CN114858774A - Method for detecting catalase activity in human serum - Google Patents

Method for detecting catalase activity in human serum Download PDF

Info

Publication number
CN114858774A
CN114858774A CN202210727446.9A CN202210727446A CN114858774A CN 114858774 A CN114858774 A CN 114858774A CN 202210727446 A CN202210727446 A CN 202210727446A CN 114858774 A CN114858774 A CN 114858774A
Authority
CN
China
Prior art keywords
fluorescence
serum sample
concentration
activity
serum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202210727446.9A
Other languages
Chinese (zh)
Inventor
李花
董娜
张爱菊
白莹
戴兴德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gansu Medical College
Original Assignee
Gansu Medical College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gansu Medical College filed Critical Gansu Medical College
Priority to CN202210727446.9A priority Critical patent/CN114858774A/en
Publication of CN114858774A publication Critical patent/CN114858774A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for detecting catalase activity in human serum, which adopts a catalytic fluorescence photometry and utilizes H 2 O 2 ‑OPD‑Fe 2+ The fluorescence release system detects the activity of catalase in human serum, a serum sample is mixed with hydrogen peroxide for reaction, o-phenylenediamine, a ferrous sulfate solution and an acetate solution are added, polyethylene glycol octyl phenyl ether is added after the serum sample and the hydrogen peroxide are fully reacted, then the mixed solution is diluted, the fluorescence intensity of the mixed solution is tested under the specific wavelength of a fluorescence photometer, the fluorescence intensity of the serum sample is tested in the same way after the serum sample is boiled, and therefore the activity of the enzyme is quantitatively determined according to the change of the fluorescence intensity. The detection method has the advantages of higher stability, more accurate result, strong sensitivity and wide detection range, and is beneficial to the development and popularization of the fluorescence detection of the medical serum catalase.

Description

Method for detecting catalase activity in human serum
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting catalase activity in human serum.
Background
Catalase (CAT) is one of important physiologically active enzymes in human body, and plays a key role in the whole process of eliminating hydrogen peroxide to prevent oxidative stress caused by human body. CAT mainly functions to decompose hydrogen peroxide, release nascent oxygen, detoxify and protect sulfydryl in vivo, thereby protecting tissue cells from damage. CAT is universally present in various cells of human body, especially in a large number of liver cells and kidney cells, and the detection of CAT activity in serum can be used as an important diagnosis basis for patients with liver diseases.
At present, several methods for measuring CAT activity are established at home and abroad, mainly including a capacity analysis method, an electric analysis method, an ultraviolet spectrophotometry method, a chromogenic photometry, a fluorescence quenching method and the like. Patent No. CN105891182A discloses a method for quantifying catalase, which detects the content of CAT based on the change of fluorescence intensity of fluorescent dye, but the fluorescent dye of 9, 10-diphenylanthracene involved therein has poor stability and easy quenching, the range of excitation wavelength is narrow, the fluorescence efficiency is low, and the sensitivity is low. Some methods are expensive in equipment, some methods need special reagents, some methods are too complex in operation, some methods are poor in repeatability and inaccurate in precision, and accurate detection results cannot be provided in time. The existing catalase fluorescence kit is evolved on the basis of an ultraviolet spectrophotometry, is simple and easy to operate, has low sensitivity and small detection range, is influenced by the chromaticity and turbidity of a sample, and is not suitable for directly detecting the catalase activity of a biological sample. Therefore, the development of a detection method with high stability, accuracy, strong sensitivity and wide detection range is a problem which needs to be solved urgently by the technical personnel in the field.
Disclosure of Invention
Aiming at the problems, the invention provides a method for detecting the catalase activity in human serum, and solves the problems of low stability, low sensitivity and narrow detection range in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention relates to a method for detecting catalase activity in human serum, which is a catalytic fluorescence photometry and utilizes H 2 O 2 -OPD-Fe 2+ The fluorescence release system for detecting the catalase activity in human serum specifically comprises the following steps:
(1) obtaining a serum sample;
(2) contacting the serum sample with H 2 O 2 Mixing, reacting at room temperature for 10-30min, sequentially adding o-phenylenediamine (OPD), a catalyst and a buffer solution, standing for reacting for 1-5min, adding a fluorescence sensitizer, diluting, selecting the emission wavelength of a fluorescence photometer to be 500-520nm, adjusting the distilled water to zero, and measuring the fluorescence intensity F;
(3) taking a fresh serum sample, carrying out enzyme boiling treatment, repeating the step (2), and measuring the fluorescence intensity F of an enzyme blank system 0 ；
(4) Calculating enzyme activity Δ F (Δ F = F) 0 - F)。
Further, the method comprises the following steps:
(1) obtaining a serum sample;
(2) mu.L of serum sample was mixed with 1.20mLH 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine, 1.00mL of catalyst and 5.00mL of buffer solution, standing for reacting for 3min, adding 1.00mL of fluorescence sensitizer, diluting to a constant volume of 50mL, selecting an emission wavelength of a fluorescence photometer to be 510nm, adjusting the emission wavelength of distilled water to zero, and measuring fluorescence intensity F;
(3) taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the step (2), and measuring the fluorescence intensity F of an enzyme blank system 0 ；
(4) Calculating enzyme activity Δ F (Δ F = F) 0 - F)。
Further, the catalyst is ferrous sulfate solution, the buffer solution is acetate solution, and the fluorescence sensitizer is polyethylene glycol octyl phenyl ether (TritonX-100).
Further, said H 2 O 2 The concentration of the compound is 1mmol/L, the concentration of the o-phenylenediamine is 1mmol/L, the concentration of the ferrous sulfate solution is 1mmol/L, the concentration of the acetate solution is 0.1mol/L, and the concentration of the TritonX-100 is 1 mmol/L.
Further, the acetate solution has a pH of 4.
Compared with the prior art, the invention has the beneficial effects that:
the method for detecting the catalase activity in human serum mainly adopts indirect fluorescenceThe photometric method is compared with the traditional detection method, the method is used for detecting Fe in ferrous sulfate solution 2+ Under the catalytic action of (3), H 2 O 2 The o-phenylenediamine is oxidized into 2, 3-Diaminophenazine (DAP), the 2, 3-diaminophenazine has fluorescence release at the emission wavelength of 510nm, the TritonX-100 integrates the functions of solubilization, sensitization and depolymerization, and the micelle action of the TritonX-100 enables the 2, 3-diaminophenazine fluorescence signal to be amplified. Compared with the existing detection method, the detection method has the advantages of higher stability, more accurate result, strong sensitivity and wide detection range, and is more suitable for popularization.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
Example 1
A method for detecting catalase activity in human serum adopts catalytic fluorescence photometry and utilizes H 2 O 2 -OPD-Fe 2+ The fluorescence release system is used for detecting the activity of catalase in human serum.
Firstly, obtaining a serum sample of a healthy male with the age of 11 years from a physical examination center without other treatment;
next, a microsampler was used to accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H 2 O 2 Mixing, reacting at room temperature for 10min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 1min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to 50mL, and selecting 930NThe emission wavelength of a fluorescence photometer is 500nm, distilled water is adjusted to zero, and the fluorescence intensity F is measured;
thirdly, taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the steps, and measuring the fluorescence intensity F of the enzyme blank system 0 ；
Finally, the enzyme activity Δ F was calculated (Δ F = F) 0 - F)。
Example 2
Serum samples from healthy males aged 11 years in example 1 were used without further treatment;
first, accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H with a microsampler 2 O 2 Mixing, reacting at room temperature for 30min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 5min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to 50mL, selecting the emission wavelength of a 930N fluorescence photometer to be 530nm, adjusting the distilled water to zero, and determining the fluorescence intensity F;
secondly, taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the steps, and measuring the fluorescence intensity F of an enzyme blank system 0 ；
Finally, the enzyme activity Δ F was calculated (Δ F = F) 0 - F)。
Example 3
Serum samples from healthy males aged 11 years in example 1 were used without further treatment;
first, accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H with a microsampler 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 3min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to a constant volume of 50mL, selecting the emission wavelength of a 930N fluorescence photometer to be 510nm, adjusting the distilled water to zero, and determining the fluorescence intensity F;
secondly, a fresh serum sample of 5 μ L is taken and enzyme-cookedPerforming death treatment, repeating the steps, and measuring the fluorescence intensity F of the enzyme blank system 0 ；
Finally, the enzyme activity Δ F was calculated (Δ F = F) 0 - F)。
Example 4
Firstly, obtaining a serum sample of a healthy female with the age of 12 years from a physical examination center without other treatment;
next, a microsampler was used to accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 3min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to a constant volume of 50mL, selecting the emission wavelength of a 930N fluorescence photometer to be 510nm, adjusting the distilled water to zero, and determining the fluorescence intensity F;
thirdly, taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the steps, and measuring the fluorescence intensity F of the enzyme blank system 0 ；
Finally, the enzyme activity Δ F was calculated (Δ F = F) 0 - F)。
Example 5
Firstly, obtaining a serum sample of a healthy male with the age of 53 years from a physical examination center without other treatment;
next, a microsampler was used to accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 3min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to a constant volume of 50mL, selecting the emission wavelength of a 930N fluorescence photometer to be 510nm, adjusting the distilled water to zero, and determining the fluorescence intensity F;
thirdly, taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the steps, and measuring the fluorescence intensity F of the enzyme blank system 0 ；
Finally, the enzyme activity Δ was calculatedF(△F = F 0 - F)。
Example 6
Firstly, obtaining a serum sample of a healthy female with the age of 50 years from a physical examination center without other treatment;
next, a microsampler was used to accurately remove 5. mu.L of serum sample and 1.20mL of 1mmol/L H 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine with the concentration of 1mmol/L, 1.00mL of ferrous sulfate solution with the concentration of 1mmol/L and 5.00mL of acetate solution with the concentration of 0.1mol/L, keeping stand for reacting for 3min, adding 1.00mL of TritonX-100 with the concentration of 1mmol/L, diluting to a constant volume of 50mL, selecting the emission wavelength of a 930N fluorescence photometer to be 510nm, adjusting the distilled water to zero, and determining the fluorescence intensity F;
thirdly, taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the steps, and measuring the fluorescence intensity F of the enzyme blank system 0 ；
Finally, the enzyme activity Δ F was calculated (Δ F = F) 0 - F)。
Comparative example 1
In the comparative example 1, a rhodamine B fluorescence quenching method was used, and a serum sample of a healthy female aged 50 years in example 6 was used first without any other treatment; then, one 50mL volumetric flask was taken, and 5. mu.L of a serum sample and 1mmol/L H was added 2 O 2 5.00mL of matrix solution, reacting in water bath at 25 ℃ for 15min, adding 1.5mL of sulfuric acid to terminate enzymatic reaction, adding 2mL of KI solution, reacting for 15min, adding 5mL of rhodamine B, diluting to constant volume with distilled water, measuring the fluorescence intensity F of the solution under the condition that the fluorescence emission wavelength is lambada em =585nm (the fluorescence excitation wavelength is lambada ex =365nm), and simultaneously finishing the absorbance F of an enzyme blank test 0 Measurement according to Δ F (Δ F = F) 0 -F) determining the catalase activity.
Comparative example 2
This comparative example 2 employed o-phenylenediamine color photometry, first using the serum sample of the healthy female of 50 years old of example 6, without further treatment; taking 5 μ L of serum sample by a micro-sampler, placing in a 50mL volumetric flask, and adding H 2 O 2 2.00mL of standard solution, reacting in water bath at 30 ℃ for 30min immediately5.00mL of buffer solution of LHCl-NaAc (pH 1.5) was added to terminate the enzymatic reaction, and Fe was added 2+ 1.50mL and 5.00mL of o-phenylenediamine, the volume is determined by distilled water, the mixture is kept stand for 3min, a 1cm cuvette is selected, the distilled water is used as a reference, and the absorbance F is measured at the wavelength of 448 nm. Simultaneously completing the absorbance F of the enzyme blank experiment 0 The CAT activity was calculated from Δ F by measurement (method as before, only the order of addition of the standard solution and the buffer was changed).
Examples 1 to 6 and comparative examples 1 and 2 were each measured 6 times in parallel, and the results are shown in Table 1.
TABLE 1 results of analysis of serum enzyme Activity
Figure DEST_PATH_IMAGE001
From the analysis of the serum enzyme activity in Table 1, it can be seen from examples 1 to 3 that the recovery rate of the sample and the relative standard deviation result are more excellent when the reaction is carried out at room temperature for 20min and the reaction is carried out at rest for 3min, and the selected emission wavelength of the 930N fluorescence photometer is 510 nm. From the results of examples 3-6, it can be seen that the catalase activity in the serum of healthy persons has a certain correlation with age, and children are higher than adults; as can be seen from the results of example 6, comparative example 1 and comparative example 2, the calculation results of the recovery rate and the relative standard deviation indicate that the detection method of the present invention has high accuracy and precision. In conclusion, the invention has the advantages of higher stability, more accurate result, strong sensitivity and wide detection range.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (5)

1. A method for detecting catalase activity in human serum is characterized in that: the detection method is a catalytic fluorescence photometry method and utilizes H 2 O 2 -OPD-Fe 2+ The fluorescence release system for detecting the catalase activity in human serum specifically comprises the following stepsThe method comprises the following steps:
(1) obtaining a serum sample;
(2) mixing the serum sample with H 2 O 2 Mixing, reacting at room temperature for 10-30min, sequentially adding o-phenylenediamine, catalyst and buffer solution, standing for reacting for 1-5min, adding a fluorescence sensitizer, diluting, selecting the emission wavelength of a fluorescence photometer to be 500-520nm, adjusting the distilled water to zero, and measuring the fluorescence intensity F;
(3) taking a fresh serum sample, carrying out enzyme boiling treatment, repeating the step (2), and measuring the fluorescence intensity F of an enzyme blank system 0 ；
(4) Calculating enzyme activity Δ F (Δ F = F) 0 - F)。
2. The method for detecting catalase activity in human serum according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) obtaining a serum sample;
(2) mu.L of serum sample was mixed with 1.20mLH 2 O 2 Mixing, reacting at room temperature for 20min, sequentially adding 1.00mL of o-phenylenediamine, 1.00mL of catalyst and 5.00mL of buffer solution, standing for reacting for 3min, adding 1.00mL of fluorescence sensitizer, diluting to a constant volume of 50mL, selecting an emission wavelength of a fluorescence photometer to be 510nm, adjusting the emission wavelength of distilled water to zero, and measuring fluorescence intensity F;
(3) taking 5 mu L of fresh serum sample, carrying out enzyme boiling treatment, repeating the step (2), and measuring the fluorescence intensity F of an enzyme blank system 0 ；
(4) Calculating enzyme activity Δ F (Δ F = F) 0 - F)。
3. The method for detecting catalase activity in human serum according to any one of claims 1-2, wherein: the catalyst is ferrous sulfate solution, the buffer solution is acetate solution, and the fluorescence sensitizer is polyethylene glycol octyl phenyl ether.
4. The method of claim 3, wherein the method comprises the steps of: said H 2 O 2 The concentration of the compound is 1mmol/L, the concentration of the o-phenylenediamine is 1mmol/L, the concentration of the ferrous sulfate solution is 1mmol/L, the concentration of the acetate solution is 0.1mol/L, and the concentration of the polyethylene glycol octyl phenyl ether is 1 mmol/L.
5. The method of claim 4, wherein the method comprises the steps of: the acetate solution had a pH of 4.
CN202210727446.9A 2022-06-25 2022-06-25 Method for detecting catalase activity in human serum Withdrawn CN114858774A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210727446.9A CN114858774A (en) 2022-06-25 2022-06-25 Method for detecting catalase activity in human serum

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210727446.9A CN114858774A (en) 2022-06-25 2022-06-25 Method for detecting catalase activity in human serum

Publications (1)

Publication Number Publication Date
CN114858774A true CN114858774A (en) 2022-08-05

Family

ID=82626382

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210727446.9A Withdrawn CN114858774A (en) 2022-06-25 2022-06-25 Method for detecting catalase activity in human serum

Country Status (1)

Country Link
CN (1) CN114858774A (en)

Similar Documents

Publication Publication Date Title
CN109266332B (en) Preparation method of ratiometric fluorescent probe for quantitatively detecting AChE and BChE in blood
CN106946902B (en) A kind of sulfur dioxide near-infrared-two-photon ratio fluorescent probe and preparation method thereof
CN112730355B (en) Cascade catalytic nano multienzyme and preparation method and application thereof
JP2014528086A (en) Measurement of lactic acid in biological fluids
CN110095444A (en) Utilize the method for carbon quantum dot detection glucose in solutions concentration
CN110878085B (en) Rapid high-selectivity hypobromous acid fluorescent probe, preparation method and application
CN112014389A (en) Ascorbic acid interference-based urine occult blood test paper and preparation method thereof
JPS584918B2 (en) Method for measuring glutamate-oxaloacetate-transaminase and glutamate-pyruvate-transaminase
CN106290346A (en) Mensuration reagent of superoxide dismutase and preparation method thereof
CN112485246A (en) Single epithelial tissue tumor cell exudate free heme color development solution and preparation method thereof
CN114858774A (en) Method for detecting catalase activity in human serum
CN102004094A (en) Method for detecting content of benzoyl peroxide in flour
CN113655006B (en) Urinary system knot Dan Chengdan risk factor detection and test system
CN101221129B (en) Sulfated bile acid enzyme fluorescence capillary analytical method and enzyme fluorescence quantitative reagent kit
CN101294192A (en) Analysis method for testing catalase activity in soil
CN108593905A (en) A kind of digoxin immune detection reagent and its preparation and detection method
CN113984689A (en) Kit for measuring glutathione reductase
CN112834493A (en) Blue fluorescent carbon dot, preparation method and application
CN110108685A (en) Using graphene quantum dot as the method for fluorescence probe detection of alkaline phosphatase concentration
CN104155269A (en) Novel method for performing in-vitro detection on blood lactic acid through fluorescent proteins Frex
CN114813557B (en) Urine para-hydroxyphenylalanine quantitative detection kit and detection method thereof
JPS5913197B2 (en) Bile acid measurement method
JP3981190B2 (en) Cholesterol determination method and reagent for determination
WO2023010613A1 (en) Relaxation nuclear magnetic resonance method for quantitatively measruing specific component in liquid biological sample
CN107132218A (en) A kind of simple detection reagent of human body cell Warburg effect and its preparation method and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication
WW01 Invention patent application withdrawn after publication

Application publication date: 20220805