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Continuous glucose monitoring using an fad-dependent glucose dehydrogenase

A glucose-dependent technology, applied in the direction of microbial determination/testing, enzymes, applications, etc., can solve problems such as impact

Pending Publication Date: 2019-11-22
KIKKOMAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This can be affected by the presence of dissolved oxygen in the measured sample

Method used

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  • Continuous glucose monitoring using an fad-dependent glucose dehydrogenase
  • Continuous glucose monitoring using an fad-dependent glucose dehydrogenase
  • Continuous glucose monitoring using an fad-dependent glucose dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0247] Unless otherwise stated, the thermal and storage stability evaluations of GDH and GOD were performed according to the following test methods.

[0248] testing method

[0249] (1) Introduction of a GDH gene derived from a microorganism belonging to the genus Mucor (Mucor) and confirmation of GDH activity in a transformant

[0250] Briefly, a GDH (MpGDH, SEQ ID NO: 1 ) from a microorganism belonging to the genus Mucor was modified by introducing mutations N66Y / N68G / C88A / Q233R / T387C / E554D / L557V / S559K into it to obtain a coding modification The gene for GDH (which may be referred to herein as MpGDH-M1, or GDH-M1). The amino acid sequence of MpGDH-M1 is shown in SEQ ID NO:3, and the base sequence is shown in SEQ ID NO:4. The DNA construct was obtained by introducing the MpGDH-M1 gene into the multiple cloning site of plasmid pUC19. More specifically, the pUC19 linearization vector included in the In-Fusion HD Cloning Kit (Clontech) was used as the pUC19 vector. The const...

Embodiment 2

[0266] Storage Stability Test of GDH / GOD Immobilized Electrode

[0267] As shown below, GDH / GOD immobilized electrodes were produced and tested for storage stability.

[0268] method of generating electrodes

[0269] 3 μl of 5% polyethyleneimine (PEI) (catalog no. 164-17821, average molecular weight 10000, from Wako, cationic polymer) was added to the carbon-based screen-printed electrode and then dried. Then, 0.5 μl of osmium polymer (catalog no. 002096, BAS) treated at 98° C. for 1 hour was added, followed by drying. Then, 3 µl of 35 mg / ml GDH / GOD was added, followed by drying. Then, 3 µl of 2% polyethylene glycol diglycidyl ether (PEGDE, average molecular weight 500, manufactured by Sigma-Aldrich) was added and stored at 4°C overnight.

[0270] * Electrodes were also produced using 1% polyacrylic acid (PA) (catalog no. 416037-100ML, average molecular weight 15000, from Sigma-Aldrich, anionic polymer) instead of PEI.

[0271] Storage Stability Test

[0272] The electro...

Embodiment 3

[0281] optimum pH

[0282] The pH optima of the glucose dehydrogenases GDH-M2 and GLD3 were evaluated.

[0283] Viability assays were performed as follows. The 2,6-dichloro-indophenol (DCIP) assay (300 mM DCIP / 1 mM PMS / 400 mM glucose) was performed by measuring the time-dependent decrease in DCIP at 520 nm. For pH 5.5 to 6.0, a citrate-sodium buffer is used, and for pH 6 to 8, a phosphate-potassium buffer is used. see Figure 7 (GDH-M2) and Figure 8 (GLD3).

[0284] By comparison, the optimum pH of GOD-3 is reported as pH 5.6 according to the manufacturer's catalog.

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Abstract

Provided is a method of continuous glucose monitoring (CGM) comprising using an FAD-GDH. The FAD-GDH is capable of retaining initial activity over a certain period of time. Also provided is a method for screening for an FAD-GDH suitable for use in CGM as well as a CGM device comprising an FAD-GDH.

Description

technical field [0001] The present invention relates to continuous glucose monitoring using FAD-dependent glucose dehydrogenases. Background technique [0002] Continuous glucose monitoring (CGM, also known as fast glucose monitoring, FGM) systems are important for the diagnosis and control of diabetes. CGM systems are capable of measuring blood glucose levels over a period of a few days to about 1-2 weeks. The CGM system includes a glucose sensor. [0003] Measurement of glucose levels can be performed with glucose oxidase (GOD). Glucose oxidase uses oxygen as an electron acceptor when oxidizing glucose. This can be influenced by the dissolved oxygen present in the measured sample. Currently, to the best of our knowledge, commercially available CGM devices are all based on glucose oxidase (GOD). Certain GODs, such as those from microorganisms belonging to the genus Aspergillus sp., such as Aspergillus niger, are known to retain enzyme activity even when placed under se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/26C12N9/04C12N15/53
CPCC12Q1/32G01N2333/90616C12Q1/54G01N33/66G01N33/68C12Y101/05G01N2333/904
Inventor 钺阳介
Owner KIKKOMAN CORP