CN104707544A - Preparation method of polygenetic upconversion magnetic coding microspheres for screening pathogenic bacteria - Google Patents
Preparation method of polygenetic upconversion magnetic coding microspheres for screening pathogenic bacteria Download PDFInfo
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Abstract
The invention relates to a preparation method of polygenetic upconversion magnetic coding microspheres for screening pathogenic bacteria. The preparation method comprises the following steps: firstly, using various rare-earth salt as raw materials, preparing rare-earth upconversion nanometer particles through adopting a solvent-thermal method, and embedding the upconversion nanometer particles obtained through adopting a high temperature swelling method and magnetic particles into polymeric microspheres. The preparation method disclosed by the invention is simple and convenient to operate, is strong in applicability, is low in cost, is good in dispersibility, is low in background fluorescent light noise, is infinite in coding capacity, and can efficiently code according to substances to be inspected. The particle diameter of the prepared polygenetic upconversion coding microspheres can be controlled in 5-10 microns, and the particle diameter is uniform. The prepared magnetic fluorescent microspheres can be quickly separated under the effect of a magnetic field within 30 minutes. The fluorescence excitation spectrum of the prepared magnetic fluorescent microspheres can be adjusted, the photochemical stability is high, and when the prepared magnetic fluorescent microspheres are exposed under an excitation light source for a long time, the fluorescence intensity is not reduced within 10 hours.
Description
Technical field
The present invention relates to the preparation method of switch magnetization coding microball on a kind of pathogenic bacteria examination polychrome, belong to medical art.
Background technology
Periodontitis is a kind of diseases associated with inflammation occurring in tooth supporting tissue.The field planting of local bacterial and bacterial plaque attachment are the initiation factor of periodontosis, and microorganism and metabolite thereof are by directly stimulating and inducing host immune response etc. to cause disorganization, cause generation and the development of disease.Current research confirms, bacterium plays principal causative effect in the development of periodontosis.Very high by bacterial periodontitis illness rate, within more than 20 year old, crowd's incidence reaches 30%-40%, and the incidence of disease of more than 35 years old crowd reaches 70%, and with advancing age, the incidence of disease is also more and more higher.The first reason causing adult's agomphosis to fall tooth has been become by bacterial periodontitis.The important indicator of Periodontal Status is evaluated in the quantity of patients with periodontal disease pathogenic bacteria and ratio change, has indispensable directive function for the disease treatment of clinician and Index for diagnosis.But due at the periodontitis ill initial stage, oral cavity pathogen has the features such as trace, kind is many, various pathogens coexists, bring very large difficulty to actual detection.
The current clinical detection for oral cavity flora and authentication method mainly contain bacterial cultivation, enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR).Although bacterial cultivation is as the goldstandard of Bacteria Detection, its cultivation cycle is long, affects caused false negative situation more, and cannot quantitatively detect in incubation by various factors.ELISA method specificity is good, and clinical practice is wide, but due to step more loaded down with trivial details, and by the restriction of antibody Percentage bound, false negative result is more, and cannot quantitatively detect.PCR method is highly sensitive, high specificity, but before and after pattern detection, step is more loaded down with trivial details, and needs special and the reagent of costliness and equipment.In addition, the physicochemical property of different pathogenic bacteria is different, adopts above detection method, still faces great difficulty in the high flux context of detection realizing various pathogens.Therefore urgently set up the new method that a kind of accurate, sensitive, high flux detects oral cavity pathogen, realize detecting while various pathogens, shorten analysis time, avoid occurring in testing process undetected.
Liquid-phase chip based on magnetic fluorescent coding microsphere is the biological molecule high flux detection technique platform of new generation after genetic chip, protein-chip.Magnetic fluorescent microspheres refers in polymer microsphere introduces magnetic nano-particle and fluorescent material, makes microballoon have magnetic property and characteristic fluorescence simultaneously, and can by flow cytometry discriminance analysis.Magnetic fluorescent microspheres surface can in conjunction with various biomolecule, the target thing molecular reaction that these biomolecule are corresponding to sample, using fluorescently-labeled mensuration substrate etc. as the reporter group detecting biologically, by the measured value of fluorescence signal, qualitative and quantitative analysis is carried out to target thing molecule.Magnetic fluorescent microspheres has boundless biomedical applications scope.Applied magnetic fluorescent microsphere carries out biomacromolecule detection, quick separating and purifying can be carried out to reactant, can detect multiple target molecules of measuring samples in a reaction tube, hole again simultaneously, the fields such as immunology detection, nucleic acid hybridization, genotyping can be widely used in.Therefore, based on the liquid-phase chip technology of magnetic fluorescent coding microsphere be realize the trace of oral cavity pathogen, high flux detects and provides good platform.
Main luminescent substance at present for the preparation of magnetic fluorescent coding microsphere is organic fluorescent dye and quanta point material.But organic fluorescent dye and quantum dot all have obvious defect as encoded luminescent material, such as organic fluorescent dye photostability is poor, and long-time placement easily photobleaching occurs and affects the accuracy of code carrier spectrum.Quantum dot as inorganic nano-crystal has higher photostability, but, quantum dot and organic fluorescent dye are all the lower conversion luminescence mechanism of short-wavelength light source excitation, the overlapping energy transferring that causes that there is absorption spectrum and emission spectrum in coding scheme between different incandescnet particle (or molecule) occurs, thus reduces optical spectrum encoded quantity and spectrum accuracy thereof.In addition, in reality detects, the large biological molecules such as albumen excite lower normal with background fluorescence in various degree short wavelength, cause the signal to noise ratio of detection signal low, thus reduce the sensitivity detected.
Up-conversion nano material, as a kind of novel fluorescence radiation material, can overcome the above-mentioned defect of fluorescent dye and quantum dot, have not easily photobleaching, Stokes displacement is large, luminous intensity is high, output wavelength can hamony, potential source biomolecule low toxin.In addition, near infrared continuous laser (980nm) is adopted to have the advantages that fluorescence background is low, signal to noise ratio is large and optical property is stable as excitaton source, effectively can improve biochemical analysis accuracy and the sensitivity of liquid-phase chip detection system, therefore, up-conversion luminescent material had great potential as a kind of emerging magnetic fluorescent coding element.At present, the correlative study of cavity pathogenic bacteria is detected based on the high flux of upper switch magnetization fluorescence-encoded micro-beads and patent has no report.
Summary of the invention
The invention provides the preparation method of switch magnetization coding microball on a kind of pathogenic bacteria examination polychrome, as shown in Figure 2:
1) synthesis of rare earth upconversion nano grain: first utilize various rare-earth salts for raw material, prepare rare earth upconversion nano grain by solvent heat.
2) switch magnetization coding microball on high temperature swelling method assembling polychrome: adopt high temperature swelling method the upper conversion nano particle prepared and magnetic-particle to be embedded in polymer microballoon.
Concrete preparation process:
1) synthesis of rare earth upconversion nano grain (UCN):
(1) be ytterbium chloride according to mass ratio: yttrium chloride: thulium chloride: erbium chloride=(10 ~ 60): (10 ~ 80): (0 ~ 3): 1
Material is joined in reactor, add deionization and raw material is dissolved completely, in magnetic agitation condition, be heated to boiling until rare earths salt becomes white solid;
(2) after water evaporate to dryness, at 50 ~ 80 DEG C, be oleic acid according to volume parts ratio: the two adds in reactor by octadecylene=1:1 ~ 3, and white solid is dissolved completely, is then warmed up to 150 ~ 180 DEG C;
(3) again adding according to ratio of quality and the number of copies is NaOH: ammonium fluoride=6:6 ~ 20, adds methyl alcohol and make it dissolve completely in the reactor the two added, wherein mass ratio=the 6:1 of NaOH and above-mentioned erbium chloride; Regulate temperature to 100 ~ 160 DEG C, adopt oil pump to vacuumize 10 ~ 40min, logical argon gas; Rapid temperature increases, to 250 ~ 300 DEG C, maintains reaction 1 ~ 3 hour;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use; The rare earth upconversion nano grain particle diameter of preparation is 20-60nm, and the macroscopical color sent is adjustable from blue light to ruddiness.
2) assembling of switch magnetization coding microball on polychrome:
(1) mass fraction carboxylic polystyrene is taken: upper conversion nano grain: magnetic nano particle is (25 ~ 100): (1 ~ 3): 1, mixes three, adds hexadecane raw material is disperseed completely, and ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 50 ~ 100 DEG C, be incubated 1 ~ 4 hour;
(3) system temperature is risen to 150 ~ 180 DEG C, be incubated 5 ~ 40 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant; Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing; On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 1.4% ~ 6.8%.
Above-mentioned carboxylic polystyrene microsphere can be adopted and prepare with the following method:
Above-mentioned carboxylic polystyrene microsphere can prepare according to any pertinent literature; Also can adopt with the following method:
0.1g sweller is added in 10gSDS solution, for subsequent use after ultrasonic emulsification; Add 30gSDS solution by 0.1g polystyrene seed microballoon (commercialization can be bought), after ultrasonic 10min makes it fully disperse, added in four-hole bottle; Then by sweller emulsion instillation four-hole bottle, swelling 6h at 30 DEG C; After adding 80gSDS solution, then add 10g styrene solution (containing the MAA of the EGDMA of 100wt%, 5wt%, all relatively and the mass percent of monomer styrene) and toluene 10g as pore-foaming agent, swelling 10h at 30 DEG C; Adding 2gPVP is dispersant, adds aqueous phase polymerization inhibitor, then adds 50mL water, polymerisation 12h at being warmed up to 80 DEG C.After being polymerized, in centrifuges, through repeatedly repeated centrifugation enrichment, suspension washing under 2000rpm, with the microballoon of secondary nucleation in removing system; Microballoon is saved backup after drying in vacuum drying oven.
The invention provides the preparation method of switch magnetization coding microball on a kind of pathogenic bacteria examination polychrome.On this, the main component of switch magnetization coding microball reagent comprises three aspects:
1) rare earth upconversion nano particle: the fluorescence producing different colours under near infrared light, for fluorescence-encoded.
2) magnetic nano-particle: produce magnetic under extraneous magnetic fields, for thing Magneto separate purifying to be checked.
3) styrene polymer microballoon: for carrying embedding rare earth upconversion nano particle and magnetic nano-particle, rich surface is used for coupling specificities antibody containing carboxyl.
On pathogenic bacteria examination polychrome, switch magnetization coding microball reagent mechanism of action figure as shown in Figure 1.On pathogenic bacteria examination polychrome, the mechanism of action of switch magnetization coding microball reagent is divided into four steps below:
1) first for subsequent use from a certain amount of level in gingival sulcus fluid of patient, usual level in gingival sulcus fluid contains a large amount of normal bacterias, cell etc., the bacterium of curing the disease (thing to be checked) simultaneously containing trace.
2) switch magnetization coding microball on polychrome is hatched with level in gingival sulcus fluid sample, switch magnetization coding microball identification in conjunction with bacterium to be checked on polychrome under the interaction of antigen, antibody.
3) by external magnetic fields, by switch magnetization coding microball on look related bacterium to be checked, together from sample, separation and purification is out.
4) finally the sample of separation and purification is carried out immunoassay, thus obtain the qualitative, quantitative data about bacterium to be checked.
The present invention assembles switch magnetization coding microball on the polychrome that obtains and has Stability Analysis of Structures, uniform particle diameter, the advantages such as multi-coloured codes, is suitable for each strain specific antibodies of coupling for concentration and separation oral cavity pathogen.
As Fig. 3 projects the display of Electronic Speculum test result, utilize solvent-thermal method to prepare rare earth upconversion nano grain particle diameter and be less than 50nm, even particle size distribution, can blue-fluorescence be sent.As the display of Fig. 4 fluorescence photo, the magnetic fluorescent microspheres uniform particle sizes of encoding with the rare earth nano grain of blue-fluorescence, fluorescence intensity is even.As the display of Fig. 5 stereoscan photograph, the rare earth up-conversion fluorescent magnetic microsphere smooth surface of preparation, uniform particle diameter.As the display of Fig. 6 fluorescence photo, macroscopical fluorescence photo of the upper conversion nano grain of the six kinds of different fluorescence colors prepared.As the display of Fig. 7 titration curve, by calculating the known up-conversion fluorescence coding microball rich surface prepared containing carboxyl, carboxyl-content is 1.8mmol/g.
The invention provides switch magnetization coding microball Reagent evaluation on a kind of pathogenic bacteria examination polychrome for the advantage of traditional fluorescent magnetic microspheres is:
Beneficial effect of the present invention:
1) on the pathogenic bacteria examination polychrome that the present invention relates to, the main advantage of switch magnetization coding microball reagent comprises: easy and simple to handle, and applicability is strong, and cost is low, good dispersion, background fluorescence noise is low, and code capacity is unlimited, can encode according to thing to be checked efficiently.
2) on the polychrome prepared by, switch magnetization coding microball particle size is controlled at 5 to 10 microns, uniform particle sizes; Prepared magnetic fluorescent microspheres can pass through magnetic fields quick separating in 30 minutes; Prepared magnetic fluorescent microspheres, fluorescence excitation spectrum is adjustable, and photochemical stable is high, and under being chronically exposed to excitation source, fluorescence intensity did not reduce in 10 hours.
Accompanying drawing explanation
Fig. 1: switch magnetization coding microball reagent mechanism of action figure on pathogenic bacteria examination polychrome;
Fig. 2: the technical scheme figure of switch magnetization coding microball preparation method of reagent thereof on pathogenic bacteria examination polychrome;
Fig. 3: utilize solvent-thermal method to prepare rare earth upconversion nano grain projection Electronic Speculum test result;
Fig. 4: the magnetic fluorescent microspheres fluorescence photo of the rare earth nano grain coding of blue-fluorescence;
Fig. 5: the rare earth up-conversion fluorescent magnetic microsphere stereoscan photograph of preparation;
Fig. 6: macroscopical fluorescence photo of the upper conversion nano grain of the six kinds of different fluorescence colors prepared;
Fig. 7: up-conversion fluorescence coding microball carboxyl titration curve.
Detailed description of the invention
In the following examples, the invention will be further elaborated, but the present invention is not limited thereto.
Embodiment 1:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 20mg ytterbium chloride, 160mg yttrium chloride and 2mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 6mL oleic acid ligand solvent and 18mL octadecylene part solvent at 50 DEG C with pipette and white solid is dissolved completely be then warmed up to 180 DEG C;
(3) then add 12mgNaOH, 40mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 120 DEG C, adopt oil pump to vacuumize 20min, logical argon gas; Rapid temperature increases, to 250 DEG C, maintains reaction 2 hours;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 40nm, and the macroscopical color sent is blue light.Fig. 3: utilize solvent-thermal method to prepare rare earth upconversion nano grain projection Electronic Speculum test result.Fig. 4: the magnetic fluorescent microspheres fluorescence photo of the rare earth nano grain coding of blue-fluorescence.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 500mg carboxylic polystyrene, the upper conversion nano grain of 20mg, 20mg magnetic nano particle, three mixed, add the high boiling hexadecane of 100mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 70 DEG C, be incubated 3 hours;
(3) system temperature is risen to 150 DEG C, be incubated 40 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 1.4%.Fig. 5: the rare earth up-conversion fluorescent magnetic microsphere stereoscan photograph of preparation
Embodiment 2:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 120mg ytterbium chloride, 20mg yttrium chloride, 6mg thulium chloride 2mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 10mL oleic acid ligand solvent and 10mL octadecylene part solvent at 80 DEG C with pipette and white solid is dissolved completely be then warmed up to 150 DEG C;
(3) then add 12mgNaOH, 12mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 100 DEG C, adopt oil pump to vacuumize 10min, logical argon gas; Rapid temperature increases, to 300 DEG C, maintains reaction 1 hour;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 20nm, and the macroscopical color sent is green glow.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 100mg carboxylic polystyrene, the upper conversion nano grain of 3mg, 1mg magnetic nano particle, three mixed, add the high boiling hexadecane of 60mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 50 DEG C, be incubated 1 hour;
(3) system temperature is risen to 160 DEG C, be incubated 25 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 4.5%.
Embodiment 3:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 90mg ytterbium chloride, 60mg yttrium chloride, 6mg thulium chloride and 3mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 8mL oleic acid ligand solvent and 16mL octadecylene part solvent at 60 DEG C with pipette and white solid is dissolved completely be then warmed up to 170 DEG C;
(3) then add 18mgNaOH, 30mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 160 DEG C, adopt oil pump to vacuumize 40min, logical argon gas; Rapid temperature increases, to 270 DEG C, maintains reaction 3 hours;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 60nm, and the macroscopical color sent is gold-tinted.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 300mg carboxylic polystyrene, the upper conversion nano grain of 20mg, 10mg magnetic nano particle, three mixed, add the high boiling hexadecane of 10mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 100 DEG C, be incubated 4 hours;
(3) system temperature is risen to 180 DEG C, be incubated 5 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 4.5%.
Embodiment 4:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 30mg ytterbium chloride, 40mg yttrium chloride, 2mg thulium chloride and 1mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 20mL oleic acid ligand solvent and 40mL octadecylene part solvent at 65 DEG C with pipette and white solid is dissolved completely be then warmed up to 165 DEG C;
(3) then add 6mgNaOH, 10mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 110 DEG C, adopt oil pump to vacuumize 10min, logical argon gas; Rapid temperature increases, to 270 DEG C, maintains reaction 1 hour;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 25nm, and the macroscopical color sent is gold-tinted.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 250mg carboxylic polystyrene, the upper conversion nano grain of 12mg, 5mg magnetic nano particle, three mixed, add the high boiling hexadecane of 35mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 60 DEG C, be incubated 1.5 hours;
(3) system temperature is risen to 160 DEG C, be incubated 8 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 3.5%.
Embodiment 5:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 45mg ytterbium chloride, 65mg yttrium chloride, 2.5mg thulium chloride and 1mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 15mL oleic acid ligand solvent and 30mL octadecylene part solvent at 55 DEG C with pipette and white solid is dissolved completely be then warmed up to 155 DEG C;
(3) then add 6mgNaOH, 15mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 160 DEG C, adopt oil pump to vacuumize 15min, logical argon gas; Rapid temperature increases, to 280 DEG C, maintains reaction 2 hours;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 28nm, and the macroscopical color sent is gold-tinted.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 240mg carboxylic polystyrene, the upper conversion nano grain of 8mg, 4mg magnetic nano particle, three mixed, add the high boiling hexadecane of 50mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 100 DEG C, be incubated 1 hour;
(3) system temperature is risen to 175 DEG C, be incubated 20 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 2.8%.
Embodiment 6:
One, the synthesis of rare earth upconversion nano grain (UCN):
(1) take 55mg ytterbium chloride, 75mg yttrium chloride, 3mg thulium chloride and 1mg erbium chloride, join in four ml waters by above-mentioned three kinds of materials, in magnetic agitation condition, ebuillition of heated is until rare earths salt becomes white solid.
(2), after water evaporate to dryness, add 15mL oleic acid ligand solvent and 15mL octadecylene part solvent at 75 DEG C with pipette and white solid is dissolved completely be then warmed up to 175 DEG C;
(3) then add 6mgNaOH, 20mg ammonium fluoride, add 10mL methyl alcohol and make it dissolve completely, regulate temperature to 140 DEG C, adopt oil pump to vacuumize 40min, logical argon gas; Rapid temperature increases, to 255 DEG C, maintains reaction 1.5 hours;
(4) acetone centrifugal purification is added after terminating, end product vacuum drying treatment, for future use.The rare earth upconversion nano grain particle diameter of preparation is 46nm, and the macroscopical color sent is gold-tinted.
Two, the assembling of switch magnetization coding microball on polychrome:
(1) take 250mg carboxylic polystyrene, the upper conversion nano grain of 25mg, 10mg magnetic nano particle, three mixed, add the high boiling hexadecane of 25mL, ultrasonic disperse is for subsequent use;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 80 DEG C, be incubated 3.5 hours;
(3) system temperature is risen to 165 DEG C, be incubated 30 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant.Centrifugal sediment is washed by washing lotion (cyclohexane and ethanol equal-volume); End product is dry, encapsulate preservation after weighing.On the polychrome prepared, the nanoparticle proportion of switch magnetization coding microball is 4.9%.
Morphologic observation, particles size and distribution measure:
Extracting sample solution is after ultracentrifugation is separated, and take out sediment, adding distil water makes dispersion on a small quantity, drips sample preparation on carbon supporting film, observes its pattern state and take pictures under transmission electron microscope.Observe under transmission electron microscope on chemotherapy rare earth and change light-operated insoluble drug release nanometer formulation particle in evenly regular spheroidal particle.The chemotherapy rare earth that experiment records changes light-operated insoluble drug release nanometer formulation even particle size distribution, controlled within the scope of 20-60nm.Obtained nano particle as shown in Figure 1.
The mensuration of microsphere surface carboxyl-content:
Adopt the carboxyl-content on Conductometric Titration Method polymer microballoon surface, concrete operations are as follows: the polystyrene microsphere 100mg accurately taking carboxylated, adds a small amount of distilled water and ultrasonic disperse; Then in suspension system, add excessive dilute NaOH solution, about the pH value to 12 of regulation system, make the carboxyl of microsphere surface and NaOH fully react 240min; With hydrochloric acid standard solution (1.01mmoL/L) titration under the stirring action of magneton, and with the potential value of the system in conductivity meter the real time measure titration process; According to potential change in titration curve, calculate the carboxyl-content of microsphere surface.Fig. 7: up-conversion fluorescence coding microball carboxyl titration curve.
Claims (4)
1. the preparation method of switch magnetization coding microball on pathogenic bacteria examination polychrome, is characterized in that step is as follows:
1) synthesis of rare earth upconversion nano grain: first utilize various rare-earth salts for raw material, prepare rare earth upconversion nano grain by solvent heat;
2) switch magnetization coding microball on high temperature swelling method assembling polychrome: adopt high temperature swelling method the upper conversion nano particle prepared and magnetic-particle to be embedded in polymer microballoon.
2. the method for claim 1, is characterized in that described step 1) method is as follows:
(1) be ytterbium chloride according to mass ratio: yttrium chloride: thulium chloride: erbium chloride=(10 ~ 60): (10 ~ 80): (0 ~ 3): material joins in reactor by 1, adding deionization makes raw material dissolve completely, in magnetic agitation condition, be heated to boiling until rare earths salt becomes white solid;
(2) after water evaporate to dryness, being cooled to 50 ~ 80 DEG C, is oleic acid with pipette according to volume parts ratio: the two adds in reactor by octadecylene=1:1 ~ 3, and white solid is dissolved completely, is then warmed up to 150 ~ 180 DEG C;
(3) again adding according to ratio of quality and the number of copies is NaOH: ammonium fluoride=6:6 ~ 20, in the reactor the two added, adds methyl alcohol and makes it dissolve completely; Wherein mass ratio=the 6:1 of NaOH and erbium chloride; Regulate temperature to 100 ~ 160 DEG C, adopt oil pump to vacuumize 10 ~ 40min, logical argon gas; Rapid temperature increases, to 250 ~ 300 DEG C, maintains reaction 1 ~ 3 hour;
(4) add acetone centrifugal purification after terminating, end product vacuum drying treatment, obtain conversion nano grain.
3. the method for claim 1, is characterized in that described step 2) method is as follows:
(1) mass fraction carboxylic polystyrene is taken: upper conversion nano grain: magnetic nano particle is (25 ~ 100): (1 ~ 3): 1, mixes three, adds hexadecane raw material is disperseed completely, ultrasonic disperse;
(2) above-mentioned solution is added be equipped with in the reaction bulb of condensing unit, pass into argon gas, and be warming up to 50 ~ 100 DEG C, be incubated 1 ~ 4 hour;
(3) system temperature is risen to 150 ~ 180 DEG C, be incubated 5 ~ 40 minutes, until chloroform volatilization is complete, be down to normal temperature rapidly afterwards; Stop reaction, take out product centrifugation, take out supernatant; With cyclohexane and ethanol equal-volume washing centrifugal sediment; End product is dry, obtain switch magnetization coding microball on polychrome.
4. on polychrome of the present invention, switch magnetization coding microball can be applicable to pathogenic bacteria examination.
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| CN106398683A (en) * | 2016-08-29 | 2017-02-15 | 上海交通大学 | Method for preparing tricolor coding microsphere composition |
| WO2017088214A1 (en) * | 2015-11-24 | 2017-06-01 | 江南大学 | Fluorescence-based biological detection system |
| CN108927079A (en) * | 2018-08-28 | 2018-12-04 | 天津大学 | Mycotoxin flux detection switch magnetization coding microball and preparation method thereof |
| CN108956982A (en) * | 2018-07-09 | 2018-12-07 | 广州华澳生物科技有限公司 | A kind of rheumatoid arthritis marker joint quantitative testing test paper and preparation method thereof |
| CN110244044A (en) * | 2019-06-13 | 2019-09-17 | 苏州百源基因技术有限公司 | A kind of rare-earths dyeing magnetic bead and its preparation and application |
| CN111426659A (en) * | 2020-03-24 | 2020-07-17 | 深圳唯公生物科技有限公司 | Magnetic fluorescent coding microsphere and preparation method thereof |
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| CN102199428A (en) * | 2011-04-11 | 2011-09-28 | 复旦大学 | Rare earth-doped upconversion nanometer crystal-based fluorescent coding microspheres and preparation method thereof |
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| CN102199428A (en) * | 2011-04-11 | 2011-09-28 | 复旦大学 | Rare earth-doped upconversion nanometer crystal-based fluorescent coding microspheres and preparation method thereof |
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| WO2017088214A1 (en) * | 2015-11-24 | 2017-06-01 | 江南大学 | Fluorescence-based biological detection system |
| CN106398683A (en) * | 2016-08-29 | 2017-02-15 | 上海交通大学 | Method for preparing tricolor coding microsphere composition |
| CN108956982A (en) * | 2018-07-09 | 2018-12-07 | 广州华澳生物科技有限公司 | A kind of rheumatoid arthritis marker joint quantitative testing test paper and preparation method thereof |
| CN108927079A (en) * | 2018-08-28 | 2018-12-04 | 天津大学 | Mycotoxin flux detection switch magnetization coding microball and preparation method thereof |
| CN110244044A (en) * | 2019-06-13 | 2019-09-17 | 苏州百源基因技术有限公司 | A kind of rare-earths dyeing magnetic bead and its preparation and application |
| CN111426659A (en) * | 2020-03-24 | 2020-07-17 | 深圳唯公生物科技有限公司 | Magnetic fluorescent coding microsphere and preparation method thereof |
| CN111426659B (en) * | 2020-03-24 | 2023-06-06 | 深圳唯公生物科技有限公司 | Magnetic fluorescent coding microsphere and preparation method thereof |
| CN113484515A (en) * | 2021-06-11 | 2021-10-08 | 江苏大学 | Method and system for rapidly detecting food-borne pathogenic bacteria |
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