CN104059626B - A kind of preparation method and applications of quantum dot-labeled heme iron - Google Patents
A kind of preparation method and applications of quantum dot-labeled heme iron Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of quantum dot-labeled protohemin.(1) oil-soluble quantum dot dehydrated alcohol is washed, then it is dissolved in chloroform with modifying reagent PEG NH2, puts in flask, ultrasonic, extract chloroform with Rotary Evaporators, add B.R. buffer, sucking-off solution, first cross micron membrane filter, then ultrafiltration, obtain QDs NH2；(2) QDs NH2 with Hemin is added in PBS than 1:20 ~ 1:80 by the amount of material, it is subsequently adding bridging agent EDC, lucifuge reaction 3h on horizontal shaker, reactant liquor is moved into super filter tube ultrafiltration again, the protohemin QDs Hemin that remaining liq i.e. product is quantum dot-labeled.QDs Hemin good stability, all confirms its inside and outside mechanism of absorption that can be used for studying heme iron at body and cell experiment, solves at present conventional isotope and marks the big defect of harm.
Description
Technical field
The present invention relates to the preparation method of a kind of protohemin, the application in terms of the preparation method of a kind of quantum dot-labeled protohemin and spike heme iron absorption in vivo and in vitro thereof, belong to biological technical field.
Background technology
Ferrum is one of trace element of needed by human, and iron in human shortage can cause the disease such as anemia, amentia.Adding up according to World Health Organization (WHO), whole world Patients with iron deficiency anemia has reached 1,000,000,000 people, and this brings the biggest threat to the healthy living of the mankind.Ferrum is mainly obtained by diet, and the ferrum in food mainly divides nonheme iron and heme iron.Small intestinal is the sole site that ferrum absorbs, and body mainly reaches iron metabolism by control iron absorption in small intestine and balances, to ensure that various physiological process is normally carried out.At present, clearly, and the intestinal absorption mechanism for heme iron is not clear for the intestinal absorption mechanism of nonheme iron.Heme iron, i.e. ferrous porphyrin, be made up of with a part Fe2+ protoporphyrin, is biology iron.Heme iron derives from the Myoglobin in meat product and hemoglobin, and such as animal livers, lean meat and fish etc., therefore the bioavailability (~ 25%) of heme iron is high, and body there are about the ferrum of 2/3 and obtains by picked-up haemachrome.Estimate according to the study, although heme iron only accounts for takes in the 1/3 of total amount, but accounts for the 2/3 of total ferrum per capita from the ferrum of haemachrome.Heme iron can directly be absorbed by intestinal epithelial cell, do not produce digestive tract to stimulate, the disease that iron deficiency is caused, treat with benefit heme iron be a kind of more efficiently means, but its mechanism of absorption is unclear, iron supplement simply, is not only therapeutically effective purpose, it is also possible to cause ferrum too much to cause Other diseases.Therefore, the mechanism of absorption research of the research of ferrum mechanism of absorption, especially heme iron becomes more and more important.
For the research of heme iron mechanism of absorption, currently mainly use isotope (55Fe, 59Fe, 14C) labelling technique, but isotope can produce very major injury to human body.In order to avoid using the injury that brings of isotope, find the injury that a kind of substitute technology brings for solving isotope labelling significant.Quantum dot (quantum dots, QDs) it is a kind of novel fluorescent labeling reagent, there is the photoluminescent property of uniqueness: fluorescence emission wavelengths is controlled, fluorescence intensity is high, good light stability, resistance to photobleaching, can realize a polynary transmitting of elementary excitation etc., has been used for the targeting such as cell, live body location, immunofluorescence label, signal transduction, living imaging probe etc. in Medical Biology.Wherein, the CdSe/ZnS quantum dot of nucleocapsid structure, there is the features such as quantum yield height, stable optical performance, be widely used in biology.And for CdSe/ZnS as fluorescence labels, the mechanism of absorption of research heme iron has no report.
Summary of the invention
It is an object of the invention to provide a kind of quantum dot (QDs, CdSe/ZnS) preparation method of labelling protohemin, product (QDs-Hemin) obtained by the method, good stability in physiological conditions, all confirm at body and cell experiment, QDs-Hemin can be used for studying the inside and outside mechanism of absorption of heme iron, solves at present conventional isotope and marks the defect big to harm.
The present invention solves its technical problem and adopts the technical scheme that such:
The present invention utilizes rotary evaporation to prepare quantum dot-labeled protohemin (QDs-Hemin), when QDs-NH2 with Hemin presses the amount of material than employing 1:20 ~ 1:80, all can prepare product.Products therefrom good stability in physiological conditions, all confirms at body and cell experiment, and QDs-Hemin can be used for studying the inside and outside mechanism of absorption of heme iron.
Concrete, the preparation method of the protohemin of quantum dot (QDs, the CdSe/ZnS) labelling of the present invention comprises the steps:
(1) oil-soluble quantum dot dehydrated alcohol is washed 1 time, condition is 12000 rpm, 5 min, then with modifying reagent PEG-NH2, it is together dissolved in chloroform, put in round-bottomed flask, ultrasonic 1 ~ 2min, extracts chloroform with Rotary Evaporators, then adds B.R. buffer in round-bottomed flask, controlling pH is 8.4, sucking-off solution, first crosses the filter membrane of 0.22 micron, places into 100
Ultrafiltration in the super filter tube of KD, i.e. obtains water miscible QDs-NH2；
(2) QDs-NH2 with Hemin obtained by step (1) is added in the PBS that pH is 7.4 in the amount of material than 1:20 ~ 1:80 ratio, it is subsequently adding bridging agent EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide), lucifuge reaction 3h on horizontal shaker, again reactant liquor is moved into super filter tube, 7000 rpm, 5 min ultrafiltration, remaining liq is exactly product quantum dot-heme iron QDs-Hemin.
What the present invention obtained has the beneficial effect that:
QDs-Hemin prepared by the present invention, for studying the inside and outside mechanism of absorption of heme iron, can reveal that the heme iron mechanism of absorption at Caco-2 cell, improve iron absorption in small intestine theoretical, enriching iron metabolism theoretical, prevention, treatment and drug development for iron in human disorder relevant disease provide theoretical basis simultaneously.
Accompanying drawing explanation
The uv absorption of Fig. 1: QDs-Hemin and fluorescent emission collection of illustrative plates.
Figure 1A is respectively the uv-visible absorption spectra of QDs-NH2, Hemin and QDs-Hemin.As can be seen from FIG., the characteristic absorption of QDs-NH2 is 579 nm, the characteristic absorption of Hemin is 390 nm and 600 nm, it is that soret band absorbs and Q band absorbs respectively, occurring in that the characteristic absorption of QDs and the soret band characteristic absorption of Hemin in product QDs-Hemin, this material the most provable is the conjugate of QDs and Hemin simultaneously.Figure 1B is the fluorescence emission peak of QDs, QDs-NH2 and QDs-Hemin, owing to-NH2 and Hemin is successfully modified on oil-soluble QDs surface, so the fluorescence emission peak of corresponding product creates red shift.
The particle diameter distribution of Fig. 2: QDs-Hemin and zeta potential diagram.
The particle diameter distribution of QDs-Hemin uses dynamic light scattering (DLS) technical measurement, as shown in Figure 2 A.It can be seen that size 450 nm of QDs-Hemin, this is owing to, in product QDs-Hemin, there is-the NH2 of residual on the surface of QDs, defines hydrogen bond between different QDs surface-NH2 and-NH2, and the dynamic light scattering particle diameter causing product is bigger.
Zeta potential is the important indicator characterizing stability of dispersions.As can be seen from Figure 2B, the Zeta potential of QDs-Hemin is-28 mV, shows that the QDs-Hemin stability using rotary evaporation to prepare is preferable.
Fig. 3: the QDs-Hemin stability in pH and NaCl.
Fig. 3 A and Fig. 3 B is QDs-Hemin fluorescence intensity in the NaCl solution (0,0.05,0.15,0.45,1 M) of the B.R. buffer that pH value is 5-9 and different ionic strength.From figure, see that QDs-Hemin is not affected by pH value and ionic strength, be applicable to living things system.
Fig. 4: the QDs-Hemin absorption in Caco-2 cell.
Caco-2 cell (people's rectum cancer cell) is all much like with small intestine epithelium in morphology and biochemical property, and its model has been widely used in the research of ectotrophic material molecule intestinal absorption.QDs-Hemin is hatched respectively Caco-2 cell 20 min, 40 min, 60 min, 80 min, then in multi-functional microplate reader, carry out intracellular fluorescence intensity detection, pole is just had to significantly increase found that start fluorescence intensity from 20 min, maximum is reached to 60 min fluorescence intensities, 80 min changes are inconspicuous, illustrate 60
Min cell has completed the absorption to QDs-Hemin.
Fig. 5: QDs-Hemin in the duodenal absorption of small intestinal.
Carry out normal kunming mice after duodenum pours into QDs-Hemin 40 min, drawing materials, cooking frozen section, with the content (color depth is different, represents that constituent content is the most different) of synchrotron radiation micro-beam x-ray fluorescence spectrometry Fe and Zn element.As can be seen from Figure 5, no matter to the Fe(in section from Hemin) location (Fig. 5 A), or to Zn(from QDs) location (Fig. 5 B), obtained collection of illustrative plates is consistent, after i.e. duodenal absorption QDs-Hemin, Fe and Zn positions altogether, and this explanation QDs-Hemin can enter duodenum, and with integral form by intestinal absorption.
Detailed description of the invention
Following example are used for illustrating the present invention, but protection scope of the present invention should not be limited by the examples.
Embodiment 1 prepares quantum dot-labeled protohemin
Oil-soluble quantum dot dehydrated alcohol is washed 1 time (12000 rpm, 5 min), then it is dissolved in chloroform with modifying reagent (PEG-NH2), puts in round-bottomed flask, ultrasonic 1 ~ 2min.Extract chloroform with Rotary Evaporators, then add B.R. buffer (pH is 8.4), sucking-off solution in round-bottomed flask, first cross the filter membrane of 0.22 micron, place into ultrafiltration in the super filter tube of 100 KD, i.e. obtain water miscible QDs-NH2.
QDs-NH2 Yu Hemin is added in PBS by 1:20, is subsequently adding bridging agent EDC(1-ethyl-3-(3-dimethylamino-propyl) carbodiimide), lucifuge reaction 3h on horizontal shaker.Again reactant liquor being moved into super filter tube, 7000 rpm, 5 min ultrafiltration, remaining liq is exactly product, i.e. the conjugate QDs-Hemin of quantum dot-protohemin.
The physiological stability experiment of embodiment 2 QDs-Hemin
Prepare the B.R. buffer that a series of pH value is 5 ~ 9, QDs-Hemin is joined in the B.R. buffer of different pH value, after standing 15h, QDs-Hemin fluorescence intensity in different pH value is tested respectively with spectrofluorophotometer, excitation wavelength is 360 nm, investigates the pH value impact on QDs-Hemin fluorescence intensity.Result shows, QDs-Hemin fluorescence intensity is not affected by pH value, can be used for living things system.
Prepare the NaCl solution that a series of concentration is 0,0.05,0.15,0.45,1 M, QDs-Hemin is joined after the solution of above-mentioned different ionic strength stands 15h, QDs-Hemin fluorescence intensity in different ionic strength solution is tested respectively with spectrofluorophotometer, excitation wavelength is 360nm, investigates the ionic strength impact on QDs-Hemin fluorescence intensity.Result shows, QDs-Hemin fluorescence intensity is not affected by ionic strength, can be used for living things system.
Embodiment 3 QDs-Hemin absorption in Caco-2 cell
Caco-2 cell uses MEM culture medium, is simultaneously introduced the hyclone of 20%, the penicillin of final concentration of 100IU/ml and 100 μ g/ml streptomycins.Cell is cultivated in CO2 incubator, and condition of culture is 37 DEG C, 5%CO2,95% air, 100% relative humidity.Within every 5 ~ 6 days, pass on according to the ratio of 1:4, within every three days, change liquid once, when cell density reaches 70% ~ 80%, change minimal medium into.It is simultaneously introduced QDs-Hemin incubated cell.Hatch Caco-2 cell 20min, 40min, 60min, 80min, then sucking-off culture fluid at 37 DEG C respectively, wash three times with PBS, enter intracellular QDs-Hemin by multi-functional microplate reader detection by quantitative.Result shows, 60
During min, cell has completed the absorption to QDs-Hemin.
Embodiment 4 QDs-Hemin is in the duodenal absorption of small intestinal
The micro-anesthetized mice of lumbar injection pentobarbital sodium, it is fixed on cystosepiment, abdominal cut, start to ligature with cord at away from stomachus pyloricus 1 ~ 2 cm, again as second position of ligation at this ligation point 3 ~ 4 cm, make duodenum circle, with syringe in intestinal circle, injection QDs-Hemin expands to intestinal circle appropriateness, 40
Draw materials after min.Paraformaldehyde fixative with 4% rinses, fixing duodenum circle, 4 DEG C of preservations.Again tissue is soaked in the sucrose of 30%, carry out freeze transverse section ,-20 DEG C
Save backup.Use the content of synchrotron radiation micro-beam x-ray fluorescence method quantitative determination QDs and Hemin.It was found that after duodenal absorption QDs-Hemin, Fe and Zn positions altogether, illustrate that QDs-Hemin can enter duodenum, and with integral form by intestinal absorption.
Above example confirms: quantum dot-labeled protohemin QDs-Hemin prepared by the present invention, in physiological conditions, good stability, can be used for studying the inside and outside mechanism of absorption of heme iron, especially for the application in terms of inside and outside spike heme iron absorption.
The contraction of the chemical substance used in the present invention is following (chemical name of material):
QDs: quantum dot
CdSe/ZnS: with cadmium selenide (CdSe) as core, zinc sulfide (ZnS) is the core-shell type quantum point of shell
Hemin: protohemin
QDs-Hemin: quantum dot-protohemin conjugate
QDs-NH2: amido modified water-soluble quantum dot
PEG-NH2: amido modified Polyethylene Glycol
EDC:1-ethyl-3-(3-dimethylamino-propyl) carbodiimide
Caco-2: people's rectum cancer cell.
Claims (2)
1. a preparation method for quantum dot-labeled protohemin, is characterized in that comprising the steps:
(1) oil-soluble quantum dot dehydrated alcohol being washed 1 time, condition is 12000 rpm, 5 min, then by itself and modification reagent PEG-NH2Together it is dissolved in chloroform, puts in round-bottomed flask, ultrasonic 1 ~ 2min, extract chloroform with Rotary Evaporators, then add the B.R. buffer that pH is 8.4, sucking-off solution in round-bottomed flask, first cross the filter membrane of 0.22 micron, place into ultrafiltration in the super filter tube of 100 KD, obtain water miscible QDs-NH2；
(2) by the QDs-NH obtained by step (1)2Add in PBS that pH be 7.4 in the amount of material than 1:20 ~ 1:80 ratio with Hemin, it is subsequently adding bridging agent 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide, lucifuge reaction 3h on horizontal shaker, again reactant liquor is moved into super filter tube, 7000 rpm, 5 min ultrafiltration, remaining liq is the heme iron QDs-Hemin that product is quantum dot-labeled.
2. an application for quantum dot-labeled protohemin, is characterized in that its application that spike heme iron absorbs in vivo and in vitro.
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| CN103063828A (en) * | 2011-10-24 | 2013-04-24 | 韩焕兴 | Method for utilizing covalence coupling to prepare photon point-antibody compound |
| WO2014015334A1 (en) * | 2012-07-20 | 2014-01-23 | Brown University | System and methods for nanostructure protected delivery of treatment agent and selective release thereof |
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| CN103063828A (en) * | 2011-10-24 | 2013-04-24 | 韩焕兴 | Method for utilizing covalence coupling to prepare photon point-antibody compound |
| WO2014015334A1 (en) * | 2012-07-20 | 2014-01-23 | Brown University | System and methods for nanostructure protected delivery of treatment agent and selective release thereof |
Non-Patent Citations (3)
| Title |
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| CdSe/ZnS Quantum Dots-G-Quadruplex/Hemin Hybrids as Optical DNA Sensors and Aptasensors;Etery Sharon等;《Analytical Chemistry》;20100901;第82卷(第17期);第7073–7077页 * |
| 不同功能化量子点与细胞的非特异性作用研究;王青等;《湖南大学学报（自然科学版）》;20100515;第37卷(第5期);第60-63页 * |
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