CN105731534A - Method for labeling titanium dioxide nanotubes with green fluorescence - Google Patents
Method for labeling titanium dioxide nanotubes with green fluorescence Download PDFInfo
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- CN105731534A CN105731534A CN201610075345.2A CN201610075345A CN105731534A CN 105731534 A CN105731534 A CN 105731534A CN 201610075345 A CN201610075345 A CN 201610075345A CN 105731534 A CN105731534 A CN 105731534A
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- C01G23/00—Compounds of titanium
- C01G23/04—Oxides; Hydroxides
- C01G23/047—Titanium dioxide
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Abstract
The invention relates to the field of fluorescence labeling, and discloses a method for labeling titanium dioxide nanotubes with green fluorescence.The method includes the steps of firstly, dissolving fluorescein isothiocyanate in dimethyl sulfoxide to prepare a solution A; secondly, adding human serum protein to a sodium carbonate aqueous solution to prepare a solution B; thirdly, dropwise adding the solution A to the solution B to prepare a solution C, conducting dialysis on the solution C, and obtaining an isothiocyanic acid labeled human serum protein solution after dialysis; fourthly, taking titanium dioxide nanotubes, dispersing the titanium dioxide nanotubes in water to prepare a solution D, adding water to thin up the isothiocyanic acid labeled human serum protein solution into a solution E, mixing the solution D with the solution E, and preparing the titanium dioxide nanotubes labeled with green fluorescence after centrifugal separation.By means of the method, the binding fastness of labels and labeled objects is high, the labels can not remarkably influence cells, and scientificity of experiment results is not influenced.
Description
Technical field
The present invention relates to fluorescent labeling field, a kind of method particularly relating to green fluorescent label titania nanotube.
Background technology
Titanium dioxide nano material has the performance of the photocatalytic degradation Organic Hazardous Compounds of excellence so that it is plays huge effect in environmental pollution improvement, also causes the increasing scientist concern to it.The photocatalytic degradation efficiency of the titanium dioxide nano material of different-shape has significant difference, and the efficiency of the photocatalytic degradation organic toxic substance of titania nanotube can be tens times more than of titania nanoparticles.
Research simultaneously for the bio-toxicity of titania nanotube is also very crucial.The interaction observing titania nanotube and cell is the Main Means of research, wherein the fluorescence staining to titania nanotube is to observe titania nanotube to be distributed situation and cell to the titania nanotube most important step of endocytosis situation at cell interior, but titania nanotube surface can not carry out the characteristic of labelling with common stain, so the fluorescence staining problem to titania nanotube that solves is to being very crucial in the researchs such as its interaction with cell.
Summary of the invention
In order to solve above-mentioned technical problem, a kind of method that the invention provides green fluorescent label titania nanotube.The present invention first prepares the human albumin of synthesis marked by fluorescein isothiocyanate by the effect of human albumin with Fluorescein isothiocyanate, then utilize the characterization of adsorption of titanium dioxide nano material and protein indirectly by marked by fluorescein isothiocyanate on titanium dioxide nano material, cell will not be made a significant impact by the method, does not affect the science of experimental result.
The concrete technical scheme of the present invention is: a kind of method of green fluorescent label titania nanotube, adopts following steps:
1) being dissolved in dimethyl sulfoxide by Fluorescein isothiocyanate and prepare to obtain solution A, keep in Dark Place, wherein Fluorescein isothiocyanate is (0.7 ~ 1.1)/(2 ~ 6) mol/mL with the amount ratio of dimethyl sulfoxide.
2) human albumin is added in the aqueous sodium carbonate that pH is 8.5 ~ 9.5, and at room temperature stir to being completely dissolved, preparing to obtain B solution, wherein human albumin and aqueous sodium carbonate amount ratio are (0.1 ~ 0.5)/(16 ~ 24) mol/mL.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, the solution A of 2 ~ 6 parts by volume is dropped in the B solution of 10 ~ 20 parts by volume, prepare to obtain C solution, then described C solution is transferred in bag filter, then described bag filter is immersed in the aqueous sodium carbonate that pH is 8.5 ~ 9.5 and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take titania nanotube and be scattered in water to be configured to the solution D of 0.5 ~ 1.5mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2 ~ 3mg/mL, obtain E solution；When lucifuge by described solution D and described E solution 1:(1 ~ 1.2 by volume) ratio mixed processing 12 ~ 60h, take solid after being then performing centrifugal separation on by mixed solution, prepare the titania nanotube of Green Marker fluorescence.
The present invention first prepares the human albumin of synthesis marked by fluorescein isothiocyanate by the effect of human albumin with Fluorescein isothiocyanate, then utilize the characterization of adsorption of titania nanotube and protein indirectly by marked by fluorescein isothiocyanate on titania nanotube, cell will not be made a significant impact by the method, does not affect the science of experimental result.
As preferably, in step 1), Fluorescein isothiocyanate is (0.8 ~ 1)/(3 ~ 5) mol/mL with the amount ratio of dimethyl sulfoxide.
As preferably, step 2) in human albumin be (0.2 ~ 0.4)/(18 ~ 22) mol/mL with aqueous sodium carbonate amount ratio.
As preferably, the volume ratio of solution A described in step 3) and described B solution is 4:15.
As preferably, the rate of addition that described solution A drops in step 3) described B solution is 0.1-0.15mL/sec.
There is agglomeration it can be avoided that solution A is added drop-wise to after B solution in dropping at a slow speed, only specifically drip at a slow speed just can make solution A with B solution uniformly, be sufficiently mixed and do not reunite.On the other hand, dropping at a slow speed, being also beneficial to human albumin can by Fluorescein isothiocyanate labelling equably, it is to avoid most of marked by fluorescein isothiocyanate are on a small amount of human albumin.
As preferably, in the dialysis procedure of step 3), the molecular cut off of described bag filter is 7000-10000；The volume ratio of described C solution and aqueous sodium carbonate is 1:(50-150).
As preferably, the titania nanotube in step 4) is through pretreatment: by titania nanotube immersion treatment in aqueous hydrochloric acid solution, be then performing centrifugal separation on obtaining the titania nanotube of pretreatment.
The present invention is in advance by titania nanotube and acid solution effect, reduce the zate current potential on titania nanotube surface, reduce the electrical charge rejection effect of human albumin and material, be conducive to the combination of material and albumen, simultaneously it is also possible that label can be evenly distributed on tagged object.
As preferably, the concentration of described aqueous hydrochloric acid solution is 1mol/L, and described titania nanotube and aqueous hydrochloric acid solution amount ratio are (4 ~ 6): (40 ~ 60) mg/mL, soak time is 4 ~ 8h, and centrifugal rotational speed is 16000 ~ 18000g.
As preferably, the centrifugal separation processes of step 4) particularly as follows: be centrifuged 4-6min by described mixed solution under the rotating speed of 18000-22000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
It is compared with the prior art, the invention has the beneficial effects as follows:
1) Fluorescein isothiocyanate is very firm with the chemical bond of human albumin, recycles the titanium dioxide nano material absorption property to protein, makes green fluorescence on titania nanotube labelling.
2) present invention promotes the combination of fluorescent agent and titania nanotube with human albumin, does not affect the accuracy of cell experiment.
3) use chemical drugs less, experimentation environmental protection.
4) method is easy, cost is low, effective.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 4mL by the Fluorescein isothiocyanate of 0.9mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.5mol is added in the aqueous sodium carbonate that pH is 9 of 20mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.13mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 9000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 9 of 100 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take titania nanotube and be scattered in water to be configured to the solution D of 1mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2.5mg/mL, obtain E solution.When lucifuge by the ratio mixed processing 36h of described solution D Yu described E solution 1:1.1 by volume, then by mixed solution centrifugal 5min under the rotating speed of 20000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Embodiment 2
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 4mL by the Fluorescein isothiocyanate of 0.9mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.5mol is added in the aqueous sodium carbonate that pH is 9 of 20mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.13mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 8000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 9 of 100 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) by the titania nanotube of 5mg immersion treatment 6h in the aqueous hydrochloric acid solution that concentration is 1mol/L of 50mL, under the rotating speed of 17000g, the titania nanotube of pretreatment then it is centrifugally separating to obtain.
5) take pretreated titania nanotube and be scattered in water to be configured to the solution D of 1mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2.5mg/mL, obtain E solution；When lucifuge by the ratio mixed processing 36h of described solution D Yu described E solution 1:1.1 by volume, then by mixed solution centrifugal 5min under the rotating speed of 20000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Embodiment 3
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 2mL by the Fluorescein isothiocyanate of 0.7mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.3mol is added in the aqueous sodium carbonate that pH is 8.5 of 16mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.1mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 7000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 8.5 of 50 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take 4mg titania nanotube immersion treatment 4h in the aqueous hydrochloric acid solution that concentration is 1mol/L of 60mL, under the rotating speed of 16000g, be then centrifugally separating to obtain the titania nanotube of pretreatment.
5) take pretreated titania nanotube and be scattered in water to be configured to the solution D of 1mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2mg/mL, obtain E solution；When lucifuge by the ratio mixed processing 12h of described solution D Yu described E solution 1:1 by volume, then by mixed solution centrifugal 4min under the rotating speed of 18000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Embodiment 4
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 6mL by the Fluorescein isothiocyanate of 1.1mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.7mol is added in the aqueous sodium carbonate that pH is 9.5 of 24mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.15mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 10000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 9.5 of 150 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take 6mg titania nanotube immersion treatment 8h in the aqueous hydrochloric acid solution that concentration is 1mol/L of 40mL, under the rotating speed of 18000g, be then centrifugally separating to obtain the titania nanotube of pretreatment.
5) take pretreated titania nanotube and be scattered in water to be configured to the solution D of 1.5mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 3mg/mL, obtain E solution；When lucifuge by the ratio mixed processing 60h of described solution D Yu described E solution 1:1.2 by volume, then by mixed solution centrifugal 6min under the rotating speed of 22000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Embodiment 5
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 3mL by the Fluorescein isothiocyanate of 0.8mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.4mol is added in the aqueous sodium carbonate that pH is 9 of 18mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.12mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 10000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 9 of 120 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take 5mg titania nanotube immersion treatment 5h in the aqueous hydrochloric acid solution that concentration is 1mol/L of 40mL, under the rotating speed of 17000g, be then centrifugally separating to obtain the titania nanotube of pretreatment.
5) take pretreated titania nanotube and be scattered in water to be configured to the solution D of 1mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2.5mg/mL, obtain E solution；When lucifuge by the ratio mixed processing 24h of described solution D Yu described E solution 1:1 by volume, then by mixed solution centrifugal 5min under the rotating speed of 21000, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Embodiment 6
A kind of method of green fluorescent label titania nanotube, adopts following steps:
1) it is dissolved in the dimethyl sulfoxide of 5mL by the Fluorescein isothiocyanate of 1mol to prepare to obtain solution A, keep in Dark Place.
2) human albumin of 0.6mol is added in the aqueous sodium carbonate that pH is 9 of 22mL, and at room temperature stir to being completely dissolved, prepare to obtain B solution.
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, solution A is dropped in B solution with the speed of 0.14mL/sec, prepare to obtain C solution, then described C solution is transferred in the bag filter that molecular cut off is 7000, then described bag filter is immersed in the aqueous sodium carbonate that pH is 9 of 80 times of quality and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling.
4) take 6mg titania nanotube immersion treatment 4 ~ 8h in the aqueous hydrochloric acid solution that concentration is 1mol/L of 50mL, under the rotating speed of 16000g, be then centrifugally separating to obtain the titania nanotube of pretreatment.
5) take pretreated titania nanotube and be scattered in water to be configured to the solution D of 1mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2.5mg/mL, obtain E solution；When lucifuge by the ratio mixed processing 48h of described solution D Yu described E solution 1:1.1 by volume, then by mixed solution centrifugal 5min under the rotating speed of 19000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
Raw materials used, equipment in the present invention, unless otherwise noted, is the conventional raw material of this area, equipment；Method therefor in the present invention, unless otherwise noted, is the conventional method of this area.
The above, be only presently preferred embodiments of the present invention, not the present invention is imposed any restrictions, every any simple modification, change and equivalent transformation above example made according to the technology of the present invention essence, all still falls within the protection domain of technical solution of the present invention.
Claims (9)
1. the method for a green fluorescent label titania nanotube, it is characterised in that adopt following steps:
1) being dissolved in dimethyl sulfoxide by Fluorescein isothiocyanate and prepare to obtain solution A, keep in Dark Place, wherein Fluorescein isothiocyanate is (0.7 ~ 1.1)/(2 ~ 6) mol/mL with the amount ratio of dimethyl sulfoxide;
2) human albumin is added in the aqueous sodium carbonate that pH is 8.5 ~ 9.5, and at room temperature stir to being completely dissolved, preparing to obtain B solution, wherein human albumin and aqueous sodium carbonate amount ratio are (0.1 ~ 0.5)/(16 ~ 24) mol/mL；
3) described solution A is transferred in syringe, described B solution is transferred in container, when lucifuge, the solution A of 2 ~ 6 parts by volume is dropped in the B solution of 10 ~ 20 parts by volume, prepare to obtain C solution, then described C solution is transferred in bag filter, then described bag filter is immersed in the aqueous sodium carbonate that pH is 8.5 ~ 9.5 and dialyses, until in bag filter solution by muddiness be changed into as clear as crystal after, take out bag filter, obtain human albumin's solution of isothiocyanic acid labelling；
4) take titania nanotube and be scattered in water to be configured to the solution D of 0.5 ~ 1.5mg/mL, then by human albumin's solution dilute concentration of described isothiocyanic acid labelling to 2 ~ 3mg/mL, obtain E solution；When lucifuge by described solution D and described E solution 1:(1 ~ 1.2 by volume) ratio mixed processing 12 ~ 60h, take solid after being then performing centrifugal separation on by mixed solution, prepare the titania nanotube of Green Marker fluorescence.
2. the method for a kind of green fluorescent label titania nanotube as claimed in claim 1, it is characterised in that in step 1), Fluorescein isothiocyanate is (0.8 ~ 1)/(3 ~ 5) mol/mL with the amount ratio of dimethyl sulfoxide.
3. the method for a kind of green fluorescent label titania nanotube as claimed in claim 2, it is characterised in that step 2) in human albumin and aqueous sodium carbonate amount ratio be (0.2 ~ 0.4)/(18 ~ 22) mol/mL.
4. the method for a kind of green fluorescent label titania nanotube as claimed in claim 1, it is characterised in that the volume ratio of solution A described in step 3) and described B solution is 4:15.
5. the method for a kind of green fluorescent label titania nanotube as described in claim 1 or 4, it is characterised in that the rate of addition that described solution A drops in step 3) described B solution is 0.1-0.15mL/sec.
6. the method for a kind of green fluorescent label titania nanotube as claimed in claim 1, it is characterised in that in the dialysis procedure of step 3), the molecular cut off of described bag filter is 7000-10000；The volume ratio of described C solution and aqueous sodium carbonate is 1:(50-150).
7. the method for a kind of green fluorescent label titania nanotube as claimed in claim 1, it is characterized in that, titania nanotube in step 4) is through pretreatment: by titania nanotube immersion treatment in aqueous hydrochloric acid solution, be then performing centrifugal separation on obtaining the titania nanotube of pretreatment.
8. the method for a kind of green fluorescent label titania nanotube as claimed in claim 7, it is characterized in that, the concentration of described aqueous hydrochloric acid solution is 1mol/L, described titania nanotube and aqueous hydrochloric acid solution amount ratio are (4 ~ 6): (40 ~ 60) mg/mL, soak time is 4 ~ 8h, and centrifugal rotational speed is 16000 ~ 18000g.
9. the method for a kind of green fluorescent label titania nanotube as described in claim 1 or 7 or 8, it is characterized in that, the centrifugal separation processes of step 4) particularly as follows: be centrifuged 4-6min by described mixed solution under the rotating speed of 18000-22000g, after isolating solid, with PBS, surplus solution is washed, then be repeatedly performed centrifugal, separate, washing, till no longer having solid to isolate.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525154A (en) * | 2009-03-26 | 2009-09-09 | 上海大学 | Method of using isosulfocyanic acid fluorescein for marking titanium-dioxide nanometer tube |
| CN104165876A (en) * | 2014-08-22 | 2014-11-26 | 江南大学 | Method for rapidly fluorescently detecting hydrogen peroxide on basis of etched nano silver triangle |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525154A (en) * | 2009-03-26 | 2009-09-09 | 上海大学 | Method of using isosulfocyanic acid fluorescein for marking titanium-dioxide nanometer tube |
| CN104165876A (en) * | 2014-08-22 | 2014-11-26 | 江南大学 | Method for rapidly fluorescently detecting hydrogen peroxide on basis of etched nano silver triangle |
Non-Patent Citations (1)
| Title |
|---|
| JUAN XU ET AL.: ""Photokilling cancer cells using highly cell-specific antibody–TiO2"", 《BIOELECTROCHEMISTRY》 * |
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