CN105731534B - A kind of method of green fluorescent label titania nanotube - Google Patents
A kind of method of green fluorescent label titania nanotube Download PDFInfo
- Publication number
- CN105731534B CN105731534B CN201610075345.2A CN201610075345A CN105731534B CN 105731534 B CN105731534 B CN 105731534B CN 201610075345 A CN201610075345 A CN 201610075345A CN 105731534 B CN105731534 B CN 105731534B
- Authority
- CN
- China
- Prior art keywords
- solution
- titanium dioxide
- dioxide nanotubes
- prepare
- human serum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 title claims abstract description 136
- 239000002071 nanotube Substances 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 17
- 108010043121 Green Fluorescent Proteins Proteins 0.000 title claims abstract description 13
- 239000000243 solution Substances 0.000 claims abstract description 152
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 67
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims abstract description 46
- 238000000502 dialysis Methods 0.000 claims abstract description 45
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 26
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 24
- 108091006905 Human Serum Albumin Proteins 0.000 claims abstract description 24
- 229910000029 sodium carbonate Inorganic materials 0.000 claims abstract description 23
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 150000002540 isothiocyanates Chemical class 0.000 claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 102000004506 Blood Proteins Human genes 0.000 claims abstract description 16
- 108010017384 Blood Proteins Proteins 0.000 claims abstract description 16
- 238000001215 fluorescent labelling Methods 0.000 claims abstract description 14
- 239000012460 protein solution Substances 0.000 claims abstract description 10
- 238000005119 centrifugation Methods 0.000 claims abstract description 8
- 238000002372 labelling Methods 0.000 claims abstract description 6
- 238000012546 transfer Methods 0.000 claims description 24
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 22
- 239000007787 solid Substances 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 abstract 2
- 238000005406 washing Methods 0.000 description 11
- 239000002086 nanomaterial Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000013033 photocatalytic degradation reaction Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 2
- 238000005054 agglomeration Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000003929 acidic solution Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 238000003933 environmental pollution control Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G23/00—Compounds of titanium
- C01G23/04—Oxides; Hydroxides
- C01G23/047—Titanium dioxide
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/10—Particle morphology extending in one dimension, e.g. needle-like
- C01P2004/13—Nanotubes
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- General Life Sciences & Earth Sciences (AREA)
- Geology (AREA)
- Inorganic Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及荧光标记领域，公开了一种绿色荧光标记二氧化钛纳米管的方法，包括：1）将异硫氰酸荧光素溶解于二甲基亚砜中配制得A溶液；2）将人血清蛋白添加到碳酸钠水溶液中配制得B溶液；3）将A溶液滴加至B溶液中配制得C溶液，将C溶液进行透析，透析后得到异硫氰酸标记的人血清蛋白溶液；4）取二氧化钛纳米管分散于水中配制成D溶液，将异硫氰酸标记的人血清蛋白溶液加水稀释得到E溶液；将D溶液与E溶液混合处理，经离心分离后制得绿色标记荧光的二氧化钛纳米管。本发明方法的标记物与标记对象的结合牢度高，且标记物不会对细胞产生显著影响，不影响实验结果的科学性。The invention relates to the field of fluorescent labeling, and discloses a method for labeling titanium dioxide nanotubes with green fluorescent light, comprising: 1) dissolving fluorescein isothiocyanate in dimethyl sulfoxide to prepare a solution A; 2) adding human serum albumin Add solution B to sodium carbonate aqueous solution to prepare solution B; 3) Add solution A dropwise to solution B to prepare solution C, dialyze solution C, and obtain isothiocyanate-labeled human serum protein solution after dialysis; 4) take Titanium dioxide nanotubes are dispersed in water to prepare solution D, and human serum protein solution labeled with isothiocyanate is diluted with water to obtain solution E; solution D and solution E are mixed, and after centrifugation, green-labeled fluorescent titanium dioxide nanotubes are prepared . The marker of the method of the present invention has high binding fastness to the marked object, and the marker will not have a significant impact on the cells, and will not affect the scientific nature of the experimental results.
Description
技术领域technical field
本发明涉及荧光标记领域，尤其涉及一种绿色荧光标记二氧化钛纳米管的方法。The invention relates to the field of fluorescent labeling, in particular to a method for green fluorescent labeling of titanium dioxide nanotubes.
背景技术Background technique
二氧化钛纳米材料具有优异的光催化降解有机有害物质的性能，使其在环境污染治理方面发挥巨大的作用，也引起越来越多的科学家对其的关注。不同形貌的二氧化钛纳米材料的光催化降解效率有显著的差异，二氧化钛纳米管的光催化降解有机有毒物质的效率可以是二氧化钛纳米颗粒的几十倍之多。Titanium dioxide nanomaterials have excellent photocatalytic degradation properties of organic harmful substances, making them play a huge role in environmental pollution control, and have attracted more and more scientists' attention. The photocatalytic degradation efficiency of titanium dioxide nanomaterials with different morphologies is significantly different, and the photocatalytic degradation efficiency of titanium dioxide nanotubes can be dozens of times that of titanium dioxide nanoparticles.
同时，对于二氧化钛纳米管的生物毒性的研究也是十分关键的。观察二氧化钛纳米管与细胞的相互作用是研究的主要手段，其中对二氧化钛纳米管的荧光染色是观察二氧化钛纳米管在细胞内部分布情况以及细胞对二氧化钛纳米管胞吞情况最重要的步骤，然而二氧化钛纳米管表面并没有与常见染色剂可进行标记的特性，所以解决对二氧化钛纳米管的荧光染色问题对其与细胞的相互作用等研究中是十分关键的。At the same time, the research on the biological toxicity of titanium dioxide nanotubes is also very critical. Observing the interaction between titanium dioxide nanotubes and cells is the main method of research, among which the fluorescent staining of titanium dioxide nanotubes is the most important step to observe the distribution of titanium dioxide nanotubes in cells and the endocytosis of titanium dioxide nanotubes by cells. However, titanium dioxide nanotubes The surface of the tube does not have the characteristics of being marked with common dyes, so it is very critical to solve the problem of fluorescent staining of titanium dioxide nanotubes and its interaction with cells.
发明内容Contents of the invention
为了解决上述技术问题，本发明提供了一种绿色荧光标记二氧化钛纳米管的方法。本发明通过人血清蛋白与异硫氰酸荧光素的作用先制备合成异硫氰酸荧光素标记的人血清蛋白，然后利用二氧化钛纳米材料与蛋白质的吸附特性间接将异硫氰酸荧光素标记在二氧化钛纳米材料上，该方法不会对细胞产生显著影响，不影响实验结果的科学性。In order to solve the above technical problems, the present invention provides a method for green fluorescent labeling titanium dioxide nanotubes. The present invention prepares and synthesizes human serum protein labeled with fluorescein isothiocyanate through the action of human serum albumin and fluorescein isothiocyanate, and then uses the adsorption properties of titanium dioxide nanomaterials and proteins to indirectly label fluorescein isothiocyanate on the On titanium dioxide nanomaterials, this method will not have a significant impact on cells, and will not affect the scientific nature of the experimental results.
本发明的具体技术方案为：一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：The specific technical scheme of the present invention is: a kind of method for green fluorescent labeling titanium dioxide nanotube, adopt following steps:
1）将异硫氰酸荧光素溶解于二甲基亚砜中配制得A溶液，避光保存，其中异硫氰酸荧光素与二甲基亚砜的用量比为(0.7~1.1)/(2~6)mol/mL。1) Prepare solution A by dissolving fluorescein isothiocyanate in dimethyl sulfoxide, and store it in the dark. The dosage ratio of fluorescein isothiocyanate to dimethyl sulfoxide is (0.7~1.1)/( 2~6) mol/mL.
2）将人血清蛋白添加到pH为8.5~9.5的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液,其中人血清蛋白与碳酸钠水溶液用量比为(0.1~0.5)/(16~24)mol/mL。2) Add human serum albumin to sodium carbonate aqueous solution with a pH of 8.5~9.5, and stir at room temperature until completely dissolved to prepare solution B, wherein the dosage ratio of human serum albumin to sodium carbonate aqueous solution is (0.1~0.5)/ (16~24)mol/mL.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将2~6体积份的A溶液滴加至10~20体积份的B溶液中，配制得C溶液，接着将所述C溶液转移至透析袋中，然后将所述透析袋浸没于pH为8.5~9.5的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution to a syringe, transfer the B solution to a container, add 2 to 6 volume parts of the A solution to 10 to 20 volume parts of the B solution under the condition of avoiding light, and prepare Obtain the C solution, then transfer the C solution to a dialysis bag, then immerse the dialysis bag in an aqueous sodium carbonate solution with a pH of 8.5 to 9.5 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent, Take out the dialysis bag to obtain isothiocyanate-labeled human serum albumin solution.
4）取二氧化钛纳米管分散于水中配制成0.5~1.5mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2~3mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1：（1~1.2）的比例混合处理12~60h，然后将混合溶液经离心分离后取固体，制得绿色标记荧光的二氧化钛纳米管。4) Disperse titanium dioxide nanotubes in water to prepare a 0.5-1.5 mg/mL D solution, then add water to dilute the isothiocyanate-labeled human serum protein solution to a concentration of 2-3 mg/mL to obtain an E solution; Under the condition of avoiding light, the D solution and the E solution are mixed according to the volume ratio of 1: (1~1.2) for 12~60h, and then the mixed solution is centrifuged and the solid is taken to prepare green-labeled fluorescent titanium dioxide nanotube.
本发明通过人血清蛋白与异硫氰酸荧光素的作用先制备合成异硫氰酸荧光素标记的人血清蛋白，然后利用二氧化钛纳米管与蛋白质的吸附特性间接将异硫氰酸荧光素标记在二氧化钛纳米管上，该方法不会对细胞产生显著影响，不影响实验结果的科学性。The present invention first prepares and synthesizes human serum protein labeled with fluorescein isothiocyanate through the action of human serum protein and fluorescein isothiocyanate, and then uses the adsorption properties of titanium dioxide nanotubes and proteins to indirectly label fluorescein isothiocyanate on the On titanium dioxide nanotubes, this method will not have a significant impact on the cells, and will not affect the scientific nature of the experimental results.
作为优选，步骤1）中异硫氰酸荧光素与二甲基亚砜的用量比为(0.8~1)/(3~5)mol/mL。Preferably, the dosage ratio of fluorescein isothiocyanate to dimethyl sulfoxide in step 1) is (0.8~1)/(3~5) mol/mL.
作为优选，步骤2）中人血清蛋白与碳酸钠水溶液用量比为(0.2~0.4)/(18~22)mol/mL。Preferably, the dosage ratio of human serum albumin to sodium carbonate aqueous solution in step 2) is (0.2-0.4)/(18-22) mol/mL.
作为优选，步骤3）中所述A溶液与所述B溶液的体积比为4:15。As a preference, the volume ratio of the A solution to the B solution in step 3) is 4:15.
作为优选，步骤3）中将所述A溶液滴加至所述B溶液的滴加速度为0.1-0.15mL/sec。Preferably, in step 3), the dropping rate of the solution A to the solution B is 0.1-0.15mL/sec.
慢速滴加能够避免A溶液滴加到B溶液后发生团聚现象，只有在特定的慢速滴加才能使A溶液与B溶液均匀、充分地混合而不发生团聚。另一方面，慢速的滴加，也有利于人血清蛋白能够被异硫氰酸荧光素均匀地标记，避免大多数异硫氰酸荧光素标记于少量的人血清蛋白上。Slow dripping can avoid agglomeration after A solution is added dropwise to B solution. Only at a specific slow rate can A solution and B solution be mixed uniformly and fully without agglomeration. On the other hand, slow dripping is also beneficial for human serum protein to be uniformly labeled by FITC, avoiding most of FITC labeling on a small amount of human serum protein.
作为优选，在步骤3）的透析过程中，所述透析袋的截留分子量为7000-10000；所述C溶液与碳酸钠水溶液的体积比为1:(50-150)。Preferably, during the dialysis process in step 3), the molecular weight cut-off of the dialysis bag is 7000-10000; the volume ratio of the C solution to the sodium carbonate aqueous solution is 1:(50-150).
作为优选，步骤4）中的二氧化钛纳米管经过预处理：将二氧化钛纳米管在盐酸水溶液中浸泡处理，然后经离心分离得到预处理的二氧化钛纳米管。Preferably, the titanium dioxide nanotubes in step 4) are pretreated: the titanium dioxide nanotubes are soaked in hydrochloric acid aqueous solution, and then centrifuged to obtain pretreated titanium dioxide nanotubes.
本发明预先将二氧化钛纳米管与酸性溶液作用，减小了二氧化钛纳米管表面的zate电位，减弱了人血清蛋白与材料的电荷排斥作用，有利于材料与蛋白的结合，同时也能够使得标记物能够均匀地分布在标记对象上。The present invention reacts titanium dioxide nanotubes with acidic solution in advance, reduces the zate potential on the surface of titanium dioxide nanotubes, weakens the charge repulsion between human serum albumin and materials, facilitates the combination of materials and proteins, and also enables markers to Evenly distributed over marked objects.
作为优选，所述盐酸水溶液的浓度为1mol/L，所述二氧化钛纳米管与盐酸水溶液用量比为(4~6):( 40~60)mg/mL，浸泡时间为4~8h，离心转速为16000~18000g。As preferably, the concentration of the aqueous hydrochloric acid solution is 1mol/L, the dosage ratio of the titanium dioxide nanotubes to the aqueous hydrochloric acid solution is (4～6):(40～60) mg/mL, the soaking time is 4～8h, and the centrifugal speed is 16000~18000g.
作为优选，步骤4）的离心分离过程具体为：将所述混合溶液在18000-22000g的转速下离心4-6min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。Preferably, the centrifugation process in step 4) specifically includes: centrifuging the mixed solution at a rotational speed of 18000-22000g for 4-6min, after separating the solid, washing the remaining solution with PBS buffer, and then repeatedly centrifuging, Separate, wash until no more solid separates out.
与现有技术对比，本发明的有益效果是：Compared with prior art, the beneficial effect of the present invention is:
1）异硫氰酸荧光素与人血清蛋白的化学键作用十分牢靠，再利用二氧化钛纳米材料对蛋白质的吸附性能，使二氧化钛纳米管标记上绿色荧光。1) The chemical bond between fluorescein isothiocyanate and human serum protein is very strong, and then use the adsorption performance of titanium dioxide nanomaterials on proteins to make the titanium dioxide nanotubes marked with green fluorescence.
2）本发明用人血清蛋白促使荧光剂与二氧化钛纳米管的结合，不影响细胞实验的准确性。2) The present invention uses human serum albumin to promote the combination of fluorescent agent and titanium dioxide nanotubes without affecting the accuracy of cell experiments.
3）使用化学药品较少，实验过程绿色环保。3) Less chemicals are used, and the experimental process is green and environmentally friendly.
4）方法简便、成本低、效果好。4) The method is simple, low in cost and good in effect.
具体实施方式detailed description
下面结合实施例对本发明作进一步的描述。The present invention will be further described below in conjunction with embodiment.
实施例1Example 1
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将0.9mol的异硫氰酸荧光素溶解于4mL的二甲基亚砜中配制得A溶液，避光保存。1) Dissolve 0.9 mol of fluorescein isothiocyanate in 4 mL of dimethyl sulfoxide to prepare solution A, and store in the dark.
2）将0.5mol的人血清蛋白添加到20mL的 pH为9的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.5mol of human serum albumin to 20mL of sodium carbonate aqueous solution with a pH of 9, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.13mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为9000的透析袋中，然后将所述透析袋浸没于100倍质量的pH为9的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution to a syringe, transfer the B solution to a container, and drop the A solution into the B solution at a rate of 0.13mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 9000, and then immerse the dialysis bag in 100 times the mass of an aqueous sodium carbonate solution with a pH of 9 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）取二氧化钛纳米管分散于水中配制成1mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2.5mg/mL，得到E溶液。在避光条件下将所述D溶液与所述E溶液按体积比1:1.1的比例混合处理36h，然后将混合溶液在20000g的转速下离心5min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。4) Disperse titanium dioxide nanotubes in water to prepare 1 mg/mL D solution, and then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 2.5 mg/mL to obtain E solution. Under light-shielding conditions, the D solution and the E solution were mixed in a volume ratio of 1:1.1 for 36 hours, and then the mixed solution was centrifuged at a speed of 20000g for 5 minutes. The solution is washed, and then centrifuged, separated, and washed repeatedly until no solids are separated.
实施例2Example 2
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将0.9mol的异硫氰酸荧光素溶解于4mL的二甲基亚砜中配制得A溶液，避光保存。1) Dissolve 0.9 mol of fluorescein isothiocyanate in 4 mL of dimethyl sulfoxide to prepare solution A, and store in the dark.
2）将0.5mol的人血清蛋白添加到20mL的 pH为9的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.5mol of human serum albumin to 20mL of sodium carbonate aqueous solution with a pH of 9, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.13mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为8000的透析袋中，然后将所述透析袋浸没于100倍质量的pH为9的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution to a syringe, transfer the B solution to a container, and drop the A solution into the B solution at a rate of 0.13mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 8000, and then immerse the dialysis bag in 100 times the mass of an aqueous sodium carbonate solution with a pH of 9 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）将5mg的二氧化钛纳米管在50mL的浓度为1 mol/L的盐酸水溶液中浸泡处理6h，然后在17000g的转速下离心分离得到预处理的二氧化钛纳米管。4) Soak 5 mg of titanium dioxide nanotubes in 50 mL of 1 mol/L hydrochloric acid aqueous solution for 6 hours, and then centrifuge at a speed of 17000 g to obtain pretreated titanium dioxide nanotubes.
5）取预处理后的二氧化钛纳米管分散于水中配制成1mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2.5mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1:1.1的比例混合处理36h，然后将混合溶液在20000g的转速下离心5min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。5) Disperse the pretreated titanium dioxide nanotubes in water to prepare a 1 mg/mL D solution, then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 2.5 mg/mL to obtain an E solution; Under the condition of avoiding light, the D solution and the E solution were mixed and treated for 36 hours according to the volume ratio of 1:1.1, and then the mixed solution was centrifuged at a speed of 20000g for 5 minutes. After the solid was separated, the remaining solution was treated with PBS buffer. Washing is carried out, and then centrifugation, separation, and washing are repeated until no solids are separated.
实施例3Example 3
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将0.7mol的异硫氰酸荧光素溶解于2mL的二甲基亚砜中配制得A溶液，避光保存。1) Dissolve 0.7 mol of fluorescein isothiocyanate in 2 mL of dimethyl sulfoxide to prepare solution A, and store in the dark.
2）将0.3mol的人血清蛋白添加到16mL的 pH为8.5的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.3mol of human serum albumin to 16mL of sodium carbonate aqueous solution with a pH of 8.5, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.1mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为7000的透析袋中，然后将所述透析袋浸没于50倍质量的pH为8.5的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution into a syringe, transfer the B solution into a container, and drop the A solution into the B solution at a rate of 0.1mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 7000, and then immerse the dialysis bag in 50 times the mass of an aqueous sodium carbonate solution with a pH of 8.5 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）取4mg二氧化钛纳米管在60mL的浓度为1 mol/L的盐酸水溶液中浸泡处理4h，然后在16000g的转速下离心分离得到预处理的二氧化钛纳米管。4) Soak 4 mg of titanium dioxide nanotubes in 60 mL of 1 mol/L hydrochloric acid aqueous solution for 4 hours, and then centrifuge at a speed of 16000 g to obtain pretreated titanium dioxide nanotubes.
5）取预处理后的二氧化钛纳米管分散于水中配制成1mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1:1的比例混合处理12h，然后将混合溶液在18000g的转速下离心4min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。5) Disperse the pretreated titanium dioxide nanotubes in water to prepare a 1 mg/mL D solution, then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 2 mg/mL to obtain an E solution; Under light conditions, the D solution and the E solution were mixed at a volume ratio of 1:1 for 12 hours, and then the mixed solution was centrifuged at a speed of 18000g for 4 minutes, and after the solid was separated, the remaining solution was treated with PBS buffer solution. Washing, and then repeated centrifugation, separation, and washing until no solids are separated.
实施例4Example 4
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将1.1mol的异硫氰酸荧光素溶解于6mL的二甲基亚砜中配制得A溶液，避光保存。1) Prepare A solution by dissolving 1.1mol of fluorescein isothiocyanate in 6mL of dimethyl sulfoxide, and store in the dark.
2）将0.7mol的人血清蛋白添加到24mL的 pH为9.5的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.7mol of human serum albumin to 24mL of sodium carbonate aqueous solution with a pH of 9.5, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.15mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为10000的透析袋中，然后将所述透析袋浸没于150倍质量的pH为9.5的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution to a syringe, transfer the B solution to a container, and drop the A solution into the B solution at a rate of 0.15mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 10,000, and then immerse the dialysis bag in 150 times the mass of an aqueous solution of sodium carbonate with a pH of 9.5 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）取6mg二氧化钛纳米管在40mL的浓度为1 mol/L的盐酸水溶液中浸泡处理8h，然后在18000g的转速下离心分离得到预处理的二氧化钛纳米管。4) Soak 6 mg of titanium dioxide nanotubes in 40 mL of 1 mol/L hydrochloric acid aqueous solution for 8 hours, and then centrifuge at a speed of 18000 g to obtain pretreated titanium dioxide nanotubes.
5）取预处理后的二氧化钛纳米管分散于水中配制成1.5mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至3mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1: 1.2的比例混合处理60h，然后将混合溶液在22000g的转速下离心6min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。5) Disperse the pretreated titanium dioxide nanotubes in water to prepare a 1.5 mg/mL D solution, then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 3 mg/mL to obtain an E solution; Under light-shielding conditions, the D solution and the E solution were mixed and treated for 60 h at a volume ratio of 1: 1.2, and then the mixed solution was centrifuged at a speed of 22000 g for 6 min. After the solid was separated, the remaining solution was treated with PBS buffer. Washing is carried out, and then centrifugation, separation, and washing are repeated until no solids are separated.
实施例5Example 5
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将0.8mol的异硫氰酸荧光素溶解于3mL的二甲基亚砜中配制得A溶液，避光保存。1) Prepare A solution by dissolving 0.8mol of fluorescein isothiocyanate in 3mL of dimethyl sulfoxide, and store in the dark.
2）将0.4mol的人血清蛋白添加到18mL的 pH为9的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.4mol of human serum albumin to 18mL of sodium carbonate aqueous solution with a pH of 9, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.12mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为10000的透析袋中，然后将所述透析袋浸没于120倍质量的pH为9的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution into a syringe, transfer the B solution into a container, and drop the A solution into the B solution at a rate of 0.12mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 10,000, and then immerse the dialysis bag in 120 times the mass of an aqueous sodium carbonate solution with a pH of 9 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）取5mg二氧化钛纳米管在40mL的浓度为1 mol/L的盐酸水溶液中浸泡处理5h，然后在17000g的转速下离心分离得到预处理的二氧化钛纳米管。4) Soak 5 mg of titanium dioxide nanotubes in 40 mL of 1 mol/L hydrochloric acid aqueous solution for 5 hours, and then centrifuge at a speed of 17000 g to obtain pretreated titanium dioxide nanotubes.
5）取预处理后的二氧化钛纳米管分散于水中配制成1mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2.5mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1:1的比例混合处理24h，然后将混合溶液在21000的转速下离心5min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。5) Disperse the pretreated titanium dioxide nanotubes in water to prepare a 1 mg/mL D solution, then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 2.5 mg/mL to obtain an E solution; Under the condition of avoiding light, the D solution and the E solution were mixed at a volume ratio of 1:1 for 24 hours, and then the mixed solution was centrifuged at a speed of 21000 for 5 minutes. After the solid was separated, the remaining solution was treated with PBS buffer. Washing is carried out, and then centrifugation, separation, and washing are repeated until no solids are separated.
实施例6Example 6
一种绿色荧光标记二氧化钛纳米管的方法，采用如下步骤：A method for green fluorescent labeling of titanium dioxide nanotubes, using the following steps:
1）将1mol的异硫氰酸荧光素溶解于5mL的二甲基亚砜中配制得A溶液，避光保存。1) Prepare solution A by dissolving 1mol of fluorescein isothiocyanate in 5mL of dimethyl sulfoxide, and store in the dark.
2）将0.6mol的人血清蛋白添加到22mL的 pH为9的碳酸钠水溶液中，并在室温下搅拌至完全溶解，配制得B溶液。2) Add 0.6mol of human serum albumin to 22mL of sodium carbonate aqueous solution with a pH of 9, and stir at room temperature until completely dissolved to prepare solution B.
3）将所述A溶液转移至注射器中，将所述B溶液转移至容器中，在避光条件下将A溶液以0.14mL/sec的速度滴加至B溶液中，配制得C溶液，接着将所述C溶液转移至截留分子量为7000的透析袋中，然后将所述透析袋浸没于80倍质量的pH为9的碳酸钠水溶液中进行透析，直至透析袋中溶液由浑浊转变为清澈透明后，取出透析袋，得到异硫氰酸标记的人血清蛋白溶液。3) Transfer the A solution into a syringe, transfer the B solution into a container, and drop the A solution into the B solution at a rate of 0.14mL/sec under light-proof conditions to prepare the C solution, and then Transfer the C solution to a dialysis bag with a molecular weight cut-off of 7000, and then immerse the dialysis bag in 80 times the mass of an aqueous sodium carbonate solution with a pH of 9 for dialysis until the solution in the dialysis bag changes from turbid to clear and transparent Finally, the dialysis bag was taken out to obtain an isothiocyanate-labeled human serum albumin solution.
4）取6mg二氧化钛纳米管在50mL的浓度为1 mol/L的盐酸水溶液中浸泡处理4~8h，然后在16000g的转速下离心分离得到预处理的二氧化钛纳米管。4) Soak 6 mg of titanium dioxide nanotubes in 50 mL of 1 mol/L hydrochloric acid aqueous solution for 4-8 hours, and then centrifuge at a speed of 16000 g to obtain pretreated titanium dioxide nanotubes.
5）取预处理后的二氧化钛纳米管分散于水中配制成1mg/mL的D溶液，再将所述异硫氰酸标记的人血清蛋白溶液加水稀释浓度至2.5mg/mL，得到E溶液；在避光条件下将所述D溶液与所述E溶液按体积比1:1.1的比例混合处理48h，然后将混合溶液在19000g的转速下离心5min，分离出固体后，用PBS缓冲液对剩余溶液进行洗涤，然后反复进行离心、分离、洗涤，直至不再有固体分离出为止。5) Disperse the pretreated titanium dioxide nanotubes in water to prepare a 1 mg/mL D solution, then dilute the isothiocyanate-labeled human serum protein solution with water to a concentration of 2.5 mg/mL to obtain an E solution; Under the condition of avoiding light, the D solution and the E solution were mixed at a volume ratio of 1:1.1 for 48h, and then the mixed solution was centrifuged at a speed of 19000g for 5min, and after the solid was separated, the remaining solution was treated with PBS buffer. Washing is carried out, and then centrifugation, separation, and washing are repeated until no solids are separated.
本发明中所用原料、设备，若无特别说明，均为本领域的常用原料、设备；本发明中所用方法，若无特别说明，均为本领域的常规方法。Raw materials used in the present invention, equipment, if not specified, are commonly used raw materials, equipment in this area; Method used in the present invention, if not specified, are conventional methods in this area.
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制，凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效变换，均仍属于本发明技术方案的保护范围。The above are only preferred embodiments of the present invention, and do not limit the present invention in any way. All simple modifications, changes and equivalent transformations made to the above embodiments according to the technical essence of the present invention still belong to the technical solution of the present invention. scope of protection.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610075345.2A CN105731534B (en) | 2016-02-03 | 2016-02-03 | A kind of method of green fluorescent label titania nanotube |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610075345.2A CN105731534B (en) | 2016-02-03 | 2016-02-03 | A kind of method of green fluorescent label titania nanotube |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105731534A CN105731534A (en) | 2016-07-06 |
| CN105731534B true CN105731534B (en) | 2017-06-23 |
Family
ID=56244827
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610075345.2A Active CN105731534B (en) | 2016-02-03 | 2016-02-03 | A kind of method of green fluorescent label titania nanotube |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105731534B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525154B (en) * | 2009-03-26 | 2011-05-04 | 上海大学 | Method of using isosulfocyanic acid fluorescein for marking titanium-dioxide nanometer tube |
| CN104165876B (en) * | 2014-08-22 | 2016-08-24 | 江南大学 | The method of rapid fluorescence detection hydrogen peroxide based on etching nanometer silver triangle |
-
2016
- 2016-02-03 CN CN201610075345.2A patent/CN105731534B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105731534A (en) | 2016-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105524441A (en) | High-polymer vesicle containing AIE (aggregation-induced emission) molecules as well as preparation method and application of high-polymer vesicle | |
| CN106834204B (en) | A kind of SFL microcarrier for cell culture and its preparation method and application | |
| CN110330672A (en) | The preparation method of poly(N-isopropylacrylamide) inverse opal hydrogel | |
| CN107325303B (en) | A kind of non-degumming silk fiber solution, preparation method and use thereof | |
| CN104789225A (en) | Quantum dots-fibroin gel fluorescence nanometer anti-fake material and preparation method thereof | |
| CN104694454B (en) | A kind of microcarrier for cell culture and its preparation method and application | |
| CN105731534B (en) | A kind of method of green fluorescent label titania nanotube | |
| CN107138736A (en) | Preparation method and application of aggregation-state phosphorescent copper nanocluster | |
| CN107462728A (en) | A kind of method for differentiating Ancient Silk Textile relic silk species based on immune vestige method | |
| CN101319101A (en) | A kind of preparation method of water-soluble nanocrystal | |
| CN109603771A (en) | A kind of preparation method of magnetic chitosan-montmorillonite nanocomposite microspheres | |
| CN1896738A (en) | Fluorescent nano-particle with surface biological function, its production and use | |
| CN114214225B (en) | A method for enriching holdfast secreted by Caulobacter crescentus | |
| CN105802605A (en) | Preparation method of fluorogold nanocomposite used for cell imaging | |
| CN105738333B (en) | A kind of method of red fluorescence mark graphene titanic oxide nano compound material | |
| CN112779000B (en) | A kind of quantum dot modified with quaternized multidentate ligand and its preparation method and application | |
| CN101008642B (en) | Preparation method of nanometer semiconductor biocompatible materials | |
| CN105731441B (en) | A kind of method of red fluorescence mark graphene oxide | |
| CN107674669A (en) | A kind of macromolecule composite coding microballoon and preparation method thereof | |
| CN106177949A (en) | A kind of fluorescence magnetic bimodal nano-carrier and preparation method | |
| CN115606589B (en) | A method for stabilizing liquid-liquid condensed phase and its application | |
| CN105861560A (en) | Preparation method and application of low-toxicity mesoporous silica gene nano-carriers | |
| CN113425853B (en) | Glutathione-modified brain-targeted zinc oxide quantum dot gene transport carrier and preparation method thereof | |
| CN1219921C (en) | High-concentration regenerated silk protein aqueous solution and preparation method thereof | |
| CN112080012B (en) | Method for packaging and releasing semiconductor fluorescent quantum dots based on hyperbranched polyamine-fatty acid supermolecule self-assembly and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |