技术领域technical field
本发明涉及生物化学技术领域，更具体地涉及一种同步X射线可见的成像标签及其制备方法。The invention relates to the technical field of biochemistry, in particular to a synchronous X-ray visible imaging label and a preparation method thereof.
背景技术Background technique
显微成像技术是细胞生命科学发展的主要推动力之一。基于同步X射线的显微技术在细胞成像领域具有独特的优势。由于X射线的波长在0.1-10nm范围内，因此其天然就是一种超分辨显微成像技术，分辨率理论上能够达到数个纳米。另外，与电子束相比，X射线对生物样品的穿透力更强，因此不需要经过切片等处理就能对完整细胞进行成像。更重要的是，X射线显微成像技术具有很好的能量分辨，能精确分辨很多元素的吸收谱。因此，结合X射线敏感的成像探针，能够实现对细胞内生物分子的高分辨识别和成像。Microscopic imaging technology is one of the main driving forces for the development of cell life science. Microscopy based on synchrotron X-rays has unique advantages in the field of cell imaging. Since the wavelength of X-rays is in the range of 0.1-10nm, it is naturally a super-resolution microscopic imaging technology, and the resolution can theoretically reach several nanometers. In addition, compared with electron beams, X-rays are more penetrating to biological samples, so intact cells can be imaged without processing such as slicing. More importantly, X-ray microscopic imaging technology has good energy resolution and can accurately distinguish the absorption spectra of many elements. Therefore, combined with X-ray-sensitive imaging probes, high-resolution recognition and imaging of intracellular biomolecules can be achieved.
在光学显微成像中，荧光标签常被用来标记生物分子。在X射线成像中，同样可以利用X射线本身的特点来开发成像标签，从而应用于制备生物探针，实现对细胞内生物分子的识别和成像。目前，研究人员已经应用X射线对金属纳米颗粒的特征吸收，在纳米颗粒上连接抗体从而对特定生物分子进行识别并成像。但是，细胞内含有大量竞争性生物分子，如何保证对感兴趣生物分子的标记特异性是一大瓶颈。因此，现阶段开发新型同步X射线可见的成像标签，实现对细胞内生物分子的精确识别和定位具有十分重要的意义。Fluorescent tags are often used to label biomolecules in optical microscopy. In X-ray imaging, the characteristics of X-rays can also be used to develop imaging tags, which can be applied to the preparation of biological probes to realize the recognition and imaging of intracellular biomolecules. At present, researchers have applied the characteristic absorption of X-rays to metal nanoparticles, and attached antibodies to the nanoparticles to identify and image specific biomolecules. However, cells contain a large number of competing biomolecules, and how to ensure the specificity of labeling biomolecules of interest is a major bottleneck. Therefore, it is of great significance to develop new synchronous X-ray visible imaging tags at this stage to realize the precise identification and positioning of intracellular biomolecules.
发明内容Contents of the invention
本发明的目的是提供一种同步X射线可见的成像标签及其制备方法，从而解决现有X射线显微成像技术中还无法实现对细胞内生物分子的精确识别和定位的问题。The purpose of the present invention is to provide a synchronous X-ray visible imaging label and its preparation method, so as to solve the problem that the existing X-ray microscopic imaging technology cannot realize the precise identification and positioning of intracellular biomolecules.
为了解决上述技术问题，本发明采用以下技术方案：In order to solve the above technical problems, the present invention adopts the following technical solutions:
根据本发明的第一方面，提供一种同步X射线可见的成像标签的制备方法，该制备方法包括以下步骤：1)将底物分子溶解于缓冲体系中；2)加入针对所述底物分子具有催化活性的酶或者小分子，混匀；3)反应一段时间至聚合物生成；以及4)将含有所述聚合物的悬液滴加在同步成像基底上，同步X射线成像观察。According to the first aspect of the present invention, there is provided a method for preparing a synchronous X-ray visible imaging label, the preparation method comprising the following steps: 1) dissolving the substrate molecule in a buffer system; 2) adding Mix the enzyme or small molecule with catalytic activity; 3) react for a period of time until the polymer is formed; and 4) drop the suspension containing the polymer on the synchronous imaging substrate, and observe with synchronous X-ray imaging.
根据本发明所提供的方法，其工作原理为：具有催化活性的酶或者小分子催化底物分子聚合，生成X射线可见的聚合物，从而制备出一种同步X射线可见的成像标签。According to the method provided by the present invention, its working principle is: an enzyme with catalytic activity or a small molecule catalyzes the polymerization of substrate molecules to generate an X-ray visible polymer, thereby preparing a synchronous X-ray visible imaging label.
步骤1)中的所述缓冲体系包括磷酸盐缓冲液(phosphate buffer saline，PBS)、二甲胂酸钠缓冲液(sodium cacodylate buffer)、三羟甲基氨基甲烷-盐酸缓冲液(Tris-HCl buffer)或其他适合相应底物分子的缓冲液。The buffer system in step 1) includes phosphate buffer saline (phosphate buffer saline, PBS), sodium cacodylate buffer (sodium cacodylate buffer), tris-hydrochloric acid buffer (Tris-HCl buffer ) or other buffers suitable for the corresponding substrate molecules.
步骤1)中所述缓冲液的pH值为7.2～7.6。其中，最佳地为7.4。The pH value of the buffer solution in step 1) is 7.2-7.6. Among them, the best is 7.4.
步骤1)中所述底物分子为3,3'-二氨基联苯胺盐酸盐(DAB)，金属增强型DAB(Metal Enhanced DAB)或EnzMet。The substrate molecule in step 1) is 3,3'-diaminobenzidine hydrochloride (DAB), metal enhanced DAB (Metal Enhanced DAB) or EnzMet.
其中所述底物分子浓度为0.2～5mM。根据不同的底物分子可选择最适的底物分子浓度。Wherein the concentration of the substrate molecule is 0.2-5 mM. According to different substrate molecules, the optimal concentration of substrate molecules can be selected.
步骤2)中所述具有催化活性的酶或者小分子为抗坏血酸过氧化物酶(APEX、APEX2)，迷你单线态氧产生蛋白(miniSOG)，辣根过氧化物酶(HRP)，曙红(eosin)，亚甲基蓝(methylene blue)，二溴荧光素(DBF)，荧光素(Fluorescein)，TAMRA，Br-TAMRA，Bromo-Cy5，AF488，AF633，ReAsH-EDT2或700DX等等。The enzymes or small molecules with catalytic activity described in step 2) are ascorbate peroxidase (APEX, APEX2), mini singlet oxygen generating protein (miniSOG), horseradish peroxidase (HRP), eosin (eosin ), methylene blue, dibromofluorescein (DBF), fluorescein (Fluorescein), TAMRA, Br-TAMRA, Bromo-Cy5, AF488, AF633, ReAsH-EDT2 or 700DX and so on.
针对具有催化活性的酶或小分子，可考虑在反应时加入光照条件。例如，对于APEX，APEX2和HRP来说，光照并非必需条件。对于miniSOG，eosin，methylene blue，DBF，Fluorescein，TAMRA，Br-TAMRA，Bromo-Cy5，AF488，AF633，ReAsH-EDT2和700DX来说，光照则为必需条件。其中的混匀方法为使用枪头吹打混匀或者漩涡振荡器振荡混匀。For enzymes or small molecules with catalytic activity, light conditions may be added during the reaction. For example, for APEX, APEX2 and HRP, lighting is not a requirement. For miniSOG, eosin, methylene blue, DBF, Fluorescein, TAMRA, Br-TAMRA, Bromo-Cy5, AF488, AF633, ReAsH-EDT2 and For the 700DX, lighting is a must. Wherein the mixing method is to use a pipette tip to blow and mix or vortex oscillator to vibrate and mix.
步骤3)中的反应温度为4～37℃。实际应用过程中根据选择的不同的酶或小分子，可选择最优的温度。The reaction temperature in step 3) is 4-37°C. In the actual application process, the optimal temperature can be selected according to the selected enzymes or small molecules.
步骤3)的反应时间为5min～2h。根据选择的不同的酶或小分子，可选择最优的反应时间。The reaction time of step 3) is 5min～2h. According to the different enzymes or small molecules selected, the optimal reaction time can be selected.
步骤4)中的所述成像基底为氮化硅窗口、铜网碳支持膜或Mylar膜。根据不同的底物分子选择最优的成像基底。氮化硅窗口厚度为50-150nm，最佳为100nm。铜网碳膜厚度为5～100nm，最佳为20nm。Mylar膜厚度为2～25μm，最佳为10μm。The imaging substrate in step 4) is a silicon nitride window, a copper mesh carbon support film or a Mylar film. Choose the optimal imaging substrate according to different substrate molecules. The thickness of the silicon nitride window is 50-150nm, preferably 100nm. The thickness of the copper mesh carbon film is 5-100nm, preferably 20nm. The thickness of the Mylar film is 2-25 μm, preferably 10 μm.
步骤4)中的所述同步X射线成像的能量为280～20000eV，根据不同的底物分子可选择不同的入射能量。其中，DAB最优成像能量为350～850eV，金属增强型DAB最优成像能量为500～8000eV。EnzMet最优成像能量为500～7000eV。The energy of the simultaneous X-ray imaging in step 4) is 280-20000 eV, and different incident energies can be selected according to different substrate molecules. Among them, the optimal imaging energy of DAB is 350-850eV, and the optimal imaging energy of metal-enhanced DAB is 500-8000eV. The optimal imaging energy of EnzMet is 500-7000eV.
步骤4)中的所述同步X射线成像的分辨率为20～200nm。The resolution of the simultaneous X-ray imaging in step 4) is 20-200 nm.
根据本发明的第二方面，还提供一种根据所述制备方法制得的同步辐射X射线可见的成像标签。According to the second aspect of the present invention, a synchrotron radiation X-ray visible imaging label prepared according to the preparation method is also provided.
本发明的积极进步效果在于：新的显微成像技术和与之相适合的分子探针对于更好地理解细胞生命过程具有十分重要的意义。但是，目前X射线显微技术多用于细胞结构成像，和该技术相适合的特异性识别细胞内重要生物靶标的分子探针仍较缺乏。本发明利用X射线具有良好的能量分辨的特点，经体外化学催化反应成功制备了同步X射线可见的成像标签，为进一步制备X射线敏感的分子探针以及实现对细胞内生物分子的特异性识别和成像打下了良好的基础。The positive progress effect of the present invention lies in that the new microscopic imaging technology and the molecular probes suitable therefor are very important for better understanding the life process of cells. However, at present, X-ray microscopy technology is mostly used for imaging cell structures, and molecular probes suitable for this technology to specifically recognize important biological targets in cells are still lacking. The invention utilizes the characteristics of good energy resolution of X-rays, and successfully prepares an imaging label visible to synchronous X-rays through in vitro chemical catalytic reaction, in order to further prepare X-ray-sensitive molecular probes and realize specific recognition of intracellular biomolecules And imaging has laid a good foundation.
附图说明Description of drawings
图1是纯化的APEX2蛋白的SDS-PAGE图，其中泳道1为标准分子量蛋白Marker，泳道2为纯化的APEX2蛋白；Figure 1 is the SDS-PAGE figure of the purified APEX2 protein, wherein lane 1 is the standard molecular weight protein Marker, and lane 2 is the purified APEX2 protein;
图2A是同步辐射X射线对DAB聚合物的成像图，图2B是同步辐射X射线对标准靶的成像图；Fig. 2A is the imaging diagram of DAB polymer by synchrotron radiation X-ray, and Fig. 2B is the imaging diagram of standard target by synchrotron radiation X-ray;
图3A、图3B、图3C、图3D分别是APEX2，HRP，miniSOG和700DX催化DAB分子生成聚合物的同步X射线成像图；Figure 3A, Figure 3B, Figure 3C, Figure 3D are APEX2, HRP, miniSOG and Simultaneous X-ray imaging of 700DX catalyzing DAB molecules to form polymers;
图4A、图4B、图4C分别是APEX2催化DAB，金属增强型DAB和EnzMet生成聚合物的同步X射线成像图。Figure 4A, Figure 4B, and Figure 4C are the simultaneous X-ray imaging images of APEX2-catalyzed DAB, metal-enhanced DAB, and EnzMet-generated polymers, respectively.
具体实施方式Detailed ways
以下结合具体实施例，对本发明做进一步说明。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。本发明所用试剂和原料均市售可得。The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. The reagents and raw materials used in the present invention are all commercially available.
本发明主要选择DAB作为底物分子代表，催化活性的酶以APEX2为主，制备了同步X射线可见的成像标签，并应用于同步X射线成像研究，以下实施例具体说明本发明的实施效果。In the present invention, DAB is mainly selected as the representative substrate molecule, and the catalytically active enzyme is mainly APEX2, and a synchronous X-ray visible imaging label is prepared and applied to synchronous X-ray imaging research. The following examples specifically illustrate the implementation effect of the present invention.
实施例1 APEX2蛋白的表达与纯化Example 1 Expression and purification of APEX2 protein
pTRC99A-APEX2质粒(Addgene plasmid#72558)购自addgene。该pTRC99A-APEX2质粒的序列如SEQ ID No：1所示。The pTRC99A-APEX2 plasmid (Addgene plasmid #72558) was purchased from addgene. The sequence of the pTRC99A-APEX2 plasmid is shown in SEQ ID No:1.
BL21-DE3大肠杆菌感受态细胞购自天根生化科技(北京)有限公司。取50ngpTRC99A-APEX2质粒加入感受态细胞，冰上放置30min，42℃热激，加入LB培养基，37℃、150rpm培养1h，取适量菌液涂布于氨苄抗性的LB琼脂糖平板上，培养过夜。挑取单克隆，接种到1mL氨苄抗性的LB培养基中，37℃、220rpm培养过夜。将1mL菌液接种到500mL氨苄抗性的LB培养基中，37℃、220rpm培养约5h，待菌液OD值达到0.6时加入420μM的IPTG和1mM的5-氨基乙酰丙酸，18℃、220rpm诱导培养16h。离心收集菌体并超声裂解。使用Ni-NTA琼脂糖亲和柱纯化并洗脱APEX2蛋白。SDS-PAGE验证APEX2蛋白的分子量和纯度。BL21-DE3 Escherichia coli competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Add 50ng of pTRC99A-APEX2 plasmid to competent cells, place on ice for 30min, heat shock at 42°C, add LB medium, culture at 37°C, 150rpm for 1h, take an appropriate amount of bacterial liquid and spread it on an ampicillin-resistant LB agarose plate, and culture overnight. A single clone was picked, inoculated into 1 mL ampicillin-resistant LB medium, and cultured overnight at 37°C and 220 rpm. Inoculate 1 mL of bacterial liquid into 500 mL of ampicillin-resistant LB medium, and incubate at 37°C, 220 rpm for about 5 hours. When the OD value of the bacterial liquid reaches 0.6, add 420 μM IPTG and 1 mM 5-aminolevulinic acid, at 18°C, 220 rpm Induction culture for 16h. Bacteria were collected by centrifugation and lysed by sonication. APEX2 protein was purified and eluted using a Ni-NTA agarose affinity column. The molecular weight and purity of APEX2 protein were verified by SDS-PAGE.
结果如图1所示，SDS-PAGE结果显示在分子量30kDa的位置有一条清晰的条带，表明该质粒成功表达了APEX2蛋白且纯度较高，可用于进一步的实验研究。The results are shown in Figure 1. The results of SDS-PAGE showed a clear band at the molecular weight of 30kDa, indicating that the plasmid successfully expressed the APEX2 protein with high purity and could be used for further experimental research.
实施例2 DAB聚合物的制备及同步X射线成像观察Example 2 Preparation of DAB Polymer and Synchronous X-ray Imaging Observation
将DAB水溶液和H2O2分别加到pH7.4的PBS缓冲液中，DAB终浓度为0.4mg/mL，H2O2终浓度为10mM，混匀。加入APEX2蛋白，终浓度为100nM。反应15min后将生成的DAB聚合物悬液滴加在氮化硅窗上。Add the DAB aqueous solution and H2 O2 to the PBS buffer at pH 7.4 respectively, the final concentration of DAB is 0.4 mg/mL, and the final concentration of H2 O2 is 10 mM, and mix well. APEX2 protein was added at a final concentration of 100 nM. After reacting for 15 minutes, the resulting DAB polymer suspension was dropped on the silicon nitride window.
DAB聚合物的X射线成像实验在上海光源BL08U1软X射线谱学显微线站进行，实验方法为软X射线透射成像。X射线经波荡器引出，经过平面光栅单色器单色化后由波带片聚焦到样品上，然后由快速正比计数探测器PMT探测透射光子。光子能量范围250-2000eV，空间分辨率为30nm。DAB聚合物悬液滴在氮化硅窗上，干燥，放置在真空样品室中，选取X射线入射能量为525eV。移动运动电机，完成DAB聚合物样品的寻找、对焦，然后进行软X射线成像，成像分辨率为30nm。The X-ray imaging experiment of DAB polymer was carried out at the BL08U1 soft X-ray spectroscopy microscope station of Shanghai Light Source, and the experimental method was soft X-ray transmission imaging. The X-rays are extracted by the undulator, monochromatized by the planar grating monochromator, and then focused onto the sample by the zone plate, and then the transmitted photons are detected by the fast proportional counting detector PMT. The photon energy range is 250-2000eV, and the spatial resolution is 30nm. The DAB polymer suspension was dropped on the silicon nitride window, dried, placed in a vacuum sample chamber, and the X-ray incident energy was selected as 525eV. Move the motion motor to complete the search and focus of the DAB polymer sample, and then perform soft X-ray imaging with an imaging resolution of 30nm.
结果如图2A、图2B所示，同步X射线显微技术能对DAB聚合物进行很好的成像，空间分辨率达到30nm，说明该聚合物具有良好的X射线吸收特性，可以作为同步X射线成像标签用于进一步的应用研究。The results are shown in Figure 2A and Figure 2B. Synchronous X-ray microscopy can image DAB polymer very well with a spatial resolution of 30nm, indicating that the polymer has good X-ray absorption properties and can be used as a synchrotron X-ray Imaging tags are used for further applied research.
实施例3 APEX2，HRP，miniSOG和700DX催化DAB分子生成聚合物及在同步X射线成像中的应用比较Example 3 APEX2, HRP, miniSOG and 700DX Catalyzed DAB Molecule to Generate Polymer and Its Application Comparison in Synchronous X-ray Imaging
APEX2体外催化DAB分子生成聚合物，方法与实施例2相同。APEX2 catalyzes DAB molecules to form polymers in vitro, and the method is the same as in Example 2.
HRP体外催化DAB分子生成聚合物，方法为：HRP购自Sigma(货号：P8375)。将DAB水溶液和H2O2分别加到pH7.4的PBS缓冲液中，DAB终浓度为0.4mg/mL，H2O2终浓度为10mM，混匀。加入HRP，使终浓度为100nM。反应15min后收集DAB聚合物。HRP catalyzes DAB molecules to form polymers in vitro, and the method is as follows: HRP is purchased from Sigma (product number: P8375). Add the DAB aqueous solution and H2 O2 to the PBS buffer at pH 7.4 respectively, the final concentration of DAB is 0.4 mg/mL, and the final concentration of H2 O2 is 10 mM, and mix well. HRP was added to a final concentration of 100 nM. The DAB polymer was collected after 15 min of reaction.
miniSOG体外催化DAB分子生成聚合物，方法为：将miniSOG的cDNA序列克隆进pBAD-Myc-His A原核表达载体骨架，构建pBAD-miniSOG质粒。pBAD-miniSOG质粒的序列如SEQ ID No：2所示。取50ng pBAD-miniSOG质粒加入感受态细胞，冰上放置30min，42℃热激，加入LB培养基，37℃、150rpm培养1h，取适量菌液涂布于氨苄抗性的LB琼脂糖平板上，培养过夜。挑取单克隆，接种到1mL氨苄抗性的LB培养基中，37℃、220rpm培养过夜。将1mL菌液接种到500mL氨苄抗性的LB培养基中，37℃、220rpm培养约5h，待菌液OD值达到0.6时加入0.2％的阿拉伯糖，37℃、220rpm诱导培养16h。离心收集菌体并超声裂解。使用Ni-NTA琼脂糖亲和柱纯化并洗脱miniSOG蛋白。miniSOG catalyzes DAB molecules to generate polymers in vitro. The method is: clone the cDNA sequence of miniSOG into the pBAD-Myc-His A prokaryotic expression vector backbone to construct the pBAD-miniSOG plasmid. The sequence of the pBAD-miniSOG plasmid is shown in SEQ ID No:2. Add 50ng of pBAD-miniSOG plasmid to competent cells, place on ice for 30min, heat shock at 42°C, add LB medium, incubate at 37°C, 150rpm for 1h, take an appropriate amount of bacterial liquid and spread it on an ampicillin-resistant LB agarose plate, Incubate overnight. A single clone was picked, inoculated into 1 mL ampicillin-resistant LB medium, and cultured overnight at 37°C and 220 rpm. Inoculate 1 mL of bacterial liquid into 500 mL of ampicillin-resistant LB medium, incubate at 37°C and 220 rpm for about 5 hours, add 0.2% arabinose when the OD value of the bacterial liquid reaches 0.6, and induce culture at 37°C and 220 rpm for 16 hours. Bacteria were collected by centrifugation and lysed by sonication. The miniSOG protein was purified and eluted using a Ni-NTA agarose affinity column.
将DAB水溶液加到pH7.4的PBS缓冲液中，DAB终浓度为0.4mg/mL，混匀。加入miniSOG，使终浓度为50nM。缓慢地向反应体系中通O2，使用150W的488nm荧光照射15min，即得DAB聚合物悬液。Add the DAB aqueous solution to the PBS buffer at pH 7.4, the final concentration of DAB is 0.4 mg/mL, and mix well. Add miniSOG to a final concentration of 50 nM. Slowly pass O2 into the reaction system, and irradiate with 150W 488nm fluorescence for 15min to obtain the DAB polymer suspension.
700DX体外催化DAB分子聚合物，方法为：700DX购自LI-COR(货号：929-70010)。将DAB水溶液加到pH7.4的PBS缓冲液中，DAB终浓度为0.4mg/mL，混匀。加入700DX，使终浓度为50nM。使用150W的700nm荧光照射15min，即得DAB聚合物悬液。 700DX catalyzes DAB molecular polymer in vitro, the method is: 700DX was purchased from LI-COR (Cat. No. 929-70010). Add the DAB aqueous solution to the PBS buffer at pH 7.4, the final concentration of DAB is 0.4 mg/mL, and mix well. join in 700DX to make a final concentration of 50 nM. Use 150W of 700nm fluorescence to irradiate for 15min to obtain the DAB polymer suspension.
上述各DAB聚合物的X射线成像观察方法与实施例2相同。The X-ray imaging observation method of each of the above DAB polymers is the same as in Example 2.
结果如图3A、3B、3C、3D所示，同步X射线成像结果显示APEX2，HRP，miniSOG和700DX均能催化DAB分子生成聚合物，但是酶APEX2，HRP，miniSOG的催化效果优于小分子700DX。The results are shown in Figure 3A, 3B, 3C, and 3D, and the simultaneous X-ray imaging results showed that APEX2, HRP, miniSOG and 700DX can catalyze DAB molecules to form polymers, but the catalytic effects of enzymes APEX2, HRP, and miniSOG are better than those of small molecules 700DX.
实施例4 APEX2催化DAB，金属增强型DAB以及EnzMet生成聚合物及在同步X射线成像中的应用比较Example 4 APEX2 catalyzed DAB, metal-enhanced DAB and EnzMet to generate polymers and their application in synchrotron X-ray imaging
APEX2体外催化DAB分子生成聚合物，方法与实施例2相同。APEX2 catalyzes DAB molecules to form polymers in vitro, and the method is the same as in Example 2.
APEX2体外催化金属增强型DAB分子生成聚合物，方法为：金属增强型DAB购自Thermofisher(货号：34065)。将金属增强型DAB溶液加到Stable Peroxide Buffer中，混匀。使其终浓度为1×。加入APEX2蛋白，使终浓度为100nM，混匀。反应15min后，即得金属增强型DAB聚合物悬液。APEX2 catalyzes metal-enhanced DAB molecules to form polymers in vitro. The method is: metal-enhanced DAB is purchased from Thermofisher (Catalog No.: 34065). Add metal-enhanced DAB solution to Stable Peroxide Buffer and mix well. Make the final concentration 1×. Add APEX2 protein to make the final concentration 100nM, and mix well. After reacting for 15 minutes, the metal-enhanced DAB polymer suspension was obtained.
APEX2体外催化EnzMet反应生成聚合物，方法为：EnzMet购自Nanoprobe(货号：6010)。将APEX2蛋白加入330μL的EnzMetTM Detect A中，混匀，终浓度为300nM。室温孵育4min后，加入330μL的EnzMetTM Detect B，混匀，室温孵育4min。加入330μL的EnzMetTMDetect C，混匀，室温反应40min，即得EnzMet聚合物悬液。APEX2 catalyzes the reaction of EnzMet in vitro to form a polymer. The method is as follows: EnzMet is purchased from Nanoprobe (product number: 6010). Add APEX2 protein to 330 μL of EnzMetTM Detect A, mix well, and the final concentration is 300 nM. After incubating at room temperature for 4 min, add 330 μL of EnzMetTM Detect B, mix well, and incubate at room temperature for 4 min. Add 330 μL ofEnzMet Detect C, mix well, and react at room temperature for 40 minutes to obtain EnzMet polymer suspension.
观察DAB聚合物、金属增强型DAB聚合物、EnzMet聚合物时，X射线入射能量分别选取525，798和606eV，其余同步X射线成像观察方法与实施例3相同。When observing DAB polymers, metal-enhanced DAB polymers, and EnzMet polymers, the incident X-ray energies were selected to be 525, 798 and 606 eV, respectively, and the rest of the synchronous X-ray imaging observation methods were the same as in Example 3.
结果如图4A、4B、4C所示，同步X射线成像结果显示APEX2可以催化DAB，金属增强型DAB以及EnzMet生成聚合物，说明这些聚合物都具有良好的X射线吸收特性，可以作为同步X射线成像标签用于进一步的应用研究。The results are shown in Figures 4A, 4B, and 4C. Synchrotron X-ray imaging results show that APEX2 can catalyze DAB, metal-enhanced DAB, and EnzMet to form polymers, indicating that these polymers have good X-ray absorption properties and can be used as synchrotron X-ray Imaging tags are used for further applied research.
以上所述的，仅为本发明的较佳实施例，并非用以限定本发明的范围，本发明的上述实施例还可以做出各种变化。即凡是依据本发明申请的权利要求书及说明书内容所作的简单、等效变化与修饰，皆落入本发明专利的权利要求保护范围。本发明未详尽描述的均为常规技术内容。What is described above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Various changes can also be made to the above embodiments of the present invention. That is to say, all simple and equivalent changes and modifications made according to the claims and description of the application for the present invention fall within the protection scope of the claims of the patent of the present invention. What is not described in detail in the present invention is conventional technical contents.
序列表sequence listing
<110> 中国科学院上海应用物理研究所<110> Shanghai Institute of Applied Physics, Chinese Academy of Sciences
<120> 一种同步X射线可见的成像标签及其制备方法<120> A Simultaneous X-ray Visible Imaging Label and Its Preparation Method
<160> 2<160> 2
<210> 1<210> 1
<211> 4925<211> 4925
<212> DNA<212>DNA
<213> 人工<213> Artificial
<220><220>
<223> pTRC99A-APEX2质粒<223> pTRC99A-APEX2 plasmid
<400> 1<400> 1
gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60
ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120
gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180
tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240
taacaatttc acacaggaaa cagaccatgg cacaccacca ccaccaccac ggaaagtctt 300taacaatttc acacaggaaa cagaccatgg cacaccacca ccaccaccac ggaaagtctt 300
acccaactgt gagtgctgat taccaggacg ccgttgagaa ggcgaagaag aagctcagag 360acccaactgt gagtgctgat taccaggacg ccgttgagaa ggcgaagaag aagctcagag 360
gcttcatcgc tgagaagaga tgcgctcctc taatgctccg tttggcattc cactctgctg 420gcttcatcgc tgagaagaga tgcgctcctc taatgctccg tttggcattc cactctgctg 420
gaacctttga caagggcacg aagaccggtg gacccttcgg aaccatcaag caccctgccg 480gaacctttga caagggcacg aagaccggtg gacccttcgg aaccatcaag caccctgccg 480
aactggctca cagcgctaac aacggtcttg acatcgctgt taggcttttg gagccactca 540aactggctca cagcgctaac aacggtcttg acatcgctgt taggcttttg gagccactca 540
aggcggagtt ccctattttg agctacgccg atttctacca gttggctggc gttgttgccg 600aggcggagtt ccctattttg agctacgccg atttctacca gttggctggc gttgttgccg 600
ttgaggtcac gggtggacct aaggttccat tccaccctgg aagagaggac aagcctgagc 660ttgaggtcac gggtggacct aaggttccat tccaccctgg aagagaggac aagcctgagc 660
caccaccaga gggtcgcttg cccgatccca ctaagggttc tgaccatttg agagatgtgt 720caccaccaga gggtcgcttg cccgatccca ctaagggttc tgaccatttg agagatgtgt 720
ttggcaaagc tatggggctt actgaccaag atatcgttgc tctatctggg ggtcacacta 780ttggcaaagc tatggggctt actgaccaag atatcgttgc tctatctggg ggtcacacta 780
ttggagctgc acacaaggag cgttctggat ttgagggtcc ctggacctct aatcctctta 840ttggagctgc acacaaggag cgttctggat ttgagggtcc ctggacctct aatcctctta 840
ttttcgacaa ctcatacttc acggagttgt tgagtggtga gaaggaaggt ctccttcagc 900ttttcgacaa ctcatacttc acggagttgt tgagtggtga gaaggaaggt ctccttcagc 900
taccttctga caaggctctt ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg 960taccttctga caaggctctt ttgtctgacc ctgtattccg ccctctcgtt gacaaatatg 960
cagcggacga agatgccttc tttgctgatt acgctgaggc tcaccaaaag ctttccgagc 1020cagcggacga agatgccttc tttgctgatt acgctgaggc tcaccaaaag ctttccgagc 1020
ttgggtttgc tgatgcctaa gatccgctag agtcgacctg caggcatgca agcttggctg 1080ttgggtttgc tgatgcctaa gatccgctag agtcgacctg caggcatgca agcttggctg 1080
ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 1140ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 1140
tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc 1200tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc 1200
gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 1260gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 1260
agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 1320agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 1320
ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 1380ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 1380
tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 1440tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 1440
ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 1500ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 1500
tttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 1560tttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 1560
ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 1620ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 1620
ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 1680ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 1680
gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 1740gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 1740
caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 1800caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 1800
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 1860ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 1860
cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 1920cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 1920
gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 1980gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 1980
taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 2040taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 2040
tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 2100tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 2100
agccatacca aacgacgagc gtgacaccac gatgcctaca gcaatggcaa caacgttgcg 2160agccatacca aacgacgagc gtgacaccac gatgcctaca gcaatggcaa caacgttgcg 2160
caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 2220caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 2220
ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 2280ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 2280
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 2340tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 2340
agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 2400agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 2400
tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 2460tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 2460
agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 2520agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 2520
gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 2580gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 2580
gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 2640gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 2640
tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 2700tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 2700
gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 2760gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 2760
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 2820accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 2820
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 2880accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 2880
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 2940gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 2940
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 3000ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 3000
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3060atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3060
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3120gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3120
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3180cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3180
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3240gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3240
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 3300gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 3300
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 3360tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 3360
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 3420cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct 3420
tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 3480tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga 3480
tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 3540tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 3540
ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 3600ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 3600
gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 3660gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 3660
tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt 3720tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt 3720
acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag 3780acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag 3780
agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg 3840agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg 3840
ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga 3900ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga 3900
aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg 3960aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg 3960
cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc 4020cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc 4020
tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca 4080tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca 4080
gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca 4140gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca 4140
atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg 4200atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg 4200
ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc 4260ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc 4260
agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc 4320agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc 4320
atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct 4380atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct 4380
cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga 4440cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga 4440
tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc 4500tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc 4500
tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg 4560tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg 4560
caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat 4620caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat 4620
acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt 4680acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt 4680
ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg 4740ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg 4740
tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca 4800tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca 4800
atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 4860atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 4860
tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gcgcgaattg 4920tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gcgcgaattg 4920
atctg 4925atctg 4925
<210> 2<210> 2
<211> 4366<211> 4366
<212> DNA<212>DNA
<213> 人工<213> Artificial
<220><220>
<223> pBAD-miniSOG质粒<223> pBAD-miniSOG plasmid
<400> 2<400> 2
aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60
tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120
aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180
attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240
atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300
taacaggagg aattaaccat ggagaaaagt ttcgtgataa ctgatccacg gctgccagac 360taacaggagg aattaaccat ggagaaaagt ttcgtgataa ctgatccacg gctgccagac 360
aatcccatca tcttcgcatc cgatggcttc ctggagctga ccgagtattc cagagaggag 420aatcccatca tcttcgcatc cgatggcttc ctggagctga ccgagtattc cagagaggag 420
atcctgggcc gcaatggccg ctttctgcag ggaccagaga cagaccaggc cacagtgcag 480atcctgggcc gcaatggccg ctttctgcag ggaccagaga cagaccaggc cacagtgcag 480
aagattcgcg atgccattag agatcagcgc gagattaccg tgcagctgat aaactacaca 540aagattcgcg atgccattag agatcagcgc gagattaccg tgcagctgat aaactacaca 540
aaaagcggga agaaattctg gaacctcctg cacctccagc ccatgaggga ccagaagggt 600aaaagcggga agaaattctg gaacctcctg cacctccagc ccatgaggga ccagaagggt 600
gagctccagt atttcatcgg agtgcagctg gatggaaagc ttgggcccga acaaaaactc 660gagctccagt atttcatcgg agtgcagctg gatggaaagc ttgggcccga acaaaaactc 660
atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttaa 720atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgagtttaa 720
acggtctcca gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta 780acggtctcca gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta 780
aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 840aatcagaacg cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg 840
tcccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 900tccccacctga ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg 900
ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 960ggtctcccca tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg 960
aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 1020aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 1020
aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga 1080aatccgccgg gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga 1080
cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 1140cgcccgccat aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt 1140
tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct 1200tttgcgtttc tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct 1200
catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 1260catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 1260
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 1320tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 1320
tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1380tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1380
ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1440ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1440
ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 1500ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 1500
cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1560cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1560
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1620ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1620
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1680tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1680
gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1740gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1740
ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1800ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1800
aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1860aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1860
acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 1920acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 1920
tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1980tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1980
cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 2040cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 2040
gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 2100gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 2100
taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 2160taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 2160
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 2220tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 2220
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2280cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 2280
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2340ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 2340
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2400accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2400
cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 2460cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 2460
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2520cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2520
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2580tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2580
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2640taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2640
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2700gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2700
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2760agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2760
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2820ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2820
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2880acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2880
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 2940caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 2940
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 3000tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 3000
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 3060tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct 3060
gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 3120gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct 3120
cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 3180cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 3180
gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 3240gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 3240
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 3300tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccggggag ctgcatgtgt 3300
cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag 3360cagaggtttt caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag 3360
gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 3420gcgaagcggc atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 3420
tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 3480tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 3480
acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 3540acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 3540
tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 3600tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 3600
gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 3660gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 3660
cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 3720cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 3720
gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg 3780gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg 3780
ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta 3840ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta 3840
atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa 3900atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa 3900
tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc 3960tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc 3960
tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc 4020tggtgcgctt catccgggcg aaagaaccccc gtattggcaa atattgacgg ccagttaagc 4020
cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc 4080cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc 4080
tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt 4140tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt 4140
cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg 4200cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg 4200
agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc 4260agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc 4260
tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga 4320tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcaggggga 4320
tcattttgcg cttcagccat acttttcata ctcccgccat tcagag 4366tcattttgcg cttcagccat acttttcata ctcccgccat tcagag 4366
| Application Number | Priority Date | Filing Date | Title |
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| CN201710228701.4ACN107064184B (en) | 2017-04-10 | 2017-04-10 | A synchronous X-ray visible imaging label and its preparation method |
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| CN201710228701.4ACN107064184B (en) | 2017-04-10 | 2017-04-10 | A synchronous X-ray visible imaging label and its preparation method |
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| CN107064184A CN107064184A (en) | 2017-08-18 |
| CN107064184Btrue CN107064184B (en) | 2019-08-06 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110836903B (en)* | 2019-11-11 | 2022-10-11 | 中国科学院上海高等研究院 | Synchronous X-ray visible multicolor imaging label and preparation method thereof |
| CN110819656B (en)* | 2019-11-11 | 2021-06-04 | 中国科学院上海高等研究院 | A kind of X-ray multicolor genetic labeling probe based on synchronous light source and its preparation method and application |
| CN114891060B (en)* | 2022-07-13 | 2022-09-30 | 深圳湾实验室 | Light activation dependent proximity labeling method for protein and application thereof |
| CN116297564B (en)* | 2023-02-27 | 2025-09-26 | 中国科学院上海高等研究院 | A synchrotron X-ray visible click chemistry imaging label and its preparation method |
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| WO2007099536A2 (en)* | 2006-03-03 | 2007-09-07 | Soreq Nuclear Research Center | Radioactive metal-bound beads |
| WO2016198609A1 (en)* | 2015-06-10 | 2016-12-15 | Danmarks Tekniske Universitet | High throughput optimization of content-loaded nanoparticles |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007099536A2 (en)* | 2006-03-03 | 2007-09-07 | Soreq Nuclear Research Center | Radioactive metal-bound beads |
| WO2016198609A1 (en)* | 2015-06-10 | 2016-12-15 | Danmarks Tekniske Universitet | High throughput optimization of content-loaded nanoparticles |
| CA2988874A1 (en)* | 2015-06-10 | 2016-12-15 | Danmarks Tekniske Universitet | High throughput optimization of content-loaded nanoparticles |
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| CN107064184A (en) | 2017-08-18 |
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