CN102514415A - Method for batch preparation of optically coded microcarrier - Google Patents
Method for batch preparation of optically coded microcarrier Download PDFInfo
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- CN102514415A CN102514415A CN2011104080143A CN201110408014A CN102514415A CN 102514415 A CN102514415 A CN 102514415A CN 2011104080143 A CN2011104080143 A CN 2011104080143A CN 201110408014 A CN201110408014 A CN 201110408014A CN 102514415 A CN102514415 A CN 102514415A
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Abstract
The invention relates to a method for batch preparation of an optically coded microcarrier. The method is characterized by comprising the following steps: a, dispersing an optical coding material into a solidifiable liquid material so as to obtain the solidifiable liquid material containing the optical coding material; b, distributing the solidifiable liquid material containing the optical coding material on substrates by using a contact printing method or a non-contact printing method and solidifying the liquid material so as to obtain optically coded microparticles; c, separating the optically coded microparticles from the substrates, collecting the optically coded microparticles and fixing a sensing sensitive material so as to obtain optically coded microcarriers; and d, repeating step a, b and c so as to respectively preparing microcarriers with different optical codes. The method provided in the invention enable great output and low cost of the prepared optically coded microcarriers to be achieved; and the optically coded microcarriers which are cheap, highly efficient and capable of synchronization detection of a plurality of biological and chemical substances are obtained.
Description
Technical field
The present invention relates to the batch preparation of optical encoding microcarrier; The liquid material that especially will contain colour substance or fluorescent material adopts contact print or off-contact printing method to be dispensed in the substrate and solidifies forming the microparticle of color or fluorescence, separated and collected optical encoding microparticle and at the fixing sensing responsive material and become the optical encoding microcarrier of microparticle from the substrate.This method has simple to operate, with low cost and characteristics that can prepared in batches.
Background technology
The suspension array chip technology is also claimed microcarrier, and it is a kind of instrument that interacts and in fluid, carry out the multi-target detection analysis through specificity between sensing responsive material fixing on the coding microparticle and testing sample.It has not only that common multi-target detection analytical technology possessed contains much information, detection time is short, required test sample volume is little and detect the low characteristics of the relative traditional detection method of cost, and no matter is to prepare also to be to use its all more other a kind of multi-target detection analytical technology-planar micro array chip technology that many outstanding advantages are arranged: bigger output, detect target arrangement, reaction and higher-quality experimental result faster more flexibly.Therefore, the research and development of suspension array chip have just more and more received showing great attention to of people and have been used widely in fields such as drug screening, health care, food security, anti-terrorisms gradually.
The suspension array chip comprises that the coding that three key technologies are the preparation of microcarrier, microcarrier and the decoding and the signal of microcarrier read.Follow deepening continuously of this area research; It is found that the flow cytometry maturation that possesses skills; Characteristics fast easy to detect; And the compatible with it advantage of fluorescence-encoded microcarrier is arranged, therefore become leading decoding and the signal reading method in the present suspension array chip, and the fluorescence-encoded optical encoding method that comprises that matches also becomes the most important coding method of microcarrier.Two fluorescence labeling microballoon technology of the Luminex company that wherein representative example is the U.S.; Under 10 kinds of variable concentrations, produce the technology of 100 kinds of codings by means of two kinds of fluorescent dyes, two fluorescence labeling microballoons of Luminex company have successfully obtained commercial application.The preparation method of optical encoding microcarrier mainly is a liquid phase synthesizing method at present, and other common method of suspension array chip microcarrier such as template and lithographic printing rule idol have appearance.Though these preparation methods can prepare the needed optical encoding microcarrier of people, on preparation speed, preparation quality and preparation cost, but be still waiting to improve to satisfy the ever-increasing related needs of people.The application will comprise the preparation of the printing technology introducing optical encoding microcarrier of contact print and off-contact printing first for this reason, in the hope of contributing for the development and the application of suspension array chip.
Summary of the invention
Technical problem:The printing preparation method who the purpose of this invention is to provide the optical encoding microcarrier is promptly with contact print technology or the further developing and using in the hope of promotion suspension array chip of off-contact printing technology prepared in batches optical encoding microcarrier.
Technical scheme:For solving the problems of the technologies described above, the invention provides a kind of batch preparation of optical encoding microcarrier, this method comprises the steps:
A, the optical encoding dispersion of materials is formed the curable liquid material that contains the optical encoding material in the curable liquid material;
B, the curable liquid material that will contain the optical encoding material are assigned in the substrate through method of contact printing or off-contact printing method and solidify liquid material to form the optical encoding microparticle;
C, from the substrate separated and collected optical encoding microparticle and fixing sensing responsive material and become the optical encoding microcarrier;
D, repeating step a, b, c prepare the microcarrier of different optical coding respectively.
Preferably, said fixedly sensing responsive material is meant in liquid material and adds the sensing responsive material and in microparticle solidification process subsequently, be fixed on the microparticle or in liquid material, add the template of sensing responsive material and solidify the back at microparticle and remove template and expose the sensing responsive material or solidify that the back connects the sensing responsive material and fixing sensing responsive material at microparticle.
Preferably, said substrate satisfy the curable liquid material at suprabasil contact angle greater than 10 degree, and the microparticle after the curable liquid material cured and the interfacial tension between substrate less than the surface of microparticle can and the surface ability of substrate.
Preferably, among the step a, said curable liquid material is to solidify through solvent evaporates deposition of solute, liquid material polymerization or liquid material to solidify.
Preferably, said optical encoding material is a kind of or surpass a kind of mixture in embedded photoluminescent material, light absorbing material and the schemochrome material.
Preferably, among the step b, said method of contact printing for through medium transfer with liquid material with situation that substrate contacts under be transferred to suprabasil method.
Preferably, said off-contact printing method for by nozzle with the discontiguous situation of substrate under liquid material is transferred to suprabasil method.
Beneficial effect:The present invention will comprise that first the printing technology of contact print and off-contact printing is used for prepared in batches optical encoding microcarrier, thereby but because the optical encoding microcarrier output of producing through this technology is big, the low optical encoding microcarrier that can obtain cheapness, the efficient multiple biology of synchronous detecting, chemical substance of cost.
The specific embodiment
The present invention will be described below with reference to accompanying drawings.
Below in conjunction with embodiment the present invention is further described.
The batch preparation of optical encoding microcarrier provided by the invention, this method comprises the steps:
A, the optical encoding dispersion of materials is formed the curable liquid material that contains the optical encoding material in the curable liquid material;
B, the curable liquid material that will contain the optical encoding material are assigned in the substrate through method of contact printing or off-contact printing method and solidify liquid material to form the optical encoding microparticle;
C, from the substrate separated and collected optical encoding microparticle and fixing sensing responsive material and become the optical encoding microcarrier;
D, repeating step a, b, c prepare the microcarrier of different optical coding respectively.
Said fixedly sensing responsive material is meant in liquid material and adds the sensing responsive material and in microparticle solidification process subsequently, be fixed on the microparticle or in liquid material, add the template of sensing responsive material and solidify the back at microparticle and remove template and expose the sensing responsive material or solidify that the back connects the sensing responsive material and fixing sensing responsive material at microparticle.
Said substrate satisfy the curable liquid material at suprabasil contact angle greater than 10 degree, and the microparticle after the curable liquid material cured and the interfacial tension between substrate less than the surface of microparticle can and the surface ability of substrate.
Among the step a, said curable liquid material is to solidify through solvent evaporates deposition of solute, liquid material polymerization or liquid material to solidify.
Said optical encoding material is a kind of or surpass a kind of mixture in embedded photoluminescent material, light absorbing material and the schemochrome material.
Among the step b, said method of contact printing for through medium transfer with liquid material with situation that substrate contacts under be transferred to suprabasil method.
Said off-contact printing method for by nozzle with the discontiguous situation of substrate under liquid material is transferred to suprabasil method.
Embodiment one:
Be that on transparency base (contact angle of solution is 80 degree), to print spacing be that 500 micron-scales are 350 microns lattice array for the aqueous solution that the monodispersed crosslinked polymethylmethacrylaparticles microballoon that is of a size of 220 nanometers of 20wt% contains 40% ethylene glycol with concentration earlier through Epson R230 ink-jet printer.Handled 1 hour down at 150 degrees centigrade after the drying at room temperature, through deionized water the green microparticle that crosslinked polymethylmethacrylaparticles makes up is washed and cleaning-drying from the transparency base then.After the green crosslinked polymethylmethacrylaparticles microparticle that is obtained handled 1 minute through ammonia plasma earlier, be placed on 1% glutaraldehyde PB buffer solution room temperature reaction 4 hours, subsequently with PBS buffer solution cleaning in 10 minutes three times.Then with 4 degrees centigrade of reactions of PBS cushioning liquid of the hepatitis A antibody of 0.2mg/ml 12 hours.Unreacted glutaraldehyde blocked with the PBS cushioning liquid reaction of 1% BSA in 2 hours.After the PBS buffer solution for cleaning, be stored in the concentration of 3-4/10 microlitres in 4 degrees centigrade the PBS buffer solution, obtain to detect the green coding microcarrier of hepatitis A.Repeat to prepare yellow color-coded microcarrier that can detect hepatitis B that makes up by the monodisperse cross-linked poly (methyl methacrylate) micro-sphere of 250 nanometers and the red-coded microcarrier that can detect third liver that makes up by the monodisperse cross-linked poly (methyl methacrylate) micro-sphere of 300 nanometers through Epson R230 ink-jet printer.
The storage liquid of storage liquid and red-coded microcarrier of respectively getting green coding microcarrier storage liquid, the yellow color-coded microcarrier of 10 microlitres before the detection is mixed and is incorporated in 37 degrees centigrade and hatched 2 hours with 180rpm speed in oscillator with testing sample, then with PBST solution cleaning three times.Add CY3 mark hepatitis A antibody, hbv antibody and the c-hepatitis antibody PBS cushioning liquid of 4 mcg/ml, hatched 1 hour, clean three times with PBS subsequently 37 degrees centigrade of speed with 60RPM.Subsequently three kinds of microcarriers are placed on the slide and at first different microcarriers are read color with decoding, under fluorescence microscope, observe the fluorescence on the different microcarriers then and contrast and to judge having or not of hepatitis A virus in the testing sample, hepatitis B and hepatitis C virus with the decoded result of fiber spectrometer through fiber spectrometer.For example, as having fluorescence then in the testing sample hepatitis A virus to be arranged on the fruit green coding microcarrier.
Embodiment two:
Prepare the toluene solution that contains the 10%2-methyl pyrrolidone of the 15wt% polystyrene (Mn=100000) of the Oil Yellow 45L that contains 0.05wt% earlier.Print by yellow polystyrene on the polypropylene screen of PET transparency base through the iP4760 of Canon ink-jet printer then and constitute disk shape microcarrier through pressure sensitive adhesive double coated support.Wherein the disk size is about 350 microns and about 500 microns at interval.Treat to immerse film in the deionized water behind the film drying and slight vibration separates polystyrene disk shape microparticle from polypropylene screen, then be dispersed in the water by yellow color-coded polystyrene disk microparticle.Thoroughly clean the back drying with deionized water and obtain the yellow color-coded microparticle that constitutes by polystyrene.After the yellow color-coded polystyrene disk shape microparticle that just obtained is handled 1 minute through ammonia plasma earlier, be placed on 1% glutaraldehyde PB buffer solution room temperature reaction 4 hours, subsequently with PBS buffer solution cleaning in 10 minutes three times.Then with 4 degrees centigrade of reactions of PBS cushioning liquid of the hepatitis A antibody of 0.2mg/ml 12 hours.Unreacted glutaraldehyde blocked with the PBS cushioning liquid reaction of 1% BSA in 2 hours.After the PBS buffer solution for cleaning, obtain to detect the yellow color-coded disk shape microcarrier of hepatitis A.Repeat to prepare the oil that passes through to add 0.05wt% that passes through to add the red-coded disk shape microcarrier that the oil red 35L of 0.05wt% makes up and can detect third liver that can detect hepatitis B through Canon's iP4760 ink-jet printer and dissolve the blue look coding disk shape microcarrier that blue 03L makes up.
The storage liquid of respectively getting three yellow color-coded microcarrier, red-coded microcarrier and blue look coding microcarrier before the detection is mixed and is incorporated in 37 degrees centigrade and hatched 2 hours with 180rpm speed in oscillator with testing sample, then with PBST solution cleaning three times.Add CY3 mark hepatitis A antibody, hbv antibody and the c-hepatitis antibody PBS cushioning liquid of 4 mcg/ml, hatched 1 hour, clean three times with PBS subsequently 37 degrees centigrade of speed with 60RPM.Subsequently three kinds of microcarriers are placed on the slide and at first different microcarriers are read color with decoding, under fluorescence microscope, observe the fluorescence on the different microcarriers then and contrast and to judge having or not of hepatitis A virus in the testing sample, hepatitis B and hepatitis C virus with the decoded result of fiber spectrometer through fiber spectrometer.For example, if on the red-coded microcarrier fluorescence is arranged then hepatitis B is arranged in the testing sample.
Embodiment three
With concentration be the aqueous solution that silicon dioxide gel that 15wt% is of a size of 200 nanometers contains 10% ethylene glycol receive with 1 through point sample instrument rise/every mode is dispensed on the surface oxidation silicon chip.After treating that solvent evaporates is intact, handled 8 hours, obtain the colloidal crystal microparticle array that forms by 200 nano silicon dioxide sols at 500 degrees centigrade.After this microparticle array cleaned 6 hours with 30% hydrogen peroxide solution and 70% concentrated sulfuric acid mixed liquor, with washed with de-ionized water and use nitrogen drying.The microparticle array that will clean was then handled 4 hours with 1% aminopropyl triethoxysilane ethanolic solution.The reaction of the glutaraldehyde PB buffer solution room temperature of 1 usefulness 1% is 4 hours then, cleans three times in 10 minutes with the PBS buffer solution subsequently.Then with 4 degrees centigrade of reactions of PBS cushioning liquid of the hepatitis A antibody of 0.2mg/ml 12 hours.Unreacted glutaraldehyde blocked with the PBS cushioning liquid reaction of 1% BSA in 2 hours.After the washed with de-ionized water, drying at room temperature.Prepare acrylamide in addition, the acrylamide pre-gathering solutions of 5wt% methylene diacrylamide and 1wt% ammonium persulfate by 10wt%.The acrylamide pre-gathering solutions received with 1 rise/every mode is dispensed on the silicon dioxide colloid crystal microparticle that has connected hepatitis A antibody through point sample instrument.The acrylamide pre-gathering solutions gets in the space of silicon dioxide colloid crystal, polymerization 12 hours under the saturated nitrogen atmosphere of tetramethylethylenediamine then.After acrylamide polymerization is accomplished, place 1% hydrofluoric acid aqueous solution to react 2 hours silicon chip to remove silica.After will the 0.1M acetic acid aqueous solution, cleaning 2 hours from the polyacrylamide microparticle that silicon chip separates, with washed with de-ionized water 3 times.Then obtain the blue polyacrylamide microcarrier responsive to hepatitis A antibody owing to have hepatitis A antibody trace.Silicon dioxide gel with 250 nanometers adopts the method for preparing yellow green polyacrylamide microcarrier responsive to hbv antibody.Silicon dioxide gel with 300 nanometers adopts the method for preparing red polypropylene acid amides microcarrier responsive to c-hepatitis antibody.Three kinds of microcarriers and testing sample reaction back are used the PBS buffer solution for cleaning, detect three kinds of microcarriers and can learn with the variation of testing sample reaction front and back absorption spectrum whether testing sample contains hepatitis A antibody, hbv antibody or c-hepatitis antibody.If for example red microcarrier spectrum with antigenic action before and after change then show that the microcarrier by the preparation of 300 nano silicons has detected c-hepatitis antibody, sample promptly to be checked contains c-hepatitis antibody.
Embodiment four
Obtain the colloid gold particle of CY3 and TMR mark at first respectively.Preparation contains collaurum, 1% polyvinyl alcohol, 1750,10% acrylamides and 5% N of 1% different proportioning CY3 and TMR mark then, and the aqueous solution of N-methylene-bisacrylamide mixes the back at the solution with preceding adding 1% ammonium persulfate (APS), and is subsequent use.On three polypropylene screens of PET transparency base, print CY3 through the iP4760 of Canon ink-jet printer respectively then and TMR mark collaurum ratio is 1:0, the hemispherical acrylamide mixed liquor drop of 1:1 and 0:1 through pressure sensitive adhesive double coated support.Place humidity greater than the nitrogen environment polymerization of 90%RH 8 hours three polypropylene screens having printed the acrylamide drop.Respectively three kinds of different hemispherical polyacrylamide microparticles are poured three containers and with washed with de-ionized water three times from polypropylene screen with deionized water subsequently.Obtain the polyacrylamide hemispherical microparticle that three kinds of sizes are about 350 microns by CY3 and/or TMR mark.The polyacrylamide hemispherical microparticle of three kinds of marks is connected HAAb, hepatitis B antibody and antibody to hepatitis C (these antibody are the sensing responsive material) respectively through glutaraldehyde promptly obtain polyacrylamide microcarrier by CY3 and/or TMR Raman coding.The Raman coding polyacrylamide hemispherical microcarrier that connected hepatitis A antibody, hbv antibody and c-hepatitis antibody and people's serum appearance take a morsel respectively behind 37 degrees centigrade of hybrid reaction 2h; Use the PBS buffer solution for cleaning, again with the fluorescently-labeled hepatitis A antibody of CY3, hbv antibody, c-hepatitis antibody solution reaction 1h and cleaning.Through the Raman spectrometer of optical fiber coupling three kinds of microcarriers are decoded through obtaining its Raman spectrum subsequently, can judge whether contain hepatitis A virus, hepatitis B or hepatitis C virus among the human serum sample through the fluorescence of analyzing different Raman coding microcarriers then.
Embodiment five
Preparation contains 1% acryloxy at 5 ' nucleic acid probe sequence of modifying, and the mean molecule quantity of the 35wt% of 0.5% pearly-lustre dye well, 0.1% azodiisobutyronitrile light trigger is 700 polyethylene glycol double methacrylate PBS cushioning liquid.Nucleic acid probe sequence is that the sensing responsive material is respectively: a:5 '-acryloxy-TTT TGA TGT AGA TGT TTT ATT AGG GTT GT-3 '; B:5 '-acryloxy-TAT TGA TGT AGA TGT TTT ATT AGG GTT GT-3 '; C:5 '-acryloxy-TCT TGA TGT AGA TGT TTT ATT AGG GTT GT-3 '; D:5 '-acryloxy-TGT TGA TGT AGA TGT TTT ATT AGG GTT GT-3 '; The solution that will contain a nucleic acid probe sequence is joined cyan pearly-lustre dyestuff, and on hydrophobic transparency base, to prepare spacing through pin type contact point sample instrument be that 500 micron-scales are about 200 microns cyan hemispherical drop.After 12 hours, using washed with de-ionized water in the irradiation of 16 watts of uviol lamps reaction under saturated steam and the protection of nitrogen gas, acquisition can detect nucleotide sequence a cyan hemispherical microcarrier.Repeat to prepare the orange hemispherical microcarrier that can detect nucleotide sequence b, can detect the purple hemispherical microcarrier of nucleotide sequence c and can detect the blue hemispherical microcarrier of nucleotide sequence d.From four kinds of microcarriers, respectively get the sodium chloride of three microcarriers and 0.2M and contain biotin in the Tris-EDTA buffer solution of 0.05% Tween-20 and be connected testing sample hybridization back and use the PBS buffer solution for cleaning.Subsequently microcarrier is further mixed at 37 degrees centigrade with the PBST buffer solution of the streptavidin that is connected with phycoerythrin and hatch 30 minutes.Clean with PBS subsequently.At first at the color coding of under fluorescence microscope, observing different microcarriers under the situation of not opening fluorescence light source, open fluorescence light source then and observe the fluorescence of different microcarriers and can judge having of nucleotide sequence a, b, c and d or do not have.
The above is merely preferred embodiments of the present invention; Protection scope of the present invention is not exceeded with above-mentioned embodiment; As long as the equivalence that those of ordinary skills do according to disclosed content is modified or changed, all should include in the protection domain of putting down in writing in claims.
Claims (7)
1. the batch preparation of an optical encoding microcarrier is characterized in that, this method comprises the steps:
The optical encoding dispersion of materials is formed the curable liquid material that contains the optical encoding material in the curable liquid material;
The curable liquid material that will contain the optical encoding material is assigned in the substrate through method of contact printing or off-contact printing method and solidifies liquid material to form the optical encoding microparticle;
C, from the substrate separated and collected optical encoding microparticle and fixing sensing responsive material and become the optical encoding microcarrier;
D, repeating step a, b, c prepare the microcarrier of different optical coding respectively.
2. the batch preparation of optical encoding microcarrier according to claim 1; It is characterized in that; Said fixedly sensing responsive material is meant, in liquid material, adds the sensing responsive material and in microparticle solidification process subsequently, is fixed on the microparticle or in liquid material, adds the template of sensing responsive material and solidify the back at microparticle and remove template and expose the sensing responsive material or solidify that the back connects the sensing responsive material and fixing sensing responsive material at microparticle.
3. the batch preparation of optical encoding microcarrier according to claim 1; It is characterized in that; Said substrate satisfy the curable liquid material at suprabasil contact angle greater than 10 degree, and the microparticle after the curable liquid material cured and the interfacial tension between substrate less than the surface of microparticle can and the surface ability of substrate.
4. the batch preparation of optical encoding microcarrier according to claim 1 is characterized in that, among the step a, said curable liquid material is to solidify through solvent evaporates deposition of solute, liquid material polymerization or liquid material to solidify.
5. the batch preparation of optical encoding microcarrier according to claim 1 is characterized in that, said optical encoding material is a kind of or surpass a kind of mixture in embedded photoluminescent material, light absorbing material and the schemochrome material.
6. the batch preparation of optical encoding microcarrier according to claim 1 is characterized in that, among the step b, said method of contact printing for through medium transfer with liquid material with situation that substrate contacts under be transferred to suprabasil method.
7. the batch preparation of optical encoding microcarrier according to claim 1 is characterized in that said off-contact printing method is for to be transferred to suprabasil method with liquid material under nozzle and the discontiguous situation of substrate.
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| CN103675258A (en) * | 2013-12-09 | 2014-03-26 | 东南大学 | Microcarrier preparation system |
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