CN102879454A - Method for detecting enzymatic activity by means of florescence and capillary electrophoresis on basis of quantum dot-polypeptide compound - Google Patents
Method for detecting enzymatic activity by means of florescence and capillary electrophoresis on basis of quantum dot-polypeptide compound Download PDFInfo
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- CN102879454A CN102879454A CN2012103670701A CN201210367070A CN102879454A CN 102879454 A CN102879454 A CN 102879454A CN 2012103670701 A CN2012103670701 A CN 2012103670701A CN 201210367070 A CN201210367070 A CN 201210367070A CN 102879454 A CN102879454 A CN 102879454A
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Abstract
The invention relates to a method for detecting enzymatic activity by means of florescence and capillary electrophoresis on the basis of a quantum dot-polypeptide compound, and belongs to the technical field of bioanalysis and nanotechnology. The method includes steps of firstly, preparing the quantum dot-polypeptide compound and labeling the polypeptide by fluorescence; and secondly, mixing enzyme with the quantum dot-polypeptide compound and then detecting enzymatic activity according to FRET(fluorescence resonance energy transfer). According to experimental results, the method is easy to operate, high in detection flexibility and short in required time. The method is a novel high-flexibility enzymatic activity detection technology improved from the conventional enzymatic activity detection technology and combined with advantages such as high flexibility of multi-wavelength fluorescence detection.
Description
Technical field
The present invention relates to bioanalysis and field of nanometer technology, particularly a kind of method that detects enzymatic activity based on the fluorescent capillary electrophoresis tube of quantum dot-polypeptide complex.
Background technology
Proteolytic enzyme (protease, proteinase) is the general designation of the enzyme of catalytic polypeptide or proteolysis, is called for short proteinase.Extensively branch is in the middle of animal, plant and bacterium, and is of a great variety, and the lysosome intensive amount of various cells is particularly abundant in the alimentary canal of animal and body.Proteinase plays an important role to metabolism and the biological regulation and control of body.
In recent years, the method that enzyme is directly or indirectly detected in the laboratory has larger development, particularly the direct-detection enzymatic activity study hotspot of experimental medicine especially.The detection of blood plasma enzyme concentration is to the diagnosis of clinical disease, and the monitoring of disease, prognosis and curative effect and assessment etc. all have more important meaning.
The direct detecting method of enzyme mainly contains chromophoric substrate method and fluorescence aptamers method etc.But chromophoric substrate method cost is high, fluorescence aptamers method substrate facile hydrolysis, complicated operation, so restricted application.Recently, Medintz has proposed a kind of fluorescent spectrometry based on quantum dot, can realize the detection of thrombin activity, for the detection of enzymatic activity provides a kind of new thinking (Medintz I.L., Clapp A.R., Brunel F.M., et al.
Nat. Mater.2006,5,581 – 588).
Capillary Electrophoresis (CE) has efficiently, at a high speed, trace, low consumption, operator scheme be many, the advantages such as the separation method exploitation is easy, owing to possess so many advantage and the macromolecular ability of separating bio, CE becomes the most rapidly one of method for separating and analyzing of development in recent years.Especially fluoroscopic examination is combined with Capillary Electrophoresis, greatly improved detectability, expanded the application of Capillary Electrophoresis.The enzyme activity assay detection method that this test is set up has overcome the defective that existing method exists, and can satisfy the mass detection needs, and the scope of application is wider, and higher accuracy and stable is preferably arranged.
Summary of the invention
The technical problem to be solved in the present invention is: in order to overcome prior art to the deficiency that enzymatic activity detects, the invention provides a kind of method that detects enzymatic activity based on the fluorescent capillary electrophoresis tube of quantum dot-polypeptide complex.
The technical solution adopted for the present invention to solve the technical problems is: a kind of method that detects enzymatic activity based on the fluorescent capillary electrophoresis tube of quantum dot-polypeptide complex, at first prepare a kind of quantum dot-polypeptide complex, the polypeptide fluorescence labeling can produce FRET (fluorescence resonance energy transfer) (FRET) between quantum dot and the fluorescence molecule.Quantum dot with after polypeptide mixes, is added respectively the enzyme of variable concentrations; Then carrying out Capillary Electrophoresis detects, detect simultaneously the fluorescence intensity of the polypeptide passage of quantum dot and fluorescence molecule mark, the variation of the polypeptide fluorescence intensity of quantum dot and fluorescence molecule mark is converted into the peptide concentration of enzyme per minute cutting as Y-axis, take enzyme concentration as X-axis, curve obtains the parameter that enzymatic activity detects; Wherein polypeptide is the polypeptide that contains enzyme recognition site to be measured, and wherein the C end is histidine-tagged sequence or halfcystine, and the N end is electronegative amino acid and uses the dye molecule mark.
During Capillary Electrophoresis described in the inventive method detected, electrophoretic buffer was preferably 25 mM borate buffers, pH9.3.Described borate buffer is through 0.22 μ m membrane filtration.
Dye molecule described in the present invention is quantum dot, TAMRA, rhodamine, Texas Red, Cy3 or Cy5, and satisfies between the dye molecule of mark biomolecule and the quantum dot FRET (fluorescence resonance energy transfer) can occur.
The inventive method can be used for detecting various hydrolytic enzyme activities, and according to different enzymes, polypeptide is the polypeptide that contains corresponding enzyme recognition site.When for example detecting fibrin ferment, described polypeptide is to contain the polypeptide that fibrin ferment is cut sequence LVPRGS.
Enzyme with after quantum dot-polypeptide complex mixes, can be detected enzymatic activity according to the variation of FRET signal.Experiment showed, that the method is simple to operate, detection sensitivity is high, and required time is short.The method is that traditional enzymatic activity detection technique is improved, in conjunction with advantages such as multi-wavelength fluoroscopic examination sensitivity height, and a kind of new highly sensitive enzymatic activity detection technique of foundation.
The invention has the beneficial effects as follows, the invention provides the method that a kind of fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex detects enzymatic activity, processing ease, detection sensitivity is high, can realize that enzymatic activity detects; Fluorescence spectrophotometer can detect a plurality of wavelength of fluorescence simultaneously, thereby has reduced error, has improved the accuracy that detects.
Description of drawings
The present invention is further described below in conjunction with drawings and Examples.
Fig. 1 is the used quantum dot of the present invention-polypeptide complex generation FRET (fluorescence resonance energy transfer) synoptic diagram.
Fig. 2 is the Capillary Electrophoresis figure that detects the fibrin ferment enzymatic activity with quantum dot-polypeptide complex QD H6-Cy5.The fibrin ferment of variable concentrations (a, 0 μ M; B, 0.034 μ M; C, 0.068 μ M; D, 0.135 μ M; E, 0.27 μ M; F, 0.54 μ M; G, 1.35 μ M) with QD H6-Cy5 37
oThe C mixing was used the Capillary Electrophoresis fluoroscopic examination after 10 minutes, detected wavelength and was respectively 612 nm(detection quantum dot) and 661 nm(detection Cy5).H6-Cy5:QD=32,?[QD]=0.5?μM.
Fig. 3 is the kinetic curve that the fibrin ferment enzymatic activity detects.
Embodiment
In conjunction with the accompanying drawings, the present invention is further detailed explanation.
The operating process of method that a kind of fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex of the present invention detects enzymatic activity is as follows:
1. at first prepare a kind of quantum dot-polypeptide complex, the polypeptide fluorescence labeling can produce FRET between quantum dot and the fluorescence molecule.Quantum dot with after polypeptide mixes, is added respectively the enzyme of variable concentrations.
2 electrophoretic buffers: used electrophoretic buffer is 25mM borate buffer (pH9.3) in this method.
3 kapillaries: used kapillary is coatings capillary pipe (internal diameter 75 μ m, length 60 cm, effective length 35 cm) not in this method.
4 electrophoresis detection: sample introduction voltage 12 KV, then sample introduction 10 s stop pressurization.Separation voltage 18 KV separate 10 min, detect simultaneously the fluorescence intensity of donor (quantum dot) and acceptor (polypeptide of fluorescence molecule mark) passage, and collection, analysis obtain corresponding testing result.
5 variations with donor and acceptor fluorescence intensity are converted into the peptide concentration of enzyme per minute cutting as Y-axis, and take enzyme concentration as X-axis, curve obtains the parameter that enzymatic activity detects.
Embodiment 1 thrombin activity detects
Instrument and the operating conditions used in the example are as follows:
Used Capillary Electrophoresis is for certainly taking capillary electrophoresis system based on fluorescent microscope, and high-voltage power supply is that Shanghai nuclear physics research institute produces; Fluorescence spectrophotometer is marine optics QE65000; The self-control microscope interfaces, with the fluorescence of microscope standard mouth by optical fiber lead-in light spectrometer.Adopt the voltage sample introduction, sample introduction voltage 12 KV, sample injection time are 10 s, and electrophoretic voltage is 18 KV, electrophoresis 10 min.
Fig. 1 is for detecting in the thrombin activity process quantum dot-polypeptide complex generation FRET (fluorescence resonance energy transfer) synoptic diagram.
Fig. 2 is for detecting the Capillary Electrophoresis figure of fibrin ferment enzymatic activity with quantum dot-polypeptide complex QD H6-Cy5.The detection wavelength that spectrometer is set is respectively 612 nm(and detects QDs) and 661nm(detection Cy5).
Experimental result shows, the concentration of fibrin ferment increases, and the FRET signal weakens gradually, so the method can be used for detecting enzymatic activity.And can change according to the fluorescence intensity of different concentration of thrombin carrying out curve fitting, calculate the parameter (Fig. 3) of enzymatic activity.
Design parameter is as shown in Table 1:
The parameter that table one distinct methods detects enzymatic activity compares
| Method | K M (μM) | V max(μM? min -1) | k cat(min -1) | k cat/K M (M -1min -1) |
| Capillary electrophoresis method | 0.21 ± 0.05 | 1.3 ± 0.1 | 0.11 | 5.4 × 10 5 |
| Fluorescent spectrometry | 0.17 ± 0.05 | 1.5 ± 0.2 | 0.13 | 7.6 × 10 5 |
And with capillary electrophoresis method and the comparison of the fluorescent spectrometry in the document used in this patent, the kinetic parameter result who obtains is close, has confirmed the accuracy of the method.
Embodiment 2 thrombin activities detect
Experiment condition is with embodiment 1, and difference is Cy5-DDDLVPRGSGP at peptide molecule
9G
2C.
Embodiment 3 proteolytic enzyme Caspase-3 are active to be detected
Experiment condition is with embodiment 1, and difference is TAMRA-DEDEVDGP at peptide molecule
9G
2H
6, DEVD is the restriction enzyme site of proteolytic enzyme Caspase-3.
Embodiment 4 proteolytic enzyme Caspase-2 are active to be detected
Experiment condition is with embodiment 1, and difference is Cy3-EEEVDVADGP at peptide molecule
9G
2H
6, VDVAD is the restriction enzyme site of proteolytic enzyme Caspase-2.
Embodiment 5 proteolytic enzyme Collagenase are active to be detected
Experiment condition is with embodiment 1, and difference is Cy3-EEERGDC at peptide molecule, and RGDC is the restriction enzyme site of proteolytic enzyme Collagenase.
Can find out that by above-mentioned five embodiment the method that a kind of fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex of the present invention detects enzymatic activity can realize that enzymatic activity detects, and highly sensitive, easy to operate, the result is accurate.
Take above-mentioned foundation desirable embodiment of the present invention as enlightenment, by above-mentioned description, the relevant staff can in the scope that does not depart from this invention technological thought, carry out various change and modification fully.The technical scope of this invention is not limited to the content on the instructions, must determine its technical scope according to the claim scope.
Claims (5)
1. method that detects enzymatic activity based on the fluorescent capillary electrophoresis tube of quantum dot-polypeptide complex, it is characterized in that: at first prepare quantum dot-polypeptide complex, the polypeptide fluorescence labeling can produce FRET (fluorescence resonance energy transfer) between quantum dot and the fluorescence molecule; Quantum dot with after polypeptide mixes, is added respectively the enzyme of variable concentrations; Then carrying out Capillary Electrophoresis detects, detect simultaneously the fluorescence intensity of the polypeptide passage of quantum dot and fluorescence molecule mark, the variation of the polypeptide fluorescence intensity of quantum dot and fluorescence molecule mark is converted into the peptide concentration of enzyme per minute cutting as Y-axis, take enzyme concentration as X-axis, curve obtains the parameter that enzymatic activity detects; Wherein polypeptide is the polypeptide that contains enzyme recognition site to be measured, and wherein the C end is histidine-tagged sequence or halfcystine, and the N end is electronegative amino acid and uses the dye molecule mark.
2. the fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex as claimed in claim 1 detects the method for enzymatic activity, it is characterized in that described Capillary Electrophoresis detects, and electrophoretic buffer is the 25mM borate buffer, pH9.3.
3. the fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex as claimed in claim 2 detects the method for enzymatic activity, and it is characterized in that: described borate buffer is through 0.22 μ m membrane filtration.
4. the fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex as claimed in claim 1 detects the method for enzymatic activity, and it is characterized in that: described enzyme is fibrin ferment; Described polypeptide is to contain the polypeptide that fibrin ferment is cut sequence LVPRGS, and wherein the C end is histidine-tagged sequence or halfcystine, and the N end is electronegative amino acid and uses the dye molecule mark.
5. the fluorescent capillary electrophoresis tube based on quantum dot-polypeptide complex as claimed in claim 1 detects the method for enzymatic activity, it is characterized in that described dye molecule is quantum dot, TAMRA, rhodamine, Texas Red, Cy3 or Cy5, and satisfy between the dye molecule of mark biomolecule and the quantum dot FRET (fluorescence resonance energy transfer) can occur.
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Cited By (9)
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| CN103149185A (en) * | 2013-02-05 | 2013-06-12 | 苏州大学 | Novel high-efficiency protease activity detecting method |
| CN103497231A (en) * | 2013-09-18 | 2014-01-08 | 常州大学 | Method for marking protein polymer by using quantum dots |
| CN105136762A (en) * | 2015-09-06 | 2015-12-09 | 常州大学 | Method for detecting enzyme kinetics in capillary |
| CN105136760A (en) * | 2015-09-06 | 2015-12-09 | 常州大学 | Rapid detection method for interaction of quantum dots and HAT labels |
| CN105241941A (en) * | 2015-09-06 | 2016-01-13 | 常州大学 | Method of quickly detecting enzyme concentration in capillary |
| CN105842210A (en) * | 2016-03-23 | 2016-08-10 | 南昌大学 | Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer |
| CN108169305A (en) * | 2017-12-25 | 2018-06-15 | 安阳师范学院 | Using hydrone as the electric signal marker and method for sensing at catalysis reactive group bottom |
| CN110044988A (en) * | 2019-04-26 | 2019-07-23 | 常州大学 | A kind of method of tortuous capillary electrophoresis detection multienzyme |
| CN111196843A (en) * | 2020-01-17 | 2020-05-26 | 常州大学 | Polypeptide ligand Cy5-H8 for modifying quantum dots |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149185A (en) * | 2013-02-05 | 2013-06-12 | 苏州大学 | Novel high-efficiency protease activity detecting method |
| CN103497231A (en) * | 2013-09-18 | 2014-01-08 | 常州大学 | Method for marking protein polymer by using quantum dots |
| CN103497231B (en) * | 2013-09-18 | 2015-04-22 | 常州大学 | Method for marking protein polymer by using quantum dots |
| CN105136760B (en) * | 2015-09-06 | 2018-08-14 | 常州大学 | A kind of method of quick detection quantum dot and the interaction of HAT labels |
| CN105136762A (en) * | 2015-09-06 | 2015-12-09 | 常州大学 | Method for detecting enzyme kinetics in capillary |
| CN105136760A (en) * | 2015-09-06 | 2015-12-09 | 常州大学 | Rapid detection method for interaction of quantum dots and HAT labels |
| CN105241941A (en) * | 2015-09-06 | 2016-01-13 | 常州大学 | Method of quickly detecting enzyme concentration in capillary |
| CN105842210A (en) * | 2016-03-23 | 2016-08-10 | 南昌大学 | Thrombin detection method based on bio-dots and Au NPs fluorescence resonance energy transfer |
| CN105842210B (en) * | 2016-03-23 | 2018-08-24 | 南昌大学 | Blood coagulation enzyme assay method based on biological quantum dot and Au NPs fluorescence resonance energy transfer |
| CN108169305A (en) * | 2017-12-25 | 2018-06-15 | 安阳师范学院 | Using hydrone as the electric signal marker and method for sensing at catalysis reactive group bottom |
| CN108169305B (en) * | 2017-12-25 | 2019-12-31 | 安阳师范学院 | Electric signal marker using water molecules as catalytic reaction substrate and sensing method |
| CN110044988A (en) * | 2019-04-26 | 2019-07-23 | 常州大学 | A kind of method of tortuous capillary electrophoresis detection multienzyme |
| CN111196843A (en) * | 2020-01-17 | 2020-05-26 | 常州大学 | Polypeptide ligand Cy5-H8 for modifying quantum dots |
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