CN102565020A - Method for quantitatively detecting protein through quantum dot resonant scattering - Google Patents
Method for quantitatively detecting protein through quantum dot resonant scattering Download PDFInfo
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- CN102565020A CN102565020A CN2012100103117A CN201210010311A CN102565020A CN 102565020 A CN102565020 A CN 102565020A CN 2012100103117 A CN2012100103117 A CN 2012100103117A CN 201210010311 A CN201210010311 A CN 201210010311A CN 102565020 A CN102565020 A CN 102565020A
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Abstract
The invention discloses a method for quantitatively detecting proteins through quantum dot resonant scattering. The method is a supplement to protein detection methods. The proteins are adsorbed by using quantum dots through an electrostatic effect, resonant scattering light can be generated within a visible light wavelength range to detect standard bovine serum albumins (BSA) and gamma-globulins, a standard curve is drawn by taking the concentration (mol/L) of the proteins as a horizontal coordinate and the highest-intensity change of scattering wavelength as a vertical coordinate, and the concentration of the proteins can be calculated through a regression equation through scattering intensity change. Moreover, the quantum dots with different grain sizes are additionally used for detecting the BSA and the gamma-globulins, good correlation between the concentration of the proteins and the scattering intensity is found, the correlation coefficient reaches 0.95 to 0.99, the gradient reaches 107, the sensitivity is higher, the stability is high, the detection time does not cause a great influence on the result and a new practical method is developed for quantitatively detecting the concentration of biological macromolecular proteins.
Description
Technical field
The invention belongs to technical field of protein detection, specifically, relate to the method for the application of quanta point resonance scattering method check and analysis protein content.
Background technology
Protein is a kind of biomacromolecule of being made up of amino acid, is the material base of vital movement, does not have life without protein.The quantitative measurement of protein has great importance for biochemical analysis and clinical medicine.Method of protein measurement commonly used has AAS (Kjeldahl, biuret method and Coomassie brilliant blue method) and fluorophotometric method, but the most sensitivity of these methods is not high, less stable.Along with the development of proteomics, people need the protein of lower biological sample of quantitative test concentration or purification, and the sensitivity of therefore seeking new detection method raising protein determination has great importance.
Light scattering is meant the optical phenomenon of on the direction beyond the incident light, observing when Ray Of Light passes through medium, and it is a kind of form of expression of electromagnetic radiation and matter interaction.Light scattering technique has crucial status in physical chemistry, colloidal chemistry, high polymer chemistry research.Because traditional light scattering technique can not satisfy the requirement of quantitative test aspect accuracy and precision, fails in analytical chemistry, to apply.Pasternack in 1993 etc. with special resonant light scattering technical research the gathering of porphyrins on nucleic acid, thereby first should technology and chemical process connect.Huang Chengzhi etc. the have utilized this technical research scattering spectrum of biomacromolecule system has been set up the new high-sensitivity analysis method of resonant light scattering technology quantitative measurement biomacromolecule.Present this technology is widely used in research and the mensuration of biomacromolecules such as nucleic acid, protein, heparin, polysaccharide.
Quantum dot has unique optical characteristics; The unique character of quantum dot is based on it self quantum effect; When grain size arrives nanoscale; The size confinement will cause size effect, quantum confined effect, macro quanta tunnel effect and surface effect, thereby derive the nanometer system of the low dimension rerum natura different with microsystem with normal sight system, show many physicochemical property that are different from the macroscopic body material.These characteristics have very wide application prospect at aspects such as nonlinear optics, magnetic medium, biology, medicine and functional materials.
In the report to existing document; Though organic dyestuff or the metallic ion indicator Analysis on Resonance and Scattering Light Spectrum to biomacromolecule protein and nucleic acid combination is arranged respectively; And the analysis of quanta point resonance scattering properties, but the bibliographical information that utilizes the quantum dot resonance scattering to come detection by quantitative protein was not arranged as yet so far.
Summary of the invention
The purpose of this invention is to provide a kind of quantum dot detection by quantitative method of protein that utilizes, can detect protein content delicately.
The quantum dot particle has scattering nature and optical characteristics, and the protein through electrostatic attraction and Van der Waals force absorption variable concentrations can produce resonance light scattering, and maximum absorption wavelength is arranged in visible wavelength range.The present invention utilizes above-mentioned characteristic and principle, in visible wavelength range, carries out synchronous fluorescence and detects, and in certain protein concentration scope, there is correlativity in the variation of the scattering strength of maximum absorption wavelength, can detect the very protein of low concentration.
Technical scheme is that a kind of quantum dot detection by quantitative method of protein that utilizes comprises the steps:
The quantum dot suspended dispersed in protein solution, is carried out synchronous fluorescence and detected under 456～466nm, measure scattering strength, and obtain protein concentration with the typical curve contrast; The amount ratio of quantum dot and protein solution is 5～10 * 10
-7Mol/ml.Preferably, the amount ratio of quantum dot and protein solution is 7～9 * 10
-7Mol/ml.The fluoroscopic examination condition is: emission and scattered grating are 5nm, and voltage is 400V.
The protein that is detected is BSA or gamma globulin.
Said quantum-dot structure comprises the SiO of kernel and pan coating
2Shell; The kernel particle diameter is 3～10nm, is ZnS/ZnO quantum dot heterojunction structure.The preparation method of this quantum dot comprises the steps:
(1) with Zn (Ac)
2And CH
3CSNH
2Add 1, in the 4-butylene glycol, ultrasonic dispersion is until forming transparent precursor solution; Zn (Ac)
2With CH
3CSNH
2Mol ratio is 1: 0.25～0.7; Zn (Ac)
2With 1,4-butylene glycol amount ratio is 0.1～0.4mol/L;
(2) the resulting precursor solution of step (1) is transferred in the hydrothermal reaction kettle, and 190～220 ℃ are reacted 16～20h down; After being cooled to room temperature, add the distilled water mixing, centrifuging is got washing of precipitate and is drying to obtain ZnS/ZnO quantum dot heterojunction structure;
(3) ZnS/ZnO quantum dot heterojunction structure is dissolved in the ethanol, adds ammoniacal liquor, stirs down to add teos solution, continues reaction 10～15 hours, gets the washing of precipitate drying; ZnS/ZnO quantum dot heterojunction structure and ethanol, ethyl orthosilicate amount ratio are 1mmol: 300～600ml: 1～2ml.
The construction method of typical curve may further comprise the steps:
Quantum dot added in the solution that protein content is 0～0.4 μ M being uniformly dispersed, detect scattering strength, is horizontal ordinate with the protein concentration, and scattering strength is changed to ordinate drawing standard curve.
The present invention utilizes quantum dot to pass through the electrostatic interaction adsorbed proteins; In visible wavelength range, can produce resonance light scattering and come examination criteria bovine serum albumin (BSA) and gamma globulin; With protein concentration (mol/L) is horizontal ordinate; Be changed to ordinate drawing standard curve with scattering wavelength maximum intensity, change and to go out protein concentration by regression equation calculation through scattering strength.The present invention also utilizes the quantum dot of different-grain diameter size that BSA and gamma globulin etc. is detected, and finds that all there are correlativity preferably in protein concentration and scattering strength, and related coefficient reaches 0.95～0.99, and slope all reaches 10
7, sensitivity is higher, and good stability, and influence not quite the result detection time, for the concentration of detection by quantitative biomacromolecule protein has been started a kind of practical new.
In visible-range, measure the synchronous scattering of different empty quantum dots earlier, prepare the standard protein solution of low concentration again, join in the different quantum dot solutions and be adsorbed on the quantum dot particle surface and measure.With the protein concentration is horizontal ordinate, is ordinate drawing standard curve with the maximum scattering Strength Changes, and then can use the concentration of calculating protein through regression equation, thereby sensitive detection method is provided for protein detection.
The invention has the beneficial effects as follows; Fluorescent characteristic and biology techniques principle in conjunction with quantum dot; Utilize the fluoroscopic examination method of protein, have highly sensitive, good stability, characteristics such as economical and practical, can be widely used in biology protein micro quantitative determination detection range; For the detection of protein content in agriculture quality testing, food nutrition standard detection, the transgenosis detection range provides good method, can obtain the result quickly and accurately.
Description of drawings
Fig. 1 is the resonance scattering figure collection of illustrative plates of No. 1 quantum dot detection BSA
Fig. 2 is the typical curve of No. 1 quantum dot detection BSA
Fig. 3 is the resonance scattering figure collection of illustrative plates of No. 2 quantum dot detection BSA
Fig. 4 is the typical curve of No. 2 quantum dot detection BSA
Fig. 5 is the resonance scattering figure collection of illustrative plates of No. 3 quantum dot detection BSA
Fig. 6 is the typical curve of No. 3 quantum dot detection BSA
Fig. 7 is the resonance scattering figure collection of illustrative plates of No. 4 quantum dot detection BSA
Fig. 8 is the typical curve of No. 4 quantum dot detection BSA
Fig. 9 is the resonance scattering figure collection of illustrative plates of No. 1 quantum dot detection gamma globulin
Figure 10 is the typical curve of No. 1 quantum dot detection gamma globulin
Figure 11 is the resonance scattering figure collection of illustrative plates of No. 2 quantum dot detection gamma globulins
Figure 12 is the typical curve of No. 2 quantum dot detection gamma globulins
Figure 13 is the resonance scattering figure collection of illustrative plates of No. 3 quantum dot detection gamma globulins
Figure 14 is the typical curve of No. 3 quantum dot detection gamma globulins
Figure 15 is the resonance scattering figure collection of illustrative plates of No. 4 quantum dot detection gamma globulins
Figure 16 is the typical curve of No. 4 quantum dot detection gamma globulins
Embodiment
Embodiment 1
1) ZnS/ZnO quantum dot heterojunction structure preparation: at first with 2mmol Zn (Ac)
2And 1.4mmolCH
3CSNH
2Add 20ml 1, in the 4-butylene glycol, ultrasonic dispersion is until forming transparent precursor solution; Then the solution that obtains is transferred in the 30mL hydrothermal reaction kettle (inner liner polytetrafluoroethylene), 200 ℃ are reacted 18h down; After treating that hydrothermal reaction kettle naturally cools to room temperature; In products therefrom, add 10～20ml distilled water mixing, the 8000rpm centrifuging, the gained deposition is cleaned with ethanol and is removed impurity three times; 60 ℃ of dry 5h promptly get ZnS/ZnO quantum dot heterojunction structure, and particle diameter is 3～4nm.
2) bag silicon step: 0.1mmol ZnS/ZnO quantum dot is dissolved in the 40mL ethanol, adds 0.5mL28wt% ammoniacal liquor mixing, continuing slowly to add 0.1mL TEOS under the stirring, continue stirring reaction 12h then.Centrifuging must precipitate, and distilled water cleans and promptly gets SiO twice
2The ZnS/ZnO quantum dot heterojunction structure of parcel, particle diameter is constant basically.
Resulting SiO
2The ZnS/ZnO quantum dot of parcel is represented with No. 1 quantum dot.
The 2mM Zn (Ac) that adds in the step 1)
2Be respectively 4,6,8mmol, all the other conditions are constant, the ZnS/ZnO quantum dot heterojunction structure particle diameter that obtains is respectively 5～6,7～8 and 9～10nm; The method of bag silicon is with step 2), resulting SiO
2The ZnS/ZnO quantum dot of parcel is represented with 2,3 and No. 4 quantum dots respectively.
Embodiment 2 is an example with the detection of bovine serum albumin (BSA)
(1) preparation of quantum dot solution and protein solution
It is 5 * 10 that 1～No. 4 quantum dot original content is
-5Mol/L carries out 60 times of dilutions with distilled water, and that carries out 15min before the use ultrasonicly makes its even suspension.
It is 10 that BSA is made into concentration
-4Mol/L, subsequent use.BSA is available from Beijing Suo Laibao biotechnology company.
(2) detect and the drawing standard curve
After treating that the instrument preheating is steadily, get 3mL wherein a kind of pure quantum dot of particle diameter in cuvette, carry out the fluorescence synchronous detection at 250-750nm place, launching grating and scattered grating all is 5nm, voltage is 400V, scattering strength is F
0
And then to add 10,20,30,40,50,60,70,80,90 and 100 μ L concentration respectively be 10
-4The BSA solution of mol/L; The detection that each step all makes zero, scattering strength is F
nThe maximum scattering wavelength of finding this ZnS and ZnO quantum dot about 461 ± 5nm, is a horizontal ordinate with the protein concentration approximately all, and the scattering strength of getting strong scattering place wavelength changes F
n-F
0Be ordinate drawing standard curve.Quantum dot to different-grain diameter detects comparison successively.
The resonance scattering collection of illustrative plates of 1～No. 4 quantum dot detection BSA and canonical plotting are shown in Fig. 1～8.1～No. 4 quantum dot detects the resonance scattering collection of illustrative plates of BSA like Fig. 1,3,5, shown in 7, and curve is respectively BSA concentration from bottom to top to be 0 and to add the collection of illustrative plates of above-mentioned different amount BSA solution.The canonical plotting of 1～No. 4 quantum dot detection BSA is like Fig. 2,4,6, shown in 8.
Embodiment 3 detects gamma globulin
(1) preparation of quantum dot solution and protein solution
It is 5 * 10 that 1～No. 4 quantum dot original content is
-5Mol/L carries out 60 times of dilutions with distilled water, ultrasonicly makes its even suspension with what before carry out 15min.
It is 10 that gamma globulin is made into concentration
-4Mol/L, subsequent use.Gamma globulin is available from Beijing Suo Laibao biotechnology company.
(2) detect and the drawing standard curve
After treating that the instrument preheating is steadily, get 3mL wherein a kind of pure quantum dot of particle diameter in cuvette, carry out the fluorescence synchronous detection at 250-750nm place, launching grating and scattered grating all is 5nm, voltage is 400V, scattering strength is F
0And then add respectively that 10,20,30,40,50,60,70 and 80 μ L concentration are 10
-4The gamma globulin of mol/L, the detection that each step all makes zero, scattering strength is F
nThe maximum scattering wavelength of finding this ZnS/ZnO quantum dot about 461nm, is a horizontal ordinate with the protein concentration approximately all, and the scattering strength of getting strong scattering place wavelength changes F
n-F
0Be ordinate drawing standard curve.Quantum dot to different-grain diameter detects comparison successively.
The resonance scattering collection of illustrative plates of 1～No. 4 quantum dot detection gamma globulin and canonical plotting are shown in Fig. 9～16.
1～No. 4 quantum dot detects the resonance scattering collection of illustrative plates of gamma globulin like Fig. 9,11, shown in 13 and 15, and curve is respectively gamma globulin from bottom to top to be 0 and to add the collection of illustrative plates of above-mentioned different amount gamma globulin solution.The canonical plotting of 1～No. 4 quantum dot detection gamma globulin is like Figure 10,12, shown in 14 and 16.
Can find out that through detecting with the drawing standard curve in the protein content certain limit, resonance scattering Strength Changes and protein concentration have correlativity preferably, protein concentration is big more, and scattering strength changes just obvious more.The variation of middle scattering strength can be found Protein content on typical curve per sample.
During check and analysis, can detect the very protein of trace, reach 10
-8The order of magnitude of mol/L; The sample lower for some protein contents can detect apace; Come detection by quantitative protein through the quanta point resonance scattering, have highly sensitive, good stability, advantage such as economical and practical provides good method for the detection for protein content in agriculture quality testing, food nutrition standard detection, the transgenosis detection range.
Claims (7)
1. one kind is utilized quantum dot detection by quantitative method of protein, it is characterized in that, comprises the steps:
The quantum dot suspended dispersed in protein solution, is carried out synchronous fluorescence and detected under 456～466nm, measure scattering strength, and obtain protein concentration with the typical curve contrast; The amount ratio of quantum dot and protein solution is 5～10 * 10
-7Mol/ml.
2. the said quantum dot detection by quantitative method of protein that utilizes of claim 1 is characterized in that said quantum-dot structure comprises the SiO of kernel and pan coating
2Shell; The kernel particle diameter is 3～10nm, is ZnS/ZnO quantum dot heterojunction structure.
3. claim 1 or the 2 said quantum dot detection by quantitative method of protein that utilize is characterized in that the preparation method of said quantum dot comprises the steps:
(1) with Zn (Ac)
2And CH
3CSNH
2Add 1, in the 4-butylene glycol, ultrasonic dispersion is until forming transparent precursor solution; Zn (Ac)
2With CH
3CSNH
2Mol ratio is 1: 0.25～0.7;
(2) the resulting precursor solution of step (1) is transferred in the hydrothermal reaction kettle, and 190～220 ℃ are reacted 16～20h down; After being cooled to room temperature, add the distilled water mixing, centrifuging is got washing of precipitate and is drying to obtain ZnS/ZnO quantum dot heterojunction structure;
(3) ZnS/ZnO quantum dot heterojunction structure is dissolved in the ethanol, adds ammoniacal liquor, stirs down to add ethyl orthosilicate, continues stirring reaction 10～15 hours, gets the washing of precipitate drying; ZnS/ZnO quantum dot heterojunction structure and ethanol, ethyl orthosilicate amount ratio are 1mol: 300～600ml: 1～2ml.
4. the said quantum dot detection by quantitative method of protein that utilizes of claim 3 is characterized in that said Zn (Ac)
2With 1,4-butylene glycol amount ratio is 0.1～0.4mol/L.
5. the said quantum dot detection by quantitative method of protein that utilizes of claim 1 is characterized in that said protein is BSA or gamma globulin.
6. the said quantum dot detection by quantitative method of protein that utilizes of claim 1 is characterized in that in the said step (1), the fluoroscopic examination condition is: emission and scattered grating are 5nm, and voltage is 400V.
7. the said quantum dot detection by quantitative method of protein that utilizes of claim 1 is characterized in that the construction method of said typical curve may further comprise the steps:
Quantum dot added in the solution that protein content is 0～0.4 μ M being uniformly dispersed, detect scattering strength, is horizontal ordinate with the protein concentration, and scattering strength is changed to ordinate drawing standard curve.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439307A (en) * | 2013-09-30 | 2013-12-11 | 桂林理工大学 | Method for rapidly detecting protein in food by using indigo dye |
| CN103604790A (en) * | 2013-11-28 | 2014-02-26 | 中南林业科技大学 | Method for quickly measuring glutelin content of rice based on quantum dot |
| CN105403539A (en) * | 2015-10-29 | 2016-03-16 | 广西师范学院 | Method for using frequency doubling scattering process for detecting hemoglobin by taking fluorescent carton spot as probe |
| CN105424658A (en) * | 2015-10-29 | 2016-03-23 | 广西师范学院 | Method for detecting trypsin with fluorescence carbon dot as probe based on frequency-doubling scattering method |
| CN105651752A (en) * | 2016-02-26 | 2016-06-08 | 国家纳米科学中心 | Detection method of amyloid protein |
| CN108754528A (en) * | 2018-06-08 | 2018-11-06 | 西北大学 | A kind of high-performance photocatalysis nano material |
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| CN1743412A (en) * | 2005-09-23 | 2006-03-08 | 上海大学 | Method for preparing II-VI family fluorescent mark semiconductor quantum point MX |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103439307A (en) * | 2013-09-30 | 2013-12-11 | 桂林理工大学 | Method for rapidly detecting protein in food by using indigo dye |
| CN103604790A (en) * | 2013-11-28 | 2014-02-26 | 中南林业科技大学 | Method for quickly measuring glutelin content of rice based on quantum dot |
| CN103604790B (en) * | 2013-11-28 | 2016-07-06 | 中南林业科技大学 | A kind of method quickly measuring rice glutelin content based on quantum dot |
| CN105403539A (en) * | 2015-10-29 | 2016-03-16 | 广西师范学院 | Method for using frequency doubling scattering process for detecting hemoglobin by taking fluorescent carton spot as probe |
| CN105424658A (en) * | 2015-10-29 | 2016-03-23 | 广西师范学院 | Method for detecting trypsin with fluorescence carbon dot as probe based on frequency-doubling scattering method |
| CN105651752A (en) * | 2016-02-26 | 2016-06-08 | 国家纳米科学中心 | Detection method of amyloid protein |
| CN108754528A (en) * | 2018-06-08 | 2018-11-06 | 西北大学 | A kind of high-performance photocatalysis nano material |
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Application publication date: 20120711 |