阅读说明：本技术 一种运载亚铁离子的荧光纳米粒子的制备方法及其在制备纳米亚铁离子复合物中的应用 (It is a kind of deliver ferrous ion fluorescent nano particles preparation method and its preparation nanometer ferrous ion compound in application ) 是由 谭明乾 王南鹰 丛爽 崔国馨 于 2019-09-12 设计创作，主要内容包括：本发明公开了一种运载亚铁离子的荧光纳米粒子的制备方法,包括以下步骤：使用乙醇对烤牛肉进行超声浸提,得粗提物溶液；采用有机溶剂对所述粗提物溶液萃取脱脂,收集水相；使用G25葡聚糖凝胶柱对所述水相进行纯化,收集365nm激发下产生荧光的洗脱液,旋蒸得浓缩液；使用ODS-BP色谱柱纯化所述浓缩液,收集荧光部分,冻干获得荧光纳米粒子。本发明制备的荧光纳米粒子能够与铁结合,将所述荧光纳米粒子与亚铁离子在醋酸钠缓冲溶液混合,30～90℃加热30min,取分子量大于3000Da的溶液冻干,得纳米亚铁离子载体复合物。本发明取材容易价格低廉,容易制取,所述纳米亚铁离子载体复合物稳定性高,具有可示踪性,跨膜性。(The invention discloses a kind of preparation methods of fluorescent nano particles for delivering ferrous ion, comprising the following steps: carries out ultrasonic extraction to roast beef using ethyl alcohol, obtains crude extract solution；Degreasing is extracted to the crude extract solution using organic solvent, collects water phase；The water phase is purified using G25 sephadex column, the lower eluent for generating fluorescence of 365nm excitation is collected, rotates to obtain concentrate；Using concentrate described in ODS-BP chromatography, fluorescence part is collected, freeze-drying obtains fluorescent nano particles.Fluorescent nano particles prepared by the present invention can mix the fluorescent nano particles with ferrous ion in conjunction with iron in sodium acetate buffer, 30~90 DEG C of heating 30min, take solution of the molecular weight greater than 3000Da to be lyophilized, obtain a nanometer ferrous ion carrier complexes.Present invention materials are easy cheap, are easy to produce, and the nanometer ferrous ion carrier complexes stability is high, have traceable property, cross-film.)

1. a kind of preparation method for the fluorescent nano particles for delivering ferrous ion, which is characterized in that comprising steps of

S1, roast beef is taken, be soaked in ethyl alcohol, 40~80Hz, 10~60min of ultrasound, suction filtration takes solution, and 40~70 DEG C of rotations are steamed It is sent to the 1/8~1/10 of the liquor capacity, obtains crude extract solution；

S2, using organic solvent, the crude extract solution described in step S1 extract degreasing, collect water phase；Wherein, the organic solvent For one or both of chloroform or ethyl acetate；

S3, it is purified using G25 sephadex column chromatography water phase described in step S2, the applied sample amount and cylinder of the water phase Long-pending ratio is 1:4mL/cm³, eluted with the deionized water of 100~250 times of column volumes, collect the lower generation fluorescence of 365nm excitation Eluent, 40~70 DEG C of rotary evaporations are concentrated into the 1/8~1/10 of the eluent original volume, obtain nanoparticle concentrate；

S4, using nanoparticle concentrate described in ODS-BP chromatography step S3, ODS-BP packing material size is 10 μm, chromatography Column internal diameter is 20mm, and length 300mm, mobile phase is the methanol aqueous solution of volume fraction 5%~20%, and the nanoparticle is dense The applied sample amount of contracting liquid is 5~15mL, and flow velocity is 16~20mL/min, and elution time is 30~120min；Detector is fluorescence inspection Device is surveyed, fluorescence spectrum excitation wavelength is 320nm, launch wavelength 398nm, collects the eluent with fluorescence, obtains fluorescence and receive Rice corpuscles aqueous solution, 40~70 DEG C of rotary evaporations to the 1/8~1/10 of the fluorescent nano particles aqueous solution original volume, freezing are dry It is dry, the fluorescent nano particles of ferrous ion must be delivered.

2. delivering the preparation method of the fluorescent nano particles of ferrous ion according to claim 1, which is characterized in that step S1 The roast beef the preparation method comprises the following steps: take beef, rub into meat stuffing, be placed in 200~280 DEG C of 10~50min of baking, obtain roasting ox Meat.

3. delivering the preparation method of the fluorescent nano particles of ferrous ion according to claim 1, which is characterized in that step S1 The w/v of the roast beef and ethyl alcohol is 1:2g/mL.

4. delivering the preparation method of the fluorescent nano particles of ferrous ion according to claim 1, which is characterized in that step S2 The extraction degreasing specifically: take crude extract solution described in step S1, the organic solvent of its 3 times of volumes is added, concussion mixes, quiet It sets to layering, removes organic solvent, phase of fetching water；Coextraction 3~5 times, the organic solvent is one in chloroform or ethyl acetate Kind or two kinds.

5. delivering the preparation method of the fluorescent nano particles of ferrous ion according to claim 1, which is characterized in that step S3 When the elution, G25 sephadex column is connected with peristaltic pump, and the revolving speed of the peristaltic pump is 40~180rpm.

6. delivering the preparation method of the fluorescent nano particles of ferrous ion according to claim 1, which is characterized in that step S4 The freeze-drying specifically: -50~50 DEG C, vacuum degree is 1~50Pa, dry 10~72h.

7. the preparation method of a kind of nanometer of ferrous ion compound, which is characterized in that comprising steps of

The fluorescent nano particles for the delivery ferrous ion for taking claim 1-8 either method to prepare are mixed with ferrous ion, are added PH5,50mM sodium acetate buffer make it dissolve, and obtain mixed liquor；The fluorescence nano grain of ferrous ion is delivered in the mixed liquor Final concentration of 2~the 10mg/mL, final concentration of 1~5mM of ferrous ion of son；The mixed liquor is placed in 30~90 DEG C of heating 30min carries out ultrafiltration centrifugation using 3000Da ultra-filtration centrifuge tube, collects the solution that molecular weight is greater than 3000Da, is freezed dry It is dry, obtain a nanometer ferrous ion compound.

8. the preparation method of nanometer ferrous ion compound according to claim 7, which is characterized in that the ferrous ion is Frerrous chloride.

9. the preparation method of nanometer ferrous ion compound according to claim 7, which is characterized in that the freeze-drying tool Body are as follows: -50~50 DEG C, vacuum degree is 1~50Pa, dry 10~72h.

10. the preparation method of nanometer ferrous ion compound according to claim 7, which is characterized in that comprising steps of

S1, beef is taken, rubs into meat stuffing, is placed in 260 DEG C of baking 30min, obtains roast beef；The roast beef 500g is taken, is soaked in In the ethyl alcohol of 1L, 70Hz ultrasound 30min, suction filtration takes solution, and 65 DEG C, 80rpm rotary evaporation to 100mL obtain crude extract solution；

S2, crude extract solution described in step S1 being taken, the ethyl acetate of its 3 times of volumes is added, concussion mixes, it stands up to layering, Upper layer ethyl acetate is removed, lower layer water phase A is taken；Take lower layer's water phase A that the ethyl acetate of its 3 times of volumes is added, concussion mixes, It stands until layering, removes upper layer ethyl acetate, take lower layer's aqueous phase B；Take lower layer's aqueous phase B that the acetic acid of its 3 times of volumes is added Ethyl ester, concussion mix, and stand until layering, removes upper layer ethyl acetate, take lower layer water phase C；

S3, it is purified using G25 sephadex column chromatography water phase C described in step S2, the applied sample amount and column of the water phase C Volume ratio is 1:4mL/cm³, eluted with the deionized water of 200 times of column volumes, when elution the G25 sephadex column and wriggling Pump is connected, and the revolving speed of the peristaltic pump is 180rpm, and collecting makes the lower eluent for generating fluorescence of 365nm excitation, 65 DEG C, 80rpm Rotary evaporation is concentrated into the 1/10 of the eluent original volume, obtains nanoparticle concentrate；

S4, using nanoparticle concentrate described in ODS-BP chromatography step S3, ODS-BP packing material size is 10 μm, chromatography Column internal diameter is 20mm, and length 300mm, mobile phase is the methanol aqueous solution of volume fraction 10%, the nanoparticle concentrate Applied sample amount be 10mL, flow velocity 20mL/min, elution time 60min；Detector is fluorescence detector, fluorescence spectrum excitation Wavelength is 320nm, launch wavelength 398nm, collects the eluent with fluorescence, obtains fluorescent nano particles aqueous solution, 65 DEG C, 80rpm rotary evaporation is to the 1/10 of the fluorescent nano particles aqueous solution original volume, -20 DEG C, vacuum degree 10Pa, freeze-drying 48h must deliver the fluorescent nano particles of ferrous ion；

S5, it takes the fluorescent nano particles 500mg for delivering ferrous ion described in step S4 to mix with 0.025g frerrous chloride, is added 50ml, pH5,50mM sodium acetate buffer make it dissolve, and obtain mixed liquor；The mixed liquor is placed in 50 DEG C of heating 30min, is made With 4 DEG C of 3000Da ultra-filtration centrifuge tube, 8000rpm ultrafiltration be centrifuged 10min, collect molecular weight be greater than 3000Da solution, -20 DEG C, Vacuum degree is 10Pa, is freeze-dried 48h, obtains a nanometer ferrous ion compound.

Technical field

The present invention relates to the preparation method of carbon nanomaterial more particularly to a kind of fluorescent nano particles for delivering ferrous ion Preparation method and its preparation nanometer ferrous ion compound in application.

Background technique

Iron is a kind of important mineral element, no matter in quantity or from importance and most important micro member One of element, and important adjustment effect is played to the functional activity of body.Iron deficiency is that the most common micronutrient of the mankind lacks Weary disease affects 10%~20% world population.In most cases, women is easy to suffer from iron-deficient during pregnancy poor Blood causes fetal growth slow；Long-term iron deficiency can lead to cerebral nervous system dysplasia, have long-term influence to immune system；Sternly It is slow that weight iron deficiency may also lead to pubertal growth.Usually solve the problems, such as this ideal method be added in the diet it is a variety of rich in iron Food, and increase the iron in diet absorption.

Common supplements-iron is divided into three classes: inorganic iron: such as ferrous sulfate, although molysite iron-content is high, easy and sulphur The chemical combination such as compound, polyphenol cause food discoloration, rotten, lipid oxidation, have brassy, have serious stimulation to make in stomach and intestine With.Long-term consumption can cause nausea,vomiting,diarrhea, especially be not easy to be completely absorbed by the body.Organic Iron: such as gluconic acid Ferrous iron, although organic chalybeate is better than inorganic chalybeate, and relatively mild, Small side effects, this kind of chalybeate in terms of bioavailability It is mostly ferrous salt, stability is poor, and price is also more expensive.Polypeptide iron: such as sea cucumber ovum peptide iron, but since production stage is more, Extraction purification complex steps, it is difficult to be widely applied.

Summary of the invention

The object of the present invention is to provide the preparation methods of the fluorescent nano particles of delivery ferrous ion, simplify tradition preparation side Formula reduces production cost, realizes that process is simple, controllable.

Present invention technical solution used for the above purpose is: a kind of fluorescent nano particles delivering ferrous ion Preparation method, comprising the following steps:

S1, roast beef is taken, be soaked in ethyl alcohol, 40~80Hz, 10~60min of ultrasound, suction filtration takes solution, 40~70 DEG C of rotations Turn to be evaporated to the 1/8~1/10 of the liquor capacity, removes ethyl alcohol, obtain crude extract solution；

S2, using organic solvent, the crude extract solution described in step S1 extract degreasing, collect water phase；Wherein, described organic Solvent is one or both of chloroform or ethyl acetate；

S3, using G25 sephadex column chromatography the water phase described in step S2 purify, the applied sample amount of the water phase and Column volume ratio is 1:4mL/cm³, eluted with the deionized water of 100~250 times of column volumes, collect the lower generation blue of 365nm excitation The eluent of the part of fluorescence, 40~70 DEG C of rotary evaporations are concentrated into the 1/8~1/10 of the eluent original volume, obtain nanoparticle Sub- concentrate；

S4, nanoparticle concentrate described in ODS-BP chromatographic purification step S3 is prepared using half, ODS-BP packing material size is 10 μm, chromatography column internal diameter is 20mm, and length 300mm, mobile phase is the methanol aqueous solution of volume fraction 5%~20%, described The applied sample amount of nanoparticle concentrate is 5~15mL, and flow velocity is 16~20mL/min, and elution time is 30~120min；Detection Device is fluorescence detector, and fluorescence spectrum excitation wavelength is 320nm, launch wavelength 398nm, collects the eluent with fluorescence, Obtain high-purity fluorescent nano particles aqueous solution, 40~70 DEG C of rotary evaporations to the fluorescent nano particles aqueous solution original volume 1/8~1/10, freeze-drying, obtained powdered substance is the fluorescent nano particles for delivering ferrous ion.

Under preferred embodiment, roast beef described in step S1 the preparation method comprises the following steps: take beef, rub into meat stuffing, be placed in 200~ 280 DEG C of 10~50min of baking, obtain roast beef；The revolving speed of the rotary evaporation is 70rpm~90rpm；The roast beef and ethyl alcohol W/v be 1:2g/ml.

Under preferred embodiment, degreasing is extracted described in step S2 specifically: is taken crude extract solution described in step S1, is added its 3 times The organic solvent of volume, concussion mix, and stand up to layering, remove organic solvent, phase of fetching water；Extraction 3~5 times until organic molten Agent color is clear；The organic solvent is one or both of chloroform or ethyl acetate.

Under preferred embodiment, G25 sephadex column described in step S3 is connected with peristaltic pump, and the revolving speed of the peristaltic pump is 40 ~180rpm.

It under preferred embodiment, is freeze-dried described in step S4 specifically: -50~50 DEG C, vacuum degree is 1~50Pa, drying 10 ~72h.

It is a further object to provide the applications of the fluorescent nano particles for delivering ferrous particle, are used to prepare Nanometer ferrous ion compound；A kind of preparation method of nanometer of ferrous ion compound, comprising steps of take the delivery ferrous from The fluorescent nano particles of son are mixed with ferrous ion, and pH5,50mM sodium acetate buffer is added and makes it dissolve, obtains mixed liquor；Institute State in mixed liquor deliver ferrous ion fluorescent nano particles final concentration of 2~10mg/mL, final concentration of the 1 of ferrous ion ~5mM；The mixed liquor is placed in 30~90 DEG C of heating 30min, ultrafiltration centrifugation is carried out using 3000Da ultra-filtration centrifuge tube, collects Molecular weight is greater than the solution of 3000Da, is freeze-dried, obtained powdered substance, as nanometer ferrous ion compound.

Under preferred embodiment, the ferrous ion is frerrous chloride；The freeze-drying specifically: -50~50 DEG C, vacuum degree For 1~50Pa, dry 10~72h；The parameter of the ultrafiltration centrifugation are as follows: 4 DEG C, 8000rpm, 10min.

A kind of preparation method of nanometer of ferrous ion compound, comprising steps of

S1, beef is taken, rubs into meat stuffing, is placed in 260 DEG C of baking 30min, obtains roast beef；The roast beef 500g is taken, is soaked It steeps in the ethyl alcohol of 1L, 70Hz ultrasound 30min, suction filtration takes solution, and 65 DEG C, 80rpm rotary evaporation to 100mL remove ethyl alcohol, obtain Crude extract solution；

S2, crude extract solution described in step S1 is taken, the ethyl acetate of its 3 times of volumes is added, concussion mixes, and is then allowed to stand straight To layering, upper layer ethyl acetate is removed, lower layer water phase A is taken；Take lower layer water phase A that the ethyl acetate of its 3 times of volumes is added, concussion is mixed It is even, it stands until layering, removes upper layer ethyl acetate, take lower layer's aqueous phase B；Take lower layer's aqueous phase B that the acetic acid second of its 3 times of volumes is added Ester, concussion mix, and stand until layering, removes upper layer ethyl acetate, take lower layer water phase C；

S3, it is purified using G25 sephadex column chromatography water phase C described in step S2, the applied sample amount of the water phase It is 1:4mL/cm with column volume ratio³, with the deionized water elutions of 200 times of column volumes (when elution the G25 sephadex column and Peristaltic pump is connected, and the revolving speed of the peristaltic pump is 180rpm), it collects and excites the lower part for generating blue-fluorescence using 365nm Eluent, 65 DEG C, 80rpm rotary evaporation be concentrated into the 1/10 of the eluent original volume, obtain nanoparticle concentrate；

S4, using nanoparticle concentrate described in ODS-BP chromatography step S3, ODS-BP packing material size is 10 μm, Chromatography column internal diameter is 20mm, and length 300mm, mobile phase is the methanol aqueous solution of volume fraction 10% (v/v), the nanoparticle The applied sample amount of sub- concentrate is 10mL, flow velocity 20mL/min, elution time 60min；Detector is fluorescence detector, fluorescence Excitation of spectra wavelength is 320nm, launch wavelength 398nm, collects the eluent with fluorescence, obtains high-purity fluorescence nano grain Sub- aqueous solution, 65 DEG C, 80rpm rotary evaporation to the fluorescent nano particles aqueous solution original volume 1/10, -20 DEG C, vacuum degree For 10Pa, freeze-drying 48h, obtained powdered substance is the fluorescent nano particles for delivering ferrous ion；

S5, it takes the fluorescent nano particles 500mg for delivering ferrous ion described in step S4 to mix with 0.025g frerrous chloride, adds Enter 50ml, pH5,50mM sodium acetate buffer to make it dissolve, obtains mixed liquor；The mixed liquor is placed in 50 DEG C of heating 30min, Using 4 DEG C of 3000Da ultra-filtration centrifuge tube, 8000rpm ultrafiltration be centrifuged 10min, collect molecular weight be greater than 3000Da solution, -20 DEG C, vacuum degree 10Pa, freeze-drying 48h, obtained powdered substance, as nanometer ferrous ion compound.

The beneficial effects of the present invention are:

The nanometer ferrous ion compound of the preparation method of nanometer ferrous ion compound of the present invention, acquisition produces valence Lattice are cheap, have super-small, highly-water-soluble, high chemical stability, surface contains the characteristics of there are many functional groups, by itself and cell Co-incubation can enter cell.Fluorescent nano particles are covalently combined with ferrous ion, are securely stablized, are shown Unique advantage can be used as a kind of novel iron supplementary.

Detailed description of the invention

Fig. 1 is the TEM figure of nanometer ferrous ion compound prepared by the embodiment of the present invention 1.

Fig. 2 is the fluorescence spectra of nanometer ferrous ion compound prepared by the embodiment of the present invention 1.

Fig. 3 is the fluorescence lifetime figure of nanometer ferrous ion compound prepared by the embodiment of the present invention 1.

Fig. 4 be the embodiment of the present invention 1 prepare nanometer ferrous ion compound 488nm excitation under cell imaging figure.

Fig. 5 be the embodiment of the present invention 1 prepare nanometer ferrous ion compound 405nm excitation under cell imaging figure.

Fig. 6 is the TEM figure that the fluorescent nano particles of ferrous ion are delivered in the roast beef of the preparation of the embodiment of the present invention 2.

Fig. 7 is the particle diameter distribution that the fluorescent nano particles of ferrous ion are delivered in the roast beef of the preparation of the embodiment of the present invention 2 Figure.

Fig. 8 is the fluorescence spectrum that the fluorescent nano particles of ferrous ion are delivered in the roast beef of the preparation of the embodiment of the present invention 2 Figure.

Fig. 9 is the fluorescence lifetime that the fluorescent nano particles of ferrous ion are delivered in the roast beef of the preparation of the embodiment of the present invention 2 Figure.

Figure 10 is 3 fluorescent nano particles metal ion stability figure of the embodiment of the present invention.

Figure 11 is 3 fluorescent nano particles various concentration salt ion stability diagram of the embodiment of the present invention.

Specific embodiment

The object of the present invention is to provide a kind of preparation methods of nanometer ferrous ion carrier in Beef Pie, simplify tradition system Standby mode reduces production cost, realizes that process is simple, controllable.

Present invention technical solution used for the above purpose is: nanometer ferrous ion carrier in a kind of Beef Pie Preparation method, comprising the following steps:

A, using beef as raw material, Minced Beef is made in beef, minced beef cutlet baking is then made into, roasted beef will be passed through Cake is soaked in 15-45min of ultrasound in dehydrated alcohol, filters removal suspension precipitating, collects liquid phase, rotary evaporation removes ethyl alcohol, obtains Crude extract.

B, water is added into crude extract to redissolve, adds organic solvent extraction degreasing, extraction is until aqueous phase solution is clear repeatedly It is clear bright, collect water phase；

C, aqueous phase solution in B is crossed into G25 sephadex column chromatography, collects the biggish part of fluorescence intensity, rotary evaporation It is concentrated to get the fluorescent nano particles aqueous solution of higher degree；

D, by the aqueous solution in half preparation chromatographic purification step C, the biggish part of fluorescence intensity is collected, high-purity is obtained Fluorescent nano particles aqueous solution, again after rotary evaporation, the powdered substance being lyophilized is to deliver ferrous ion Fluorescent nano particles；

E, fluorescent nano particles powder is mixed with ferrous ion, pH 5,50mM sodium acetate buffer, which is added, keeps its molten Solution, is put into 30-90 DEG C of water bath with thermostatic control 30min, is put into 3000Da ultra-filtration centrifuge tube later, and 4 DEG C of 8000rpm are centrifuged 10 minutes, The substance that molecular weight is greater than 3000Da is collected, the powdered substance being lyophilized, as nanometer ferrous ion carrier are compound Object.

Beef used is chuck or brisket meat or sirloin or sirloin or sirloin or beef tendon meat in the step A.

It is 200-280 DEG C that temperature is baked in the step A, and cooking time is 10-50min, the matter of Beef Pie and ethyl alcohol Amount volume ratio is 1:2, and it is 60 DEG C -70 DEG C that the condition of rotary evaporation removing ethyl alcohol, which is temperature, and revolving speed is 70rpm -90rpm.

Organic solvent is one or more of n-hexane, chloroform, methylene chloride or ethyl acetate in the step B, slightly The volume ratio of extract solution and organic solvent is 1:3, and it is 60 DEG C -70 that the condition of rotary evaporation removing organic solvent, which is temperature, DEG C, revolving speed is 70rpm -90rpm.

Wriggling flow rate pump is 40-180rpm in the step C.

It during partly preparing chromatography eluant in the step D, is eluted using water and methanol, water and methanol ratio are 9:1, stream Speed is 20mL/min.

The preferred frerrous chloride of step E ferrous ions, nanoparticle and ferrous iron concentration are respectively 2-10mg/ in solution ML and 1-5mM, freeze-drying time are 96h-120h.

The fluorescent nano particles extracted from baked food are cheap, have super-small, highly-water-soluble, and high chemistry is steady Qualitative, surface contains the characteristics of there are many functional groups, it can be entered cell with cell co-culture.Fluorescent nano particles and Asia Iron ion covalently combines, and securely stablizes, molecular weight is small more than polypeptide, shows unique advantage.

With reference to embodiments, a specific embodiment of the invention is described in more details, so as to more preferably Ground understands the advantages of the solution of the present invention and its various aspects.However, specific embodiments described below and embodiment are only It is for illustrative purposes, rather than limiting the invention.