CN105136760B - A kind of method of quick detection quantum dot and the interaction of HAT labels - Google Patents
A kind of method of quick detection quantum dot and the interaction of HAT labels Download PDFInfo
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- CN105136760B CN105136760B CN201510560728.4A CN201510560728A CN105136760B CN 105136760 B CN105136760 B CN 105136760B CN 201510560728 A CN201510560728 A CN 201510560728A CN 105136760 B CN105136760 B CN 105136760B
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Abstract
The present invention relates to the methods of a kind of quick detection quantum dot of field of nano biotechnology and the interaction of HAT labels, it is characterized in that the labels of fluorescent dye ATTO 590 interact with quantum dot (QDs) in capillary containing HAT tag polypeptides, by the polypeptide of quantum dot and dye marker by different mol ratio example successively injection capillary, measure the ratio between receptor sense channel (625nm) and the peak area of donor sense channel (565nm) and the relationship containing HAT tag polypeptide molar concentrations, draw the matched curve of peak area ratio concentration, pass through interaction of both capillary electrophoresis detections in capillary.The present invention provides a kind of methods that can accurate, quickly, sensitively detect quantum dot and the interaction of HAT labels, and easy to operate, repeatability height has further expanded application of the quantum dot probe in field of bioanalysis.
Description
Technical field
The present invention relates to field of nano biotechnology, and in particular to a kind of quickly detection quantum dot and HAT label phase interactions
Method.
Background technology
Quantum dot (QDs) is used as a kind of novel fluorescent material, has that exciting light spectrum width, emission spectrum be narrow and symmetrical, color
The advantages that adjustable, anti-light bleaching and long fluorescence lifetime, gradually makes it be used widely in bioanalysis detection.
Currently, in biomarker it is most widely used be metal organic solvent method synthesis CdSe/ZnS quantum dots.But
It is that its oil-soluble limits its biologic applications.Therefore, hydrophilic ligand exchange, the encapsulation of amphipathic ligand, silane coating etc. can be passed through
Fat-soluble quantum dot is converted to water-soluble quantum dot by method, to keep its surface-functionalized.Water-soluble quantum dot can with polypeptide,
Albumen and DNA prepare quantum dot fluorescence probe by the method for covalent coupling or Electrostatic Absorption.
Quantum dot is combined with fluorescent polypeptide, when the distance between they are less thanWhen radius, it may occur that non-radiative
Energy transfer, i.e. fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET).To
By fluoroscopic examination, the interaction between can analyzing.
On the other hand, Capillary Electrophoresis as a kind of high-resolution, highly sensitive, high-throughput and low sample consumption differential from
Technology has broad application prospects in field of bioanalysis.Meanwhile by the way that fluoroscopic examination is combined with Capillary Electrophoresis,
Detection limit is substantially increased, the application of capillary has been expanded.And can to detect biomolecule in real time micro for the method in capillary
Variation, it is sensitiveer than the method for extracapillary, convenient, rapid.
HAT labels are natural histidine affinity tag, sequence KDHLIHNVHKEFHAHAHNK, wherein carrying 3 positive electricity
Lotus is easy to happen electrostatic adsorption with the negative electrical charge of quantum dot surface, assembles so as to cause quantum dot, which has limited HAT marks
The application of label.
This method marks the polypeptide containing HAT labels with quantum dot (QDs) in capillary by fluorescent dye (ATTO 590)
Interaction in pipe passes through by the polypeptide of quantum dot and dye marker by different mol ratio example successively micro injection capillary
Fluorescent capillary electrophoresis tube detects the interaction in capillary between the two, FRET occurs, and measure receptor sense channel
The ratio between (625nm) and the peak area of donor sense channel (565nm) and the relationship containing HAT tag polypeptide molar concentrations, are painted
The matched curve of peak area ratio-concentration processed.
Invention content
The technical problem to be solved by the present invention is to there is no the deficiency of good detection HAT stamp methods in the prior art, provide
A kind of method of quick detection quantum dot and the interaction of HAT labels.
In order to solve the above technical problems, the technical solution adopted in the present invention is, it is a kind of quickly to detect quantum dot and HAT marks
The method for signing interaction, which is characterized in that steps are as follows：
(1) mark the polypeptide containing HAT labels with quantum dot (QDs) in capillary by fluorescent dye (ATTO 590)
Interaction passes through fluorescence by the polypeptide of quantum dot and dye marker by different mol ratio example successively micro injection capillary
Interaction of both capillary electrophoresis detections in capillary.
(2) and the ratio between peak area for measuring receptor sense channel (625nm) and donor sense channel (565nm) with containing
The relationship of HAT tag polypeptide molar concentrations draws the matched curve of peak area ratio-concentration.
Further, the polypeptide and Loading sequence of the quanta point biological probe in capillary are advanced electrophoretic velocity
Slow quantum dot, then into the fast fluorescent polypeptide of electrophoretic velocity.
Preferably, the HAT polypeptides of the fluorochrome label, the peptide C end of dye marker has HAT sequence labels can
To be coupled QDs, N-terminal is coupled fluorescent dye ATTO 590, and it is existing that fluorescence resonance energy transfer can be generated between QDs and fluorescent dye
As.
Further, the quantum dot is the quantum dot containing Zn.
Preferably, the quantum dot is CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
Advantageous effect obtained by the present invention is, provided by the invention to can be used for one kind quickly detection quantum dot and HAT marks
The method for signing interaction, easy to operate, repeatability is high, has further expanded quantum dot probe answering in field of bioanalysis
With.
Description of the drawings
Fig. 1：Electrophoretogram (solid line, 565nm, the QDs sense channels of ATTO 590-HAT and the QDs self assembly in capillary；
Dotted line, 590 sense channel of 625nm, ATTO), (a) QDs, (b) 1:1(ATTO 590-HAT:QDs, similarly hereinafter), (c) 2:1, (d)
4:1, (e) 8:1,(f)16:1.
Fig. 2：S625/S565Value and ATTO 590-HAT molar concentrations relationship.
Specific implementation mode
The present invention will be described further with regard to following embodiment, however, it should be noted that these embodiments are only to illustrate
It is used, and is not necessarily to be construed as the limitation that the present invention is implemented.
Embodiment 1
ATTO 590-HAT and interactions of the CdSe/ZnS QDs in capillary
1, fat-soluble QDs is converted to water-soluble QDs through GSH
By 18mg GSH, 5mg KOH, 250 μ L methanol mixings take 40 μ L mixed solutions that the 200 fat-soluble quantum dots of μ L are added
In, vibrate 30min.After oscillation, 200 μ L 1mM NaOH are added, fat-soluble quantum dot is transferred in water phase.In taking-up
Layer quantum dot, is added 1mL methanol and 30 μ L NaCl (30mg/mL) are precipitated, and is dissolved in borate buffer (pH 7.4,10mM).Instead
Multiple precipitation twice, is finally dissolved in 200 μ L boron buffer solutions (pH 7.4,10mM).
2, Peptide systhesis and label
Polypeptide DDSSGGKDHLIHNVHKEFHAHAHNK is synthesized using Fmoc solid-phase synthesis.With HBTU/HOBt (1:1)
Fmoc-protected amino acid is activated, 30min is coupled；It is deprotected with 20% piperidines of basic solvent, exposes amino；Using HBTU and
HOBt activates the carboxyl on next amino acid, with the amino coupled on resin, forms peptide bond；It repeats the above steps, follows repeatedly
Ring adds amino acid, until synthesis is completed.It is deprotected again with 20% piperidines, amino is exposed, with EDC/HOBt (1:1) it activates
The carboxyl of ATTO-590 carries out the label of dyestuff.With cutting reagent (TFA, dithioglycol, water and TIS, 94:2.5:2.5:1,v/
V) polypeptide is cut down from resin, then passes through ice ether and precipitate, precipitation is collected by centrifugation, is isolated and purified by HPLC, it is cold
Freeze the final products ATTO 590-HAT being dried to obtain.
3, the assembling of quanta point biological probe
ATTO 590-HAT and QDs is respectively according to molar ratio 1:1,2:1,4:1,8:1,16:In 1 micro injection capillary,
Sample injection time is respectively 10s, and sampling interval 20s passes through the interaction of both capillary electrophoresis detections.With with ATTO
The increase of 590-HAT and QDs molar ratios, FRET are gradually increased (Fig. 1).
4, curve matching
It is logical by being detected with QDs donors to the ATTO 590-HAT receptor sense channel peak areas under the conditions of different mol ratio
Road peak area ratio (S625/S565), matched curve (Fig. 2) is drawn out, obtains curvilinear equation y=-0.0012x2+0.8888x+
0.0814。
Embodiment 2
Cy5-HAT and interactions of the CdSe/ZnS QDs in capillary
Step 1-3 is the same as embodiment 1.
4, the curve matching of interaction variation
By to the Cy5-HAT receptor sense channel peak areas and QDs donor sense channels peak under the conditions of different mol ratio
Area ratio (S625/S565), matched curve is drawn out, obtains curvilinear equation y=-0.0008x2+0.0.0742x+0.1703。
It is enlightenment with above-mentioned desirable embodiment according to the present invention, through the above description, relevant staff is complete
Various change and modification can be carried out without departing from the scope of the technological thought of the present invention' entirely.This invention it is technical
Range is not limited to the contents of the specification, it is necessary to determine its technical scope according to right.
Claims (3)
1. a kind of method of quick detection quantum dot and the interaction of HAT labels, it is characterised in that：Fluorescent dye ATTO590 marks
Note interacts with quantum dot (QDs) in capillary containing HAT tag polypeptides, and the peptide C end of the dye marker has
HAT sequence labels, N-terminal are coupled fluorescent dye ATTO590, successively by different mol ratio example by the polypeptide of quantum dot and dye marker
In micro injection capillary, by the ratio between peak area for measuring receptor sense channel 625nm and donor sense channel 565nm with
Relationship containing HAT tag polypeptide molar concentrations draws the matched curve of peak area ratio-concentration, passes through fluorescent capillary electrophoresis tube
Detect the interaction in capillary between the two.
2. the method for a kind of quick detection quantum dot according to claim 1 and the interaction of HAT labels, feature exist
In the HAT tag polypeptides and quantum dot of the dye marker are nanoliter level.
3. the method for a kind of quick detection quantum dot according to claim 1 and the interaction of HAT labels, feature exist
In the quantum dot is the quantum dot containing Zn, is CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
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Citations (3)
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|---|---|---|---|---|
| CN102879454A (en) * | 2012-09-27 | 2013-01-16 | 常州大学 | Method for detecting enzymatic activity by means of florescence and capillary electrophoresis on basis of quantum dot-polypeptide compound |
| US8476083B1 (en) * | 2012-05-18 | 2013-07-02 | The United States Of America, As Represented By The Secretary Of The Navy | Spectro-temporal optical encoding of information using a time-gated fluorescence resonance transfer (FRET) |
| CN104237181A (en) * | 2014-09-02 | 2014-12-24 | 常州大学 | Detection method for interaction of proteins and quantum dots in capillary tube |
Family Cites Families (1)
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| WO2012071428A2 (en) * | 2010-11-22 | 2012-05-31 | Solulink, Inc. | Methods and/or use of oligonucleotide conjugates for assays and detections |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8476083B1 (en) * | 2012-05-18 | 2013-07-02 | The United States Of America, As Represented By The Secretary Of The Navy | Spectro-temporal optical encoding of information using a time-gated fluorescence resonance transfer (FRET) |
| CN102879454A (en) * | 2012-09-27 | 2013-01-16 | 常州大学 | Method for detecting enzymatic activity by means of florescence and capillary electrophoresis on basis of quantum dot-polypeptide compound |
| CN104237181A (en) * | 2014-09-02 | 2014-12-24 | 常州大学 | Detection method for interaction of proteins and quantum dots in capillary tube |
Non-Patent Citations (3)
| Title |
|---|
| Preferential Binding of a Novel Polyhistidine Peptide Dendrimer Ligand on Quantum Dots Probed by Capillary Electrophoresis;jianhao Wang等;《analytical chemistry》;20110705;第83卷;摘要，第6324页右栏，第63245页右栏，Scheme 1，图1 * |
| Protein A Detection Based on Quantum Dots-Antibody Bioprobe Using Fluorescence Coupled Capillary Electrophoresis;Lin Qiu等;《Int. J. Mol. Sci》;20140124;第15卷;全文 * |
| 基于靶向多肽的量子点探针用于结肠癌肿瘤组织识别;王建浩等;《科学通报》;20130111;第58卷(第7期);全文 * |
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