CN100580428C - Spectrophotometric method for detecting trace amount horseradish peroxidase - Google Patents
Spectrophotometric method for detecting trace amount horseradish peroxidase Download PDFInfo
- Publication number
- CN100580428C CN100580428C CN200710049975A CN200710049975A CN100580428C CN 100580428 C CN100580428 C CN 100580428C CN 200710049975 A CN200710049975 A CN 200710049975A CN 200710049975 A CN200710049975 A CN 200710049975A CN 100580428 C CN100580428 C CN 100580428C
- Authority
- CN
- China
- Prior art keywords
- horseradish peroxidase
- hrp
- concentration
- mol
- test tube
- 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.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a method to detect a trace amount of horse radish peroxidase by using cationic surfactant association particle spectrophotometry. HAc-NaAc buffer solution, KI, HRP, and H2O2 are added in a test tube having a lid in turn; after the mixture is shaken up, the tube is arranged in a warm bath for reaction; TPB is added, and then the mixture is shaken up; the volume of the mixture is fixed; the solution got is poured in a quartz sampling cell, and the quartz sampling cell is arranged on an ultraviolet-visible pectrophotometer to measure the absorbance at the position of 308nm; the concentration of horse radish peroxidase is got according to the standard curve. The method has the advantages that KI is used as the hydrogen donor substrate of HRP, which is innocuous and available; the HRP catalyzed hydrogen acceptor substrate H2O2 has specificity. The method makes use of the HRP catalyzed reaction, in which H2O2 oxidizes KI, to measure HRP activity, and the method has high sensitivity and excellent selectivity; the equipments are simple, and just the ultraviolet-visible pectrophotometer and the thermostatic water bath; the operation by adopting the method is simple and easy; the cost of the reagents is low.
Description
Technical field:
The present invention relates to detect the method for trace amount horseradish peroxidase, particularly a kind of method that detects trace amount horseradish peroxidase with cationic surfactant association microparticle spectrophotometric method.
Background technology:
Horseradish peroxidase (Horseradish Peroxidase is called for short HRP) is a kind of very important peroxidase, in fields such as analytical chemistry, environmental science, clinical chemistry, organic syntheses important use is arranged all.The content of measuring HRP in free HRP and the various HRP mark can be measured the amount of antibody in body fluid and other tissue fluid or antigen indirectly, has crucial meaning for the diagnosis of immunology and histochemical research and clinical disease.
Based on HRP catalysis H
2O
2Phenyl amines, phenol analog derivative that oxidation is colourless generate quinoid or azo class dimerization or poly product with color, fluorescence or electrochemical activity, and available spectrophotometric method, fluorescence method or electrochemical method detect HPR activity and H
2O
2, and be widely used in enzyme linked immunological and enzyme analysis.
The method of existing mensuration HRP activity mainly contains spectrophotometric method, fluorophotometric method, chemoluminescence method, and enzyme linked immunosorbent assay analysis method, ELFA method, galvanochemistry enzyme-linked immunosorbent assay etc.Wherein the sensitivity of volt-ampere enzyme-linked immunosorbent assay is higher, but operation is more loaded down with trivial details.Mainly there are two shortcomings in said method: the first, and used substrate mostly is phenyl amines, phenol analog derivative, and less stable sees that light or air are very easily oxidized, and more not enough is that the phenyl amines substrate has carcinogenesis, and its application is restricted.The second, HRP a kind ofly lacks the enzyme of specific oxidation to hydrogen donor substrate (as phenol, amine, ascorbic acid etc.), can the multiple substrate of catalytic oxidation, thereby substrate analogue also has interference to measuring.Therefore, new substrate and the new method of research HRP are significant.
Summary of the invention:
The objective of the invention is provides a kind of method that detects trace amount horseradish peroxidase with cationic surfactant association microparticle spectrophotometric method for overcoming the deficiencies in the prior art.
The object of the invention adopts following technical scheme to realize:
The present invention has studied cationic surfactant bromotetradecane yl pyridines (TPB)-HRP-KI-H
2O
2The photometry feature and the analytical applications thereof of system.Under acid condition, HRP can catalysis H
2O
2With excessive I
-Reaction generates I
3 -, I
3 -Form association microparticle (TPB-I with cationic surfactant TPB etc.
3)
n, there is absorption effect in this association microparticle, and HRP concentration is linear with the absorbance at TPB system 304nm place within the specific limits.Set up in view of the above a kind of highly sensitive, selectivity good, the photometry of easy fast measuring trace HR.
The present invention adopts cationic surfactant association microparticle spectrophotometric method to detect trace amount horseradish peroxidase.Concrete detection method comprises the steps:
1. in the tool plug test tube of 5.0mL, add HAc-NaAc buffer solution, KI and HRP successively;
2. in the tool plug test tube of step 1, add H again
2O
2, shake up;
3. the test tube with step 2 places tepidarium, adds TPB again, shakes up, and is settled to 2.5mL with distilled water
4. above-mentioned gained solution being poured in the quartz colorimetric utensil in right amount, placed on the ultraviolet-visible pectrophotometer scanning to obtain the absorption spectrum of system, is blank not add the absorption intensity that HRP records;
5. above-mentioned gained solution is poured in the quartz colorimetric utensil, placed ultraviolet-visible pectrophotometer to measure the absorbance at 304nm place, the drawing standard working curve is according to typical curve: A
Abs=0.0801c+0.1252, R
2=0.9981, can try to achieve the concentration of horseradish peroxidase in 0.4～3.2ng/mL scope.
In the described tool plug of step 1 test tube, the HAc-NaAc buffer solution of Jia Ruing successively, its pH value is 4.6～5.2, volume 0.2～0.5mL, the KI concentration of adding is 4 * 10
-3Mol/L～6 * 10
-3Mol/L, the HRP concentration of adding is 0.1 μ g/mL, volume is 10～80 μ L;
The H of the described adding of step 2
2O
2Concentration is 1.0 * 10
-5～3.0 * 10
-5Mol/L;
The described bath temperature of step 3 is 20～30 ℃, and the water-bath time is 25～50min, and TPB concentration is 2.0 * 10
-5～6.0 * 10
-5Mol/L;
The described mensuration wavelength of step 5 is 304nm.
Principle of the present invention is: in the HAc-NaAc of slant acidity buffer solution, as H
2O
2When existing, but HRP catalysis H
2O
2Produce OH, the oxidable excessive I of OH
-Generate I
2, excessive I
-With I
2Can be in conjunction with forming I
3 -, and TPB can with I
3 -In conjunction with forming (I
3 --TPB)
nThe association particulate.This particulate be the analysis showed that with NaNo-ZS90 nano particle size and Zeta potential analyser the mean grain size of this particulate is in 700～800nm scope.Can produce absorption effect because of this particulate has bigger particle diameter, and this particle size is relevant with the concentration of HRP, in view of the above, can set up the new method of indirect determination HRP vigor.Its key reaction is as follows:
H
2O
2→·OH (1)
·OH+I
-→OH
-+I
2 (2)
I
2+I
-→I
3 - (3)
nTPB+nI
3 -→(I
3 --TPB)
n (4)
Advantage of the present invention is:
1:KI is as the hydrogen donor substrate of HRP, and this reagent is nontoxic to be easy to get;
2:HRP catalysis hydrogen acceptor substrate H
2O
2Have specificity, this method is utilized HRP catalysis H
2O
2The HRP activity is measured in the reaction of oxidation KI, and it is good that method has selectivity, the characteristics of highly sensitive (reaching pg/mL);
3: equipment is simple, only needs a ultraviolet-visible pectrophotometer and constant temperature water bath;
4: easy and simple to handle, reagent cost is cheap.
Description of drawings:
Fig. 1 is embodiment of the invention KI-HRP-H
2O
2The ultraviolet-visible absorption spectroscopy figure of-TPB system, wherein a:pH4.6-4.80 * 10
-3Mol/L KI-1.51 * 10
-5Mol/L H
2O
2-4.00 * 10
-5Mol/L TPB; B:pH4.6-4.80 * 10
-3Mol/L KI-1.51 * 10
-5Mol/L H
2O
2-4.00 * 10
-5Mol/L TPB-0.40ng/mL HRP; C:pH4.6-4.80 * 10
-3Mol/L KI-1.51 * 10
-5Mol/L H
2O
2-4.00 * 10
-5Mol/LTPB-2.40ng/mL HRP; D:pH4.6-4.80 * 10
-3Mol/L KI-1.51 * 10
-5Mol/L H
2O
2-4.00 * 10
-5Mol/L TPB-3.20ng/mL HRP.
Embodiment:
Below in conjunction with embodiment and accompanying drawing the present invention is further described:
The method that adopts cationic surfactant association microparticle spectrophotometric method to detect trace amount horseradish peroxidase may further comprise the steps:
1. in the tool plug test tube of 5.0mL, add pH successively and be 4.6 HAc-NaAc buffer solution 0.2mL, 4.80 * 10
-3The KI of mol/L, the HRP of 0.1 μ g/mL, 10～80 μ L;
2. in the solution of step 1, add 1.51 * 10
-5Mol/L H
2O
2, shake up;
3. behind 25 ℃ of water-bath 30min, add 4.0 * 10 again
-5Mol/L TPB shakes up, and is settled to 2.5mL with distilled water;
4. above-mentioned gained solution being poured in the quartz colorimetric utensil, placed on the ultraviolet-visible pectrophotometer scanning to obtain the absorption spectrum of system, is blank not add the photon absorbing intensity that HRP records, at the 304nm place, according to typical curve A
Abs=0.0801c+0.1252, R
2=0.9981, can try to achieve the concentration of horseradish peroxidase in the 0.4-3.2ng/mL scope.
KI-HRP-H among the figure
2O
2The ultraviolet spectrogram of-TPB system shows: (TPS-I
3)
nThe association particulate can produce absorption effect, and maximum absorption wavelength is chosen in the 304nm place and measures at the 304nm place, and along with the increase of HRP concentration, the absorption intensity of this association particulate increases.
Though abovely embodiment of the present invention is described, those skilled in the art will appreciate that and above-mentionedly can make numerous variations and modification to embodiment that scope of the present invention is only limited by appended claims only for for example by example.
Claims (1)
1. detect the method for trace amount horseradish peroxidase, it is characterized in that: adopt cationic surfactant association microparticle spectrophotometric method to detect trace amount horseradish peroxidase, its detection method comprises the steps:
(1) in tool plug test tube, add HAc-NaAc buffer solution, KI and horseradish peroxidase successively, wherein, described tool plug test tube is 5.0mL, and the HAc-NaAc pH value of buffer solution of adding is 4.6～5.2, volume 0.2～0.5mL, and the KI concentration of adding is 4 * 10
-3Mol/L～6 * 10
-3Mol/L, the horseradish peroxidase concentration of adding is 0.1 μ g/mL, volume is 10～80 μ L;
(2) in the tool plug test tube of step (1), add H again
2O
2, shake up, wherein the H of Jia Ruing
2O
2Concentration is 1.0 * 10
-5～3.0 * 10
-5Mol/L;
(3) test tube with step (2) places the tepidarium reaction, add cationic surfactant bromotetradecane yl pyridines again, shake up, be settled to 2.5ml with distilled water, wherein, bath temperature is 20～30 ℃, and the water-bath time is 25～50min, and the bromotetradecane yl pyridines concentration of adding is 2.0 * 10
-5～6.0 * 10
-5Mol/L;
(4) above-mentioned gained solution being poured in the quartz colorimetric utensil in right amount, placed on the ultraviolet-visible pectrophotometer scanning to obtain the absorption spectrum of system, is blank not add the photon absorbing intensity that horseradish peroxidase records; Measure the absorbance at 304nm place, according to gained experimental result drawing standard working curve, according to this curve A
Abs=0.0801c+0.1252, R
2=0.9981, can try to achieve the concentration of horseradish peroxidase in 0.4～3.2ng/mL scope, wherein A
AbsBe the absorbance at 304nm place, c is the concentration of horseradish peroxidase, and R is a related coefficient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710049975A CN100580428C (en) | 2007-09-04 | 2007-09-04 | Spectrophotometric method for detecting trace amount horseradish peroxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200710049975A CN100580428C (en) | 2007-09-04 | 2007-09-04 | Spectrophotometric method for detecting trace amount horseradish peroxidase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101201316A CN101201316A (en) | 2008-06-18 |
| CN100580428C true CN100580428C (en) | 2010-01-13 |
Family
ID=39516586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200710049975A Expired - Fee Related CN100580428C (en) | 2007-09-04 | 2007-09-04 | Spectrophotometric method for detecting trace amount horseradish peroxidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100580428C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105675793A (en) * | 2014-11-20 | 2016-06-15 | 中国科学院青岛生物能源与过程研究所 | Peroxidase-imitating material and application thereof |
| CN105498874B (en) * | 2016-01-29 | 2017-05-24 | 中国农业大学 | Chip, system and method for detecting peroxidase concentration, as well as chip production method |
| CN105784660B (en) * | 2016-04-05 | 2018-10-19 | 广西师范学院 | Utilize the method for water-soluble InP/ZnS QDs probe in detecting horseradish peroxidase concentration |
| CN109187466A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | It is a kind of to be catalyzed TMB-H with compatible reaction regulation carbon dots2O2The method of reaction product fluorescent spectrometry detection biotin |
| CN109238998B (en) * | 2018-10-16 | 2021-09-03 | 常州大学 | Method for detecting peroxide value of grease by using phenols as substrates |
-
2007
- 2007-09-04 CN CN200710049975A patent/CN100580428C/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| 催化分光光度法测定辣根过氧化物酶新方法研究. 魏永锋，阎宏涛.光谱学与光谱分析，第21卷第5期. 2001 |
| 催化分光光度法测定辣根过氧化物酶新方法研究. 魏永锋，阎宏涛.光谱学与光谱分析，第21卷第5期. 2001 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101201316A (en) | 2008-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Yang et al. | Portable aptasensor of aflatoxin B1 in bread based on a personal glucose meter and DNA walking machine | |
| Ge et al. | Advanced sensing technologies of phenolic compounds for pharmaceutical and biomedical analysis | |
| Alex et al. | Review of recent developments (2018–2020) on acetylcholinesterase inhibition based biosensors for organophosphorus pesticides detection | |
| CN100580428C (en) | Spectrophotometric method for detecting trace amount horseradish peroxidase | |
| Choi et al. | Chromogenic and fluorogenic signaling of sulfite by selective deprotection of resorufin levulinate | |
| Goodey et al. | Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities | |
| Swager et al. | Introduction: chemical sensors | |
| Germain et al. | Turn-on fluorescence detection of H2O2 and TATP | |
| Li et al. | Measurement of atmospheric hydrogen peroxide and hydroxymethyl hydroperoxide with a diffusion scrubber and light emitting diode− liquid Core waveguide-based fluorometry | |
| Wu et al. | Homogeneous, unmodified gold nanoparticle-based colorimetric assay of hydrogen peroxide | |
| Greenawald et al. | Development of an inexpensive RGB color sensor for the detection of hydrogen cyanide gas | |
| Chen et al. | Novel colorimetric method for simultaneous detection and identification of multimetal ions in water: Sensitivity, selectivity, and recognition mechanism | |
| Kumari et al. | Fe-TAML encapsulated inside mesoporous silica nanoparticles as peroxidase mimic: femtomolar protein detection | |
| Gründler | Chemical sensors | |
| Garreffi et al. | Highly sensitive and selective fluorescence sensor based on nanoporous silicon-quinoline composite for trace detection of hydrogen peroxide vapors | |
| CN109187470B (en) | Aptamer-mediated silver-doped carbon dot catalysis H2O2Method for measuring lead by reaction with TMB (tetramethylbenzidine) through fluorescence spectrum | |
| Deng et al. | Chemiluminescent ion sensing platform based on ionophores | |
| Michel et al. | Integrated sensor for continuous flow electrochemiluminescent measurements of codeine with different ruthenium complexes | |
| Huang et al. | Colorimetric and photothermal dual-mode immunoassay of aflatoxin B1 based on peroxidase-like activity of Pt supported on nitrogen-doped carbon | |
| Kumar et al. | Organic cation receptor for colorimetric lateral flow device: detection of zearalenone in food samples | |
| Verma et al. | Single-drop and nanogram level determination of sulfite (SO32−) in alcoholic and nonalcoholic beverage samples based on diffuse reflectance fourier transform infrared spectroscopic (DRS-FTIR) analysis on KBr matrix | |
| Lu et al. | Remote detection of HCN, HF, and nerve agent vapors based on self-referencing, dye-impregnated porous silicon photonic crystals | |
| Silva et al. | Optical sensor for sulfur dioxide determination in wines | |
| Wang et al. | A visual cardiovascular biomarker detection strategy based on distance as readout by the coffee-ring effect on microfluidic paper | |
| Ganjali et al. | Lanthanide recognition: A dysprosium (III) selective fluorimetric bulk optode |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100113 Termination date: 20100904 |