FI20245266A1 - Glucose oxidase conjugated hydrogel - Google Patents
Glucose oxidase conjugated hydrogelInfo
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- FI20245266A1 FI20245266A1 FI20245266A FI20245266A FI20245266A1 FI 20245266 A1 FI20245266 A1 FI 20245266A1 FI 20245266 A FI20245266 A FI 20245266A FI 20245266 A FI20245266 A FI 20245266A FI 20245266 A1 FI20245266 A1 FI 20245266A1
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Abstract
The present disclose concerns glucose oxidase conjugated hydrogels, in particular to hydrogels featuring a bifunctional polymer and glucose oxidase that are covalently attached to each other by photo-crosslinking.
Description
GLUCOSE OXIDASE CONJUGATED HYDROGEL
The disclosure relates to glucose oxidase conjugated hydrogels, in particular to hydrogels featuring a bifunctional polymer and glucose oxidase which are covalently attached to each other by photo-crosslinking.
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.
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.
The amount, activity, and stability of the glucose oxidase need to be carefully
N optimized for the analytical performance, reproducibility, and shelf-life of CGMs.
N
& A specific problem with CGMs is enzyme migration. Enzyme migration may lead to
O biocompatibility issues as the enzyme can diffuse and reach the skin where it may
I 25 cause skin irritation and sensitization in the patient. The migration also decreases
W analytical performance since there is less enzyme in the biosensor to react with, and & thus detect glucose. Furthermore, enzyme migration lowers the reproducibility and
N shelf-life of the biosensor.
Al
Accordingly, there is need for accurate, stable, and specific biosensors for glucose monitoring.
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.
Accordingly, it is an object of the present invention to provide a method for producing a glucose oxidase conjugated hydrogel, the method comprising steps of a) reacting glucose oxidase with an oxidizing reagent to generate an aldehyde group on the glucose oxidase, b) contacting the glycose 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
S c) subjecting the conjugate to crosslinking reaction thereby producing the 8 25 glucose oxidase conjugated hydrogel.
S It is also an object of the present invention to provide a glucose oxidase conjugated
E- hydrogel obtainable by the method of claim 1.
S It is still an object of the present invention to provide a method for coating at least a
N working electrode of a biosensor, the method comprising applying the glucose
N 30 oxidase containing hydrogel obtainable by the method of claim 1 at least onto the working electrode.
It is still an object of the present invention to provide a method for coating at least a working electrode of a biosensor, the method comprising dispensing the polyethylene glycol acrylate-glucose oxidase conjugate obtainable by the step b) of the method of claim 1 at least onto the working electrode and subjecting the conjugate to a crosslinking reaction.
It is still an object of the present invention to provide a biosensor comprising a working electrode coated with the glucose oxidase conjugated hydrogel obtainable by the method according to claim 1.
It is still an object of the present disclosure to provide a use of the glucose oxidase conjugated hydrogel obtainable by the method of claim 1 as a coating of a working electrode of a biosensor.
Exemplifying and non-limiting embodiments of the invention are described in accompanied dependent claims.
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.
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.
N
O
3 Exemplifying and non-limiting embodiments of the invention and their advantages = 25 are explained in greater detail below with reference to the accompanying drawings, 2 in which:
O
N figure 1 shows an exploded view of part of an exemplary electrochemical sensor < . so
N 100 suitable for glucose monitoring,
Al 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 hydrogel of the present disclosure and a working electrode of the state of art.
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.
According to one aspect the present invention concerns a method for producing glucose oxidase conjugated hydrogel. The principle of the method is shown in figures 2 and 3.
According to 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 o-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.
According to another embodiment the method comprises the following steps
S a) reacting glucose oxidase 10 with an oxidizing reagent A to generate an 0 aldehyde group on the glucose oxidase 20, 2 25 b) reacting the glucose oxidase aldehyde group with a bifunctional polyethylene 2 glycol B’ comprising an a-aminooxy group and an o-acrylate group to form a © an oxime bond between the aldehyde group of the glucose oxidase and the & aminooxy group of the bifunctional polyethylene glycol thereby generating a
N polyethylene glycol acrylate-glucose oxidase conjugate 30b, and
N 30 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. 5 The oxidizing reagent can be any oxidizing reagent known to oxidize cis-diols. A particular oxidizing reagent is 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.
According to 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.
The bifunctional polyethylene glycol used for conjugation with the aldehyde group of the glucose oxidase includes an a-hydrazide group and an o-acrylate group or an a-aminooxy group and an w-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). + The crosslinking of step c) includes subjecting the conjugate of step b) to a
S photoinitiator typically ranging between 1-50 mM in the presence of light such as UV & light or visible/blue light. An exemplary photoinitiator is lithium phenyl (2,4,6- re 25 — trimethylbenzoyl) phosphinate (LAP) at a concentration of 5 mM. Wavelength is z typically 365-395 nm for UV light or 405-450 nm for blue/visible light. The a © crosslinking produces glucose oxidase conjugated hydrogel having intra- and & intermolecular crosslinks. The number of crosslinks can be adjusted by tuning the
N amount of number of acrylate tethers produced in step b) and the enzyme
N 30 concentration in the crosslinking reaction.
According to a particular embodiment the crosslinking is performed on surface of an electrode of a biosensor. According to this embodiment 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. According to the method 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.
According to one aspect the present disclosure concerns a glucose oxidase conjugated hydrogel obtainable by the method disclosed above.
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.
According to one embodiment the method includes applying desired amount of the glucose oxidase conjugated hydrogel on the electrode.
According to another embodiment the method includes i) dispensing an admixture comprising the glucose oxidase conjugate disclosed above and a photoinitiator at least on the working electrode, 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. + The latter approach is preferable since it provides better adhesion of the glucose
N
S oxidase conjugated hydrogel to the electrode. 3 According to another aspect the present disclosure concerns use of the glucose 3 25 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
O coating at least the working electrode of the biosensor. However, the glucose
N
3 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. According to 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.
EXPERIMENTAL
Preparation of polyethylene glycol acrylate-glucose oxidase conjugate
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- 5000 Da. An exemplary ratio was 200:1 when hydrazide-PEG-acrylate of molecular — weight 3400 Da was conjugated to aldehyde modified glucose oxidase.
Dispending on the electrodes
The dispensing volume/area (uL/cm?) was varied between 25-120. An exemplary ratio was 55 for a working electrode of area 0.64 cm?. Dispensing was designed as a uniform continuous layer or to create a specific pattern of droplets on the working electrode.
Crosslinking
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 1-50 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
N working electrode to cover the surface, and either UV light (365-395 nm) or blue a light (405-450 nm) was irradiated. The irradiation time varied between 15 — 180 3 seconds depending on the system. An exemplary crosslinking was performed using = 25 — UV light irradiation of 90 seconds in the presence of 2 mM LAP as the photoinitiator. & The irradiation intensity or dose varied between 250-4000 mJ/cm?.
O
& Comparison to state of art o
O The performance of the sensor 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 dropcasting on the working electrode and placing a hydrogel layer on top. Results are shown in figure 4. The sensor 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 pM at 20 °C).
The advantages of the present invention can be summarized as follows i. Covalent attachment of the enzyme into the hydrogel prevents enzyme migration. i. 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. = Analytical performance is improved because the enzyme is bound in the hydrogel that acts as an analyte transport layer where glucose can diffuse efficiently. After reacting with the enzyme, hydrogen peroxide can be detected at the electrodes due to the vicinity of the bound enzyme to the electrode surface. iv. 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. v. The easy 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.
S
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Claims (12)
1. 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 glycose 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 N in an amount greater than 1:1 molar ratio, preferably greater than 100:1 molar a ratio to the glucose oxidase. 2 25
4. The method according to any one of claims 1 to 3 wherein the oxidizing reagent z is periodate, preferably sodium periodate.
W
5. The method according to any one of claims 1 to 4 wherein step a) includes one & or more of
X 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.
6. The method according to any one of claims 1 to 5 wherein the crosslinking reaction includes subjecting to a photoinitiator.
7. The method according to claim 6 wherein the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, and the subjecting is in presence of UV light of 365-395 nm or visible/blue light of 405-450 nm.
8. The method according to any one of claims 1 to 7 wherein the subjecting of step c) is on surface of an electrode of a biosensor.
9. Glucose oxidase conjugated hydrogel obtainable by the method according to any one of claims 1 to 7.
10. 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 / on said electrode, or 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.
11. The method according to claim 10 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.
12. Use of the glucose oxidase conjugated hydrogel obtainable by the method according to any one of claims 1 to / as a coating of an electrode of a biosensor. N 13. A biosensor comprising at least a working electrode coated with the glucose a 25 oxidase conjugated hydrogel obtainable by the method according to any one 3 of claims 1 to 7, 10, or 11. O I = O O N LO < N O N
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI20245266A FI20245266A1 (en) | 2024-03-05 | 2024-03-05 | Glucose oxidase conjugated hydrogel |
| PCT/FI2025/050072 WO2025186507A1 (en) | 2024-03-05 | 2025-02-17 | Glucose oxidase conjugated hydrogel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FI20245266A FI20245266A1 (en) | 2024-03-05 | 2024-03-05 | Glucose oxidase conjugated hydrogel |
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| FI20245266A1 true FI20245266A1 (en) | 2025-09-06 |
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| Application Number | Title | Priority Date | Filing Date |
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| FI20245266A FI20245266A1 (en) | 2024-03-05 | 2024-03-05 | Glucose oxidase conjugated hydrogel |
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| FI (1) | FI20245266A1 (en) |
| WO (1) | WO2025186507A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006080017A2 (en) * | 2005-01-26 | 2006-08-03 | Ramot At Tel Aviv University Ltd. | Biologically active silver-coated proteins |
| EP3093029A1 (en) * | 2009-07-27 | 2016-11-16 | Baxalta GmbH | Blood coagulation protein conjugates |
| US20170315077A1 (en) * | 2016-04-28 | 2017-11-02 | Medtronic Minimed, Inc. | In-situ chemistry stack for continuous glucose sensors |
| US20190177683A1 (en) * | 2017-12-07 | 2019-06-13 | Indiana University Research And Technology Corporation | Stabilization of enzyme-immobilized hydrogels for extended hypoxic cell culture |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7432069B2 (en) * | 2005-12-05 | 2008-10-07 | Sontra Medical Corporation | Biocompatible chemically crosslinked hydrogels for glucose sensing |
-
2024
- 2024-03-05 FI FI20245266A patent/FI20245266A1/en unknown
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- 2025-02-17 WO PCT/FI2025/050072 patent/WO2025186507A1/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006080017A2 (en) * | 2005-01-26 | 2006-08-03 | Ramot At Tel Aviv University Ltd. | Biologically active silver-coated proteins |
| EP3093029A1 (en) * | 2009-07-27 | 2016-11-16 | Baxalta GmbH | Blood coagulation protein conjugates |
| US20170315077A1 (en) * | 2016-04-28 | 2017-11-02 | Medtronic Minimed, Inc. | In-situ chemistry stack for continuous glucose sensors |
| US20190177683A1 (en) * | 2017-12-07 | 2019-06-13 | Indiana University Research And Technology Corporation | Stabilization of enzyme-immobilized hydrogels for extended hypoxic cell culture |
Non-Patent Citations (4)
| Title |
|---|
| a document cited to establish the publication date of document D7 * |
| AC-PEG-HZ. Catalog number HE009019. Product information page [online]. Biopharma PEG, 22.1.2020. [retrieved 1.10.2024] Retrieved from <https://www.biochempeg.com/product/AC-PEG-HZ.html> * |
| Aminooxy-PEG-Acrylates. Catalog number R-AC-0004. Product information page [online], Ruixibio, 24.10. 2017. [retrieved 1.10.2024] Retrieved from <https://www.ruixibiotech.com/pts/aminooxy-peg-acrylates> * |
| ZHANG S. ET AL. : "Covalent attachment of glucose oxidase to an Au electrode modified with gold nanoparticles for use as glucose biosensor", BIOELECTROCHEMISTRY, vol. 67, no. 1, 2005, pages 15 - 22, XP004943866, DOI: 10.1016/j.bioelechem.2004.12.002 * |
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| Publication number | Publication date |
|---|---|
| WO2025186507A8 (en) | 2025-10-02 |
| WO2025186507A1 (en) | 2025-09-12 |
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| In | Contact: Department Analytical Biochemistry, University of Potsdam, Potsdam, Germany. |