Enzyme electrode and sensor for glucose measurement and use thereof

Abstract

The present invention relates to an enzyme electrode for glucose measurement. The enzyme electrode comprises an electrode substrate layer and a sensing layer covering the electrode substrate layer, wherein the sensing layer comprises: an NAD(P)(H)-dependent glucose oxidoreductase and an NAD(P)(H) oxidoreductase. The enzyme electrode further comprises a first polymer layer covering the sensing layer, the first polymer layer being used for protecting the sensing layer and / or restricting the permeation of glucose and / or an interferant. The enzyme electrode further comprises NAD(P)(H) which is located on the sensing layer; and / or an NAD(P)(H) derivative which is a polymer covalently bound to NAD(P)(H) and located on the sensing layer and / or the first polymer layer. The enzyme electrode of the present invention can achieve stable, accurate and / or anti-interference continuous measurement of the blood glucose level.

Description

Enzyme electrode, sensor for glucose detection and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of electrochemistry, in particular to an enzyme electrode, a sensor for glucose detection and application thereof. BACKGROUND

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

[0003] With the rising incidence of diabetes, blood glucose monitoring has become an indispensable part of the daily management of diabetic patients. Traditional blood glucose detection methods, such as finger prick blood sampling, are accurate but invasive and inconvenient, limiting their application in continuous blood glucose monitoring. To solve this problem, continuous glucose monitoring (CGM) technology has emerged, which provides continuous, real-time blood glucose level monitoring through minimally invasive or non-invasive methods, thereby helping patients better manage blood glucose.

[0004] Patent CN116698945A discloses a new continuous blood glucose monitoring technology, which uses an enzyme electrode system. The core components of the enzyme electrode system 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 electronic mediators, improving the stability of the system and eliminating the biological toxicity that may be caused by the leakage of electronic mediators.

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

[0006] Therefore, the present application is proposed.

[0007] SUMMARY

[0008] The present application aims to achieve stable, accurate, and / or anti-interference continuous detection of blood glucose levels.

[0009] The object of the present application can be achieved by the following technical solutions.

[0010] In a first aspect, the present application provides an enzyme electrode for glucose detection, 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.

[0011] 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 interferents.

[0012] The enzyme electrode further comprises: NAD(P)(H) located in the sensing layer; and / or 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.

[0013] The enzyme electrode of the present application uses a polymer layer to cover the sensing layer, which has the following effects: fixing and stabilizing the enzymes and / or cofactors (NAD(P)(H) and / or NAD(P)(H) derivative) on the surface of the sensing layer; and / or avoiding direct contact of the sensing layer with the detection environment, reducing the mass transfer of glucose, oxygen, etc., thereby reducing the interference of the detection environment on the sensing layer. In some cases, the enzyme electrode of the present application comprises NAD(P)(H) derivative, which is a NAD(P)(H) covalent polymer covalently bound to the polymer, more stable than NAD(P)(H), not easy to lose, provides NAD(P)(H) for the sensing layer, plays a complementary or substitutive role for 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 NAD(P)(H) derivative can also play a role in embedding and stabilizing the enzymes and / or NAD(P)(H).

[0014] 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 interferents, and the second polymer layer does or does not comprise the NAD(P)(H) derivative.

[0015] In some specific embodiments, the enzyme electrode further comprises a support substrate, and the electrode substrate is located on the support substrate.

[0016] In some embodiments, the NAD(P)(H) covalently bound polymer comprises a NAD(P)(H) cross-linked polymer or a NAD(P)(H) grafted polymer; preferably comprises a NAD(P)(H) cross-linked PEI, a NAD(P)(H) cross-linked CMC or a NAD(P)(H) grafted PVP.

[0017] In some embodiments, the NAD(P)(H) cross-linked PEI has a chemical structure as shown below:

[0018] wherein n1 represents an integer from 5 to 50, preferably 11; and n2 represents an integer from 1 to 200, preferably 6.

[0019] In some embodiments, the NAD(P)(H) cross-linked CMC has a chemical structure as shown below:

[0020] wherein n represents an integer from 200 to 3000, preferably 400.

[0021] In some embodiments, the NADP + grafted PVP has a chemical structure as shown below:

[0022] wherein x = 0.05 to 0.3, y = 0.05 to 0.5, z = 0.3 to 0.8, and x + y + z = 1; preferably, x = 0.7, y = 0.1, and z = 0.2.

[0023] In some embodiments, the first polymer layer and / or the second polymer layer is selected from a polymer dissolution membrane or an in-situ synthesized polymer membrane; preferably, the polymer layer is selected from a PEI cross-linked membrane, a TPU membrane, a PU membrane, a PVP membrane, a chitosan membrane, a NAD(P)(H) cross-linked polymer membrane or a NAD(P)(H) grafted polymer membrane.

[0024] In some embodiments, the sensing layer further comprises a cross-linking agent; preferably, the cross-linking agent is selected from one or more of an epoxy cross-linking agent, glutaraldehyde and polyethylene glycol; more preferably, the cross-linking agent is selected from PEG or PEGDGE.

[0025] In some embodiments, the NAD(P)(H)-dependent glucose oxidoreductase is a NADP + -dependent glucose oxidase or a NADP + -dependent glucose dehydrogenase, the NADP + oxidoreductase is a diaphorase.

[0026] In some embodiments, the NAD(P)(H) is one or more of NADH, NADPH, NAD + and NADP + In some embodiments, the NAD(P)(H) is one or more of NADH, NADPH, NAD + and NADP + In some embodiments, the NAD(P)(H) is NADP + , and the NAD(P)(H) derivative is NADP + derivative.

[0027] In a second aspect, the present application further provides a method for preparing the enzyme electrode of any one of the preceding aspects, 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.

[0028] In some embodiments, the sensing layer is formed by dip coating or drop coating, and the at least first polymer layer and / or the second polymer layer is formed by dip coating.

[0029] In a third aspect, the present application further provides a sensor for glucose detection, wherein the sensor comprises the enzyme electrode of any one of the preceding aspects as a working electrode, and further comprises a counter electrode and a reference electrode.

[0030] In some embodiments, the counter electrode is a carbon electrode, and the reference electrode is an Ag / Cl reference electrode.

[0031] In a fourth aspect, the present application further provides use of the enzyme electrode or the sensor of any one of the preceding aspects in the preparation of a continuous glucose detection instrument.

[0032] In a fifth aspect, the present application further provides a detection method, the method comprising continuous glucose detection using the sensor of any one of the preceding aspects.

[0033] In summary, the present application has at least one of the following advantages:

[0034] (1) The enzyme electrode comprises the first polymer layer and / or the second polymer layer, which has the following effects: fixing and stabilizing the enzyme and / or the co-factor (NAD(P)(H) and / or NAD(P)(H) derivative) on the surface of the sensing layer; and / or, avoiding direct contact of the sensing layer with the detection environment, reducing the mass transfer of glucose, oxygen, etc., thereby reducing the interference of the detection environment on the sensing layer.

[0035] (2) In some cases, the enzyme electrode of the present application comprises NAD(P)(H) derivatives, which are NAD(P)(H) covalent polymers, covalently bound on the polymer, more stable than NAD(P)(H), not easy to lose, providing NAD(P)(H) for the sensing layer, playing a complementary or replacement role for NAD(P)(H), thereby solving the problem of unstable detection results caused by the loss of NAD(P)(H).

[0036] (3) The polymer structure of NAD(P)(H) derivatives can also play a role in embedding and stabilizing NAD(P)(H) for enzymes and / or small molecules.

[0037] (4) In some cases, the NAD(P)(H) derivative is NADP(P)(H) cross-linked PEI, PEI is negatively charged, and has an adsorption effect on NAD + or NADP + , which is conducive to the stability of NAD + or NADP + . BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1, glucose sensor electrode (TPU polymer layer) glucose titration curve;

[0039] Figure 2, the corresponding linear relationship diagram of Figure 1;

[0040] Figure 3, stability of glucose sensor electrode (TPU polymer layer) in 15 days at 37°C (5mM glucose environment);

[0041] Figure 4, glucose titration curve of glucose sensor electrode (TPU polymer layer) on day 1 and day 15;

[0042] Figure 5, the corresponding linear relationship diagram of Figure 4;

[0043] Figure 6, glucose sensor electrode (PVP-NADP polymer layer) glucose titration curve;

[0044] Figure 7, the corresponding linear relationship diagram of Figure 6;

[0045] Figure 8, stability of glucose sensor electrode (PVP-NADP polymer layer) in 10mM glucose solution;

[0046] Figure 9, glucose titration curve of glucose sensor electrode (PVP-NADP polymer layer) Day1-Day35;

[0047] Figure 10, the corresponding linear relationship diagram of Figure 9;

[0048] Figure 11, comparison of sensor performance with and without TPU polymer layer;

[0049] Figure 12, comparison of sensor performance with and without immobilized NADP + in TPU polymer layer;

[0050] Figure 13, comparison of sensor performance with and without PVP-NADP polymer layer;

[0051] Figure 14, chemical structure of PVP-NADP graft polymer;

[0052] Figure 15, glucose titration curve of glucose sensor electrode (NADP + derivative dissolving membrane);

[0053] Figure 16, linear relationship graph corresponding to Figure 15;

[0054] Figure 17, glucose titration curve of glucose sensor electrode (NADP + derivative crosslinked membrane);

[0055] Figure 18, linear relationship graph corresponding to Figure 17;

[0056] Figure 19, structural schematic diagram of PEGDGE;

[0057] Figure 20, structural schematic diagram of PEI, wherein m represents an integer greater than 0, which can represent an integer in the range of 5-50, for example, 6;

[0058] Figure 21, electrochemical test results of two kinds of glucose sensor electrodes (control group, experimental group) overnight on Day 0;

[0059] Figure 22, test of 12-day stability of glucose sensor (experimental group);

[0060] Figure 23, structural schematic diagram of PEI grafting NADP, wherein n1 represents an integer of 5-50, preferably 11; n2 represents an integer of 1-200, preferably 6;

[0061] Figure 24, structural schematic diagram of CMC grafting NADP, wherein n represents an integer of 200-3000, preferably 400;

[0062] Figure 25, electrochemical test results of two kinds of glucose sensor electrodes (PEI grafting NADP, CMC grafting NADP);

[0063] Figure 26, test results of current change of three kinds of sensor probes when galactose is used as an external interference;

[0064] Figure 27, test results of current change of three kinds of sensor probes when hydroxyurea is used as an external interference;

[0065] Figure 28, current change test results of three sensor probes as an added interferent when ascorbic acid is an added interferent;

[0066] Figure 29, current change test results of three sensor probes as an added interferent when xylose is an added interferent.

[0067] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings. DETAILED DESCRIPTION

[0068] Enzyme electrode

[0069] The present application provides an enzyme electrode for glucose detection, which 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.

[0070] 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 interferents.

[0071] The enzyme electrode further comprises: NAD(P)(H) in the sensing layer; and / or NAD(P)(H) derivatives, which are polymers covalently bound to NAD(P)(H), in the sensing layer and / or the first polymer layer.

[0072] Electrode substrate layer

[0073] The electrode substrate layer is used to fix or accommodate enzymes and allow electrons to flow through it to the enzyme during detection.

[0074] In some embodiments, the electrode base layer has electrical conductivity, and / or, has one or more of physical adsorption, chemical adsorption, electrostatic adsorption to the enzyme, thereby allowing the enzyme to be placed on and inside the surface of the electrically conductive base without loss. In some embodiments, the electrode base can be selected from a metal material or a carbon material. As a metal material, for example, gold (Au), platinum (Pt), silver (Ag), and palladium (Pd) can be mentioned. As a carbon material, for example, carbon, as carbon, for example, graphite, carbon nanotube, graphene, mesoporous carbon, etc. can be mentioned. In some specific embodiments, the electrode base layer is selected from noble metal nanoparticles, nanocarbon, conductive polymer, and nanocomposite, such as ITO, graphene, carbon nanotube, gold electrode, silver electrode, conductive hydrogel, or a conductor or semiconductor doped with nanoelectrode.

[0075] Sensing layer

[0076] The sensing layer provides an enzyme for the catalytic reaction of glucose, which includes 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 yellow enzyme.

[0077] In some embodiments, the sensing layer further includes NAD(P)(H) and / or NAD(P)(H) derivative.

[0078] In some embodiments, the sensing layer includes NAD(P)(H) but not NAD(P)(H) derivative. In some specific embodiments, the enzyme electrode includes the NAD(P)(H) derivative, which is 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 includes only NAD(P)(H) but not the NAD(P)(H) derivative.

[0079] In some embodiments, the sensor layer does not include NAD(P)(H) and / or NAD(P)(H) derivative, which is provided by other layer structures of the electrode, for example, by the first and / or second polymer layer.

[0080] In some embodiments, the sensing layer includes one or more cross-linking agents.

[0081] In some embodiments, the sensing layer is coated with an enzyme cocktail comprising 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 NAD(P)(H)-dependent glucose oxidase or glucose dehydrogenase has a concentration of 5 mg / mL to 50 mg / mL, e.g., 10 mg / mL to 40 mg / mL, e.g., 15 mg / mL to 25 mg / mL in the enzyme cocktail. In some specific embodiments, the NAD(P)(H) oxidoreductase has a concentration of 1 mg / mL to 8 mg / mL, e.g., 2 mg / mL to 8 mg / mL, e.g., 5 mg / mL to 8 mg / mL in the enzyme cocktail. In some specific embodiments, the enzyme cocktail further comprises NAD(P)(H) and / or NAD(P)(H) derivative, which has a concentration of 5 mg / mL to 75 mg / mL, e.g., 15 mg / mL to 60 mg / mL, e.g., 30 mg / mL to 40 mg / mL in the enzyme cocktail. In some specific embodiments, the NAD(P)(H) derivative is carboxymethylcellulose (CMC) substituted NADP + with a concentration of 20-115 mg / mL. In another example, the NAD(P)(H) derivative is PEI side chain derivative with a concentration of 20-115 mg / mL. In some embodiments, the enzyme cocktail further comprises one or more cross-linking agents, which has a concentration of 5 mg / mL to 20 mg / mL, e.g., 5 mg / mL to 10 mg / mL, e.g., 15 mg / mL to 20 mg / mL in the enzyme cocktail.

[0082] NAD(P)(H) and / or NAD(P)(H) derivative

[0083] In the present disclosure, NAD(P)(H) refers to NAD + , NADH, NADP + , and NADPH. NAD(H) refers to NAD + and NADH. NADP(H) refers to NADP + and NADPH.

[0084] In the present disclosure, NAD(P)(H) derivative refers to one or more of NAD + , NADH, NADP + , NADPH covalently linked to other molecules to form a derivative.

[0085] In some embodiments, the enzyme electrode does not comprise NAD(P)(H) derivative, and NAD(P)(H) provides the co-factor. In some embodiments, the enzyme electrode does not comprise NAD(P)(H), and the NAD(P)(H) derivative replaces NAD(P)(H) to provide the co-factor. In some embodiments, the enzyme electrode comprises both NAD(P)(H) and NAD(P)(H) derivative, and both of them together provide the co-factor.

[0086] 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.

[0087] In some specific embodiments, the NAD(P)(H) derivative is a NAD(P)(H) cross-linked CMC, which is cross-linked from NAD(P)(H) and CMC, for example, NADP cross-linked CMC cross-linked from NADP and CMC, or NADH cross-linked CMC cross-linked from NADH and CMC. In some specific embodiments, the NAD(P)(H) cross-linked CMC is cross-linked from NADP and CMC, and the CMC has a molecular weight of 90000-700000, for example, 250000. In some specific embodiments, the NAD(P)(H) cross-linked CMC has a chemical structure as shown below: + In some specific embodiments, the NAD(P)(H) derivative is a NAD(P)(H) cross-linked CMC, which is cross-linked from NAD(P)(H) and CMC, for example, NADP cross-linked CMC cross-linked from NADP and CMC, or NADH cross-linked CMC cross-linked from NADH and CMC. In some specific embodiments, the NAD(P)(H) cross-linked CMC is cross-linked from NADP and CMC, and the CMC has a molecular weight of 90000-700000, for example, 250000. In some specific embodiments, the NAD(P)(H) cross-linked CMC has a chemical structure as shown below:

[0088] wherein n represents an integer of 200-3000, preferably 400.

[0089] In some specific embodiments, the NAD(P)(H) cross-linked CMC is located in the sensing layer and / or the first polymer layer.

[0090] In some specific embodiments, the NAD(P)(H) derivative is a NAD(P)(H) cross-linked PEI, which is cross-linked from the side chain of NAD(P)(H) and boric acid and PEI, for example, NADP cross-linked PEI cross-linked from NADP and boric acid and PEI, or NADH cross-linked PEI cross-linked from NADH and boric acid and PEI. In some specific embodiments, the PEI can be branched PEI 800, PEI 1800, PEI 3000, PEI 10000, PEI 25000, PEI 70000, or PEI 100000. In some specific embodiments, the NAD(P)(H) cross-linked PEI has a chemical structure as shown below:

[0091] wherein n represents an integer of 200-3000, preferably 400.

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

[0093] In some embodiments, the NAD(P)(H) derivative can also be a NAD(P)(H) grafted polymer, such as a NAD(P)(H) grafted PVP. The NAD(P)(H) grafted PVP can be film formed by dissolution or cross-linking. In some specific embodiments, the NADP + The grafted PVP has a chemical structure as shown below:

[0094] wherein x = 0.05-0.3, y = 0.05-0.5, z = 0.3-0.8, and x+y+z = 1; preferably, x = 0.7, y = 0.1, and z = 0.2.

[0095] Cross-linking agent

[0096] The cross-linking agent is used to initiate at least partial cross-linking of the cross-linking precursor, to form a stable structure. In some embodiments, the cross-linking agent is selected from the group consisting of an epoxy cross-linking agent, glutaraldehyde, PEG, PEGDGE, and PEI. In some specific embodiments, the sensing layer comprises a cross-linking agent; in particular, the cross-linking agent is added to the enzyme mixture, to form an interaction with the components in the enzyme mixture to stabilize the system, which can include covalent bond, charge adsorption, hydrogen bond, etc. In some specific embodiments, the cross-linking agent is polyethylene glycol (e.g., PEG400), which forms hydrogen bond with the components in the system (e.g., enzyme, NAD(P)(H), and / or NAD(P)(H)). In some specific embodiments, the cross-linking agent is PEI, which generates charges to adsorb and stabilize the components in the system (e.g., enzyme, NAD(P)(H), and / or NAD(P)(H)) by in-situ cross-linking. In the previously mentioned example, the NAD(P)(H) and / or NAD(P)(H) form a grafted product with the PVP polymer through covalent bond, to form a polymer layer to stabilize the system.

[0097] Polymer layer

[0098] The enzyme electrode of the present application comprises a first polymer layer overlying the sensing layer. The polymer layer is used to protect the sensing layer, and / or, to limit the permeation of glucose and / or interferents.

[0099] In some specific embodiments, the enzyme electrode further comprises a second polymer layer overlying the first polymer layer, to limit the permeation of glucose and / or interferents.

[0100] In some specific embodiments, the enzyme electrode has only one polymer layer, which is the first polymer layer, and does not have a second polymer layer, and the polymer layer is used to protect the sensing layer, and to limit the permeation of glucose and interferents.

[0101] In some embodiments, the polymer layer comprises NAD(P)(H) derivatives. In some specific embodiments, the NAD(P)(H) derivatives are located in the first polymer layer; in some specific embodiments, the NAD(P)(H) derivatives are located in the first and second polymer layers; in some specific embodiments, the NAD(P)(H) derivatives are located in the sensing layer and the first polymer layer. For detailed description of NAD(P)(H) derivatives, see “NAD(P)(H) and / or NAD(P)(H) derivatives”.

[0102] In some embodiments, the polymer layer is formed by dissolution or in-situ synthesis.

[0103] In some embodiments, the polymer layer is selected from a polymer dissolution film, an in-situ synthesized polymer film or an NAD(P)(H) grafted polymer film; preferably, from a PEI cross-linked film, a TPU film, a PU film, a PVP film, a chitosan film, an NAD(P)(H) cross-linked polymer or an NAD(P)(H) grafted polymer film.

[0104] Supporting substrate

[0105] The supporting substrate is used to provide support for the electrode substrate and other layer structures overlaid on the electrode substrate. The electrode supporting substrate can be conductive or insulating, including but not limited to metals (such as titanium and its alloys) and bioceramics (such as calcium phosphates (e.g. HAp and a, b-tricalcium phosphate [a, b]-TCP)), calcium carbonate, bioactive glass and glass-ceramics, alumina and zirconia) and natural (such as collagen, gelatin, silk fibroin [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, etc. The hardness of the substrate is not limited, when the enzyme electrode is applied to an implantable glucose detector, the electrode supporting substrate preferably uses a slightly soft material to improve the wearing comfort; when the enzyme electrode is applied to a test paper, the electrode supporting substrate preferably uses a slightly hard material.

[0106] Sensor

[0107] The present application provides a sensor for glucose detection, the sensor comprising the enzyme electrode of any one of the preceding. In some embodiments, the sensor is a three-electrode chemical system, the enzyme electrode as the working electrode, the sensor further comprising 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.

[0108] Preparation method

[0109] Step one, printing (preferably screen printing) electrode substrate layer of working electrode, counter electrode and reference electrode onto a support substrate; Step two, coating enzyme mixture on the surface of the working electrode to form a sensing layer; and, Step three, forming a first polymer layer on the surface of the sensing layer; and, optionally, Step four, forming a second polymer layer on the surface of the first polymer layer.

[0110] The process of enzyme mixture immobilization on the working electrode can be achieved by pull-up dip coating or drop coating method. In some embodiments, the enzyme mixture is drop coated. For example, a volume of 5 μΐ of enzyme mixture is drop coated on the working electrode of the substrate using a drop coating device, and then left to dry in air. The drying time is 20-80 minutes, for example 30 minutes or 60 minutes. The temperature of the standing can be 20-40 °C, for example 25 °C, or 40 °C.

[0111] In some embodiments, the enzyme mixture is applied by pull-up dip coating. For example, a pull-up machine dips the top end of the substrate electrode into the enzyme mixture, pulls it out of the enzyme mixture, and leaves it to dry for a period of time, and repeats multiple times, and finally left to dry in air. The dipping depth is 2.5 to 3.5 mm, preferably actually 3 mm, and the pull-up rate is 1500-3000 μιη / s. The interval time can be 60-150 s, preferably 100 s, and the number of repetitions is 10-30.

[0112] In some embodiments, the enzyme mixture needs to be dried after being added to the electrode by dip coating. The drying time is 20-80 minutes, for example 30 minutes or 60 minutes. The temperature of the standing is 20-40 °C, for example 25 °C, or 40 °C.

[0113] The first polymer layer can be formed by pull-up dip coating of a first polymer layer cross-linking solution and then curing. In some embodiments, the first polymer layer cross-linking solution contains PEI and PEGDGE. The content of PEI in the cross-linking solution is in the range of 10% to 20%, and the mass ratio is 15 mg / mL; the content of PEGDGE in the cross-linking solution is 50-200 mg / mL, and the content of NAD(P)(H) in the cross-linking solution is in the range of 10-100 mg / mL, which can be 30-80 mg / mL, or 50-70 mg / mL. The number of pull-ups of the cross-linking solution is in the range of 10-30. The drying method is room temperature drying for 2-12 hours.

[0114] The first polymer layer can also be directly dissolved from a high molecular polymer. Such high molecular polymers include PU, PVP, TPU, NAD(P)(H) grafted film, chitosan film. In some embodiments, the first polymer layer is formed by dissolving NAD(P)(H) grafted polymer or directly dissolving a high molecular polymer. In some specific embodiments, the NADP +The graft polymer solution is coated on the electrode surface by dip coating and left to dry. NADP + The graft polymer can be used in the solution at a concentration of 35 mg / mL, 60 mg / mL, 100 mg / mL, preferably 60 mg / mL. The standing time can be 8-24 hours, for example, 8-12 hours, or 20-24 hours. The standing temperature can be 20-50 °C, for example, 25-45 °C, or 30-40 °C. In some embodiments, the first polymer layer is directly dissolved from the high molecular polymer TPU, and the TPU content in the solution is in the range of 6%-12% by mass ratio at 60 mg / mL-120 mg / mL.

[0115] In some specific embodiments, the protective layer is formed by cross-linking the NAD(P)(H) graft polymer. In one embodiment, the cross-linking agent is PEGDGE, and the NADP + The graft polymer is mixed to form a cross-linking solution, which is coated on the electrode surface by dip coating and left to dry. NADP + The graft polymer can be used in the solution at a concentration of 35 mg / mL, 60 mg / mL, 100 mg / mL (60 mg / mL in practice). The cross-linking agent can be PEGDGE, PEI, or PEG. The cross-linking agent can be used at a concentration of 50 mg / mL, 65 mg / mL, 85 mg / mL, preferably 100 mg / mL. The standing time after mixing can be 0.1-0.8 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 dip coated on the electrode can be 8-24 h, for example, 8-12 h, or 20-24 h. The standing temperature can be 20-50 °C, for example, 20-40 °C, or 35-50 °C.

[0116] In some embodiments, the first polymer layer can be formed by in-situ cross-linking. In one specific embodiment, the cross-linking precursor is PEI, and the cross-linking agent is PEG and PEGDGE. In one specific embodiment, the cross-linking precursor is PVP-NADP, and the cross-linking agent is PEGDGE, PEG, and PEI. By adjusting the side chain polymerization, the overall function of the polymer layer, such as hydrophilicity or hydrophobicity, oxygen permeability, etc., can be affected.

[0117] In some embodiments, the first polymer layer can be prepared by dip coating. A dip coating machine immerses the top end of the substrate electrode into a film solution, then pulls it out of the film solution to dry for a period of time, and repeats multiple times, and finally dries in air. The immersion depth is 2.5 to 3.5 mm, preferably 3 mm, and the pulling rate is 500 to 3000 pm / 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 dip coating, light-protected drying is performed on the surface of the electrode. The drying time is 10 to 24 hours, for example, it can be 12 hours, or it can be 20 hours. The standing temperature can be 20-40°C, for example, it can be 25°C, or it can be 40°C.

[0118] Optionally, the method of the present application further comprises forming a second polymer layer on the surface of the first polymer layer, which can be formed by direct dissolution, in-situ cross-linking, dip coating, etc. For example, the second polymer layer can also be directly dissolved from a high molecular polymer. Such high molecules include PU, PVP, TPU, 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 is 6%-12%, with a mass ratio of 60 mg / mL-120 mg / mL.

[0119] Method of use

[0120] The enzyme electrode and sensor of the present application are based on direct electron transfer of NAD(P)(H)-dependent glucose oxidase, and are used for detecting glucose concentration in solution. One of the use scenarios is to implant the electrode tip of the sensor into the subcutaneous tissue fluid to test the glucose level, and to perform real-time continuous measurement.

[0121] In embodiments, the test solution of the sensor electrode is a human simulation solution, the environmental temperature is 37°C, and it can also be used in a low-oxygen environment under normal oxygen levels.

[0122] 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 through a wire. The electrochemical workstation records the real-time current by chronoamperometry (CA). The applied voltage of chronoamperometry can be selected in the range of -0.1 to +0.05 V, for example, -0.1 V, +0.05 V. The measurement data acquisition frequency can be selected as 1 per second.

[0123] 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 by remote signal transmission, such as Bluetooth signal. In one embodiment, the measurement chip uses a fixed -0.1V voltage to drive the sensor electrode measurement, collects one data point every 0.5 seconds, and transmits data to the receiving host every 1 minute.

[0124] Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are for illustration only and should not be construed as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be purchased on the market.

[0125] Example description

[0126] The SBF solution referred to in the following examples refers to a human body simulation solution, the formula of which is as follows: pH 7.4, containing NaCl 135 mM, KCl 5 mM, MgCl2 1.5 mM, CaCl2 2.5 mM, Na2HPO4 1 mM, Na2SO4 0.5 mM, NaHCO3 4.2 mM, Tris 5 mM. The buffer used in the enzyme mixture solution referred to in the following examples is SBF solution. The manufacturers, models and important parameters of some reagents referred to in the following examples are as follows: NADP + (SHANGHAI SHANGKE, SZ-3002·Na, 1184-16-3); polyethyleneimine (PEI) (MACKLIN, 9002-98-6, M.W. 3000); polyethylene glycol diglycidyl ether (PEGDGE) (MACKLIN, 72207-80-8, Mn = 500); sodium carboxymethyl cellulose (CMC-Na) (MACKLIN, 004-32-4, 50-200 mPa.s); 3-aminobenzoic acid (MACKLIN, 30418-59-8, 98%); 4-carboxybenzoic acid (MACKLIN, 14047-29-1, 97%); MES (MACKLIN, 1266615-59-1, 99%); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (MACKLIN, 7365-45-9, 99%); 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide (EDC) (MACKLIN, 25952-53-8, 98.5%); N-hydroxysuccinimide (NHS) (MACKLIN, 6066-82-6, 99%); triethanolamine (MACKLIN, 102-71-6, 98%), boric acid (MACKLIN, 10043-35-3, 99.99%); glucose dehydrogenase (Shenzhen Jinhe Biological, Oxzyme-201); diaphorase (Shenzhen Jinhe Biological, Oxzyme-2101).

[0127] Preparation and performance test of the sensor (TPU polymer layer) of Example 1

[0128] The enzyme electrode sensor containing TPU polymer layer was prepared as follows: (1) The electrode substrate of the sensor was formed by screen printing, which was a three-electrode system including a working electrode, a counter electrode and a reference electrode. Then, the enzyme mixture was prepared according to the formulation described in Table 2, and the enzyme mixture was coated on 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. After the enzyme mixture was coated on the electrode, it was dried at 40 °C for 30 minutes. (2) In a clean room of 100,000 levels and an environment containing tetrahydrofuran vapor, the TPU polymer was dissolved into a film solution using 90% tetrahydrofuran, and the formulation is shown in Table 3. (3) The film solution was uniformly coated on the glucose sensor containing the sensing layer by dip coating. The immersion depth was 3.0 mm, the pulling rate was 600 μm / s, the interval was 30 minutes, and the number of repetitions was 10. After the dip coating was completed, the electrode surface was dried at 25 °C in the dark for 12 hours. The sensor electrode that can be tested and used was obtained.

[0129] The prepared sensor electrode was placed in a constant temperature SBF solution, and a constant voltage chronoamperometry test was performed at a voltage of -0.05 V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, magnetic stirring (800 revolutions / minute) was started. After the reaction started, the current was stable, and a glucose concentration solution was added for titration, so that the glucose concentration in the reaction solution increased in a gradient, ranging from 0 to 20 mM (Figure 1). The change of the reaction rate corresponding to the change of the glucose concentration was reflected by the reaction current, and the corresponding relationship between the glucose titration concentration and the reaction current was plotted (as shown in Figure 2). Table 1 summarizes the calibration curve slope, linear intensity and sensitivity of the sensor. The results show that the sensor containing TPU as the polymer layer can reflect a linear relationship with glucose and has a continuous response.

[0130] This example also tested the stability of the sensor electrode in a SBF solution containing 5 mM glucose at a voltage of -0.05 V for 15 consecutive days. The test results are shown in Figure 3, and the sensor containing TPU has good stability when working continuously for a long time (15 days).

[0131] This example also tested the sensor in a SBF solution for 15 consecutive days. The titration curve and linear relationship on the first day (Day 0) and the fifteenth day (Day 14) are shown in Figures 4-5, and the curve slope, R 2 and sensitivity are shown in Table 4. The results indicate that the electrode can maintain good glucose detection activity, sensitivity and stability for a long time (15 days).

[0132] Table 1 Calibration curve information

[0133] Table 2 Sensing layer formulation

[0134] Table 3 Polymer layer formulation

[0135] Table 4 Calibration curve information

[0136] Example 2 Preparation and performance testing of a sensor (PVP-NADP polymer layer)

[0137] The enzyme electrode sensor containing PVP-NADP polymer layer was prepared as follows: (1) ethylene pyridine and initiator were mixed and dissolved in N,N- dimethylformamide (DMF) with a mass ratio of 400:1, and heated in a water bath at 60°C for 10h. The reaction product was separated by precipitation with methanol solution to obtain a first intermediate product. Then, the first intermediate product was dissolved in DMF with bromoacetic acid at a mass ratio of 1:1, and heated in a water bath at 80°C for 18h. The reaction product was separated by precipitation with ethyl acetate solution to obtain a second intermediate product. Finally, the second intermediate product was dissolved in DMF with nicotinamide adenine dinucleotide phosphate (NADP + ) at a mass ratio of 10:1, and heated in a water bath at 70°C for 5h. The reaction product was separated and purified by methanol solution, and dried to obtain PVP-NADP graft polymer. The structure is shown in Figure 14. (2) 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. An enzyme mixture was prepared according to the formulation shown in Table 6, and the enzyme mixture was coated on the surface of the working electrode by dip coating, to form a sensing layer. The immersion depth was 2.5mm, the pulling rate was 1000pm / s, the interval time was 60s, and the number of repetitions was 10. After the enzyme mixture was dip coated on the electrode, it was dried at 40°C for 30 minutes. (3) A PVP-NADP polymer layer was formed on the surface of the sensing layer by dip coating, and the formulation of the polymer layer is shown in Table 7 (wherein the PVP-NADP graft polymer was prepared by the method described in step (1)). The immersion depth was 3.0mm, the pulling rate was 600pm / s, the interval was 30 minutes, and the number of repetitions was 10. After dip coating, the electrode surface was dried at 25°C in the dark for 12 hours. The sensor electrode ready for testing was obtained.

[0138] The prepared sensor electrode was placed in a constant temperature SBF solution, and chronoamperometry test was carried out under the control of an electrochemical workstation (Ivium, Palmsense, etc.) with a constant voltage of -0.05V. At the same time, magnetic stirring (800 revolutions / minute) was started. After the reaction started, the current was stable, and a concentrated glucose solution was added for titration.

[0139] The results are shown in Figures 6-7. Figures 6-7 show the titration curves of the sensor under different concentrations of glucose (0-20mM) within 4h, and Table 5 summarizes the calibration curve slope, linear strength and sensitivity of the sensor. The test results show that the sensor using PVP-NADP as the polymer layer can reflect a linear relationship and a continuous response to glucose.

[0140] This embodiment also tests the stability of the sensor in a 10mM glucose-containing SBF solution within 35 days (see Figure 8 for details) and the glucose titration curve and linear relationship within 35 consecutive days (see Figures 9-10 for details). Figures 9-10 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 embodiment proves that the sensor has good stability under long-term work, and the linearity and sensitivity of the glucose concentration response are almost unchanged.

[0141] Table 5 Calibration curve information

[0142] Table 6 Sensor layer formulation

[0143] Table 7 Polymer layer formulation

[0144] Table 8 Calibration curve information

[0145] Example 3 Sensor performance comparison (Example 1 sensor vs. bare electrode)

[0146] The performance of the sensor electrode (containing TPU polymer layer) shown in Example 1 and the bare sensor electrode (without TPU polymer layer) was compared (chronoamperometry, voltage -0.05V). Figure 11 shows the comparison of the output current of the sensor containing TPU polymer layer and the bare electrode sensor after sensing glucose, with the maximum current of each being 1. The presence of TPU polymer layer makes the sensor electrode have a wider linear range, which is more suitable for the working environment of continuous blood glucose monitoring.

[0147] Example 4 Sensor performance comparison (Example 1 sensor vs. NADP-free sensor +

[0148] ​The sensor electrode shown in Example 1 (sensing layer on the working electrode with immobilized NADP + ) was compared with a sensor (same as the electrode shown in Example 1 except that the sensing layer on the working electrode did not have NADP + ) by chronoamperometry (voltage -0.05 V). The results are shown in Figure 12. Figure 12 shows the current response of the two types of sensing layer electrodes in a solution containing 5 mM glucose and 50 μM NADP + over 24 h. As shown, the electrode without NADP + in the sensing layer did not respond to the external glucose, indicating that the TPU polymer layer limited the permeation of NADP + molecules.

[0149] Example 5 Sensor Performance Comparison (Example 2 Sensor vs. Bare Electrode)

[0150] The performance of the sensor electrode with the PVP-NADP polymer layer shown in Example 2 was compared with that of a sensor electrode without the PVP-NADP polymer layer (bare electrode) by chronoamperometry (voltage -0.05 V). Figure 13 shows the current output of the sensor with and without the PVP-NADP polymer layer after exposure to glucose, with the maximum current of each being 1. The presence of the PVP-NADP polymer layer allowed the sensor electrode to have a wider linear range, which is more suitable for the working environment of continuous blood glucose monitoring.

[0151] Example 6 Sensor (PVP-NADP Polymer Layer-TPU Polymer Layer) Preparation and Performance Test

[0152] The enzyme electrode sensor containing PVP-NADP polymer layer and TPU polymer layer was prepared by the following method: (1) Firstly, the electrode substrate of the sensor electrode was formed by screen printing, which was a three-electrode system including working electrode, counter electrode and 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 pm / s. The interval time was 60 s, and the number of repetitions was 10. After the enzyme mixture was dip-coated on the electrode, it was dried at 40°C for 30 minutes. (2) A layer of PVP-NADP polymer layer (first polymer layer) was formed on the surface of the sensing layer by dip coating. The formula of the first polymer layer is shown in Tables 9 and 10 (wherein the preparation method of PVP-NADP graft polymer is described in Example 2), which corresponds to the dissolution and cross-linking film formation methods, respectively. The immersion depth was 3.0 mm, the pulling rate was 1000 pm / s, the interval was 10 minutes, and the number of repetitions was 5. After dip coating, the electrode surface was dried at 25°C in the dark for 12 hours. (3) A layer of TPU polymer layer (second polymer layer) was formed on the surface of the first polymer layer by dip coating. The formula of the second polymer layer is shown in Tables 9-10. The immersion depth was 4.0 mm, the pulling rate was 600 pm / s, the interval was 30 minutes, and the number of repetitions was 10. After dip coating, the electrode surface was dried at 25°C in the dark for 12 hours. The sensor electrode ready for testing was obtained.

[0153] The prepared sensor electrode was placed in a constant temperature SBF solution and tested by chronoamperometry method with voltage-0.05V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). At the same time, magnetic stirring (800 rpm) was started. After the reaction started, the current was stable, and a concentrated glucose solution was added for titration.

[0154] Figure 15 is the titration curve of the sensor electrode containing the first polymer layer formed by dissolution under different concentrations of glucose conditions; Figure 16 is the linear relationship between the corresponding sensor signal and different concentrations of glucose. Figure 17 is the titration curve of the sensor electrode containing the first polymer layer formed by cross-linking under different concentrations of glucose conditions; Figure 18 is the linear relationship between the corresponding sensor signal and different concentrations of glucose. The experimental results show that, without NADP + in the sensing layer, PVP-NADP as the first polymer layer can provide the required NADP + , and TPU as the second polymer layer can reduce the sensing concentration of the enzyme layer for glucose. Both of them can enhance the stability of the enzyme, limit the NADP + molecule, and widen the linear range of measurement.

[0155] Table 9 Sensor electrode structure composition (dissolution)

[0156] Table 10 Sensor Electrode Structure Composition (Crosslinking)

[0157] Example 7: Fabrication and Performance Testing of the Sensor (NADP Crosslinked PEI Polymer Layer)

[0158] The 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. The electrode substrate was a three-electrode system including a working electrode, a counter electrode, and a reference electrode. Second, the enzyme mixture was dip-coated onto the surface of the working electrode to form a sensing layer. The enzyme mixture formulation is shown in Table 11. The dip depth was 2.5 mm, and the pull-out rate was 1000 μm / s. The interval was 60 s, and the number of repetitions was 10. After the enzyme mixture was dip-coated onto 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. The polymer layer formulation is shown in Table 12. The dip depth was 3.0 mm, the pull-out rate was 600 μm / s, the interval was 30 minutes, and the number of repetitions was 10. After dip-coating, 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.

[0159] The prepared sensor electrode was placed in a constant-temperature SBF solution, and a constant-voltage chronoamperometry test was performed at -0.05V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). Magnetic stirring (800 rpm) was simultaneously activated. Figure 21 shows a comparison of the two sensor electrodes in a solution containing 5 mM glucose over 18 hours. This example demonstrates that NADP can be effectively immobilized on the electrode using PEGDEG (Figure 19) and PEI (Figure 20). + According to the ratios shown in Table 11-12, mixing the enzyme solution containing PEGDGE and the membrane solution containing PEI forms a water-insoluble film on the electrode surface. This process requires a relatively long curing time. Once fully cured, the resulting film is insoluble in water, and no swelling was observed in water. This method does not have special requirements for temperature and humidity during the curing process. After film formation, the PEI and NADP in the film layer... + Ion adsorption can make NADP + The membrane was immobilized in a water-insoluble layer. The membrane was then placed over the electrode, and a substrate was added for chronoamperometry testing. The results showed that the immobilized NADP... + The membrane has electrochemical activity and good stability (see Figures 21-22 for details).

[0160] Figure 21 is a comparison of the control group and the experimental group at the time of testing on day 0. The control group, i.e., without the formation of a polymer layer using PEGDGE and PEI, was directly immersed on the electrode, and the experimental group used the same concentration of NADP + when cross-linked. The results show that, without cross-linking, the current returned to zero overnight; the experimental group, i.e., NADP cross-linked using PEGDGE and PEI to form a film + The overnight current plot after cross-linking shows that, after overnight, the current only decreased by 5 nA, and the performance was retained by 83.3%. The control group returned to zero after overnight. Figure 22 is the stability test of the experimental group every day. The test did not add NADP + , only glucose was added as the substrate, and the current was basically stable. From day 1 to day 12, the current was retained by 96.3%.

[0161] Table 11 Sensing layer formula

[0162] Table 12 Polymer layer formula

[0163] Example 8 Preparation and performance test of sensor (NADP cross-linked PEI added to sensing layer-TPU polymer film, or NADP cross-linked CMC added to sensing layer-TPU polymer film)

[0164] The sensor containing the NADP cross-linked PEI polymer layer was prepared as follows: (1) Preparation of NADP cross-linked PEI: prepare solutions of different proportions of PEGDGE, PEI, and 3-APBA, with 10% ethanol aqueous solution as the solvent; take equal volumes of the three solutions, magnetically stir 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 solution color 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 dialysis 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 a 0.5%-2% solution of CMC-Na, with MES buffer (pH = 5-6) as the solvent; prepare EDC and NHS solutions, with MES buffer (pH = 5-6) as the solvent; mix the above three solutions according to a certain proportion, magnetically stir until completely mixed; add a certain amount of NADP +Solution (solvent is boric acid solution with pH = 7-8); after mixing, stir the solution overnight, add a certain amount of triethanolamine solution, stir for 30 min; transfer the above solution into a dialysis bag (molecular weight 1000), and dialyze with deionized water 3-4 times; after dialysis, transfer the solution in the dialysis bag into 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. The electrode substrate is a three-electrode system including the working electrode, the counter electrode and the reference electrode. Next, 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 crosslinked material prepared in step (1) or (2). The immersion depth is 2.5 mm, and the pulling rate is 1000 μm / s. The interval is 60 s, and the number of repetitions is 10. After the enzyme mixture is dip-coated onto 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 formulation 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 the repetition is 10. After dip-coating, 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.

[0165] The prepared sensor electrode was placed in a constant-temperature SBF solution, and a constant-voltage chronoamperometry test was performed at -0.05V under the control of an electrochemical workstation (Ivium, Palmsense, etc.). Simultaneously, a magnetic stirrer (800 rpm) was activated. After the reaction started and the current stabilized, 5 mM glucose was added, and the current feedback of the sensor electrode was observed. In this embodiment, linear polyethyleneimine (PEI) and NAD(P)+ were cross-linked with an epoxy compound to form NADP as shown in Figure 23. + The derivatives (PEI-NADP) and the NADP derivative (CMC-NADP) formed by crosslinking carboxymethyl cellulose (CMC) with NAD(P)+, as shown in Figure 24, were used to prepare electrodes and corresponding sensors as shown in Table 13. Electrochemical tests were performed, and the results are shown in Figure 25. These results reflect the effects of these two NADP derivatives. + The derivatives possess NADP + Its catalytic effect can be used in this reaction system, especially in the sensing layer.

[0166] Table 13 Electrode Structure Composition of PEI-NAD(P)+ Derivatives

[0167] Example 9: Comparison of Sensor Anti-interference Performance (Example 1 Sensor vs. Abbott Probe, Silicon-based Probe)

[0168] Performance comparison of the sensor of Example 1 with commercially available probes (Abbott, FreeStyle Libre) and probes (silicon-based, silicon-based GS1) (constant potential chronoamperometry, voltage -0.05 V): each sensor electrode was placed in a constant temperature 37 degrees Celsius SBF solution, and chronoamperometry test was performed under the control of an electrochemical workstation (Ivium, Palmsense, etc.) at a constant voltage of +0.05 V. At the same time, magnetic stirring (800 rpm) was started. After the reaction started, 6.66 mM glucose was added, and when the current was stable, the recommended concentration of interferent was added to the solution, and the change in feedback current was observed.

[0169] This example compares the working conditions and current responses of three probes in chronoamperometry in a human body simulation solution containing glucose under the interference conditions of the registration review guidelines. The specific test results are shown in Figures 26-29 and Table 14. Figures 26-29 are the current change test results when galactose, hydroxyurea, ascorbic acid and xylose are added as interferents. The chart data shows that the sensor described in Example 1 of the present application has almost no current fluctuation compared to the other two sensor probes under the condition of added interference, and exhibits superior anti-interference performance.

[0170] Table 14

[0171] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to part or all of the technical features; and 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 application.

Claims

1. An enzyme electrode for glucose detection, characterized by, 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 permeation of glucose and / or interferents; The enzyme electrode further comprises: NAD(P)(H) in the sensing layer; and / or a derivative of NAD(P)(H) which is a polymer covalently binding NAD(P)(H), 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 permeation of glucose and / or interferents, the second polymer layer further comprising or not comprising the derivative of NAD(P)(H).

3. The enzyme electrode according to claim 1, wherein The enzyme electrode further comprises a support substrate, and the electrode substrate is located on the support substrate.

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

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

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

7. The electrode of claim 4, wherein The NAD(P)(H)-grafted PVP has a chemical structure as shown below: Wherein, 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 is selected from polymer dissolution membrane or in-situ synthesized polymer membrane; preferably, the polymer layer is selected from PEI cross-linked membrane, TPU membrane, PU membrane, PVP membrane, chitosan membrane, NAD(P)(H) cross-linked polymer membrane or NAD(P)(H) grafted polymer membrane.

9. The enzyme electrode according to claim 1, wherein The sensing layer further comprises a cross-linking agent; preferably, the cross-linking agent is selected from epoxy cross-linking agent, glutaraldehyde, polyethylene glycol, PEG or PEGDGE.

10. The enzyme electrode according to claim 1, wherein 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, 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 comprises 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.

12. A sensor for glucose detection, characterized by, The sensor comprises the enzyme electrode of any one of claims 1-10 as a working electrode, and further comprises a counter electrode and a reference electrode.

13. The sensor of claim 12, wherein, 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 for the manufacture of an apparatus for the continuous detection of glucose.

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

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