CN101907625A - Method for preparing quantum dot immune fluorescent probe - Google Patents
Method for preparing quantum dot immune fluorescent probe Download PDFInfo
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- CN101907625A CN101907625A CN2010102202758A CN201010220275A CN101907625A CN 101907625 A CN101907625 A CN 101907625A CN 2010102202758 A CN2010102202758 A CN 2010102202758A CN 201010220275 A CN201010220275 A CN 201010220275A CN 101907625 A CN101907625 A CN 101907625A
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Abstract
The invention provides a method for preparing a quantum dot immune fluorescent probe. A monoclonal antibody fluorescent probe marked by a water-soluble quantum dot is covalently connected with a monoclonal antibody at the tail end of a carboxylation-modified quantum dot; and generally, each quantum dot can be connected with one or more antibody molecules. The preparation method comprises the following steps of: firstly, preparing a mercaptopropanoic acid-modified quantum dot in a water solution by using a hydrothemal method; and then connecting the carboxylation-modified quantum dot with antibody protein by using a covalent coupling method to obtain the quantum dot immune fluorescent probe with favorable fluorescent characteristic and excellent bioactivity. Compared with the prior art, the invention has the advantages that the probe has favorable dispersibility, strong specificity, high fluorescent intensity and photobleaching resistance and can be applied to the fields of immunological detection, cellular signal transduction, fluorescent imaging technologies of biological cells or tissues and other relevant bio-medical researches. The method is simple and easy to apply and can be finished in a general biological and chemical laboratory.
Description
Technical field
The present invention relates to a kind of preparation method of quantum dot immune fluorescent probe, belong to nanomaterial science, bioanalysis chemistry, nano biological medical domain.
Background technology
Quantum dot (quantum dots QDs), claims semiconductor nano again, its radius less than or near the exciton Bohr radius, mainly constitute by II-VI family element (as CdTe, CdSe) or III-V family element (as GaAs, InP).Quantum dot has caused the extensive concern of scientific circles in recent years because of having unique optics and electrology characteristic, the absorption spectrum that it is extensive and continuous in the ultraviolet-visible optical range, the multi-color marking function that narrow and symmetrical fluorescence emission spectrum makes it to have singly and excites, pilosity is penetrated.Quantum dot also has the peculiar property of good light stability, fast light bleaching, makes it be expected to replace traditional organic fluorescent dye, obtains the widespread use in biomedical fluorescence labeling field.
Quantum dot need combine with biomolecule such as biotin/avidin, antibody, oligonucleotides earlier usually in the application of biomedical marker field, forms biological fluorescent labeling, adds in the sample in conjunction with respective ligand, antigen or the nucleic acid of desiring mark again.Quantum dot can pass through the direct and antibodies of Electrostatic Absorption method with combining of antibody, though this method is simple, in conjunction with reliable inadequately, environmental factors such as pH, temperature, ionic strength change can make quantum dot-antibody complex uncoupling.Directly in preparation process, a large amount of amino or carboxyl have been introduced on its surface at the water-soluble quantum dot of aqueous phase preparation, therefore can form chemical covalent coupling by amino in these function group and the antibody protein molecule or carboxyl, obtain stable quantum dot-antibody fluorescence probe
Summary of the invention
Use in biomedical sector for expanding quantum dot, the invention provides and a kind of water-soluble carboxyl quantum dot is used for the method that mark has bioactive antibody protein and then prepares immune fluorescent probe, this method can be connected carboxylated quantum dot effectively with antibody protein, and keep the biologically active of antibody well, can be applicable to biomarker analysis field, for example based on the antigen/antibody quantitative test of measuring fluorescence intensity based on the antigen and antibody specific combination, Enzyme Linked Immunoadsorbent Assay, location in the born of the same parents of specific protein in the labeled cell, research nucleic acid hybridization etc.
Quantum dot immune fluorescent probe of the present invention is at the specific antibody of the terminal connection of carboxylated quantum dot, can connect one or an above antibody molecule usually, and number can be adjusted by the ratio of control quantum dot and antibody.
Wherein the core of carboxylated quantum dot is any one among CdTe, CdSe, CdTe/ZnS, the CdSe/ZnS, and is that aqueous phase directly prepares, with the water-soluble quantum dot of mercaptopropionic acid or other carboxylic sulfhydryl compound modifications.Used antibody can be a domain at a kind of albumen or certain albumen, and perhaps the monoclonal antibody of one section polypeptide also can be at the polyclonal antibody from different plant species albumen.
A kind of preparation method of quantum dot immune fluorescent probe is characterized in that adopting following steps:
A) be 0.2mol/L with concentration, the pH value is phosphate buffer dissolving 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the N-carbonyl succinimide of 7.2-7.6, be mixed with carboxyl activator, the concentration of (3-dimethylaminopropyl) carbodiimide of 1-ethyl-3-in the activator and N-carbonyl succinimide is 50mg/mL, it is in the commercially available carboxylated quantum dot of 1mg/mL that this activator was added to concentration in 1: 8 by volume, at room temperature slight vibration makes activated carboxylic form active ester, make quantum dot sedimentation, abandoning supernatant through the supercentrifuge separation;
B) toward steps A) quantum dot precipitation in add A) in the phosphate buffer that uses, make quantum dot be suspended into A again in slight vibration on the vortex oscillator) volume after adding activator, supercentrifuge separates and is resuspended again, repeats like this 2 times, and high speed centrifugation makes the quantum dot sedimentation once more;
C) adding concentration is the antibody-solutions of 1mg/mL, slight vibration makes quantum dot be suspended into steps A again in antibody-solutions) in add volume behind the activator, and antibody is fully contacted with the quantum dot of activated carboxylic, the reaction of generation covalent coupling, at room temperature place 1h, every the slight vibration of 20min once, 1h is after low speed centrifuge separates the quantum dot agglomerate sedimentation that makes formation, and the gained supernatant is quantum dot immune fluorescent probe.
The present invention directly prepares on the basis of water-soluble quantum dot technology in the hydro-thermal method that development in recent years is got up, modify the quantum dot surface with mercaptopropionic acid as stabilizing agent, make it to have a large amount of carboxyl function groups, utilize EDC and NHS or one of them as carboxyl and amino coupling agent, antibody is connected the quantum dot surface reliably.This method is simple, safe and reliable, need not specific apparatus reagent, all can finish at most of biologies, chemical laboratory, is fit to be applied to biomedical labeled analysis field.
Description of drawings
Fig. 1 is for being the photo of quantum dot immune fluorescent probe.
Fig. 2 is the TEM image of quantum dot immune fluorescent probe.
Fig. 3 combines the fluorescent microscope photo behind the antigen of elisa plate bottom for quantum dot immune fluorescent probe.
Embodiment
Embodiment 1:
The NaBH4 of 0.4g and 0.08g tellurium powder are added in the 50mL conical flask, add the 5mL deionized water, logical nitrogen 20min, jam-pack flexible glue plug inserts a syringe needle immediately, and magnetic agitation 2h obtains the NaHTe solution of milky prepared fresh.Get the CdCl that 0.5mL concentration is 200mmol/L
2Aqueous solution, 0.5mL the concentration freshly prepared mercaptopropionic acid aqueous solution that is 200mmol/L, add in another 50mL conical flask, add the 14ml deionized water, dripping concentration is the NaOH solution of 1mol/L, adjusting pH is 12.0, logical nitrogen 20min in above-mentioned solution, jam-pack flexible glue plug immediately, insert a syringe needle,, inject the conical flask that the Cd precursor liquid is housed fast with the NaHTe solution 0.2mL of 1mL syringe extraction prepared fresh, conical flask is put into 96 ℃ of water-baths heat 1h, take out and naturally cool to room temperature; Open the flexible glue plug of the conical flask that reaction mixture is housed, with the solution five equilibrium in two 15mL centrifuge tubes, in two centrifuge tubes, add and the isopyknic absolute ethyl alcohol of supernatant respectively again, when having treated that floccus produces, with the speed centrifuging 10min of 3000rpm/min, the sucking-off supernatant repeats absolute ethanol washing, step with centrifugal separation 2 times, after for the last time supernatant being removed centrifuge tube is positioned in the thermo-humidistat cabinet and spends the night, obtain CdTe quantum dot dry powder.
Take water-soluble quantum dot as follows: the quantum dot dry powder that will redissolve with PBS is with 2% the pure nitric acid dilution 100 of top grade, 000 times, according to the described method of the standard WS-T 174-1999 of the Ministry of Public Health, with the concentration of Cd in the quantitative quantum dot of GFAAS (graphite furnace atomic absorption spectrometry), and be converted into the concentration of CdTe.With the pH value is 7.2-7.6, and the PBS of 0.2M dilutes quantum dot solution to 1.0mg/mL, vibrating dispersion 20s on the vortex oscillator, and it is standby to get 1mL.
If antibody obtains for purifying voluntarily, then should avoid adding Sodium azide as antiseptic, can use proclin300 as antiseptic, whole mass percentage concentration is 0.02%; If antibody is commercial product, then should prevent that Sodium azide from combining with the carboxyl of activation using after to the PBS dialysed overnight under 4 ℃ of conditions, and then with its deactivation, the combination of obstruction antibody.The concentration of antibody can be adjusted as required, preferred 0.1-5mg/mL, and more preferably 0.5-2mg/mL is used for the coupling with quantum dot.
EDC and NHS are mixed with the solution that concentration is 50mg/mL respectively, and for guaranteeing the efficient of coupling, coupling agent solution should fresh preparation.
Get 2 1.5mL centrifuge tubes, in each centrifuge tube, add the scattered quantum dot of 100 μ L, 540 μ L deionized waters successively, 240 μ L PBS and 60 μ L EDC and 60 μ L NHS solution.Centrifuge tube is wrapped up with tinfoil, be placed on the shaking table lucifuge reaction 30min under the room temperature.
Take out centrifuge tube, place supercentrifuge, 12000rpm centrifuging 5min makes the quantum dot sedimentation, abandoning supernatant under the room temperature.Add 1.0mL PBS, the resuspended quantum dot of vortex vibration 20s, high speed centrifugation separates once more afterwards, repeats 2 times, removes remaining coupling agent in the solution.
Adding 200 μ L concentration is the antibody-solutions that 1.0mg/mL does not contain Sodium azide, adds to be equipped with in the centrifuge tube of quantum dot, and vibration evenly disperses quantum dot in antibody-solutions gently on the vortex oscillator.Wrap up centrifuge tube with masking foil, be placed on the shaking table oscillating reactions 1h under the room temperature.
Take out centrifuge tube, place low speed centrifuge, the centrifugal 5min of 3000rpm, the precipitation that removal may form, the gained supernatant is water-soluble quantum dot-antibody mediated immunity fluorescence probe.
Quantum dot immune fluorescent probe is observed by transmission electron microscope (TEM), can be seen that quantum dot has good monodispersity (Fig. 2) in the prepared probe.With the corresponding antigen bag by elisa plate, add quantum dot immune fluorescent probe combination with it, wash plate 3 times with the cleansing solution and the PBS that contain Tween-20 respectively again, under fluorescent microscope, observe afterwards, can be clear that the fluorescence (Fig. 3) of probe, show that probe has good biologically active.Further adopt the ELISA method to measure tiring of antibody in the probe, the result can reach 106, prove that the biologically active of antibody has obtained well keeping in the coupling reaction that probe prepares.
Claims (2)
1. the preparation method of a quantum dot immune fluorescent probe is characterized in that adopting following steps:
A) be 0.2mol/L with concentration, the pH value is phosphate buffer dissolving 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the N-carbonyl succinimide of 7.2-7.6, be mixed with carboxyl activator, the concentration of (3-dimethylaminopropyl) carbodiimide of 1-ethyl-3-in the activator and N-carbonyl succinimide is 50mg/mL, it is in the commercially available carboxylated quantum dot of 1mg/mL that this activator was added to concentration in 1: 8 by volume, at room temperature slight vibration makes activated carboxylic form active ester, make quantum dot sedimentation, abandoning supernatant through the supercentrifuge separation;
B) toward steps A) quantum dot precipitation in add A) in the phosphate buffer that uses, make quantum dot be suspended into A again in slight vibration on the vortex oscillator) volume after adding activator, supercentrifuge separates and is resuspended again, repeats like this 2 times, and high speed centrifugation makes the quantum dot sedimentation once more;
C) adding concentration is the antibody-solutions of 1mg/mL, slight vibration makes quantum dot be suspended into steps A again in antibody-solutions) in add volume behind the activator, and antibody is fully contacted with the quantum dot of activated carboxylic, the reaction of generation covalent coupling, at room temperature place 1h, every the slight vibration of 20min once, 1h is after low speed centrifuge separates the quantum dot agglomerate sedimentation that makes formation, and the gained supernatant is quantum dot immune fluorescent probe.
2. preparation method according to claim 1 is characterized in that: antibody is at different albumen, perhaps albumen domain, and the perhaps monoclonal antibody of one section polypeptide, or at the polyclonal antibody from different plant species albumen.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102262157A (en) * | 2011-07-07 | 2011-11-30 | 清华大学深圳研究生院 | Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby |
| CN102288765A (en) * | 2011-07-07 | 2011-12-21 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN102942175A (en) * | 2012-11-23 | 2013-02-27 | 中国科学院长春光学精密机械与物理研究所 | Application of carbon nanodots as water-soluble ratiometric fluorescent probe |
| CN103487574A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | Method for marking immune globulin by quantum dot |
| CN103513025A (en) * | 2012-06-21 | 2014-01-15 | 复旦大学附属华山医院 | Stomach cancer targeting quantum dots, and preparation method and application thereof |
| CN109097021A (en) * | 2017-06-20 | 2018-12-28 | Tcl集团股份有限公司 | The method for improving near-infrared water-soluble copper sulphur indium quantum dot quantum efficiency |
| CN109813887A (en) * | 2019-02-19 | 2019-05-28 | 吉林省爱诺德生物工程有限公司 | A kind of immunological response real-time detection method |
| CN110333341A (en) * | 2019-07-04 | 2019-10-15 | 浙江理工大学 | A method of silk fabric cultural relics sample is identified based on protein biochip technology |
| CN111197110A (en) * | 2020-01-16 | 2020-05-26 | 浙江农林大学 | RPA-FLD method for detecting feline panleukopenia virus |
| CN113916846A (en) * | 2020-07-10 | 2022-01-11 | 中国科学院福建物质结构研究所 | Fluorescent quantum dot nanoprobe, preparation method thereof and application thereof in detecting circulating tumor cells |
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102262157A (en) * | 2011-07-07 | 2011-11-30 | 清华大学深圳研究生院 | Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby |
| CN102288765A (en) * | 2011-07-07 | 2011-12-21 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN102288765B (en) * | 2011-07-07 | 2013-12-04 | 清华大学深圳研究生院 | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit |
| CN102262157B (en) * | 2011-07-07 | 2014-02-05 | 清华大学深圳研究生院 | Quantum dot-based method for immunofluorescence of clenbuterol hydrochloride and special kit thereby |
| CN103513025B (en) * | 2012-06-21 | 2016-08-10 | 复旦大学附属华山医院 | Stomach cancer target quantum dot and its preparation method and application |
| CN103513025A (en) * | 2012-06-21 | 2014-01-15 | 复旦大学附属华山医院 | Stomach cancer targeting quantum dots, and preparation method and application thereof |
| CN102942175A (en) * | 2012-11-23 | 2013-02-27 | 中国科学院长春光学精密机械与物理研究所 | Application of carbon nanodots as water-soluble ratiometric fluorescent probe |
| CN102942175B (en) * | 2012-11-23 | 2014-11-19 | 中国科学院长春光学精密机械与物理研究所 | Application of carbon nanodots as water-soluble ratiometric fluorescent probe |
| WO2015054939A1 (en) * | 2013-10-16 | 2015-04-23 | 深圳市大爱医疗科技有限公司 | Method of labeling immunoglobulin using quantum dots |
| CN103487574B (en) * | 2013-10-16 | 2015-06-17 | 深圳市金准生物医学工程有限公司 | Method for marking immune globulin by quantum dot |
| CN103487574A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | Method for marking immune globulin by quantum dot |
| CN109097021A (en) * | 2017-06-20 | 2018-12-28 | Tcl集团股份有限公司 | The method for improving near-infrared water-soluble copper sulphur indium quantum dot quantum efficiency |
| CN109813887A (en) * | 2019-02-19 | 2019-05-28 | 吉林省爱诺德生物工程有限公司 | A kind of immunological response real-time detection method |
| CN110333341A (en) * | 2019-07-04 | 2019-10-15 | 浙江理工大学 | A method of silk fabric cultural relics sample is identified based on protein biochip technology |
| CN111197110A (en) * | 2020-01-16 | 2020-05-26 | 浙江农林大学 | RPA-FLD method for detecting feline panleukopenia virus |
| CN113916846A (en) * | 2020-07-10 | 2022-01-11 | 中国科学院福建物质结构研究所 | Fluorescent quantum dot nanoprobe, preparation method thereof and application thereof in detecting circulating tumor cells |
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Application publication date: 20101208 |