CN102961761A - Quantum dot targeting probe for colorectal cancer tumor tissue identification and preparation method thereof - Google Patents
Quantum dot targeting probe for colorectal cancer tumor tissue identification and preparation method thereof Download PDFInfo
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- CN102961761A CN102961761A CN2012104333419A CN201210433341A CN102961761A CN 102961761 A CN102961761 A CN 102961761A CN 2012104333419 A CN2012104333419 A CN 2012104333419A CN 201210433341 A CN201210433341 A CN 201210433341A CN 102961761 A CN102961761 A CN 102961761A
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Abstract
The invention discloses a method for preparing a quantum dot targeting probe (TCP-1-H6) for colorectal cancer tumor tissue identification and belongs to the field of nano biotechnology. A novel polypeptide ligand comprises histidine sequences (1) for combining with quantum dots, a sequence (2) for providing a polypeptide rigidity structure and a targeting sequence (3), wherein various sequences are combined through peptide bonds. The bifunctional ligand is simple in synthesis method, the N end is combined with the quantum dots through six histidine sequences, the stability of the ligand and the quantum dots is greatly improved, and the polypeptide ligand can serve as a nano fluorescence probe to be applied to colorectal cancer tumor marking.
Description
Technical field
The present invention relates to the nanometer biotechnology field, be specifically related to a kind of quantum dot targeted probes for the identification of colon cancer tumor tissues and preparation method thereof.
Background technology
Quantum dot (QDs) is a kind of zero dimension semiconductor nanocrystal, almost spherical, diameter 1 ~ 12nm, can be scattered in water or organic solvent and form colloid, due to approaching exciton (electron-hole pair) the Bohr radius that even is less than the corresponding semiconductor phase material of the size of QDs, the electronics and the hole that while being stimulated, produce are limited in narrow and small three dimensions, thereby show quantum effect, with traditional organic dyestuff, compare, QDs has more excellent spectral quality, as exciting light spectrum width and continuous distribution, and emission spectra is symmetric and width is narrow, color is adjustable, and photochemical stability is high, be difficult for photodissociation (Marcel BJ, Mario M, Peter G, et al.
science1998,281,2013-2016).These superior performances all are widely applied the aspect such as cell imaging and biological specific markers QDs in vivo, and it also will become very important a kind of probe tool in the molecular cell imaging research day by day.What therefore, have a high performance water-soluble quantum dot syntheticly has been subject to close attention.
At present, most widely used in biomarker is the synthetic fat-soluble CdSe/ZnS quantum dot of metal organic solvent method.Therefore, fat-soluble QDs must be changed into to water miscible QDs and just can carry out biomarker.And fat-soluble QDs is changed into to the most frequently used method of water miscible QDs, be first to use sulfydryl micromolecule (as TGA, mercaptopropionic acid, glutathion, dimercaptosuccinic acid etc.) to replace the part on fat-soluble QDs surface, then by coupling agent and biomolecule coupling; The method of the QDs labelling biomolecule that another kind is commonly used is and contains histidine-tagged polypeptide or protein binding.The affine coordinative role of metal of the Zn atom on QDs surface and polyhistidine residue, the proteins and peptides that contains histidine residues can directly be received the Zn atom on QDs surface, this method has good stability, becomes gradually a kind of method commonly used in biomarker.(Sapsford K. E., Pons T., Medintz I. L., the et al. such as Sapsford
j. Phys.Chem. C 2007, 111,11528-11538) systematically to have studied containing the interaction between histidine polypeptide and QDs and binding constant etc., the association rate of 6 histidine sequences and QDs is approximately 100 seconds, K
d -1be about 1 nM.Therefore, between quantum dot and the polypeptide that contains His-tag in conjunction with being to stablize very much and rapidly.
TCP-1 (ring type polypeptide cyclo (1,9)-CTPSPFSHC) combines and filters out a kind of novel cancer target polypeptide by display technique of bacteriophage in body and in-situ junction intestinal cancer animal model.This polypeptide can not be attached to normal structure (Li Z J, Wu W K K, Ng S S M, et al. in conjunction with colon tumor new life's vascular cell (CD31+ positive cell) in vivo specifically
j Control Release, 2010,148:292 – 302).
Therefore, we have prepared a kind of bifunctional TCP-1-H6 polypeptide, the targeting of the existing TCP-1 of this peptide species contain again one histidine-tagged, avoid quantum dot and polypeptide in conjunction with being subject to the impact of coupling agent, thereby but obtain a kind of targeting quantum dot probe of specific recognition colon cancer tumor.
Summary of the invention
The technical problem to be solved in the present invention is: poor and be subject to the problem such as coupling agent impact in order to overcome existing polypeptide ligand and QDs combination stability, limit its universal use in biological field.For solving the problems of the technologies described above, the invention provides a kind of bifunctional peptide molecule, the speed of being combined with QDs is fast, and good stability, can well realize the targeting labelling of colon cancer tumor.
The technical solution adopted for the present invention to solve the technical problems is: a kind of quantum dot targeted probes for the identification of colon cancer tumor tissues, comprise for the histidine sequence (1) in conjunction with quantum dot, the sequence (2) that the polypeptide rigid structure is provided and targeting sequence (3), combine with peptide bond between each sequence.Described histidine sequence (1) forms (H6) by 6-8 histidine; The sequence of described rigid structure is 9-11 proline sequence; Described targeting sequence (3) is TCP-1, after making quantum dot and polypeptide being connected, has the colon cancer tumor-targeting function.
Quantum dot of the present invention is the quantum dot that contains Zn, as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
The invention also discloses the preparation method of the above-mentioned quantum dot targeted probes for the identification of colon cancer tumor tissues, comprise the following steps: at first polypeptide is mixed according to the ratio of 18-36:1 with quantum dot, shake after 10 minutes, the histidine sequence of polypeptide is combined with the Zn on quantum dot surface, remove unnecessary peptide molecule by ultrafiltration again, obtain quantum dot-polypeptide fluorescent probe.
After adopting above-mentioned technical scheme, the beneficial effect that the present invention obtains is, the targeting bifunctional polypeptides part that can be used for the identification of colon cancer tumor provided by the invention, synthetic method is simple, repeatable high, the speed of being combined with quantum dot is fast, and stability is high, solve existing quantum dot polypeptide ligand stability deficiency, further expanded the application in tumor imaging as namo fluorescence probe of quantum dot-polypeptide complex.
The accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is the structural representation of modifying the New-type bifunctional polypeptide ligand (TCP-1-H6) of QDs; In figure, 1 is to be to provide the sequence, the 3rd of polypeptide rigid structure, targeting sequence in conjunction with the histidine sequence, 2 of QDs.
Fig. 2 is the electrophoresis pattern of different proportion TCP-1-H6:QD: a, QDs; B, 9:1; C, 18:1; D, the 36:1 electrophoresis pattern.
Fig. 3 be Colon 26 cells respectively with TCP-1-H6 QD (A), QDs (B) and TP-1-H6 QD (C) mix the co-focusing imaging figure of hatching after 4 h, scale 20 μ m.
Fig. 4 is TCP-1-H6 QD identification colon cancer tumor tissues.A, the imaging of TCP-1-H6 QD colon tumor tissue; B, the imaging of TCP-1-H6 QD tumor tissues adds 150 μ M TCP-1-H6 simultaneously; C, the imaging of TCP-1-H6 QD normal colonic tissue; D, the imaging of TCP-1-H6 QD cerebral tissue; E, the imaging of quantum dot tumor tissues.Amplification: 400 *.
The specific embodiment
Embodiment
The present invention will be described further with regard to following examples, but will be appreciated that, these embodiment are the use for illustrating only, and should not be interpreted as restriction of the invention process.
Method of the present invention adopts conventional solid phase Fmoc method; be that the single amino acid of being protected by Fmoc on solid-phase resin goes to expose amino after protection; form peptide bond by amino acid whose carboxyl in condensation reaction and solution, thereby aminoacid is connected on resin, peptide chain is extended to the N end from the C end.
1, stock:
(1) resin be connected molecule: the resin that solid phase Fmoc method is selected is Rink Amide-ChemMatrix
?resin.This resin has extraordinary swellability, can make between peptide chain to carry out better condensation reaction, and have enough cyberspaces to meet ever-increasing peptide chain.Employing HBTU, is fixed to peptide molecule on resin as being connected molecule with HOBt.
(2) monomer: synthetic monomer used is the a-amino acid through chemical modification.
2, reactions steps:
The first step, first aminoacid is covalently bound to resin
Add suitable condensing agent as HBTU and HOBt, fat is amino acid whose fixing to complete altogether to make protected aminoacid c-terminus and resin formation;
Second step, go protection
Adopt basic solvent 20% piperidines to remove the Fmoc on amino, expose amino.
The 3rd step, activation and crosslinked
Adopt activator HBTU and HOBt to activate the carboxyl on next amino, crosslinked with the amino on resin, form peptide bond.
The 4th step, repeat second step and the 3rd step, and iterative cycles adds single amino acid, until synthesized.
3, synthetic post processing:
(1) eluting and deprotection: with deprotection agent trifluoroacetic acid (TFA), peptide chain is eluted from branch, and the deprotection base.
(2) HPLC analyzes purification, and lyophilizing is preserved.
(3) annulation.Take the straight-chain polypeptide CTPSPFSHCGP obtained
9g
2h
62mg, at buffer (the 0.1M NaHCO of pH9.4
3, 0.8mL; 0.1M Na
2cO
3, 0.2 mL) in, to shake 4 hours, lyophilizing is preserved.
By said method, obtain peptide sequence TCP-1-H6 (cyclo (1,9)-CTPSPFSHCGP
9g
2h
6), see Fig. 1.
4, quantum dot and polypeptide coupling
Polypeptide is mixed according to the ratio of 18:1 with quantum dot, after shaking 10 minutes, with ultrafiltration, remove unnecessary peptide molecule, obtain quantum dot-polypeptide fluorescent probe, and confirmed (Fig. 2, curve c) by capillary electrophoresis.
5, colon cancer tumor marker
Colon 26 cells mix and hatch with TCP-1-H6 QD respectively, and the fluorescence imaging result as shown in Figure 3.The experimental result confirmation, the fluorescence of quantum dot has very large the coincidence with the fluorescence of DAPI, and this shows that TCP-1-H6 QD mainly is present in nucleus.Quantum dot fluorescence imaging clearly ground shows that TCP-1-H6 QD can enter in colon 26 cells by specificity, and the targeting of TCP-1-H6 guiding quantum dot enters in cell (Fig. 3).
In addition, experimental result also confirms, TCP-1-H6 QD can specific recognition colon cancer tumor tissue section (Fig. 4 A), but can not identify normal tissue such as colon and cerebral tissue (Fig. 4 C and Fig. 4 D).In competitive assay below, tumor tissues and TCP-1-H6 QD and 150 μ M TCP-1-H6 are mixed together.What is interesting is, TCP-1-H6 QD is marked tumor tissue (Fig. 4 B) clearly still.These results show that TCP-1-H6-QD is after tumor tissues is combined, because the multivalence effect can not competed by the TCP-1-H6 of high concentration.In addition, do not have the quantum dot of labeling polypeptide to mix experiment (Fig. 4 E) in contrast with tumor tissues, do not find obvious quantum dot fluorescence.Therefore, these experiments clearly show that TCP-1-H6 QD can be specifically in conjunction with the colon cancer tumor tissues, and can greatly improve by multivalence its labeling effciency that interacts.
The polypeptide synthesis step is with embodiment 1.Synthetic polypeptide ligand sequence is as follows:
cyclo(1,?9)-CTPSPFSHCGP
10G
2H
7。
The polypeptide synthesis step is with embodiment 1.Synthetic polypeptide ligand sequence is as follows:
cyclo(1,?9)-CTPSPFSHCGP
11G
2H
8
The above-mentioned foundation desirable embodiment of the present invention of take is enlightenment, and 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 description, must determine its technical scope according to the claim scope.
Claims (4)
1. the quantum dot targeted probes for colon cancer tumor tissues identification, it is characterized in that this probe comprises for the histidine sequence (1) in conjunction with quantum dot, the sequence (2) that the polypeptide rigid structure is provided and targeting sequence (3), combines with peptide bond between each sequence; Described histidine sequence (1) is comprised of 6-8 histidine sequence; It is described that the sequence (2) of polypeptide rigid structure is provided is 9-11 proline sequence; The peptide sequence TCP-1 that described targeting sequence (3) is the colon cancer tumor-targeting.
2. a kind of quantum dot targeted probes for colon cancer tumor tissues identification according to claim 1, it is characterized in that: described quantum dot is the quantum dot that contains Zn.
3. a kind of quantum dot targeted probes for colon cancer tumor tissues identification according to claim 1, it is characterized in that: the quantum dot of the described Zn of containing is CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
4. the method for the preparation of the quantum dot targeted probes of colon cancer tumor tissues identification, it is characterized in that, at first polypeptide to be mixed according to the ratio of 18-36:1 with quantum dot, shake after 10 minutes, the histidine sequence of polypeptide is combined with the Zn on quantum dot surface, remove unnecessary peptide molecule by ultrafiltration again, obtain quantum dot-polypeptide fluorescent probe.
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Cited By (5)
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CN105126127A (en) * | 2015-10-14 | 2015-12-09 | 常州大学 | Preparation method of colon cancer nuclear magnetic resonance multifunctional contrast agent |
CN106008671A (en) * | 2016-05-31 | 2016-10-12 | 常州大学 | Novel annular polypeptide ligand used for modifying quantum dots |
CN106046115A (en) * | 2016-05-31 | 2016-10-26 | 常州大学 | Novel peptide ligand modifying quantum dots |
CN110045109A (en) * | 2018-01-15 | 2019-07-23 | 中国医学科学院药物研究所 | A kind of application of polypeptide in clinical early stage colorectal cancer and diagnosing precancerous disease |
CN111196843A (en) * | 2020-01-17 | 2020-05-26 | 常州大学 | Polypeptide ligand Cy5-H8 for modifying quantum dots |
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JIANHAO WANG ET AL.: "Preferential Binding of a Novel Polyhistidine Peptide Dendrimer Ligand on Quantum Dots Probed by Capillary Electrophoresis", 《ANAL. CHEM.》, vol. 83, no. 16, 5 July 2011 (2011-07-05) * |
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Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105126127A (en) * | 2015-10-14 | 2015-12-09 | 常州大学 | Preparation method of colon cancer nuclear magnetic resonance multifunctional contrast agent |
CN105126127B (en) * | 2015-10-14 | 2017-11-10 | 常州大学 | A kind of preparation method of the multi-functional contrast agent of colon cancer nuclear magnetic resonance |
CN106008671A (en) * | 2016-05-31 | 2016-10-12 | 常州大学 | Novel annular polypeptide ligand used for modifying quantum dots |
CN106046115A (en) * | 2016-05-31 | 2016-10-26 | 常州大学 | Novel peptide ligand modifying quantum dots |
CN106046115B (en) * | 2016-05-31 | 2019-05-28 | 常州大学 | A kind of novel polypeptide ligand for modifying quantum dot |
CN106008671B (en) * | 2016-05-31 | 2019-07-09 | 常州大学 | A kind of novel cyclic polypeptide ligand for modifying quantum dot |
CN110045109A (en) * | 2018-01-15 | 2019-07-23 | 中国医学科学院药物研究所 | A kind of application of polypeptide in clinical early stage colorectal cancer and diagnosing precancerous disease |
CN111196843A (en) * | 2020-01-17 | 2020-05-26 | 常州大学 | Polypeptide ligand Cy5-H8 for modifying quantum dots |
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