) derivative, which is a covalent polymer of NAD(P)(H) that is covalently bound to the polymer, is more stable than NAD(P)(H), and is not easily lost. It provides NAD(P)(H) for the sensing layer, supplements or replaces NAD(P)(H), thereby solving the problem of unstable detection results caused by the loss of NAD(P)(H).

[0038] (3) The polymer structure of NAD(P)(H) derivatives can also encapsulate and stabilize enzymes and / or small molecules of NAD(P)(H).

[0039] (4) In some cases, the NAD(P)(H) derivative is NADP(P)(H) cross-linked PEI, which is negatively charged and has a negative charge on NAD. + or NADP + Has adsorption effect, which is beneficial to NAD + or NADP + stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 , Glucose titration curve of glucose sensor electrode (TPU polymer layer);

[0041] Figure 2 , Figure 1 The corresponding linear relationship graph;

[0042] Figure 3 , stability of glucose sensor electrode (TPU polymer layer) within 15 days at 37°C (5mM glucose environment);

[0043] Figure 4 , Glucose titration curves of the glucose sensor electrode (TPU polymer layer) on the 1st and 15th days;

[0044] Figure 5 , Figure 4 The corresponding linear relationship graph;

[0045] Figure 6 , Glucose titration curve of glucose sensor electrode (PVP-NADP polymer layer);

[0046] Figure 7 , Figure 6 The corresponding linear relationship graph;

[0047] Figure 8 , stability of glucose sensor electrode (PVP-NADP polymer layer) in 10 mM glucose solution;

[0048] Fig. 9 , glucose sensor electrode (PVP-NADP polymer layer) Day1-Day35 glucose titration curve;

[0049] Fig.10 , Fig. 9 The corresponding linear relationship graph;

[0050] Fig.11 ,Comparison of sensor performance with and without TPU polymer layer;

[0051] Fig.12 , whether there is fixed NADP in the TPU polymer layer + Comparison of sensor performance;

[0052] Fig.13 ,Comparison of sensor performance with and without PVP-NADP polymer layer;

[0053] Fig.14 , chemical formula structure of PVP-NADP grafted polymer;

[0054] Fig.15 、Glucose sensor electrode (NADP + Glucose titration curve of derivative dissolution film;

[0055] Fig.16 , Fig.15 The corresponding linear relationship graph;

[0056] Fig.17 、Glucose sensor electrode (NADP + Glucose titration curve of derivative cross-linked membrane);

[0057] Fig.18 , Fig.17 The corresponding linear relationship graph;

[0058] Fig.19 , Schematic diagram of PEGDGE structure;

[0059] Fig. 20 , PEI structure diagram, wherein m represents an integer greater than 0, and can represent an integer in the range of 5 to 50, for example, 6;

[0060] Fig.21 , electrochemical test results of two glucose sensor electrodes (control group, experimental group) on Day 0 overnight;

[0061] Fig. 22 , 12-day stability test of glucose sensor (experimental group);

[0062] Fig.23, schematic diagram of the structure of PEI grafted with NADP, wherein n1 represents an integer of 5 to 50, preferably 11; n2 represents an integer of 1 to 200, preferably 6;

[0063] Fig.24 , schematic diagram of the structure of CMC grafted with NADP, wherein n represents an integer of 200 to 3000, preferably 400;

[0064] Fig.25 , electrochemical test results of two glucose sensor electrodes (PEI grafted NADP, CMC grafted NADP);

[0065] Fig.26 , the test results of the current changes of the three sensor probes when galactose was used as an external interference;

[0066] Fig. 27 , when hydroxyurea is used as an external interfering substance, the current change test results of the three sensor probes when they are used as external interfering substances;

[0067] Fig.28 , when ascorbic acid is used as an external interference, the current change test results of the three sensor probes when they are used as external interferences;

[0068] Fig.29 , and the current change test results of the three sensor probes when xylose is used as an external interferent.

[0069] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0070] Enzyme Electrode

[0071] The present invention provides an enzyme electrode for glucose detection, the enzyme electrode comprising an electrode substrate layer and a sensing layer covering the electrode substrate layer, wherein the sensing layer comprises: NAD(P)(H)-dependent glucose oxidoreductase and NAD(P)(H) oxidoreductase;

[0072] The enzyme electrode further comprises a first polymer layer covering the sensing layer, for protecting the sensing layer and / or for limiting the penetration of glucose and / or interfering substances;

[0073] The enzyme electrode further comprises: NAD(P)(H), which is located in the sensing layer; and / or, a NAD(P)(H) derivative, which is a polymer covalently bound to NAD(P)(H), located in the sensing layer and / or the first polymer layer.

[0074] Electrode base layer

[0075] The electrode substrate layer is used to fix or hold the enzyme and allow electrons to flow through it and be conducted to the enzyme during detection.

[0076] In some embodiments, the electrode substrate layer has electrical conductivity, and / or has one or more of physical adsorption, chemical adsorption, and electrostatic adsorption to the enzyme, so that the enzyme is placed on the surface and inside of the conductive substrate without being lost. In some embodiments, the electrode substrate can be selected from a metal material or a carbon material. As metal materials, for example, gold (Au), platinum (Pt), silver (Ag) and palladium (Pd) can be cited. As carbon materials, for example, carbon can be cited, and as carbon, for example, graphite, carbon nanotubes, graphene, mesoporous carbon, etc. can be cited. In some specific embodiments, the electrode substrate layer is selected from noble metal nanoparticles, nanocarbon, conductive polymers and nanocomposites, such as ITO, graphene, carbon nanotubes, gold electrodes, silver electrodes, conductive hydrogels or conductors or semiconductors doped with nanoelectrodes.

[0077] Sensing layer

[0078] The sensing layer provides an enzyme for glucose catalysis reaction, the enzyme comprising NAD(P)(H) dependent glucose oxidoreductase and NAD(P)(H) oxidoreductase. In some embodiments, the NAD(P)(H) dependent glucose oxidoreductase is selected from NAD(P)(H) dependent glucose oxidase or glucose dehydrogenase. In some specific embodiments, the NAD(P)(H) dependent glucose dehydrogenase is a dehydrogenase of classification 1.1.1.47. In some specific embodiments, the NAD(P)(H) oxidoreductase is a diaphorase.

[0079] In some embodiments, the sensing layer further comprises NAD(P)(H) and / or NAD(P)(H) derivatives.

[0080] In some embodiments, the sensing layer includes NAD(P)(H) but not NAD(P)(H) derivatives. In some specific embodiments, the enzyme electrode includes the NAD(P)(H) derivatives, which are provided by other layer structures of the electrode, for example, by the first and / or second polymer layer. In some specific embodiments, the enzyme electrode only includes NAD(P)(H) but not the NAD(P)(H) derivatives.

[0081] In some embodiments, the sensor layer does not include NAD(P)(H) and / or NAD(P)(H) derivatives, and the NAD(P)(H) and / or NAD(P)(H) derivatives are provided by other layer structures of the electrode, for example, by the first and / or second polymer layer.

[0082] In some embodiments, the sensing layer includes one or more cross-linking agents.

[0083] In some embodiments, the sensing layer is coated with an enzyme mixture, and the enzyme mixture includes NAD(P)(H)-dependent glucose oxidoreductase (e.g., NAD(P)(H)-dependent glucose oxidase or glucose dehydrogenase) and NAD(P)(H) oxidoreductase. In some specific embodiments, the concentration of the NAD(P)(H)-dependent glucose oxidase or glucose dehydrogenase in the enzyme mixture is 5 mg / mL to 50 mg / mL, such as 10 mg / mL to 40 mg / mL, such as 15 mg / mL to 25 mg / mL. In some specific embodiments, the concentration of the NAD(P)(H) oxidoreductase in the enzyme mixture is 1 mg / mL to 8 mg / mL, such as 2 mg / mL to 8 mg / mL, such as 5 mg / mL to 8 mg / mL. In some specific embodiments, the enzyme mixture further comprises NAD(P)(H) and / or NAD(P)(H) derivatives, and the concentration of NAD(P)(H) and / or NAD(P)(H) derivatives in the enzyme mixture is 5 mg / mL to 75 mg / mL, such as 15 mg / mL to 60 mg / mL, such as 30 mg / mL to 40 mg / mL. In some specific embodiments, the NAD(P)(H) derivative is NADP of carboxymethyl cellulose (CMC). + The substitution product has a concentration of 20-115 mg / mL. In another embodiment, the NAD(P)(H) derivative is a PEI side chain derivative, and its concentration is 20-115 mg / mL. In some embodiments, the enzyme mixture also includes one or more cross-linking agents, and its concentration in the enzyme mixture is 5 mg / mL to 20 mg / mL, such as 5 mg / mL to 10 mg / mL, such as 15 mg / mL to 20 mg / mL.

[0084] NAD(P)(H) and / or NAD(P)(H) derivatives

[0085] In the present invention, NAD(P)(H) is NAD + , NADH, NADP + and NADPH. NAD(H) is a general term for NAD + and NADH, NADP(H) is NADP+ and NADPH.

[0086] In the present invention, NAD(P)(H) derivatives refer to NAD + , NADH, NADP + , one or more of NADPH are covalently linked to other molecules to form derivatives.

[0087] In some embodiments, the enzyme electrode does not include NAD(P)(H) derivatives, and the cofactor is provided by NAD(P)(H). In some embodiments, the enzyme electrode does not include NAD(P)(H), and the NAD(P)(H) derivative replaces NAD(P)(H) to provide the cofactor. In some embodiments, the enzyme electrode includes both NAD(P)(H) and NAD(P)(H) derivatives, and the cofactor is provided by both.

[0088] In some embodiments, the NAD(P)(H) derivative is a polymer covalently bound to NAD(P)(H). In some specific embodiments, the polymer covalently bound to NAD(P)(H) is a NAD(P)(H) cross-linked polymer or a NAD(P)(H) grafted polymer.

[0089] In some specific embodiments, the NAD(P)(H) derivative is formed by cross-linking NAD(P)(H) with CMC, wherein NAD(P)(H) cross-links CMC, for example, + It is cross-linked with carboxymethyl cellulose (CMC) via a cross-linking agent PEGDGE. Optionally, the molecular weight of the carboxymethyl cellulose is 90,000 to 700,000, for example, 250,000. Optionally, the NAD(P)(H) derivative has a chemical structure as shown below:

[0090]

[0091] Here, n represents an integer of 200 to 3000, and is preferably 400.

[0092] In some specific embodiments, the NAD(P)(H) cross-linked CMC is located in the sensing layer and / or the first polymer layer.

[0093] In some specific embodiments, the NAD(P)(H) derivative is NAD(P)(H) cross-linked PEI, for example, formed by cross-linking the side chain of NAD(P)(H) with boric acid and PEI. Optionally, the PEI can be branched PEI 800, PEI1800, PEI 3000, PEI 10000, PEI 25000, PEI 70000, PEI 100000. In some specific embodiments, the NAD(P)(H) cross-linked PEI has the following chemical formula structure:

[0094]

[0095] Here, n represents an integer of 200 to 3000, and is preferably 400.

[0096] In some specific embodiments, the NAD(P)(H) cross-linked PEI is located in the first polymer layer.

[0097] In some embodiments, the NAD(P)(H) derivative may also be a NAD(P)(H) grafted polymer, such as NAD(P)(H) grafted PVP. The NAD(P)(H) grafted PVP may be formed into a film by dissolution or cross-linking. In some specific embodiments, the NADP + Grafted PVP has the following chemical structure:

[0098]

[0099] Among them, x=0.05~0.3, y=0.05~0.5, z=0.3~0.8, x+y+z=1; preferably, x=0.7, y=0.1, z=0.2.

[0100] Crosslinking agent

[0101] The crosslinking agent is used to induce at least partial crosslinking of the crosslinking precursor to achieve the effect of forming a stable structure. In some embodiments, the crosslinking agent is selected from epoxy crosslinking agent, glutaraldehyde, PEG, PEGDGE and PEI. In some specific embodiments, the sensing layer includes a crosslinking agent; specifically, the crosslinking agent is added to the enzyme mixture, and a force is formed with the components in the enzyme mixture to stabilize the system, and the force may include covalent bonds, charge adsorption, hydrogen bonds, etc. In some specific embodiments, the crosslinking agent is polyethylene glycol (e.g., PEG400), which forms hydrogen bonds with components in the system (e.g., enzymes, NAD(P)(H) and / or NAD(P)(H)). In some specific embodiments, the crosslinking agent is PEI, which generates charges through in situ crosslinking, adsorbs and stabilizes components in the system (e.g., enzymes, NAD(P)(H) and / or NAD(P)(H)). In one of the aforementioned embodiments, NAD(P)(H) and / or NAD(P)(H) are grafted with PVP polymer via covalent bonds to form a polymer layer to stabilize the polymer layer.

[0102] Polymer layer

[0103] The enzyme electrode of the present invention comprises a first polymer layer covering the sensing layer, wherein the polymer layer is used to protect the sensing layer and / or to limit the penetration of glucose and / or interfering substances.

[0104] In some specific embodiments, the enzyme electrode further comprises a second polymer layer covering the first polymer layer, for limiting the penetration of glucose and / or interfering substances.

[0105] In some specific embodiments, the enzyme electrode has only one polymer layer, including only the first polymer layer but not the second polymer layer, and the polymer layer is used to protect the sensing layer and limit the penetration of glucose and interferents.

[0106] In some embodiments, the polymer layer includes a NAD(P)(H) derivative. In some embodiments, the NAD(P)(H) derivative is located in the first polymer layer; in some embodiments, the NAD(P)(H) derivative is located in the first polymer layer and the second polymer layer; in some embodiments, the NAD(P)(H) derivative is located in the sensing layer and the first polymer layer. For a detailed description of NAD(P)(H) derivatives, see "NAD(P)(H) and / or NAD(P)(H) derivatives".

[0107] In some embodiments, the polymer layer is formed by dissolution or in situ synthesis.

[0108] In some embodiments, the polymer layer is selected from a polymer dissolution film, an in situ synthesized polymer film or a NAD(P)(H) grafted polymer film; preferably, selected from a PEI cross-linked film, a TPU film, a PU film, a PVP film, a chitosan film, a NAD(P)(H) cross-linked polymer or a NAD(P)(H) grafted polymer film.

[0109] Support substrate

[0110] The support substrate is used to provide support for the electrode substrate and other layer structures covering the electrode substrate. The electrode support substrate can be conductive or insulating, including but not limited to metals (such as titanium and its alloys) and bioceramics (such as calcium phosphate (such as HAp and α, β-tricalcium phosphate [α, β]-TCP]), calcium carbonate, bioactive glass and glass ceramics, alumina and zirconium oxide) and natural (such as collagen, gelatin, silk protein [SF], chitosan, hyaluronic acid [HA], gellan gum [GG] and its derivatives, alginate and synthetic polymers (such as polyurethane [PU] and polycaprolactone [PCL]) and combinations thereof. The hardness of the substrate is not limited. When the enzyme electrode is applied to an implantable glucose detector, the electrode support substrate preferably uses a softer material to improve wearing comfort; when the enzyme electrode is applied to a test strip, the electrode support substrate preferably uses a harder material.

[0111] sensor

[0112] The present invention provides a sensor for grape detection, the sensor comprising any of the above enzyme electrodes. In some embodiments, the sensor is a three-electrode chemical system, the enzyme electrode is used as a working electrode, and the sensor further comprises a counter electrode and a reference electrode. In some specific embodiments, the counter electrode is a carbon electrode, and the reference electrode is an Ag / Cl reference electrode.

[0113] Preparation method

[0114] Step 1: print (preferably screen print) the electrode base layer of the working electrode, the counter electrode and the reference electrode onto a supporting substrate; Step 2: apply the enzyme mixture on the surface of the working electrode to form a sensing layer; and, Step 3: form a first polymer layer on the surface of the sensing layer; and, optional Step 4: form a second polymer layer on the surface of the first polymer layer.

[0115] The process of fixing the enzyme mixture on the working electrode can be achieved by a pull-up dip coating or a drip coating method. In some embodiments, the enzyme mixture is drip coated. For example, a 5 μL volume of the enzyme mixture is dripped onto the substrate working electrode using a drip coating device, and then left to dry in the air. The drying time is 20 to 80 minutes, such as 30 minutes or 60 minutes. The standing temperature can be 20 to 40°C, such as 25°C, or 40°C.

[0116] In some embodiments, the enzyme mixture is applied by dipping and pulling. For example, a pulling machine immerses the top of the substrate electrode into the enzyme mixture, then takes out the enzyme mixture and leaves it to dry for a period of time, repeats this process several times, and finally leaves it to dry in the air. The immersion depth is 2.5 to 3.5 mm, preferably 3 mm, and the pulling rate is 1500 to 3000 μm / s. The interval time can be 60 to 150 s, preferably 100 s, and the number of repetitions is 10 to 30 times.

[0117] In some embodiments, the enzyme mixture needs to be dried after being added to the electrode by dip coating. The drying time is 20 to 80 minutes, such as 30 minutes or 60 minutes. The standing temperature is 20 to 40°C, such as 25°C, or 40°C.

[0118] The first polymer layer can be formed by pulling and dipping the first polymer layer cross-linking liquid and then curing it. In some embodiments, the first polymer layer cross-linking liquid contains PEI and PEGDGE. The PEI content in the cross-linking liquid ranges from 10% to 20%, and the mass ratio is 15 mg / mL; the PEGDGE content in the cross-linking liquid is 50-200 mg / mL, and the NAD(P)(H) content in the cross-linking liquid ranges from 10-100 mg / mL, which can be 30-80 mg / mL, or 50-70 mg / mL. The number of pulls of the cross-linking liquid ranges from 10 to 30. The drying method is dried at room temperature for 2-12 hours.

[0119] The first polymer layer can also be directly dissolved from a high molecular polymer. Such high polymers include PU, PVP, TPU, NAD(P)(H) grafted membranes, and chitosan membranes. In some embodiments, the first polymer layer is formed by dissolving a NAD(P)(H) grafted polymer or directly dissolved from a high molecular polymer. In some specific embodiments, NADP + The grafted polymer solution is coated on the electrode surface by dip coating and then left to dry. +The amount of grafted polymer in the dissolution solution can be 35 mg / mL, 60 mg / mL, 100 mg / mL, preferably 60 mg / mL. The standing time is 8 to 24 hours, for example, 8 to 12 hours, or 20 to 24 hours. The standing temperature can be 20 to 50°C, for example, 25 to 45°C, or 30 to 40°C. In some embodiments, the first polymer layer is directly dissolved from the high molecular polymer TPU, and the TPU content in the dissolution solution ranges from 6% to 12%, and the mass ratio is 60 mg / mL to 120 mg / mL.

[0120] In some specific embodiments, the protective layer is formed by cross-linking the NAD(P)(H) grafted polymer. + The grafted polymers are mixed to form a cross-linking solution, which is then coated on the electrode surface by dip coating and then left to dry. + The amount of grafted polymer in the cross-linking solution can be 35 mg / mL, 60 mg / mL, 100 mg / mL (actually 60 mg / mL). The type of cross-linking agent can be PEGDGE, PEI or PEG. The amount of cross-linking agent can be 50 mg / mL, 65 mg / mL, 85 mg / mL, preferably 100 mg / mL. The standing time after the two are mixed can be 0.1 to 0.8 h, preferably 0.25 h, for example, 0.25 h, 0.5 h or 0.75 h. The standing time after the cross-linking solution is dipped into the electrode is 8 to 24 h, for example, 8 to 12 h, or 20 to 24 h. The standing temperature can be 20 to 50 ° C, for example, 20 to 40 ° C, or 35 to 50 ° C.

[0121] In some embodiments, the first polymer layer can be formed by in-situ crosslinking. In a specific embodiment, the crosslinking precursor is PEI, and the crosslinking agent is PEG and PEGDGE. In a specific embodiment, the crosslinking precursor is PVP-NADP, and the crosslinking agent is PEGDGE, PEG and PEI. By adjusting the side chain polymerization, the overall function of the polymer layer can be affected, such as hydrophilicity, hydrophobicity, oxygen permeability, etc.

[0122] In some embodiments, the first polymer layer can be prepared by pulling and dipping. The pulling machine immerses the top of the base electrode into the film liquid, then pulls out the film liquid and lets it stand for a period of time to dry, and repeats this process many times, and finally lets it stand in the air to dry. The immersion depth is 2.5 to 3.5 mm, preferably 3 mm, and the pulling rate is 500 to 3000 μm / s. The interval time can be 10 minutes to 60 minutes, preferably 30 minutes, and the number of repetitions can be 5 to 50, preferably 9 times. After the dip coating is completed, the electrode surface is dried in the dark. The drying time is 10 to 24 hours, for example, 12 hours or 20 hours. The standing temperature can be 20-40°C, for example, 25°C or 40°C.

[0123] Optionally, the method of the present invention further includes forming a second polymer layer on the surface of the first polymer layer, and the second polymer layer can refer to the first polymer layer and be formed by direct dissolution, in-situ crosslinking, pull-up dip coating, etc. For example, the second polymer layer can also be directly dissolved from a high molecular polymer. Such polymers include PU, PVP, TPU, and chitosan film. In some embodiments, the first polymer layer is directly dissolved from a high molecular polymer TPU, and the TPU content in the dissolution solution ranges from 6% to 12%, and the mass ratio is 60 mg / mL to 120 mg / mL.

[0124] How to use

[0125] The enzyme electrode and sensor of the present invention is based on the direct electron transfer of NAD(P)(H)-dependent glucose oxidoreductase to detect the glucose concentration in the solution. One of its use scenarios is to implant the electrode tip of the sensor into the subcutaneous tissue fluid to test the glucose level in real time and continuously measure it.

[0126] In an embodiment, the test solution of the sensor electrode is a human body simulation fluid, the ambient temperature is 37 degrees Celsius, and the oxygen level is normal. It can also be used in a low oxygen environment.

[0127] In some embodiments, the reaction signal of the sensor is tested and the signal is automatically collected by an electrochemical workstation. The electrochemical workstation is connected to the three-electrode system of the sensor electrode by a wire. The electrochemical workstation records the real-time current by chronoamperometry (CA). The applied voltage of the chronoamperometry can be in the range of -0.1 to +0.05V, for example -0.1V, +0.05V. The measurement data acquisition frequency can be selected as once per second.

[0128] In some other embodiments, the sensor response signal is measured by an electronic circuit chip chronoamperometry to record temporary data. The temporary data is automatically and periodically transmitted to the processing device via remote signal transmission, such as a Bluetooth signal. In one embodiment, the measurement chip uses a fixed -0.1V voltage to drive the sensor electrode for measurement, collects a data point every 0.5 seconds, and transmits data to the receiving host every 1 minute.

[0129] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0130] Example Description

[0131] The SBF solution involved in the following examples refers to a human body simulation solution, and its formula is as follows: pH 7.4, containing NaCl 135mM, KCl 5mM, MgCl2 1.5mM, CaCl2 2.5mM, Na2HPO4 1mM, Na2SO4 0.5mM, NaHCO3 4.2mM, Tris 5mM. The buffer used in the enzyme mixture involved in the following examples is SBF solution. The manufacturers, models and important parameters of some reagents involved in the following examples are as follows: NADP +(Shanghai Shangke, SZ-3002·Na, 1184-16-3); polyethyleneimine (PEI) (McLean, 9002-98-6, MW3000); polyethylene glycol diglycidyl ether (PEGDGE) (McLean, 72207-80-8, Mn=500); sodium carboxymethyl cellulose (CMC-Na) (McLean, 004-32-4, 50-200mPa.s); 3-aminophenylboronic acid (McLean, 30418-59-8, 98%); 4-carboxyphenylboronic acid (McLean, 14047-29-1, 97%); MES (McLean, 1266615-59-1, 99%); N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (McLean, 7365-45-9, 99%); 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (McLean, 25952-53-8, 98.5%); N-hydroxysuccinimide (NHS) (McLean, 6066-82-6, 99%); triethanolamine (McLean, 102-71-6, 98%), boric acid (McLean, 10043-35-3, 99.99%); glucose dehydrogenase (Shenzhen Jinhe Biotechnology, Oxzyme-201); diaphorase (Shenzhen Jinhe Biotechnology, Oxzyme-2101).

[0132] Example 1 Preparation and performance testing of sensor (TPU polymer layer)

[0133] An enzyme electrode sensor containing a TPU polymer layer was prepared as follows: (1) The electrode substrate of the sensor was formed by screen printing, and the electrode substrate was a three-electrode system including a working electrode, a counter electrode and a reference electrode. Afterwards, an enzyme mixture was prepared according to the formula described in Table 2, and the enzyme mixture was applied to the surface of the working electrode by dip coating to form a sensing layer. The immersion depth was 2.5 mm, and the pulling rate was 1000 μm / s. The interval time was 60 s, and the number of repetitions was 10 times. After the enzyme mixture was dip-coated onto the electrode, it was dried at 40°C for 30 minutes. (2) In a 100,000-class clean room and an environment containing tetrahydrofuran vapor, the TPU polymer was dissolved in 90% tetrahydrofuran to form a membrane solution, and the formula was shown in Table 3. (3) The membrane solution was evenly coated on the glucose sensor containing the sensing layer by the dip-coating method. The immersion depth was 3.0 mm, the pulling rate was 600 μm / s, the interval was 30 minutes, and it was repeated 10 times. After the dip coating is completed, the electrode surface is dried at 25°C in the dark for 12 hours to obtain a sensor electrode that can be used for testing.

[0134] Place the prepared sensor electrode in a constant temperature SBF solution and perform a constant voltage chronoamperometry test at a voltage of -0.05V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, start magnetic stirring (800 rpm). After the reaction starts, wait for the current to stabilize, add a concentrated glucose solution for titration, so that the glucose concentration gradient in the reaction solution increases, ranging from 0 to 20mM ( Figure 1 The change in reaction rate is fed back by the reaction current, corresponding to the change in glucose concentration, and the corresponding relationship between the glucose titration concentration and the reaction current is plotted (such as Figure 2 ). The calibration curve slope, linear strength and sensitivity of the sensor are summarized in Table 1. The results show that the sensor containing TPU as the polymer layer can reflect a linear relationship and continuously respond to glucose.

[0135] This example also conducted a stability test on the sensor electrode at a voltage of -0.05 V for 15 consecutive days in a SBF solution containing 5 mM glucose. Figure 3 As shown, the sensor containing TPU has good stability when working continuously for a long time (15 days).

[0136] In this embodiment, the sensor was tested in SBF solution for 15 consecutive days. The titration curves and linear relationships on Day 0 and Day 14 are shown in Figure 1. Figure 4-5 As shown, the slope of the curve, R 2 The results show that the electrode can maintain good glucose detection activity, sensitivity and stability over a long period of time (15 days).

[0137] Table 1 Calibration curve information

[0138] Glucose Sensor Slope 1.22 <![CDATA[R 2 ]]> 0.9920 Sensitivity 1.193

[0139] Table 2 Sensing layer formula

[0140]

[0141]

[0142] Table 3 Polymer layer formulation

[0143] TPU 100 mg Tetrahydrofuran 10 mL

[0144] Table 4 Calibration curve information

[0145] Slope <![CDATA[R 2 ]]> Sensitivity Day 1 (day0) 1.22 0.9920 1.19 Day 15 (day 14) 1.34 0.9903 1.32

[0146] Example 2 Preparation and performance testing of sensor (PVP-NADP polymer layer)

[0147] An enzyme electrode sensor containing a PVP-NADP polymer layer was prepared as follows: (1) vinylpyridine and an initiator were fully mixed and dissolved in N,N-dimethylformamide (DMF) at a mass ratio of 400:1, and heated in a water bath at 60°C for 10 hours. The reaction product was separated by precipitation with a methanol solution to obtain a first intermediate product. Then, the first intermediate product and bromoacetic acid were dissolved in DMF at a mass ratio of 1:1, and heated in a water bath at 80°C for 18 hours. The reaction product was separated by precipitation with an ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product was mixed with nicotinamide adenine dinucleotide phosphate (NADP + ) was dissolved in DMF at a mass ratio of 10:1, and heated in a 70°C water bath for 5 h. The reaction product was separated and purified by methanol solution, and the PVP-NADP grafted polymer was obtained after drying. Its structure is as follows Fig.14 As shown. (2) The electrode substrate of the sensor electrode is formed by screen printing, and the electrode substrate is a three-electrode system including a working electrode, a counter electrode and a reference electrode. The enzyme mixture is prepared according to the formula shown in Table 6, and the enzyme mixture is coated on the surface of the working electrode by dip coating to form a sensing layer. The immersion depth is 2.5 mm, and the pulling rate is 1000 μm / s. The interval time is 60 s, and the number of repetitions is 10 times. After the enzyme mixture is dip-coated on the electrode, it is dried at 40°C for 30 minutes. (3) On the surface of the sensing layer, a layer of PVP-NADP polymer layer is formed by dip coating, and the formula of the polymer layer is shown in Table 7 (wherein the PVP-NADP grafted polymer is prepared by the method described in step (1)). The immersion depth is 3.0 mm, the pulling rate is 600 μm / s, the interval is 30 minutes, and it is repeated 10 times. After the dip coating is completed, the electrode surface is dried at 25°C in the dark for 12 hours. A sensor electrode that can be tested and used is obtained.

[0148] Place the prepared sensor electrode in a constant temperature SBF solution and perform a constant voltage chronoamperometry test at a voltage of -0.05 V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, start magnetic stirring (800 rpm). After the reaction starts, wait until the current is stable, and add a concentrated glucose solution for titration.

[0149] The results are as follows Figure 6-7 shown. Figure 6-7 The titration curve of the sensor at different concentrations of glucose (0-20 mM) within 4 h is shown, and the calibration curve slope, linear strength and sensitivity of the sensor are summarized in Table 5. The test results show that the sensor using PVP-NADP as the polymer layer can reflect a linear relationship and continuous response to glucose.

[0150] This example also tested the stability of the sensor in SBF solution containing 10 mM glucose for 35 days (see Figure 8 ) and the glucose titration curve and linear relationship over 35 consecutive days (see Figure 9-10 ). Figure 9-10 The results show that the sensor can continuously and stably respond to glucose for 35 days and has a linear relationship. Table 8 also shows the linear relationship and sensitivity of the sensor. This example proves that the sensor has good stability under long-term operation, and the linearity and sensitivity of the response to glucose concentration are almost unchanged.

[0151] Table 5 Calibration curve information

[0152] Glucose Sensor Slope 2.07 R2 0.9994 Sensitivity 2.065

[0153] Table 6 Sensing layer formula

[0154] Concentration (mg / mL) Volume (μL) Final concentration (mg / mL) Diaphorase 33.2 2 2.2 Glucose dehydrogenase 20 14 9.3 <![CDATA[NADP + ]]> 76.5 14 35.7 Total volume 30

[0155] Table 7 Polymer layer formulation

[0156] PVP-NADP Graft Polymer 60 mg Anhydrous ethanol 0.95 mL HEPES (10 mM, pH = 8) 0.05 mL

[0157] Table 8 Calibration curve information

[0158] Slope <![CDATA[R 2 ]]> Sensitivity Day 1 2.07 0.9994 2.065 Day 7 1.93 0.9992 1.978 Day 14 2.25 0.9961 2.205 Day 20 1.92 0.9963 1.953 Day 30 1.91 0.9996 1.892 Day 35 1.77 0.9996 1.757

[0159] Example 3 Sensor Performance Comparison (Example 1 Sensor vs. Bare Electrode)

[0160] The performance of the sensor electrode (containing a TPU polymer layer) and the bare sensor electrode (not containing a TPU polymer layer) shown in Example 1 were compared (constant voltage chronoamperometry, voltage -0.05 V). Fig.11 The comparison of the output current of the sensor containing TPU polymer layer and bare electrode after sensing glucose is shown. With the maximum current of each being 1, the presence of the TPU polymer layer enables the sensor electrode to have a wider linear range that is more suitable for the working environment of continuous blood glucose monitoring.

[0161] Example 4 Sensor Performance Comparison (Example 1 Sensor vs No NADP + sensor)

[0162] The sensor electrode (the sensing layer on the working electrode is fixed with NADP) shown in Example 1 was measured by constant voltage chronoamperometry (voltage -0.05 V). + ) and the sensing layer on the working electrode has no NADP + The performance of the sensor (other electrodes are the same as those shown in Example 1) was tested and compared. The test results are shown in Fig.12 shown. Fig.12The results show that the 24 h in the presence of 5 mM glucose and 50 μM NADP + Comparison of the current responses of the two sensing layer electrodes in a solution of . As shown in the figure, the sensing layer does not contain NADP + The electrode has no signal response to external glucose, which shows that the TPU polymer layer is effective in limiting NADP + The penetration of molecules is restricted.

[0163] Example 5 Sensor Performance Comparison (Example 2 Sensor vs. Bare Electrode)

[0164] The performance of the sensor electrode containing the PVP-NADP polymer layer shown in Example 2 and the sensor electrode without the PVP-NADP polymer layer (bare electrode) were tested and compared (constant voltage chronoamperometry, voltage -0.05V). Fig.13 The comparison of the output current of sensors containing PVP-NADP polymer layer and those without such polymer layer after sensing glucose is shown. Taking the respective maximum current as 1, the presence of PVP-NADP polymer layer can make the sensor electrode have a wider linear range that is more suitable for the working environment of continuous blood glucose monitoring.

[0165] Example 6 Preparation and Performance Testing of Sensor (PVP-NADP Polymer Layer-TPU Polymer Layer)

[0166] An enzyme electrode sensor containing a PVP-NADP polymer layer and a TPU polymer layer was prepared as follows: (1) First, the electrode substrate of the sensor electrode was formed by screen printing, and the electrode substrate was a three-electrode system including a working electrode, a counter electrode and a reference electrode. Secondly, the enzyme mixture was coated on the surface of the working electrode by dip coating to form a sensing layer. The enzyme mixture formula is shown in Tables 9-10. The immersion depth was 2.5 mm, and the pulling rate was 1000 μm / s. The interval time was 60 s, and the number of repetitions was 10 times. After the enzyme mixture was dip coated on the electrode, it was dried at 40°C for 30 minutes. (2) On the surface of the sensing layer, a layer of PVP-NADP polymer layer (first polymer layer) was formed by dip coating. The formula of the first polymer layer is shown in Tables 9 and 10 (the preparation method of the PVP-NADP grafted polymer is shown in Example 2), corresponding to the dissolution and cross-linking film-forming methods, respectively. The immersion depth was 3.0 mm, the pulling rate was 1000 μm / s, the interval was 10 minutes, and it was repeated 5 times. After the dip coating is completed, the electrode surface is dried at 25°C in the dark for 12 hours. (3) A TPU polymer layer (second polymer layer) is formed on the surface of the first polymer layer by dip coating. The formula of the second polymer layer is shown in Table 9-10. The immersion depth is 4.0 mm, the pulling rate is 600 μm / s, the interval is 30 minutes, and it is repeated 10 times. After the dip coating is completed, the electrode surface is dried at 25°C in the dark for 12 hours. A sensor electrode that can be tested and used is obtained.

[0167] Place the prepared sensor electrode in a constant temperature SBF solution and perform a constant voltage chronoamperometry test at a voltage of -0.05 V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, start magnetic stirring (800 rpm). After the reaction starts, wait until the current is stable, and add a concentrated glucose solution for titration.

[0168] Fig.15 is a titration curve of a sensor electrode containing a first polymer layer formed by dissolution under conditions of different glucose concentrations; Fig.16 It is the linear relationship between its corresponding sensor signal and different concentrations of glucose. Fig.17 is a titration curve of a sensor electrode containing a first polymer layer formed in a cross-linking manner under conditions of different glucose concentrations; Fig.18 The corresponding sensor signal is linearly related to different concentrations of glucose. The experimental results show that when the sensing layer does not contain NADP + In this case, PVP-NADP as the first polymer layer can provide the NADP required for the reaction. + , TPU as the second polymer layer can reduce the sensitivity of the enzyme layer to glucose. The two together enhance the stability of the enzyme and + The molecular limit is reduced and the linear range of the measurement is broadened.

[0169] Table 9 Sensor electrode structure composition (dissolution)

[0170]

[0171] Table 10 Sensor electrode structure composition (cross-linking)

[0172]

[0173]

[0174] Example 7 Preparation and Performance Testing of Sensor (NADP Cross-linked PEI Polymer Layer)

[0175] A sensor containing a NADP-crosslinked PEI polymer layer was prepared as follows: (1) First, the electrode substrate of the sensor electrode was formed by screen printing, and the electrode substrate was a three-electrode system including a working electrode, a counter electrode and a reference electrode. Secondly, the enzyme mixture was coated on the surface of the working electrode by dip coating to form a sensing layer. The enzyme mixture formula is shown in Table 11. The immersion depth was 2.5 mm, and the pulling rate was 1000 μm / s. The interval time was 60 s, and the number of repetitions was 10 times. After the enzyme mixture was dip coated on the electrode, it was dried at 40°C for 30 minutes. (2) On the surface of the sensing layer, a PEI-PEGDGE polymer layer was formed by dip coating, and the formula of the polymer layer was shown in Table 12. The immersion depth was 3.0 mm, the pulling rate was 600 μm / s, the interval was 30 minutes, and it was repeated 10 times. After the dip coating was completed, the electrode surface was dried at 25°C in the dark for 10 hours. A sensor electrode that can be tested and used was obtained.

[0176] The prepared sensor electrode was placed in a constant temperature SBF solution, and a constant voltage chronoamperometry test was performed under the control of an electrochemical workstation (Ivium, Palmsense, etc.) at a voltage of -0.05 V. At the same time, magnetic stirring (800 rpm) was turned on. Fig.21 The comparison of two sensor electrodes in a solution containing 5 mM glucose over 18 h is shown. Fig.19 ) and PEI( Fig. 20 ), which can effectively fix NADP on the electrode + . According to the ratio shown in Table 11-12, the enzyme solution containing PEGDGE and the membrane solution containing PEI are mixed to form a water-insoluble membrane on the electrode surface. This process requires a long time to solidify. After the complete solidification, the formed membrane is insoluble in water and no swelling phenomenon in water is observed. There is no special requirement for temperature and humidity during the solidification process of this method. After the membrane is formed, the PEI and NADP in the membrane layer + Ion adsorption can make NADP+ The membrane is covered on the electrode and the substrate is added to perform the chronoamperometry test. + The film layer is electrochemically active and has good stability (see Figure 21-22 ).

[0177] Fig.21 This is a comparison between the control group and the experimental group on day 0. The control group, that is, without forming a polymer layer with PEGDGE and PEI, directly soaked the electrode with NADP at the same concentration as that used in the experimental group for cross-linking. + , using glucose as substrate for testing. The results showed that if there was no cross-linking, the current would return to zero overnight; the experimental group, that is, using PEGDGE and PEI to form a film to cross-link NADP + The overnight chronoamperometry shows that the current dropped by only 5nA overnight, and the performance was retained by 83.3%. The current of the control group returned to zero after overnight. Fig. 22 This is the daily stability test for the experimental group. No NADP is added during the test. + , only glucose was added as substrate, and the current was basically stable. From the 1st day to the 12th day, the current retained 96.3%.

[0178] Table 11 Sensing layer formula

[0179]

[0180] Table 12 Polymer layer formulation

[0181] Solution Solvents concentration Polyethyleneimine (PEI) HEPES 200mg / mL <![CDATA[NADP + ]]> HEPES 40mg / mL

[0182] Example 8 Preparation and Performance Test of Sensor (NADP Cross-linked PEI Added to Sensing Layer-TPU Polymer Membrane, or NADP Cross-linked CMC Added to Sensing Layer-TPU Polymer Membrane)

[0183] The sensor containing NADP cross-linked PEI polymer layer was prepared as follows: (1) Preparation of NADP cross-linked PEI: Prepare PEGDGE, PEI, and 3-APBA solutions in different proportions, with the solvent being 10% ethanol aqueous solution; Take equal volumes of the three solutions and mix them, stir them magnetically at room temperature until there is no stratification, then add a certain amount of NADP +Solution (solvent is boric acid solution with pH=7-8); continue to stir the mixed solution until the color of the solution changes from clear and transparent to brownish yellow, transfer the solution to a dialysis bag (molecular weight 1000), dialyze with deionized water for 3-5 times, transfer the dialyzed product to a freeze dryer for freeze drying; the brownish yellow powder after freeze drying is the PEI-NADP derivative. (2) Preparation of NADP cross-linked CMC: prepare CMC-Na into a 0.5%-2% solution, using MES buffer (pH=5-6) as the solvent; prepare EDC and NHS solutions, using MES buffer (pH=5-6) as the solvent; mix the above three solutions in a certain proportion, and stir magnetically until completely mixed; add a certain amount of NADP + solution (the solvent is a boric acid solution with a pH of 7 to 8); the mixed solution is stirred overnight, a certain amount of triethanolamine solution is added, and stirred for 30 minutes; the above solution is transferred to a dialysis bag (molecular weight 1000), and dialyzed with deionized water 3-4 times; after the dialysis is completed, the solution in the dialysis bag is transferred to a freeze dryer for freeze drying, and the dried powder sample is the CMC-NADP derivative. (3) The electrode substrate of the sensor electrode is formed by screen printing, and the electrode substrate is a three-electrode system including a working electrode, a counter electrode and a reference electrode. Secondly, the enzyme mixture is coated on the surface of the working electrode by dip coating to form a sensing layer. The enzyme mixture formula is shown in Table 13 (where NADP + The derivative is the cross-linked product prepared in step (1) or (2). The immersion depth is 2.5 mm, and the pulling rate is 1000 μm / s. The interval time is 60 s, and the number of repetitions is 10 times. After the enzyme mixture is dip-coated on the electrode, it is dried at 40°C for 30 minutes. (4) On the surface of the sensing layer, a TPU polymer layer is formed by dip-coating. The formula of the polymer layer is shown in Table 10. The immersion depth is 3.0 mm, the pulling rate is 600 μm / s, the interval is 30 minutes, and it is repeated 10 times. After the dip-coating is completed, the electrode surface is dried at 25°C in the dark for 12 hours. A sensor electrode that can be tested and used is obtained.

[0184] The prepared sensor electrode was placed in a constant temperature SBF solution and tested by constant voltage chronoamperometry at a voltage of -0.05 V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, magnetic stirring (800 rpm) was turned on. After the reaction started, when the current was stable, 5 mM glucose was added and the current feedback of the sensor electrode was observed. In this embodiment, linear polyethyleneimine (PEI) was cross-linked with NAD(P)+ through an epoxy compound to form Fig.23 NADP + Derivatives (PEI-NADP), and cross-linked with NAD(P)+ by carboxymethyl cellulose (CMC) Fig.24The NADP derivative (CMC-NADP) shown in Table 13 was prepared, and the electrodes and corresponding sensors shown in Table 13 were prepared, and electrochemical tests were performed. The test results are shown in Fig.25 This result reflects that these two NADP + The derivatives have NADP + The catalytic effect can be used in the reaction system, especially in the sensing layer.

[0185] Table 13 PEI-NAD(P)+ derivative electrode structure composition

[0186]

[0187] Example 9 Comparison of Sensor Anti-interference Performance (Example 1 Sensor vs. Abbott Probe, Silicon-based Probe)

[0188] The sensor shown in Example 1 was compared with commercially available probes (Abbott, FreeStyle Libre) and probes (silicon-based, silicon-based GS1) (constant voltage chronoamperometry, voltage -0.05V): Each sensor electrode was placed in a SBF solution at a constant temperature of 37 degrees Celsius, and a constant voltage chronoamperometry test with a voltage of +0.05V was performed under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, magnetic stirring (800 rpm) was turned on. After the reaction started, 6.66mM glucose was added, and when the current was stable, the recommended concentration of interference was added to the solution, and the change in feedback current was observed.

[0189] In this example, under the interference conditions under the registration review guidelines, the chronoamperometry was performed in a human body simulation fluid containing glucose to compare the working conditions and current responses of the three probes. Figure 26-29 , as shown in Table 14. Figure 26-29 The current change test results when galactose, hydroxyurea, ascorbic acid and xylose are added as external interferences are shown in the chart. The data in the chart show that the sensor described in Example 1 of the present invention has almost no current fluctuation when external interference is added, compared with the other two sensor probes, showing better anti-interference performance.

[0190] Table 14

[0191]

[0192] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An enzyme electrode for glucose detection, characterized in that: The enzyme electrode comprises an electrode substrate layer and a sensing layer covering the electrode substrate layer, wherein the sensing layer comprises: NAD(P)(H)-dependent glucose oxidoreductase and NAD(P)(H) oxidoreductase; The enzyme electrode further comprises a first polymer layer covering the sensing layer, for protecting the sensing layer and / or for limiting the penetration of glucose and / or interfering substances; The enzyme electrode further comprises: NAD(P)(H), which is located in the sensing layer; and / or a derivative of NAD(P)(H), which is a polymer covalently bound to NAD(P)(H), located in the sensing layer and / or the first polymer layer.

2. The enzyme electrode according to claim 1, characterized in that The enzyme electrode further comprises a second polymer layer covering the first polymer layer for limiting the penetration of glucose and / or interfering substances, and the second polymer layer further comprises or does not comprise the NAD(P)(H) derivative.

3. The enzyme electrode according to claim 1, characterized in that The enzyme electrode further comprises a supporting substrate, and the electrode substrate is located on the supporting substrate.

4. The enzyme electrode according to any one of claims 1 to 3, characterized in that: The polymer covalently bound to NAD(P)(H) includes a NAD(P)(H) cross-linked polymer or a NAD(P)(H) grafted polymer; preferably includes NAD(P)(H) cross-linked PEI, NAD(P)(H) cross-linked CMC or NAD(P)(H) grafted PVP; more preferably, the NAD(P)(H) cross-linked PEI is located in the first polymer layer, the NAD(P)(H) cross-linked CMC is located in the sensing layer and / or the first polymer layer, and the NAD(P)(H) grafted PVP is located in the first polymer layer and / or the second polymer layer.

5. The enzyme electrode according to claim 4, characterized in that The NAD(P)(H) cross-linked PEI has a chemical structure as shown below: Herein, n1 represents an integer of 5 to 50, preferably 11; n2 represents an integer of 1 to 200, preferably 6.

6. The enzyme electrode according to claim 4, characterized in that The NAD(P)(H) cross-linked CMC has a chemical structure as shown below: Here, n represents an integer of 200 to 3000, and is preferably 400.

7. The electrode according to claim 4, characterized in that The NAD(P)(H)-grafted PVP has the chemical structure shown below: Among them, x=0.05~0.3, y=0.05~0.5, z=0.3~0.8, x+y+z=1; preferably, x=0.7, y=0.1, z=0.

2.

8. The enzyme electrode according to claim 1 or 2, characterized in that: The first polymer layer and / or the second polymer layer are selected from polymer dissolution membranes or in-situ synthesized polymer membranes; preferably, the polymer layer is selected from PEI cross-linked membranes, TPU membranes, PU membranes, PVP membranes, chitosan membranes, NAD(P)(H) cross-linked polymer membranes or NAD(P)(H) grafted polymer membranes.

9. The enzyme electrode according to claim 1, characterized in that The sensing layer further comprises a crosslinking agent; preferably, the crosslinking agent is selected from epoxy crosslinking agent, glutaraldehyde, polyethylene glycol, PEG or PEGDGE.

10. The enzyme electrode according to claim 1, characterized in that The NADP + The NAD(P)(H)-dependent glucose oxidoreductase is an NAD(P)(H)-dependent glucose oxidase or an NAD(P)(H)-dependent glucose dehydrogenase, and the NAD(P)(H) oxidoreductase is a diaphorase.

11. A method for preparing the enzyme electrode according to any one of claims 1 to 10, characterized in that: The method includes forming the sensing layer on the surface of the electrode substrate, and forming the first polymer layer on the surface of the sensing layer. Optionally, the method also includes forming the second polymer layer on the surface of the first polymer layer.

12. A sensor for glucose detection, characterized in that: The sensor comprises the enzyme electrode according to any one of claims 1 to 10 as a working electrode, and further comprises a counter electrode and a reference electrode.

13. The sensor according to claim 12, characterized in that The counter electrode is a carbon electrode, and the reference electrode is an Ag / Cl reference electrode.

14. Use of the enzyme electrode according to any one of claims 1 to 10 or the sensor according to claim 12 or 13 in preparing a continuous detection instrument for glucose.

15. A detection method, characterized in that: The method comprises using the sensor according to claim 12 or 13 to continuously detect glucose.

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