CN103694392B - A kind of preparation method of carboxyl polystyrene copolymerization fluorescent microsphere - Google Patents
A kind of preparation method of carboxyl polystyrene copolymerization fluorescent microsphere Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of carboxyl polystyrene copolymerization fluorescent microsphere; it is that dehydrated alcohol, initiator azodiisobutyronitrile, polyethylene of dispersing agent ketopyrrolidine are placed in water and obtain clear transparent solutions; styrene, acrylic acid and pi-allyl fluorescent dye it is simultaneously introduced in solution; carry out copolyreaction under nitrogen protection, obtain carboxyl polystyrene copolymerization fluorescent microsphere.The present invention uses dispersion polymerization processes, and the carboxyl polystyrene copolymerization fluorescent microsphere prepared has good monodispersity and stable fluorescence property.
Description
Technical field
The present invention relates to the preparation method of a kind of fluorescent microsphere, particularly relate to the preparation method of a kind of carboxyl polystyrene copolymerization fluorescent microsphere.
Background technology
Fluorescent microsphere has a good application prospect.Research shows, it can be widely applied to detection and the analysis aspect of the correction of fluorescent instrument, biomolecule and synthetic molecules, the most surface-functionalized fluorescent microsphere, at biomedicine field, there is potential using value, such as gene sequencing and expression, protein sequencing, medical diagnosis on disease and drug screening etc..Meanwhile, at the aspect such as environmental monitoring and food test, also fluorescent microsphere is had substantial amounts of demand.
CN 102115508A discloses the synthetic method of a kind of micro-size polymer fluorescent microspheres, and first it prepare high-quality quantum dot, then uses diffuse-aggregate method, is coated on by quantum dot in polystyrene micron ball, can obtain the fluorescent microsphere of 1-10 m.But in the fluorescent microsphere prepared of the method, quantum dot easily leaks, and can cause the pollution of sample in use.
Document (Materials Letters, 2008,
62,2603-2606) report, with VCz, dansyl chloride allylamine and styrene as raw material, uses diffuse-aggregate method can prepare fluorescent microsphere, but this fluorescent microsphere does not has functional group, be difficult to be combined with subsequent biological.
It is known that, polystyrene microsphere has good dispersion, specific surface area is big, have biologically inert, do not dissolved by common solvent or swelling, and have the specific physiques such as surface respond, the binding ability with the materials such as protein, dyestuff, parent's ligand, is suitable as the carrier of fluorescent material in bioanalysis.By styrene and the fluorescent dye copolymerization with double bond, polystyrene fluorescence polymer material can be prepared, its stable morphosis and stable and efficient luminous efficiency, organism is marked, detects and the application of the aspect such as screening is a lot of.But, according to document [Journal of Materials
Chemistry, 2009,19,2018-2025] report, the luminescent dye molecule with double bond is relatively big, exists a certain degree of sterically hindered, have from inhibition in polymerization process, the double bond fluorescent dye being simultaneously introduced can destroy the polymerizing condition that the most blank microsphere is stable, is not easily formed the microsphere of uniform particle diameter.
Summary of the invention
It is an object of the invention to provide a kind of preparation method with carboxy functional group, good monodispersity, carboxyl polystyrene copolymerization fluorescent microsphere that fluorescence property is good and stable.
The preparation method of the carboxyl polystyrene copolymerization fluorescent microsphere that the present invention provides is to be obtained fluorescent microsphere by styrene, acrylic acid with pi-allyl fluorescent dye copolymerization; its concrete preparation method is: dehydrated alcohol, initiator azodiisobutyronitrile, polyethylene of dispersing agent ketopyrrolidine is placed in water and obtains clear transparent solutions; styrene, acrylic acid and pi-allyl fluorescent dye it is simultaneously introduced in solution; carry out copolyreaction under nitrogen protection, obtain carboxyl polystyrene copolymerization fluorescent microsphere；Wherein, described pi-allyl fluorescent dye includes pi-allyl rhodamine B, pi-allyl fluorescein or pi-allyl Nile red, and the addition of pi-allyl fluorescent dye is the 0.25～0.8% of styrene quality.
In above-mentioned copolyreaction, described acrylic acid consumption is the 3.5～5.5% of styrene quality；The consumption of initiator azodiisobutyronitrile is the 2～2.5% of styrene quality；The consumption of polyethylene of dispersing agent ketopyrrolidine is the 3.5～7% of styrene quality；Described copolyreaction under nitrogen protection, reacts 12～24h in 65～70 DEG C.
Carboxyl polystyrene copolymerization fluorescent microsphere priority deionized water that above-mentioned preparation method is obtained by the present invention and ethanol cyclic washing, until after cleaning mixture no longer detects fluorescence, obtaining final fluorescent microsphere product in 50 DEG C of vacuum drying.
The present invention uses diffuse-aggregate method, carries out copolymerization by having the styrene monomer of double bond, acrylic monomers and pi-allyl fluorescent dye monomer, has prepared carboxyl polystyrene copolymerization fluorescent microsphere.
Compared with prior art, the invention have the advantages that 1, use the pi-allyl fluorescent dye with polymerizable groups and styrene and acrylic acid direct polymerization, make fluorescent dye be evenly distributed in microsphere, thus ensure that higher microsphere whole lighting efficiency；Make fluorescent dye be stable in the presence of in microsphere, it is not easy to leakage, cancellation, do not result in test sample and pollute；Fluorescent microsphere is made to have carboxy functional group.2, dispersion copolymerization method is used to prepare fluorescent microsphere, the fluorescent microsphere uniform particle diameter (about 2 m) therefore prepared, there is good monodispersity.3, the present invention uses one kettle way to prepare fluorescent microsphere, makes to prepare the easy and simple to handle of fluorescent microsphere.4, the present invention uses micro-reaction to prepare fluorescent microsphere, makes to prepare the with low cost of fluorescent microsphere.
Accompanying drawing explanation
Fig. 1 is the fluorescence emission spectrogram of the carboxyl polystyrene copolymerization fluorescent microsphere that the embodiment of the present invention provides.
Fig. 2 is carboxyl polystyrene microsphere and the infrared absorption spectroscopy comparison diagram of carboxyl polystyrene copolymerization fluorescent microsphere of embodiment of the present invention offer.
Fig. 3 is the SEM figure of the blank microsphere obtained by the embodiment of the present invention and fluorescent microsphere.
Fig. 4 is the shows fluorescent microscopy images of the fluorescent microsphere obtained by the embodiment of the present invention.
Detailed description of the invention
Technical solution of the present invention is further elaborated with embodiment below in conjunction with the accompanying drawings.
Embodiment 1
1, the synthesis of pi-allyl rhodamine B: by document (J. Mater. Chem., 2009,
19,2018-2025) method, by rhodamine B 240g, potassium carbonate 0.73g, 3-bromopropene 2.65g puts in reaction bulb, add solvent DMF 50ml, catalyst iodine trace, hydroquinone of polymerization retarder trace, under the conditions of anhydrous and oxygen-free, 71 DEG C of reaction 25h, synthesis of allyl rhodamine B, thick product obtains sterling through column chromatography.
2, the preparation of carboxyl polystyrene copolymerization fluorescent microsphere: take azodiisobutyronitrile 0.06g, polyvinylpyrrolidone 0.18g, dehydrated alcohol 9.6ml and water 0.4ml respectively, be placed in microscale reactor, ultrasonic clear transparent solutions.Add styrene 3.02g, acrylic acid 0.15g, pi-allyl rhodamine B 15mg, letting nitrogen in and deoxidizing, seal.It is placed in constant temperature oscillator, under the conditions of 70 DEG C, reacts 12h.Priority deionized water and ethanol cyclic washing thus obtained microsphere, till no longer detecting fluorescence in cleaning mixture, 50 DEG C of vacuum drying, obtain carboxyl polystyrene copolymerization fluorescent microsphere.
Comparative example 1
Take azodiisobutyronitrile 0.06g, polyvinylpyrrolidone 0.18g, dehydrated alcohol 9.6ml and water 0.4ml, be placed in microscale reactor, ultrasonic clear transparent solutions.Add styrene 3.02g and acrylic acid 0.15g, letting nitrogen in and deoxidizing, seal.It is placed in constant temperature oscillator, under the conditions of 70 DEG C, reacts 12h.Priority deionized water and ethanol cyclic washing thus obtained microsphere, 50 DEG C of vacuum drying, obtain carboxyl polystyrene blank microsphere.
Embodiment 2
1, the synthesis of pi-allyl fluorescein: by document (J. Mater. Chem., 2009,
19,2018-2025) method, puts into fluorescein 2.00g, 3-bromopropene 2.42g and Anhydrous potassium carbonate 4.97g in reaction bulb, add solvent DMF 50ml, catalyst iodine trace, hydroquinone of polymerization retarder trace, under the conditions of anhydrous and oxygen-free, 71 DEG C of reaction 25h, obtain pi-allyl fluorescein yellow crude, after Gossypol recrystallized from chloroform, obtain sterling through column chromatography.
2, the preparation of carboxyl polystyrene copolymerization fluorescent microsphere: take azodiisobutyronitrile 0.07g, polyvinylpyrrolidone 0.21g, dehydrated alcohol 9ml and water 1.2ml respectively, be placed in microscale reactor, ultrasonic clear transparent solutions.Add styrene 3.0g, acrylic acid 0.11g, pi-allyl fluorescein 9mg, letting nitrogen in and deoxidizing, seal.It is placed in constant temperature oscillator, under the conditions of 70 DEG C, reacts 12h.Priority deionized water and ethanol cyclic washing thus obtained microsphere, till no longer detecting fluorescence in cleaning mixture, 50 DEG C of vacuum drying, obtain carboxyl polystyrene copolymerization fluorescent microsphere.
Embodiment 3
1, the synthesis of pi-allyl Nile red: by document (J. Mater. Chem., 2009,
19,2018-2025) method, will between N, N-diethylaminophenol 1.65g, concentrated hydrochloric acid 3.40ml and water 1.70ml put in reaction bulb, under ice bath, stirring is to dissolving, and controls temperature at 0 DEG C, is slowly added dropwise sodium nitrite in aqueous solution (NaNO wherein20.78g, H2O 5.9mL), 30min adds, 0～5 DEG C of reaction 4h, filters to obtain crude hydrochloride product, is vacuum dried 8h at 50 DEG C.Being dissolved in by thick product in the ethanol of 25ml boiling, be cooled to 40 DEG C, being slowly added to ether until there being crystallization to occur, mixture places refrigerator 24h, filters to obtain khaki acicular crystal 5-(lignocaine)-2-nitrosophenol.
5-(lignocaine)-2-nitrosophenol 0.50g, 1,6-dihydroxy naphthlene 0.33g and the DMF 40ml being dried being put in reaction bulb, be heated with stirring to backflow, after reaction 4h, decompression boils off DMF, obtains crude product 1.24g.Pillar layer separation purifies to obtain sterling 9-(lignocaine)-2-hydroxyl-5H-benzo [a] azophenlyene-5-ketone.
By 9-(lignocaine)-2-hydroxyl-5H-benzo [a] azophenlyene-5-ketone 0.26g, 3-bromopropene 0.28g, Anhydrous potassium carbonate 0.54g and methanol 7.30ml puts in reaction bulb, it is heated with stirring to backflow, reaction 48h obtains crude product, and dry method loading purifies to obtain sterling pi-allyl Nile red through pillar layer separation.
2, the preparation of carboxyl polystyrene copolymerization fluorescent microsphere: take azodiisobutyronitrile 0.057g, polyvinylpyrrolidone 0.17g, dehydrated alcohol 8.4ml and water 1.1ml respectively, be placed in microscale reactor, ultrasonic clear transparent solutions.Add styrene 2.85g, acrylic acid 0.11g, pi-allyl Nile red 18mg, letting nitrogen in and deoxidizing, seal.It is placed in constant temperature oscillator, under the conditions of 70 DEG C, reacts 12h.Priority deionized water and ethanol cyclic washing thus obtained microsphere, till no longer detecting fluorescence in cleaning mixture, 50 DEG C of vacuum drying, obtain carboxyl polystyrene copolymerization fluorescent microsphere.
Fig. 1 is the fluorescence emission spectrogram of carboxyl polystyrene copolymerization fluorescent microsphere 3% alcohol dispersion liquid of embodiment 1,2,3 preparation, by Fig. 1 a (embodiment 1) it can be seen that under 556nm exciting light, the maximum emission wavelength of this fluorescent microsphere is 575nm；By Fig. 1 b (embodiment 2) it can be seen that under 457nm exciting light, the maximum emission wavelength of this fluorescent microsphere is 526nm；By Fig. 1 c (embodiment 3) it can be seen that under 547nm exciting light, the maximum emission wavelength of this fluorescent microsphere is 596nm.
In Fig. 2, figure a is the infrared absorpting light spectra of reference examples 1 carboxyl polystyrene blank microsphere, and figure b is the infrared absorpting light spectra of embodiment 1 carboxyl polystyrene copolymerization fluorescent microsphere.From figure a it is observed that 1702cm-1Place is the stretching vibration peak of C=O base, 3446cm-1For the stretching vibration peak of hydroxyl in carboxylic acid, the existence of microsphere surface carboxyl is described；1489cm-1And 1448cm-1Place is the C-C stretching vibration peak on phenyl ring, 700cm-1And 758cm-1For the out-of-plane bending vibration peak of c h bond, 3019,3060 and 2921cm on phenyl ring-1Stretching vibration peak for c h bond unsaturated on phenyl ring.Above-mentioned figure a is provable is successfully prepared carboxyl polystyrene blank microsphere.Comparing infrared spectrogram b and infrared spectrogram a of carboxyl polystyrene blank microsphere of carboxyl polystyrene copolymerization fluorescent microsphere, the two is essentially the same, and this is owing in fluorescent microsphere, fluorescent dye content is few, can not symbolize the characteristic peak of fluorescent dye in infrared spectrum.
Fig. 3 is the SEM figure of reference examples 1 carboxyl polystyrene blank microsphere and embodiment 1 carboxyl polystyrene copolymerization fluorescent microsphere.From Fig. 3 a it can be seen that the blank microsphere of preparation has good sphericity and monodispersity is good, lay a good foundation for preparation copolymerization fluorescent microsphere.From Fig. 3 b it can be seen that the copolymerization fluorescent microsphere of preparation has essentially identical particle diameter and dispersibility with blank microsphere.
In Fig. 4, a be carboxyl polystyrene rhodamine B copolymerization fluorescent microsphere photo, b be carboxyl polystyrene fluorescein copolymerization fluorescent microsphere photo, c be carboxyl polystyrene Nile red copolymerization fluorescent microsphere photo.
Claims (2)
1. the preparation method of a carboxyl polystyrene copolymerization fluorescent microsphere; it is to be obtained fluorescent microsphere by styrene, acrylic acid with pi-allyl fluorescent dye copolymerization; it is characterized in that using following method to prepare: dehydrated alcohol, initiator azodiisobutyronitrile, polyethylene of dispersing agent ketopyrrolidine are placed in water and obtain clear transparent solutions; styrene, acrylic acid and pi-allyl fluorescent dye it is simultaneously introduced in solution; under nitrogen protection; carry out copolyreaction 12～24h in 65～70 DEG C, obtain carboxyl polystyrene copolymerization fluorescent microsphere；Wherein, described pi-allyl fluorescent dye includes pi-allyl rhodamine B, pi-allyl fluorescein or pi-allyl Nile red, the addition of pi-allyl fluorescent dye is the 0.25～0.8% of styrene quality, acrylic acid consumption is the 3.5～5.5% of styrene quality, the consumption of initiator azodiisobutyronitrile is the 2～2.5% of styrene quality, and the consumption of polyethylene of dispersing agent ketopyrrolidine is the 3.5～7% of styrene quality.
The preparation method of carboxyl polystyrene copolymerization fluorescent microsphere the most according to claim 1, it is characterized in that the described carboxyl polystyrene copolymerization fluorescent microsphere priority deionized water obtained and ethanol cyclic washing, until cleaning mixture no longer detects fluorescence, 50 DEG C of vacuum drying.
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