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(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>367702</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Neodymium (III) acetate hydrate (Nd(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>325805</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Ytterbium (III) acetate hydrate (Yb(CH3CO2)3)</td>
			<td>Sigma Aldrich</td>
			<td>326011</td>
			<td>99.9% trace metals basis</td>
		</tr>
		<tr>
			<td>Yttrium(III) acetate hydrate (Y(CH3CO2)3)</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 (NH4F)</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-NH2)</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&#39;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 (Ac4ManNAz)</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-N3)</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

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

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

科研

化学

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

硒化镉/硫化镉棒和纳米四脚合成种子

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

的吲哚酚苷的检测糖苷酶活动的合成

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

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 ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

镉免费的InP /硫化锌量子点的合成适合生物医学应用

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

金纳米棒基于对苯二酚合成

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Measurements of Long-range Electronic Correlations During Femtosecond Diffraction Experiments Performed on Nanocrystals of Buckminsterfullerene

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the results of femtosecond X-ray free electron laser (XFEL) experiments. An increasing number of experimental observations have shown that although nuclear motion can be negligible, given a short enough incident pulse duration, electronic motion cannot be ignored. The current and widely accepted models assume that although electrons undergo dynamics driven by interaction with the pulse, their motion could largely be considered &#39;random&#39;. This would then allow the supposedly incoherent contribution from the electronic motion to be treated as a continuous background signal and thus ignored. The original aim of our experiment was to precisely measure the change in intensity of individual Bragg peaks, due to X-ray induced electronic damage in a model system, crystalline C60. Contrary to this expectation, we observed that at the highest X-ray intensities, the electron dynamics in C60 were in fact highly correlated, and over sufficiently long distances that the positions of the Bragg reflections are significantly altered. This paper describes in detail the methods and protocols used for these experiments, which were conducted both at the Linac Coherent Light Source (LCLS) and the Australian Synchrotron (AS) as well as the crystallographic approaches used to analyse the data.

We describe an experiment designed to probe the electronic damage induced in nanocrystals of Buckminsterfullerene (C60) by intense, femtosecond pulses of X-rays. The experiment found that, surprisingly, rather than being stochastic, the X-ray induced electron dynamics in C60 are highly correlated, extending over hundreds of unit cells within the crystals1.

在纳米富勒的飞秒衍射实验的长程电子关联的测量

The precise details of the interaction of intense X-ray pulses with matter are a topic of intense interest to researchers attempting to interpret the ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

结合电喷雾、自组装和溶剂型结构控制制备球形和蜗杆状胶束纳米晶

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.