Name: synthesis of core-shell lanthanide-doped upconversion nanocrystals for cellular applications
Uploaded: 2017-11-10T13:00:00.000+00:00
Duration: 13 min 51 s
Description: Watch this Scientific Journal Video about Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications at JoVE.com

这项工作得到了部分支持由台大-MUV NAM/16001, RG110/16 (S), (rg 11/13) 和 (rg 35/15) 授予在南洋理工大学, 新加坡和国家自然科学基金 (自然科学基金) (No. 51628201)。

警告: 请在使用前查阅所有相关的材料安全数据表 (MSDS)。在高温 (〜290和 #176 C) 中进行 UCNs 的合成时, 请使用所有适当的安全做法, 包括使用工程控制 (油烟罩) 和个人防护设备 ( 例如 、安全护目镜、手套、实验室工作服全长长裤, 和闭脚趾鞋). 
 1. NaYF 的合成 4 : yb/tm/钕 (30/0.5/1%) @NaYF 4 : nd (20%) 核壳纳米晶 <ol> <li> 合成 NaYF 4 : yb/tm/钕 (30/0.5/1%) 核心纳米结构 <ol>稀土 (CH 3 CO 2 ) 3 , 氢氧化钠, NH 4 F 甲醇库存解决方案 注: rare-earth 稀土离子包括钇 (Y)、镱 (Yb)、铥 (Tm) 和钕 (Nd)。
		<ol> <li> 溶解500毫克的乙酸钇 (III) 水合物, 在5毫升甲醇 (100 毫克/毫升), 250 毫克镱 (iii) 水合乙酸5毫升甲醇 (50 毫克/毫升), 10 毫克铥 (iii) 水合物在1毫升甲醇 (10 毫克/毫升), 和10毫克的乙酸钕 (iii) 水合物在1毫升 metha(10 毫克/毫升) 在玻璃小瓶与超声波清洗浴 2 min. </li>
			<li> 将400毫克氢氧化钠放入50毫升的离心管中, 用20毫升甲醇与超声波清洗液结合, 制备氢氧化钠溶液 (20 毫克/毫升). </li>
			<li> 合并600毫克 nh 4 F 在50毫升离心管与30毫升甲醇与超声波清洗浴准备 NH 4 f 库存解决方案 (20 毫克/毫升). 
			注: 将镧系络合物、氢氧化钠和 NH 4 F 放入玻璃瓶中, 用膜密封, 然后将其存放在4和 #176 的冰箱中; 直到需要为止。准备好的甲醇库存解决方案每2周更换一次. </li>
		</ol> </li> <li> 准备 NaYF 4 : Yb/Tm/Nd 核心纳米结构 <ol> 吸管3毫升的油酸和7毫升的 1-十八到50毫升三颈烧瓶中. <li>
			<li> 合并1.089 毫升的 Y (CH 3 CO 2 ) 3 库存解决方案, 0.608 毫升的 Yb (ch 3 co 2 ) 3 库存解决方案, 83.6 & #181; Tm (ch 3 co 2 ) 3 库存解决方案,128.5 和 #181; Nd (CH 3 CO 2 ) 3 库存解决方案放入烧瓶中. </li>
			<li> 将温度计 (0-360 和 #176; C 范围) 放入烧瓶中, 并让其尖端接触溶液。将烧瓶放在玻璃容器中, 甲基硅油. </li>
			<li> 将溶液加热到100和 #176; 用热板将甲醇蒸发掉。将烧瓶与双真空/气歧管连接至 Schlenk 线, 以除去残余甲醇, 并在搅拌时将反应混合物保持在真空下 2-3 分钟. </li>
			<li> 将温度提高到150和 #176; C 保持这种温度为60分钟. 在合成过程中保持 700 rpm 的搅拌速度. 
			注: 设置适中的搅拌速度, 避免溅出溶液. </li>
			<li> 停止热板并在室温下保持烧瓶, 以使溶液冷却缓慢. 
			注意: 可以在此暂停协议. </li>
			<li> 将2毫升氢氧化钠-甲醇库存溶液和2.965 毫升 NH 4 F-甲醇库存溶液合并为15毫升离心管。收紧管与它的上限和混合的解决方案由强大的涡流为 5 s. </li>
			<li> 将氢氧化钠-NH 4 F 混合物慢慢地放入烧瓶中, 由玻璃吸管超过5分钟. </li>
			<li> 将混合物的温度提高到50和 #176; C 并保持此温度为30分钟. 
			注: 将温度设置在不超过50和 #176; C 因为高温会促进晶体的成核和生长. </li>
			<li> 增加温度 (~ 100 和 #176; C) 将甲醇蒸发, 并将烧瓶与双真空/气歧管 Schlenk 线连接, 以去除残余甲醇, 并将反应混合物置于真空下 2-3 分钟. </li>
			<li> 将塞的位置切换为用氮气填充烧瓶. </li>
			<li> 将温度提高到290和 #176; c 的升温速率为5和 #176; c/分钟保持反应混合物在290和 #176; c 为1.5 小时. </li>
			<li> 停止热板, 取出烧瓶, 使反应混合物在室温下缓慢降温, 同时搅拌. 
			注意: 注意高温 (和 #62; 400 和 #176; C), 避免皮肤接触时严重灼伤. </li>
			<li> 将烧瓶中的混合物转化为50毫升离心管。用30毫升乙醇冲洗烧瓶, 并将溶液转移到离心管上. </li>
			<li> 4000 和 #215 离心产品; 在室温下为8分钟, 并丢弃上清液. </li>
			<li> 将10毫升正己烷添加到离心管中, 并 re-disperse 产品与超声 (60 赫, 240 W) 为 2 min. </li>
			<li> 在试管中加入30毫升的乙醇。在4000和 #215 旋转产品; g 为8分钟, 然后丢弃上清. </li>
			<li> 用5毫升正己烷将离心管底部的固态产品重新分散。将解决方案存储在4和 #176 的冰箱中, 用于下一步的步骤. 
			注: 用 808 nm 激光 (2 W) 照射溶液, 测试核壳 UCNs 的转换发射. </li>
		</ol> </li> </ol> </li> <li> 准备 NaYF 4 : Yb/Tm/钕 (30/0.5/1%) @NaYF 4 : nd (20%) 核壳纳米晶 <ol> <li> 将油酸3毫升和7毫升 1-十八合并为50毫升三颈烧瓶。添加1.082 毫升的 Y (ch 3 co 2 ) 3 库存解决方案和 2.87 ml (ch 3 co 2 ) 3 库存解决方案放入瓶中. </li>
		<li> 添加获得的核心纳米结构 (80 毫克5毫升的正己烷从步 1.1.2.18) 进入烧瓶, 而搅拌. </li>
		<li> 将温度计 (0-360 和 #176; C 范围) 放入烧瓶中, 并让其尖端接触混合物。将烧瓶放在玻璃容器中, 甲基硅油. </li>
		<li> 将混合物加热100和 #176; C 在热板的顶部以蒸发甲醇和己烷。将烧瓶与双真空/气歧管连接至 Schlenk 线, 以去除残余溶剂, 并在搅拌时将反应混合物保持在真空下 2-3 分钟. </li>
		<li> 将温度提高到150和 #176; C 并将其保持为60分钟, 在合成中保持 700 rpm 的搅拌速度. 
		注: 设置适中的搅拌速度, 避免溅出溶液. </li>
		<li> 停止热板, 取出烧瓶, 使溶液在室温下缓慢降温. 
		注意: 可以在此暂停协议. </li>
		<li> 吸管2毫升氢氧化钠-甲醇库存溶液和2.965 毫升 NH 4 F-甲醇库存溶液成15毫升离心管。收紧管与它的上限和混合的解决方案由强大的涡流为 5 s. </li>
		<li> 将混合物放入烧瓶中, 由玻璃吸管慢慢超过5分钟. </li>
		<li> 将混合物的温度提高到50和 #176; c 并将其保持在50和 #176; c 为 30 min. 
		注: 设置温度不高于50和 #176; C 因为高温会促进晶体的成核和生长. </li>
		<li> 增加温度 (~ 100 和 #176; C) 将甲醇蒸发, 并将烧瓶与双真空/气歧管 Schlenk 线连接, 以去除残余甲醇, 使反应混合物在真空状态下保持 2-3 分钟. </li>
		<li> 将塞的位置切换为用氮气填充烧瓶. </li>
		<li> 将温度提高到290和 #176; C在5和 #176 的加热速率; c/分钟保持反应混合物在290和 #176; c 为 1.5 h. </li>
		<li> 停止热板, 取出烧瓶, 使反应混合物在室温下缓慢降温, 同时搅拌. 
		注意: 注意高温 (和 #62; 400 和 #176; C), 避免皮肤接触时严重灼伤. </li>
		<li> 将烧瓶中的混合物转化为50毫升离心管。用30毫升乙醇冲洗烧瓶, 并将溶液转移到离心管上. </li>
		<li> 4000 和 #215 离心产品; 在室温下为8分钟, 并丢弃上清液. </li>
		<li> 将10毫升正己烷添加到离心管中, 并 re-disperse 产品与超声 (60 赫, 240 W) 为 2 min. </li>
		<li> 在试管中加入30毫升的乙醇。在4000和 #215 旋转产品; g 为8分钟, 然后丢弃上清. </li>
		<li> 用5毫升正己烷将离心管底部的固态产品重新分散。将解决方案存储在4和 #176 的冰箱中, 直到需要为止. 
		注: 用 808 nm 激光 (2 W) 照射溶液, 测试核壳 UCNs 的转换发射. </li>
	</ol> </li> </ol> 2。生物相容性 UCNs 纳米结构的合成 <ol> <li> 制备无配体 UCNs 纳米粒子 <ol> <li> 将30毫升乙醇与在50毫升离心管中制备的油酸 UCNs 溶液 (步骤 1.2.18) 结合在一起。将混合物离心4000和 #215; 在室温下为8分钟, 并丢弃上清液. </li>
		<li> 结合10毫升酸性水溶液 (pH = 4), 由 HCl (0.1 M) 与沉淀在50毫升离心管和溶解沉淀由超声 (60 赫, 240 W) 为 30 min. </li>
		<li> 将溶液在2小时剧烈搅拌的玻璃瓶中转移. </li>
		<li> 用30毫升乙醚萃取水溶液, 除去油酸, 重复三次. </li>
		<li> 在混合醚层中加10毫升水, 轻轻摇动. </li>
		<li> 收集水相一起 (20 毫升) 和添加20毫升丙酮与涡流 5 s. </li>
		<li> 在3.5万和 #215 向下旋转产品; g 为10分钟, 然后丢弃上清。在2毫升水中溶解沉淀. </li>
	</ol> </li> <li> 聚合物改性 UCNs (临机 UCNs) <ol> <li> 结合200毫克聚丙烯酸 (临机局, 兆瓦 = 1800) 与20毫升水超声 (60 赫, 240 W) 为 20 min. 在临机局溶液中加入上述无配体 UCNs剧烈搅拌. </li>
		<li> 将 pH 值调整为 7.4, 氢氧化钠溶液 (1 米) 与超声 (60 赫, 240 W) 为 30 min. 在室温下搅拌时, 将混合物保持在24小时. </li>
		<li> 用离心法收集沉淀3.5万和 #215; g 为10分钟, 重新悬浮在10毫升水中的产品超声 (60 赫, 240 W) 为 5 min 和离心机再次在3.5万和 #215; g 为 10 min. 重复此步骤三次. </li>
		<li> 在8毫升水中用超声 (60 赫, 240 W) 将该产品重新分散到5分钟, 将溶液存储在 ~ 4 和 #176 的冰箱中, 直到需要为止. </li>
	</ol> </li> <li> 功能性 DBCO-UCNs 纳米粒子 <ol> <li> 在3.5万和 #215 下离心制得 PAA@UCNs (1 毫克); g 为10分钟, 在1毫升干 DMF 中超声 (60 赫, 240 W) 为1分钟重新悬浮沉淀。并在3.5万和 #215 再次离心; g 为10分钟重复此步骤两次. </li>
		<li> 在200和 #181 中溶解沉淀; 在玻璃瓶中干 DMF添加 HOBT (12.2 毫克), 14 毫克), DBCO-NH 2 (5 毫克), 和 DIPEA (16 和 #181; L) 在瓶与磁性搅拌为 24 h. </li>
		<li> 3.5万和 #215 离心收集产品; g 为 10 min. 在1毫升亚砜中除去上清和 re-suspend 沉淀, 再在3.5万和 #215 离心机; g 为 10 min. 重复此步骤三次. </li>
		<li> 在使用前将产品分散在0.2 毫升的亚甲基亚砜中, 并存储在4和 #176 的冰箱中. </li>
	</ol> </li> </ol> 3。DBCO-UCNs 在活细胞膜通道调节中的 Bioapplications 性研究 <ol> <li> 在 Dulbecco 和 #39 中培养 HEK293 细胞 (DMEM), 辅以 10% FBS、100单位/毫升青霉素、100和 #181; 链霉素和在湿式恒温箱中保持 5% CO 2 , 在37和 #176; c. 种子1和 #215; 10 5 单元格中1毫升 DMEM/井在 12-井板和保持它在孵化器为 24 h. </li>
	<li> 将质粒 (pCAGGS-ChR2-Venus、1和 #181; g) 与 P3000 转染试剂 (2 和 #181 相结合; l) 在离心管 a 中的最小基本介质 (100 和 #181; l), 并在 B 管中添加转染试剂 (1.5 和 #181; l) 在 (100 和 #181; l) 中。</li> <li> 在室温下孵育10分钟的管 A 和 B 中的溶液混合物. </li>
	<li> 将解决方案与400和 #181 相结合;用1毫升无血清 DMEM 两次冲洗细胞. </li>
	<li> 添加600和 #181; 将混合物转染为12井板的每一个井, 并在37和 #176 孵育细胞; C 孵化 4 h. 去除培养基, 用1毫升 DMEM 冲洗两次. </li>
	<li> 添加1毫升 DMEM, 包含1和 #181; Ac 4 ManNAz (50 mM 在亚甲基亚砜) 在井中, 并保持它在孵化器2天. </li>
	<li> 取出介质, 用1毫升 PBS 清洗一次。在12井板的每个井中加入1毫升胰蛋白酶-EDTA (0.25%) 溶液, 孵育在37和 #176; C 直到细胞分离 (~ 2 min)。在1和 #215 的共焦皿中重新培养细胞; 10 5 单元格/1 mL DMEM 中的单元格过夜. </li>
	<li> 删除介质, 并添加1毫升新鲜 DMEM 与2和 #181; DBCO-UCNs (50 毫克/毫升) 在37和 #176 的菜2小时;对于共聚焦成像, 清洗细胞两次与 DMEM 和增加1和 #181; L 鲁比罗-N 3 (10 mM) 为 30 min. </li>
	<li> 细胞内钙分析, 孵育1和 #181 的混合物; l Rhod-3 (10 毫米)、10和 #181; l 磺 (250 mm) 和10和 #181; l 在0.1% 毫升 DMEM 介质中装载缓冲器 (Pluronic 表面活性剂多元醇, 1 w/v), 在黑暗中为30分钟. </li>
	<li> 用无血清 DMEM 冲洗细胞, 用 808 nm 近红外光照射, 剂量为0.8 瓦/厘米 2 , 20 分钟 (5 分钟后5分钟, 光照). </li>
	<li> 在共焦显微镜上记录成像结果 (激光光源: 561 nm 激光器; 探测器范围: 610/75 nm; 镜头: 100x 1.4 NA 油, 曝光时间: 100 毫秒)。在每个井中加入1毫升胰蛋白酶-EDTA (0.25%) 溶液, 孵育在37和 #176; C 直到细胞分离 (~ 2 min)。在1毫升 PBS 中重新悬浮细胞流式细胞仪 (FCM) 分析 (前: 561 nm, Em: 582/15 nm). </li>
</ol>

<ol>
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我们没有什么要透露的。

本文提出了一种合成核壳镧系稀土掺杂的转换纳米晶 (UCNs) 及其表面改性的方法, 并用功能基进行了细胞应用。这种新型纳米材料具有优异的光学性能, 可以通过光子转换过程在近红外光激励下发出紫外线和可见光。在本协议中, 核壳 UCNs 纳米结构 (NaYF4: Yb/Tm/钕 (30/0.5/1%) @NaYF4: nd (20%)) 是在油酸和 1-十八的混合物中采用高温沉淀法制备的。TEM 图像表明, 这些核心和核壳纳米晶的形貌是...

近年来, 镧系掺杂的转换纳米晶 (UCNs) 被广泛应用于生物医学应用中的传统有机染料和量子点的替代品, 主要以其优异的化学和光学性能为基础,包括很好的生物相容性, 高抗漂白, 和窄带宽发射1,2,3。更重要的是, 他们可以作为一个有前途的 nanotransducer 与优秀的组织穿透深度在体内将近红外线 (近红外) 激发转化为从紫外线, 可见, 和红外光谱的广泛范围的排放, 通过光子转换进程4,5。这些独特的性质使镧系掺杂的 UCNs 作为一种特别有前途的生物传感、生物医学成像和疾病的载体 theranostics6,7,8。
UCNs 的一般成分主要是基于掺杂的镧系离子在含有增敏剂的绝缘主机基体中 (例如、Yb3 +、Nd3 +) 和一个激活器 (如、Tm3 +、Er3 +、Ho3 +)在水晶之内均匀地9。纳米晶体的不同光发射归因于镧系掺杂的 4f轨道内的局部电子跃迁, 这是由于它们的阶梯状布置的能级10。因此, 对多组分镧系掺杂的合成 UCNs 的尺寸和形貌进行精确控制是十分关键的。通过正确的研究, 为制备镧系掺杂的稀土 UCNs, 包括热分解、高温沉淀、热液合成、溶胶-凝胶处理、等, 都建立了良好的方法.11,12,13在这些方法中, 高温沉淀法是 UCNs 合成的最常用和最方便的方法之一, 可以严格控制, 以制备出所需的均匀形状的高质量纳米晶体, 并在相对较短的反应时间和低成本14中的大小分布。然而, 这种方法合成的大多数纳米结构, 主要是以油酸和油等疏水性配体为上限, 由于水溶液中疏水性配基溶解度的限制, 通常会阻碍它们的进一步 bioapplication。15. 因此, 有必要进行适当的表面改性技术, 以在生物应用中, 在体外UCNs, 在体内, 并。
本文介绍了通过高温沉淀法合成核壳 UCNs 纳米结构的详细实验过程和化 UCNs 表面生物相容性聚合物的可行性改造技术进一步的蜂窝应用。此 UCNs nanoplatform 将三镧系离子 (Yb3 +、Nd3 +和 Tm3 +) 并入纳米晶体中, 以获得强蓝光发射 (~ 480 nm), 在近红外光激励下在 808 nm, 这有更大的穿透深度活体组织。众所周知, Nd3 +掺杂的 UCNs 在这一光谱窗口 (808 nm) 上显示了最小的吸水和过热效应, 与常规 UCNs 相比, 980 nm 辐照后的16,17, 18. 此外, 为了利用生物系统中的 UCNs, UCNs 表面的疏水性配体 (油酸) 首先被超声在酸性溶液中去除19。然后再用一种生物相容聚合物 (聚丙烯酸, 临机局) 对无配体 UCNs 进行进一步的修饰, 以获得在水溶液中的很大溶解度20。此外, 作为细胞应用的概念证明, 亲水 UCNs 是进一步功能化的分子配体 (二苄 cyclooctyne, DBCO) 的具体定位在 N3标记细胞膜。在近红外光 (808 nm) 辐照下, upconverted 蓝色发射在 480 nm 可以有效地激活 light-gated 通道蛋白, channelrhodopsins-2 (ChR2), 在细胞表面上, 从而促进阳离子 (如, Ca2 +离子) 流入在活细胞的细胞膜上。
该视频协议为镧系掺杂的 UCNs 合成、生物相容表面修饰和活细胞中的 UCNs bioapplication 提供了一种可行的方法。在纳米晶体生长所用的合成技术和化学试剂上的任何差异都将影响细胞实验中使用的最终 UCNs 纳米结构的大小分布、形态和转换发光 (伦敦) 光谱。这一详细的视频协议是为了帮助这一领域的新研究人员提高 UCNs 的重复性与高温沉淀方法, 并避免最常见的错误, UCNs 生物相容性表面修改进一步蜂窝应用。

<table><tbody><tr><td>1-Octadecene</td><td>Sigma Aldrich</td><td>O806</td><td>Technical grade</td></tr><tr><td>oleic acid</td><td>Sigma Aldrich</td><td>364525</td><td>Technical grade</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412</td><td>Technical grade</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>A405</td><td>Technical grade</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A18</td><td>Technical grade</td></tr><tr><td>Hexane</td><td>Sigma Aldrich</td><td>H292</td><td>Technical grade</td></tr><tr><td>Thulium (III) acetate hydrate (Tm(CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>367702</td><td>99.9% trace metals basis</td></tr><tr><td>Neodymium (III) acetate hydrate (Nd(CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>325805</td><td>99.9% trace metals basis</td></tr><tr><td>Ytterbium (III) acetate hydrate (Yb(CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>326011</td><td>99.9% trace metals basis</td></tr><tr><td>Yttrium(III) acetate hydrate (Y(CH&lt;sub&gt;3&lt;/sub&gt;CO&lt;sub&gt;2&lt;/sub&gt;)&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>326046</td><td>99.9% trace metals basis</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma Aldrich</td><td>S5881</td><td>reagent grade</td></tr><tr><td>Ammonium fluoride (NH&lt;sub&gt;4&lt;/sub&gt;F)</td><td>Sigma Aldrich</td><td>338869</td><td>ACS reagent</td></tr><tr><td>Hydrogen chloride (HCl)</td><td>Fisher Scientific</td><td>A144</td><td>reagent grade</td></tr><tr><td>polyacrylic acid (PAA)</td><td>Sigma Aldrich</td><td>323667</td><td>average Mw 1800</td></tr><tr><td>1-Hydroxybenzotriazole hydrate (HOBT)</td><td>Sigma Aldrich</td><td>54802</td><td>ACS reagent</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)</td><td>Sigma Aldrich</td><td>E7750</td><td>commercial grade</td></tr><tr><td>Dibenzocyclooctyne-amine (DBCO-NH&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>761540</td><td>ACS reagent</td></tr><tr><td>N,N-Diisopropylethylamine (DIPEA)</td><td>Sigma Aldrich</td><td>D125806</td><td>ACS reagent</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>BP231</td><td>Technical grade</td></tr><tr><td>HEK293 cell line</td><td>ATCC</td><td>CRL-1573</td><td>human embryonic kidney</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>F1051</td><td>ACS reagent</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15140122</td><td>10,000 U/mL</td></tr><tr><td>plasmid (pCAGGS-ChR2-Venus)</td><td>Addgene</td><td>15753</td><td>Plasmid sent as bacteria in agar stab</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>11965092</td><td>High glucose</td></tr><tr><td>opti-Modified Eagle Medium (MEM)</td><td>Thermo Fisher</td><td>51985034</td><td>Reduced Serum Media</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Thermo Fisher</td><td>L3000015</td><td>Lipid-Based Transfection</td></tr><tr><td>N-Azidoacetylmannosamine, Acetylated (Ac&lt;sub&gt;4&lt;/sub&gt;ManNAz)</td><td>Sigma Aldrich</td><td>A7605</td><td>ACS reagent</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Thermo Fisher</td><td>25200056</td><td>Phenol red</td></tr><tr><td>Rhod-3 AM Calcium Imaging Kit</td><td>Thermo Fisher</td><td>R10145</td><td>Fluorescence dye</td></tr><tr><td>5-carboxytetramethylrhodamine-azide (Rhod-N&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma Aldrich</td><td>760757</td><td>Azide-fluor 545</td></tr><tr><td>Confical dish</td><td>ibidi GmbH</td><td>81158</td><td>Glass Bottom, 35 mm</td></tr><tr><td>50 ml conical centrifuge tubes</td><td>Greiner Bio-One</td><td>227261</td><td>Polypropylene</td></tr><tr><td>15 ml conical centrifuge tubes</td><td>Greiner Bio-One</td><td>188271</td><td>Polypropylene</td></tr><tr><td>1.5 ml conical microcentrifuge tubes</td><td>Greiner Bio-One</td><td>616201</td><td>Polypropylene</td></tr><tr><td>Phenylmethyl silicone oil</td><td>Clearco Products</td><td>63148-52-7</td><td>Less than 320 degrees Celsius</td></tr><tr><td>Glass thermometer</td><td>GH Zeal</td><td>L0111/10</td><td>From -10 to 360 degrees Celsius</td></tr><tr><td>12-well plate</td><td>Sigma Aldrich</td><td>Z707775</td><td>Polystyrene</td></tr></tbody></table>

synthesis of core-shell lanthanide-doped upconversion nanocrystals for cellular applications

镧系掺杂的转换纳米晶 (UCNs) 近年来因其具有良好的可控光学特性而备受关注, 它可以吸收近红外 (近红外线) 光, 并能将其转化为多路多路的排放, 跨越了广泛的区域, 从紫外线到可见光到近红外。本文介绍了高温沉淀合成核壳 UCNs 的详细实验程序, 该方法将不同的镧系离子加入纳米晶, 有效转化深层穿透近红外激发 (808nm) 成强烈的蓝色发射在 480 nm。通过对生物相容聚合物 (聚丙烯酸、临机局) 的表面改性控制, 制备的 UCNs 在缓冲液中具有很大的溶解度。亲水纳米晶体进一步功能化的特定配体 (二苄基 cyclooctyne, DBCO) 的地方在细胞膜上。在近红外光 (808 nm) 辐照下, upconverted 蓝光发射可以有效地激活细胞膜上的 light-gated 通道蛋白, 并专门调节细胞质中的阳离子 (例如, Ca2 +) 的流入。该协议提供了一种可行的方法, 合成核壳镧系稀土掺杂 UCNs 和后续的生物相容表面改性进一步的蜂窝应用。

在图 1和图 2中显示了核壳系镧系掺杂的 UCNs 的合成过程。分别收集了核和核壳 UCNs 纳米结构的透射电子显微镜 (TEM) 和高分辨率透射电镜 (HRTEM) 图像 (图 1)。通过在酸性溶液中去除 UCNs 表面的疏水性油酸, 用亲水聚合物 (临机) 进行改性, 制备了无配体 UCNs。然后, 羧基盖的临机局-UCNs 是功能化的 DBCO-NH2</su...

Watch this Scientific Journal Video about Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications at JoVE.com

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

提出了一种用于合成核-壳镧系稀土掺杂转换纳米晶 (UCNs) 的协议, 以及它们在近红外线 (近红外) 光照下对通道蛋白调控的细胞应用。

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

synthesis-core-shell-lanthanide-doped-upconversion-nanocrystals-for

Universidad San Sebastian

Research

JoVE Journal

Chemistry

15.2K 次观看.  Nanyang Technological University. 提出了一种用于合成核-壳镧系稀土掺杂转换纳米晶 (UCNs) 的协议, 以及它们在近红外线 (近红外) 光照下对通道蛋白调控的细胞应用。

视频: Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Integrating a Triplet-triplet Annihilation Up-conversion System to Enhance Dye-sensitized Solar Cell Response to Sub-bandgap Light

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents and hence higher efficiencies. Photon up-conversion by way of triplet-triplet annihilation (TTA-UC) is an attractive technique for using these otherwise wasted low energy photons to produce photocurrent, while not interfering with the photoanodic performance in a deleterious manner. Further to this, TTA-UC has a number of features, distinct from other reported photon up-conversion technologies, which renders it particularly suitable for coupling with DSC technology. In this work, a proven high performance TTA-UC system, comprising a palladium porphyrin sensitizer and rubrene emitter, is combined with a high performance DSC (utilizing the organic dye D149) in an integrated device. The device shows an enhanced response to sub-bandgap light over the absorption range of the TTA-UC sub-unit resulting in the highest figure of merit for up-conversion assisted DSC performance to date.

An integrated device, incorporating a dye-sensitized solar cell and triplet-triplet annihilation up-conversion unit was produced, affording enhanced light harvesting, from a wider section of the solar spectrum. Under modest irradiation levels a significantly enhanced response to low energy photons was demonstrated, yielding a record figure of merit for dye-sensitized solar cells.

集成的三重态 - 三重态湮灭上转换系统，提高染料敏化太阳能电池的响应子带隙光

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents ...

科研

工程学

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

A&#39;即插即用&#39;的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

化学

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large extinction coefficients which can be tuned across the visible spectrum. Research into the plasmonic enhancement of optical transitions has become popular, due to the possibility of altering and in some cases improving photo-absorption or emission properties of nearby chromophores such as molecular dyes or quantum dots. The electric field of the plasmon can couple with the excitation dipole of a chromophore, perturbing the electronic states involved in the transition and leading to increased absorption and emission rates. These enhancements can also be negated at close distances by energy transfer mechanism, making the spatial arrangement of the two species critical. Ultimately, enhancement of light harvesting efficiency in plasmonic solar cells could lead to thinner and, therefore, lower cost devices. The development of hybrid core/shell particles could offer a solution to this issue. The addition of a dielectric spacer between a gold nanoparticles and a chromophore is the proposed method to control the exciton plasmon coupling strength and thereby balance losses with the plasmonic gains. A detailed procedure for the coating of gold nanoparticles with CdS and ZnS semiconductor shells is presented. The nanoparticles show high uniformity with size control in both the core gold particles and shell species allowing for a more accurate investigation into the plasmonic enhancement of external chromophores.

The synthesis of uniform gold nanoparticles coated with semiconductor shells of CdS or ZnS is performed. The semiconductor coating is conducted by first depositing a silver sulfide shell and exchanging the silver cations for zinc or cadmium cations.

合成，表征和混合金/硫化镉和Au / ZnS核/壳纳米颗粒功能化

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

生物工程

Hyperspectral Imaging as a Tool to Study Optical Anisotropy in Lanthanide-Based Molecular Single Crystals

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based molecular single crystals. As representative example, we chose a single crystal of the heterodinuclear Ln-based complex [TbEu(bpm)(tfaa)6] (bpm=2,2&#8217;-bipyrimidine, tfaa&#8211; =1,1,1-trifluoroacetylacetonate) exhibiting bright visible emission under UV excitation. HSI is an emerging technique that combines 2-dimensional spatial imaging of a luminescent structure with spectral information from each pixel of the obtained image. Specifically, HSI on single crystals of the [Tb-Eu] complex provided local spectral information unveiling variation of the luminescence intensity at different points along the studied crystals. These changes were attributed to the optical anisotropy present in the crystal, which results from the different molecular packing of Ln3+ ions in each one of the directions of the crystal structure. The HSI herein described is an example of the suitability of such technique for spectro-spatial investigations of molecular materials. Yet, importantly, this protocol can be easily extended for other types of luminescent materials (such as micron-sized molecular crystals, inorganic microparticles, nanoparticles in biological tissues, or labelled cells, among others), opening many possibilities for deeper investigation of structure-property relationships. Ultimately, such investigations will provide knowledge to be leveraged into the engineering of advanced materials for a wide range of applications from bioimaging to technological applications, such as waveguides or optoelectronic devices.

Here, we present a protocol to obtain luminescent hyperspectral imaging data and to analyze optical anisotropy features of lanthanide-based single crystals using a Hyperspectral Imaging System.

高光谱成像作为研究基于兰塔尼的分子单晶光学自给性的工具

In this work, we describe a protocol for a novel application of hyperspectral imaging (HSI) in the analysis of luminescent lanthanide (Ln3+)-based ...

Triplet Fusion Upconversion Nanocapsule Synthesis

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has significant implications for producing high energy light beyond a material's surface. However, the deployment of UC materials has been stymied due to poor material solubility, high concentration requirements, and oxygen sensitivity, ultimately resulting in reduced light output. Toward this end, nanoencapsulation has been a popular motif to circumvent these challenges, but durability has remained elusive in organic solvents. Recently, a nanoencapsulation technique was engineered to tackle each of these challenges, whereupon an oleic acid nanodroplet containing upconversion materials was encapsulated with a silica shell. Ultimately, these nanocapsules (NCs) were durable enough to enable triplet fusion upconversion-facilitated volumetric three-dimensional (3D) printing. By encapsulating upconversion materials with silica and dispersing them in a 3D printing resin, photopatterning beyond the surface of the printing vat was made possible. Here, video protocols for the synthesis of upconversion NCs are presented for both small-scale and large-scale batches. The outlined protocols serve as a starting point for adapting this encapsulation scheme to multiple upconversion schemes for use in volumetric 3D printing applications.

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

三重融合上转换纳米胶囊合成

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has ...

Synthesis of Single-Crystalline Core-Shell Metal-Organic Frameworks

Because of their designability and unprecedented synergistic effects, core-shell metal-organic frameworks (MOFs) have been actively examined recently. However, the synthesis of single-crystalline core-shell MOFs is very challenging, and thus a limited number of examples have been reported. Here, we suggest a method of synthesizing single-crystalline HKUST-1@MOF-5 core-shells, which is HKUST-1 at the center of MOF-5. Through the computational algorithm, this pair of MOFs was predicted to have the matched lattice parameters and chemical connection points at the interface. To construct the core-shell structure, we prepared the octahedral- and cubic-shaped HKUST-1 crystals as a core MOF, in which the (111) and (001) facets were mainly exposed, respectively. Via the sequential reaction, the MOF-5 shell was well-grown on the exposed surface, showing a seamless connect interface, which resulted in the successful synthesis of single-crystalline HKUST-1@MOF-5. Their pure phase formation was proved by optical microscopic images and powder X-ray diffraction (PXRD) patterns. This method presents the potential of and insights into the single-crystalline core-shell synthesis with different kinds of MOFs.

Here, we demonstrate a protocol for the two-step synthesis of single-crystalline core-shells using a non-isostructural metal-organic framework (MOF) pair, HKUST-1 and MOF-5, which have well-matched crystal lattices.

单晶核壳金属有机骨架的合成

Because of their designability and unprecedented synergistic effects, core-shell metal-organic frameworks (MOFs) have been actively examined recently. ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

持久性发光纳米颗粒的水热合成

Synthesis of Persistent Luminescent Nanoparticles for Rewritable Displays and Illumination Applications

Persistent luminescent nanoparticles (PLNPs) possess the capabilities to maintain extended longevity and robust emission even after the excitation has ceased. PLNPs have been widely used across various domains, including information displays, data encryption, biological imaging, and artistic decoration with sustained and vivid luminosity, providing boundless possibilities for a variety of innovative technology and artistic projects. This protocol focuses on an experimental procedure for the hydrothermal synthesis of PLNPs. The successful synthesis of enduring luminescent nanomaterials with Mn2+ or Cr3+ serving as a luminescent center in Zn2GeO4: Mn (ZGO: Mn) or ZnGa2O4: Cr highlights the universality of this synthetic method. On the other hand, the optical properties of ZGO: Mn can be changed by adjusting the pH of precursor solutions, demonstrating the tunability of the protocol. When charged with ultraviolet (UV) at a wavelength of 365 nm for 3 min and then stopped, PLNPs exhibit the remarkable capacity to generate afterglow efficiently and consistently, which makes them ideal for making two-dimensional rewritable displays and three-dimensional transparent, luminous artworks. This protocol outlined in this paper provides a feasible method for the synthesis of persistent luminescent nanoparticles for further illumination and imaging applications, opening up novel prospects for the fields of science and art.

作者聚焦：利用功能纳米材料推进生物成像和治疗

A protocol is presented for the synthesis of persistent luminescent nanomaterials (PLNPs) and their potential applications in rewritable displays and artistic processing utilizing the afterglow effect under ultraviolet light (365 nm) irradiation.

用于可擦写显示器和照明应用的持久性发光纳米颗粒的合成

Persistent luminescent nanoparticles (PLNPs) possess the capabilities to maintain extended longevity and robust emission even after the excitation has ...

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

摘要

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此视频中的章节

集成的三重态 - 三重态湮灭上转换系统，提高染料敏化太阳能电池的响应子带隙光

A'即插即用'的方法来创建水分散性Nanoassemblies包含两亲聚合物，有机染料和上变频纳米粒子

合成，表征和混合金/硫化镉和Au / ZnS核/壳纳米颗粒功能化

纳米谐波再生研究

高光谱成像作为研究基于兰塔尼的分子单晶光学自给性的工具

三重融合上转换纳米胶囊合成

单晶核壳金属有机骨架的合成

转换纳米粒子介导的激酶活化远程触发细胞内信号转导的集成系统

用于可擦写显示器和照明应用的持久性发光纳米颗粒的合成

用于可擦写显示器和照明应用的持久性发光纳米颗粒的合成