This work was financially supported by Swiss National Science Foundation (NRP 62 &#8211; Smart Materials). Also acknowledged is the support of B. Hanselmann, K. Kehl, U. Sch&#252;tz and B. Leuthold.

1. Plasma-initiated Polymerization
<ol>
	<li>Preparing of monomer solution.
	<ol>
		<li>Dissolve HEMA (100 ml; 0.718 mol) in 200 ml water and wash 3x with hexane (100 ml) in a separating funnel. Saturate the aqueous phase with sodium chloride and extract the HEMA with diethyl ether (50 ml). Dry the organic phase over MgSO4 and remove the solvent in vacuo (100 mbar, 40 &#176;C). Distill the HEMA under reduced pressure (15 mbar; 99 &#176;C).</li>
		<li>Prepare a 0.62 M methanolic solution of the inhibitor-free HEMA produced in Section 1.1.1. Pour 30 ml of the solution into a one-necked flask and eliminate oxygen by bubbling Ar through the solution for 1 hr.</li>
	</ol>
	</li>
	<li>Surface-induced polymerization.
	<ol>
		<li>Position two polycarbonate membranes next to each other into the plasma chamber (Figure 2). Place the shiny side of the membrane pointing towards the gas phase.</li>
		<li>Connect the plasma chamber to a high vacuum (20 mbar) for 5 min. Close the valve to the vacuum and open the other valve, which is connected to argon and oxygen gas and purge the chamber with this mixture for 2 hr with 15 sccm argon and 2.5 sccm oxygen.</li>
		<li>Initiate the plasma and reduce the power to the desired power (for polycarbonate membrane: 12 W) and treat the membranes for 4 min with the plasma. Connect the monomer solution with the chamber by opening the corresponding valve. Switch off the plasma and evacuate the chamber.</li>
		<li>Connect the monomer solution with the chamber by opening the corresponding valve and pour the solution into the chamber. Ensure that the membranes are covered with the monomer solution. Open the valve connected to the argon and store the reaction mixture for 12 hr at 20 &#176;C (conditioned room).</li>
		<li>Remove the monomer solution. Wash the membranes with methanol in an ultrasonic bath for 5 min. Repeat the washing procedure with water.</li>
		<li>Dry the membrane in vacuo over molecular sieves for 2 hr.</li>
	</ol>
	</li>
</ol>
2. Postmodification of Coated Membranes
<ol>
	<li>Prepare a solution of spirobenzopyran 1 (Figure 1) (100 mg; 0.27 mmol), N,N-dicyclohexylcarbodiimide (DCC) (55 mg; 0.27 mmol) and dimethyl aminopyridine (DMAP) (33 mg, 0.27 mmol) in tert-butylmetylether (TBME) (12 ml).</li>
	<li>Place a protecting stirrer bar and a protecting grid into a round-bottom flask. Dry the flask and flood the flask with argon.</li>
	<li>Pour the solution into the flask, followed by the coated membrane.</li>
	<li>Stir gently at room temperature for 12 hr.</li>
	<li>Remove the solution and wash the membrane with tert-butylmetylether in an ultrasonic bath for 5 min. Repeat the washing procedure with ethanol and water.</li>
	<li>Dry the membrane in vacuo over molecular sieves for 2 hr.</li>
</ol>
3. Surface Tension Measurements
<ol>
	<li>Fix the membrane with a standard tape on a metal O-ring. Position a drop of nanopure water (3.3 &#956;l) on that part of the membrane surface, which is not in contact with the O-ring. Measure the contact angle (CA) at 5 different spots of the membrane.</li>
	<li>For testing the long term stability of the samples, measure the contact angles at three different spots of the membranes after 0, 1, 2, 3, 7, 14, and 21 days.</li>
</ol>
4. Permeability Tests of the Photochromic Membranes
<ol>
	<li>Fill the receptor chamber of the Franz diffusion cell with water (12 ml).</li>
	<li>Fix the membrane in a Franz diffusion cell. Ensure that the membrane is in contact with the water of the receptor chamber. Fill the donor chamber (the chamber on top of the membrane) with an aqueous caffeine solution (20 mM; 3.0 ml). Irradiate the membrane from the top of the donor chamber with white-light (Figure 3). Collect samples (200 &#956;l) from the receptor cell; for track-edged polycarbonate membranes with a pore diameter of 200 nm, collect samples every 10 min.</li>
	<li>Repeat the experiment as described in step 4.2. but irradiate the membrane with UV-light (366 nm, 80 W/m2) during the entire permeability test.</li>
	<li>Determination of the caffeine concentrations of the collected samples.
	<ol>
		<li>Plot a calibration curve with 15 different caffeine concentrations (between 0.05 mg/ml and 1.5 mM/L) using a UV/Vis spectrometer. Calibrate at 293 nm.</li>
		<li>Determine the concentration of each of the collected samples using the calibration curve.</li>
		<li>Plot the determined concentration vs. the time of the collected samples. Make a linear fit through the points and determine &#916;c from the slope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>d'Agostino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Approaches to Plasma Production and Control. , (2008).</li><li>Liston, E. M., Martinu, L., Wertheimer, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+surface+modification+of+polymers+for+improved+adhesion:+a+critical+review.">Plasma surface modification of polymers for improved adhesion: a critical review.</a> J. Adh. Sci. Technol. 7 (10), 1091-1127 (1993).</li><li>Siow, K. S., Britcher, L., Kumar, S., Griesser, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+Methods+for+the+Generation+of+Chemically+Reactive+Surfaces+for+Biomolecule+Immobilization+and+Cell+Colonization+-+A+Review.+Process.">Plasma Methods for the Generation of Chemically Reactive Surfaces for Biomolecule Immobilization and Cell Colonization - A Review. Process.</a> Polymers. 3 (6-7), 392-418 (2006).</li><li>Hossain, M. M., Hegemann, D., Herrmann, A. S., Chabrecek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+angle+determination+on+plasma-treated+poly(ethylene+terephthalate)+fabrics+and+foils.">Contact angle determination on plasma-treated poly(ethylene terephthalate) fabrics and foils.</a> Appl. Polymer Sci. 102 (2), 1452-1458 (2006).</li><li>Guimond, S., Hanselmann, B., Amberg, M., Hegemann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+functionalization+of+textiles:+Specifics+and+possibilities.">Plasma functionalization of textiles: Specifics and possibilities.</a> Pure Appl. Chem. 82 (6), 1239-1245 (2010).</li><li>Lymberopoulos, D. P., Economou, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+simulation+of+glow+discharge+plasma+reactors.">Modeling and simulation of glow discharge plasma reactors.</a> Journal of Vacuum Sci. Technol. A Vacuum Surf. Films. 12 (4), 1229-1236 (1994).</li><li>Hegemann, D., Brunner, H., Oehr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+treatment+of+polymers+for+surface+and+adhesion+improvement.">Plasma treatment of polymers for surface and adhesion improvement.</a> Nuclear Instr. Methods Phys. Res. B Interact. Atoms. 208, 281-286 (2003).</li><li>&#216;iseth, S. K., Krozer, A., Kasemo, B., Lausmaa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+spin-coated+high-density+polyethylene+films+by+argon+and+oxygen+glow+discharge+plasma+treatments.">Surface modification of spin-coated high-density polyethylene films by argon and oxygen glow discharge plasma treatments.</a> Appl. Surf. Sci. 202 (1-2), 92-103 (2002).</li><li>Choi, W. -. K., Koh, S. -. K., Jung, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+chemical+reaction+between+polycarbonate+and+kilo-electron-volt+energy+Ar[sup+++]+ion+in+oxygen+environment.">Surface chemical reaction between polycarbonate and kilo-electron-volt energy Ar[sup + ] ion in oxygen environment.</a> J. Vacuum Sci. Technol. A Vacuum Surf. Films. 14 (4), 2366-2371 (1996).</li><li>Kitova, S., Minchev, M., Danev, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RF+plasma+treatment+of+polycarbonate+substrates.">RF plasma treatment of polycarbonate substrates.</a> Optoelectron. Adv. Mater. 7 (5), 2607-2612 (2005).</li><li>Friedrich, J. F., Mix, R., Schulze, R. D., Meyer-Plath, A., Joshi, R., Wettmarshausen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+plasma+techniques+for+polymer+surface+modification+with+monotype+functional+groups.">New plasma techniques for polymer surface modification with monotype functional groups.</a> Plasma Process. Polymers. 5 (5), 407-423 (2008).</li><li>Baumann, L., 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=Tuning+the+resistance+of+polycarbonate+membranes+by+plasma-induced+graft+surface+modification.">Tuning the resistance of polycarbonate membranes by plasma-induced graft surface modification.</a> Appl. Surf. Sci. 268, 450-457 (2013).</li><li>Hegemann, D., Hossain, M. M., Balazs, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanostructured+plasma+coatings+to+obtain+multifunctional+textile+surfaces.">Nanostructured plasma coatings to obtain multifunctional textile surfaces.</a> Prog. Org. Coatings. 58 (2-3), 237-240 (2007).</li><li>Gengenbach, T., Vasic, Z., Li, S., Chatelier, R., Griesser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+restructuring+and+oxidation+to+the+aging+of+the+surface+of+plasma+polymers+containing+heteroatoms.">Contributions of restructuring and oxidation to the aging of the surface of plasma polymers containing heteroatoms.</a> Plasmas Polymers. 2 (2), 91-114 (1997).</li><li>Gengenbach, T. R., Chatelier, R. C., Griesser, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Ageing+of+Plasma-deposited+Polymer+Films:+Global+Analysis+of+X-ray+Photoelectron+Spectroscopy+Data.">Characterization of the Ageing of Plasma-deposited Polymer Films: Global Analysis of X-ray Photoelectron Spectroscopy Data.</a> Interface Anal. 24 (4), 271-281 (1996).</li><li>Hirotsu, T., Nakajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+ethanol+permseparation+by+pervaporation+throught+the+plasma+graft+copolymeric+membranes+of+acrylic+acid+and+acrylamide.">Water ethanol permseparation by pervaporation throught the plasma graft copolymeric membranes of acrylic acid and acrylamide.</a> Appl. Polymer Sci. 36 (1), 177-189 (1988).</li><li>Baumann, L., de Courten, D., Wolf, M., Rossi, R. M., Scherer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-Responsive+Caffeine+Transfer+through+Porous+Polycarbonate.">Light-Responsive Caffeine Transfer through Porous Polycarbonate.</a> Appl. Mater. Interf. 5 (13), 5894-5897 (2013).</li><li>Minkin, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-,+thermo-,+solvato-,+and+electrochromic+spiroheterocyclic+compounds.">Photo-, thermo-, solvato-, and electrochromic spiroheterocyclic compounds.</a> Chem. Rev. 104 (5), 2751-2776 (2004).</li><li>Berkovic, G., Krongauz, V., Weiss, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiropyrans+and+spirooxazines+for+memories+and+switches.">Spiropyrans and spirooxazines for memories and switches.</a> Chem. Rev. 100 (5), 1741-1753 (2000).</li><li>Vlassiouk, I., Park, C. -. D., Vail, S. A., Gust, D., Smirnov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Nanopore+Wetting+by+a+Photochromic+Spiropyran:+A+Light-Controlled+Valve+and+Electrical+Switch.">Control of Nanopore Wetting by a Photochromic Spiropyran: A Light-Controlled Valve and Electrical Switch.</a> Lett. 6 (5), 1013-1017 (2006).</li><li>Baumann, L., 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=Development+of+light-responsive+porous+polycarbonate+membranes+for+controlled+caffeine+delivery.">Development of light-responsive porous polycarbonate membranes for controlled caffeine delivery.</a> RSC Adv. 3 (45), 23317-23326 (2013).</li><li>Nicoletta, F. P., Cupelli, D., Formoso, P., De Filpo, G., Colella, V., Gugliuzza, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Responsive+Polymer+Membranes:+A+Review.">Light Responsive Polymer Membranes: A Review.</a> Membranes. 2 (1), 134-197 (2012).</li></ol>

The authors have nothing to disclose.

The plasma process produces a purple gas, which is caused by ionized argon. An orange color would indicate the presence of undesired nitrogen from a leak. The plasma process does not only form radicals on the surface but also etches the membrane7,12. Too much etching can change the pore diameter significantly, which would influence the permeability of the membrane. The controlled reaction conditions of the presented setup allow enhancing the reproducibility of the plasma-initiated grafting process. Nevertheles...

Plasma modification of materials has become an important process in many industrial fields. Cleaning of surfaces and functionalization of surfaces without changing the bulk property of the material has made the plasma treatment an essential process in surface science1-8.
Plasma treatment of polymers results in homolytic bond cleavage. This leads to an edging of the polymeric material and to the formation of radical rich surfaces. By using plasma containing oxygen molecules, the surface becomes oxygen rich and thus more hydrophilic9-11. However, the hydrophilicity of the surfaces is not stable over time12. In order to enhance the long-term stability, the plasma treated surface can be chemically modified after or during the plasma process13-15. This treatment is normally performed by adding a reactive monomer species into the gas phase during the plasma process; these monomers then polymerize from the created radicals of the polymer surface. If the chemical treatment is performed with a nonvolatile monomer, the polymer grafting has to take place after the plasma modification. In order to perform a controlled grafting after the radicals are formed on the surface, a plasma setup is described, which allows the plasma-initiated surface-induced polymerization from the surface in solution under controlled conditions12,16.
The presentation focuses on the modification of track-edged polymer membranes12,17. By modifying the surface tension of these membranes, the permeability rate can be varied12. This clean and fast process allows the creation of very thin layers (&#60;5 nm), which cover the entire membrane surface without changing the bulk property of the polymer membrane. Due to the edging during the plasma process, the pore diameters of the track-edged membranes augment slightly12. The edging rate is depending on the polymer and has a linear behavior.
When using monomers with reactive functional groups, the grafted polymers can be further functionalized. This is demonstrated by the postmodification of a PHEMA-grafted membrane with a carboxylic acid functionalized spiropyran. This results in a photochromic surface, since spiropyran is known to transform into a merocyanine species when irradiated with UV-light. The spiropyran form can be reestablished by irradiating the merocyanine form with visible light (Figure 1)18,19. Since the merocyanine form is more polar than the spiropyran state, the surface tension of the coating can be triggered with light20. The change in surface tension influences the permeability resistance of the membrane towards aqueous solutions. The set-up how to perform the permeability tests of these light-responsive membranes will be shown and the significant change in permeability resistance (decrease in permeability resistance by 97%) is demonstrated. Such a membrane can be integrated in a drug delivery setup or in smart sensing systems.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> 
Figure 1. Photoisomerization of spirobenzopyran compound 1.

<table><tbody><tr><td>2-Hydroxyethyl methacrylate, 97%</td><td>Sigma-Aldrich</td><td>128635</td><td></td></tr><tr><td>Hexane 99%</td><td>Biosolve</td><td></td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;, anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methanol, 99%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>14262</td><td>dried over molecular sieves</td></tr><tr><td>N,N-Dicylcohexylcarbodiimide, 99%</td><td>Sigma-Aldrich</td><td>D8002</td><td></td></tr><tr><td>Dimethyl aminopyridine, 99%</td><td>Sigma-Aldrich</td><td>107700</td><td></td></tr><tr><td>Tert-butylmethylether, 98%</td><td>Fluka</td><td>306975</td><td></td></tr><tr><td>Polycarbonate membrane</td><td>Whatman</td><td></td><td>Nanopore Track Etched (TE) (1.0 &amp;mu;m, 0.2 &amp;mu;m, 0.1 &amp;mu;m, 50 nm, 30 nm, and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td></tr><tr><td>Caffeine (reagent plus)</td><td>Sigma-Aldrich</td><td>C0750</td><td></td></tr><tr><td>Franz diffusion cell (12 ml)</td><td>SES-Analysesysteme</td><td>6C010015</td><td>15 mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbar and clamp</td></tr><tr><td>UV-Lamp</td><td></td><td></td><td>UV irradiation (366 nm, 15 W/m&lt;sup&gt;2&lt;/sup&gt;)</td></tr><tr><td>White light lamp</td><td></td><td></td><td>White light irradiation (500 W bulb)</td></tr><tr><td>UV/Vis spectrophotometer</td><td>Varian 50Bio/50MPR</td><td></td><td></td></tr><tr><td>Polyester membranes</td><td>Sterlitech</td><td>PET0225100</td><td>Polyester Membrane Filters, 0.2 &amp;mu;m pore diameter, 25 mm diameter</td></tr><tr><td>Polyvinylidene fluoride membranes</td><td>Millipore</td><td></td><td>PVDF Membranes Durapore (0.22 &amp;mu;m pore diameter; 47 mm membrane diameter)</td></tr><tr><td>Argon (99.9995%)</td><td>Alphagaz</td><td></td><td></td></tr><tr><td>Dressler Cesar RF Power Generator</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Multi Gas Controller 647C</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Mass-Flow controllers</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Vacuubrand RE 2.5 rotary vane vacuum pump</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Contact angle measurement device</td><td>Kr&amp;uuml;ss</td><td>G10</td><td></td></tr><tr><td>Balances</td><td>Mettler Toledo</td><td>AB204-S and Mettler ME30</td><td></td></tr></tbody></table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

The etch rate can be followed by weighing the membrane after different periods of time. As can be seen from Figure 4, the etch rate follows for polyester, polyvinylidene fluoride, and polycarbonate membranes a linear etch rate, which can be determined from the slope of the linear correlation of the etch time versus mass loss. As shown in Figure 4, the polycarbonate membranes show the lowest etch rate of all the three polymer membranes. One consequence of the etching is the chang...

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Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

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Bioengineering

11.7K Views.  Empa, Swiss Federal Laboratories for Materials Science and Technology. A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

Video: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Chemistry

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Engineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Biochemistry

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...