Name: Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level (Video) | JoVE Journal
Uploaded: 2016-09-26T14:00:00.000+00:00
Duration: 6 min 55 s
Description: Learn to characterize diffusive motion of cyclic polymers at the single molecule level using fluorescence microscopy. This method reveals heterogeneous diffusion patterns hidden in bulk measurements for polymer physics research.

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5 minutes

This work was supported by a Grant-in-Aid for Scientific Research No. 22750122 (S.H.), No. 26288099 (T.Y.), and No. 23350050 (Y.T.) of the Japan Society for the Promotion of Science. S.H. is grateful for The Kurata Memorial Hitachi Science and Technology Foundation. The research reported in this publication was supported by the King Abdullah University of Science and Technology (S.H.).

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<li>Habuchi, S. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHabuchi%2C+S+%22Topological+Polymer+Chemistry%3A+Progress+of+Cyclic+Polymers+in+Syntheses%2C+Properties+and+Functions%22">Topological Polymer Chemistry: Progress of Cyclic Polymers in Syntheses, Properties and Functions</a>. Tezuka, Y. , World Scientific. 265-290 (2013).</li>
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<li>Kusumi, A., Sako, Y., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Confined+lateral+diffusion+of+membrane-receptors+as+studied+by+single-particle+tracking+%28nanovid+microscopy%29+-+effects+of+calcium-induced+differentiation+in+cultured+epithelial-cells%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Confined lateral diffusion of membrane-receptors as studied by single-particle tracking (nanovid microscopy) - effects of calcium-induced differentiation in cultured epithelial-cells.</a> Biophys. J. 65 (5), 2021-2040 (1993).</li>
<li>Schutz, G. J., Schindler, H., Schmidt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-molecule+microscopy+on+model+membranes+reveals+anomalous+diffusion%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Single-molecule microscopy on model membranes reveals anomalous diffusion.</a> Biophys. J. 73 (2), 1073-1080 (1997).</li>
<li>Vrljic, M., Nishimura, S. Y., Brasselet, S., Moerner, W. E., McConnell, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+diffusion+of+individual+class+II+MHC+membrane+proteins+in+cells%5BTitle%5D)+AND+%22Biophys.+J%22%5BJournal%5D)">Translational diffusion of individual class II MHC membrane proteins in cells.</a> Biophys. J. 83 (5), 2681-2692 (2002).</li>
<li>Margineanu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photophysics+of+a+water-soluble+rylene+dye%3A+Comparison+with+other+fluorescent+molecules+for+biological+applications%5BTitle%5D)+AND+%22J.+Phys.+Chem.+B%22%5BJournal%5D)">Photophysics of a water-soluble rylene dye: Comparison with other fluorescent molecules for biological applications.</a> J. Phys. Chem. B. 108 (32), 12242-12251 (2004).</li>
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<li>Habuchi, S., Oba, T., Vacha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-beam+single-molecule+defocused+fluorescence+imaging+reveals+local+anisotropic+nature+of+polymer+thin+films%5BTitle%5D)+AND+%22Phys.+Chem.+Chem.+Phys%22%5BJournal%5D)">Multi-beam single-molecule defocused fluorescence imaging reveals local anisotropic nature of polymer thin films.</a> Phys. Chem. Chem. Phys. 13 (15), 6970-6976 (2011).</li>
<li>Zettl, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-Diffusion+and+Cooperative+Diffusion+in+Semidilute+Polymer+Solutions+As+Measured+by+Fluorescence+Correlation+Spectroscopy%5BTitle%5D)+AND+%22Macromolecules%22%5BJournal%5D)">Self-Diffusion and Cooperative Diffusion in Semidilute Polymer Solutions As Measured by Fluorescence Correlation Spectroscopy.</a> Macromolecules. 42 (24), 9537-9547 (2009).</li>
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</ol>

The authors have nothing to disclose.

The 4-armed and 8-shaped polymers were prepared via the ESA-CF protocol (Figure 1), which is a critical step for the synthesis.12,24 Monofunctional and bifunctional linear poly(THF)s with N-phenylpiperidinium end groups were synthesized according to the previous procedure.11 The ion exchange was carried out by reprecipitation of an acetone solution of a polymer precursor with triflate counteranions into an aqueous solution containing an excess amount of carboxylate.
<p ...

Cyclic polymers are unique in that they do not have chain ends. They often exhibit unusual behaviors which is distinct from their linear counterpart, including increased thermal stability of polymer micelles by a linear-to-cyclic conversion,1,2 and spatial organization of DNA in bacterial cells by a loop formation.3 Topological interactions between the cyclic chains are believed to be the critical factor for such unusual behaviors.4,5 Therefore, characterizing the motion and relaxation of cyclic polymers under entangled conditions has been an important research topic in polymer science for decades.6
Cyclic polymer dynamics has been investigated using both synthetic and naturally occurring molecules by means of ensemble averaged experimental methods such as nuclear magnetic resonance (NMR), light scattering, and viscosity measurements.7-9 However, these studies often suffer from impurity molecules in the samples.10 Furthermore, spatiotemporal heterogeneities of the motion of individual molecules caused by inherent structural heterogeneity of entangled polymers are often hidden behind the ensemble averaging in these studies. In order to characterize molecular level dynamics of cyclic polymers, a synthesis method that provides high purity cyclic polymers and an experimental and analysis methods that allow for quantitative characterization of molecular motion at the single molecule level have to be developed. Here, we will show a method to synthesize high-purity cyclic and dicyclic poly(THF)s that incorporate a fluorophore unit using an electrostatic self-assembly and covalent fixation (ESA-CF) process11-13 and a method to analyze the motion of the individual fluorophore-incorporated polymer chains using a combination of mean-squared displacement (MSD) and cumulative distribution function (CDF) analyses.
A proper data processing has been shown to be essential for the accurate characterization of the diffusive motion. With an adequate MSD and CDF analyses, a multiple-mode diffusion of the cyclic and dicyclic polymers in the melt and semi-dilute solution of the linear polymer chains has been revealed,14-16 suggesting the significant effects of the topological states of the polymers on the diffusive motion of the chains under entangled conditions.17 While the experimental and analytical approaches to characterize the motion of the cyclic polymers are described in this protocol, the same method can be used to quantitatively characterize the diffusive motion in many other heterogeneous systems. The approach would be especially suitable when multiple diffusion components existing in the samples are to be analyzed.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>THF</td><td>Godo</td><td></td><td></td></tr><tr><td>Wakosil C-300</td><td>Wako Pure Chemical Industries</td><td></td><td></td></tr><tr><td>Acetone</td><td>Godo</td><td></td><td></td></tr><tr><td>Toluene</td><td>Godo</td><td></td><td></td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-Hexane</td><td>Godo</td><td></td><td></td></tr><tr><td>CHCl&lt;sub&gt;3&lt;/sub&gt;</td><td>Kanto Chemical</td><td></td><td></td></tr><tr><td>Bio-Beads S-X1</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Methyl triflate</td><td>Nacalai Tesque</td><td></td><td></td></tr><tr><td>Triflic anhydride</td><td>Nacalai Tesque</td><td></td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Wako Pure Chemical Industries</td><td></td><td></td></tr><tr><td>Ethanol</td><td>Wako Pure Chemical Industries</td><td></td><td></td></tr><tr><td>Poly(tetrahydrofuran)</td><td>Aldrich</td><td></td><td></td></tr><tr><td>Chloroform</td><td>Wako Pure Chemical Industries</td><td></td><td></td></tr><tr><td>Immersion oil</td><td>Cargille</td><td>Type 37 / Type A</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Neck 100-ml round-bottom flask</td><td></td><td></td><td></td></tr><tr><td>Flask</td><td></td><td></td><td></td></tr><tr><td>Beaker</td><td></td><td></td><td></td></tr><tr><td>Funnel</td><td></td><td></td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td></td><td></td></tr><tr><td>Reflux condenser</td><td></td><td></td><td></td></tr><tr><td>Syringe</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td></td></tr><tr><td>Magnetic stirrer</td><td></td><td></td><td></td></tr><tr><td>Rotary evaporator</td><td></td><td></td><td></td></tr><tr><td>Microscope cover slips (24 x 24 mm, No. 1)</td><td>Matsunami Glass</td><td>CO22241</td><td></td></tr><tr><td>Staining jar</td><td>AS ONE Corporation</td><td>1-7934-01</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>VWR International&amp;nbsp;</td><td>142-0047</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Ar-Kr ion laser</td><td>Coherent</td><td>Innova 70C</td><td></td></tr><tr><td>Berek compensator</td><td>Newport</td><td>5540</td><td></td></tr><tr><td>Excitation filter</td><td>Semrock</td><td>LL01-488-12.5</td><td></td></tr><tr><td>Dichloric mirror</td><td>Omega optical</td><td>500DRLP</td><td></td></tr><tr><td>Emission filter</td><td>Semrock</td><td>BLP01-488R-25</td><td></td></tr><tr><td>Lens and mirror</td><td>Thorlabs</td><td></td><td></td></tr><tr><td>EM-CCD camera</td><td>Andor Technology</td><td>iXon</td><td></td></tr><tr><td>Objective lens (100X, N.A. = 1.3)</td><td>Olympus</td><td>UPLFLN 100XOP</td><td></td></tr><tr><td>Objective heater</td><td>Bioptechs</td><td></td><td></td></tr><tr><td>Preparative GPC</td><td>Japan Analytical Industry</td><td>LC-908</td><td></td></tr></tbody></table>

synthesis of cyclic polymers and characterization of their diffusive motion in the melt state at the single molecule level

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

The perylene diimide-incorporated 4-armed star and 8-shaped dicyclic poly(THF)s were synthesized using the electrostatic self-assembly and covalent fixation (ESA-CF) process (Figure 1, Figure 2). Time-lapse single-molecule fluorescence images were measured for the 4-armed (Figure 3a) and 8-shaped (Figure 3b) polymers. The time-lapse fluorescence images (Figure 3) show spatially isolated bright and sharp s...

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

The overall goal of this experiment is to characterize diffusional motion of topological polymers, especially cyclic polymers, under entangled conditions at the single molecule level. This method can help answer key questions in the field of polymer physics, such as topology-dependent spacio-temporal dynamics of polymers. The main advantage of this technique is that heterogeneous diffusion can be quantitatively characterized at the single-chain level, which is usually hidden behind on some level.To begin this procedure, dissolve perylene diimide salt in 150 milliliters of water, then dissolve monofunctional poly(THF)in four millimeters of acetone. Add the acetone solution drop-wise to the vigorously stirred aqueous solution. Collect the formed precipitate by vacuum filtration.Next, prepare a five milligram per milliliter solution of the precipitate in toluene. Reflux the solution for four hours. After allowing the solution to cool, completely remove the solvent under reduced pressure by rotary evaporation.When finished, dissolve the residue in a 2:1 mixture of n-hexane:acetone and filter the resulting solution through a plug of silica gel. Then add the filtered solution to ice cold water to precipitate the product. To prepare the polymer melt sample, add 100 microliters of non-labeled linear poly(THF)to a glass bottle and heat it to approximately 25 degrees celsius using a hair dryer.Prepare a 10 to the minus six molars solution of the fluorophore incorporated polymer solution in chloroform. Then add one microliter of this solution to 100 microliters of the non-labeled linear poly(THF)melt. After thoroughly mixing the sample with a pipette tip, evaporate the chloroform by heating with a hair dryer.Using a micro-pipette place 10 microliters of the sample on a cleaned cover slip. Place another cleaned cover slip on the sample and gently press the two cover slips together using plastic tweezers. Next, attach an objective heater to the objective lens of an inverted microscope and set the temperature to 30 degrees celsius.Place one drop of immersion oil on the lens and mount the sample on the microscope stage. Ensure that a sample thickness of approximately 10 micrometers is obtained by checking the axial position of the bottom and top surface of the sample. Then adjust the focus of the microscope to a few micrometers above the bottom surface of the sample.Following this, apply an electron multiplying or EM gain to a CCD camera in order to obtain a high quality fluorescent image of the single fluorophore. Now set a region of interest using the software controlling the camera. To optimize the experimental conditions, adjust the illumination area of the sample to approximately 20 micrometers in diameter using the diaphragm inserted in the excitation beam path.Set the excitation laser power at the sample to four to eight milliwatts by manually selecting an appropriate neutral density filter inserted in the excitation beam path. Finally, record 500 to 1000 fluorescence image sequences of the fluorophore incorporated polymer in the melt state at a 100 to 200 hertz frame rate. Time-lapse single molecule florescence images were measured for the four-armed and eight-shaped polymers and show spatially isolated bright and sharp spots due to the incorporation of the highly fluorescent perylene diimide fluorophore into the chains.The frequency histograms of the diffusion coefficient determined by mean-squared displacement analysis display broad distributions resulting from both the statistical error of the analysis and heterogeneity of the diffusion. The frequency histograms show clear deviations from the homogeneous diffusion model, which demonstrates heterogeneous diffusion of the polymer molecules. The single and double Gaussian models hit the experimentally obtained cumulative distribution functions well and demonstrate that the diffusion of the formed polymer is described by the broad distribution of the diffusion coefficient, whereas the eight-shaped polymer displays two distinct diffusion modes.Once mastered, the synthesis of polymers can be done in six hours and the single molecule immersion experiment can be done in several hours if they are performed properly. After it's development, this technique paved the way for researchers in the field of polymer science to explore polymer dynamics in crowded environments. After watching this video, you should have a good understanding of how to characterize diffusive motion of topological polymers under entangled condition at the single-chain level.Don't forget that working with lasers and organic solvents can be extremely hazardous and precautions such as laser safety and review of the MSDS should always be taken while performing this procedure.

synthesis-cyclic-polymers-characterization-their-diffusive-motion

Research

JoVE Journal

Chemistry

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

The goal of this protocol is to initiate polymerization using dynamic sulfur bonds in poly(S-divinylbenzene) at mild temperatures (90 °C) without using solvents. Terpolymers are characterized by GPC, DSC and 1H NMR, and tested for changes in solubility.

Synthesis of Terpolymers at Mild Temperatures Using Dynamic Sulfur Bonds in Poly(S-Divinylbenzene)

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of ε-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

Here, we describe protocols for the preparation of trans-cyclobutane fused cyclooctenes (tCBCO), their polymerization to prepare depolymerizable olefinic polymers, and the depolymerization of these polymers under mild conditions. Additionally, protocols for the preparation of depolymerizable networks and compression molding of rigid linear plastics based on this system are described.

Depolymerizable Olefinic Polymers Based on Fused-Ring Cyclooctene Monomers

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

This protocol describes the use of amide coupling reactions of isonicotinic acid and diaminoalkanes to form bridging ligands suitable for use in the synthesis of multinuclear platinum complexes, which combine aspects of the anticancer drugs BBR3464 and picoplatin.

Amide Coupling Reaction for the Synthesis of Bispyridine-based Ligands and Their Complexation to Platinum as Dinuclear Anticancer Agents

Learn to characterize diffusive motion of cyclic polymers at the single molecule level using fluorescence microscopy. This method reveals heterogeneous diffusion patterns hidden in bulk measurements for polymer physics research.

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

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