核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| 1-Octadecene | Sigma Aldrich | O806 | Technical grade |
| oleic acid | Sigma Aldrich | 364525 | Technical grade |
| Methanol | Fisher Scientific | A412 | Technical grade |
| Ethanol | Fisher Scientific | A405 | Technical grade |
| Acetone | Fisher Scientific | A18 | Technical grade |
| Hexane | Sigma Aldrich | H292 | Technical grade |
| Thulium (III) acetate hydrate (Tm(CH3CO2)3) | Sigma Aldrich | 367702 | 99.9% trace metals basis |
| Neodymium (III) acetate hydrate (Nd(CH3CO2)3) | Sigma Aldrich | 325805 | 99.9% trace metals basis |
| Ytterbium (III) acetate hydrate (Yb(CH3CO2)3) | Sigma Aldrich | 326011 | 99.9% trace metals basis |
| Yttrium(III) acetate hydrate (Y(CH3CO2)3) | Sigma Aldrich | 326046 | 99.9% trace metals basis |
| Sodium hydroxide (NaOH) | Sigma Aldrich | S5881 | reagent grade |
| Ammonium fluoride (NH4F) | Sigma Aldrich | 338869 | ACS reagent |
| Hydrogen chloride (HCl) | Fisher Scientific | A144 | reagent grade |
| polyacrylic acid (PAA) | Sigma Aldrich | 323667 | average Mw 1800 |
| 1-Hydroxybenzotriazole hydrate (HOBT) | Sigma Aldrich | 54802 | ACS reagent |
| 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) | Sigma Aldrich | E7750 | commercial grade |
| Dibenzocyclooctyne-amine (DBCO-NH2) | Sigma Aldrich | 761540 | ACS reagent |
| N,N-Diisopropylethylamine (DIPEA) | Sigma Aldrich | D125806 | ACS reagent |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | BP231 | Technical grade |
| HEK293 cell line | ATCC | CRL-1573 | human embryonic kidney |
| Fetal Bovine Serum (FBS) | Sigma Aldrich | F1051 | ACS reagent |
| Penicillin-Streptomycin | Thermo Fisher | 15140122 | 10,000 U/mL |
| plasmid (pCAGGS-ChR2-Venus) | Addgene | 15753 | Plasmid sent as bacteria in agar stab |
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher | 11965092 | High glucose |
| opti-Modified Eagle Medium (MEM) | Thermo Fisher | 51985034 | Reduced Serum Media |
| Lipofectamine 3000 Transfection Reagent | Thermo Fisher | L3000015 | Lipid-Based Transfection |
| N-Azidoacetylmannosamine, Acetylated (Ac4ManNAz) | Sigma Aldrich | A7605 | ACS reagent |
| Trypsin-EDTA (0.25%) | Thermo Fisher | 25200056 | Phenol red |
| Rhod-3 AM Calcium Imaging Kit | Thermo Fisher | R10145 | Fluorescence dye |
| 5-carboxytetramethylrhodamine-azide (Rhod-N3) | Sigma Aldrich | 760757 | Azide-fluor 545 |
| Confical dish | ibidi GmbH | 81158 | Glass Bottom, 35 mm |
| 50 ml conical centrifuge tubes | Greiner Bio-One | 227261 | Polypropylene |
| 15 ml conical centrifuge tubes | Greiner Bio-One | 188271 | Polypropylene |
| 1.5 ml conical microcentrifuge tubes | Greiner Bio-One | 616201 | Polypropylene |
| Phenylmethyl silicone oil | Clearco Products | 63148-52-7 | Less than 320 degrees Celsius |
| Glass thermometer | GH Zeal | L0111/10 | From -10 to 360 degrees Celsius |
| 12-well plate | Sigma Aldrich | Z707775 | Polystyrene |
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