CN102206487B - Carboxylation beta-cyclodextrin modified low-toxicity functional quantum dot and preparation method thereof - Google Patents
Carboxylation beta-cyclodextrin modified low-toxicity functional quantum dot and preparation method thereof Download PDFInfo
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- CN102206487B CN102206487B CN 201110100075 CN201110100075A CN102206487B CN 102206487 B CN102206487 B CN 102206487B CN 201110100075 CN201110100075 CN 201110100075 CN 201110100075 A CN201110100075 A CN 201110100075A CN 102206487 B CN102206487 B CN 102206487B
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Abstract
The invention relates to a carboxylation beta-cyclodextrin modified low-toxicity functional quantum dot and a preparation method thereof, belonging to the field of specific molecular recognition diagnostic reagent. In the invention, the preparation method of the carboxylation beta-cyclodextrin modified low-toxicity functional quantum dot comprises the steps that: a functional group carboxyl is connected on beta-cyclodextrin through a chemical modification method to obtain carboxy-beta-cyclodextrin; under the action of 1-ethyl-3-(3-dimethyl propylamine) carbodiimide hydrochloride and N-hydroxysuccinimide, activated folic acid is firstly coupled with amino-terminated poly (ethylene glycol) and then coupled with the carboxy-beta-cyclodextrin to obtain folic acid-beta-cyclodextrin; silver, zinc and other low-toxicity elements are utilized as raw materials to prepare an oil soluble infrared quantum dot, and the folic acid-beta-cyclodextrin is utilized to perform water-soluble modification on the oil soluble infrared quantum dot to obtain the carboxylation beta-cyclodextrin modified low-toxicity functional quantum dot. The quantum dot prepared by the method disclosed by the invention has good water solubility and low toxicity, and the emission spectrum is in a near infrared area, and the quantum dot can be used for specific fluorescence detection on tumors because of being coupled with the folic acid.
Description
Technical field
The specificity fluorescent that hypotoxicity functional quantum point that a kind of carboxylated method beta-cyclodextrin is modified and preparation method thereof, the functional quantum point of preparation can be used for tumor markers etc. detects, and belongs to specific molecular identifying and diagnosing reagent field.
Background technology
Folic acid is the necessary VITAMIN of cell (the especially cell of molecular marker for increased proliferation), and it participates in a carbon shift reaction of multiple pathways metabolism.The cell traffic of folic acid is by two kinds of transmembrane proteins, and namely the reductibility folate carrier of low-affinity and the folacin receptor of high-affinity are finished.Confirmed that at present folacin receptor is in kinds of tumor cells surface overexpression, as ovarian cancer, cervical cancer, carcinoma of endometrium, mammary cancer, lung cancer, brain tumor, ependymoma etc., and only limiting to some, the expression in most healthy tissuess is difficult to enter sanguimotor epithelial cell teleblem.Folic acid contains α and two carboxyls of γ, be easy to modify, have reduced immunogenicity, small volume, high chemical stability and biological stability simultaneously, high tumour perviousness, easily be combined with medicine, the advantage such as good and low cost with the consistency of organic or aqueous solvent is so the research of folate-mediated cancer target has obtained developing rapidly.
The fluorescent probe that is used for biomolecules and cell marking and identification comprises organic molecular fluorescence probe and inorganic molecule fluorescent probe two classes.Deficiencies such as although the kind of organic molecule fluorescent probe is many, it is little to exist the Stoke displacement, and easy photobleaching and quantum yield are low.The inorganic molecule fluorescent probe mainly be quantum dot (Quantum Dots, QDs).QDs refer to radius less than or close to the semiconductor nano crystal grain of exciton Bohr radius, particle diameter is generally 1-10nm, is generally the compound that is made of II-VI family or III-V family element.Quantum dot is as a kind of novel fluorescence dyestuff, have a lot of uniquenesses and good optical characteristics, comprise the excitation spectrum that (1) is continuous and wide, and the fluorescence spectra position can be regulated and control by the particle diameter that changes QDs, so only just can excite the QDs of multiple different colours fluorescence with a kind of excitation light source of wavelength, carry out multiplex fluorescence and detect.(2) emmission spectrum is narrow, can manifest multiple different colours simultaneously and zero lap.(3) the Stokes displacement is big, can avoid emission spectrum and excitation spectrum overlapping, detects thereby allow to carry out spectroscopy under the situation of low signal intensity.(4) anti-photobleaching ability is strong, and the height light stability of this anti-photobleaching is very important for the research of the long-time imaging of needs.(5) light stability height is convenient to obtain not have the good optical physics characteristics such as fluorescent signal of background interference.(6) fluorescent yield height, intensity is strong, and the fluorescent brightness that single quantum dot shows is 10-20 times of organic fluorescent dye.Therefore, quantum dot demonstrates its unique application prospect in a plurality of research fields.
The difference of the solvent that adopts during according to synthetic quantum dot, the quantum dot synthetic method can be summed up as two big classes, and a kind of method is to utilize the precursor thermolysis to synthesize in high boiling organic solvent, the product fluorescence efficiency height that obtains, even, the good stability of size.Another kind is to utilize stablizer directly synthetic in the aqueous solution, and the distribution of sizes of product is wide, and fluorescence efficiency is lower.General the synthetic of quantum dot carries out in organic solvent, formed hydrophobic shell in the outside of nonmetal nuclear, and feasible synthetic quantum dot has hydrophobicity and can not biologically use.In order to make them have biocompatibility, often they are carried out functionalization, or it is carried out second layer bag quilt, can strengthen its lasting stability and suspension character water-soluble, nuclear, make quantum dot have better biological activity.
Can be obtained the quantum dot of high degree of specificity by the bag of modifying quantum dot, be used for diagnosis (molecular imaging) and treat (bio-carrier) etc.But their genotoxic potential just is their bag by last, is broken away from nuclear if these wrap, no matter discharge nuclear (CdTe, CdSe) or the single cadmium metal (Cd) of molectron, and all can toxigenicity.Perhaps, some quantum dot capsulating materials itself just have cytotoxicity, as acetate methanol (MAA).Simultaneously, quantum dot may be degraded under illumination and oxidizing condition, and therefore stability seems most important in its commercialization process.
The near infrared fluorescent probe diagnosis of molecule in vivo plays a part key in identifying.Because near-infrared band (600 ~ 800nm) light wave has been avoided water in the body, the optimum absorb wavelength of main absorptive tissue such as aerobic and anaerobic oxyphorase, thereby have good biological tissue's penetrativity, and to biological tissue's not damaged.Therefore the water-soluble quantum dot that has near-infrared luminous characteristic is more suitable for bioanalysis and uses.
Cyclodextrin (Cyclodextrin, be called for short CD) is the general name of a series of cyclic oligosaccharides of generating under the cyclomaltodextrin glucanotransferase effect that is produced by genus bacillus of amylose starch, contains 6～12 D-glucopyranose units usually.Wherein study morely and what have important practical usage is the molecule that contains 6,7,8 glucose units, be called α-, β-, γ-Huan Hujing.According to the result of X-line crystalline diffraction, infrared spectra and spectral analysis of the nuclear magnetic resonance, determine that each D (+)-Glucopyranose of formation cyclodextrin molecular is chair conformation.Each glucose unit all is combined into ring with 1,4-glycosidic link.Can not rotate freely owing to connect the glycosidic link of glucose unit, cyclodextrin is not cylindric molecular structure but slightly tapered annulus.Wherein, the primary hydroxyl of cyclodextrin has surrounded the osculum of taper, and its secondary hydroxyl has surrounded big mouthful of taper.Form slightly tapered hollow cylinder three-dimensional ring structure.Beta-cyclodextrin (β-CD) molecular structure is shown below:
Inner chamber is hydrophobic because the outer rim of cyclodextrin is hydrophilic, thereby it can provide a hydrophobic combining site as enzyme, as main body (Host) the various suitable objects of envelope (Guest), as organic molecule, mineral ion and gas molecule etc.In three kinds of cyclodextrin, because α-CD molecule cavity hole is less, can only wrap the guest species that connects than small molecules usually, range of application is less; The molecule of γ-CD cavity is big, but its production cost height, industrial can not mass production, its application is restricted; The molecule cavity of β-CD is moderate, applied range, and production cost is low, is the maximum cyclodextrin product of present industrial use.
According to the constructional feature of beta-cyclodextrin, can the hydrogen atom of its structural primary hydroxyl or secondary hydroxyl be replaced, perhaps replace primary hydroxyl or/and secondary hydroxyl, or eliminate the hydrogen atom on the carbon atom of 6-position, open one or more C by partial oxidation
2-C
3Singly-bound.Method by chemically modified is incorporated into β-CD with water soluble groups such as amino, carboxyls, can greatly improve its character, thereby enlarges use range.
Folic acid is the synthetic necessary VITAMIN of biological nucleic acid, especially to proliferative cell.The cell traffic of folic acid is by two kinds of transmembrane proteins reduced form folate carrier that is low-affinity and the folacin receptor (FR) of high-affinity.Confirmed FR in the kinds of tumor cells overexpression, when the folic acid intake was limited, the ability of tumour cell picked-up folic acid was much better than.And the expression of FR in the newborn healthy tissues of majority only limits to some and is difficult to enter sanguimotor epithelial cell teleblem.Just because of the characteristic that FR expresses, such as having high-affinity (Kd=10
-10MolL
-1), reduced immunogenicity is easy to modify, volume little (Mr=441.4), and height chemical stability and biological stability, with the physiological compatibility of organic solvent, advantages such as low cost are developed rapidly the research of folate-mediated targeted tumour.The chemical name of folic acid is N-(4-((2-amino-4-oxo-1; 4-dihydro-6-pteridine) benzoyl methylamino-))-L-L-glutamic acid; contain two carboxyls of α and γ, experimental results show that the conjugate of deriving by the γ-carboxyl of folic acid has the binding ability higher with folacin receptor.
Folic acid is dissolved in an amount of anhydrous dimethyl sulphoxide (DMSO), after adding the activation of 1-ethyl-3-(3-dimethyl propylamine) carbodiimide hydrochloride (EDCI) and N-hydroxy-succinamide (NHS), splash into and be dissolved in the beta-cyclodextrin that has connected carboxyl among the anhydrous DMSO and at room temperature reacted 24 ~ 36 hours, can obtain folic acid-beta-cyclodextrin coupled product.
Coupling the cyclodextrin of folic acid have good water-solubility, utilize it to come the quantum dot of near infrared emission is modified, not only can improve the water-soluble of quantum dot, increase the consistency with organism, and because folic acid overexpression in kinds of tumor cells, the characteristic of quantum dot near infrared emission can be avoided UV-light to the damage of organism, and can be deep into organism inside, uses so the quantum dot after modifying can be used as the probe of tumour cell.
Summary of the invention
Purpose of the present invention: the hypotoxicity functional quantum point that the carboxylated method beta-cyclodextrin of the present invention is modified is in order to improve the biocompatibility of quantum dot, to reduce its bio-toxicity, improving the sensitivity that it detects tumor markers as near infrared fluorescent probe simultaneously.
Technical scheme of the present invention: the hypotoxicity functional quantum point that a kind of carboxylated method beta-cyclodextrin is modified, connected by chemical process by beta-cyclodextrin and to go up carboxyl, more further with folacin coupled formation folic acid-beta-cyclodextrin; Described quantum dot is the hypotoxicity functional quantum point, and the element of forming the hypotoxicity functional quantum point is silver, zinc and indium; This quantum dot is modified by folic acid-beta-cyclodextrin and is obtained the hypotoxicity functional quantum point that beta-cyclodextrin is modified;
The hypotoxicity functional quantum point that this beta-cyclodextrin is modified consist of Yin Li ︰ Yin Li ︰ Xin Li ︰ NaS
2CN (C
2H
5)
2The mol ratio of ︰ folic acid-beta-cyclodextrin is 0.9-0.95 ︰ 0.9-0.95 ︰ 0.1-0.2 ︰ 3.5-4.5 ︰ 4-6.
The preparation method of the hypotoxicity functional quantum point that described carboxylated method beta-cyclodextrin is modified is characterized in that step is as follows:
(1) preparation of carboxyl-beta-cyclodextrin
The beta-cyclodextrin and the 55-65mL deionized water that add 4.5-5.5mmol in container add 8-10mmol KOH beta-cyclodextrin are all dissolved under the magnetic agitation situation.Be warming up to 45-55 ℃ gradually, add the carboxylated reagent of 9-11mmol, be warming up to 85-95 ℃ and kept 1 hour under the continuous stirring condition, stop heating then; After the question response system is cooled to room temperature, pH with dilute sulphuric acid conditioned reaction system is 5-6, add the 100-200mL dehydrated alcohol, utilize the neutral alumina post to carry out the purifying chromatography, adopt 55%-65% ethanol as eluent, collect elutriant, concentrate eluant final vacuum drying obtains carboxyl-beta-cyclodextrin;
(2) coupling of carboxyl-beta-cyclodextrin and folic acid prepares folic acid-beta-cyclodextrin
The activation of a, folic acid: add 4.5-5.5mmol folic acid and be dissolved in the 11-13mL anhydrous dimethyl sulphoxide adding 5.5-6.5mmol 1-ethyl-3-(3-dimethyl propylamine under continuous agitation condition in container) carbodiimide hydrochloride and 5.5-6.5mmol N-hydroxy-succinamide priming reaction are 4 hours;
Folic acid after the activation is added drop-wise in the anhydrous dimethyl sulphoxide solution of the two amino polyoxyethylene glycol that contain 180-220mg, and reaction is 10 hours under the lucifuge condition, by the method purifying of silica gel column chromatography, obtains folic acid-polyethylene glycol conjugation thing then;
The coupling of b, folic acid-polyethylene glycol conjugation thing and carboxymethyl-beta-cyclodextrin
Carboxyl-the beta-cyclodextrin that in 45-55mL anhydrous dimethyl sulphoxide solution, adds 1.8-2.2 mmol step (1) preparation, dissolving back adding 1.8-2.2mmol 1-ethyl-3-(3-dimethyl propylamine) carbodiimide hydrochloride and 1.8-2.2mmol N-hydroxy-succinamide priming reaction are 6 hours, then activation solution is added drop-wise in the above-mentioned damping fluid that contains folic acid-polyethylene glycol conjugation thing, reaction is 24 hours under the lucifuge condition; The crude product of reaction obtains folic acid-beta-cyclodextrin by the method purifying of silica gel column chromatography;
(3) preparation of quantum dot
The preparation of a, presoma: with 0.9-0.95mmol AgNO
3, 0.9-0.95mmol In (NO
3)
3, 0.1-0.2mmol Zn (NO
3)
2Be dissolved in the deionized water of 3-5mL, again with 3.5-4.5mmol NaS
2CN (C
2H
5)
2Be dissolved in the 2.5-7.5 deionized water solution, magnetic agitation 20-40min removed moisture wherein after both mixed, and used deionized water and the 8-12mL methanol wash of 10-14mL successively, obtained quantum dot presoma N,N-Diethyldithiocarbamic Acid;
The preparation of b, oil soluble quantum dot: under the nitrogen protection condition, step a gained quantum dot presoma N,N-Diethyldithiocarbamic Acid slowly is heated to 170-180 ℃, and keep this temperature 40-60min, then to wherein adding the 2-4mL oleyl amine, continue to stop heating behind the heating 2-4min, and be cooled to room temperature gradually;
The purification process of c, quantum dot: step b gained oil soluble quantum dot at the centrifugal 2-5min of 500-2500r/min, is got supernatant liquor, to wherein adding 8-12mL methyl alcohol, obtain the quantum dot of purifying behind the stirring 1-5min with the vacuum filtration mode;
(4) functional modification of quantum dot
The purifying quantum dot of getting step (3) gained is dissolved in the 1.5-8.5mL chloroform soln, step (2) gained folic acid-beta-cyclodextrin is dissolved in the 1.5-6.5mL deionized water, mix the back and stir 3-10min, 500-2500r/min is centrifugal, and the water intaking layer namely gets the hypotoxicity functional quantum point that the carboxylated method beta-cyclodextrin of product is modified.
Described carboxylated reagent is the 2-sodium chloroacetate, 3-chloropropionic acid sodium, 3-bromo-propionic acid sodium, 4-chloro-butyric acid sodium, 4-bromo-butyric acid sodium.
The application of the hypotoxicity functional quantum point that described carboxylated method beta-cyclodextrin is modified, described quantum dot detects as the specificity fluorescent that probe is used for tumor markers.
Beneficial effect of the present invention: the present invention utilizes silver, hypotoxicity elements such as zinc are as raw material, prepare oil-soluble near-infrared quantum dots, and with the beta-cyclodextrin of coupling folic acid it is carried out water miscible modification, obtain the hypotoxicity functional quantum point that beta-cyclodextrin is modified.This functional quantum point has good water-solubility, and hypotoxicity, emmission spectrum be in the near-infrared region, and since coupling folic acid, the specificity fluorescent that can be used for tumour detects.
Description of drawings
The quantum dot that the carboxylated method beta-cyclodextrin of Fig. 1 is modified is to the mark image of folacin receptor positive tumor cell.
The quantum dot that the carboxylated method beta-cyclodextrin of Fig. 2 is modified is to the mark image of the negative tumour cell of folacin receptor.
Embodiment
The following embodiment that provides only illustrates for the present invention is made further, and has no intention specification sheets is made any restriction.
Embodiment 1
(1) preparation of carboxymethyl-beta-cyclodextrin
In three mouthfuls of round-bottomed flasks of 250mL, add the beta-cyclodextrin of 5mmol, add the 60mL deionized water, under the magnetic agitation situation, add 9mmol KOH so that beta-cyclodextrin all dissolves.Be warming up to 50 ℃ gradually, add 10mmol 2-sodium chloroacetate, be warming up to 90 ℃ and kept 1 hour under the continuous stirring condition, stop heating then.After the question response system is cooled to room temperature, be 5-6 with the pH of dilute sulphuric acid conditioned reaction system, add the 150mL dehydrated alcohol, utilize the neutral alumina post to carry out the purifying chromatography, 60% ethanol is collected elutriant as eluent, concentrate eluant final vacuum drying obtains carboxymethyl-beta-cyclodextrin.
(2) coupling of carboxymethyl-beta-cyclodextrin and folic acid prepares folic acid-beta-cyclodextrin
The activation of a, folic acid: add 5mmol folic acid and be dissolved among the anhydrous DMSO of 12mL adding 6mmol 1-ethyl-3-(3-dimethyl propylamine under continuous agitation condition in the there-necked flask of dried and clean) carbodiimide hydrochloride (EDCI) and 6mmol N-hydroxy-succinamide (NHS) priming reaction are 4 hours.
Folic acid after the activation is added drop-wise to the two amino polyoxyethylene glycol (PEG-bis-Amine that contains 200mg, molecular weight is 4000) anhydrous DMSO solution in, reaction is 10 hours under the lucifuge condition, by the method purifying of silica gel column chromatography, obtains folic acid-polyethylene glycol conjugation thing then.
The coupling of b, folic acid-polyethylene glycol conjugation thing and carboxymethyl-beta-cyclodextrin
The carboxymethyl-beta-cyclodextrin that in the anhydrous DMSO solution of 50mL, adds the preparation of 2 mmol steps (1), dissolving back adding 2mmol 1-ethyl-3(3-dimethyl propylamine) carbodiimide hydrochloride (EDCI) and 2mmol N-hydroxy-succinamide (NHS) priming reaction are 6 hours, then activation solution is added drop-wise in the above-mentioned damping fluid that contains folic acid-polyethylene glycol conjugation thing, reaction is 24 hours under the lucifuge condition.The crude product of reaction obtains folic acid-beta-cyclodextrin by the method purifying of silica gel column chromatography.
(3) preparation of purifying quantum dot
The preparation of a, presoma: with 0.9mmol AgNO
3, 0.9mmol In (NO
3)
3, 0.2mmol Zn (NO
3)
2Deionized water solution and 4mmol NaS
2CN (C
2H
5)
2Deionized water solution mix, remove moisture wherein behind the magnetic agitation 30min, and use deionized water and the 10mL methanol wash of 12mL respectively, obtain quantum dot presoma N,N-Diethyldithiocarbamic Acid.
The preparation of b, oil soluble quantum dot: the quantum dot presoma N,N-Diethyldithiocarbamic Acid with preparation under the nitrogen protection condition slowly is heated to 180 ℃; and keep this temperature 45min; to wherein adding the 3mL oleyl amine, continue to stop heating behind the heating 3min, and be cooled to room temperature gradually then.
The purification process of c, quantum dot: with step b gained oil soluble quantum dot centrifuging and taking supernatant liquor, add 10mL methyl alcohol therein, remove by filter the large particulate matter of generation then, obtain the purifying quantum dot.
(4) functional modification of quantum dot
The purifying quantum dot of getting step (3) gained is dissolved in the 2mL chloroform soln, stir certain hour with the aqueous solution 2mL of step (2) gained folic acid-beta-cyclodextrin conjugate, centrifugal, the water intaking layer namely gets the hypotoxicity functional quantum point that the carboxylated method beta-cyclodextrin of product is modified.
Embodiment 2
(1) preparation of propyloic-beta-cyclodextrin
In three mouthfuls of round-bottomed flasks of 250mL, add the beta-cyclodextrin of 5mmol, add the 60mL deionized water, under the magnetic agitation situation, add 9mmol KOH so that beta-cyclodextrin all dissolves.Be warming up to 50 ℃ gradually, add 10mmol 3-chloropropionic acid sodium, be warming up to 90 ℃ and kept 1 hour under the continuous stirring condition, stop heating then.After the question response system is cooled to room temperature, be 5-6 with the pH of dilute sulphuric acid conditioned reaction system, add the 150mL dehydrated alcohol, utilize the neutral alumina post to carry out the purifying chromatography, 60% ethanol is collected elutriant as eluent, concentrate eluant final vacuum drying obtains propyloic-beta-cyclodextrin.
(2) coupling of propyloic-beta-cyclodextrin and folic acid prepares folic acid-beta-cyclodextrin
The activation of a, folic acid: add 5mmol folic acid and be dissolved among the anhydrous DMSO of 12mL adding 6mmol 1-ethyl-3-(3-dimethyl propylamine under continuous agitation condition in the there-necked flask of dried and clean) carbodiimide hydrochloride (EDCI) and 6mmol N-hydroxy-succinamide (NHS) priming reaction are 4 hours.
Folic acid after the activation is added drop-wise to the two amino polyoxyethylene glycol (PEG-bis-Amine that contains 200mg, molecular weight is 4000) anhydrous DMSO solution in, reaction is 10 hours under the lucifuge condition, by the method purifying of silica gel column chromatography, obtains folacin coupled polyoxyethylene glycol then.
B, folacin coupled polyoxyethylene glycol and the coupling of propyloic-beta-cyclodextrin
Propyloic-the beta-cyclodextrin that in the anhydrous DMSO solution of 50mL, adds the preparation of 2 mmol steps (1), dissolving back adding 2mmol 1-ethyl-3-(3-dimethyl propylamine) carbodiimide hydrochloride (EDCI) and 2mmol N-hydroxy-succinamide (NHS) priming reaction are 6 hours, then activation solution is added drop-wise in the above-mentioned damping fluid that contains folate-conjugated polyethylene glycol, reaction is 24 hours under the lucifuge condition.The crude product of reaction obtains folic acid-beta-cyclodextrin by the method purifying of silica gel column chromatography.
(3) preparation of purifying quantum dot
The preparation of a, presoma: with 0.95mmol AgNO
3, 0.95mmol In (NO
3)
3, 0.1mmol Zn (NO
3)
2Deionized water solution and 4mmol NaS
2CN (C
2H
5)
2Deionized water solution mix, remove moisture wherein behind the magnetic agitation 30min, and use deionized water and the 10mL methanol wash of 12mL respectively, obtain quantum dot presoma N,N-Diethyldithiocarbamic Acid.
The preparation of b, oil soluble quantum dot: the quantum dot presoma N,N-Diethyldithiocarbamic Acid with step a preparation under the nitrogen protection condition slowly is heated to 180 ℃; and keep this temperature 40min; then to wherein adding the 3mL oleyl amine; continue to stop heating behind the heating 3min, and be cooled to room temperature gradually.
The purification process of c, quantum dot: with step b gained oil soluble quantum dot centrifuging and taking supernatant liquor, add 10mL methyl alcohol therein, remove by filter the large particulate matter of generation then, obtain the quantum dot of purifying.
(4) functional modification of quantum dot
The purifying quantum dot of getting step (3) gained is dissolved in the 8mL chloroform soln, stir certain hour with the aqueous solution 6mL of step (2) gained folic acid-beta-cyclodextrin conjugate, centrifugal, the water intaking layer namely gets the hypotoxicity functional quantum point that the carboxylated method beta-cyclodextrin of product is modified.
Embodiment 3
Folic acid-beta-cyclodextrin is modified the quantum yield test of quantum dot
Folic acid-beta-cyclodextrin is modified the fluorescence quantum yield of quantum dot and is at room temperature measured, the aqueous solution (fluorescence quantum yield is 0.98) with rhodamine B is that reference solution is as reference, measure rhodamine B and folacin coupled beta-cyclodextrin respectively and modify the integration fluorescence intensity of quantum dot identical wavelength excites under and the absorbance at this wavelength place, the formula calculating quantum yield below the substitution:
(the quantum yield of Φ-test substance; Φ
F, calThe quantum yield of-reference substance; The integral area of S-fluorescence emission spectrum; The absorbancy of A-solution under wavelength selected; The specific refractory power of n-solution; The excitation wavelength of λ ex-test substance; S
CalThe integral area of-reference substance fluorescence emission spectrum; A
CalThe absorbancy of-reference solution under selected wavelength; n
CalThe specific refractory power of-reference solution; λ
ExcalThe excitation wavelength of-reference material.）
The aqueous solution fluorescence quantum yield of reference material rhodamine B is 0.98, and the quantum yield that calculates embodiment 1 is 0.070, and adopting the quantum yield of the embodiment 1 after folacin coupled beta-cyclodextrin is modified is 0.025.The quantum yield of embodiment 2 is 0.061, and adopting the quantum yield of the embodiment 2 after folacin coupled beta-cyclodextrin is modified is 0.015.Adopting folacin coupled beta-cyclodextrin to modify the quantum yield decrease to some degree of back quantum dot, may be that the quantum dot surface imperfection increases in the modification, thereby has influenced the fluorescence property of quantum dot.
Embodiment 4
Folic acid-beta-cyclodextrin is modified quantum dot to the mark of tumour cell
Cultivate the positive HeLa Cells of folacin receptor and negative human lung carcinoma cell, had digestive transfer culture is in 96 porocyte culture plates, at 37 ℃ of cell culture incubators, 5% CO
2Cultivated 12 hours.Respectively cultured cells is taken to from incubator on the Bechtop then, slowly washes 1-2 time with the PBS that sterilized, impurity such as flush away cell debris.Modify quantum dot with the folic acid-beta-cyclodextrin of preparation among the embodiment 1 and do fluorescent mark, bovine serum albumin (BSA) is used for stablizing unlabelled folic acid-beta-cyclodextrin and modifies quantum dot.Put into the incubator incubation 1-2 hour.Wash cell 3 times with phosphoric acid buffer then, place under the near infrared imaging system and observe, as depicted in figs. 1 and 2.Because the HeLa Cells of the folacin receptor positive has a large amount of folic acid to take in, stronger fluorescent signal is arranged as can be seen, the human lung carcinoma cell of folacin receptor feminine gender does not then have tangible fluorescent signal, illustrate that folic acid-beta-cyclodextrin modification quantum dot has higher affinity to the tumour cell of overexpression folacin receptor, can specific marked tumor cell.
By above-mentioned each embodiment and specific embodiment the present invention is made an explanation, but skilled person in the art will appreciate that under the situation that does not depart from aim of the present invention and scope, can make various modifications, change and replacement to the present invention.
Claims (2)
1. the preparation method of the hypotoxicity functional quantum point modified of a carboxylated method beta-cyclodextrin, it is characterized in that the hypotoxicity functional quantum point that described carboxylated method beta-cyclodextrin is modified is: connected by chemical process by beta-cyclodextrin and go up carboxyl, more further with folacin coupled formation folic acid-beta-cyclodextrin; Described quantum dot is the hypotoxicity functional quantum point, and the element of forming the hypotoxicity functional quantum point is silver, zinc and indium; This quantum dot is modified by folic acid-beta-cyclodextrin and is obtained the hypotoxicity functional quantum point that beta-cyclodextrin is modified;
The hypotoxicity functional quantum point that this beta-cyclodextrin is modified consist of Yin Li ︰ Yin Li ︰ Xin Li ︰ NaS
2CN (C
2H
5)
2The mol ratio of ︰ folic acid-beta-cyclodextrin is 0.9-0.95 ︰ 0.9-0.95 ︰ 0.1-0.2 ︰ 3.5-4.5 ︰ 4-6; Its preparation process is as follows:
(1) preparation of carboxyl-beta-cyclodextrin
The beta-cyclodextrin and the 55-65mL deionized water that add 4.5-5.5mmol in container add 8-10mmol KOH beta-cyclodextrin are all dissolved under the magnetic agitation situation; Be warming up to 45-55 ℃ gradually, add the carboxylated reagent of 9-11mmol, be warming up to 85-95 ℃ and kept 1 hour under the continuous stirring condition, stop heating then; After the question response system is cooled to room temperature, pH with dilute sulphuric acid conditioned reaction system is 5-6, add the 100-200mL dehydrated alcohol, utilize the neutral alumina post to carry out the purifying chromatography, adopt 55%-65% ethanol as eluent, collect elutriant, concentrate eluant final vacuum drying obtains carboxyl-beta-cyclodextrin;
(2) coupling of carboxyl-beta-cyclodextrin and folic acid prepares folic acid-beta-cyclodextrin
The activation of a, folic acid: add 4.5-5.5mmol folic acid and be dissolved in the 11-13mL anhydrous dimethyl sulphoxide adding 5.5-6.5mmol 1-ethyl-3-(3-dimethyl propylamine under continuous agitation condition in container) carbodiimide hydrochloride and 5.5-6.5mmol N-hydroxy-succinamide priming reaction are 4 hours;
Folic acid after the activation is added drop-wise in the anhydrous dimethyl sulphoxide solution of the two amino polyoxyethylene glycol that contain 180-220mg, and reaction is 10 hours under the lucifuge condition, by the method purifying of silica gel column chromatography, obtains folic acid-polyethylene glycol conjugation thing then;
The coupling of b, folic acid-polyethylene glycol conjugation thing and carboxyl-beta-cyclodextrin
Carboxyl-the beta-cyclodextrin that in 45-55mL anhydrous dimethyl sulphoxide solution, adds 1.8-2.2 mmol step (1) preparation, dissolving back adding 1.8-2.2mmol 1-ethyl-3-(3-dimethyl propylamine) carbodiimide hydrochloride and 1.8-2.2mmol N-hydroxy-succinamide priming reaction are 6 hours, then activation solution is added drop-wise in the above-mentioned damping fluid that contains folic acid-polyethylene glycol conjugation thing, reaction is 24 hours under the lucifuge condition; The crude product of reaction obtains folic acid-beta-cyclodextrin by the method purifying of silica gel column chromatography;
(3) preparation of quantum dot
The preparation of a, presoma: with 0.9-0.95mmol AgNO
3, 0.9-0.95mmol In (NO
3)
3, 0.1-0.2mmol Zn (NO
3)
2Be dissolved in the deionized water of 3-5mL, again with 3.5-4.5mmol NaS
2CN (C
2H
5)
2Be dissolved in the 2.5-7.5 deionized water solution, magnetic agitation 20-40min removed moisture wherein after both mixed, and used deionized water and the 8-12mL methanol wash of 10-14mL successively, obtained quantum dot presoma N,N-Diethyldithiocarbamic Acid;
The preparation of b, oil soluble quantum dot: under the nitrogen protection condition, step a gained quantum dot presoma N,N-Diethyldithiocarbamic Acid slowly is heated to 170-180 ℃, and keep this temperature 40-60min, then to wherein adding the 2-4mL oleyl amine, continue to stop heating behind the heating 2-4min, and be cooled to room temperature gradually;
The purification process of c, quantum dot: step b gained oil soluble quantum dot at the centrifugal 2-5min of 500-2500 r/min, is got supernatant liquor, to wherein adding 8-12mL methyl alcohol, obtain the quantum dot of purifying behind the stirring 1-5min with the vacuum filtration mode;
(4) functional modification of quantum dot
The purifying quantum dot of getting step (3) gained is dissolved in the 1.5-8.5mL chloroform soln, step (2) gained folic acid-beta-cyclodextrin is dissolved in the 1.5-6.5mL deionized water, mix the back and stir 3-10min, 500-2500r/min is centrifugal, and the water intaking layer namely gets the hypotoxicity functional quantum point that the carboxylated method beta-cyclodextrin of product is modified.
2. the preparation method of the hypotoxicity functional quantum point of modifying according to the described carboxylated method beta-cyclodextrin of claim 1 is characterized in that described carboxylated reagent is the 2-sodium chloroacetate, 3-chloropropionic acid sodium, 3-bromo-propionic acid sodium, 4-chloro-butyric acid sodium, 4-bromo-butyric acid sodium.
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| CN104250552B (en) * | 2013-06-25 | 2016-06-22 | 华东理工大学 | The method of water-soluble CdS quantum dot-beta cyclodextrin clathrate prepared by supercritical carbon dioxide |
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| CN109825284B (en) * | 2019-01-23 | 2022-01-25 | 东莞理工学院 | Ethylenediamine-beta-cyclodextrin modified Mn doped ZnS quantum dot and preparation method and application thereof |
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