CN105784660A - Method for detecting concentration of horseradish peroxidase by utilizing water-soluble InP/ZnS QDs probe - Google Patents
Method for detecting concentration of horseradish peroxidase by utilizing water-soluble InP/ZnS QDs probe Download PDFInfo
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- CN105784660A CN105784660A CN201610205731.9A CN201610205731A CN105784660A CN 105784660 A CN105784660 A CN 105784660A CN 201610205731 A CN201610205731 A CN 201610205731A CN 105784660 A CN105784660 A CN 105784660A
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a method for detecting concentration of horseradish peroxidase by utilizing a water-soluble InP/ZnS QDs probe. The method comprises the following steps: step 1, preparing horseradish peroxidase standard solutions which have different concentration and contain water-soluble InP/ZnS QDs; detecting the fluorescence intensity of the standard solutions to obtain fluorescence spectrograms of the standard solutions; establishing a linear relation of a difference value between the fluorescence intensity of the standard solution with the concentration of the horseradish peroxidase of zero and the fluorescence intensity of all the standard solutions and the concentration of the horseradish peroxidase; step 2, preparing a horseradish peroxidase sample solution containing the water-soluble InP/ZnS QDs and detecting the fluorescence intensity of the standard solution; and determining the concentration of the horseradish peroxidase in the sample solution through the linear solution. By taking the InP/ZnS QDs as the probe and utilizing the property of quenching the fluorescence of the water-soluble InP/ZnS QDs of the horseradish peroxidase, the concentration of the horseradish peroxidase is detected; and a detection process is simple and convenient, high in sensitivity and low in detection limit, and online and in-situ rapid and sensitive detection of the concentration of the horseradish peroxidase can be realized.
Description
Technical field
The present invention relates to a kind of method detecting horseradish peroxidase concentration, particularly relate to a kind of method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration.
Background technology
Horseradish peroxidase is a kind of glycoprotein, and the content that the origin of its title is because in Radix Cochleariae officinalis this enzyme is significantly high, and it is currently used a kind of marker enzyme, for traget antibody or other oroteins, can be used for detection and localization, can be used for quantitative assay.Horseradish peroxidase is widely used in the fields such as work, agriculture, doctor, environmental protection, can use the biochemical diagnosis test kits such as the blood glucose of this enzyme preparation, triglyceride, cholesterol and enzyme exempts from the enzyme labelled antibody energy during ELISA measures.
In recent years, along with quantum dot synthetic technology is constantly progressive and quantum dot going deep in field application such as life sciences, solaode, optics, the impact that environment produces is received the concern of more and more people by its toxicity.Therefore, traditional IIB-VI quantum dot such as CdTe, CdSe etc., although its technical development comparative maturity, but the latent defect containing this toxic element of Cd, will greatly limit the application in its future.By comparison, III-V quantum dot has relatively low toxicity, not wherein InP quantum dot especially prominent (not containing the toxic element such as Cd, Hg, As, Se), its spectral region covers visible and near infrared region (500-850nm), and having relatively small particle size, these features are all not available for the II-VI quantum dots such as traditional CdSe.Excellent biocompatibility and optical property make InP/ZnS quantum dot be more suitable for being applied in the fields such as bioanalysis.
Summary of the invention
It is an object of the present invention to provide the new method of a kind of water solublity InP/ZnSQDs probe in detecting horseradish peroxidase, the method is simple to operate, it is quick and highly sensitive to detect, and can carry out the highly sensitive identification of horseradish peroxidase in solution.
A further object of the invention is research water solublity InP/ZnSQDs new opplication in horseradish peroxidase Concentration Testing.
Technical scheme provided by the invention is:
A kind of method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, including:
Step one, preparation variable concentrations the standard solution of horseradish peroxidase of containing water-soluble InP/ZnSQDs, detect the fluorescence intensity of described standard solution, obtain the fluorescence spectrum figure of described standard solution, set up the linear relationship between the difference of the fluorescence intensity of the standard solution that horseradish peroxidase concentration is zero and the fluorescence intensity of all standard solution and horseradish peroxidase concentration；
Step 2, preparation containing water-soluble InP/ZnSQDs the sample solution of horseradish peroxidase, detect the fluorescence intensity of described sample solution, determine the concentration of horseradish peroxidase in described sample solution by described linear relationship.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the method preparing described standard solution in described step one is:
The original solution of the horseradish peroxidase of preparation variable concentrations, take the original solution of different volumes, and the buffer solution that adds the water solublity InP/ZnSQDs of same volume and different volumes wherein respectively is equal to the volume of mixed liquor, obtain the standard solution of equal-volume variable concentrations.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the method preparing described sample solution in described step 2 is:
A certain amount of water solublity InP/ZnSQDs is added in unknown horseradish peroxidase solution, make identical with the concentration of water solublity InP/ZnSQDs in described standard solution, the buffer solution added and prepare described standard solution identical type, to equal with the volume of described standard solution, obtains described sample solution.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described step 2 being determined, in described sample solution, the method for the concentration of horseradish peroxidase is:
The fluorescence intensity of standard solution that horseradish peroxidase concentration is zero is brought in described linear relationship with the difference of the fluorescence intensity of described sample solution, obtains the concentration of horseradish peroxidase in described sample solution.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described buffer solution is pH value is the phosphate buffered solution of 7.5.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the excitation wavelength detecting described standard solution and described sample solution fluorescence intensity is 388nm.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the volume of described standard solution and described sample solution is 3mL.
Preferably, in the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described standard solution and in described sample solution the concentration of water solublity InP/ZnSQDs be 23.04nM.
The present invention at least includes following beneficial effect:
The present invention using water solublity InP/ZnSQDs as probe, utilize the characteristic of horseradish peroxidase quencher water solublity InP/ZnSQDs fluorescence, horseradish peroxidase concentration is detected, detection process is simple and convenient, highly sensitive, detection is limit low, it may be achieved the online original position rapid sensitive detection of horseradish peroxidase concentration in actual sample.
Part is embodied by the further advantage of the present invention, target and feature by description below, and part is also by by being understood by those skilled in the art the research of the present invention and practice.
Accompanying drawing explanation
Fig. 1 be the horseradish peroxidase of variable concentrations of the present invention standard solution in after water solublity InP/ZnSQDs probe reacts with horseradish peroxidase, the fluorescence spectrum figure obtained when excitation wavelength is 388nm；
Fig. 2 is horseradish peroxidase concentration of the present invention be the fluorescence intensity of the standard solution of zero and variable concentrations horseradish peroxidase standard solution fluorescence intensity difference and horseradish peroxidase concentration between linear relationship curve.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to description word.
Embodiment 1
One, linear relationship is set up
The original solution of the horseradish peroxidase of preparation variable concentrations, and add the water solublity InP/ZnSQDs of same volume wherein respectively, i.e. water solublity indium phosphide/ZnS quantum dots, then it is diluted to 3mL by phosphate buffered solution, preparing the standard solution of the horseradish peroxidase of a series of equal-volume variable concentrations, in several standard solution, the concentration of horseradish peroxidase is different, and the concentration of water solublity InP/ZnSQDs is identical, wherein, in standard solution, the concentration of horseradish peroxidase is followed successively by 0,7 × 10-9mol/L、1.5×10-8mol/L、2.5×10-8mol/L、3×10-8mol/L、6×10-8mol/L、1×10-7mol/L、2×10-7Mol/L and 4 × 10-7Mol/L, its number respectively a, b, c, d, e, f, g, h and i, in standard solution, the concentration of water solublity InP/ZnSQDs is 23.04nM；
After in the standard solution of the horseradish peroxidase of variable concentrations, water solublity InP/ZnSQDs probe reacts with horseradish peroxidase, with spectrofluorophotometer under the excitation wavelength of 388nm, detect the fluorescence intensity of above-mentioned standard solution respectively, obtain fluorescence spectrum figure as shown in Figure 1, from fluorescence spectrum figure, read the fluorescence intensity level corresponding to standard solution of the horseradish peroxidase of variable concentrations.
In fluorescence spectrum figure, the fluorescence intensity of the standard solution that horseradish peroxidase concentration is zero is vertical coordinate with the difference of the fluorescence intensity of the horseradish peroxidase standard solution of variable concentrations, in standard solution, the concentration of horseradish peroxidase is abscissa, draw standard curve as shown in Figure 2, thus the linear relationship drawn between difference and the horseradish peroxidase concentration of the fluorescence intensity of standard solution that horseradish peroxidase concentration is zero and the fluorescence intensity of variable concentrations horseradish peroxidase standard solution, linear equation Y=0.01+8.13X, R2=0.999, the detection limit of horseradish peroxidase can be reached 2 × 10-10Mol/L, detects the concentration of horseradish peroxidase in sample solution with this linear relationship.
Two, the mensuration of horseradish peroxidase concentration in sample solution
Separately take the original solution of the horseradish peroxidase of certain volume, and it is added thereto to the water solublity InP/ZnSQDs of certain volume, then it is diluted to 3mL by phosphate buffered solution, obtains sample solution, wherein, in sample solution, the concentration of water solublity InP/ZnSQDs is 23.04nM.With spectrofluorophotometer under the excitation wavelength of 388nm, the fluorescence intensity of detection sample solution, the fluorescence intensity of standard solution that horseradish peroxidase concentration is zero is brought in linear equation with the difference of the fluorescence intensity of sample solution, calculates and obtain the concentration of horseradish peroxidase in sample solution.
Embodiment 2
A kind of method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, including:
Step one, preparation variable concentrations the standard solution of horseradish peroxidase of containing water-soluble InP/ZnSQDs, detect the fluorescence intensity of described standard solution, obtain the fluorescence spectrum figure of described standard solution, set up the linear relationship between the difference of the fluorescence intensity of the standard solution that horseradish peroxidase concentration is zero and the fluorescence intensity of all standard solution and horseradish peroxidase concentration, as shown in Figure 2；
Step 2, preparation containing water-soluble InP/ZnSQDs the sample solution of horseradish peroxidase, detect the fluorescence intensity of described sample solution, determine the concentration of horseradish peroxidase in described sample solution by described linear relationship.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the method preparing described standard solution in described step one is:
The original solution of the horseradish peroxidase of preparation variable concentrations, take the original solution of different volumes, and the buffer solution that adds the water solublity InP/ZnSQDs of same volume and different volumes wherein respectively is equal to the volume of mixed liquor, obtain the standard solution of equal-volume variable concentrations.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the method preparing described sample solution in described step 2 is:
A certain amount of water solublity InP/ZnSQDs is added in unknown horseradish peroxidase solution, make identical with the volume of water solublity InP/ZnSQDs in described standard solution, the buffer solution added and prepare described standard solution identical type, to equal with the volume of described standard solution, obtains described sample solution.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described step 2 being determined, in described sample solution, the method for the concentration of horseradish peroxidase is:
The fluorescence intensity of standard solution that horseradish peroxidase concentration is zero is brought in described linear relationship with the difference of the fluorescence intensity of described sample solution, obtains the concentration of horseradish peroxidase in described sample solution.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described buffer solution is pH value is the phosphate buffered solution of 7.5.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the excitation wavelength detecting described standard solution and described sample solution fluorescence intensity is 388nm.After in the standard solution of the horseradish peroxidase of variable concentrations, water solublity InP/ZnSQDs probe reacts with horseradish peroxidase, the fluorescence spectrum figure obtained when excitation wavelength is 388nm, as shown in Figure 1.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, the volume of described standard solution and described sample solution is 3mL.
In the described method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, described standard solution and in described sample solution the concentration of water solublity InP/ZnSQDs be 23.04nM.
Although embodiment of the present invention are disclosed as above, but listed utilization that it is not restricted in description and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, it is easily achieved other amendment, therefore, under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to specific details and shown here as the embodiment with description.
Claims (8)
1. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration, it is characterised in that including:
Step one, preparation variable concentrations the standard solution of horseradish peroxidase of containing water-soluble InP/ZnSQDs, detect the fluorescence intensity of described standard solution, obtain the fluorescence spectrum figure of described standard solution, set up the linear relationship between the difference of the fluorescence intensity of the standard solution that horseradish peroxidase concentration is zero and the fluorescence intensity of all standard solution and horseradish peroxidase concentration；
Step 2, preparation containing water-soluble InP/ZnSQDs the sample solution of horseradish peroxidase, detect the fluorescence intensity of described sample solution, determine the concentration of horseradish peroxidase in described sample solution by described linear relationship.
2. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 1, it is characterised in that the method preparing described standard solution in described step one is:
The original solution of the horseradish peroxidase of preparation variable concentrations, take the original solution of different volumes, and the buffer solution that adds the water solublity InP/ZnSQDs of same volume and different volumes wherein respectively is equal to the volume of mixed liquor, obtain the standard solution of equal-volume variable concentrations.
3. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 2, it is characterised in that the method preparing described sample solution in described step 2 is:
A certain amount of water solublity InP/ZnSQDs is added in unknown horseradish peroxidase solution, make identical with the concentration of water solublity InP/ZnSQDs in described standard solution, the buffer solution added and prepare described standard solution identical type, to equal with the volume of described standard solution, obtains described sample solution.
4. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 3, it is characterised in that determine in described step 2 that in described sample solution, the method for the concentration of horseradish peroxidase is:
The fluorescence intensity of standard solution that horseradish peroxidase concentration is zero is brought in described linear relationship with the difference of the fluorescence intensity of described sample solution, obtains the concentration of horseradish peroxidase in described sample solution.
5. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 4, it is characterised in that described buffer solution is pH value is the phosphate buffered solution of 7.5.
6. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 5, it is characterised in that the excitation wavelength detecting described standard solution and described sample solution fluorescence intensity is 388nm.
7. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 6, it is characterised in that the volume of described standard solution and described sample solution is 3mL.
8. the method utilizing water solublity InP/ZnSQDs probe in detecting horseradish peroxidase concentration as claimed in claim 7, it is characterised in that described standard solution and in described sample solution the concentration of water solublity InP/ZnSQDs be 23.04nM.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106556584A (en) * | 2016-11-15 | 2017-04-05 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting thimet concentration |
| CN106596478A (en) * | 2016-11-15 | 2017-04-26 | 广西师范学院 | Method of utilizing InP/ZnS QDs probe to detect dimethoate concentration |
| CN106770081A (en) * | 2016-11-15 | 2017-05-31 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting parathion-methyl concentration |
| CN106770082A (en) * | 2016-11-15 | 2017-05-31 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting parathion concentration |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106556584A (en) * | 2016-11-15 | 2017-04-05 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting thimet concentration |
| CN106596478A (en) * | 2016-11-15 | 2017-04-26 | 广西师范学院 | Method of utilizing InP/ZnS QDs probe to detect dimethoate concentration |
| CN106770081A (en) * | 2016-11-15 | 2017-05-31 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting parathion-methyl concentration |
| CN106770082A (en) * | 2016-11-15 | 2017-05-31 | 广西师范学院 | Using the method for InP/ZnS QDs probe in detecting parathion concentration |
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