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KR-20260064050-A - Digitonin-Treated Mitochondria-Lactate Oxidase Electrode for Enhanced Lactate Detection and Manufacturing method Thereof

KR20260064050AKR 20260064050 AKR20260064050 AKR 20260064050AKR-20260064050-A

Abstract

The present invention relates to an electrode for detecting lactate comprising a digitonin-treated mitochondrial lactate oxidase and a method for manufacturing the same. By functionalizing the enzyme layer using an electron transfer medium and a crosslinking agent based on this enzyme, and by applying digitonin-treated mitochondria, the electrode operates with high sensitivity at low detection concentrations and has the effect of being customizable for the user.

Inventors

  • 김창준
  • 셀바라잔 바르시니

Assignees

  • 경상국립대학교산학협력단

Dates

Publication Date
20260507
Application Date
20241031

Claims (12)

  1. Conductive substrate; and An electrode for detecting lactate, comprising an enzyme layer located on a substrate and including mitochondria treated with digitonin, lactate oxidase, and a crosslinking agent.
  2. In paragraph 1, An electrode for detecting lactic acid, characterized in that the conductive substrate is one or more selected from the group consisting of gold, aluminum, platinum, nickel, graphene, silver nanowire film, carbon, metal grid, and indium tin oxide.
  3. In paragraph 1, The above-mentioned digitonin-treated mitochondria is an electrode for detecting lactate, characterized by being prepared by treating 1 µl of digitonin into mitochondria in an amount corresponding to 1 to 5 mg of mitochondrial protein.
  4. In paragraph 1, An electrode for detecting lactic acid, characterized in that the crosslinking agent is one or more selected from the group consisting of gelatin, silk fibroin, cellulose, dextran, cyclodextran, alginate, chitosan, and agar.
  5. In paragraph 1, An electrode for detecting lactate, characterized in that, in the enzyme layer above, mitochondria treated with digitonin, lactate oxidase, and a crosslinking agent are included in a volume ratio of 1:0.1:0.1 to 1:2:2.
  6. A lactic acid detection sensor comprising an electrode for detecting lactic acid according to claim 1.
  7. In paragraph 6, The above-described lactic acid detection sensor is characterized by comprising: a substrate; an electrode for detecting lactic acid disposed on the upper surface of the substrate; and a reference electrode disposed on the upper surface of the electrode for detecting lactic acid, spaced apart from the electrode for detecting lactic acid.
  8. A method for manufacturing an electrode for detecting lactate, comprising the step of preparing a mixture containing mitochondria treated with digitonin, lactate oxidase, and a crosslinking agent, and applying it onto a conductive substrate to prepare an enzyme layer.
  9. In paragraph 8, A method for manufacturing an electrode for detecting lactic acid, characterized in that the crosslinking agent is one or more selected from the group consisting of gelatin, silk fibroin, cellulose, dextran, cyclodextran, alginate, chitosan, and agar.
  10. In paragraph 8, The above-mentioned digitonin-treated mitochondria are prepared by adding a digitonin solution to a mitochondrial solution, A method for manufacturing an electrode for detecting lactate, characterized by using 1 μl of digitonin to treat mitochondria in an amount corresponding to 1 to 5 mg of mitochondrial protein.
  11. In paragraph 8, A method for manufacturing an electrode for detecting lactic acid, characterized in that the mitochondria treated with the above-mentioned digitonin, lactate oxidase, and crosslinking agent are a mixture mixed in a volume ratio of 1:0.1:0.1 to 1:2:2.
  12. In paragraph 8, A method for manufacturing a lactic acid detection sensor, characterized by further including a step of drying at 10 to 30 ℃ for 0.1 to 5 hours after the step of manufacturing the enzyme layer.

Description

Digitonin-Treated Mitochondria-Lactate Oxidase Electrode for Enhanced Lactate Detection and Manufacturing Method Thereof The present invention relates to an electrode for detecting lactate comprising a digitonine-treated mitochondrial lactate oxidase and a method for manufacturing the same. Because human sweat contains many molecules, ions, and biomarker substances related to health status and disease, sweat-based wearable sensors capable of non-invasively detecting them have recently been attracting attention. Most commercially available wearable medical and healthcare devices currently use only indicators such as heart rate and body temperature, which are relatively easy to monitor, because it is difficult to measure and quantify chemical biomarkers obtained from biological samples such as blood and sweat. Among the body's chemical indicators, lactic acid is present in both blood and sweat and is an important biomarker that reflects not only the oxygen supply to muscles but also the intensity of physical activity and the oxygen supply status of the muscles, as it is produced during the breakdown of glucose in the absence of oxygen. Currently, sensors utilizing lactate oxidase electrodes, which convert lactate to pyruvate, are the most widely used for electrochemical biosensing to detect lactate. However, in the case of lactate oxidase electrodes, pyruvate accumulates over time, gradually inhibiting the activity of the lactate oxidase. Furthermore, sensitivity decreases due to interference caused by pyruvate, making continuous and convenient measurement difficult. Accordingly, as the need for an accurate and rapid lactic acid detection method and a corresponding high-performance sensor is increasing, the present invention has strived to complete a lactic acid detection sensor having a new structure to solve the aforementioned problems and provide better lactic acid detection performance. As a result, it has succeeded in completing a lactic acid detection sensor that is practical, economical, and capable of maintaining excellent detection performance for a long time. FIG. 1 is a schematic diagram showing the structure of an electrode for detecting lactic acid according to the present invention. In the drawing, ★ represents digitonin. Figure 2 illustrates the manufacturing process of an electrode for detecting lactic acid according to the present invention. Figure 3 illustrates a detection system for analyzing the electrochemical characteristics of an electrode for detecting lactic acid. Figure 4 is a confocal fluorescence microscope image of Saccharomyces cerevisiae cells and mitochondria according to Preparation Example 1. Figure 4a shows stained Saccharomyces cerevisiae cells, Figure 4b shows unstained Saccharomyces cerevisiae cells, Figure 4c shows stained mitochondria, and Figure 4d shows unstained mitochondria. Figure 5 shows the Cryo-TEM results for mitochondria (a) obtained from Preparation Example 1 and mitochondria (b) obtained from Preparation Example 2. FIG. 6 is the FT-IR spectrum for the lactic acid detection electrode of Example 1 (Lox-D-Mito-Gel) (a), and the lactic acid detection electrodes of Comparative Example 1 and Comparative Example 4 (LOx-Gel (c), LOx-Mito-Gel (b)). As a control, an electrode with gelatin immobilized on a gold electrode was used (d). Figure 7 shows the results of PCA on the lactic acid detection electrode of Example 1 (LOx-D-Mito-Gel) and the lactic acid detection electrodes of Comparative Example 1 and Comparative Example 4 (LOx-Gel, LOx-Mito-Gel). As a control, an electrode with gelatin immobilized on a gold electrode was used (gelatin). Figure 8 shows the cyclic voltammograms of the mitochondrial electrode (Mito-Gel) prepared from Comparative Example 2. a is the cyclic voltammogram performed under conditions with pyruvate, and b is the cyclic voltammogram performed under conditions without pyruvate. The interpolated graph is the peak current graph over time for each condition. Figure 9 shows the cyclic voltammograms of the mitochondrial electrode (D-Mito-Gel) modified with digitonin prepared from Comparative Example 3. a is the cyclic voltammogram performed under conditions with pyruvate, and b is the cyclic voltammogram performed under conditions without pyruvate. The interpolated graph is the peak current graph over time for each condition. FIG. 10 shows the cyclic voltammograms (CV) of the lactic acid detection electrode (LOx-Gel) prepared from Comparative Example 1. a is the CV curve performed under conditions with lactic acid, and b is the CV curve performed under conditions without lactic acid. The interpolated graph is the peak current graph over time for each condition. FIG. 11 shows the cyclic voltammograms of the lactic acid detection electrode (LOx-Mito-Gel) prepared from Comparative Example 4. a is the cyclic voltammogram performed under conditions with lactic acid, and b is the cyclic voltammogram performed under conditions without lactic acid. The int