Cell-based biosensor, preparation method thereof and method of evaluating oxidation resistance by employing same

A technology of cell sensor and anti-oxidation ability, applied in biochemical equipment and methods, screening of compounds, detection of programmed cell death, etc., can solve the problem of inability to fully simulate the cell microenvironment, incomparable test results, and large differences in subjects and other issues, to achieve the effect of shortening the research cycle, short training cycle, and small differences

Active Publication Date: 2017-12-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The mechanism cannot be explained by a single chemical reaction process
Moreover, in vivo experiments on macromolecular organisms cannot completely simulate the cellular microenvironment, and animal experiments are not only complicated but also subject to large differences, the test results cannot be compared

Method used

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  • Cell-based biosensor, preparation method thereof and method of evaluating oxidation resistance by employing same
  • Cell-based biosensor, preparation method thereof and method of evaluating oxidation resistance by employing same
  • Cell-based biosensor, preparation method thereof and method of evaluating oxidation resistance by employing same

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preparation example Construction

[0046] a), preparation of acidified manganese dioxide: MnO 2 Dissolve in concentrated H in an ultrasonic (ultrasonic condition: 100KHz, time 5min) bath 2 SO 4 and concentrated HNO 3 (the massfraction of concentrated sulfuric acid ≥ 70%, the massfraction of concentrated nitric acid is about 68%) obtains mixed solution in the combined acid solution, then mixed solution is stirred 3-5h in ice bath (0 ℃), centrifugal, washing and After drying, a-MnO was obtained 2 (acidified manganese dioxide) nanomaterials;

[0047] b), preparation of modified electrode: a-MnO 2 Nanomaterials are dispersed in chitosan solution under ultrasonic (ultrasonic conditions: 100KHz, time 8min) to obtain a suspension with a solid phase concentration of 0.9-1.1mg / mL, and then 8-12μL of the suspension is dropped on the surface of a dry gold electrode Above, the modified electrode was obtained after drying at 4-6°C;

[0048] c), the preparation of the mixed gel: the graphene oxide is mixed with the DME...

Embodiment 1

[0055] Example 1 Control group

[0056] Positive control: put cell sensor one in Tyrode buffer at 37°C (Tyrode solution preparation: 1000ml Tyrode solution contains: NaCl 8.0g, 10% KCl 2.0ml (0.2g), 10% MgSO 4 .7H 2 O 2.6ml (0.26g), 5% NaH 2 PO 4 .2H 2 O1.3ml (0.065g), NaHCO 3 1.0g, 1M CaCl 2 1.8ml (0.2g), glucose 1.0g), add PMA to make the concentration 100ng / mL, let it stand for 10 minutes, wash 3 times with PBS buffer (phosphate buffered saline) of pH=7.4, and then the cells Sensor one is connected to the electrochemical workstation, in the presence of 1.0mM Fe(CN) 6 3- The cell sensor in the solution is detected by electrochemical method, and the current signal I is recorded 0 = 84.6μA;

[0057] Blank: Cell sensor three was placed in Tyrode buffer at 37 °C, washed 3 times with PBS buffer after 10 minutes, and then connected to the electrochemical workstation, in the presence of 1.0 mM Fe(CN) 6 3- The cell sensor 3 was detected electrochemically in the solution...

Embodiment 2

[0058] Embodiment 2 Lactobacillus plantarum NJAU-01

[0059] 1) Preparation of bacterial solution: Lactobacillus plantarum NJAU-01 isolated from Chinese traditional fermented ham was used to inoculate fermented sausage as the initial culture. It was identified by API 50CHL kit (bioMérieux Inc., France) and 16S rDNA sequencing analysis. Lactobacillus plantarum NJAU-01 strain was grown in MRS broth at 37°C for 18h. First, bacterial cells were harvested, washed and resuspended in deionized water. Adjust the cell concentration to 10 5 , 10 6 , 10 7 , 10 8 , 10 9 and 10 10 CFU / mL. Next, the cells were incubated with 1 mg / ml lysozyme for 30 min at 37°C and then sonicated in an ice bath. After removing cell debris by centrifugation (8000 g, 10 min, 4° C.), the supernatant was obtained as Lactobacillus plantarum NJAU-01 bacterial liquid.

[0060] 2), pretreatment: put Lactobacillus plantarum NJAU-01 bacterial liquid and cell sensor II in Tyrode buffer at 37°C, then add PMA t...

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Abstract

The invention relates to a cell-based biosensor, a preparation method thereof and a method of evaluating oxidation resistance by employing the same. By taking macrophage as a sensing medium and electrochemical impedance as an index, the cell-based biosensor breaks through constraint of conventional detection and evaluation, thereby providing a brand-new path for evaluating the oxidation resistance. The cell-based biosensor comprises a naked gold electrode and macrophage RAW264.7 fixed on the naked gold electrode. By way of establishing a cell model, oxidative stress damage of the macrophage RAW264.7 is simulated, and research on the oxidation resistance of the macrophage by antioxidant substances is lucubrated, so that cell-based biosensor is of significant meaning on research of antioxidant substances by the antioxidant substances, and is particular suitable for researching the antioxidant action of the antioxidant substances on a human body.

Description

technical field [0001] The invention relates to the field of antioxidant capacity measurement, in particular to a novel method for evaluating antioxidant capacity based on a cell sensor using an electrochemical method. Using macrophages as the sensing medium, a 3D macrophage electrochemical sensor was constructed to evaluate the antioxidant capacity. Background technique [0002] The human body is in contact with the outside world, including factors such as breathing, pollution, and ultraviolet radiation, which constantly generate free radicals in the human body. Oxidation reactions that occur during normal physiological activities in the body involve oxidative active substances. When free radicals and the new free radicals induced by them exceed the body's antioxidant capacity, excessive accumulation of oxygen free radicals in cells will cause oxidation. The state of stress, which will lead to the accumulation of oxidative damage to macromolecular substances (proteins, lip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48G01N27/327
CPCG01N27/3275G01N27/48G01N33/5055G01N33/5438G01N33/82G01N2800/7009C12Q1/025G01N27/3272G01N27/3278G01N27/60G01N2500/10G01N27/4166
Inventor 张万刚葛庆丰蒋栋磊葛攀玮周光宏
Owner NANJING AGRICULTURAL UNIVERSITY
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