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EP-4735875-A1 - METHOD OF MANUFACTURING AN ENZYME-ELECTRODE AND ENZYME-ELECTRODE

EP4735875A1EP 4735875 A1EP4735875 A1EP 4735875A1EP-4735875-A1

Abstract

The present invention relates to a method for manufacturing an enzyme electrode, wherein said method comprises the steps of: i) preparing a carbon particle suspension comprising carbon black and a non-conducting cationic polymer; ii) preparing a mixture comprising an enzyme and the carbon particle suspension; iii) applying the mixture to a conductive surface of an electrode; and iv) drying the mixture applied to the conductive surface of the electrode.

Inventors

  • Schulz, Christopher
  • Felice, Alfons

Assignees

  • DirectSens GmbH

Dates

Publication Date
20260506
Application Date
20240624

Claims (20)

  1. 1. A method for manufacturing an enzyme electrode, wherein said method comprises the steps of: i. preparing a carbon particle suspension comprising carbon black and a nonconducting cationic polymer; ii. preparing a mixture comprising an enzyme and the carbon particle suspension; iii. applying the mixture to a conductive surface of an electrode; and iv. drying the mixture applied to the conductive surface of the electrode.
  2. 2. The method of claim 1 , wherein the carbon black is graphitized carbon black.
  3. 3. The method of claim 1 or 2, wherein the non-conducting cationic polymer is polyethyleneimine (PEI), diethylaminoethyl dextran (DEAE), polylysine, or polydiallyldimethylammonium chloride (PDADMAC).
  4. 4. The method of any one of claims 1 to 3, wherein in the carbon particle suspension the ratio of carbon black: non-conducting cationic polymer is in the range of 10:1 to 1 :10.
  5. 5. The method of any one of claims 1 to 4, wherein the carbon particle suspension further comprises water.
  6. 6. The method of claim 5, wherein the carbon particle suspension has a concentration in the range of 1 to 10 % (w/v).
  7. 7. The method of any one of claims 1 to 6, wherein the carbon particle suspension is prepared by a sonication treatment.
  8. 8. The method of claim 7, wherein a probe sonicator is used for sonication treatment.
  9. 9. The method of claim 7 or 8, wherein the sonication treatment is performed for at least 60 seconds.
  10. 10. The method of any one of claims 7 to 9, wherein the sonication treatment is performed at a power density of 0.4 W/cm 3
  11. 11 . The method of any one of claims 7 to 10, wherein the sonication treatment is an ultrasonic treatment.
  12. 12. The method of any one of claims 1 to 11 , wherein agglomerates are removed prior to preparing the mixture comprising the carbon particle suspension and the enzyme.
  13. 13. The method of any one of claims 1 to 12, wherein the conductive surface is gold, platinum, or carbon.
  14. 14. The method of any one of claims 1 to 13, wherein the enzyme is a direct electron transfer (DET) enzyme.
  15. 15. The method of claim 14, wherein the DET enzyme is DET enzyme having analyte oxidizing activity.
  16. 16. The method of claim 15, wherein the analyte oxidizing activity is glucose oxidizing activity, lactose oxidizing activity, or lactate oxidizing activity.
  17. 17. The method of any one of claims 14 to 16, wherein the DET enzyme is cellobiose dehydrogenase (CDH), flavocytochrome b2 (FCb2), or a functional variant thereof.
  18. 18. The method of any one of claims 1 to 17, wherein in the mixture the ratio of enzyme:carbon particle suspension is 1 :4.
  19. 19. An enzyme electrode comprising an analyte sensing layer, wherein said analyte sensing layer is a single layer comprising carbon black, a non-conducting cationic polymer, and an enzyme.
  20. 20. The enzyme electrode of claim 19, wherein carbon black is graphitized carbon black.

Description

METHOD OF MANUFACTURING AN ENZYME-ELECTRODE AND ENZYME-ELECTRODE FIELD OF THE INVENTION The present invention relates to the field of enzyme electrodes and their manufacturing. The invention further relates to the use of enzyme electrodes for the detection and quantification of analytes, in particular to methods and means for the detection and/or quantification of analytes with enzyme-based methods. BACKGROUND OF THE INVENTION Enzyme electrodes are used in several different industries. Thereby, these electrodes are incorporated into biosensors for the detection of analytes. In food analytics and healthcare, carbohydrates such as glucose or lactate can be detected by biosensors using enzyme modified electrodes. In general, biosensors are analytical devices that leverage the high specificity of biological recognition elements, which are typically enzymes. Thereby, a biosensor consists of a biological recognition element e.g., an enzyme, which is able to specifically interact with a target molecule and a transducer able to convert this interaction into a measurable signal. In biosensors, enzymes are connected to electrodes and electric currents are measured that are proportional to the enzymes’ substrate concentration in a sample. Biosensors are easy to operate, highly specific and do not need expensive and heavy equipment like it is the case for HPLC or NMR. In general, three generations of biosensors are known. A biosensor based on an oxidase such as LOx is dependent on oxygen and is a first-generation biosensor. In second-generation biosensors, redox mediators other than the O2/H2O2 pair are applied to transfer electrons from the enzyme to the electrode. In contrast, third-generation biosensors are based on direct electron transfer from enzyme to electrode. Thereby, the enzymes used in these third-generation biosensors are characterized by direct electron transfer (DET) capability to the electrode. Cellobiose dehydrogenase (CDH), flavocytochrome b2 (FCb2), and flavin adenine dinucleotide glucose dehydrogenase (FADGDH) are examples of such DET-capable enzymes. One of the major challenges in this field is the material of the electronic circuits and the manufacturing of enzyme modified electrodes. While most applications are based on screen-printed carbon-based electrodes, small embodiments rely on the use of gold or other metals, that can be processed at very small scales. Thereby, enzyme interaction with e.g., gold-based circuits, yields only in very small signals, that are not useful for commercial applications. Different approaches for manufacturing enzyme modified electrodes have been developed in the field. Thereby, also different materials have been used. The manufacturing of electrodes equipped with a carbon black-enzyme ink has been reported. For example, Shimizu, H. and Tsugawa, W. (2012) described glucose monitoring by a Direct Electron Transfer electrode using an electrode equipped with a carbon-enzyme ink comprising a carbon ink. Thereby, the carbon ink comprises a carbon black, an anionic polymer (Nation), and water. US2020/024631A1 discloses an electrode made from carbon black, with a polymer matrix containing PEI and enzymes deposited on the electrode. A method for manufacturing a bioelectrode consisting of carbon black and polyethyleneimine (PEI) was described by Jayapiriya et al. (2023). Carbon black and PEI are applied to the electrode as a solution, and following a drying step, antibodies are immobilized onto the dried layer. Ibanez-Redin et al. (2020) describe a method to produce a bioelectrode using 3D printing to produce a composite of carbon black, PEI and glucose oxidase. US2022/133190A1 discloses an electrode comprising cellobiose dehydrogenase coupled to the neutral compound polyvinyl alcohol in the presence of ketjen black which is deposited on a carbon electrode, comprising an additional layer of a polycationic composition is disposed over the analyte modulating layer. However, prepared carbon inks as described in the literature are often not well dispersed consisting of too large particles leading to sedimentation over time and clogging issues of liquid dispensing devices impeding manufacturability. Limited stability of the enzyme after mixing with the carbon ink also is an issue due to incompatibility of the enzyme with certain ink ingredients. Thus, there is an urgent need in the art for methods of manufacturing electrodes and methods of manufacturing enzyme modified electrodes which provide improved signals on various electrode materials including also usually hard to modify metallic surfaces. SUMMARY OF THE INVENTION It is the objective of the present invention to provide methods of manufacturing enzyme modified electrodes which provide improved signals. The objective is solved by the subject matter of the present invention. According to the invention there is provided a method for manufacturing an enzyme electrode, wherein said method comprises the steps of: i. preparing