CN101799416A - Method for detecting induced pluripotent stem cell - Google Patents
Method for detecting induced pluripotent stem cell Download PDFInfo
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- CN101799416A CN101799416A CN 201010120881 CN201010120881A CN101799416A CN 101799416 A CN101799416 A CN 101799416A CN 201010120881 CN201010120881 CN 201010120881 CN 201010120881 A CN201010120881 A CN 201010120881A CN 101799416 A CN101799416 A CN 101799416A
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Abstract
The invention relates to a method for detecting an induced pluripotent stem cell in the technical field of detection, which comprises the following steps of: preparing magnetic nano-particles and water-soluble quantum dots; generating silicon dioxide microspheres by hydrolyzing tetraethyl orthosilicate and preparing fluorescent magnetic nano silicon spheres by adopting an inverse microemulsion method; carrying out surface modification on the fluorescent magnetic nano silicon spheres and then coupling an antibody to obtain the fluorescent magnetic nano silicon spheres coupled with the antibody; preparing a microarray chip by using internal control protein as a positive control, using bovine serum albumin as a negative control and utilizing the antibody for resisting a specific marker expressed on the surface of the induced pluripotent stem cell; and detecting a sample by utilizing the microarray chip and then carrying out fluorescence labeling on the microarray chip by utilizing the fluorescent magnetic nano silicon spheres coupled with the antibody to obtain a detecting result. The method of the invention can realize the simultaneous detection of a plurality of specific molecules on a single sample and has little sample usage quantity, high sensitivity and specificity, low detecting cost, simple operation, high automation degree and high detecting speed.
Description
Technical field
The present invention relates to a kind of detection method of technical field of bioengineering, specifically is a kind of method that detects induced multi-potent stem cells.
Background technology
The combination that is constituted behind the materials such as quantum dot binding antibody, antigen or DNA is called as bioprobe or fluorescence probe.But this class fluorescence probe only can only be done qualitative or quantitative test to the biomolecule of institute's mark, does not possess desire is made the function that the biomolecule of qualitative or quantitative test is gathered or separated.Easily characteristics such as functionalization are very noticeable because of it has good superparamagnetism and surface for magnetic nano-particle, and it can compound antibody, antigen or immunoglobulin (Ig).The object that separates with desire combines in vivo, carry out immune magnetic and separate under the effect in magnetic field, but itself does not possess the function of mark.Mark with separate the important process that becomes in the modern biomedical engineering field, if mark can be finished in a step with the work that separates, will greatly promote the development of bio-science technology.Therefore, the composite nano particle that integrates magnetic property and luminescent properties receives much concern with its good prospects for application.Biochip technology has been widely used in fields, forward position such as gene expression, functional genome, protein group rapidly in more than ten years development recently.Especially in recent years, related field extends to applied occasion rapidly from the research occasion, has brought into play more and more important effect at aspects such as clinical examination, medical diagnosis on disease, drug screenings.Microarray is a kind of extensive, high-throughout important analysis technology that can be used for biological study.In recent years, the protein microarray technology demonstrates great potential in functional study, medical diagnosis on disease and the drug development of protein group.The principal element that influences the protein microarray analytical performance comprises specificity, immobilization of protein technology and the detection method etc. of protein molecule identification, and wherein fixing the and detection method of protein is the most basic technology of carrying out protein microarray research.Crystallization---the microarray Enzyme-multiplied immune technique of successfully having developed chip technology and elisa technique in conjunction with Enzyme-multiplied immune technique commonly used clinically, this technology is keeping on the high basis of the methodological maturation of ELISA, convenience, automaticity, has characteristics such as the high flux, low cost of chip technology, high parallel, microminiaturization again.In the clinical diagnosis, can carry out the detection of multiple disease indicators simultaneously to single sample; Amount of samples is little; Sensitivity and reliability are higher; The detection cost is low, the automaticity height; Be beneficial to large-scale promotion application.On the basis of enzyme linked immunosorbent assay technology and microarray technology, form detection technique system one microarray Enzyme-multiplied immune technique of new generation finally, set up the protein chip technology platform.The protein chip detection technique has incomparable advantage on fast detecting, multinomial order joint-detection, it will lead the technological revolution of external diagnosis reagent industry.
Induced multi-potent stem cells (induced pluripotent stem cells, iPS cell) is to utilize carrier that different transcription factors are changed in the body cell of differentiation, and making its reprogrammed is a kind of cell type of similar embryonic stem cell.In recent years, the ips cell becomes the research focus of life science, and the breakthrough of the ips cell huge hope that has been the basic medical research of many difficult disease and clinical practice aspect band is in the stem-cell research field, epigenetics research and biomedical research field all caused strong repercussion.The evaluation of ips cell must meet the condition of stem cell versatility, have powerful self ability and differentiation potential, identify that at present the versatility means of ips cell have: the expression of morphological criteria, marker molecule, growth characteristics, potentiality of development, epigenetics feature.The ips cell is determined and can be analyzed by the expression of marker molecule, present existing ripe means are to carry out the evaluation of surface antigen by immunofluorescence technique, direct staining method and indirect staining are arranged: (1) direct staining method is that the specific fluorescence antibody with mark directly is added on the antigen specimen, dyeing through uniform temperature and time, flush away is not participated in the unnecessary fluorescence antibody of reaction, just can see the specificity junction mixture that tested antigen and fluorescence antibody form and the fluorescence that sends under fluorescent microscope.The advantage of direct staining method is: the specificity height, and easy and simple to handle, than faster.(2) indirect staining is to react with known unlabelled specific antibody (first antibody) and antigen specimen earlier, behind the effect certain hour, the unreacted antibody of flush away, antiantibody with mark is antiglobulin antibody (second antibody) and antigen specimen reaction again, if reaction has taken place mutually in the antigen-antibody in the first step, then antibody is fixed or combines with fluorescein-labeled antiantibody, form Ag-Ab-antiantibody compound, the unreacted mark antiantibody of flush away again, visible fluorescence under fluorescent microscope.The advantage of indirect staining is to check unknown antigen, also can check unknown antibody; With a kind of antiglobulin antibody of mark, can with the antibodies of all animals identical on kind, check various unknown antigen or antibody, the susceptibility height.
Through the literature search of prior art being found Yamanaka etc. delivers " Induction of Pluripotent Stem Cells from Adult HumanFibroblasts by Defined Factors " (inducing human fibroblasts to be reassembled as the research of multipotential stem cell by specific transcription factor) on 861～872 pages of " cell " (cell) 2007 the 131st phases of magazine, propose in this article ips cell surface specific marker thing to be identified by immunofluorescence technique, its weak point is: because the factor of participation reaction is more, the possibility of being disturbed is also bigger, result of determination is difficult sometimes, complex operation, contrast morely, the time is long; The antibody cost height of band fluorescent dye; The organic fluorescent dye fluorescence intensity is low; Fluorescence meeting cancellation in short time, organic fluorescent dye toxicity is big.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of method that detects induced multi-potent stem cells is provided.Method of the present invention can realize simultaneously single sample being carried out the detection of multiple specific molecular; Amount of samples is little; Sensitivity and specificity height; The detection cost is low; Simple to operate, the automaticity height; Detection speed is fast.
The present invention relates to a kind of method that detects induced multi-potent stem cells, comprise the steps:
Step 1 adopts conventional method to prepare magnetic nano-particle and water-soluble quantum dot;
Step 2 is got positive tetraethyl orthosilicate (TEOS) hydrolysis and is generated silicon dioxide microsphere, adopts the prepared by reverse microemulsion method fluorescent magnetic nano silicon spheres;
Step 5 is utilized the micro-array chip test sample of step 4, with the fluorescent magnetic nano silicon spheres of the coupling antibody of step 3 gained micro-array chip is carried out fluorescence labeling afterwards, testing result.
In the step 1, described preparation magnetic nano-particle is specially and adopts the coprecipitation preparation, with Fe
2+With Fe
3+Be to mix at 1: 2 in molar ratio, add the aqueous solution of the NaOH of 1.5M afterwards, vigorous stirring, nitrogen protection, 80 ℃ be reaction 1h down, magnetic separates, magnetic nano-particle.
In the step 1, described water-soluble quantum dot is the CdTe quantum dot.
The preparation method of described CdTe quantum dot is specially: at Cd
2+Add mercaptoacetic acid in the aqueous solution of salt, regulating pH with the aqueous solution of the NaOH of 1M is 11, adds the aqueous solution of NaHTe under nitrogen protection, the magnetic agitation, 100 ℃ of 1～24h that reflux down, the CdTe quantum dot.
In the step 2, described reverse microemulsion process is specially: magnetic nano-particle is mixed with water-soluble quantum dot, join in the oil phase microemulsion solution of cyclohexane, vigorous stirring gets the water-in-oil microemulsion reaction system, is 27% NH with volume fraction
3H
2It is alkalescence that O regulates the pH value, adds positive tetraethyl orthosilicate (TEOS) hydrolysis reaction 24h then, and magnetic separates, and gets fluorescence magnetic particle silicon ball.
In the step 3, described surface modification is specially, and fluorescence magnetic particle silicon ball is dispersed in the ethanol, and 80 ℃ of oil baths add silane coupling agent down, stirring reaction 3h, and magnetic separates, and obtains amidized fluorescent magnetic nano silicon spheres; Afterwards amidized fluorescent magnetic nano silicon spheres is dispersed in the DMF solution, adds excessive succinic anhydride, 25 ℃ of following stirring reaction 24h, magnetic separate, the fluorescent magnetic nano silicon spheres after the modification.
Described silane coupling agent is aminopropyl triethoxysilane (APTS).
In the step 4, described preparation micro-array chip is specially: will resist the antibody of the specific marker thing of induced multi-potent stem cells surface expression to be sprayed onto the detection zone of 4 * 4 micro array structures on the nitrocellulose filter, simultaneously with confidential reference items albumen as positive control area, BSA is as negative control, with the neutral protein sealing, drying, the nitrocellulose filter that will be solidified with detection zone and check plot then is assembled on the macromolecule water uptake paper, be assembled at last in the plastic casing and place, promptly.
In the step 5, described that testing result is specially: earlier with the wetting chip of PBST, add testing sample again, wash film with PBST afterwards, remove the antigen that does not connect, add the fluorescent magnetic nano silicon spheres of coupling antibody again, wash film with PBST once more, place under the uviol lamp and observe,, then can under ultraviolet irradiation, can form the distinguishable colored detection zone of naked eyes if contain the antigen of being caught in the testing sample by the corresponding antibodies on the nitrocellulose filter.
Compared with prior art, the present invention has following beneficial effect: fluorescent magnetic nano silicon spheres is a kind of composite nano material, use its fluorescence property to solve easy cancellation of traditional organic fluorescent dye and the low problem of fluorescence intensity, its magnetic property can separation and purification not the specific marker thing antibody of the anti-ips cell surface expression of coupling disturb to avoid pattern detection produced; Has good biological safety simultaneously; A sample to be checked can carry out the detection of 4 species specificity labels simultaneously on a chip, and possess feminine gender and positive control area simultaneously, therefore can realize simultaneously single sample being carried out the detection of multiple specific molecular, simple to operate, fast detecting, time saving and energy saving; This detection method amount of samples is little, and it is low to detect cost, the thing micro-to be checked in can enriched sample, and recall rate and specificity all are higher than traditional immunofluorescence technique; The link of detection method participation reaction of the present invention is less, and the possibility of being disturbed is also less, therefore has high sensitivity; Micro-array chip preparation technology is simple, and is economical and practical.
Description of drawings
Fig. 1 is the micro-array biochip structural representation among the embodiment 1;
Fig. 2 is the micro-array biochip structural representation among the embodiment 2.
Embodiment
Following example will the invention will be further described in conjunction with the accompanying drawings.Present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Ips cell surface expression specificity oct4, sox2, nanog, AP detection of antigens
Detection method is analyzed oct4, sox2, and nanog, people's oct4 is selected in the expression of AP antigen in the ips cell for use, sox2, nanog, the AP monoclonal antibody is as detection means, and anti-BSA monoclonal antibody is as negative control, and anti-beta-tubulin monoclonal antibody is as positive control.
Step 1 takes by weighing 1.35g (0.005mol) FeCl
36H
2O and 0.69 (0.0025mol) FeSO
47H
2O is dissolved in the deionized water of 50ml, the NaOH that under nitrogen protection, 80 ℃ environment, adds 20ml1.5M, behind the vigorous stirring reaction 1h, isolate black precipitate with the method that magnetic separates, and be water washing 3 times with deionization, get magnetic nano-particle, magnetic nano-particle being dispersed in forming concentration in the 20ml deionized water afterwards is 8mg/ml solution;
Step 2 is got 1.1417g (5mmol) CdCl
22.5H
2In the O dissolving 110ml water, adding 1.1053g (12mmol) TGA again stirs, regulate pH to 11 with 1MNaOH, use nitrogen protection again, add the freshly prepd NaHTe aqueous solution of 2.5mmol, this reaction system is heated to 100 ℃, backflow 4h, obtaining the fluorescent emission wavelength is 570nm, and concentration is the yellow CdTe water-soluble quantum dot that the surface of 4mg/ml has carboxyl;
Step 5, with the phosphate buffer of pH 7.60.01mol/L specific marker thing (oct4 with the ips cellular expression, sox2, nanog, AP) corresponding antibody dilution is 1mg/ml, as shown in Figure 1, be sprayed onto with the Bio-Dot point sample instrument on the nitrocellulose filter of 0.45um, the antibody-solutions that sprays 1nL by the every pin of computer control high speed is on the nitrocellulose filter of 4 * 4 structures to microarray, be sprayed on the upper left of 4 * 4 micro array structures and lower-right diagonal position orientation with the positive contrast of anti-beta-tubulin antibody simultaneously, be sprayed on the upper right of 4 * 4 micro array structures and lower-left diagonal orientation with the negative contrast of anti-BSA antibody, then with the PBS sealing that contains 10mg/ml, 37 ℃ of dryings, at last nitrocellulose filter is assembled on the macromolecule water uptake paper and put into plastic casing and be placed on 4 ℃ stand-by;
Step 6, to contain the wetting micro-array chip of PBS damping fluid (PBST) 100ul of 0.05%Tween20, after treating that PBST infiltrates through nitrocellulose filter, add 200ul sample to be checked, to be infiltrated go in the film after, add 3 removals of PBST washing and do not connect antigen, the oct4 that is connected with that adds 200ul more respectively, sox2, nanog, the fluorescent magnetic nano silicon spheres of AP antibody, with PBST solution washing film three times, with ultraviolet lantern irradiation micro-array chip, test result can distinguish by naked eyes, as containing in the sample to be checked by the oct4 of the antibody capture on the nitrocellulose filter then, sox2, nanog, the yellow detection circle of the distinguishable positive of naked eyes zone then can appear in AP antigen on micro-array chip; And no matter whether contain oct4 in the sample to be checked, sox2, nanog, AP antigen, beta-tubulin will be trapped in the zone that is coated with anti-beta-tubulin and form the positive contrast of macroscopic yellow encircled, then yellow circle can not occur at positive region as chip failure; And negative control area in no case color development area can occur.
Embodiment 2
Ips cell surface expression specificity SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 detection of antigens
Detection method is analyzed SSEA-3, SSEA-4, TRA-1-60, the expression of TRA-1-81 antigen in the ips cell, select anti-SSEA-3 for use, SSEA-4, TRA-1-60, the monoclonal antibody of TRA-1-81 is as detection means, and anti-BSA monoclonal antibody is as negative control, and anti-GAPDH monoclonal antibody is as positive control;
Step 1 takes by weighing 1.35g (0.005mol) FeCl
36H
2O and 0.69 (0.0025mol) FeSO
47H
2O is dissolved in the deionized water of 50ml, the NaOH that under nitrogen protection, 80 ℃ environment, adds 20ml1.5M, behind the vigorous stirring reaction 1h, isolate black precipitate with the method that magnetic separates, and be water washing 3 times with deionization, get magnetic nano-particle, magnetic nano-particle being dispersed in forming concentration in the 20ml deionized water at last is 8mg/ml solution;
Step 2,1.1417g (5mmol) CdCl
22.5H
2In the O dissolving 110ml water, adding 1.1053g (12mmol) TGA again stirs, regulate pH to 11 with 1MNaOH, use nitrogen protection again, add the freshly prepd NaHTe aqueous solution of 2.5mmol, this reaction system is heated to 100 ℃, backflow 4h, obtaining the fluorescent emission wavelength is 570nm, and concentration is the yellow CdTe water-soluble quantum dot that the surface of 4mg/ml has carboxyl;
Step 5, with the phosphate buffer of pH 7.60.01mol/L specific marker thing (SSEA-3 with the ips cellular expression, SSEA-4, TRA-1-60, TRA-1-81) corresponding antibody dilution is 1mg/ml, as shown in Figure 2, be sprayed onto with the Bio-Dot point sample instrument on the nitrocellulose filter of 0.45um, the antibody-solutions that sprays 1nL by the every pin of computer control high speed is on the nitrocellulose filter of 4 * 4 structures to microarray, be sprayed on the upper left of 4 * 4 micro array structures and lower-right diagonal position orientation with the positive contrast of anti-GAPDH antibody simultaneously, be sprayed on the upper right of 4 * 4 micro array structures and lower-left diagonal orientation with the negative contrast of anti-BSA antibody, then with the PBS sealing that contains 10mg/ml, 37 ℃ of dryings, at last nitrocellulose filter is assembled on the macromolecule water uptake paper and put into plastic casing and be placed on 4 ℃ stand-by;
Step 6, to contain the wetting micro-array chip of PBS damping fluid (PBST) 100ul of 0.05%Tween20, after treating that PBST infiltrates through nitrocellulose filter, add 200ul sample to be checked, to be infiltrated go in the film after, add 3 removals of PBST washing and do not connect antigen, the SSEA-3 that is connected with that adds 200ul more respectively, SSEA-4, TRA-1-60, the fluorescent magnetic nano silicon spheres of TRA-1-81 antibody, with PBST solution washing film three times, with ultraviolet lantern irradiation micro-array chip, test result can distinguish by naked eyes, as containing in the sample to be checked by the SSEA-3 of the antibody capture on the nitrocellulose filter then, SSEA-4, TRA-1-60, the yellow detection circle of the distinguishable positive of naked eyes zone then can appear in TRA-1-81 antigen on micro-array chip; And no matter whether contain SSEA-3 in the sample to be checked, SSEA-4, TRA-1-60, TRA-1-81 antigen, GAPDH albumen all will be trapped in the zone that is coated with anti-GAPDH antibody and form the positive contrast of macroscopic yellow encircled, then yellow circle can not occur at positive region as chip failure; And negative control area in no case color development area can occur.
Claims (9)
1. a method that detects induced multi-potent stem cells is characterized in that, comprises the steps:
Step 1, preparation magnetic nano-particle and water-soluble quantum dot;
Step 2 is got positive tetraethyl orthosilicate hydrolysis and is generated silicon dioxide microsphere, adopts the prepared by reverse microemulsion method fluorescent magnetic nano silicon spheres;
Step 3, the surface modification fluorescent magnetic nano silicon spheres, coupling antibody afterwards, the fluorescent magnetic nano silicon spheres of coupling antibody;
Step 4, with the positive contrast of confidential reference items albumen, the negative contrast of bovine serum albumin(BSA) utilizes the Antibody Preparation micro-array chip of the specific marker thing of anti-induced multi-potent stem cells surface expression;
Step 5 is utilized the micro-array chip test sample of step 4, with the fluorescent magnetic nano silicon spheres of the coupling antibody of step 3 gained micro-array chip is carried out fluorescence labeling afterwards, testing result.
2. the method for detection induced multi-potent stem cells according to claim 1 is characterized in that, in the step 1, described preparation magnetic nano-particle is specially and adopts the coprecipitation preparation, with Fe
2+With Fe
3+Be to mix at 1: 2 in molar ratio, add the aqueous solution of the NaOH of 1.5M afterwards, stir, nitrogen protection, 80 ℃ be reaction 1h down, and magnetic separates, magnetic nano-particle.
3. the method for detection induced multi-potent stem cells according to claim 1 is characterized in that, in the step 1, described water-soluble quantum dot is the CdTe quantum dot.
4. the method for detection induced multi-potent stem cells according to claim 3 is characterized in that, the preparation method of described CdTe quantum dot is specially: at Cd
2+Add mercaptoacetic acid in the aqueous solution of salt, regulating pH with the aqueous solution of the NaOH of 1M is 11, adds the aqueous solution of NaHTe under nitrogen protection, the magnetic agitation, 100 ℃ of 1～24h that reflux down, the CdTe quantum dot.
5. the method for detection induced multi-potent stem cells according to claim 1, it is characterized in that, in the step 2, described reverse microemulsion process is specially: magnetic nano-particle is mixed with water-soluble quantum dot, join in the oil phase microemulsion solution of cyclohexane, vigorous stirring gets the water-in-oil microemulsion reaction system, is 27% NH with volume fraction
3H
2It is alkalescence that O regulates the pH value, adds positive tetraethyl orthosilicate hydrolysis reaction 24h then, and magnetic separates, and gets fluorescent magnetic nano silicon spheres.
6. the method for detection induced multi-potent stem cells according to claim 1, it is characterized in that, in the step 3, described surface modification is specially, fluorescent magnetic nano silicon spheres is dispersed in the ethanol, and 80 ℃ of oil baths add silane coupling agent, stirring reaction 3h down, magnetic separates, and obtains amidized fluorescent magnetic nano silicon spheres; Afterwards amidized fluorescent magnetic nano silicon spheres is dispersed in the DMF solution, adds excessive succinic anhydride, 25 ℃ of following stirring reaction 24h, magnetic separate, the fluorescent magnetic nano silicon spheres after the modification.
7. the method for detection induced multi-potent stem cells according to claim 6 is characterized in that, described silane coupling agent is an aminopropyl triethoxysilane.
8. the method for detection induced multi-potent stem cells according to claim 1, it is characterized in that, in the step 4, described preparation micro-array chip is specially: will resist the antibody of the specific marker thing of induced multi-potent stem cells surface expression to be sprayed onto the detection zone of 4 * 4 micro array structures on the nitrocellulose filter, simultaneously with confidential reference items albumen as positive control area, BSA is as negative control, seal with neutral protein, dry, the nitrocellulose filter that will be solidified with detection zone and check plot then is assembled on the macromolecule water uptake paper, be assembled at last in the plastic casing and place, promptly.
9. the method for detection induced multi-potent stem cells according to claim 1, it is characterized in that, in the step 5, described that testing result is specially: earlier with the wetting chip of PBST, add testing sample again, wash film with PBST afterwards, remove the antigen that does not connect, the fluorescent magnetic nano silicon spheres that adds coupling antibody again, wash film with PBST once more, place under the uviol lamp and observe,, then can under ultraviolet irradiation, can form the distinguishable colored detection zone of naked eyes if contain the antigen of being caught in the testing sample by the corresponding antibodies on the nitrocellulose filter.
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