CN1975420B - Producing method of functional quantum point used for specific recognizating tumor cell - Google Patents
Producing method of functional quantum point used for specific recognizating tumor cell Download PDFInfo
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- CN1975420B CN1975420B CN2006100982706A CN200610098270A CN1975420B CN 1975420 B CN1975420 B CN 1975420B CN 2006100982706 A CN2006100982706 A CN 2006100982706A CN 200610098270 A CN200610098270 A CN 200610098270A CN 1975420 B CN1975420 B CN 1975420B
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- quantum dot
- tumor cell
- folic acid
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Abstract
A method for preparing functional quanta point which is used for specially identifying the tumor cell belongs to the nanometer material and biological technical field. The method includes the following steps: (1) preparing the CdS quanta point; (2) selecting small molecular compound with mercapto for surface modification; (3) functionally modifying the quanta point surface: after the modification to the quanta point surface in the last step, adding DMSO solutions of N-hydroxysuccinimide and 1-(3-dimethylamino propyl)-3-ethylcarbodiimide to form a high activity intermediate which can react with the amino in the subsequently added folic acid. The water solubility is improved by modifying the quanta point surface through the small molecular containing the mercapto. The fluorescent quanta point can specially identify and express the tumor cell with the high level folic acid acceptor via an effective method and by combining the folic acid and the quanta points, thereby being used for a fluorescent probe in cell biological study.
Description
Affiliated technical field
A kind of method for preparing that is used as the functional quantum point of specific recognition tumor cell belongs to the present invention and is nano material and biological technical field.
Background technology
Tumor is the harm whole mankind's a No.1 disease.The early diagnosis of tumor, early treatment and targeted therapy are the contents that people pay close attention to always and study.The conventional fluorescent dyestuff that is used for RESEARCH ON CELL-BIOLOGY exist much can't customer service limitation, strict like excitation wavelength, fluorescence efficiency is low, emission wavelength and excitation wavelength overlap easily, light stability is poor, or the like, brought a lot of inconvenience to biological study.
Along with the development of technology, the research and development center of gravity of nanotechnology shifts to function, application, device from the synthetic of single nano material gradually.Wherein, in the interface field of nanotechnology and biotechnology, quantum dot has obtained good application.Mostly the method for preparing of the quantum dot of reporting in the document is the little micelle assay of anti-phase, and people are many with containing Cd
2+Ion and S
2-Ion solution prepares the CdS quantum dot, for example, Heesun Yang, paul HHolloway, app.phy.lett.2003,82,1965. or the like.Though be feasible, not good enough on the uniformity of response time and product, exist the monodispersity and the severe reaction conditions of efficient and gained quantum dot, problems such as reactant toxicity.Prepare in oil phase because quantum dot is many, and biotic environment is a water, so before functionalization, will carry out surface modification the quantum dot of gained.Water miscible quantum dot is existing report in document, like Daniel R.Larson et al.Science, 2003,300,1434-1436. or the like.Quantum dot as biological study will have the specific recognition function to different types of cell, and existing quantum dot can not satisfy this requirement aspect the surface-functionalized modification, in dyeing course, has the unspecific staining problem.
Summary of the invention
The object of the present invention is to provide a kind of method for preparing that is used as the functional quantum point of specific recognition tumor cell,, reduce the intensity of unspecific staining as much as possible, adapted to the needs of RESEARCH ON CELL-BIOLOGY to improve the selectively targeted function of quantum dot.
A kind of method for preparing that is used as the functional quantum point of specific recognition tumor cell is applied to prepare detect and expresses the tumor cell that high-level folacin receptor is arranged on the cell membrane, it is characterized in that being made up of following steps:
(1), the preparation of CdS quantum dot: adopt the little glue bundle body method of anti-phase under the room temperature, and with H
2S is feeding H as the S source
2Before the S gas, in system, lead to 5-10 minute nitrogen earlier, the oxygen in the system of draining is through the particle diameter of cadmium salt and surfactant concentrations control quantum dot in little micelle volume;
(2), the micromolecular compound of selecting to have sulfydryl carries out surface modification to quantum dot;
(3), the surface-functionalized modification of quantum dot: process (2) carries out the surface of quantum dot with washing with acetone 3-5 time, washing after the modification, and centrifugal, oven dry hockets; Then; 5mg quantum dot powder is dispersed in the 5-10mL phosphate buffer; Add the N-hydroxy-succinamide of 20-30 μ L0.05M and the inferior maple solution of dimethyl of 1-(3-dimethylamino-propyl)-3-ethyl carbon two imido respectively, stirred 30-60 minute, add the inferior maple solution of dimethyl of the folic acid of 20-30 μ L 0.05M; Stir reaction 12-24 hour; With deionized water reactant was dialysed 5-8 hour at last, unreacted micromolecule is removed; The purpose of the N-hydroxy-succinamide of adding 20-30 μ L 0.05M and the inferior maple solution of dimethyl of 1-(3-dimethylamino-propyl)-3-ethyl carbon two imido is to make and forms the higher intermediate of a kind of specific activity in the system, can react with the amino on the folic acid that adds subsequently.
The preparation of above-mentioned CdS quantum dot can be adopted conventional methods such as hydro-thermal method, and the saline solution that can adopt S is as the S source.If but adopt H
2S is as the S source, promptly at room temperature with H
2S gas feeds in the microemulsion system, with the preparation quantum dot, can improve the preparation process, makes the simple of preparation condition change, improves product uniformity and controllability.
If feeding H
2Before the S gas, in system, lead to 5-10 minute nitrogen earlier, the oxygen in the system of can draining prevents H
2S gas is oxidized.
If in the preparation process of CdS quantum dot, control the particle diameter that Cd salt and surfactant concentrations in little micelle volume can be controlled quantum dot.
The functional quantum point of method for preparing is applied to detect expresses the tumor cell that high-level folacin receptor is arranged on the cell membrane.
Compare with existing quantum dot, use H
2S is as the S source, feeds the nitrogen oxygen in the reaction system of draining, and preparation is convenient, and is quick, and reaction unit is not harsh with the reaction condition requirement.Want to make quantum dot that target cell is carried out specific stain, the present invention has overcome the deficiency of prior art, and the whole process of preparation does not need harsh appointed condition, simple and safe operation, and materials safety, cost is low.When the surface-functionalized modification of quantum dot, select folic acid as the quantum dot surface targets to functional molecular.Folic acid is one of vigorous primary raw material of metabolism in the tumor cell, and it combines and get into cell to participate in cellular metabolism with the folacin receptor on tumor cell membrane surface specifically, and the folacin receptor level of the surface of cell membrane of tumor cell is higher than normal cell far away.So folacin receptor is as the target function acceptor molecule of the medicine of many treatment tumors.The present invention has successfully modified the surface of prepared CdS quantum dot to folic acid, make quantum dot its have the function of specific recognition folacin receptor.
Description of drawings
Fig. 1: the photo of the transmission electron microscope of used CdS quantum dot in the cell recognition, the about 5nm of particle diameter.
Fig. 2: the UV, visible light optical absorption spectra of quantum dot.
Fig. 3: the fluorescence spectrum of quantum dot under the burst of ultraviolel of 380nm, near the fluorescence emission peak its 519nm is obvious, and we survey the fluorescence basis of tumor cell just.
Fig. 4: the infared spectrum of the CdS quantum dot of modifying without TGA.
Fig. 5: the infrared absorption pattern of the CdS quantum dot after modifying through TGA (can confirm that from the absworption peak of Fig. 4 and Fig. 5 TGA successfully modified on the quantum dot).
Fig. 6: with the quantum dot and the HepG2 of process modified with folic acid do not cultivate the fluorescence photo after 1 hour altogether.
Fig. 7: with the quantum dot and the HepG2 of process modified with folic acid do not cultivate the fluorescence photo after 3 hours altogether.
Fig. 8: with the quantum dot and the HepG2 of process modified with folic acid do not cultivate the fluorescence photo after 6 hours altogether.
Fig. 9: quantum dot and HepG2 with through modified with folic acid cultivate the fluorescence photo after 1 hour altogether.
Figure 10: quantum dot and HepG2 with through modified with folic acid cultivate the fluorescence photo after 3 hours altogether.
Figure 11: quantum dot and HepG2 with through modified with folic acid cultivate the fluorescence photo after 6 hours altogether.
The specific embodiment
Embodiment 1:
One, the preparation of quantum dot
(1) solution of preparing A OT and normal heptane
The n-heptane solution 30mL of the AOT of preparation 0.1M.
(2) configuration of reverse micelle system
In above-mentioned solution, add CdCl
2Aqueous solution, make that the concentration of water in system is 0.1M.Add CdCl
2Aqueous solution after, with ultrasonic dispersing 30 minutes,, so just obtained the little micelle volume of anti-phase up to the system clear.
(3) reaction generates the CdS quantum dot
Under the room temperature, feed 5 minutes nitrogen to the little micelle volume of above-mentioned anti-phase, the oxygen in the eliminating system, under the situation of magnetic agitation, system is gone into competent H in above-mentioned little micelle volume then
2S gas.Accomplish when the system color no longer changes just explanation reaction, quantum dot is successfully preparation.
2. TGA surface modification
Above-mentioned reactant is added acetone carry out breakdown of emulsion, wash the surfactant on quantum dot surface off.With acetone, centrifugal, 5 times repeatedly.Oven dry obtains the quantum dot powder then.
In the 5mg semiconductor-quantum-point, add the TGA of 5mL, the room temperature lower magnetic force stirred 12 hours.
3. the surface-functionalized modification of quantum dot: promptly quantum dot is connected with folic acid.
In said process, stir after 12 hours, with washing with acetone 3 times (wash, centrifugal; Oven dry hockets), then it is dispersed in the 5mL phosphate buffer, add the N-hydroxy-succinamide of 25 μ L0.05M and the inferior maple solution of dimethyl of 1-(3-dimethylamino-propyl)-3-ethyl carbon two imido respectively; After stirring half an hour; Add the inferior maple solution of dimethyl of the folic acid of 25 μ L 0.05M, stir, reacted 12 hours.With deionized water reactant was dialysed 5 hours at last, unreacted micromolecule is removed.
Like particle diameter and the finishing design sketch of Fig. 1-Fig. 5, show that this method has successfully prepared high-quality quantum dot for the functional quantum point of present embodiment preparation.
Two, cell dyeing implementation process and observation thereof
Utilize conventional cell culture processes; Select expression such as HeLa, HepG2, KB cell that the tumor cell and normal epithelial cell and above-mentioned quantum dot co-cultivation of high-level folacin receptor (FR) are arranged; Observe with laser confocal microscope then; Through the fluorescence intensity of cell under more different cell categories, the different incubation time, proved conclusively tumor cell specific adsorption and the internalization of quantum dot has been time dependence (6 hours states that reach capacity).
Like Fig. 6-11 is the result of the test of HepG2, confirmed should technology effectiveness and reliability.
Embodiment 2:
The maximum creativeness of method of the present invention is this step of the surface-functionalized modification of quantum dot (being that quantum dot is connected with folic acid).This step is:
Through the micromolecular compound that contains sulfydryl the quantum dot of preparation is carried out with washing with acetone 3-5 time, washing after the surface, centrifugal, oven dry hockets; Then; 5mg quantum dot powder is dispersed in the 5-10mL phosphate buffer; Add the inferior maple solution of dimethyl of 1-(3-dimethylamino-propyl)-3-ethyl carbon two imido of 0.05M of the inferior maple solution of dimethyl and same volume of the N-hydroxy-succinamide of 20-30 μ L 0.05M respectively, stirred 30-60 minute, add the dimethyl Asia maple solution of the folic acid of 20-30 μ L 0.05M; Stir reaction 12-24 hour.With deionized water reactant was dialysed 5-8 hour at last, unreacted micromolecule is removed.
Claims (2)
1. method for preparing as the functional quantum point of specific recognition tumor cell is characterized in that being made up of following steps:
(1), the preparation of CdS quantum dot: adopt the little glue bundle body method of anti-phase under the room temperature, and with H
2S is feeding H as the S source
2Before the S gas, in system, lead to 5-10 minute nitrogen earlier, the oxygen in the system of draining is through the particle diameter of cadmium salt and surfactant concentrations control quantum dot in little micelle volume;
(2), the micromolecular compound of selecting to have sulfydryl carries out surface modification to quantum dot;
(3), the surface-functionalized modification of quantum dot: the surface of quantum dot was carried out after the modification through a last step, with washing with acetone 3-5 time, washing, centrifugal, dry and hocket; Then; 5mg quantum dot powder is dispersed in the 5-10mL phosphate buffer; The inferior maple solution of dimethyl of 1-(3-dimethylamino-propyl)-3-ethyl carbon two imido of the 0.05M of the inferior maple solution of dimethyl of the N-hydroxy-succinamide of adding 20-30 μ L 0.05M and same volume stirred 30-60 minute in phosphate buffer; In above-mentioned system, add the inferior maple solution of dimethyl of the folic acid of 20-30 μ L 0.05M afterwards again, stir, reaction 12-24 hour; With deionized water reactant was dialysed 5-8 hour at last, unreacted micromolecule is removed.
2. the method for preparing of the functional quantum point of specific recognition tumor cell according to claim 1 is characterized in that: this method preparation and be applied to detect through the quantum dot of surface-functionalized modification and express the tumor cell that high-level folacin receptor is arranged on the cell membrane.
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| CN102274002B (en) * | 2011-04-26 | 2014-02-26 | 中国药科大学 | In-situ tumor nondestructive detection kit and preparation method thereof |
| CN102818826B (en) * | 2012-07-21 | 2014-09-17 | 上海师范大学 | Electrochemical cell-based biosensor based on nanometer Ag@BSA biomimetic interface and preparation method thereof |
| CN104650856B (en) * | 2015-02-26 | 2017-01-04 | 江汉大学 | A kind of preparation method of cadmiumsulfide quantum dot solution |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6689338B2 (en) * | 2000-06-01 | 2004-02-10 | The Board Of Regents For Oklahoma State University | Bioconjugates of nanoparticles as radiopharmaceuticals |
| CN1869692A (en) * | 2005-05-23 | 2006-11-29 | 武汉大学 | Three-function nano-ball |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6689338B2 (en) * | 2000-06-01 | 2004-02-10 | The Board Of Regents For Oklahoma State University | Bioconjugates of nanoparticles as radiopharmaceuticals |
| CN1869692A (en) * | 2005-05-23 | 2006-11-29 | 武汉大学 | Three-function nano-ball |
Non-Patent Citations (3)
| Title |
|---|
| Xun Wang,et al.A general strategy for nanocrystal synthesisi.《NATURE》.2005,第437卷(第7055期),121-124. * |
| 谢海燕.基于量子点的生物医学功能纳米材料的制备与应用.《中国博士学位论文全文数据库 工程科技I辑》.2004,1.1量子点的性质、制备与应用. * |
| 陈良，等.一种表面修饰的CdS量子点的合成与特征分析.《微纳电子技术》.2005,(第11期),522-525. * |
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