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Bioengineering

生物应用近红外发光金纳米簇的合成

Published: March 22, 2020
doi:
10.3791/60388
, , , , , ,
1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, 2Department of Chemical Physics and Optics, Faculty of Mathematics and Physics,Charles University, 3Department of Cell Biology, Faculty of Science,Charles University, 4First Faculty of Medicine,Charles University

Summary

介绍了一种可靠且易于重现的方法，用于制备可功能的近红外发光金纳米簇，并通过流细胞学和共聚焦激光扫描显微镜直接检测HeLa细胞内部。

Abstract

在过去十年中，荧光金纳米簇（AuNCs）在生物应用中越来越受欢迎，并致力于其开发。在该协议中，详细介绍了一种最近开发的制备水溶性、生物相容性和胶体稳定近红外发光AuNCs的方法。这种室温自下而上的化学合成提供了易于功能的AuNCs，在水溶液中用三氯酸和三醇改性聚乙烯乙二醇盖住。合成方法既不需要有机溶剂或附加配体交换，也不需要丰富的合成化学知识来繁殖。由此产生的AuNCs提供免费表面碳碱酸，可与具有自由胺组的各种生物分子一起进行功能化，而不会对AuNCs的光致发光特性产生不利影响。还描述了HeLa细胞对AuNC摄取的流细胞程定量和共聚焦显微成像的快速、可靠的过程。由于斯托克斯发生大移位，因此，为了有效检测AuNC的近红外光致发光，必须正确设置流式细胞学和共聚焦显微镜中的滤波器。

Introduction

在过去十年中，超小型（±2 nm）光致发光金纳米簇（PL AuNC）已成为基础研究和实际,,应用11、2、3、4、5、6、7、8、9、102的有前途的探针。10,8,9,5,6,7,34其许多理想的特性包括高光稳定性、可调发射最大值、长发射寿命、大斯托克斯移位、低毒性、良好的生物相容性、肾间隙和方便的生物结合。PL AuNCs 可以提供从蓝色到近红外 （NIR） 光谱区域的光致发光，具体取决于星团11内的原子数和表面配体12的性质。NIR（650-900 nm）发射AuNCs对于细胞和组织的长期体外和体内成像特别有希望，因为它们提供高信噪比，因为与内在自荧光的最小重叠、散射和吸收较弱以及NIR光13、14,14的组织渗透率高。

近年来，利用Au-S共价相互作用的各种方法已经开发出来，以制备含有各种含Thiol配体的NIR-PL AuNCs13、15、16、17。13,15,16,17对于生物医学应用，AuNCs 必须使用生物成分进行功能化，以促进结合相互作用。因此，高胶体稳定性且在水溶剂中易于功能的 AuN 是极可取的。本协议的总体目标是描述先前报告的18种AuNCs制备，其表面具有可功能的碳化物酸组，在水环境中详细采用三氯酸和聚乙烯乙二醇（PEG），并与遵循酸-a胺耦合法的具有原胺的分子结合。由于合成的方便性和高可重复性，这种协议可以由非化学背景的研究人员使用和调整。

AuNC 在生物医学研究中的应用的关键要求之一是能够观察和测量细胞内的 AuNC。在监测纳米粒子被细胞接受的方法中，流细胞学（FCM）和共聚焦激光扫描显微镜（CLSM）提供了稳健、高通量的方法，能够快速测量大量细胞中荧光纳米材料的内化。这里还介绍了FCM和CLSM直接测量和分析细胞内PL AuN的方法，无需额外的染料。

Protocol

1. 制备近红外发射AuNC （1） 加入7.8毫克（37.8μmol）三基酸（TA）和60μL的2米NaOH到23.4 mL的超纯水（25°C的电阻率18.2 MΩ.cm），搅拌（至少1000 rpm），直到完全溶解（±15-20分钟）。为了更快地溶解 TA，将混合物声波。对于合成，建议使用新鲜准备的 TA 解决方案。 将10.2 μL的HAuCl4+3H2O（470mg/mL）的水溶液添加到溶液中。 15分钟后，在剧烈搅拌（至少1，0004 rp…

Representative Results

NIR PL AuNC 是在 TA 存在的情况下从 Au3+准备的，然后在 AuNC 表面上绑定了三醇端接的 PEG （MW 2，000），以按照图 1所示的工作流获得1。在1和3（氨基丙基）三磷酸（TPP）溴化物之间的阿米里质耦合提供2。正如预期的那样，吸收光谱（图2a）表明，AuNCs 1和2没有特征的表面质粒带，并且…

Discussion

使用自下而上的方法合成了排放近红外的AuNCs，其中黄金前体溶液（HAuCl4）用合适的三醇配体处理，然后减少Au3+。水溶液中的金属离子的减少往往聚集并产生大纳米粒子，而不是超小的NCs21。为了制备超小（±2 nm）的PL AuN，对合成条件进行了调整，以防止形成大颗粒，促进超小簇的形成。用来盖住AuNC表面的配体的性质在影响粒子12、22、23、24、25、26、27、28、29、…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢阿尔兹贝塔·马格多列诺娃在流细胞学方面的帮助。作者承认GACR项目Nr.18-12533S的财政支持。显微镜由欧洲区域发展基金和捷克共和国国家预算共同资助的共聚焦和荧光显微镜实验室进行，项目没有。CZ.1.05/4.1.00/16.0347 和 CZ.2.16/3.1.00/21515，并得到捷克-生物成像大型 RI 项目 LM2015062 的支持。

Materials

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride TCI Chemicals D1601 https://www.tcichemicals.com/eshop/en/eu/commodity/D1601/;jsessionid=3AD046E5389206AAE33C8AAB5036CDD6?gclid=CjwKCAjwiZnnBRBQEiwAcWKfYrO69K6Np3tYeSsAouqGndUvzzsy1hStBPuHG-X3cpTIsAqq9z0cDBoC76MQAvD_BwE
Bovine serum albumin Sigma-Aldrich A4161 https://www.sigmaaldrich.com/catalog/product/sigma/a4161?lang=en&region=CZ
Disodium hydrogen phosphate dihydrate PENTA s.r.o. 15130-31000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_281.pdf
DL-Thioctic acid, 98% Alfa Aesar L04711 https://www.alfa.com/en/catalog/L04711/
Hydrochloric acid 35% PENTA s.r.o. 19350-11000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_512.pdf
Hydrogen tetrachloroaurate(III) trihydrate, ACS, 99.99% (metals basis), Au 49.0% min Alfa Aesar 36400 https://www.alfa.com/en/catalog/036400/
O-(2-Mercaptoethyl)-O′-methylpolyethylene glycol 2000 Sigma-Aldrich 743127 https://www.sigmaaldrich.com/catalog/product/aldrich/743127?lang=en&region=CZ
Potassium chloride PENTA s.r.o. 16200-31000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_346.pdf
Sodium borohydride Sigma-Aldrich 452882 https://www.sigmaaldrich.com/catalog/product/aldrich/452882?lang=en&region=CZ&gclid=CjwKCAjwiZnnBRBQEiwAcWKfYuoZKvdK_fH24F1gGugG4pamF2FFZLd36YyZmRTdGgkbm5SbyGP0jBoCoo0QAvD_BwE
Sodium chloride PENTA s.r.o. 16610-31000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_376.pdf
Sodium dihydrogenphosphate dihydrate PENTA s.r.o. 12330-31000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_124.pdf
Sodium hydroxide pellets PENTA s.r.o. 15740-31000 https://www.pentachemicals.eu/soubory/specifikace/specifikace_307.pdf
XTT (sodium 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) Thermo Fisher Scientific X12223 https://www.thermofisher.com/order/catalog/product/X12223#/X12223
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References

  1. Wang, Y., Chen, J., Irudayaraj, J. Nuclear Targeting Dynamics of Gold Nanoclusters for Enhanced Therapy of HER2+ Breast Cancer. ACS Nano. 5 (12), 9718-9725 (2011).
  2. Chen, L. Y., Wang, C. W., Yuan, Z., Chang, H. T. Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Analytical Chemistry. 87 (1), 216-229 (2015).
  3. Dongyun, C., Zhentao, L., Li, N., Lee, J. Y., Xie, J., Lu, J. Jianmei Amphiphilic Polymeric Nanocarriers with Luminescent Gold Nanoclusters for Concurrent Bioimaging and Controlled Drug Release. Advanced Functional Materials. 23 (35), 4324-4331 (2013).
  4. Tan, X., Jin, R. Ultrasmall metal nanoclusters for bio-related applications. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 5 (6), 569-581 (2013).
  5. Yuan, X., Luo, Z., Yu, Y., Yao, Q., Xie, J. Luminescent Noble Metal Nanoclusters as an Emerging Optical Probe for Sensor Development. Chemistry – An Asian Journal. 8 (5), 858-871 (2013).
  6. Zheng, K., Setyawati, M. I., Leong, D. T., Xie, J. Antimicrobial Gold Nanoclusters. ACS Nano. 11 (7), 6904-6910 (2017).
  7. Li, Q., et al. Design and mechanistic study of a novel gold nanocluster-based drug delivery system. Nanoscale. 10 (21), 10166-10172 (2018).
  8. Zhang, X. D., et al. Ultrasmall Au10-12(SG)10-12 Nanomolecules for High Tumor Specificity and Cancer Radiotherapy. Advanced Materials. 26 (26), 4565-4568 (2014).
  9. Zhang, X. D., et al. Ultrasmall Glutathione-Protected Gold Nanoclusters as Next Generation Radiotherapy Sensitizers with High Tumor Uptake and High Renal Clearance. Scientific Reports. 5, 8669 (2015).
  10. Zhang, X. D., et al. Enhanced Tumor Accumulation of Sub-2 nm Gold Nanoclusters for Cancer Radiation Therapy. Advanced Healthcare Materials. 3 (1), 133-141 (2014).
  11. Zheng, J., Zhang, C., Dickson, R. M. Highly Fluorescent, Water-Soluble, Size-Tunable Gold Quantum Dots. Physical Review Letters. 93 (7), 077402 (2004).
  12. Wu, Z., Jin, R. On the Ligand’s Role in the Fluorescence of Gold Nanoclusters. Nano Letters. 10 (7), 2568-2573 (2010).
  13. Lin, C. A. J., et al. Synthesis, Characterization, and Bioconjugation of Fluorescent Gold Nanoclusters toward Biological Labeling Applications. ACS Nano. 3 (2), 395-401 (2009).
  14. Yang, L., Shang, L., Nienhaus, G. U. Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells. Nanoscale. 5 (4), 1537-1543 (2013).
  15. Mishra, D., et al. Aqueous Growth of Gold Clusters with Tunable Fluorescence Using Photochemically Modified Lipoic Acid-Based Ligands. Langmuir. 32 (25), 6445-6458 (2016).
  16. Wu, Z., Gayathri, C., Gil, R. R., Jin, R. Probing the Structure and Charge State of Glutathione-Capped Au25(SG)18 Clusters by NMR and Mass Spectrometry. Journal of the American Chemical Society. 131 (18), 6535-6542 (2009).
  17. Stamplecoskie, K. G., Kamat, P. V. Size-Dependent Excited State Behavior of Glutathione-Capped Gold Clusters and Their Light-Harvesting Capacity. Journal of the American Chemical Society. 136 (31), 11093-11099 (2014).
  18. Pramanik, G., et al. Gold nanoclusters with bright near-infrared photoluminescence. Nanoscale. 10 (8), 3792-3798 (2018).
  19. Salvati, A., et al. Quantitative measurement of nanoparticle uptake by flow cytometry illustrated by an interlaboratory comparison of the uptake of labelled polystyrene nanoparticles. NanoImpact. 9, 42-50 (2018).
  20. Zhang, C. J., et al. Mechanism-Guided Design and Synthesis of a Mitochondria-Targeting Artemisinin Analogue with Enhanced Anticancer Activity. Angewandte Chemie. 128 (44), 13974-13978 (2016).
  21. Shang, L., Dong, S., Nienhaus, G. U. Ultra-small fluorescent metal nanoclusters: Synthesis and biological applications. Nano Today. 6 (4), 401-418 (2011).
  22. Higaki, T., et al. Controlling the Atomic Structure of Au30 Nanoclusters by a Ligand-Based Strategy. Angewandte Chemie International Edition. 55 (23), 6694-6697 (2016).
  23. Li, G., et al. Tailoring the Electronic and Catalytic Properties of Au25 Nanoclusters via Ligand Engineering. ACS Nano. 10 (8), 7998-8005 (2016).
  24. Kim, A., Zeng, C., Zhou, M., Jin, R. Surface Engineering of Au36(SR)24 Nanoclusters for Photoluminescence Enhancement. Particle & Particle Systems Characterization. 34 (8), 1600388 (2017).
  25. Chevrier, D. M., et al. Molecular-Scale Ligand Effects in Small Gold–Thiolate Nanoclusters. Journal of the American Chemical Society. 140 (45), 15430-15436 (2018).
  26. Yuan, X., Goswami, N., Chen, W., Yao, Q., Xie, J. Insights into the effect of surface ligands on the optical properties of thiolated Au25 nanoclusters. Chemical Communications. 52 (30), 5234-5237 (2016).
  27. Yuan, X., Goswami, N., Mathews, I., Yu, Y., Xie, J. Enhancing stability through ligand-shell engineering: A case study with Au25(SR)18 nanoclusters. Nano Research. 8 (11), 3488-3495 (2015).
  28. Jiang, J., et al. Oxidation at the Core-Ligand Interface of Au Lipoic Acid Nanoclusters That Enhances the Near-IR Luminescence. The Journal of Physical Chemistry C. 118 (35), 20680-20687 (2014).
  29. Padelford, J. W., Wang, T., Wang, G. Enabling Better Electrochemical Activity Studies of H2O-Soluble Au Clusters by Phase Transfer and a Case Study of Lipoic-Acid-Stabilized Au22. ChemElectroChem. 3 (8), 1201-1205 (2016).
  30. Wang, T., Wang, D., Padelford, J. W., Jiang, J., Wang, G. Near-Infrared Electrogenerated Chemiluminescence from Aqueous Soluble Lipoic Acid Au Nanoclusters. Journal of the American Chemical Society. 138 (20), 6380-6383 (2016).
  31. Aldeek, F., Muhammed, M. A. H., Palui, G., Zhan, N., Mattoussi, H. Growth of Highly Fluorescent Polyethylene Glycol- and Zwitterion-Functionalized Gold Nanoclusters. ACS Nano. 7 (3), 2509-2521 (2013).
  32. Oh, E., Susumu, K., Goswami, R., Mattoussi, H. One-Phase Synthesis of Water-Soluble Gold Nanoparticles with Control over Size and Surface Functionalities. Langmuir. 26 (10), 7604-7613 (2010).
  33. Nair, L. V., Nazeer, S. S., Jayasree, R. S., Ajayaghosh, A. Fluorescence Imaging Assisted Photodynamic Therapy Using Photosensitizer-Linked Gold Quantum Clusters. ACS Nano. 9 (6), 5825-5832 (2015).
  34. Porret, E., et al. Hydrophobicity of Gold Nanoclusters Influences Their Interactions with Biological Barriers. Chemistry of Materials. 29 (17), 7497-7506 (2017).
  35. Shang, L., et al. One-Pot Synthesis of Near-Infrared Fluorescent Gold Clusters for Cellular Fluorescence Lifetime Imaging. Small. 7 (18), 2614-2620 (2011).
  36. Wu, M., et al. Solution NMR Analysis of Ligand Environment in Quaternary Ammonium-Terminated Self-Assembled Monolayers on Gold Nanoparticles: The Effect of Surface Curvature and Ligand Structure. Journal of the American Chemical Society. 141 (10), 4316-4327 (2019).
  37. Gao, X., Cui, Y., Levenson, R. M., Chung, L. W. K., Nie, S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nature Biotechnology. 22 (8), 969-976 (2004).
  38. Bartczak, D., Kanaras, A. G. Preparation of Peptide-Functionalized Gold Nanoparticles Using One Pot EDC/Sulfo-NHS Coupling. Langmuir. 27 (16), 10119-10123 (2011).
  39. Dutta, D., Sailapu, S. K., Chattopadhyay, A., Ghosh, S. S. Phenylboronic Acid Templated Gold Nanoclusters for Mucin Detection Using a Smartphone-Based Device and Targeted Cancer Cell Theranostics. ACS Applied Materials & Interfaces. 10 (4), 3210-3218 (2018).
  40. Retnakumari, A., et al. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia. Nanotechnology. 22 (28), 285102 (2011).
  41. Pyo, K., et al. Highly Luminescent Folate-Functionalized Au22 Nanoclusters for Bioimaging. Advanced Healthcare Materials. 6 (16), 1700203 (2017).
  42. Fernández, T. D., et al. Intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells. Biomaterials. 43, 1-12 (2015).

Tags

Gold NanoclustersNear-infrared EmittingPhotoluminescentThiol-functionalized LigandAmine-functionalized LigandBiosensingBio-imagingUltrafiltrationPolyethylene GlycolEDC HCLCell Culture
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Pramanik, G., Keprova, A., Valenta, J., Bocan, V., Kvaková, K., Libusova, L., Cigler, P. Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications. J. Vis. Exp. (157), e60388, doi:10.3791/60388 (2020).
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