Glucose oxidase conjugated hydrogel

Abstract

The present disclose concerns glucose oxidase conjugated hydrogels, in particular to hydrogels (40a) featuring a bifunctional polymer and glucose oxidase that are covalently attached to each other by photo-crosslinking.

Description

[0001] GLUCOSE OXIDASE CONJUGATED HYDROGEL

[0002] FIELD

[0003] The disclosure relates to glucose oxidase conjugated hydrogels for wearable biosensors, in particular to hydrogels featuring a bifunctional polymer and glucose oxidase which are covalently attached to each other by photo-crosslinking.

[0004] BACKGROUND

[0005] Continuous glucose monitors (CGMs) are medical devices used for the management of diabetes. They track glucose levels in the body continuously or at frequent intervals, offering a dynamic view of the patient's blood glucose changes. With real-time information on blood glucose levels, patients may make more informed decisions about their medication, insulin dosage, diet, and activities. The widespread use of CGMs has been enabled by advancements in the field of glucose biosensors that have led to very high sensitivity, accuracy, and user- friendliness.

[0006] Figure 1 shows an exploded view of part of an exemplary electrochemical sensor 101 suitable for glucose monitoring. In the figure, only the anodic compartment 102 is shown. It comprises a working electrode 102a, a counter electrode 102b and a reference electrode 102c printed on a substrate 103, and an interface 104 configured to be in contact with skin of a subject. The counter electrode and the reference electrode are typically made of Ag / AgCl. An exemplary working electrode is a carbon electrode cast with glucose oxidase.

[0007] The amount, activity, and stability of glucose oxidase need to be carefully optimized for the analytical performance, reproducibility, and shelf-life of CGMs.

[0008] A specific problem with CGMs is enzyme migration, which may lead to biocompatibility issues as the enzyme can diffuse to the skin causing irritation and sensitization in the patient. The migration also decreases analytical performance since there is less enzyme in the biosensor to react with and thus detect glucose. Furthermore, enzyme migration lowers the reproducibility and shelf-life of the biosensor.

[0009] Another problem with CGMs is the diffusion of glucose to the sensing electrode. Invasive biosensors using needles or microneedle arrays to penetrate the skin use several layers to limit glucose diffusion and prevent interferents from reaching the biosensor surface, which increases initialization and response times to glucose level changes. Finally, a third problem in current CGMs is the effect of electrical artefacts because the biosensors rely on measuring electrical signals produced at the sensing electrode. The biosensor interface with the skin should be designed to minimize drift, capacitive noise and electrical impedance that adversely impact accuracy and operational stability of CGMs.

[0010] Accordingly, there is a need for accurate and stable biosensors that also provide fast and specific detection of glucose in CGMs.

[0011] SUMMARY

[0012] The following presents a simplified summary to provide a basic understanding of some aspects of different invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to a more detailed description of exemplifying and non-limiting embodiments of the invention.

[0013] A first aspect the present disclosure is to provide a method for producing a glucose oxidase conjugated hydrogel, the method comprising the steps of: a) reacting glucose oxidase with an oxidizing reagent to generate an aldehyde group on the glucose oxidase, b) contacting the glucose oxidase aldehyde group with i. bifunctional polyethylene glycol comprising a hydrazide group and an acrylate group to form a hydrazone bond between the hydrazide group of the bifunctional polyethylene glycol and the aldehyde group of the glucose oxidase or ii. bifunctional polyethylene glycol comprising an aminooxy group and an acrylate group to form an oxime bond between the aminooxy group of the bifunctional polyethylene glycol and the aldehyde group of the glucose oxidase thereby generating a polyethylene glycol acrylate-glucose oxidase conjugate, and c) subjecting the conjugate to crosslinking reaction thereby producing the glucose oxidase conjugated hydrogel.

[0014] A second aspect of the present disclosure is to provide a glucose oxidase conjugated hydrogel obtainable by the method of the first aspect. A third aspect of the present disclosure to is provide a method for coating at least a working electrode of a biosensor, the method comprising applying the glucose oxidase containing hydrogel obtainable by the method of the first aspect at least on the working electrode.

[0015] A fourth aspect of the present disclosure is to provide a method for coating at least a working electrode of a biosensor, the method comprising the steps of: i. dispensing a mixture of the glucose oxidase conjugate obtainable by step b) of the method of the first aspect and a photoinitiator onto said electrode, and ii. subjecting the mixture to crosslinking reaction thereby providing an electrode coated with a glucose oxidase conjugated hydrogel.

[0016] A fifth aspect of the present disclosure is to provide a biosensor comprising a working electrode coated with the glucose oxidase conjugated hydrogel obtainable by the method of the first aspect, the third aspect or the fourth aspect.

[0017] A sixth aspect of the present disclosure is to provide a use of the glucose oxidase conjugated hydrogel obtainable by the method of the first aspect as a coating of a working electrode of a biosensor.

[0018] Exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims.

[0019] Exemplifying and non-limiting embodiments of the invention both as to constructions and to methods of operation, together with additional objects and advantages thereof, are best understood from the following description of specific exemplifying embodiments when read in conjunction with the accompanying drawings.

[0020] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of un-recited features. The features recited in dependent claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of “a” or “an”, i.e., a singular form, throughout this document does not exclude a plurality.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Exemplifying and non-limiting embodiments of the invention and their advantages are explained in greater detail below with reference to the accompanying drawings, in which: figure 1 shows an exploded view of part of an exemplary electrochemical sensor 100 suitable for glucose monitoring, figures 2 and 3 show schematic presentations of the method for producing hydrogels according to non-limiting exemplary embodiments of the present disclosure, and figure 4 shows a comparison of the performance of a biosensor including a working electrode coated with glucose oxidase containing hydrogels of the present disclosure and a working electrode of the state of art.

[0023] DESCRIPTION

[0024] The specific examples provided in the description below should not be construed as limiting the scope and / or the applicability of the accompanied claims. Lists and groups of examples provided in the description below are not exhaustive unless otherwise explicitly stated.

[0025] According to one aspect the present disclosure concerns a method for producing glucose oxidase conjugated hydrogel. The principle of the method is shown in figures 2 and 3.

[0026] In one embodiment the method comprises the following steps: a) reacting glucose oxidase 10 with an oxidizing reagent A to generate an aldehyde group on the glucose oxidase 20, b) reacting the glucose oxidase aldehyde group with a bifunctional polyethylene glycol B comprising an a-hydrazide group and an co-acrylate group to form a hydrazone bond between the aldehyde group of the glucose oxidase and the hydrazide group of the bifunctional polyethylene glycol thereby generating a polyethylene glycol acrylate-glucose oxidase conjugate 30a, and c) subjecting the conjugate to crosslinking reaction C thereby producing the glucose oxidase conjugated hydrogel 40a.

[0027] In another embodiment the method comprises the following steps: a) reacting glucose oxidase 10 with an oxidizing reagent A to generate an aldehyde group on the glucose oxidase 20, b) reacting the glucose oxidase aldehyde group with a bifunctional polyethylene glycol B’ comprising an a-aminooxy group and an co-acrylate group to form an oxime bond between the aldehyde group of the glucose oxidase and the aminooxy group of the bifunctional polyethylene glycol thereby generating a polyethylene glycol acrylateglucose oxidase conjugate 30b, and c) subjecting the conjugate to crosslinking reaction C thereby producing the glucose oxidase conjugated hydrogel 40b. The amount of oxidizing reagent should be sufficient to produce the desired amounts of aldehyde groups to the glucose oxidase. Typically, the oxidizing agent is present in amounts greater than a 1 : 1 molar ratio, preferably greater than a 100 : 1 molar ratio to the molar amount of glucose oxidase.

[0028] The oxidizing reagent can be any oxidizing reagent known to oxidize cis-diols. A particular oxidizing reagent periodate, preferably sodium periodate. The concentration of the sodium periodate is typically from 0.01 mM to 500 mM, such as 10 mM. pH of the oxidation reaction is typically in the range of 5 to 7.4, such as 5.5. The time for oxidizing reaction is typically from 1 h to 2 h at 20 °C.

[0029] In a preferable embodiment the excess of oxidizing reagent is neutralized and removed from the reaction mixture before step b). An exemplary neutralization reagent is glycerol, such as 0.1 M aqueous glycerol.

[0030] The bifunctional polyethylene glycol used for conjugation with the aldehyde group of the glucose oxidase includes an a -hydrazide group and an co-acrylate group or an a-aminooxy group and an co-acrylate group. The bifunctional polyethylene glycol suitable for the method has preferably a molecular weight of from 500 Da to 5000 Da, such as 2000 Da. The bifunctional polyethylene glycol is preferably used in a molar amount in excess of the amount of aldehydes present in the oxidized glucose oxidase. The molar ratio of the bifunctional polyethylene glycol and the aldehyde groups is typically from 10:1 to 2000:1. The excess of the bifunctional polyethylene glycol is preferably removed from the reaction mixture before step c).

[0031] The crosslinking produces glucose oxidase conjugated hydrogel having intra- and intermolecular crosslinks. The number of crosslinks can be adjusted by tuning the amount of number of acrylate tethers produced in step b) and the enzyme concentration in the crosslinking reaction.

[0032] In an embodiment the crosslinking of step c) comprises subjecting the conjugate of step b) to a photoinitiator. The concentration of the photoinitiator is typically 1-50 mM, and the crosslinking is performed in the presence of light such as UV light or visible / blue light.

[0033] In an exemplary embodiment the crosslinking of step c) is performed in the presence of 1- 10 mM, such as 5 m lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate at is in the presence of UV light of wavelength 365-395 nm or visible / blue light of wavelength 405-450 nm. In a particular embodiment the crosslinking is performed on surface of an electrode of a biosensor. Accordingly, a mixture of the conjugate of step b) and a photoinitiator is dispensed on the electrode, and the mixture is subjected to UV or visible / blue light. At least the working electrode is coated, but coating of the reference electrode and the counter electrode by the glucose oxidase conjugated hydrogel can be performed also.

[0034] The present disclosure also concerns a glucose oxidase conjugated hydrogel obtainable by the method disclosed above.

[0035] According to another aspect the present disclose concerns method for coating an electrode of a biosensor with the glucose oxidase conjugated hydrogel obtainable as disclosed above. At least the working electrode is coated with the glucose oxidase conjugated hydrogel. It is also possible to coat the counter electrode and the reference electrode with the glucose oxidase conjugated hydrogel.

[0036] In one embodiment the method comprises applying desired amount of the glucose oxidase conjugated hydrogel on the electrode.

[0037] In another embodiment the method comprises the steps of: i) dispensing an admixture comprising the glucose oxidase conjugate disclosed above and a photoinitiator at least on a working electrode of a biosensor, and ii) subjecting the admixture to light such as UV or visible / blue light thereby providing a working electrode coated with glucose oxidase conjugated hydrogel.

[0038] An exemplary photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate. The subjecting is typically performed the presence of UV light of 365-395 nm or visible / blue light of 405-450 nm. An exemplary concentration of the photoinitiator is 1-10 mM.

[0039] The latter approach is preferable since it provides better adhesion of the glucose oxidase conjugated hydrogel to the electrode.

[0040] According to another aspect the present disclosure concerns use of the glucose oxidase conjugated hydrogel obtainable by the method disclosed above as a coating of an electrode of a biosensor. The glucose oxidase conjugated hydrogel is used for coating at least a working electrode of the biosensor. However, the glucose oxidase conjugated hydrogel can be used for coating the working electrode, the reference electrode, and the counter electrode of the biosensor. According to still another aspect the present disclosure concerns a biosensor comprising at least a working electrode coated with the glucose oxidase conjugated hydrogel obtainable by the method disclosed above.

[0041] In one embodiment not only the working electrode but also the reference electrode and the counter electrode of the biosensor are coated with the glucose oxidase conjugated hydrogel.

[0042] EXPERIMENTAL

[0043] Preparation of polyethylene glycol acrylate-glucose oxidase conjugate

[0044] The conjugation of bifunctional polyethylene glycol (PEG) to the enzyme-aldehyde was optimized at different molar ratios as 10:1, 25:1, 50:1, 125:1, 200:1, 500:1, and 2000:1 depending on the molecular weight of PEG used between the range of 500-20 000 Da. An exemplary molar ratio was 200:1 when hydrazide-PEG-acrylate of molecular weight 3400 Da was conjugated to aldehyde modified glucose oxidase.

[0045] Dispending on the electrodes

[0046] The dispensing volume / area (pL / cm2) was varied between 25-120. An exemplary ratio was 55 for a working electrode of area 0.5 cm2. Dispensing was designed as a uniform continuous layer or to create a specific pattern of droplets on the working electrode.

[0047] Crosslinking

[0048] The crosslinking reaction included the acrylate functionalized PEGylated glucose oxidase enzyme in concentration range between 10-100 mg / mL and a photoinitiator in concentration range between 1-10 mM. Exemplary concentration of the acrylate functionalized PEGylated glucose oxidase enzyme and the photoinitiator were 20 mg / L and 5 mM, respectively. Upon mixing, the solution was dispensed on the working electrode to cover the surface, and irradiated by either UV light (365-395 nm) or blue light (405-450 nm). The irradiation time varied between 15 - 180 seconds depending on the system. An exemplary crosslinking was performed using UV light irradiation of 90 seconds in the presence of 2 mM LAP as the photoinitiator. The irradiation intensity or dose varied between 50-4000 mJ / cm2.

[0049] Comparison to state of art

[0050] The performance of the sensors according to the present disclosure was compared with a state-of-art sensor where the glucose oxidase enzyme is encapsulated in a BSA-Chitosan mixture followed by drop casting on the working electrode and placing a hydrogel layer on top. Results are shown in figure 4. The sensors of the present disclosure showed two times better sensitivity towards detecting glucose by the electrochemical method performed at the exactly similar conditions (the glucose range of 5-100 iiM at 20 °C).

[0051] The advantages of the present disclosure can be summarized as follows: i. Covalent bonding of the enzyme into the hydrogel prevents enzyme migration. ii. Covalent bonding improves biocompatibility since the amount of enzyme in contact with the skin is significantly reduced preventing irritation and sensitization. PEG chains attached to the enzyme make the enzyme less immunogenetic, preventing skin reactions. iii. The oxidation of the glucose oxidase to generate aldehyde groups improves the tunability of PEG grafting density. iv. Analytical performance is improved because the enzyme is uniformly bound in the hydrogel to form a single very thin sensing layer on the electrode surface. Thus, the diffusion of glucose through the hydrogel is fast and the reaction to form hydrogen peroxide happens in the close vicinity to the electrode surface where it can be detected quickly. Thus, the response time is faster. v. The fast reaction between glucose and the enzyme in the thin hydrogel layer helps to avoid charge build-up on electrode surface, thereby also reduces capacitive effects that helps to improve background noise in the sensor signal and avoid time-dependant drift in sensor response. vi. The simple biosensor design comprising a thin hydrogel sensing layer results in low electrical impedance at the electrode surface. One thin sensing layer helps to avoid complex interactions between analytes and the hydrogel materials, thereby helping to improve the analyte stability. The enzymatic reaction between glucose and glucose oxidase produces hydrogen peroxide, which is electrochemically detected at the working electrode surface. As the hydrogen peroxide has short shelf-life, the hydrogel design improves the analyte stability. vii. Reproducibility and shelf-life of the biosensor are improved since there is minimal enzyme loss due to migration and the amount of enzyme from sensor to sensor varies less. viii. The tunability of the enzyme-conjugated hydrogel matrix mechanics allows conforming the biosensor design criteria such as better adhesion to sensor substrate, robust mechanics for reliable performance, and molecular design to optimize the analytical performance. ix. Manufacturing complexity of biosensors is significantly lower as the method of the present disclosure requires one-step photo crosslinking reaction on the electrodes, thus avoiding multiple layer formations using several materials.

[0052] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0053] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment

[0054] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0055] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention. While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below

[0056] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e., a singular form, throughout this document does not exclude a plurality.

Claims

CLAIMS1. A method for producing a glucose oxidase conjugated hydrogel, the method comprising the steps of: a) reacting glucose oxidase with an oxidizing reagent to generate an aldehyde group on the glucose oxidase; b) contacting the glucose oxidase aldehyde group with i. a bifunctional polyethylene glycol comprising a hydrazide group and an acrylate group to form a hydrazone bond between the hydrazide group of bifunctional polyethylene glycol and the aldehyde group of the glucose oxidase or ii. a bifunctional polyethylene glycol comprising an aminooxy group and an acrylate group to form an oxime bond between the aminooxy group of the bifunctional polyethylene glycol and the aldehyde group of the glucose oxidase, thereby generating a polyethylene glycol acrylate-glucose oxidase conjugate, and c) subjecting said polyethylene glycol acrylate-glucose oxidase conjugate to crosslinking reaction thereby producing the glucose oxidase conjugated hydrogel.

2. The method according to claim 1, wherein molar ratio of said bifunctional polyethylene glycol and the aldehyde group is from 10:1 to 2000:1.

3. The method according to claim 1 or 2, wherein the oxidizing reagent is present in an amount greater than 1 : 1 molar ratio to the glucose oxidase.

4. The method according to any one of claims 1 to 3, wherein the oxidizing reagent is present in an amount greater than 100:1 molar ratio to the glucose oxidase.

5. The method according to any one of claims 1 to 4, wherein the oxidizing reagent is periodate.

6. The method according to claim 5, wherein the periodate is sodium periodate.

7. The method according to any one of claims 1 to 6, wherein step a) includes one or more of: i. concentration of the oxidizing reagent is from 0.01 mM to 500 mM, ii. pH of the oxidizing reaction is from 5 to 7.4, and iii. time of the oxidizing reaction is from 1 h to 2 h at 20 °C.

8. The method according to any one of claims 1 to 7, wherein the crosslinking reaction comprises subjecting to a photoinitiator.

9. The method according to claim 8, wherein the photoinitiator is lithium phenyl (2,4,6- trimethylbenzoyl) phosphinate, and wherein the subjecting of step c) is in the presence of UV light of wavelength 365-395 nm or visible / blue light of wavelength 405-450 nm, and the concentration of the photoinitiator is 1-10 mM.

10. The method according to claim 8 or 9, wherein in step c) concentration of the polyethylene glycol acrylate-glucose oxidase conjugate is 10- 100 mg / mL and / or pH is from 5 to 7.4.

11. The method according to any one of claims 1 to 10, wherein the subjecting of step c) is on surface of an electrode of a biosensor.

12. Glucose oxidase conjugated hydrogel obtainable by the method according to any one of claims 1 to 11.

13. A method for coating at least a working electrode of a biosensor, the method comprising applying the glucose oxidase conjugated hydrogel obtainable by the method according any one of claims 1 to 11 on said electrode.

14. A method for coating at least a working electrode of a biosensor, the method comprising the steps of: i. dispensing a mixture of the glucose oxidase conjugate obtainable by step b) of claim 1 and a photoinitiator onto said electrode, and ii. subjecting the mixture to crosslinking reaction thereby providing an electrode coated with a glucose oxidase conjugated hydrogel.

15. The method according to claim 14, wherein the photoinitiator is lithium phenyl (2,4,6- trimethylbenzoyl) phosphinate, and the subjecting is the presence of UV light of 365- 395 nm or visible / blue light of 405-450 nm, and the concentration of the photoinitiator is 1-10 mM.

16. Use of the glucose oxidase conjugated hydrogel obtainable by the method according to any one of claims 1 to 11 as a coating of an electrode of a biosensor.

17. A biosensor comprising at least a working electrode coated with the glucose oxidase conjugated hydrogel obtainable by the method according to any one of claims 1 to 11, 13 to 15.

Images