CN109897118A - The preparation method and application of the rice bran polysaccharide iron of fluorescent marker - Google Patents
The preparation method and application of the rice bran polysaccharide iron of fluorescent marker Download PDFInfo
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- CN109897118A CN109897118A CN201910170508.9A CN201910170508A CN109897118A CN 109897118 A CN109897118 A CN 109897118A CN 201910170508 A CN201910170508 A CN 201910170508A CN 109897118 A CN109897118 A CN 109897118A
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Abstract
The present invention discloses a kind of preparation method and application of the rice bran polysaccharide iron of fluorescent marker, is related to tracer technique field.The preparation method of the rice bran polysaccharide iron of the fluorescent marker includes: rice bran polysaccharide iron and rhodamine B is soluble in water, addition aprotic solvent, formation mixed solution；Mixed solution is dialysed, reservation liquid is obtained；Dehydrated alcohol is added and is retained in liquid, must be precipitated；By washing of precipitate, the rice bran polysaccharide iron of fluorescent marker is obtained after dry.The present invention can be reacted this characteristic using amino active in reducing end under neutral in rice bran polysaccharide iron molecule and rhodamine B, prepare rice bran polysaccharide iron-rhodamine B compound, i.e. the rice bran polysaccharide iron of fluorescent marker, method is simple, operability is good.And the rice bran polysaccharide iron of the fluorescent marker of preparation is applied in tracer rice bran polysaccharide iron, quick, convenient and fast method is provided for tracer rice bran polysaccharide iron, solves the problems, such as that rice bran polysaccharide iron is difficult to tracer.
Description
Technical field
The present invention relates to tracer technique field, in particular to the preparation method of the rice bran polysaccharide iron of a kind of fluorescent marker and answer
With.
Background technique
Rice bran polysaccharide iron bioactivity is furtherd investigate to the deep processing of China's agricultural resource and higher value application is reinforced, pushes rice
Application and development of the chaff polyferose in food, drug have extremely important realistic meaning.But due to big portion's rice bran polysaccharide iron
Without chromophoric group altogether in molecular structure, tracer and detection cannot be carried out by simple ultra-violet absorption spectrum or fluorescence spectrum,
Currently, mainly passing through high performance liquid chromatograph (HPLC) and differential refraction detector (RID), matter in the research of rice bran polysaccharide iron
It composes detector (MSD) or evaporative light scattering detector (ELSD) combination and analysis detection, but this kind of detecting instrument is carried out to polyferose
It is expensive, it is high and complicated for operation with liquid chromatograph used time operating cost, it seriously limits the research of rice bran polysaccharide iron and opens
The efficiency of hair.
Summary of the invention
The main object of the present invention is to propose a kind of preparation method and application of the rice bran polysaccharide iron of fluorescent marker, it is intended to be solved
The problem of being certainly difficult to tracer rice bran polysaccharide iron.
To achieve the above object, the present invention proposes a kind of preparation method of the rice bran polysaccharide iron of fluorescent marker, the fluorescence
The preparation method of the rice bran polysaccharide iron of label, comprising the following steps:
Rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed solution；
The mixed solution is dialysed, reservation liquid is obtained；
Dehydrated alcohol is added in the reservation liquid, stands, centrifugation, must precipitate；
The precipitating is successively washed with 95% ethyl alcohol, dehydrated alcohol, using the rice for obtaining fluorescent marker after freeze-drying
Chaff polyferose.
Preferably, described that rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed
In the step of closing solution, the mass ratio of the rice bran polysaccharide iron and rhodamine B is (10~1): 1.
Preferably, described that rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed
In the step of closing solution, the volume ratio of the water and aprotic solvent is 10:(1~2.5).
Preferably, described that rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed
In the step of closing solution, the aprotic solvent is pyridine or triethylamine.
Preferably, described that rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed
Close solution the step of include:
Rice bran polysaccharide iron and rhodamine B accurately are weighed, and the rice bran polysaccharide iron and rhodamine B is soluble in water, then plus
Enter aprotic solvent, is protected from light stirring, reaction 22~form mixed solution afterwards for 24 hours at room temperature.
Preferably, the described the step of mixed solution is dialysed, liquid must be retained, includes:
The mixed solution is placed in bag filter, under light protected environment, flowing water is dialysed into dialyzate without the Luo Dan that dissociates
Bright B detection；
Wherein, the bag filter is the bag filter that molecular cut off is 1000Da.
Preferably, described that dehydrated alcohol is added in the reservation liquid, it stands, centrifugation, in the step of obtaining precipitating,
The additional amount of the dehydrated alcohol and the volume ratio for retaining liquid are (2.5~4): 1；And/or
When the standing, temperature is 0~4 DEG C；And/or
The time of repose be 12~for 24 hours.
Preferably, described that rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed
Before the step of closing solution, further includes:
Into rice bran polysaccharide aqueous solution be added sodium hydroxide solution to pH be 8~9；
In the environment of pH is 8~9, in 60~70 DEG C of water-baths, liquor ferri trichloridi is added to the rice bran polysaccharide water
Reaction forms reaction solution in solution, until there is red-brown precipitation in the reaction solution；
The reaction solution is centrifuged, supernatant is taken, dehydrated alcohol is added in Xiang Suoshu supernatant, stands 12 through 0~4 DEG C
After~14h, it is centrifuged taking precipitate；
The sediment alcohol is washed, is dried, rice bran polysaccharide iron is obtained；
Wherein, the volume ratio of the dehydrated alcohol and the supernatant is (2.5~4): 1.
Moreover, it relates to which the rice bran polysaccharide iron of the fluorescent marker of above-mentioned preparation method preparation is in tracer rice bran polysaccharide
In application.
In technical solution of the present invention, amino active in reducing end under neutral in rice bran polysaccharide iron molecule and rhodamine B is utilized
Can react this characteristic, prepare rice bran polysaccharide iron-rhodamine B compound, i.e. the rice bran polysaccharide iron of fluorescent marker, side
Method is simple, operability is good.And the rice bran polysaccharide iron of the fluorescent marker has ultraviolet characteristic absorption peak at 545 ± 5nm, 575
There is apparent fluorescence intensity at ± 5nm, there is fluorescence, therefore, the present invention proposes to utilize the rice bran polysaccharide iron of fluorescent marker
This characteristic is applied on tracer rice bran polysaccharide iron, provides solution for tracer rice bran polysaccharide iron, and apply fluorescent marker
Rice bran polysaccharide iron tracer rice bran polysaccharide iron method it is quick, convenient, the detection device to be used is common and testing cost is low
It is honest and clean, it is easy to spread.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the process signal of an embodiment of the preparation method of the rice bran polysaccharide iron of fluorescent marker provided by the invention
Figure；
Fig. 2 is the process signal of another embodiment of the preparation method of the rice bran polysaccharide iron of fluorescent marker provided by the invention
Figure；
Fig. 3 is the infrared spectrogram of product and rice bran polysaccharide iron made from Examples 1 to 4 and rhodamine B；
Fig. 4 is the rice bran polysaccharide iron of fluorescent marker and the fluorescence spectra of rhodamine B made from Examples 1 to 4；
Fig. 5 be fluorescent marker made from Examples 1 to 4 rice bran polysaccharide iron and rice bran polysaccharide iron and rhodamine B it is ultraviolet
Abosrption spectrogram；
Fig. 6 is the fluorescence spectra of the rice bran polysaccharide iron of the fluorescent marker of cellular uptake in embodiment 5；
Fig. 7 is the fluorescence imaging figure that the cell of rice bran polysaccharide iron of fluorescent marker is taken in embodiment 5；
Fig. 8 is the fluorescence imaging that the cell of the rice bran polysaccharide iron of fluorescent marker and the compound of DNA is taken in embodiment 6
Figure.
The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Since in big portion's rice bran polysaccharide iron molecule structure, without chromophoric group altogether, simple ultra-violet absorption spectrum cannot be passed through
Or fluorescence spectrum carries out tracer and quantitative analysis, currently, mainly passing through high performance liquid chromatograph in the research of rice bran polysaccharide iron
(HPLC) with differential refraction detector (RID), mass detector (MSD) or evaporative light scattering detector (ELSD) combination to polysaccharide
Iron carries out analysis detection, but this kind of detecting instrument is expensive, high and complicated for operation with liquid chromatograph used time operating cost,
Seriously limit the efficiency of rice bran polysaccharide iron research and development.
In consideration of it, utilizing rice bran polysaccharide the invention proposes a kind of preparation method of the rice bran polysaccharide iron of fluorescent marker
Amino active in reducing end under neutral and rhodamine B reacts this characteristic in iron molecule, and having prepared one kind can be with fluorescence
The rice bran polysaccharide iron of the fluorescent marker of tracer.Now in conjunction with the one of the preparation method of the rice bran polysaccharide iron of fluorescent marker shown in FIG. 1
The flow diagram of embodiment is described, the preparation method of the rice bran polysaccharide iron of the fluorescent marker the following steps are included:
Step S110, rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixing
Solution.
In the present embodiment, the rice bran polysaccharide iron and the mass ratio of rhodamine B are preferably (10~1): 1；The water and
The volume ratio of aprotic solvent is preferably 10:(1~2.5)；The aprotic solvent is preferably pyridine or triethylamine.It is non-proton molten
Agent can provide alkaline environment, and promote the combination of rice bran polysaccharide iron and rhodamine B as catalyst, and additional amount is not used
More, with solvent, i.e., subject to the amount of water, the amount of water then need to only guarantee that rice bran polysaccharide iron and rhodamine B are sufficiently dissolved.
When step S110 is implemented, it should be noted that the control of every experiment condition, to improve rice bran polysaccharide iron and rhodamine B
Percentage bound, step S110 includes: accurately to weigh rice bran polysaccharide iron and rhodamine B in one embodiment of the invention,
And the rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, is protected from light stirring, reaction 22 at room temperature
~form mixed solution afterwards for 24 hours.Rhodamine B and part aprotic solvent are light sensitive, degradable, and being protected from light stirring can be improved instead
Product yield reduces by-products content.
In addition, used rice bran polysaccharide iron can be by by rice bran polysaccharide and iron ion network when step S110 is implemented
Conjunction obtains, in another embodiment of the invention, referring to Fig. 2, before i.e. step S110 further include:
Step S110a, into rice bran polysaccharide aqueous solution be added sodium hydroxide solution to pH be 8~9；
Step S110b, in the environment of pH is 8~9, in 60~70 DEG C of water-baths, liquor ferri trichloridi is added to described
Reaction forms reaction solution in rice bran polysaccharide aqueous solution, until there is red-brown precipitation in the reaction solution；
Step S110c, the reaction solution is centrifuged, takes supernatant, dehydrated alcohol is added in Xiang Suoshu supernatant, through 0
After~4 DEG C of 12~14h of standing, it is centrifuged taking precipitate；
Step S110d, the sediment alcohol washed, dried, obtain rice bran polysaccharide iron.
Wherein, the volume ratio of the dehydrated alcohol and the supernatant is (2.5~4): 1.
Under alkaline environment, rice bran polysaccharide can occur complex reaction with molysite and generate rice bran polysaccharide-iron complex, wherein
Molysite can be any one soluble inorganic salt of iron, in the present embodiment, preferably liquor ferri trichloridi.Rice bran polysaccharide-
Iron complex is insoluble in Organic Alcohol, and the dehydrated alcohol under special low temperature can be such that it precipitates simultaneously from solution using this characteristic
It separates.
Step S120, the mixed solution is dialysed, obtains reservation liquid.
In above-mentioned steps S110 after reaction, in mixed solution in addition to reaction product, there is also some small molecules
Impurity and free rhodamine B, therefore to clean, removal of impurities mode can be chromatography, UF membrane or dialysis, compare and
Speech is dialysed easy to operate and inexpensive, in the present embodiment, selects dialysis.In the specific implementation, step S120 includes: by institute
It states mixed solution to be placed in bag filter, under light protected environment, flowing water is dialysed into dialyzate without the rhodamine B detection that dissociates.Wherein,
The bag filter is preferably the bag filter that molecular cut off is 1000Da, and the rice bran polysaccharide of 1000Da is greater than with molecular cut off
Iron-rhodamine B compound, small molecular weight impurity and free rhodamine B, which then enter in dialyzate, to be discharged, therefore dialysis time is to dialyse
It is dwell time when being detected in liquid without the rhodamine B that dissociates, detection can be fluorescence detection without the method for the rhodamine B that dissociates,
If dialyzate does not occur fluorescent absorption peak, that is, illustrate in dialyzate without the rhodamine B that dissociates.
Step S130, dehydrated alcohol is added in the reservation liquid, stands, centrifugation, must precipitates.
Step S140, by it is described precipitating successively washed with 95% ethyl alcohol, dehydrated alcohol, using after freeze-drying obtain fluorescence
The rice bran polysaccharide iron of label.
In step S130, the additional amount of the dehydrated alcohol and the volume ratio for retaining liquid are (2.5~4): 1；It is described
When standing, temperature is 0~4 DEG C；The time of repose be 12~for 24 hours.
Rice bran polysaccharide iron-rhodamine B compound, i.e. the rice bran polysaccharide iron of fluorescent marker are insoluble in dehydrated alcohol, and 0~
Solubility is lower at 4 DEG C, the rice bran polysaccharide iron of fluorescent marker can be made quickly, fully to sink using the method for the alcohol precipitation under low temperature
It forms sediment, separation, washing.
Further, the invention further relates to the rice bran polysaccharide iron of the fluorescent marker of above-mentioned preparation method preparation in tracer rice bran
Application in polysaccharide.
Studies have shown that the rice bran polysaccharide iron of fluorescent marker has ultraviolet characteristic absorption peak at 545 ± 5nm, in 575 ± 5nm
There is apparent fluorescent absorption peak at place, has fluorescence.Therefore, pass through ultraviolet spectrophotometry and fluorescence detection, it is possible to authenticate glimmering
The rice bran polysaccharide iron of signal, so that it is determined that its distribution situation in the cell, the rice bran polysaccharide iron of tracer fluorescent marker, in turn
Achieve the purpose that tracer rice bran polysaccharide iron.
In practical application, can first convert rice bran polysaccharide iron to the rice bran polysaccharide iron of fluorescent marker can specifically lead to
Above-mentioned preparation method is crossed, the rice bran polysaccharide iron of fluorescent marker is prepared using rice bran polysaccharide iron as raw material, then will have been taken in glimmering
Prepare liquid is made in the cell of the compound of the rice bran polysaccharide iron of signal or the rice bran polysaccharide iron of fluorescent marker and DNA, passes through
Fluorescence detection can show the compound of the rice bran polysaccharide iron of fluorescent marker or the rice bran polysaccharide iron of fluorescent marker and DNA
Distribution situation in the cell.In the specific implementation, it can be carried out according to the following steps:
Step S210, with complete medium by the rice bran polysaccharide dissolved ferric iron of fluorescent marker at sample solution.
Step S220, when cell growth reaches 70-80% convergence degree, after pancreatin digestion process, 24 are uniformly laid on
In orifice plate, so that every hole cell number is about 5 × 104It is a, it is subsequently placed in 37 DEG C, 5%CO2Under conditions of cultivate for 24 hours.
Step S230, the above-mentioned sample solution of complete medium in above-mentioned 24 orifice plate is replaced, is placed under the same terms
Continue after cultivating a period of time.Two holes are chosen, solution therein is sucked out, with phosphate buffered saline solution rinse, then use pancreatin
Then digestion process uses complete medium cell dispersion, obtains prepare liquid.
Step S240, take above-mentioned prepare liquid in ELISA Plate, not add the hole of sample solution as blank control, setting excitation
Wavelength 540nm measures the fluorescent value at launch wavelength 575nm.
Cellular uptake amount maximum time point can also be chosen, cell climbing sheet is prepared according to above-mentioned same method, is placed in
Under the microscope, under green exciting light, the rice bran polysaccharide iron of fluorescent marker is aobvious red for fluorescence microscopy.
Certainly, other than tracer is by the rice bran polysaccharide iron of cellular uptake, the rice bran polysaccharide iron of fluorescent marker can also be answered
For distribution feelings of the tracer rice bran polysaccharide iron in other extracellular solution (such as mixed solution or reaction system solution)
Condition, that is, after converting rice bran polysaccharide iron to the rice bran polysaccharide iron of fluorescent marker, pass through fluorescence detection or uv-spectrophotometric
Method detects solution.
Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that
Following embodiment is only used to explain the present invention, is not intended to limit the present invention.
Embodiment 1
Be added dropwise into rice bran polysaccharide aqueous solution 10% sodium hydroxide solution to pH be 8, and pH be 8~9 in the environment of, in
Under 70 DEG C of water bath condition, 10% liquor ferri trichloridi is added drop-wise to reaction in above-mentioned rice bran polysaccharide aqueous solution and forms reaction solution,
Until there is red-brown precipitation in reaction solution.Reaction solution is centrifuged, the anhydrous of 3 times of supernatant volumes is added into supernatant
Ethyl alcohol after standing 14h at 0 DEG C, is centrifuged taking precipitate, sediment alcohol is washed, is dried, rice bran polysaccharide iron is obtained, spare.
It takes above-mentioned rice bran polysaccharide iron 20mg, rhodamine B 20mg to be dissolved in 10ml water, 2ml pyridine is then added, at room temperature
It is protected from light and is stirred to react, reaction forms mixed solution afterwards for 24 hours；Mixed solution is placed in the bag filter that molecular cut off is 1000Da
In, under light protected environment, flowing water is dialysed into dialyzate without after the rhodamine B detection that dissociates, and stops dialysis, and go bail for and stay liquid；By 3
The dehydrated alcohol for retaining liquid product again, which is added, to be retained in liquid, and standing 12h is protected from light at 4 DEG C, and centrifugation obtains dark red precipitate；It will be dark
After red precipitate is successively washed with 95% ethyl alcohol, dehydrated alcohol, be freeze-dried fluorescent marker rice bran polysaccharide iron.
Embodiment 2
Be added dropwise into rice bran polysaccharide aqueous solution 10% sodium hydroxide solution to pH be 9, and pH be 8~9 in the environment of, in
Under 60 DEG C of water bath condition, 10% liquor ferri trichloridi is added drop-wise to reaction in above-mentioned rice bran polysaccharide aqueous solution and forms reaction solution,
Until there is red-brown precipitation in reaction solution.Reaction solution is centrifuged, the anhydrous of 4 times of supernatant volumes is added into supernatant
Ethyl alcohol after standing 12h at 4 DEG C, is centrifuged taking precipitate, sediment alcohol is washed, is dried, rice bran polysaccharide iron is obtained, spare.
It takes above-mentioned rice bran polysaccharide iron 200mg, rhodamine B 20mg to be dissolved in 10ml water, 2.5ml pyridine is then added, in room
It is protected from light and is stirred to react under temperature, form mixed solution after reacting 22h；Mixed solution is placed in the dialysis that molecular cut off is 1000Da
In bag, under light protected environment, flowing water is dialysed into dialyzate without after the rhodamine B detection that dissociates, and stops dialysis, and go bail for and stay liquid；
The dehydrated alcohol that 2.5 times retain liquid product is added and is retained in liquid, standing is protected from light at 0 DEG C for 24 hours, centrifugation obtains dark red precipitate；
After dark red precipitate is successively washed with 95% ethyl alcohol, dehydrated alcohol, be freeze-dried fluorescent marker rice bran polysaccharide iron.
Embodiment 3
Be added dropwise into rice bran polysaccharide aqueous solution 10% sodium hydroxide solution to pH be 8.6, and pH be 8~9 in the environment of,
Under 65 DEG C of water bath conditions, 10% liquor ferri trichloridi is added drop-wise to reaction in above-mentioned rice bran polysaccharide aqueous solution and is formed and reacts molten
Liquid, until there is red-brown precipitation in reaction solution.Reaction solution is centrifuged, 2.5 times of supernatant volumes are added into supernatant
Dehydrated alcohol, after standing 13h at 2 DEG C, be centrifuged taking precipitate, sediment alcohol washed, is dried, rice bran polysaccharide iron is obtained, it is spare.
It takes above-mentioned rice bran polysaccharide iron 100mg, rhodamine B 20mg to be dissolved in 10ml water, 1ml pyridine is then added, in room temperature
Under be protected from light and be stirred to react, form mixed solution after reacting 23h；Mixed solution is placed in the bag filter that molecular cut off is 1000Da
In, under light protected environment, flowing water is dialysed into dialyzate without after the rhodamine B detection that dissociates, and stops dialysis, and go bail for and stay liquid；By 4
The dehydrated alcohol for retaining liquid product again, which is added, to be retained in liquid, and standing 16h is protected from light at 2 DEG C, and centrifugation obtains dark red precipitate；It will be dark
After red precipitate is successively washed with 95% ethyl alcohol, dehydrated alcohol, be freeze-dried fluorescent marker rice bran polysaccharide iron.
Embodiment 4
Be added dropwise into rice bran polysaccharide aqueous solution 10% sodium hydroxide solution to pH be 8, and pH be 8~9 in the environment of, in
Under 70 DEG C of water bath condition, 10% liquor ferri trichloridi is added drop-wise to reaction in above-mentioned rice bran polysaccharide aqueous solution and forms reaction solution,
Until there is red-brown precipitation in reaction solution.Reaction solution is centrifuged, the anhydrous of 3 times of supernatant volumes is added into supernatant
Ethyl alcohol after standing 12h at 4 DEG C, is centrifuged taking precipitate, sediment alcohol is washed, is dried, rice bran polysaccharide iron is obtained, spare.
It takes above-mentioned rice bran polysaccharide iron 50mg, rhodamine B 20mg to be dissolved in 10ml water, 2ml triethylamine is then added, in room temperature
Under be protected from light and be stirred to react, reaction forms mixed solution afterwards for 24 hours；Mixed solution is placed in the bag filter that molecular cut off is 1000Da
In, under light protected environment, flowing water is dialysed into dialyzate without after the rhodamine B detection that dissociates, and stops dialysis, and go bail for and stay liquid；By 3
The dehydrated alcohol for retaining liquid product again, which is added, to be retained in liquid, and standing 13h is protected from light at 4 DEG C, and centrifugation obtains dark red precipitate；It will be dark
After red precipitate is successively washed with 95% ethyl alcohol, dehydrated alcohol, be freeze-dried fluorescent marker rice bran polysaccharide iron.
Product made from Examples 1 to 4 is verified.
(1) it characterizes
Infrared detection is carried out to product made from rice bran polysaccharide iron, rhodamine B and Examples 1 to 4 respectively, records infrared figure
Spectrum is as shown in Figure 3.
From figure 3, it can be seen that the infrared signature absorption peak of rhodamine B is 1508cm-1、1409cm-1、1008cm-1, rice
All there is not infrared signature absorption peak in these wavelength in chaff polyferose, and product is in 1504cm made from Examples 1 to 4-1、
1410cm-1、1008cm-1There is infrared signature absorption peak in place, illustrates that rhodamine B has successfully been combined with rice bran polysaccharide iron,
The preparation method of the rice bran polysaccharide iron of fluorescent marker i.e. proposed by the present invention synthesizes successfully.
(2) fluorescence spectrum
Fluorescence detection is carried out to the rice bran polysaccharide iron of fluorescent marker made from rhodamine B and Examples 1 to 4 respectively, takes one
Determine concentration rhodamine B and embodiment 1-4 made from fluorescent marker rice bran polysaccharide ferrous solution, set with sepectrophotofluorometer
Excitation wavelength 540nm, measures the fluorescent value at 560-640nm, and record fluorescence spectrum is as shown in Figure 4.
Figure 4, it is seen that the rice bran polysaccharide iron of fluorescent marker made from rhodamine B and Examples 1 to 4 exists
There is maximum fluorescence intensity in the vicinity 575nm, illustrates still there is fluorescence after rhodamine B is integrated on rice bran polysaccharide iron, that is,
The rice bran polysaccharide iron of fluorescent marker can be analyzed by fluorescence detection.
(3) ultraviolet detection
Purple is carried out respectively to the rice bran polysaccharide iron of fluorescent marker made from rice bran polysaccharide iron, rhodamine B and Examples 1 to 4
Outer detection.Take the rice bran polysaccharide iron of fluorescent marker made from certain density rice bran polysaccharide iron, rhodamine B and embodiment 1-4 molten
Liquid, with the absorbance value at ultraviolet specrophotometer measurement 500-600nm.It is as shown in Figure 5 to record ultra-violet absorption spectrum.
From figure 5 it can be seen that there is apparent characteristic absorption peak at 550nm in rhodamine B, made from Examples 1 to 4
Equally there is faint characteristic absorption peak at 550nm in the rice bran polysaccharide iron of fluorescent marker, and spy does not occur in rice bran polysaccharide iron
Absorption peak is levied, illustrates that rhodamine B has successfully been combined with rice bran polysaccharide iron in the rice bran polysaccharide iron of fluorescent marker, and fluorescent marker
Rice bran polysaccharide iron can be analyzed by ultraviolet spectrophotometry.
(4) cytotoxicity detects
The fluorescent marker rice bran polysaccharide iron pair prepared in Examples 1 to 4 under same concentration is measured using thiazolyl blue (MTT) method
The influence of Hela cell survival rate.Plating density of the Hela cell in 96 orifice plates is 1x104/ hole is placed in 37 DEG C, 5%CO2Item
It is cultivated under part for 24 hours, when cell growth reaches 70-80% convergence degree, the sample that concentration is 0.5mg/mL is added into each hole respectively
100 μ L of product solution, under the same terms stimulation cell for 24 hours after, the MTT solution of 10 μ L 5mg/mL is added, continues to put under the same terms
After setting 4h, cell culture fluid is sucked, the crystal of 100 μ L dmso solution cells generation is added in each hole, is surveyed with microplate reader
Determine the absorbance value A1 at 570nm.Be not loaded sample wells cell survival rate be 100%, absorbance value A0.Cell survival rate
It is calculated by formula 1, the results are shown in Table 1.
Formula 1: cell survival rate (%)=A1/A0 × 100
The cytotoxicity of fluorescent marker rice bran polysaccharide iron obtained in 1 embodiment 1-4 of table
| Hela cell survival rate (%) | |
| Embodiment 1 | 95.6 |
| Embodiment 2 | 97.7 |
| Embodiment 3 | 96.2 |
| Embodiment 4 | 96.8 |
As can be seen from Table 1, when the fluorescent marker rice bran polysaccharide concentration of iron of each embodiment preparation is 0.5mg/mL, Hela is thin
The cell survival rate of born of the same parents is 95% or more, illustrates the cytotoxicity of the fluorescent marker rice bran polysaccharide iron prepared by the method very
It is low, it is especially advantageous for tracer and observation when rice bran polysaccharide iron carries out cell experiment to rice bran polysaccharide iron portion in the cell.
Embodiment 5
It by the fluorescent marker rice bran polysaccharide dissolved ferric iron prepared in embodiment 1-4 is 0.1mg/mL at concentration with complete medium
Sample solution.When cell growth reaches 70-80% convergence degree, with pancreatin digestion process, and equably it is laid in 24 orifice plates,
So that every hole cell number is about 5 × 104It is a, it is subsequently placed in 37 DEG C, 5%CO2Under conditions of cultivate for 24 hours.
Complete medium in above-mentioned 24 orifice plate is replaced with sample solution, is placed under the same terms and continues to cultivate.Respectively
In 1,2,3,4,6,8,10,12h, two holes are chosen, and culture solution is sucked out, with phosphate buffered saline solution rinse 3-5 times, then
Cell dissociation is handled with pancreatin, then with 500 μ L complete medium cell dispersions, prepare liquid is made.
It takes 100 μ L prepare liquids in ELISA Plate, not add the hole of sample solution measurement as blank control, sets excitation wavelength
540nm measures the fluorescent value at launch wavelength 575nm.The fluorescence intensity level for recording measurement is as shown in Figure 6.Meanwhile choosing cell
At intake maximum time point, cell climbing sheet is prepared after the same method, be placed under fluorescence microscope (× 100) and observe,
Under green exciting light, the result that Hela cell absorbs fluorescent marker rice bran polysaccharide iron is as shown in Figure 7.
As seen from Figure 6, fluorescent marker rice bran polysaccharide iron made from embodiment 1-4 can by Hela cellular uptake,
And the trend that intake gradually increases is showed as time increases, the maximum value of cellular uptake and guarantor are finally reached in 8h
It is fixed to keep steady.
Picture (Fig. 7) under fluorescence microscope shows that (white bright spot is to show fluorescence to same result in figure
Fluorescent marker rice bran polysaccharide iron), and as seen in Figure 7 fluorescent marker rice bran polysaccharide iron by main after Hela cellular uptake
In cytoplasm, illustrates that rice bran polysaccharide iron enters and be concentrated mainly on cytoplasm in cell, the amount in nucleus is less.
Embodiment 6
It by the fluorescent marker rice bran polysaccharide dissolved ferric iron prepared in embodiment 1-4 is 0.5mg/mL at concentration with complete medium
Fluorescent marker rice bran polysaccharide ferrous solution.When cell growth reaches 70-80% convergence degree, with pancreatin digestion process, uniformly
It is laid in 24 orifice plates for being placed with cell climbing sheet, so that every hole cell number is about 5 × 104It is a, it is placed in 37 DEG C, 5%CO2Under conditions of
Culture is for 24 hours.It takes the above-mentioned fluorescent marker rice bran polysaccharide ferrous solution of 10 μ L in EP pipe, 1 μ g DNA solution is added, be uniformly mixed, room
Temperature places 30min and obtains complex solution.
24 orifice plates are taken out, complete medium is replaced with complex solution, is placed under the same terms and cultivates 4h, then to each hole
Middle that the 4' that 10 μ L concentration are 100ng/mL is added, 6- diamidino -2-phenylindone (DAPI) solution continues to train under the same terms
Support 30min, be sucked out complex solution, clean cell surface 2-3 times with phosphate buffered saline solution, be placed in fluorescence microscope (×
100) it is observed under, under uv excitation light, result of the Hela cell to the compound intake of fluorescent marker rice bran polysaccharide iron and DNA
As shown in Figure 8.
From figure 8, it is seen that the nucleus of Hela cell is presented blue under uv excitation light after core dyestuff DAPI dyeing
Color, fluorescent marker rice bran polysaccharide iron form red complex (part that white circle is sketched the contours in figure) after agglomerating DNA, and main point
Cloth is around nucleus.
The above is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, for this field
For technical staff, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc. should all be included within the scope of the present invention.
Claims (9)
1. a kind of preparation method of the rice bran polysaccharide iron of fluorescent marker, which comprises the following steps:
Rice bran polysaccharide iron and rhodamine B is soluble in water, aprotic solvent is added, reaction forms mixed solution；
The mixed solution is dialysed, reservation liquid is obtained；
Dehydrated alcohol is added in the reservation liquid, stands, centrifugation, must precipitate；
The precipitating is successively washed with 95% ethyl alcohol, dehydrated alcohol, it is more using the rice bran for obtaining fluorescent marker after freeze-drying
Sugared iron.
2. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described that rice bran is more
In the step of sugared iron and rhodamine B are soluble in water, add aprotic solvent, and reaction forms mixed solution, the rice bran polysaccharide
Iron and the mass ratio of rhodamine B are (10~1): 1.
3. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described that rice bran is more
In the step of sugared iron and rhodamine B are soluble in water, add aprotic solvent, and reaction forms mixed solution, the water and non-matter
The volume ratio of sub- solvent is 10:(1~2.5).
4. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described that rice bran is more
It is described non-proton molten in the step of sugared iron and rhodamine B are soluble in water, add aprotic solvent, and reaction forms mixed solution
Agent is pyridine or triethylamine.
5. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described that rice bran is more
Sugared iron and rhodamine B are soluble in water, add aprotic solvent, react the step of forming mixed solution and include:
Rice bran polysaccharide iron and rhodamine B accurately are weighed, and the rice bran polysaccharide iron and rhodamine B is soluble in water, added non-
Proton solvent is protected from light stirring, reaction 22~form mixed solution afterwards for 24 hours at room temperature.
6. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described described to mix
Closing the step of solution dialyses, must retain liquid includes:
The mixed solution is placed in bag filter, under light protected environment, flowing water is dialysed into dialyzate without the rhodamine B inspection that dissociates
Out；
Wherein, the bag filter is the bag filter that molecular cut off is 1000Da.
7. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described by anhydrous second
In the step of alcohol is added in the reservation liquid, stands, centrifugation, obtains precipitating,
The additional amount of the dehydrated alcohol and the volume ratio for retaining liquid are (2.5~4): 1；And/or
When the standing, temperature is 0~4 DEG C；And/or
The time of repose be 12~for 24 hours.
8. the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in claim 1, which is characterized in that described that rice bran is more
Before the step of sugared iron and rhodamine B are soluble in water, add aprotic solvent, and reaction forms mixed solution, further includes:
Into rice bran polysaccharide aqueous solution be added sodium hydroxide solution to pH be 8~9；
In the environment of pH is 8~9, in 60~70 DEG C of water-baths, liquor ferri trichloridi is added to the rice bran polysaccharide aqueous solution
Middle reaction forms reaction solution, until there is red-brown precipitation in the reaction solution；
The reaction solution is centrifuged, supernatant is taken, dehydrated alcohol is added in Xiang Suoshu supernatant, through 0~4 DEG C of standing 12~
After 14h, it is centrifuged taking precipitate；
The sediment alcohol is washed, is dried, rice bran polysaccharide iron is obtained；
Wherein, the volume ratio of the dehydrated alcohol and the supernatant is (2.5~4): 1.
9. fluorescent marker prepared by the preparation method of the rice bran polysaccharide iron of fluorescent marker as described in any one of claims 1 to 8
Application of the rice bran polysaccharide iron in tracer rice bran polysaccharide iron.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0665087A (en) * | 1992-08-12 | 1994-03-08 | Kanei:Kk | Antipruritic for external use |
| CN105777925A (en) * | 2016-03-14 | 2016-07-20 | 宋逍 | Preparation method of acanthopanax obovatus Hoo polysaccharide iron and application thereof |
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2019
- 2019-03-06 CN CN201910170508.9A patent/CN109897118A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0665087A (en) * | 1992-08-12 | 1994-03-08 | Kanei:Kk | Antipruritic for external use |
| CN105777925A (en) * | 2016-03-14 | 2016-07-20 | 宋逍 | Preparation method of acanthopanax obovatus Hoo polysaccharide iron and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| XIANHU PAN: "Rice bran polysaccharide-metal complexes showed safe antioxidant activity in vitro", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 潘显虎等: "荧光标记的米糠多糖及其细胞摄入研究", 《食品研究与开发》 * |
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