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Measurement method for dehydrogenase and substrate concentration thereof, electron mediator, and measurement reagent for dehydrogenase including said electron mediator and for substrate thereof

一种电子中介、测定方法的技术，应用在微生物的测定/检验、生物测试、生物化学设备和方法等方向，能够解决不容易使用、试剂溶液安定性差等问题

Inactive Publication Date: 2016-12-14
DOJINDO LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the cytotoxicity test, by using lipoamide dehydrogenase, an enzyme that performs electron transfer instead of a low-molecular electron mediator, the electron transfer of living cells is inhibited, and the concentration or measurement of dehydrogenase derived from dead cells in the presence of living cells The method of activity is also suggested, but the reagent solution composed of the enzyme used has poor stability and is not easy to use

Method used

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  • Measurement method for dehydrogenase and substrate concentration thereof, electron mediator, and measurement reagent for dehydrogenase including said electron mediator and for substrate thereof
  • Measurement method for dehydrogenase and substrate concentration thereof, electron mediator, and measurement reagent for dehydrogenase including said electron mediator and for substrate thereof
  • Measurement method for dehydrogenase and substrate concentration thereof, electron mediator, and measurement reagent for dehydrogenase including said electron mediator and for substrate thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Synthesis of Ⅰ-1

[0065] [chemical formula 8]

[0066]

[0067] The 5-Methyl-1-carboxybutyl phenazine derivative (1) was synthesized according to the method already reported (International Publication No. 2009 / 118157). 40mg (50μmol) of N 6 ―2AE―NAD (Tongjin Institute of Chemistry) was dissolved in 1ml of 100mmol / L phosphate buffer solution (pH 7.4), and 40mg (209μmol) of WSC (1-Ethyl-3-(3-dimethylaminopro pyl)carbodiimide hydrochloride) and 85.8 mg (209 μmol) of compound 1 was reacted at room temperature for 20 hours. The reaction mixture was purified by reverse-phase HPLC, and dried under reduced pressure in a desiccator to obtain 12 mg of a red solid of I-1.

Embodiment 2

[0068] Embodiment 2: Synthesis of Ⅰ-4

[0069] [chemical formula 9]

[0070]

[0071] The 5-Ethyl-1-carboxybutyl phenazine derivative (2) was synthesized according to the method already reported (International Publication No. 2009 / 118157). 44.8mg (57μmol) of N 6 ―2A E―NADP (Tongjin Institute of Chemistry) was dissolved in 1ml of 100mmol / L phosphate buffer solution (pH 7.4), and 40mg (209μmol) of WSC (1-Ethyl-3-(3-dimethylaminopro pyl)carbodiimide hydrochloride) and 88.7 mg (209 μmol) of compound 2 was reacted at room temperature for 20 hours. The reaction mixture was purified by reverse-phase HPLC, and dried under reduced pressure in a desiccator to obtain 15 mg of a red solid of I-4.

Embodiment 3

[0072] Embodiment 3: Synthesis of Ⅰ-6

[0073] [chemical formula 10]

[0074]

[0075] The 5-Ethyl-1-carboxybutyl phenazine derivative (2) was synthesized according to the method already reported (International Publication No. 2009 / 118157). Dissolve 40mg (301μmol) of aspartic acid in 1ml of 100mmol / L phosphate buffer solution (pH 7.4), add 40mg (209μmol) of WSC (1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and 88.7 mg (209 μmol) of compound 2 was reacted at room temperature for 20 hours. The reaction mixture was purified by reverse-phase HPLC, and dried under reduced pressure in a desiccator to obtain 45 mg of a red solid of I-6.

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Abstract

Provided are: a measurement method for measuring a dehydrogenase in the presence of live cells and the substrate concentration thereof, said measurement method being characterized by using an electron mediator represented, for example, by structural formula (I) and resistant to reduction by live cells; the electron mediator indicated by structural formula (I); and a measurement reagent for a dehydrogenase including the electron mediator and for the substrate thereof. (I) A-L-M (In structural formula (I), A indicates a structural site containing at least one type of anionic group, M indicates a structural site where electron transfer occurs, and L indicates a linker site that links A and M.

Description

technical field [0001] The invention belongs to the technical field of component determination in cell culture fluid and blood, and in particular relates to a dehydrogenase and a method for determining the concentration of dehydrogenase and its substrate useful in the presence of living cells. The electron mediator used depends on the assay reagent for the dehydrogenase and its substrate containing the electron mediator. Background technique [0002] The concentration of substrates such as glucose in cell culture fluid and blood is measured using a detection series using substrate-specific enzymes such as oxidase and dehydrogenase. In particular, the measurement series using dehydrogenase is not affected by oxygen in the atmosphere, so the utility value is high. After dehydrogenase reacts as a substrate, auxiliary enzymes such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are reduced (NAD + →NADH, NADP + →NADPH). The re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/32C07C211/52C12Q1/06
CPCC12Q1/32G01N2333/904G01N33/52
Inventor 岩本正史田中智也渡辺荣治志贺匡宣
Owner DOJINDO LAB
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