可控合成和高度统一聚荧光追踪（<em>ñ</em> -isopropylacrylamide）微凝胶
Summary
非搅拌沉淀聚合提供了一种快速，可重复的成型方法来刺激敏感聚（N- -isopropylacrylamide）粒径分布窄的微粒凝胶的合成。在这个协议中合成，光散射表征和在宽视场显微镜设置这些微粒凝胶的单个粒子荧光跟踪被证明。
Abstract
刺激敏感聚（N -isopropylacrylamide）（PNIPAM）微凝胶具有前瞻性的各种实际应用和基础研究使用。在这项工作中，我们使用单一粒子由一个快速无搅拌沉淀聚合过程跟踪的荧光标记PNIPAM微凝胶作为调谐微粒凝胶大小展示。这种方法非常适合原型新的反应的组合物和条件或用于不需要大量产品的应用程序。微凝胶合成，颗粒大小和结构测定动态和静态光散射在协议中详细介绍。它表明在加入官能共聚单体可具有在颗粒成核和结构有很大的影响。通过宽视场荧光显微镜单粒子跟踪允许标记的示踪剂微粒凝胶的扩散的非标记的微凝胶的浓缩矩阵调查，系统不易受调查其他方法，如动态光散射。
Introduction
刺激敏感聚（N -isopropylacrylamide）（PNIPAM）微凝胶1,2吸引过去二十年的不断兴趣，因为他们在各种智能应用的潜力。证明用例包括可切换的乳液稳定剂3-8，微透镜9中，为了便于细胞收集10,11细胞培养基材，以及低分子量化合物的智能的载体和其它生物医学用途12。从一个基础研究点这些颗粒已被证明是调查受试者有用诸如胶体相互作用13-15和聚合物-溶剂相互作用16-18。
成功使用PNIPAM微凝胶以及它们的衍生物中的任何给定的应用的典型地需要在粒度分布的平均粒径和宽的知识。对实验结果的涉及PNIPAM微正确解释凝胶，颗粒结构，这可以通过官能共聚单体的影响，必须已知。动态和静态光散射（DLS和SLS，分别）是唯一适用于因为这些方法是快速和相对容易使用的获取这些信息;它们探查颗粒性能非侵入在其天然环境中（分散液）。 DLS和SLS还收集广大避免由于样本量小，是典型的显微镜方法而产生的偏见颗粒的数据。因此，这项工作的第一目的是介绍关于对从业人员新的胶体特性的光散射很好的做法。
典型地，沉淀聚合在实验室规模进行，并找到合适的反应条件对于特定的颗粒属性可以是费力的和所需要的合成的许多重复。在对比大批量合成，无搅拌沉淀聚合19,20是芳APID程序，其中不同的反应物组合物的批次可以聚合粒径分布窄的同时屈服颗粒。同时聚合减少实验误差，产量大意味着正确的反应条件可快扩大该反应被发现。因此，我们的第二个目的是要证明在原型和在不需要大量产品的应用程序的非搅拌沉淀聚合的有用性。
合成与表征的不同方面走到了一起，在胶体相互作用的研究荧光标记的PNIPAM微凝胶的应用实例。这里我们使用高度精确的单粒子跟踪调查标记的示踪剂微凝胶在未标记的基质微凝胶分散在很宽的基质浓度范围扩散和解决集中胶体分散的笼效应。宽视场荧光显微镜是非常适合FOR此目的，因为它可以表征中的大量潜在的不同的矩阵种类的几示踪分子的特定行为。这是相对于技术如DLS，SLS和流变学，其测量的系统总体平均性能，因此无法解析在一个大系统的小数目的探针粒子的行为。此外，在该具体例常规的光散射方法不能也利用由于高粒子浓度，这会导致强烈的多次散射无效任何标准分析。自动数据处理和统计方法使用使整个系统的行为也对单粒子跟踪，当通过大样本平均分析。
Protocol
1.微凝胶合成注：N -isopropylacrylamide（NIPAM）从正己烷重结晶。原样使用其它试剂。 聚（NIPAM）矩阵微凝胶的常规合成批在过滤245毫升溶解1.8克NIPAM和24毫克N，N'-bisacrylamide（BIS）（0.2微米的再生纤维素（RC）的膜过滤器）在装有回流冷凝器的500毫升三颈圆底烧瓶中双蒸水，搅拌器和橡胶隔片。 插入温度计和120mm的针头穿过隔膜的氮气输入。 </…
Representative Results
PNIPAM微粒凝胶颗粒的批次中从而最终颗粒体积的数量，而且，在成核阶段20疏水共聚单体染料甲基丙烯酰硫代若丹明B通过减少在间歇的粒子数密度影响成核过程中的反应初确定。为两个不同的初始NIPAM浓度颗粒浓度的降低可以被看作是在折叠状态与染料浓度的增加，平均最终颗粒的体积增加，在图1所示的?…
Discussion
少量官能共聚单体的加成可对PNIPAM衍生微凝胶的粒度和结构的显著效果。同时小规模试管聚合是考虑到这种变化的好方法，有助于迅速找到目标粒度合适的反应剂组合物根据需要扩大该反应。该颗粒的质量是约指数依赖当热分解引发剂的聚合温度，例如KPS，使用20，因此，人们需要建立反应器中良好的再现内稳定和精确的温度…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for financial support within the Sonderforschungsbereich SFB 985 “Functional Microgels and Microgel Systems”.
Materials
| Acetone | VWR Chemicals | KRAF13455 | |
| Bisacrylamid | AppliChem | A3636 | |
| n-Hexane | Merck | 104374 | |
| N-Isopropylacrylamide | Fisher Scientific | AC412785000 | recrystallized from n-hexane |
| Methacryloxyethyl thiocarbamoyl rhodamine B | Polysciences | 23591 | |
| Potassium peroxodisulfate | Merck | 105091 | |
| Silicone oil 47 V 350 | VWR Chemicals | 83851 | |
| Toluene | Sigma Aldrich | 244511 | |
| F12 Refrigerated/heating circulator | Julabo | 9116612 | |
| Microscope | Olympus | IX83 | |
| XY(Z) Piezo System | Physik Instrumente | P-545.3R7 | |
| 100x Oil immersion objective | Olympus | UPLSAPO | |
| QuadLine Beamsplitter | AHF Analysentechnik | F68-556T | |
| Cobolt Jive 150 laser | Cobolt | 0561-04-01-0150-300 | |
| Multimode Fiber | Thorlabs | UM22-600 | |
| iXON Ultra 897 EMCCD camera | Andor | DU-897U-CS0-BV | |
| Laser goniometer | SLS Systemtechnik | Mark III | |
| CF40 Cryo-compact circulator | Julabo | 9400340 | |
| Laser goniometer system | ALV GmbH | ALV / CGS-8F | |
| Multi-tau corretator | ALV GmbH | ALV-7004 | |
| Light scattering electronics | ALV GmbH | ALV / LSE 5004 | |
| Photon counting module | PerkinElmer | SPCM-CD2969 | 2 units in pseudo cross-correlation mode |
| 633 nm HeNe Laser | JDS Uniphase | 1145P | |
| F32 Refrigerated/heating circulator | Julabo | 9312632 |
References
- Pelton, R. Temperature-sensitive aqueous microgels. Adv. Colloid Interfac. 85, 1-33 (2000).
- Pich, A., Richtering, W. Microgels by Precipitation Polymerization: Synthesis, Characterization and Functionalization. Adv. Polym. Sci. 234, 1-37 (2010).
- Richtering, W. Responsive Emulsions Stabilized by Stimuli-Sensitive Microgels: Emulsions with Special Non-Pickering Properties. Langmuir. 28 (50), 17218-17229 (2012).
- Wiese, S., Spiess, A. C., Richtering, W. Microgel-Stabilized Smart Emulsions for Biocatalysis. Angew. Chem. Int. Edit. 52 (2), 576-579 (2012).
- Schmitt, V., Ravaine, V. Surface compaction versus stretching in Pickering emulsions stabilised by microgels. Curr. Opin. Colloid In. 18 (6), 532-541 (2013).
- Wellert, S., Richter, M., Hellweg, T., von Klitzing, ., R, Y., Hertle, Responsive Microgels at Surfaces and Interfaces. Z. Phys. Chem. 229 (7-8), 1-26 (2015).
- Li, Z., Harbottle, D., Pensini, E., Ngai, T., Richtering, W., Xu, Z. Fundamental Study of Emulsions Stabilized by Soft and Rigid Particles. Langmuir. 31 (23), 6282-6288 (2015).
- Deshmukh, O. S., van den Ende, D., Stuart, M. C., Mugele, F., Duits, M. H. G. Hard and soft colloids at fluid interfaces: Adsorption, interactions, assembly & rheology. Adv. Colloid Interfac. 222, 215-227 (2015).
- Serpe, M. J., Kim, J., Lyon, L. A. Colloidal Hydrogel Microlenses. Adv. Mater. 16 (2), 184-187 (2004).
- Schmidt, S., Zeiser, M., Hellweg, T., Duschl, C., Fery, A., Möhwald, H. Adhesion and Mechanical Properties of PNIPAM Microgel Films and Their Potential Use as Switchable Cell Culture Substrates. Adv. Func. Mater. 20 (19), 3235-3243 (2010).
- Xia, Y., He, X., et al. Thermoresponsive Microgel Films for Harvesting Cells and Cell Sheets. Biomacromolecules. 14 (10), 3615-3625 (2013).
- Guan, Y., Zhang, Y. PNIPAM microgels for biomedical applications: from dispersed particles to 3D assemblies. Soft Matter. 7 (14), 6375 (2011).
- Yunker, P. J., Chen, K., Gratale, M. D., Lohr, M. A., Still, T., Yodh, A. G. Physics in ordered and disordered colloidal matter composed of poly(N-isopropylacrylamide) microgel particles. Rep. Prog. Phys. 77 (5), 056601-056629 (2014).
- Lohr, M. A., Still, T., et al. Vibrational and structural signatures of the crossover between dense glassy and sparse gel-like attractive colloidal packings. Phys. Rev. E. 90 (6), 062305 (2014).
- Dreyfus, R., Xu, Y., Still, T., Hough, L. A., Yodh, A. G., Torquato, S. Diagnosing hyperuniformity in two-dimensional, disordered, jammed packings of soft spheres. Phys. Rev. E. 91 (1), 012302-012312 (2015).
- Kojima, H., Tanaka, F. Reentrant volume phase transition of cross-linked poly(N-isopropylacrylamide) gels in mixed solvents of water/methanol. Soft Matter. 8 (10), 3010-3011 (2012).
- Hofmann, C. H., Plamper, F. A., Scherzinger, C., Hietala, S., Richtering, W. Cononsolvency Revisited: Solvent Entrapment by N-Isopropylacrylamide and N, N-Diethylacrylamide Microgels in Different Water/Methanol Mixtures. Macromolecules. 46 (2), 523-532 (2013).
- Bischofberger, I., Calzolari, D. C. E., Trappe, V. Co-nonsolvency of PNiPAM at the transition between solvation mechanisms. Soft Matter. 10 (41), 8288-8295 (2014).
- Virtanen, O. L. J., Richtering, W. Kinetics and particle size control in non-stirred precipitation polymerization of N-isopropylacrylamide. Colloid Polym. Sci. 292 (8), 1743-1756 (2014).
- Virtanen, O. L. J., Ala-Mutka, H. M., Richtering, W. Can the Reaction Mechanism of Radical Solution Polymerization Explain the Microgel Final Particle Volume in Precipitation Polymerization of N-Isopropylacrylamide?. Macromol. Chem. Phys. 216 (13), 1431-1440 (2015).
- Glatter, O. A new method for the evaluation of small-angle scattering data. J. Appl. Crystallogr. 10 (5), 415-421 (1977).
- Svergun, D. I. Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25 (4), 495-503 (1992).
- Glatter, O. Convolution Square Root of Band-Limited Symmetrical Functions and Its Application to Small-Angle Scattering Data. J. Appl. Crystallogr. 14, 101-108 (1981).
- Glatter, O., Hainisch, B. Improvements in Real-Space Deconvolution of Small-Angle Scattering Data. J. Appl. Crystallogr. 17, 435-441 (1984).
- Cheezum, M. K., Walker, W. F., Guilford, W. H. Quantitative Comparison of Algorithms for Tracking Single Fluorescent Particles. Biophys. J. 81 (4), 2378-2388 (2001).
- Wöll, D., Kölbl, C., Stempfle, B., Karrenbauer, A. A novel method for automatic single molecule tracking of blinking molecules at low intensities. Phys. Chem. Chem. Phys. 15 (17), 6196-6205 (2013).
- Saxton, M. J., Jacobson, K. Single-particle tracking: Applications to membrane dynamics. Annu. Rev. Bioph. Biom. 26, 373-399 (1997).
- Pusey, P. N., van Megen, W. Detection of small polydispersities by photon correlation spectroscopy. J. Chem. Phys. 80 (8), 3513 (1984).
- Stieger, M., Pedersen, J. S., Richtering, W., Lindner, P. Small-angle neutron scattering study of structural changes in temperature sensitive microgel colloids. J. Chem. Phys. 120 (13), 6197-6206 (2004).
- Wu, X., Pelton, R. H., Hamielec, A. E., Woods, D. R., McPhee, W. The kinetics of poly(N-isopropylacrylamide) microgel latex formation. Colloid Polym. Sci. 272, 467-477 (1994).
- Weeks, E. R., Weitz, D. A. Subdiffusion and the cage effect studied near the colloidal glass transition. Chem. Phys. 284 (1-2), 361-367 (2002).
- Ernst, D., Köhler, J., Weiss, M. Probing the type of anomalous diffusion with single-particle tracking. Phys. Chem. Chem. Phys. 16 (17), 7686-7691 (2014).
- . FitIt! (Version 1.1.4) Available from: https://www.github.com/ovirtanen/fitit (2015)
- Provencher, S. W. A Constrained Regularization Method For Inverting Data Represented By A Linear Algebraic or Integral Equations. Comput. Phys. Commun. 27 (3), 213-227 (1982).
- Holtzer, L., Meckel, T., Schmidt, T. Nanometric three-dimensional tracking of individual quantum dots in cells. Appl. Phys. Lett. 90 (5), 053902-053904 (2007).
- Diezmann, A. V., Lee, M. Y., Lew, M. D., Moerner, W. E. Correcting field-dependent aberrations with nanoscale accuracy in three-dimensional single-molecule localization microscopy. Optica. 2 (11), 985-989 (2015).
- Lindner, P., Zemb, T. . Neutrons, X-rays and Light: Scattering Methods Applied to Soft Condensed Matter. , (2002).
Citer Cet Article
Virtanen, O. L. J., Purohit, A., Brugnoni, M., Wöll, D., Richtering, W. Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels. J. Vis. Exp. (115), e54419, doi:10.3791/54419 (2016).