Method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence

An anti-oxidative ability, allophycocyanin technology, applied in fluorescence/phosphorescence, measurement device, material analysis by optical means, etc., can solve the problem of inaccurate measurement results

Inactive Publication Date: 2018-02-27
SHANGHAI OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Fluorescein is by far the most commonly used method due to its high stability and low cost, however the fluorescent indicator emits light in the short to medium wavelength range and is affected by a static background signal from the sample matrix in the 400-550nm range , resulting in inaccurate final measurement results

Method used

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  • Method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence
  • Method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence
  • Method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence

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Experimental program
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preparation example Construction

[0017] (2) Preparation of Allophycocyanin (APC) Solution

[0018] Measure 10 μL of APC (10 mg / ml) and dissolve it in 500 μL of buffer solution at a concentration of 144 nM, cover it with tin foil and store it in a refrigerator at 4°C.

[0019] (3) Preparation of AAPH stock solution

[0020] Weigh 0.174g AAPH and dissolve it in 5ml buffer solution with a concentration of 128nM, cover it with tin foil and store it in a refrigerator at 4°C.

[0021] (4) Preparation of Trolox stock solution

[0022] Measure 5.26 μL of vitamin E solution (800 mg / ml) and dissolve it in 100 ml of absolute ethanol at a concentration of 100 μM, cover it with tin foil and store it in a refrigerator at 4°C.

[0023] (5) Preparation of actual samples

[0024] The sample was diluted and dissolved with buffer, and centrifuged at 14000 r / min for 15 minutes. The supernatant was taken, covered with tin foil and stored in a refrigerator at 4°C.

[0025] (6) Establishment of standard curve and detection of ...

example 1

[0036] Add 150 μL of PBS buffer, 15 μL of 144nM APC solution, 15 μL of dry red wine diluent (diluted 1500 times) to one well of the 96-well ELISA plate, let stand at 37°C for 15 minutes, then immediately add 20 μL of AAPH solution. Immediately place the plate in the multipurpose plate reader and start the assay. Before each reading, the plate is shaken automatically. Fluorescence was recorded every 5 min for 2 h. Three parallel determinations were performed simultaneously.

example 2

[0038] Add 150 μL PBS buffer solution, 15 μL 144nM APC solution, 15 μL grape juice diluent (diluted 1000 times) to one well of the 96-well ELISA plate in turn, let stand at 37°C for 15 minutes, then immediately add 20 μL AAPH solution. Immediately place the plate in the multipurpose plate reader and start the assay. Before each reading, the plate is shaken automatically. Fluorescence was recorded every 5 min for 2 h. Three parallel determinations were performed simultaneously.

[0039] sample

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Abstract

The invention belongs to the field of rapid food detection and discloses a method for detecting oxidation resistance by utilizing long-wavelength allophycocyanin fluorescence. The method comprises thefollowing steps: with allophycocyanin as a fluorescence indicator, competing free radicals released by AAPH with an antioxidant so as to cause delay of fluorescence decay time of allophycocyanin, with a decay curve without the antioxidant as a blank, calculating an AUC (Area Under the Curve) difference of the two, establishing a standard curve, and performing actual sample detection. The method disclosed by the invention has the advantages that allophycocyanin belongs to a long-wavelength fluorescence indicator, has the excitation wavelength of 653nm and emission wavelength of 668nm and can avoid interference of autofluorescence matters in an actual sample; and allophycocyanin has high pH stability and is non-sensitive to internal and external quenching. According to the advantages, the detection sensitivity is greatly improved.

Description

Technical field: [0001] The invention belongs to the field of rapid detection of food and relates to a method for detecting antioxidant capacity by using long-wavelength allophycocyanin fluorescence. Background technique: [0002] ORAC is an abbreviation for Oxygen Radical Absorbance Capacity, which is also called antioxidant capacity. The ORAC analysis method is based on the principle that free radicals destroy fluorescent probes and cause changes in fluorescence intensity. The water-soluble analogue of vitamin E, Trolox (dro-2,5,7,8-tetramethylchroman-2-carboxylic acid) is used as a quantitative standard. Fluorescence microplate analyzer for analysis. The change in fluorescence intensity reflects the degree of free radical damage. In the presence of antioxidants, it can inhibit the fluorescence changes caused by free radicals. The degree of inhibition reflects its antioxidant capacity against free radicals. [0003] B-phycoerythrin was used as a fluorescent indicator i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/643
Inventor 吴继魁宋人君徐蓁禾罗磊张俊玲
Owner SHANGHAI OCEAN UNIV
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