CN105043831A - Nano material capable of trapping erythrocytes - Google Patents
Nano material capable of trapping erythrocytes Download PDFInfo
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- CN105043831A CN105043831A CN201510388120.8A CN201510388120A CN105043831A CN 105043831 A CN105043831 A CN 105043831A CN 201510388120 A CN201510388120 A CN 201510388120A CN 105043831 A CN105043831 A CN 105043831A
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Abstract
The invention discloses a biological compatible nano substrate-based capture method of rare cells in blood (including, but not be limited to circulating tumor cells, circulating fibrocytes, and fetal nucleated red blood cells). According to a preparation method, controllable hydrolysis and hydrothermal reaction are adopted, layered nano particles are synthesized via two-step synthesis, and a precursor solution is prepared; a glass substrate is subjected to spin coating so as to realized film forming, and a single layer nano substrate with excellent light transmittance performance is obtained via sintering; and the surface of the nano substrate is modified with an antibody with high specificity via a series of matured chemical modification processes. According to the biological compatible nano substrate material-based capture method, high efficiency capture of fetal nucleated red blood cells is realized via interaction of the nano substrate with cell surfaces enhanced by the rough surface of the nano substrate, and antigen antibody specific binding. Fetal umbilical cord blood is taken as a research object; the biological compatible nano substrate material-based capture method is developed based on cellular immunofluorescence staining; biological compatible nano particles are used for capturing and detection of the rare cells in blood; and a new through is provided for capturing of the rare cells in blood with nano materials. The biological compatible nano substrate material-based capture method possesses promising application potential in research of the rare cells in blood.
Description
Technical field:
The invention belongs to technical field of biological material, relate to and a kind ofly compatible nano particle substrate can catch the method for rare cell in blood based on biology.
Background technology:
Fetal nucleated red blood is a distinctive mark of fetal hemopoiesis system and fetus peripheral blood circulation, is the rare cell of one in maternal blood.Fetal blood circulation is set up in after fertilization 3 weekend, and its red blood cell is mainly derived from yolk bag.When full-term pregnancy, the blood cell source of 90% is in marrow.Erythroblast does not exist only in the blood circulation of fetus, and can enter maternal peripheral blood by placental barrier, and its quantity reduces gradually along with pregnant Zhou Zengjia.Fetal nucleated red blood has various features: 1. do not have nucleated red blood cell in normal human peripheral blood, if occur, belongs to pathological phenomenon; 2. mononuclearcell is belonged to, containing fetus full gene group; 3. surface has more relatively special antigen composition as CD71, CD36 and glycophorin (GPA) etc.; 4. in gravid woman's peripheral blood, content is less, between 1: 105 ~ 1: 109; 5. life cycle is short, and about 90 day postpartum disappeared very soon in female blood, not by previous gestation effect during for diagnosing.The special nature of erythroblast, for the prediction of some pathological pregnancy and diagnosis provide new approaches, wherein, the fetal nucleated red blood examination fetal genetic disease in the female blood of antenatal utilization, has become the Non-invasive Prenatal Diagnosis technology that new development is in recent years got up.But because in maternal peripheral blood, fetal nucleated red blood content is very micro-, be difficult to be directly used in diagnosis, must first through enrichment, therefore to the sorting of erythroblast with to catch be an important research topic.
At present, conventional cell sorting techniques is flow cytometry, but this technology use apparatus expensive, bulky, need professional to operate, and the large usage quantity of cell, is difficult in laboratory and hospital's penetration and promotion.The operation of Magnetic activated cell sorting method is relatively simple, and expense relative moderate, the used time is short, and its principle is under the effect of externally-applied magnetic field, by the cell separation of the labeling of monoclonal antibody in conjunction with magnetic particle out.But the purity that the method is separated is not high, except the target cell needing to be separated, lymphocyte and some monocytes also may be activated, so the selection of antibody seems most important during sorting.Unicellular micromanipulation partition method identifies target cell from morphology under the microscope, and with micromanipulator by its one by one picking go out, then analyze.This technology separating purity is high, but technical difficulty is large, and instrument and equipment requires high, not easily promotes.
In the recent period, utilize the application of microflow control technique target acquisition cell more and more extensive, the method first carries out specific surface treatment to base material makes it to have nanostructured, then carries out surface chemical modification and specific antibodies is coupled to target acquisition cell in substrate.The size that nano material is therefore special and structure and show that small-size effect, high-specific surface area etc. are many is different from general properties of materials, increase the contact probability of cell to be measured and target spot, simultaneously, the rough surface of nano material can reduce electrostatic force thus reduce repulsive interaction, increase the adhesiveness of target cell, greatly improve the bioaccumulation efficiency of cell to be measured, the low Young modulus of nano material is also conducive to catching of cell.Utilizing nano material to catch cell is a kind of comparatively feasible method, and erythroblast is as a kind of special cells in cell, also can use for reference correlation technique and catch it.
Summary of the invention:
For this reason, the invention provides a kind of can solve the problem or at least can partly solve the problem (comprise based at the bottom of biological compatibility nano based but be not limited only to Silicon, SiO2, MnO2, TiO2, ZnO, ZrO2, SnO2, hydroapatite particles and polystyrene microsphere etc.) (comprise for cell rare in blood but be not limited only to circulating tumor cell, Circulating fibrocyte, fetal nucleated red blood etc.) catching method.This method can realize specific isolation from whole blood and catch out rare cell, and to be dyeed identification of cell by fluorescin.Specifically comprise the steps:
Preparing biology can compatible nano particle base material step: after the butyl titanate of use is hydrolyzed in deionized water; Synthesized by Hydrothermal Method particle; then according to the nanoparticulate dispersed of 1g synthesis to the lauric acid of 0.05g; after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing; precursor liquid is made in absolute ethyl alcohol dilution; again by after precursor liquid rotary coating to clean glass surface, 500 DEG C of high annealings, 15 minutes sintering film forming.
Chemical modification step: after first using washes of absolute alcohol substrate surface in modification, at room temperature soak 1 hour and 45 minutes respectively with MPTMS and GMBS of 4% again, then the Streptavidin of 20ug/ml is dripped, spend the night at 4 DEG C, drip the antibody of 10ug/ml after cleaning and at room temperature place 2 hours.
Cell capture step: utilize density－gradient centrifuga－tion method sub-argument from fresh blood to go out the monocyte containing erythroblast and be distributed in phosphate buffer (PBS), then be added drop-wise to and modified in the substrate of antibody, in 37 DEG C of cell culture incubators, leave standstill 1 hour.
Cell dyeing and authentication step: after completing cell capture, use paraformaldehyde to fix successively, and tritonX-100 punching and Blocking blockade after process, dropping immunofluorescence protein dyestuff, and lucifuge is spent the night preservation at 4 DEG C.Re-use DAPI and redye nucleus.Then observation of cell coloration result under fluorescent microscope is placed in.
According to the present invention is based on biology can in the blood of compatible nano particle substrate rare cell capture method can effectively from fresh blood separating trap go out target cell; and the cell of catching is identified by immunofluorescence protein staining, directly can also carry out other to it and analyze.
Alternatively, according to method of the present invention, also comprise:
Observation and recording step: the result of catching in chip is observed and record, the result after immunofluorescence Identification of Fusion Protein is observed and record.
Alternatively, according to the inventive method, also comprise:
First glass cleaning step: the glass used in preparation nano based bottom material is 3:1 in dense H2SO4 and H2O2(volume ratio) soak 1.5 hours, then by the clean post-drying of deionized water rinsing in mixed solution.
Alternatively, according to the inventive method, also comprise:
Cleaning step in substrate modification: chip material uses absolute ethyl alcohol and DMSO(dimethyl sulfoxide after modifying MPTMS respectively) clean three times, clean three times with DMSO and PBS respectively after then modifying GMBS.
Alternatively, according to the inventive method, also comprise:
Collecting and treatment step of fresh blood: collect fresh blood and be kept in time containing EDTA(anti-coagulants) heparin tube, and in 6 hours process.Then be slowly added drop-wise to the surface of lymphocyte separation medium after mixing according to the ratio of 1:1 with PBS, be next placed in horizontal speed centrifuge 400g centrifugal treating 30 minutes, then take out mononuclear cell layer and be distributed in PBS solution stand-by.
Alternatively, according to the inventive method, also comprise:
Observation step after cell capture: after completing cell capture process, cleans three times gently with PBS, to remove the cell of cell and the non-specific adsorption do not captured; Then (keep cell complete, in order to avoid clasmatosis affects subsequent analysis) when keeping chip moistening and be placed into basis of microscopic observation cell capture situation and record.
Alternatively, according to the inventive method, also comprise:
Cell type identification result and recording step: the chip after dyeing is placed on fluorescent microscope, observation of cell coloration result, and utilize CCD to take lower result.
The present invention is based at the bottom of biological compatibility nano based and (comprise but be not limited only to Silicon, SiO2, MnO2, TiO2, ZnO, ZrO2, SnO2, hydroapatite particles and polystyrene microsphere etc.) (comprise for cell rare in blood but be not limited only to circulating tumor cell, Circulating fibrocyte, fetal nucleated red blood etc.) catching method tool have the following advantages, be mainly manifested in: (1) utilizes hydrolysis and water heat transfer to have the nano particle of good biological compatibility, then rotary coating is in glass surface high annealing sintering film forming, preparation method is simple, cheap, and the efficient capture of synthesis particle size realization to cell can be regulated and controled, (2) utilize the Interaction enhanced capture effect of nanometer base chip rough surface structure and cell surface compound structure, in substrate, modified antibodies realizes specific target acquisition cell simultaneously, (3) from fresh blood, specific isolation catches out target cell, and ensures that cell monolayer is distributed on base chip, facilitates subsequent cell research and analysis, (4) utilize immunofluorescence kinds of staining methods for protein to carry out analysis and identification to the cell that chip captures, the validity of rare cell is caught in simultaneous verification nano particle substrate for specificity from whole blood.
Accompanying drawing explanation
By reading hereafter detailed description of the preferred embodiment, various other advantage and benefit will become cheer and bright for those of ordinary skill in the art.Accompanying drawing only for illustrating the object of preferred implementation, and does not think limitation of the present invention.And in whole accompanying drawing, represent identical parts by identical reference symbol.Wherein in the accompanying drawings, the multiple identical parts of alphabetic flag instruction after reference number, when making a general reference these parts, by its last alphabetic flag of omission.In the accompanying drawings:
Fig. 1 show according to a kind of embodiment of the inventive method can the operational flowchart of fetal nucleated red blood catching method of compatible TiO2 nano particle substrate based on biology;
Fig. 2 shows the schematic diagram of the TiO2 nano particle substrate chip according to a kind of embodiment of the inventive method;
Fig. 3 shows the pictorial diagram of the TiO2 nano particle substrate chip according to a kind of embodiment of the inventive method;
Fig. 4 shows and schemes according to the SEM of the TiO2 nano particle substrate chip of a kind of embodiment of the inventive method;
Fig. 5 shows and schemes according to the AFM of the TiO2 nano particle substrate chip of a kind of embodiment of the inventive method;
Fig. 6 shows the schematic diagram of the nano particle substrate after according to the modified antibodies of a kind of embodiment of the inventive method;
Fig. 7 shows and from fetal cord whole blood, catches the schematic diagram of fetal nucleated red blood cell according to the TiO2 nano particle substrate chip that utilizes of a kind of embodiment of the inventive method;
Fig. 8 shows the Ming field design sketch utilizing TiO2 nano particle substrate chip to catch monocyte isolated in fetal cord whole blood according to the inventive method;
Fig. 9 shows the number of the fetal nucleated red blood caught from fresh Cord blood according to the inventive method and identify.
Embodiment
The invention provides many applicable creative concepts, this creative concept can be reflected in a large number of in concrete context.The embodiment described in following embodiment of the present invention only as the exemplary illustration of specific implementation of the present invention, and does not form limitation of the scope of the invention.Below in conjunction with accompanying drawing and concrete embodiment, the invention will be further described.
One embodiment of the present invention according to Fig. 1 utilizes biologically the substrate of compatible TiO2 nano particle can catch the method for fetal nucleated red blood.First TiO2 nanometer substrate preparation manipulation S1100 is entered: after the butyl titanate of use is hydrolyzed in deionized water, Synthesized by Hydrothermal Method particle 200, the TiO2 Granular composite synthesized according to 1g is to the lauric acid of 0.05g, after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing, the precursor liquid that TiSP concentration is 5mg/ml is made with absolute ethyl alcohol dilution, again by after precursor liquid rotary coating to clean glass 100 surface, 300 DEG C of high annealing sintering film forming, Fig. 3 shows the pictorial diagram of TiO2 nano particle substrate chip, Fig. 4 shows the SEM of TiO2 nano particle substrate chip, Fig. 5 shows the AFM figure of TiO2 nano particle substrate chip, then enter TiO2 nanometer substrate chemical and modify operation S1200: after first using washes of absolute alcohol substrate surface in modification, at room temperature soak 1 hour and 45 minutes respectively with MPTMS and GMBS of 4% again, then the Streptavidin of 20ug/ml is dripped, spend the night at 4 DEG C, drip the antibody of 10ug/ml after cleaning and at room temperature leave standstill 2 hours, the antibody 201, Fig. 6 that formation is combined with nano particle shows the schematic diagram that chip is modified, nanometer substrate enters cell capture operation S1300: utilize density－gradient centrifuga－tion method to isolate the monocyte containing erythroblast from fresh Cord blood and be distributed in phosphate buffer (PBS), then be added drop-wise to and modified in the substrate of antibody, 1 hour is left standstill in 37 DEG C of cell culture incubators, erythroblast 300 is identified by antibody 201 and combines with it, then catching of fetal nucleated red blood is achieved after using PBS to wash the cell do not captured with physisorption, Fig. 7 shows the schematic diagram that nanometer base chip is caught fetal nucleated red blood, Fig. 8 shows the Ming field design sketch of TiO2 nanometer base chip to cell capture, next immunofluorescent staining authentication step S1400 is entered: after completing cell capture process, after the cell captured being fixed, punching, blockading, then prepare immunofluorescence protein dyestuff, drip and keep in Dark Place at 4 DEG C 8 hours after pigmented section.Then dripping DAPI(concentration in use is 0.1ug/ml) redye nucleus, complete immunofluorescent staining process; Then the identification and analysis to cellular identification result and recording step S1500 is entered: observe cell results and record (as analyzed and taking pictures), Fig. 9 shows the inventive method analyzes the fetal nucleated red blood captured number to 12 cord blood samples; Finally enter instrument cleaning step S1600, observed rear deionized water and ethanol rinse substrate, so far step S1600 terminates, erythroblast catch and qualification completes.
Biology according to one embodiment of the present invention compatible nano particle substrate can catch fetal nucleated red blood method; the concentration of precursor liquid TiSP is preferably 4-6mg/ml; the roughness preferably 80 ~ 90nm of the TiO2 nanometer film substrate of preparation; the concentration preferably 0.09 ~ 0.11mol/L of phosphate buffer; the preferred biotinylatedanti-CD147 of antibody, its working concentration is preferably 10ug/ml.
The present invention is based at the bottom of biological compatibility nano based and (comprise but be not limited only to Silicon; SiO2, MnO2, TiO2; ZnO; ZrO2, SnO2, hydroapatite particles and polystyrene microsphere etc.) (comprise for cell rare in blood but be not limited only to circulating tumor cell; Circulating fibrocyte; fetal nucleated red blood etc.) catching method to relate to parameter more, therefore specific embodiment is only as the exemplary illustration to implementation of the present invention, and does not form limiting the scope of the invention.Below the biology provided using one embodiment of the present invention can the compatible TiO2 nano particle substrate specific operation process of catching fetal nucleated red blood method be described further as embodiment.
According to the technological parameter of nanometer substrate fabrication method of the present invention, design following examples and exemplary illustration is carried out to the present invention.
Embodiment 1
One embodiment of the present invention according to Fig. 1 utilizes biologically the substrate of compatible TiO2 nano particle can catch the method for fetal nucleated red blood, first the preparation manipulation S1100 of TiO2 nano particle is entered: after first utilizing hydrolysis hydro-thermal, prepare TiO2 nano particle precursor liquid, the butyl titanate used is hydrolyzed in deionized water, then the nano particle 200 that water heat transfer particle diameter is 200-500nm is utilized, the TiO2 Granular composite synthesized according to 1g is to the lauric acid of 0.05g, after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing, being diluted in absolute ethyl alcohol and making TiSP concentration is 5mg/ml precursor liquid, and then get the clean glass of a slice 100, be 3:1 in dense H2SO4 and H2O2(volume ratio) soak 1.5 hours in mixed solution, then dry on platforms at 120 ° of C after clean with a large amount of deionized water rinsing and dry, again by after precursor liquid rotary coating to clean glass surface, 500 DEG C of high annealings sintered film forming after 15 minutes, obtain the nanometer film substrate that roughness is 85nm, Fig. 2 shows the schematic diagram of TiO2 nano particle substrate chip in glass substrate, Fig. 4 shows the SEM of TiO2 nano particle substrate chip, Fig. 5 shows the AFM figure of TiO2 nano particle substrate chip, then enter TiO2 nanometer substrate chemical and modify operation S1200: after first using washes of absolute alcohol substrate surface in modification, after with absolute ethyl alcohol MPTMS being diluted to the concentration of 4% again, by substrate as wherein, soak one hour under room temperature, washes of absolute alcohol three times are used again after taking-up, then dimethyl sulfoxide (DMSO) (DMSO) is used to clean 3 times, the concentration dilution of GMBS is become 1mg/ml by following DMSO, washed substrate is continued to be placed in GMBS, soak 45 minutes under room temperature, 3 times are cleaned with DMSO after taking-up, 3 times are cleaned again with PBS, after drying up, chip cutting is become the little chip of 1cm*1cm, then each little chip drips the Streptavidin that 20ul concentration is 20ug/ml, place 6 hours at 4 DEG C, be the antibody of 10ug/ml by dropping 20ul concentration after PBS cleaning and at room temperature preserve 2 hours, form the antibody 201 be combined with nano particle, substrate after the modification obtained as shown in Figure 6, catch erythroblast with the substrate after modification and enter operation S1300: the surface being slowly added drop-wise to lymphocyte separation medium after the Cord blood of the puerpera collected and PBS being mixed according to the ratio of 1:1, next 400g centrifugal treating is placed in horizontal speed centrifuge 30 minutes, the cell dispersal obtained becomes suspending liquid in 1mlPBS, then being added drop-wise to by cell suspending liquid has modified in the substrate of antibody, 1 hour is left standstill in 37 DEG C of cell culture incubators, there is specific recognition and caught by substrate in fetal nucleated red blood 300 antigen and substrate antibody 201, then three times are rinsed gently with PBS, to remove the cell of cell and the non-specific adsorption do not captured, next enter immunofluorescent staining authentication step S1400: after completing cell capture process, use paraformaldehyde solution to fix successively to cell on chip, tritonX-100 solution punches, and Blockingsolution blockades.Then drip immunofluorescence protein dyestuff, then natural drying after cleaning, is then placed into basis of microscopic observation immunofluorescent staining result, and analyzes and record; Finally enter instrument cleaning step S1600, observed rear deionized water and ethanol rinse substrate, so far step S1600 terminates, and utilizes nano material to catch rare cell and completes.
Embodiment 2
One embodiment of the present invention according to Fig. 1 utilizes biologically the substrate of compatible TiO2 nano particle can catch the method for fetal nucleated red blood, first the preparation manipulation S1100 of TiO2 nano particle is entered: after first utilizing hydrolysis hydro-thermal, prepare TiO2 nano particle precursor liquid, the butyl titanate used is hydrolyzed in deionized water, then the nano particle 200 that water heat transfer particle diameter is 200-500nm is utilized, the TiO2 Granular composite synthesized according to 1g is to the lauric acid of 0.05g, after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing, being diluted in absolute ethyl alcohol and making TiSP concentration is 5mg/ml precursor liquid, , and then get the clean glass of a slice 100, be 3:1 in dense H2SO4 and H2O2(volume ratio) soak 1.5 hours in mixed solution, then dry on platforms at 120 ° of C after clean with a large amount of deionized water rinsing and dry, again by after precursor liquid rotary coating to clean glass surface, 500 DEG C of high annealings, 15 minutes sintering film forming, obtain the nanometer film substrate that roughness is 85nm, Fig. 2 shows the schematic diagram of TiO2 nano particle substrate chip in glass substrate, Fig. 4 shows the SEM of TiO2 nano particle substrate chip, Fig. 5 shows the AFM figure of TiO2 nano particle substrate chip, then enter TiO2 nanometer substrate chemical and modify operation S1200: after first using washes of absolute alcohol substrate surface in modification, after with absolute ethyl alcohol MPTMS being diluted to the concentration of 4% again, by substrate as wherein, soak one hour under room temperature, washes of absolute alcohol three times are used again after taking-up, then dimethyl sulfoxide (DMSO) (DMSO) is used to clean 3 times, the concentration dilution of GMBS is become 1mg/ml by following DMSO, washed substrate is continued to be placed in GMBS, soak 45 minutes under room temperature, 3 times are cleaned with DMSO after taking-up, 3 times are cleaned again with PBS, after drying up, chip cutting is become the little chip of 1cm*1cm, then each little chip drips the Streptavidin that 20ul concentration is 20ug/ml, place more than 6 hours at 4 DEG C, be the antibody of 10ug/ml by dropping 20ul concentration after PBS cleaning and at room temperature place 2 hours, form the antibody 201 be combined with nano particle, substrate after the modification obtained as shown in Figure 6, catch erythroblast with the substrate after modification and enter operation S1300: the surface being slowly added drop-wise to lymphocyte separation medium after the Cord blood of the puerpera collected and PBS being mixed in the ratio of 1:1, next 400g centrifugal treating is placed in horizontal speed centrifuge 30 minutes, the cell dispersal obtained mixes in 1mlPBS, then after diluting 10 times with PBS, get 1ml cell suspending liquid to be added drop-wise to and to have modified in the substrate of antibody, 1 hour is left standstill in 37 DEG C of cell culture incubators, there is specific recognition and caught by substrate in fetal nucleated red blood 300 antigen and substrate antibody 201, then three times are rinsed gently with PBS, to remove the cell of cell and the non-specific adsorption do not captured, next enter immunofluorescent staining authentication step S1400: after completing cell capture process, use paraformaldehyde solution to fix successively to cell on chip, tritonX-100 solution punches, and Blockingsolution blockades.Then drip immunofluorescence protein dyestuff, then natural drying after cleaning, is then placed into basis of microscopic observation immunofluorescent staining result, and analyzes and record; Finally enter instrument cleaning step S1600, observed rear deionized water and ethanol rinse substrate, so far step S1600 terminates, and utilizes nano material to catch rare cell and completes.
Embodiment 3
One embodiment of the present invention according to Fig. 1 utilizes biologically the substrate of compatible TiO2 nano particle can catch the method for fetal nucleated red blood, first the preparation manipulation S1100 of TiO2 nano particle is entered: after first utilizing hydrolysis hydro-thermal, prepare TiO2 nano particle precursor liquid, the butyl titanate used is hydrolyzed in deionized water, then the nano particle 200 that water heat transfer particle diameter is 200-500nm is utilized, the TiO2 Granular composite synthesized according to 1g is to the lauric acid of 0.05g, after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing, being diluted in absolute ethyl alcohol and making TiSP concentration is 5mg/ml precursor liquid, and then get the clean glass of a slice 100, be 3:1 in dense H2SO4 and H2O2(volume ratio) soak 1.5 hours in mixed solution, then dry on platforms at 120 ° of C after clean with a large amount of deionized water rinsing and dry, again by after precursor liquid rotary coating to clean glass surface, 500 DEG C of high annealings sintered film forming after 15 minutes, obtain the nanometer film substrate that roughness is 85nm, Fig. 2 shows the schematic diagram of TiO2 nano particle substrate chip in glass substrate, Fig. 4 shows the SEM of TiO2 nano particle substrate chip, Fig. 5 shows the AFM figure of TiO2 nano particle substrate chip, then enter TiO2 nanometer substrate chemical and modify operation S1200: after first using washes of absolute alcohol substrate surface in modification, after with absolute ethyl alcohol MPTMS being diluted to the concentration of 4% again, by substrate as wherein, soak one hour under room temperature, washes of absolute alcohol three times are used again after taking-up, then dimethyl sulfoxide (DMSO) (DMSO) is used to clean 3 times, the concentration dilution of GMBS is become 1mg/ml by following DMSO, washed substrate is continued to be placed in GMBS, soak 45 minutes under room temperature, 3 times are cleaned with DMSO after taking-up, 3 times are cleaned again with PBS, after drying up, chip cutting is become the little chip of 1cm*1cm, then each little chip drips the Streptavidin that 20ul concentration is 20ug/ml, place more than 6 hours at 4 DEG C, be the biotinylatedanti-CD147 antibody of 10ug/ml by dropping 20ul concentration after PBS cleaning and at room temperature place 2 hours, form the antibody 201 be combined with nano particle, substrate after the modification obtained as shown in Figure 6, catch erythroblast with the substrate after modification and enter operation S1300: the surface being slowly added drop-wise to lymphocyte separation medium after the Cord blood of the puerpera collected and PBS being mixed according to the ratio of 1:1, next 400g centrifugal treating is placed in horizontal speed centrifuge 30 minutes, the cell dispersal obtained mixes in 1mlPBS, then after diluting 100 times with PBS, get 1ml cell suspending liquid to be added drop-wise to and to have modified in the substrate of antibody, 1 hour is left standstill in 37 DEG C of cell culture incubators, there is specific recognition and caught by substrate in fetal nucleated red blood 300 antigen and substrate antibody 201, then three times are rinsed gently with PBS, to remove the cell of cell and the non-specific adsorption do not captured, next enter immunofluorescent staining authentication step S1400: after completing cell capture process, use paraformaldehyde solution to fix successively to cell on chip, tritonX-100 solution punches, and Blockingsolution blockades.Then drip immunofluorescence protein dyestuff, then natural drying after cleaning, is then placed into basis of microscopic observation immunofluorescent staining result, and analyzes and record; Finally enter instrument cleaning step S1600, observed rear deionized water and ethanol rinse substrate, so far step S1600 terminates, and utilizes nano material to catch rare cell and completes.
Biological the substrate of compatible TiO2 nano particle the method for fetal nucleated red blood can be caught according to utilizing in embodiment of the present invention 1-3, can isolate in MNS from Cord blood simple and effectively catch fetal nucleated red blood, the clear glass substrate of chip is convenient to carry out real-time in-situ observation to the cell of catching simultaneously; Captured cell can keep good completing property, and carries out follow-up cell type identification experiment;
By the pictorial diagram of the TiO2 nano particle substrate chip of unmodified antibody in the glass substrate shown in Fig. 3, can find out that the substrate of TiO2 nanometer film has good light transmission, be convenient to analyze on inverted microscope and light field observation.
Schematic diagram is caught to what isolate monokaryon in fetal cord whole blood by the TiO2 nano particle substrate chip shown in Fig. 7; can find out that specific recognition occurs the antibody 201 that fetal nucleated red blood 300 antigen and substrate are modified on nano particle 200, thus realize catching.
In addition, utilize biology to have carried out recording (shown in Fig. 8) by the fetal nucleated red blood that obtains of the compatible TiO2 nano particle substrate method of catching fetal nucleated red blood to embodiment 1, and the target cell number of catching and identify is added up (as Fig. 9).
The design sketch of catching is shown by Fig. 8, also can finds out that monocyte cell is trapped in substrate, illustrate that this nano particle substrate chip can catch cell effectively.
2409 ± 537 (mean ± s.d.) by the statistics of the fetal nucleated red blood of catching from 12 parts of fresh Cord blood samples and identifying according to the inventive method shown in Fig. 9.
It should be noted, the present invention will be described instead of limit the invention for above-described embodiment, and those skilled in the art can design alternative embodiment when not departing from claims scope.In the claims, any reference symbol between bracket should be configured to limitations on claims.Word " comprises " not to be got rid of existence and does not arrange element in the claims or step.
Claims (10)
1. one kind based on the catching method being used for rare cell in blood at the bottom of biological compatibility nano based, it is characterized in that: adopt the method for hydrolysis and hydro-thermal to prepare the nanometer film substrate of controllable particle diameter, make to modify fetal erythrocyte specific antibody on chemically at the bottom of nano based, then after covering upper process, umbilical cord blood is unicellular, realize catching the specificity of fetal nucleated red blood at the bottom of nano based, and adopt the detection of immunofluorescence protein staining to catch cell, included concrete preparation process is as follows:
(1) utilize hydrolysis and hydro-thermal method to prepare nano particle precursor liquid, and by the concentration of nano particle in diluted precursor liquid, regulate and control the roughness at the bottom of nano based, then precursor liquid rotary coating is sintered film forming at glass surface;
(2) utilize chemical modification method to modify streptavidin in nanometer film substrate, then drip the antibody with biotin;
(3) use density－gradient centrifuga－tion method, from new blood, isolate monocyte;
(4) MNS is added to the substrate being modified with antibody hatches and catches;
(5) identification of cell type.
2. method according to claim 1, also comprises:
Observe and recording step: cell results is caught to novel antibodies and observes and record and the analytic record of cellular identification result.
3. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle, it is characterized in that: the butyl titanate Synthesized by Hydrothermal Method particle in acid condition used in described step (1), then according to the nanoparticulate dispersed of 1g synthesis to the lauric acid of 0.05g, after the ethyl cellulose of 0.2g and the terpinol of 10ml and alcohol mixed solution (volume ratio is 1:1) mixing, precursor liquid is made with absolute ethyl alcohol dilution, again by after precursor liquid rotary coating to clean glass surface, 500 DEG C of high annealings, 15 minutes sintering film forming.
4. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: also by the concentration of nano particle in diluted precursor liquid in described step (1); the roughness of regulation and control at the bottom of nano based, and control at about 85nm as excellent.
5. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: the base material used in described step (2) is prepared in (1), and the chip being cut into 1cm*1cm is stand-by.
6. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: described step (2) chemical modification process is; use MPTMS successively, after GMBS and Streptavidin process, drip antibody.
7. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: the fresh blood used in described step (3) is after getting blood; load immediately and be added with in the heparin tube of EDTA, and processed in 6 hours.
8. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: described step (3) Midst density gradient centrifugation is slowly added drop-wise on the interface of lymphocyte separation medium by fresh blood sample; then centrifugal treating 30 minutes under 400g, takes out monocyte wherein after blood layering.
9. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle, it is characterized in that: be constant-temperature incubation 1 hour in 37 DEG C of cell culture incubators in described step (4).
10. be used for the catching method containing cell in blood according to described in claim 1 based on the substrate of biological compatibility nano particle; it is characterized in that: in cellular identification process, cell is fixed successively in described step (5); punch and blockade and be treated to, then drip specific immunofluorescence albumen and be used for cellular identification.
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| CN201510388120.8A CN105043831A (en) | 2015-07-03 | 2015-07-03 | Nano material capable of trapping erythrocytes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753573A (en) * | 2018-06-07 | 2018-11-06 | 武汉大学 | The method for being captured in micro-fluidic chip and identifying fetal nucleated red blood |
| TWI764359B (en) * | 2019-11-06 | 2022-05-11 | 精拓生技股份有限公司 | Composite material film for expansion of circulating tumor cells ex vivo and preparation method thereof, method and kit for expansion of circulating tumor cells ex vivo, method for detecting drug effect, and cryopreservation solution |
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