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.).

1. Synthesis of Monofunctional and Bifunctional Poly(THF)
<ol>
	<li>Monofunctional poly(THF)
	<ol>
		<li>Flame dry a 2-neck 100-ml round-bottom flask. Vacuum and fill the flask with nitrogen (3 cycles).</li>
		<li>Add distilled tetrahydrofuran (THF) (50 ml) to the flask. Put the flask in a water bath at 20 &#176;C and equilibrate the temperature.</li>
		<li>Add methyl triflate (0.5 mmol) to the flask by a syringe. Stir the mixture for 5-10 min at 20 &#176;C.</li>
		<li>Add N-phenyl pyrrolidine (4-6 equiv.) to the flask by a syringe. Stir the mixture for 30-60 min.</li>
		<li>Completely remove the solvent under reduced pressure (ca. 100 Torr). Dissolve the residue in 3-5 ml of acetone. Add the acetone solution into 300-500 ml of n-hexane. Filter the precipitate and dry it under reduced pressure.</li>
	</ol>
	</li>
	<li>Bifunctional poly(THF)
	<ol>
		<li>Flame dry a 2-neck 100-ml round-bottom flask. Vacuum and fill the flask with nitrogen (3 cycles).</li>
		<li>Add distilled THF (50 ml) to the flask. Put the flask in a water bath at 20 &#176;C and equilibrate the temperature.</li>
		<li>Add triflic anhydride (0.3 mmol) to the flask by a syringe. Stir the mixture for 5-10 min at 20 &#176;C.</li>
		<li>Add N-phenyl pyrrolidine (4-6 equiv.) to the flask by a syringe. Stir the mixture for 30-60 min.</li>
		<li>Completely remove the solvent under reduced pressure (ca. 100 Torr). Dissolve the residue in 3-5 ml of acetone. Add the acetone solution to 300-500 ml of n-hexane. Filter the precipitate and dry it under reduced pressure.</li>
	</ol>
	</li>
</ol>
2. Synthesis of Perylene Diimide-incorporated 4-armed Star and 8-shaped Dicyclic Poly(THF)
<ol>
	<li>Armed star poly(THF)
	<ol>
		<li>Ion exchange
		<ol>
			<li>Dissolve perylene diimide tetracarboxylate sodium salt in water (10 mg/ml, 150 ml). Dissolve monofunctional poly(THF) in acetone (160 mg/ml, 4 ml). Add the acetone solution dropwise into the vigorously stirred aqueous solution. Collect the formed precipitate by filtration.</li>
			<li>Repeat the above procedure with the recovered precipitate (2.1.1.1) four times.</li>
		</ol>
		</li>
		<li>Covalent fixation
		<ol>
			<li>Dissolve the obtained precipitate in toluene (5 mg/ml). Reflux the solution for 4 hr.</li>
			<li>Completely remove the solvent under reduced pressure (ca. 100 Torr). Filter the residue through a plug of silica gel with n-hexane/acetone (2/1 vol/vol). Add the solution into ice-cooled water (300-500 ml) to precipitate the product. Collect the precipitate by filtration.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dicyclic 8-shaped poly(THF)
	<ol>
		<li>Ion exchange
		<ol>
			<li>Dissolve perylene diimide tetracarboxylate sodium salt in water (6 mg/ml, 50 ml). Dissolve bifunctional poly(THF) (0.5 g) in 30-50 ml of acetone. Add the acetone solution dropwise into the vigorously stirred aqueous solution at 0 &#176;C. Collect the formed precipitate by filtration.</li>
			<li>Repeat the above procedure with the recovered precipitate (2.2.1.1).</li>
		</ol>
		</li>
		<li>Covalent fixation
		<ol>
			<li>Dissolve the obtained precipitate in toluene (0.05 g/L). Reflux the solution for 4 hr.</li>
			<li>Completely remove the solvent under reduced pressure (ca. 100 Torr). Add toluene to partially dissolve the residue. Re-precipitate into 300-500 ml of n-hexane.</li>
			<li>Filter the formed precipitate through a plug of silica gel with n-hexane/acetone (2/1 vol/vol). Re-precipitate into 300-500 ml of water.</li>
			<li>Purify the formed precipitate by column chromatography18 using a polystyrene gel. Further purify the crude product by preparative gel permeation chromatography (GPC)19 with an eluent of CHCl3 to remove byproducts by monitoring refractive index (RI) and UV detectors.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Single-molecule Fluorescence Imaging Experiment
<ol>
	<li>Sample preparation
	<ol>
		<li>Cleaning of microscope cover slips
		<ol>
			<li>Place No. 1.5 24 x 24 mm microscope cover slips in a staining jar.</li>
			<li>Add 1 M potassium hydride solution (100 ml) into the jar and sonicate for 15 min. Pour off the potassium hydroxide solution by decantation and rinse the cover slips with ultra-pure water for several times. Add spectroscopic grade ethanol (100 ml) into the jar and sonicate for 15 min.</li>
			<li>Pour off the ethanol by decantation and rinse the cover slips with ultra-pure water for several times. After pouring off the ultra-pure water by decantation, repeat the step 3.1.1.2.</li>
			<li>Add ultra-pure water to the jar and sonicate for 15 min. Rinse the cover slips with ultra-pure water for several times. Take out the cover slips from the jar by a plastic tweezers and dry them by either dry air or dry nitrogen.</li>
		</ol>
		</li>
		<li>Preparation of polymer melt samples14,15
		<ol>
			<li>Add 100 &#181;l of non-labeled linear poly(THF) in a glass bottle and heat it to a temperature above the melting point (approximately 25 &#176;C) using a hair dryer.</li>
			<li>Dissolve the fluorophore-incorporated polymer (linear, 4-armed star, cyclic, or 8-shaped dicyclic synthesized in 2.1 and 2.2) in chloroform (1 ml, 10-6 M). Add 1 &#181;l of the solution to the 100 &#181;l of the melt of the non-labeled linear poly(THF).</li>
			<li>After thoroughly mixing the sample with a pipette tip, evaporate chloroform by heating the sample using a dryer. 
			NOTE: This provides a melt of the non-labeled linear poly(THF) containing 10-8 M of the fluorophore incorporated polymers.</li>
			<li>Take 10 &#181;l of the sample using a micro-pipette and drop it on a cleaned cover slip. Put another cleaned cover slip on the sample and sandwich the sample between the two cover slips.</li>
			<li>Press the sample gently using a plastic tweezers.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Wide-field fluorescence imaging setup15
	<ol>
		<li>Introduction of an excitation laser (488 nm) into the back port of the inverted microscope
		<ol>
			<li>Insert an excitation bandpass filter and polarizer into the beam path.</li>
			<li>Expand the beam to approximately 1 cm in diameter by a beam expander.</li>
			<li>Insert a quarter wave plate into the beam path. Set the optical axis of the wave plate at 45 degree with respect to that of the polarizer. Alternatively, insert a Berek compensator and set the optical delay to &#955;/4.</li>
			<li>Insert a diaphragm in the excitation beam path to adjust the beam size.</li>
			<li>Before introducing the laser beam into the back port of the inverted optical microscope, insert a focusing lens (plan-convex lens, focal length &#8776; 300 mm) at a position where the laser beam out of the objective lens is collimated.</li>
		</ol>
		</li>
		<li>After reflecting the laser beam using a dichroic mirror mounted on a filter cube, introduce the laser beam to the sample through a high numerical aperture (NA) objective lens (e.g., NA 1.3, 100X magnification, oil immersion).</li>
		<li>Attach an objective heater to the objective lens and set the temperature to 30 &#176;C.</li>
		<li>Mounting the sample on the stage of the inverted microscope
		<ol>
			<li>Drop one drop of immersion oil on the objective lens and mount the sample on the microscope state.</li>
			<li>Ensure that the sample thickness of approximately 10 &#181;m is obtained by checking the axial position of the bottom and top surface of the sample.</li>
			<li>Adjust the focus of the microscope to a few micrometers above the bottom surface of the sample.</li>
		</ol>
		</li>
		<li>Obtain circularly polarized excitation light under the objective lens
		<ol>
			<li>Insert a polarizer into the collimated laser beam out of the objective lens.</li>
			<li>Record the intensity of the laser transmitted through the polarizer by inserting a power meter after the polarizer. Record the transmitted laser power at different polarization angles by rotating the polarizer.</li>
			<li>If the transmitted laser power is not constant at all polarization angles, slightly rotate the quarter wave plate or Berek compensator inserted in the excitation beam path.</li>
			<li>Repeat step 3.2.5.2 and 3.2.5.3 until the constant transmitted laser power is obtained at all polarization angles. Ensure that the circularly polarized light is obtained at the sample.</li>
		</ol>
		</li>
		<li>Setup the EM (electron multiplying)-charge coupled device (CCD) camera
		<ol>
			<li>Attach the EM-CCD camera to the side port of the microscope and connect it to the image acquisition software.</li>
			<li>If necessary, synchronize the camera exposure to a mechanical shutter or acousto-optical tunable filter inserted in the excitation beam path by sending the transistor-transistor logic (TTL) signals generated by the EM-CCD camera to the devices. Alternatively, synchronize the camera exposure to the laser output by sending the TTL signals generated by the EM-CCD camera to the laser. 
			NOTE: The latter option is applicable only when a solid-state laser whose output power can be modulated by input transistor-transistor logic (TTL) signals is used for the experiment.</li>
			<li>Apply an EM gain (typically approximately 300) to the CCD camera using software controlling the camera in order to obtain a high quality fluorescence image of the single fluorophore.</li>
			<li>Set a region of interest (ROI) (typically 128 x 128 pixels at the center of the field of view) using software controlling the camera. 
			NOTE: This allows for the imaging experiments at the frame rates of 100 - 200 Hz in the frame transfer mode, which is required for visualizing the motion of the fluorophore-incorporated polymer chains in the melt sample.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Running the experiment
	<ol>
		<li>Optimizing the experimental conditions
		<ol>
			<li>Adjust the illumination area of the sample to approximately 20 &#181;m in diameter using the diaphragm inserted in the excitation beam path.</li>
			<li>Set the excitation laser power at the sample to 4 - 8 mW by manually selecting an appropriate neutral density (ND) filter inserted in the excitation beam path. 
			NOTE: This provides the mean laser power of 1 - 2 kW cm-2 at the sample.</li>
			<li>Record fluorescence images of the sample at the frame rates of 100 - 200 Hz. If the fluorescence intensity obtained from the individual fluorophore-incorporated polymers is too low, gradually increase the excitation power using the ND filter until reaching approximately 100 mW at the sample.</li>
			<li>If the quality of the single-molecule fluorescence image is still not satisfactory, check the fluorescence impurities in the sample by recording fluorescence images of a pure melt of the non-labeled poly(THF). In case a high fluorescence background is observed, use different non-labeled poly(THF).</li>
			<li>If the density of the fluorescence spot obtained from the fluorophore-incorporated polymers in the melt is too high to spatially isolate them (this causes errors in the analysis of the diffusive motion), decrease the concentration of the fluorophore-incorporated polymers in the sample until spatially isolated spots are observed.</li>
			<li>If the density of the fluorescence spot obtained from the fluorophore-incorporated polymers in the melt is too low (this causes a low throughput of the imaging experiment), increase the concentration of the fluorophore-incorporated polymers in the sample until an appropriate density of the fluorescence spot is reached.</li>
			<li>If the fluorescence images obtained from the fluorophore-incorporated polymers in the melt are blurred, increase the frame rates of the imaging acquisition. 
			NOTE: This often requires a smaller ROI, typically 64 x 64 pixels.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Image acquisition
	<ol>
		<li>Once the experimental conditions are optimized, leave the mounted sample on the microscope stage for an hour so that the sample reaches equilibrium conditions.</li>
		<li>Record 500 - 1,000 fluorescence image sequences of the fluorophore-incorporated polymers in the melt state at a 100 - 200 Hz frame rate. If the default file format is not TIFF, convert all the image sequences to the TIFF format.</li>
	</ol>
	</li>
</ol>
4. Analysis of the Diffusive Motion
<ol>
	<li>Mean-squared displacement (MSD) analysis
	<ol>
		<li>Crop the fluorescence image sequences in such a way that each image sequence contains a single and well-focused diffusing fluorophore-incorporated polymer using image processing software, such as ImageJ.</li>
		<li>When the cropped image sequences contain more than 10 frames, split the image sequences into multiple sequences such that each sequence consists of 10 frames.</li>
		<li>Determine the positions of the molecules in each image sequences accurately by two-dimensional Gaussian fitting of the images.</li>
		<li>Determine the diffusion coefficient (D) of individual molecules by mean-squared displacement (MSD) analysis of the diffusion trajectories (i.e., time-dependent positions of the molecule) using an equation20 
		<img alt="Equation1" src="/files/ftp_upload/54503/54503equation1.jpg" /> 
		where xi and yi are the positions of the molecule in the image frame i, and n denotes the frame number with the time lapse &#916;t from frame i.</li>
		<li>Plot the diffusion coefficients in a frequency histogram. 
		NOTE: Typically, the histogram is constructed from more than 100 molecules.</li>
	</ol>
	</li>
	<li>Cumulative distribution function (CDF) analysis 
	NOTE: A CDF, P(r2, i&#916;t) corresponds to the cumulative probability of finding the diffusing molecules within a radius r from the origin after a certain time lag i&#916;t.
	<ol>
		<li>Calculate the squared-displacement occurring during time lags of 1&#916;t, 2&#916;t, &#183;&#183;&#183;&#183;, i&#916;t for all the diffusion trajectories obtained in 4.1.3. 
		NOTE: These calculations give total mi squared-displacements for the time lags of i&#916;t.</li>
		<li>Calculate numbers of the squared-displacements (li) within total mi data set that are smaller than r2 at different r2 values (0 &#60; r2 &#60; &#8734;). Normalized li vs r2 plots correspond to the CDF, P(r2, i&#916;t).</li>
	</ol>
	</li>
	<li>Analysis of CDFs with distinct diffusion models 
	Note: The obtained CDFs are fitted by distinct diffusion models; homogeneous diffusion model, multiple diffusion modes in which the D distribution is described by a Gaussian (single Gaussian model), and multiple diffusion modes in which the D distribution is described by multiple Gaussian (multiple Gaussian model).
	<ol>
		<li>In the homogeneous diffusion model, determine a mean D by fitting the CDF using an equation21 
		<img alt="Equation2" src="/files/ftp_upload/54503/54503equation2.jpg" /> 
		NOTE: Any deviation from the equation suggests the heterogeneous diffusion of the molecule.</li>
		<li>In the single Gaussian model, determine the probability distribution of D described by a Gaussian (f(D)) by fitting the CDF using15 
		<img alt="Equation3" src="/files/ftp_upload/54503/54503equation3.jpg" /> 
		<img alt="Equation4" src="/files/ftp_upload/54503/54503equation4.jpg" /> 
		where A, w, and D0 are the amplitude, width, and center of the Gaussian.</li>
		<li>In the double Gaussian model, determine the probability distribution of jth component of D described by a Gaussian (f(D)) by fitting the CDF using14 
		<img alt="Equation5" src="/files/ftp_upload/54503/54503equation5.jpg" /> 
		<img alt="Equation6" src="/files/ftp_upload/54503/54503equation6.jpg" /> 
		where Aj is the fraction of each diffusion component, and &#945;j, wj, and D0j are the amplitude, width, and center of the jth component of the Gaussian.</li>
	</ol>
	</li>
	<li>Calculation of the theoretical probability distribution of diffusion coefficient 
	NOTE: The probability distributions of D occurring due to the statistical errors (p(D)dD) are calculated for the different diffusion models; homogeneous diffusion model, multiple diffusion modes in which the D distribution is described by a Gaussian (single Gaussian model), and multiple diffusion modes in which the D distribution is described by multiple Gaussian (multiple Gaussian model).
	<ol>
		<li>In the homogeneous diffusion model, calculate the statistical probability distribution of D using an equation22 
		<img alt="Equation7" src="/files/ftp_upload/54503/54503equation7.jpg" /> 
		where N is the number of the data points in a diffusion trajectory (N = 10, see 4.1.2), D0 is the mean diffusion coefficient (determined by the CDF analysis, see 4.2.3.1), and D is the experimentally obtained diffusion coefficient for an individual trajectory.</li>
		<li>In the single Gaussian diffusion model, calculate the statistical probability distribution of D using an equation15 
		<img alt="Equation8" src="/files/ftp_upload/54503/54503equation8.jpg" /> 
		where f(D) denotes probability distribution of D determined by the CDF analysis (see 4.2.3.2), and D0 is the mean diffusion coefficient (determined by the CDF analysis, see 4.2.3.2).</li>
		<li>In the double Gaussian diffusion model, calculate the statistical probability distribution of D using an equation14 
		<img alt="Equation9" src="/files/ftp_upload/54503/54503equation9.jpg" /> 
		where f(Dj) denotes probability distribution of the jth component of D (Dj) determined by the CDF analysis (see 4.2.3.3), and D0j is the mean diffusion coefficient of the jth component (determined by the CDF analysis, see 4.2.3.3).</li>
	</ol>
	</li>
</ol>

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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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+microscopy+on+model+membranes+reveals+anomalous+diffusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+diffusion+of+individual+class+II+MHC+membrane+proteins+in+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photophysics+of+a+water-soluble+rylene+dye:+Comparison+with+other+fluorescent+molecules+for+biological+applications.">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><li>Tezuka, Y., Oike, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+and+covalent+fixation+for+topological+polymer+chemistry.">Self-assembly and covalent fixation for topological polymer chemistry.</a> Macromol. Rapid Commun. 22 (13), 1017-1029 (2001).</li><li>Deres, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Origin+of+Heterogeneity+of+Polymer+Dynamics+near+the+Glass+Temperature+As+Probed+by+Defocused+Imaging.">The Origin of Heterogeneity of Polymer Dynamics near the Glass Temperature As Probed by Defocused Imaging.</a> Macromolecules. 44 (24), 9703-9709 (2011).</li><li>Flier, B. M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+Diffusion+in+Thin+Polymer+Films+As+Observed+by+High-Temperature+Single-Molecule+Fluorescence+Microscopy.">Heterogeneous Diffusion in Thin Polymer Films As Observed by High-Temperature Single-Molecule Fluorescence Microscopy.</a> J. Am. Chem. Soc. 134 (1), 480-488 (2012).</li><li>Habuchi, S., Oba, T., Vacha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-beam+single-molecule+defocused+fluorescence+imaging+reveals+local+anisotropic+nature+of+polymer+thin+films.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Diffusion+and+Cooperative+Diffusion+in+Semidilute+Polymer+Solutions+As+Measured+by+Fluorescence+Correlation+Spectroscopy.">Self-Diffusion and Cooperative Diffusion in Semidilute Polymer Solutions As Measured by Fluorescence Correlation Spectroscopy.</a> Macromolecules. 42 (24), 9537-9547 (2009).</li><li>Kirstein, J., Platschek, B., Jung, C., Brown, R., Bein, T., Brauchle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploration+of+nanostructured+channel+systems+with+single-molecule+probes.">Exploration of nanostructured channel systems with single-molecule probes.</a> Nat. Mater. 6 (4), 303-310 (2007).</li></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 border="0" cellpadding="0" cellspacing="0" width="846">
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			<td height="21" style="height:21px;width:320px;">Materials</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">THF</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Wakosil C-300</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Acetone</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Toluene</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">n-Hexane</td>
			<td style="width:240px;">Godo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">CHCl3</td>
			<td style="width:240px;">Kanto Chemical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Bio-Beads S-X1</td>
			<td style="width:240px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Methyl triflate</td>
			<td style="width:240px;">Nacalai Tesque</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Triflic anhydride</td>
			<td style="width:240px;">Nacalai Tesque</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Potassium Hydroxide</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ethanol</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Poly(tetrahydrofuran)</td>
			<td style="width:240px;">Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Chloroform</td>
			<td style="width:240px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Immersion oil</td>
			<td style="width:240px;">Cargille</td>
			<td style="width:119px;">Type 37 / Type A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;"></td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Equipment</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">2-Neck 100-mL round-bottom flask</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Flask</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Beaker</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Funnel</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Filter paper</td>
			<td style="width:240px;">Whatman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Reflux condenser</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Syringe</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Water bath</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Magnetic stirrer</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Rotary evaporator</td>
			<td style="width:240px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Microscope cover slips (24 x 24 mm, No. 1)</td>
			<td style="width:240px;">Matsunami Glass</td>
			<td style="width:119px;">CO22241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Staining jar</td>
			<td style="width:240px;">AS ONE Corporation</td>
			<td style="width:119px;">1-7934-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ultrasonic cleaner</td>
			<td style="width:240px;">VWR International&#160;</td>
			<td style="width:119px;">142-0047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Inverted microscope</td>
			<td style="width:240px;">Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Ar-Kr ion laser</td>
			<td style="width:240px;">Coherent</td>
			<td style="width:119px;">Innova 70C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Berek compensator</td>
			<td style="width:240px;">Newport</td>
			<td align="right" style="width:119px;">5540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Excitation filter</td>
			<td style="width:240px;">Semrock</td>
			<td style="width:119px;">LL01-488-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dichloric mirror</td>
			<td style="width:240px;">Omega optical</td>
			<td style="width:119px;">500DRLP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Emission filter</td>
			<td style="width:240px;">Semrock</td>
			<td style="width:119px;">BLP01-488R-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Lens and mirror</td>
			<td style="width:240px;">Thorlabs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EM-CCD camera</td>
			<td style="width:240px;">Andor Technology</td>
			<td style="width:119px;">iXon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Objective lens (x100, N.A. = 1.3)</td>
			<td style="width:240px;">Olympus</td>
			<td style="width:119px;">UPLFLN 100XOP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Objective heater</td>
			<td style="width:240px;">Bioptechs</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Preparative GPC</td>
			<td style="width:240px;">Japan Analytical Industry</td>
			<td style="width:119px;">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

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...

Watch this Scientific Journal Video about Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level at JoVE.com

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

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 ...

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

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JoVE Journal

Chemistry

7.8K Views.  King Abdullah University of Science and Technology (KAUST). A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

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

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

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

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.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

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

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

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&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

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

Amide coupling reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. Isonicotinic acid, or its derivatives, are coupled to variable length diaminoalkane chains under an inert atmosphere in anhydrous DMF or DMSO with the use of a weak base, triethylamine, and a coupling agent, 1-propylphosphonic anhydride. The products precipitate from solution upon formation or can be precipitated by the addition of water. If desired, the ligands can be further purified by recrystallization from hot water. Dinuclear platinum complex synthesis using the bispyridine ligands is done in hot water using transplatin. The most informative of the chemical characterization techniques to determine the structure and gross purity of both the bispyridine ligands and the final platinum complexes is 1H NMR with particular analysis of the aromatic region of the spectra (7-9 ppm). The platinum complexes have potential application as anticancer agents and the synthesis method can be modified to produce trinuclear and other multinuclear complexes with different hydrogen bonding functionality in the bridging ligand.

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 reactions can be used to synthesize bispyridine-based ligands for use as bridging linkers in multinuclear platinum anticancer drugs. ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

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.

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...