CN104209071B - A kind of preparation method of biological polysaccharide polymer fluorescent microsphere - Google Patents
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Abstract
nullThe invention discloses the preparation method of a kind of biological polysaccharide polymer fluorescent microsphere，It is characterized in being dispersed in 0.1～1mL ultra-pure water the polystyrene fluorescent microsphere of 1 weight portion，Under agitation，It is added drop-wise in the diallyl dimethyl amide hydrochloride 1.0～4.0mg/mL of 3～10mL，It is centrifuged after continuously stirred 1～4h，Remove supernatant，Clean once with ultra-pure water，After centrifugal collection, vibrating dispersion is in 1～2mL ultra-pure water，It is added drop-wise under stirring in the polysaccharide polyelectrolyte macromolecular solution 1.0～6.0mg/mL of 5～50mL labellings or unmarked fluorescence molecule，It is centrifuged after continuously stirred 1～4h，After removing supernatant，Precipitation is scattered in pure water，Centrifugal collecting precipitate，Distribute it to again in ultra-pure water，Obtain polysaccharide high molecular fluorescent microballoons.This biological polysaccharide polymer fluorescent microsphere particle diameter is 1～10 μm, and fluorescence exciting wavelength is 350～800nm, has good biocompatibility and stability.
Description
Technical field:
The present invention relates to the preparation method of a kind of biological polysaccharide polymer fluorescent microsphere.Belong to field of biomedical polymer materials.
Background technology
Fluorescent microsphere can launch the organic or inorganic fluorescent material of fluorescence owing to being enriched in single microsphere, luminous intensity and
Fluorescence sensitivity is the highest, thus raw at Clinical Laboratory and diagnosis, immunoassay, blood circulation and cytology research etc.
Thing medical domain has obtained extremely being widely applied (Macromolecules 2011,44,4801；Macromol.Chem.Phys.
2005,206,2440).In the various characteristics of microsphere, the key factor affecting its application is the size of microsphere, dispersibility
And pattern, in addition, for its biocompatibility requirement, also seem particularly significant (Macromolecules 2005,38,
8300)。
In recent years, the polystyrene fluorescent microsphere synthesizing monodispersed preliminary dimension has had breakthrough progress, preparation micro-
Ball has pattern rule, uniform particle diameter, good surface respond and is prone to the advantages such as functionalization (European Polymer
Journal 2009,45,550), in fields such as immunological technique and disease early diagnosiss, there is the highest research and using value.
The preparation of polystyrene fluorescent microsphere, it is common that by physical absorption or the method for chemical bonding, embedding or surface inside microsphere
Connect fluorescein molecule.It is a kind of method simple, direct that the method utilizing physical absorption prepares fluorescent microsphere, but physics is inhaled
The enrichment on microsphere of the attached fluorescence molecule is unstable, and fluorescein adsorption efficiency is low.Chemical bonding prepares fluorescent microsphere phase
For the method (Journal of Colloid and Interface Science 2011,363,137) of physical absorption, have an advantage in that
Fluorescence molecule is the surface by being covalently bonded to microsphere, and its physicochemical properties are the most more stable, are difficult to from microsphere surface
Dissociate.But the fluorescein molecule of part instability is exposed at microsphere surface, is easily affected by the surrounding environment.It addition, polyphenyl
Ethylene microsphere surface lacks the capsulating material of one layer of biocompatibility.
Summary of the invention
The present invention seeks to provide the preparation method of a kind of high molecular fluorescent microballoons for prior art deficiency, its feature
It is electronegative with surface and marked the polystyrene microsphere of fluorescence molecule as template, by self-assembling technique, will be with on the contrary
Saccharide polyelectrolyte (such as dextran sulfate, hyaluronic acid, the alginic acid) continuous precipitation of electric charge in microsphere surface charged layer,
It is self-assembled into functional microsphere, is prepared as the fluorescent microsphere of polysaccharide polymer parcel.This fluorescent microsphere is stably dispersed in aqueous phase,
Optical property is stable, and has good biocompatibility, and does not reacts with conventional polymer, nor affects on antibiosis
Element titer, the great advantage of this method is the macromole (such as antibody) of various difference in functionalitys can be attached to assembled layers,
The microsphere prepared has good application prospect in terms of flow cytometer and cell marking.
The object of the invention is realized by techniques below measure, and wherein said raw material number, in addition to specified otherwise, is parts by weight.
The preparation method of high molecular fluorescent microballoons comprises the following steps:
1) preparation of surface carboxyl groups polystyrene microsphere
(1) by initiator 0.04～0.06 weight portion, polyvinylpyrrolidone 0.04～0.3 weight portion, polyethyleneglycol octyl group
Phenyl ether 0.05～0.1 weight portion and styrene 1 weight portion are dissolved in dehydrated alcohol: water=100: the mixing of 0～25 volume ratios is molten
Liquid 4～8 parts, add in the reactor with agitator, thermometer and reflux condensate device, and stirring is mixed evenly, to instead
Answer system to lead to nitrogen deoxidation treatment, then aforesaid reaction vessel is placed in oil bath, be heated to 40～80 DEG C of reactions 1～2h；
(2) styrene 1 weight portion and allyl acid 0.02～0.06 weight portion are dissolved in dehydrated alcohol: water=100: 0～25 volumes
The mixed solution 4 of ratio～8 parts, add in another reactor, and reaction system leads to nitrogen deoxidation treatment, and be heated to 40～
80℃；Reaction 15～90min, adds in aforesaid reaction vessel by styrene and the allyl acid solution of heat, continues reaction 10～24
H, is removed by centrifugation unreacted monomer, and is again dispersed in the phosphate buffer solution of pH=6～8.4, it is thus achieved that surface
Carboxylic polystyrene microsphere；
2) preparation of high molecular fluorescent microballoons
(1) preparation of fluorescent microsphere
The surface carboxyl groups polystyrene microsphere solution of 1 weight portion is placed in reactor, adds and Surfaces of Polystyrene Microparticles
Carboxyl molar equivalent is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and the Surfaces of Polystyrene Microparticles of 1～10
Carboxyl molar equivalent is the N-hydroxysuccinimide of 1～10, is stirred at room temperature 10～60min, will be containing amino-functional
Fluorescence molecule 0.001～0.01 molar equivalent of group adds reaction system, and continues stirring reaction 6～24h, through bag filter
(MWO=6000～8000) dialysis removes salt and unreacted fluorescence molecule, it is thus achieved that fluorescent microsphere；
(2) the high molecular microsphere of surface modified polysaccharide is prepared by LBL self-assembly
The polystyrene microsphere of 1 weight portion is dispersed in 0.1～1mL ultra-pure water, under agitation, be added drop-wise to concentration be 1.0～
4.0mg/mL, volume is in the diallyl dimethyl amide hydrochloride of 3～10mL, continuously stirred 1～4h, from
The heart removes supernatant, cleans once with ultra-pure water, centrifugal collecting precipitation, is scattered in 1～2mL ultra-pure water, under stirring again
Being added drop-wise to concentration is 1.0～6.0mg/mL, and volume is that the polysaccharide of 5～50mL labellings or unmarked fluorescence molecule is birdsed of the same feather flock together electrolysis
In matter macromolecular solution, continuously stirred 1～4h, centrifugal segregation supernatant, precipitation is redispersed in pure water, centrifugal collection
Precipitate, is then distributed to precipitate in ultra-pure water, it is thus achieved that the high molecular microsphere of surface modified polysaccharide.
Described initiator is any one in AMBN, azodiisobutyronitrile or potassium peroxydisulfate.
The fluorescence molecule of described amido functional group is fluorescein, rhodamine, any one in Cy5, launches the scope of wavelength
Contain 350～800nm.
It is template that described two step dispersion copolymerization methods prepare carboxylated polystyrene microsphere, and mean diameter is 1～10 μm.
The high molecular microsphere of surface modified polysaccharide that high molecular fluorescent microballoons preparation method prepares.
Modified polysaccharide high molecular microsphere in surface is used for flow cytometer and cell marking.
Structural characterization and performance test:
1. use scanning electron microscope that pattern, the granularmetric analysis of surface carboxyl groups polyethylene microsphere are tested, the most as shown in Figure 1,
Result shows: the surface carboxyl groups polyethylene microsphere pattern rule of preparation, and in single dispersion；Its uniform particle diameter, point
Cloth is narrower.
2. use spectrofluorophotometer that the fluorescence spectrum of the polystyrene fluorescent microsphere of surface FITC and Cy5 functionalization is divided
Analysis test, the most as shown in Figure 3,
Result shows: the polystyrene fluorescent microsphere of surface FITC and Cy5 functionalization is respectively at maximum excitation/transmitting wavelength
There is at 488/525nm and 633/670nm stronger relative intensity of fluorescence.
3. use laser co-focusing that the fluorescence distribution analysis of the polystyrene fluorescent microsphere of surface FITC and Cy5 functionalization is surveyed
Examination, the most as shown in Figure 4,
Result shows: the fluorescence distribution of the polystyrene fluorescent microsphere of surface FITC and Cy5 functionalization at microsphere surface, this
It is primarily due to fluorescence molecule by the way of chemical bonding, is attached to Surfaces of Polystyrene Microparticles.
4. the change of the current potential of the high molecular ball of surface modified polysaccharide that LBL self-assembly is prepared by employing Malvern nano particle size instrument
Fractional analysis is tested, the most as shown in Figure 5,
Result shows: the success of LBL self-assembly, when the electrolyte diallyl dimethyl amine of positively charged is inhaled as ground floor
When being attached to originally on electronegative polystyrene microsphere, the current potential on its surface has become about+38mV, the second strata cloudy from
What the assembling of sub-dextran sulfate made surface charge becomes-46mV.
5. use the flow cytometer being equipped with 488nm argon ion laser glimmering to the polystyrene of surface FITC functionalization
The applied analysis test of light microsphere, the most as shown in Figure 6,
Result shows: the polystyrene fluorescent microsphere of surface FITC functionalization is compared to polystyrene microsphere, and its positive is
100%, its fluorescence intensity can meet the flow cytometer requirement to it.
Present invention have the advantage that
1. the present invention is simple to operate, regulates and controls ball size according to raw material rate of charge, and microsphere thickness of the shell and component are led to easily
Number of times and the material therefor of crossing change self assembly circulation control, and meanwhile, the size and shape of shell is by the yardstick of core used in advance
Determine.
2. the high molecular fluorescent microballoons that prepared by the present invention, particle size distribution is homogeneous, have good surface respond, it is easy to merit
Energyization；
3. the polysaccharide high molecular fluorescent microballoons that prepared by the present invention, relatively polystyrene microsphere has more preferable biocompatibility；
4. the polysaccharide high molecular fluorescent microballoons that prepared by the present invention, can realize multiple fluorescence-encoded, and the scope launching wavelength contains
350～800nm.Fluorescent microsphere has good optical property, is widely used in the analysis of flow cytometer；
5. the polysaccharide high molecular fluorescent microballoons that prepared by the present invention, can protect the fluorescein molecule of instability, from surrounding
Impact.
Accompanying drawing illustrates:
Fig. 1 (a) is the scanning electron microscope of the polystyrene microsphere of the surface carboxyl groups of 1.1 μm particle diameters,
Fig. 1 (b) is the scanning electron microscope of the polystyrene microsphere of the surface carboxyl groups of 2.1 μm particle diameters
Fig. 1 (c) is the scanning electron microscope of the polystyrene microsphere of the surface carboxyl groups of 2.6 μm particle diameters
Fig. 1 (d) is the scanning electron microscope of the polystyrene microsphere of the surface carboxyl groups of 3.6 μm particle diameters
Fig. 2 is the self assembly schematic diagram with polystyrene microsphere as template.
Fig. 3 (a) is the fluorescence spectrum figure of the polystyrene fluorescent microsphere of surface FITC functionalization.
Fig. 3 (b) is the fluorescence spectrum figure of the polystyrene fluorescent microsphere of surface C y5 functionalization.
Fig. 4 (a) is the laser co-focusing figure of the polystyrene fluorescent microsphere of surface FITC functionalization.
Fig. 4 (b) is the laser co-focusing figure of the polystyrene fluorescent microsphere of surface C y5 functionalization.
Fig. 5 is the polystyrene microsphere change at LBL self-assembly polyelectrolyte macromolecule after-potential.
Fig. 6 is the flow cytometer showed figure of the polystyrene fluorescent microsphere of surface FITC functionalization.
Specific implementation method:
By the following examples the present invention is specifically described, it is necessary to it is pointed out here that be that the present embodiment is served only for this
Bright it is further described, it is impossible to being interpreted as limiting the scope of the invention, the person skilled in the art in this field can root
Some nonessential improvement and adjustment are made according to present disclosure.
Embodiment 1
1. the preparation of the polystyrene microsphere of surface carboxyl groups
Two step dispersion copolymerization methods are used to prepare the polystyrene microsphere of surface carboxyl groups.First by initiator AMBN
(AMBN) 0.12g, stabilizer polyvinylpyrrolidone PVP360 (Mw=360000g/mol) 0.135g, help
Stabilizer T X3050.15g, and styrene monomer 3.0g is dissolved in dehydrated alcohol 16.15mL and 0.85mL ultra-pure water mixes
In solution, adding in the reactor with agitator, thermometer and reflux condensate device, stirring is mixed evenly, to reactant
The logical nitrogen deoxidation treatment of system, is then placed in aforesaid reaction vessel in oil bath, is heated to 60 DEG C of reaction 1h；By styrene 3g
In the mixed solution of the ultra-pure water being dissolved in dehydrated alcohol 7.6mL and 0.4mL with allyl acid 0.06g, add another reactor
In, reaction system is led to nitrogen deoxidation treatment, and is heated to 60 DEG C；Reaction 60min, by molten to styrene and the allyl acid of heat
Liquid adds in aforesaid reaction vessel, continues reaction 18h, removes unreacted monomer by centrifugal (3000rpm, 20min),
And be again dispersed in the phosphate buffer solution of pH=7.4, it is thus achieved that surface carboxyl groups polystyrene microsphere；Pass through scanning electron microscope
Observing, its size is 1.1 ± 0.1 μm, and is distributed homogeneous, as shown in Fig. 1 (a)；
2. prepared by the labelling of polystyrene fluorescent microsphere
Measure the polystyrene microsphere solution (60mg/mL) of 5mL surface carboxyl groups in reactor, add 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) 0.54mg and N-hydroxysuccinimide (NHS) 1.62
Mg, at ambient temperature after stirring 15min, the rhodamine 0.3mg that end contains amino functional group adds reaction system,
Stirring reaction 10h under room temperature condition.Salt and unreacted fluorescence is removed through bag filter (MWO=6000～8000) dialysis
Molecule, it is thus achieved that fluorescent microsphere；
3. modify surface characteristics of microspheres by layer-by-layer
The schematic diagram of LBL self-assembly such as Fig. 2.5mg polystyrene microsphere is dispersed in 0.1mL ultra-pure water, at magnetic force
Under stirring condition, it is added drop-wise in the diallyl dimethyl amide hydrochloride (PDDA, 2.0mg/mL) of 3mL,
Continuously stirred 2h, centrifugal segregation supernatant, cleans once with ultra-pure water, centrifugal collecting precipitation, is scattered in 2mL ultra-pure water
In, it is added drop-wise in the dextran sulphate solution (4.0mg/mL) of 15mL labelling or unmarked fluorescence molecule many under stirring again
Saccharide polyelectrolyte macromolecular solution, continuously stirred 2h, centrifugal segregation supernatant, precipitation is redispersed in pure water, from
The heart collects precipitate, is then distributed in ultra-pure water by precipitate, it is thus achieved that the high molecular microsphere of surface modified polysaccharide.
Embodiment 2
1. the preparation of the polystyrene microsphere of surface carboxyl groups
Two step dispersion copolymerization methods are used to prepare the polystyrene microsphere of surface carboxyl groups.First by initiator AMBN
(AMBN) 0.175g, stabilizer polyvinylpyrrolidone PVP55 (Mw=55000g/mol) 0.7g, help stable
Agent TX3050.245g, and styrene monomer 3.125g is dissolved in dehydrated alcohol 13mL, adds with agitator, temperature
In the reactor of meter and reflux condensate device, stirring is mixed evenly, and reaction system is led to nitrogen deoxidation treatment, then by above-mentioned
Reactor is placed in oil bath, is heated to 60 DEG C of reaction 1h；Styrene 3.125g and allyl acid 0.125g is dissolved in anhydrous
In ethanol 13mL, add in another reactor, reaction system is led to nitrogen deoxidation treatment, and is heated to 60 DEG C；Reaction
90min, adds in aforesaid reaction vessel by styrene and the allyl acid solution of heat, continues reaction 18h, after completion of the reaction, logical
Cross centrifugal (2000rpm, 20min) and remove unreacted monomer, and be again dispersed in buffer solution.By scanning electricity
Sem observation, its size is 2.1 ± 0.2 μm, and is distributed homogeneous, as shown in Fig. 1 (b).
2. prepared by the labelling of polystyrene fluorescent microsphere
Measure the polystyrene microsphere solution (60mg/mL) of 5mL surface carboxyl groups in reactor, add 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) 6.54mg and N-hydroxysuccinimide (NHS) 11.56
Mg, at ambient temperature after stirring 15min, the Cy50.3mg that end contains amino functional group adds reaction system,
Stirring reaction 10h under room temperature condition.Salt and unreacted fluorescence is removed through bag filter (MWO=6000～8000) dialysis
Molecule, it is thus achieved that fluorescent microsphere；Fig. 3 (b) is the fluorescent microsphere of Cy5 labelling emission spectrum under excitation wavelength 635nm.
Fig. 4 (b) is the laser co-focusing figure of the fluorescent microsphere of Cy5 labelling.
3. modify surface characteristics of microspheres by layer-by-layer
5mg polystyrene microsphere is dispersed in 0.2mL ultra-pure water, under the conditions of magnetic agitation, is added drop-wise to 5mL's
In diallyl dimethyl amide hydrochloride (PDDA, 1.0mg/mL), continuously stirred 2h, centrifugal segregation supernatant
Liquid, cleans once with ultra-pure water, centrifugal collecting precipitation, is scattered in 2mL ultra-pure water, is added drop-wise to 25mL under stirring again
Polysaccharide polyelectrolyte macromolecular solution in the dextran sulphate solution (2.0mg/mL) of labelling or unmarked fluorescence molecule, holds
Continuous stirring 2h, centrifugal segregation supernatant, precipitation is redispersed in pure water, centrifugal collecting precipitate, then by precipitate
It is distributed in ultra-pure water, it is thus achieved that the high molecular microsphere of surface modified polysaccharide.
Example 3
1. the preparation of the polystyrene microsphere of surface carboxyl groups
Two step dispersion copolymerization methods are used to prepare the polystyrene microsphere of surface carboxyl groups.First by initiator AMBN
(AMBN) 0.125g, stabilizer polyvinylpyrrolidone PVP55 (Mw=55000g/mol) 0.5g, help stable
Agent TX3050.175g, and styrene monomer 3.125g is dissolved in dehydrated alcohol 25mL, adds with agitator, temperature
In the reactor of meter and reflux condensate device, stirring is mixed evenly, and reaction system is led to nitrogen deoxidation treatment, then by above-mentioned
Reactor is placed in oil bath, is heated to 60 DEG C of reaction 1h；Styrene 3.125g and allyl acid 0.125g is dissolved in anhydrous
In ethanol 25mL, add in another reactor, reaction system is led to nitrogen deoxidation treatment, and is heated to 60 DEG C；Reaction
60min, adds in aforesaid reaction vessel by styrene and the allyl acid solution of heat, continues reaction 18h, after completion of the reaction, logical
Cross centrifugal (1000rpm, 20min) and remove unreacted monomer, and be again dispersed in buffer solution.By scanning electricity
Sem observation, its size is 2.6 ± 0.2 μm, and is distributed homogeneous, as shown in Fig. 1 (c).
2. prepared by the labelling of polystyrene fluorescent microsphere
Measure the polystyrene microsphere solution (60mg/mL) of 5mL surface carboxyl groups in reactor, add 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) 6.54mg and N-hydroxysuccinimide (NHS) 11.56
Mg, at ambient temperature after stirring 15min, the FITC 0.3mg that end contains amino functional group adds reaction system,
Stirring reaction 10h under room temperature condition.Salt and unreacted fluorescence is removed through bag filter (MWO=6000～8000) dialysis
Molecule, it is thus achieved that fluorescent microsphere；Its photoluminescent property is characterized by spectrofluorophotometer and copolymerization Jiao, and Fig. 3 (a) is FITC
The fluorescent microsphere of labelling emission spectrum under excitation wavelength 488nm, Fig. 4 (a) is the fluorescent microsphere of FITC labelling
Laser co-focusing figure.
3. modify surface characteristics of microspheres by layer-by-layer
5mg polystyrene microsphere is dispersed in 1mL ultra-pure water, under the conditions of magnetic agitation, is added drop-wise to the poly-of 10mL
In diallydimethyl amide hydrochloride (PDDA, 4.0mg/mL), continuously stirred 4h, centrifugal segregation supernatant,
Clean once with ultra-pure water, centrifugal collecting precipitation, be scattered in 2mL ultra-pure water, under stirring, be added drop-wise to 50mL labelling again
Or polysaccharide polyelectrolyte macromolecular solution in the dextran sulphate solution (6.0mg/mL) of unmarked fluorescence molecule, persistently stir
Mix 2h, centrifugal segregation supernatant, precipitation is redispersed in pure water, centrifugal collecting precipitate, then precipitate is disperseed
In ultra-pure water, it is thus achieved that the high molecular microsphere of surface modified polysaccharide.By measuring the knot of the variation monitoring self assembly of microsphere current potential
Really, as shown in Figure 5.
Example 4
1. the preparation of the polystyrene microsphere of surface carboxyl groups
Two step dispersion copolymerization methods are used to prepare the polystyrene microsphere of surface carboxyl groups.First by initiator AMBN
(AMBN) 0.125g, stabilizer polyvinylpyrrolidone PVP55 (Mw=55000g/mol) 0.375g, help steady
Determine agent TX3050.175g, and styrene monomer 3.125g is dissolved in dehydrated alcohol 15mL, add with agitator, temperature
In the reactor of degree meter and reflux condensate device, stirring is mixed evenly, and reaction system is led to nitrogen deoxidation treatment, then by upper
State reactor to be placed in oil bath, be heated to 60 DEG C of reaction 1h；Styrene 3.125g and allyl acid 0.187g is dissolved in nothing
In water-ethanol 15mL, add in another reactor, reaction system is led to nitrogen deoxidation treatment, and is heated to 60 DEG C；Instead
Answer 60min, styrene and the allyl acid solution of heat are added in aforesaid reaction vessel, continuation reaction 18h, after completion of the reaction,
Remove unreacted monomer by centrifugal (1000rpm, 20min), and be again dispersed in buffer solution.By scanning
Electron microscopic observation, its size is 3.6 ± 0.2 μm, and is distributed homogeneous, as shown in Fig. 1 (d).
2. prepared by the labelling of polystyrene fluorescent microsphere
Measure the polystyrene microsphere solution (60mg/mL) of 5mL surface carboxyl groups in reactor, add 1-(3-bis-
Methylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) 3.54mg and N-hydroxysuccinimide (NHS) 7.08mg,
At ambient temperature after stirring 15min, the FITC 0.3mg that end contains amino functional group adds reaction system, room temperature
Under the conditions of stirring reaction 10h.Salt and unreacted fluorescence molecule is removed through bag filter (MWO=6000-8000) dialysis,
Obtain fluorescent microsphere；
3. modify surface characteristics of microspheres by layer-by-layer
5mg polystyrene microsphere is dispersed in 0.2mL ultra-pure water, under the conditions of magnetic agitation, is added drop-wise to 5mL's
In diallyl dimethyl amide hydrochloride (PDDA, 2.0mg/mL), continuously stirred 1h, centrifugal segregation supernatant
Liquid, cleans once with ultra-pure water, centrifugal collecting precipitation, is scattered in 1mL ultra-pure water, is added drop-wise to 25mL under stirring again
Polysaccharide polyelectrolyte macromolecular solution in the dextran sulphate solution (4.0mg/mL) of labelling or unmarked fluorescence molecule, holds
Continuous stirring 2h, centrifugal segregation supernatant, precipitation is redispersed in pure water, centrifugal collecting precipitate, then by precipitate
It is distributed in ultra-pure water, it is thus achieved that the high molecular microsphere of surface modified polysaccharide.
Application example 1
By the high molecular fluorescent microsphere of surface modified polysaccharide of preparation, being scattered in ultra-pure water, concentration is about 106Individual/mL,
Being placed in flow cytometer detection, its excitation wavelength is 488nm, and microsphere number is set to 50000, and thinks labelling
The high molecular microsphere of surface modified polysaccharide of fluorescence is matched group.Result is obtained, as shown in Figure 6 through data analysis.Streaming is thin
Born of the same parents' instrument analysis result shows, the fluorescent microsphere of FITC labelling, can meet the flow cytometer detection to it.
Claims (6)
1. the preparation method of a biological polysaccharide polymer fluorescent microsphere, it is characterised in that the method comprises the following steps:
1) preparation of surface carboxyl groups polystyrene microsphere
(1) by initiator 0.04～0.06 weight portion, polyvinylpyrrolidone 0.04～0.3 weight portion, Value 3608 0.05～0.1 weight portion and styrene 1 weight portion are dissolved in dehydrated alcohol: water=100: the mixed solution 4 of 0～25 volume ratios～8 parts, add in the reactor with agitator, thermometer and reflux condensate device, stirring is mixed evenly, reaction system is led to nitrogen deoxidation treatment, then aforesaid reaction vessel is placed in oil bath, is heated to 40～80 DEG C of reaction 1～2 h；
(2) styrene 1 weight portion and allyl acid 0.02～0.06 weight portion are dissolved in dehydrated alcohol: water=100: the mixed solution 4 of 0～25 volume ratios～8 parts, add in another reactor, reaction system is led to nitrogen deoxidation treatment, and is heated to 40～80 DEG C；Reaction 15～90 min, the styrene of heat and allyl acid solution are added in step (1) in the reactor with agitator, thermometer and reflux condensate device, continue reaction 10～24 h, unreacted monomer is removed by centrifugation, and be again dispersed in pH=6～8.4 phosphate buffer solution in, it is thus achieved that surface carboxyl groups polystyrene microsphere；
2) preparation of biological polysaccharide polymer fluorescent microsphere
(1) preparation of polystyrene fluorescent microsphere
The surface carboxyl groups polystyrene microsphere solution of 1 weight portion is placed in reactor, add and 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride that Surfaces of Polystyrene Microparticles carboxyl molar equivalent is 1～10 and N-hydroxysuccinimide that Surfaces of Polystyrene Microparticles carboxyl molar equivalent is 1～10, it is stirred at room temperature 10～60 min, fluorescence molecule 0.001～0.01 molar equivalent containing amido functional group is added reaction system, and continue stirring reaction 6～24 h, dialyse through MWO=6000～8000 bag filters and remove salt and unreacted fluorescence molecule, obtain polystyrene fluorescent microsphere；
(2) biological polysaccharide polymer microsphere is prepared by LBL self-assembly
The polystyrene fluorescent microsphere of 1 weight portion is dispersed in 0.1～1 mL ultra-pure water, under agitation, being added drop-wise to concentration is 1.0～4.0 mg/mL, volume is in the diallyl dimethyl amide hydrochloride of 3～10 mL, continuously stirred 1～4 h, centrifugal segregation supernatant, clean once with ultra-pure water, centrifugal collecting precipitation, it is scattered in 1～2 mL ultra-pure waters, being added drop-wise to concentration under stirring again is 1.0～6.0 mg/mL, volume is in the polysaccharide polyelectrolyte macromolecular solution of 5～50 mL labellings or unmarked fluorescence molecule, continuously stirred 1～4 h, centrifugal segregation supernatant, precipitation is redispersed in pure water, centrifugal collecting precipitate, then precipitate is distributed in ultra-pure water, obtain biological polysaccharide polymer fluorescent microsphere.
2. the preparation method of biological polysaccharide polymer fluorescent microsphere as claimed in claim 1, it is characterised in that initiator is any one in AMBN, azodiisobutyronitrile or potassium peroxydisulfate.
3. the preparation method of biological polysaccharide polymer fluorescent microsphere as claimed in claim 1, it is characterized in that the fluorescence molecule containing amido functional group is fluorescein, the scope launching wavelength contains 350～800 nm, including Fluorescein isothiocyanate (FITC), rhodamine, any one in Cy5.
4. the preparation method of biological polysaccharide polymer fluorescent microsphere as claimed in claim 1, it is characterised in that the polystyrene microsphere of the surface carboxyl groups of preparation, its mean diameter is controlled in the range of 1～10 μm, and particle diameter narrow distribution.
5. the high molecular fluorescent microballoons of the surface modified biological polysaccharide that the preparation method of biological polysaccharide polymer fluorescent microsphere as described in one of Claims 1 to 4 prepares; it has good biocompatibility, and its surface biological polysaccharide can protect the fluorescence molecule of instability.
6. the high molecular fluorescent microballoons of surface modified biological polysaccharide is used for flow cytometer and cell marking as claimed in claim 5.
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| CN105758835B (en) * | 2016-05-04 | 2018-03-27 | 成都爱兴生物科技有限公司 | A kind of homogeneous immunoassay POCT detection methods and the system using the detection method |
| CN106699930A (en) * | 2016-12-01 | 2017-05-24 | 沈阳化工研究院有限公司 | Oil mark polymer and preparation method and application thereof |
| CN108084316A (en) * | 2017-12-15 | 2018-05-29 | 中北大学 | A kind of preparation method of carboxylated porous crosslinked polystyrene copolymerization fluorescent microsphere |
| CN108531550A (en) * | 2018-02-01 | 2018-09-14 | 武汉尚码生物科技有限公司 | Marking and identifying method of particle carrier and application thereof |
| CN108579633B (en) * | 2018-03-29 | 2020-09-22 | 山东大学 | Preparation method of rare earth nano particle/polystyrene composite microsphere |
| CN110305241A (en) * | 2019-05-13 | 2019-10-08 | 长春国科医工科技发展有限公司 | A kind of quantum dot fluorescence coding microball and preparation method thereof based on fluorescence resonance energy transfer analysis |
| CN115109580A (en) * | 2022-05-11 | 2022-09-27 | 上海大学 | Polystyrene AIE dye fluorescence encoding microsphere material with carboxyl coated on outer layer, and preparation method and application thereof |
| CN116218513A (en) * | 2023-03-03 | 2023-06-06 | 杭州谱康医学科技有限公司 | FITC-labeled fluorescent microsphere, preparation method and application |
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| CN101824191B (en) * | 2010-04-29 | 2011-09-14 | 苏州大学 | Macromolecular fluorescent microspheres and preparation method thereof |
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