This research was supported by Kansas State University. A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is sponsored at Oak Ridge National Laboratory by the Scientific User Facilities Division, Office of Basic Energy Sciences and U.S. Department of Energy.

1. PGMA-b-PVDMA Synthesis20
<ol>
	<li>Synthesis of PGMA macro-chain transfer agent (Macro-CTA)
	<ol>
		<li>Use a 250-mL round-bottom reaction &#64258;ask equipped with a polytetrafluoroethylene-coated magnetic stir bar.</li>
		<li>Combine 14.2 g of glycidyl methacrylate GMA (142.18 g/mol) with 490.8 mg of 2-cyano-2-propyl dodecyl trithiocarbonate (CPDT) (346.63 g/mol), and 87.7 mg of 2,2&#8242;-azobis (4-methoxy-2,4-dimethyl valeronitrile) (V-70) (308.43 g/mol) (molar ratio of CPDT: V-70 = 5:1), and benzene (100 mL) into air free round bottom flask.</li>
		<li>Degas the reaction mixture using argon and stir for 30 min. Subsequently put the solution in a temperature-controlled oil bath at 30 &#176;C and react for 18 h. 
		NOTE: The targeted molecular weight for the Macro-CTA is 10,000 g/mol. 18 hours was determined to be the time necessary to reach reasonable conversion. The color of the polymer solution is transparent light yellow.</li>
		<li>After 18 h, terminate the reaction by submerging the round bottom flask in liquid N2.</li>
		<li>Precipitate the polymer by pouring the light-yellow solution of polymer/benzene (~100 mL) into 400 mL of hexane.</li>
		<li>Stir the mixture for 5 min. Precipitate will be settled at the bottom of the beaker and is recovered by filtration.</li>
		<li>Dry the precipitate overnight under vacuum. Then dilute it in 400 mL of tetrahydrofuran (THF). Re-precipitate in hexane.</li>
		<li>Dry this new precipitate again with argon overnight. 
		NOTE: Macro-CTA is a fine yellow powder. The product yield of the reaction will be ~43.8%. The Mn of the PGMA Macro-CTA is 7,990 g/mol with a polydispersity (PDI) of 1.506 (MW = 12,030 g/mol).</li>
	</ol>
	</li>
	<li>Synthesis of PGMA-b-PVDMA
	<ol>
		<li>Fractionally distill the VDMA under reduced pressure, and reserve the middle fraction (~70%) for use. 
		NOTE: This is required to remove polymerization inhibitor. The distillation apparatus is attached to a Schlenk line and the air seal valve is partially opened to the vacuum line. Minimal heat is applied using a varistat and heating mantle until the VDMA monomer begins distilling over at a rate of 1 drop per second.</li>
		<li>Combine the 2-Vinyl-4,4- dimethyl azlactone (VDMA) (139.15 g/mol)monomer (10.436 g) with the PGMA-macroCTA (1.669 g), V-70 (14.5 mg; molar ratio of PGMA-macroCTA: V-70 = 3:1) and benzene (75.0 mL) in a single-neck 250-mL round-bottom reaction &#64258;ask equipped with a Teflon-coated magnetic stir bar. 
		NOTE: Molecular weight information, PVDMA: 139.15 g/mol, PGMA-macroCTA: 12,030 g/mol, Benzene: 78.11 g/mol.</li>
		<li>Degas the mixture with high purity argon and stir for 30 min, and then put in oil bath at 32 &#176;C for 18 h.</li>
		<li>Terminate the reaction by submerging the round bottom flask in liquid N2.</li>
		<li>Precipitate the polymer three times into hexane and dry it at room temperature under vacuum.</li>
		<li>Characterize the molecular weight and PDI of the product by using size exclusion chromatography (S) (see the Table of Materials) according to the procedure in Lokitz et al.20.The size exclusion chromatograph (S) is equipped with three PLgel 5 &#181;m mixed-C columns (300 x 7.5 mm) in series, a refractive index detector (Wavelength= 880 nm), a photodiode array detector, multi-angle light scattering (MALS) detector (Wavelength= 660 nm), and a viscometer (see the Table of Materials). 
		NOTE: All experiments performed in this manuscript used product with PGMA and PVDMA block lengths of 56 and 175, respectively. The molecular weight of the block copolymer was 37,620 g/mol and the PDI was 1.16.</li>
	</ol>
	</li>
</ol>
2. Generation of Parylene Stencil Patterns Over Silicon Substrates
<ol>
	<li>Parylene coating
	<ol>
		<li>Sonicate silicon wafers in 50% wt. acetone in water for 5 min followed by sonication in 50% wt. isopropanol (IPA) in water for 5 min.</li>
		<li>Rinse silicon wafers with deionized (DI) water and blow dry with nitrogen gas.</li>
		<li>Deposit 80 nm and 1 &#181;m thick parylene N on 4-inch silicon wafers using a parylene coater (see the Table of Materials). 
		NOTE: Characterize the thickness of parylene films by using a surface profilometer (see the Table of Materials).
		<ol>
			<li>Calibrate parylene film thickness with parylene dimer mass for each individual parylene coating system. 
			NOTE: In the current system, ~80 mg and ~1000 mg parylene N dimer was required to obtain 80 nm and 1 &#181;m film thickness, respectively (based on the calibration curve obtained).</li>
			<li>Use the following settings during operation of the parylene coater: pressure: 80 mTorr, duration: 1 h, furnace temperature: 690 &#176;C, vaporizer temperature: 160 &#176;C.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Photolithography
	<ol>
		<li>Bake wafers in an oven at 100 &#176;C for 20 min; then let wafers sit for another 3 min at room temperature. 
		NOTE: Additional wait time improves adhesion of the photoresist.</li>
		<li>Add 2 mL of positive photoresist&#160;(see the Table of Materials) and dispense at the center of the parylene-coated wafer. Spin coat the wafers at 3000 rpm for 30 s. 
		NOTE: Spin coating must be done under the hood.</li>
		<li>Wait 1 min, bake wafer on a hot plate at 105 &#176;C for 1 min.</li>
		<li>Load photomask in a mask alignment system (see the Table of Materials). Expose wafers to UV light (&#955;=325 nm) for 10 s with a dosage of 65 mJ/cm2.</li>
		<li>Let the wafers sit for another 5 min at room temperature.</li>
		<li>Develop wafers by submerging in developer&#160;(see the Table of Materials) solution for 2 min. Rinse the wafers with deionized water, and then dry with N2. Do this under the hood. 
		NOTE: After developing, photoresist appears completely removed from areas exposed to UV. Use an optical microscope (see the Table of Materials) to verify the wafers.</li>
	</ol>
	</li>
	<li>Reactive ion etching
	<ol>
		<li>Use a reactive ion etching (RIE) tool (see the Table of Materials) to etch developed wafers with oxygen plasma.</li>
		<li>Apply an oxygen flow rate of 50 cm3/min at a chamber pressure of 20 mTorr.</li>
		<li>For a parylene film thickness of 1 &#181;m, use RF power of 50 W and inductively coupled plasma (ICP) power of 500 W for 100 s was to remove exposed parylene from patterned areas. This corresponded to a parylene etch rate of 1.0-1.15 &#181;m/min.</li>
		<li>For a parylene thickness of 80 nm, use RF power of 50 W and ICP power of 200 W for 55 s to remove exposed parylene from patterned areas. This corresponds to a parylene etch rate of 570-620 nm/min. 
		NOTE: For efficient parylene removal, determine the parylene etch rate for each RIE system.</li>
		<li>Inspect etched substrates with an optical microscope. The silicon surface will appear shiny after the parylene is completely removed from exposed regions.</li>
		<li>Verify etch depth using a surface profilometer (see the Table of Materials).</li>
	</ol>
	</li>
</ol>
3. Parylene Lift-off Procedure
<ol>
	<li>Preparation of polymer solutions
	<ol>
		<li>Dissolve PGMA-b-PVDMA into chloroform (1% wt.). Chloroform should be anhydrous to prevent hydrolysis of azlactone groups. 
		NOTE: Chloroform is the preferred solvent because it has a high degree of solubility for the polymer, allowing for more uniform surface deposition of single polymer chains compared to other organic solvents25.</li>
	</ol>
	</li>
	<li>Cleaning parylene stencils with the plasma cleaner
	<ol>
		<li>Turn on the plasma cleaner (see the Table of Materials) main power and put the parylene-coated substrates in the plasma cleaner chamber.</li>
		<li>Turn on the vacuum pump and evacuate the air in the chamber until the pressure gauge is less than 400 mTorr.</li>
		<li>Slightly open the metering valve and allow the air to enter to the plasma cleaner until the pressure gauge shows 800-1000 mTorr.</li>
		<li>Select RF with Hi mode and expose the substrates for 3 min.</li>
		<li>At the end of process, turn off the RF power and vacuum pump.</li>
		<li>Turn off the plasma cleaner and remove the substrates. 
		NOTE: After plasma cleaning, the surface shows hydrophilic behavior (Figure 1B) . The water contact angle of bare silicon surfaces before and after plasma cleaning are 27&#176; &#177; 2&#176; and 0&#176;, respectively.</li>
	</ol>
	</li>
	<li>Spin-coating of PGMA-b-PVDMA, annealing and sonication over the parylene stencils
	<ol>
		<li>Immediately spin-coat the substrates with 100 &#181;L of 1% wt. PGMA-b-PVDMA in anhydrous chloroform at 1500 rpm, for 15 s using a spin coater (see the Table of Materials). 
		NOTE: Perform spin-coating within 1-2 s of pipetting the polymer solution to minimize film non-uniformity caused by rapid chloroform evaporation.</li>
		<li>Anneal the polymer films at 110 &#176;C in a vacuum oven (see the Table of Materials) for 18 h. 
		NOTE: Annealing allows for polymer microphase segregation and surface attachment of the GMA block to the surface26.
		<ol>
			<li>After the annealing, characterize the polymer coating by measuring the contact angle of substrates. Surfaces show a contact angle of 75&#176;&#177; 1&#176; (Figure 1C)20.</li>
		</ol>
		</li>
		<li>Sonicate the substrates in 20 mL of acetone or chloroform for 10 min to remove the parylene layer and any physisorbed polymer. 
		NOTE: Use the following sonication conditions: ultra sonic power, 284 W; Operating frequency, 40 kHz (see the Table of Materials). 
		NOTE: Parylene can also be peeled off the substrate by applying a piece of Scotch tape at the edge of the substrate then pulling the tape away27.</li>
		<li>Store the substrates under vacuum in a desiccator until characterization.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57562/57562fig1.jpg" /> 
Figure 1: Contact angle measurements for treated silicon substrates. (A) Bare silicon, (B) Plasma-cleaned silicon, (C) Spin-coated silicon with PGMA-b-PVDMA (after annealing and sonication in chloroform). <a href="https://www.jove.com/files/ftp_upload/57562/57562fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. PGMA-b-PVDMA Interface-Directed Assembly Procedure
NOTE: This procedure can be performed on substrates containing either a chemically inert background (section 4.1), or a biologically inert background (section 4.2), depending on the application.
<ol>
	<li>Preparation of chemically inert background on silicon substrates
	<ol>
		<li>Use oxygen plasma cleaner to clean the bare silicon (section 3.2).</li>
		<li>Pipette 100 &#181;L of trichloro(1H,1H,2H,2H-perfluorooctyl) silane (TPS) onto a Petri dish and place the silicon substrates inside a vacuum desiccator next to the Petri dish.</li>
		<li>Apply vacuum (-750 Torr) for 1 h for chemical vapor deposition (CVD). 
		CAUTION: TPS is highly toxic and the CVD process should be performed inside a fume hood. 
		NOTE: After 1 h the substrate shows hydrophobic behavior. A contact angle of 109&#176;&#177; 3&#176; is typically measured after the CVD process. The thickness of the TPS film is 1.5 &#177; 0.5 nm. 
		NOTE: TPS blocks reaction of the reactive surface oxide with PGMA-b-PVDMA.</li>
		<li>Coat the wafers with parylene (1 &#181;m thickness). Perform photolithography and reactive ion etching to generate parylene patterns (section 2) and to etch away the TPS layer in the exposed regions.</li>
	</ol>
	</li>
	<li>Preparation of polyethylene glycol (PEG) background on silicon substrates.
	<ol>
		<li>Use the oxygen plasma cleaner for 3 min to clean the bare silicon substrates (section 3.2).</li>
		<li>Perform CVD of TPS for 1 h (section&#160;4.1.2).</li>
		<li>Immerse substrates into a 0.7% wt/v solution of Pluronic F-127 in ultrapure water for 18 h to generate a PEG layer on the surface28,29. 
		NOTE: Pluronic contains a hydrophobic polypropylene oxide (PPO) polymer block between&#160;two PEG chains. The PPO block anchors the polymer to the TPS surface while the PEG chains are exposed to solution28.</li>
		<li>Wash and rinse the substrate for 5 min with 100 mL of ultrapure water.</li>
		<li>Deposit 80 nm and 1 &#181;m thick parylene N on 4-inch silicon wafers using a parylene coater.</li>
		<li>Perform photolithography and reactive ion etching to generate parylene patterns (section 2).</li>
	</ol>
	</li>
	<li>Sonication, spin-coating of PGMA-b-PVDMA polymer, and annealing the substrates
	<ol>
		<li>Sonicate chemically inert (TPS) substrates (section 4.1) or PEG-functional substrates (section 4.2) for 10 min in acetone to remove the parylene layer.</li>
		<li>Spin-coat the sonicated substrate with 100 &#181;L of 1% wt. PGMA-b-PVDMA in anhydrous chloroform at 1500 rpm for 15 s.</li>
		<li>Anneal the polymer films at 110 &#176;C under vacuum for 18 h.</li>
		<li>Sonicate the substrates in acetone or chloroform for 10 min to remove physisorbed polymer present in background regions on the surface.</li>
		<li>Store the substrates in a vacuum desiccator until further use.</li>
	</ol>
	</li>
</ol>
5. Custom PGMA-b-PVDMA Micro-Contact Printing (&#956;CP)
<ol>
	<li>PDMS stamp fabrication
	<ol>
		<li>Fabricate the silicon masters according to the standard photolithography procedure30. Use CVD process (section 4.1.2) to deposit anti-adhesive TPS onto the silicon masters. 
		NOTE: The silicon mold should be treated with TPS the first time it is used, and re-applied after it has been used 5-10 times.</li>
		<li>Perform standard soft lithography methods for fabrication of stamps (PDMS precursor to curing agent mass ratio 10:1)31. 
		NOTE: Stamps used in this study consist of micropillar arrays (diameter = 5-50 &#181;m, height = 20 &#181;m).</li>
		<li>Cut out a single stamp. Clean the stamp by sonicating for 10 min in HCl (1 M), 5 min in acetone, followed by 5 min in ethanol.</li>
		<li>Dry the stamps in a convection oven at 80 &#176;C for 20 min to remove residual organic solvent.</li>
	</ol>
	</li>
	<li>Microcontact printing of PGMA-b-PVDMA onto silicon substrates
	<ol>
		<li>Deposit TPS onto the surface of PDMS stamps using the CVD process (section 4.1.2). 
		NOTE: The TPS layer is used to prevent coupling of the polymer to the stamp surface. 
		NOTE: Contact angle measurements can be used to characterize stamps after TPS adsorption, as shown in Figure 2 (Inset A, B).</li>
		<li>Dissolve the PGMA-b-PVDMA polymer into anhydrous chloroform at a concentration of 0.25-1% wt.</li>
		<li>Submerge the stamps into 5 mL of the polymer solution for 3 min.</li>
		<li>Plasma clean 2&#215;2 cm bare silicon substrates for 3 min to clean surface for coupling with the PGMA blocks (section 3.2).</li>
		<li>Take out the polymer-coated stamps from the polymer solution. 
		NOTE: Stamps must be used for printing while they are still wet and a layer of solution exists over them.</li>
		<li>Put inked stamp directly on silicon substrate.</li>
		<li>Use a manual drill press stand (see the Table of Materials) (Figure 3) to press the polymer-coated stamps onto the silicon surface to promote pattern transfer. Immediately apply the stamp to the substrate (within 1-2 s) after taking out the coated stamps from polymer solution. 
		NOTE: Both the silicon and the PDMS stamp can be placed on double-sided tape backing to minimize PDMS stamp deformation due to non-uniform or high pressure stamping32.</li>
		<li>Apply conformal contact between polymer-inked stamp and silicon substrate for 1 min. Use the estimated pressure of 75 g/cm2(7.35 kPa) to press.</li>
		<li>Gently separate the stamp from the silicon surface.</li>
		<li>Anneal the printed silicon substrates immediately in a vacuum oven at 110 &#176;C for 18 h.</li>
		<li>Sonicate the printed silicon substrates in acetone or chloroform for 10 min to remove any physically-adsorbed PGMA-b-PVDMA and then dry with N2.
		<ol>
			<li>Perform surface characterization analysis for both PDMS stamp (after printing step) and printed-silicon (after annealing and sonication steps) to verify the successful transfer of PGMA-b-PVDMA. 
			NOTE: Surface profilometer and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) analysis could be used to analyze the printed-silicon substrate and PDMS stamp, respectively.</li>
		</ol>
		</li>
		<li>Store the substrates under vacuum in a desiccator until characterization.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57562/57562fig2.jpg" /> 
Figure 2: ATR-FTIR measurements for treated PDMS stamps (Relative intensity). (Inset A) Contact angle measurements for bare PDMS stamp. (Inset B) Contact angle measurements for TPS treated PDMS stamp. <a href="https://www.jove.com/files/ftp_upload/57562/57562fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57562/57562fig3.jpg" /> 
Figure 3: Setup for &#956;CP of PGMA-b-PVDMA solutions onto silicon substrates. The procedure includes use of a (A)&#160;manual drill press, (B) a TPS-functionalized PDMS stamp coated with the PGMA-b-PVDMA polymer, (C) a plasma cleaned 2&#215;2 cm silicon substrate, and (D) double-sided tape.

The authors have nothing to disclose.

This article presents three approaches to patterning PGMA-b-PVDMA, each with its set of advantages and drawbacks. The parylene lift-off method is a versatile method for patterning block co-polymers at micro to nanoscale resolution, and has been used as a deposition mask in other patterning systems33,34,35. Due to its relatively weak surface adhesion, the parylene stencil can be easily removed from the surface by sonicat...

Developing fabrication techniques that allow for versatile and precise control of chemical and biological surface functionality is desirable for a variety of applications, from capture of environmental contaminants to development of next generation biosensors, implants, and tissue engineering devices1,2. Functional polymers are excellent materials for tuning surface properties through &#34;grafting from&#34; or &#34;grafting to&#34; techniques3. These approaches allow for control of surface reactivity based on the chemical functionality of the monomer and molecular weight of the polymer4,5,6. Azlactone-based polymers have been intensely studied in this context, as azlactone groups rapidly couple with different nucleophiles in ring-opening reactions. This includes primary amines, alcohols, thiols and hydrazine groups, thereby providing a versatile route for further surface functionalization7,8. Azlactone-based polymer films have been employed in different environmental and biological applications including analyte capture9,10, cell culture6,11, and anti-fouling/anti-adhesive coatings12. In many biological applications, patterning azlactone polymer films at nano to micrometer length scales is desirable to facilitate spatial control of biomolecule presentation, cellular interactions, or to modulate surface interactions13,14,15,16,17,18. Therefore, fabrication methods should be developed to offer high pattern uniformity and well-controlled film thickness, without compromising chemical functionality19.
Recently, Lokitz et al. developed a PGMA-b-PVDMA block copolymer which was capable of manipulating surface reactivity. PGMA blocks couple to oxide-bearing surfaces, yielding high and tunable surface densities of azlactone groups20.&#160;Previously reported methods for patterning this polymer for creation of biofunctional interfaces used traditional top-down photolithography approaches that generated non-uniform polymer films with background regions contaminated with residual photoresist material, causing high levels of non-specific chemical and biological interactions21,22,23. Here, attempts to passivate background regions caused cross-reaction with azlactone groups, compromising polymer reactivity. Considering these limitations, we recently developed techniques for patterning brush (~90 nm) or highly crosslinked (~1-10 &#956;m) films of PGMA-b-PVDMA into chemically or biologically inert backgrounds in a manner that completely preserves the chemical functionality of the polymer24. These presented methods utilize parylene lift-off, interface-directed assembly (IDA), and custom microcontact printing (&#956;CP) techniques. Highly detailed experimental methods for these patterning approaches, as well as critical film characterizations and challenges and limitations associated with each technique are presented here in written and video format.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol, &amp;ge; 99.5%</td><td>Sigma-Aldrich</td><td>459844</td><td>-</td></tr><tr><td>HCL, 1.019 N in H2O</td><td>Fluka Analytical</td><td>318949</td><td>-</td></tr><tr><td>Acetone, &amp;ge; 99.5%</td><td>Sigma-Aldrich</td><td>320110</td><td>-</td></tr><tr><td>Benzene, &amp;ge; 99.9%</td><td>Sigma-Aldrich</td><td>270709</td><td>-</td></tr><tr><td>Isopropanol, ACS reagent, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>190764</td><td></td></tr><tr><td>Hexane</td><td>Fisher Chemical</td><td>H292-4</td><td>-</td></tr><tr><td>Argon</td><td>Matheson Gas</td><td>G1901175</td><td>-</td></tr><tr><td>Tetrahydrofuran (THF), &amp;ge; 99.9%</td><td>Sigma-Aldrich</td><td>401757</td><td>-</td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td>-</td></tr><tr><td>Polydimethyl Siloxane (PDMS) Slygard 184</td><td>Dow Corning</td><td>4019862</td><td>-</td></tr><tr><td>Trichloro (1H,1H,2H,2H-perfluorooctyl) silane (TPS), 97%</td><td>Sigma-Aldrich</td><td>448931</td><td>It is toxic. Work with it under hood</td></tr><tr><td>Anhydrous Chloroform, &amp;ge; 99%</td><td>Sigma-Aldrich</td><td>372978</td><td>-</td></tr><tr><td>Positive Photoresist AZ1512</td><td>MicroChemicals</td><td>AZ 1512</td><td>amber-red liquid, density 1.083 g/cm3, spin coating step should be done under the hood</td></tr><tr><td>Developer AZ 300 MIF</td><td>MicroChemicals</td><td>AZ300 MIF</td><td>clear colourless liquid with slight amine odor and density of 1 g/cm3</td></tr><tr><td>1,2-Vinyl-4,4- dimethyl azlactone (VDMA)</td><td>Isochem North America, LLC</td><td>VDMA</td><td>-</td></tr><tr><td>2-cyano-2-propyl dodecyl trithiocarbonate (CPDT)</td><td>Sigma-Aldrich</td><td>723037</td><td>-</td></tr><tr><td>2,2&amp;prime;-Azobis (4methoxy-2,4-dimethyl valeronitrile) (V-70)</td><td>Wako Specialty Chemicals</td><td>CAS NO. 15545-97-8, EINECS No. 239-593-8</td><td>-</td></tr><tr><td>Parylene N</td><td>Specialty Coating Systems</td><td>15B10004</td><td>-</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Parylene Coater</td><td>Specialty Coating Systems</td><td>SCS Labcoater (PDS 2010)</td><td>-</td></tr><tr><td>Mask alignment system</td><td>Neutronix Quintel</td><td>NXQ8000</td><td>-</td></tr><tr><td>Oxygen Plasma Etcher</td><td>Oxford Instruments</td><td>Plasma Lab System 100</td><td>-</td></tr><tr><td>Surface Profilometer</td><td>Veeco</td><td>Dektak 150</td><td>Scan type was standard hill. Scan duration and force were 120 s and 1 mg, respectively.</td></tr><tr><td>Brightfield Upright Microscope</td><td>Olympus Corporation</td><td>BX51</td><td>-</td></tr><tr><td>Oxygen Plasma&amp;nbsp; Cleaner</td><td>Harrick Plasma</td><td>PDC-001-HP</td><td>-</td></tr><tr><td>Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR)</td><td>Perkin Elmer</td><td>ATR-FTIR 100</td><td>-</td></tr><tr><td>Atomic Force Microscopy (AFM)</td><td>PicoPlus</td><td>Picoplus atomic force microscope</td><td>Veeco MLCT-E cantilevers with a 0.5 N/m spring constant. Scan speeds varied between 0.25 and 1 Hz.</td></tr><tr><td>Scanning Electron Microscopy (SEM)</td><td>Hitachi Science Systems Ltd., Tokyo, Japan</td><td>-</td><td>-</td></tr><tr><td>Rotary Tool Workstation</td><td>Dremel</td><td>Model 220-01</td><td>-</td></tr><tr><td>Spin Coater</td><td>Smart Coater</td><td>SC100</td><td>-</td></tr><tr><td>Vacuum Oven</td><td>Yamato Scientific Co.</td><td>PCD-C6(5)000)</td><td>-</td></tr><tr><td>Size Exclusion Chromatography (SEC)</td><td>Waters Alliance 2695 Separations Module</td><td>720004547EN</td><td>-</td></tr><tr><td>Refractive Index (RI) detector</td><td>Waters</td><td>Model 2414</td><td>-</td></tr><tr><td>Photodiode Array Detector</td><td>Waters</td><td>Model 2996, 716001286</td><td>-</td></tr><tr><td>Multi-angle Light Scattering (MALS) Detector</td><td>Wyatt Technology</td><td>miniDAWN TREOS II</td><td>-</td></tr><tr><td>Viscometer</td><td>Wyatt Technology</td><td>Viscostar</td><td>-</td></tr><tr><td>PLgel 5 &amp;micro;m mixed-C columns (300 x 7.5 mm)</td><td>Agilent</td><td>5 &amp;micro;m mixed-C columns</td><td>-</td></tr><tr><td>Ellipsometer</td><td>J. A. Woollam</td><td>alpha-SE</td><td>Cauchy model, PGMA and PVDMA layers had refractive indices of 1.50 and 1.52 at 632 nm</td></tr><tr><td>Ultrasonic Sonicator</td><td>Fischer Scientific</td><td>FS-110H</td><td>-</td></tr></tbody></table>

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.

Contact angle measurements can be used to evaluate the functionalization of silicon with PGMA-b-PVDMA. Figure 1 depicts the contact angle of the silicon substrate during the different processing steps. Hydrophilic behavior of the plasma cleaned silicon substrate is shown in Figure 1B. The contact angle after polymer spin coating and annealing is 75&#176; &#177; 1&#176;(Figure 1C)...

Watch this Scientific Journal Video about Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers at JoVE.com

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

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 ...

fabricating-reactive-surfaces-with-brush-like-crosslinked-films

Research

JoVE Journal

Engineering

8.2K Views.  Kansas State University. Oppervlakte fabricage methoden voor patroon afzetting van nanometer dik borstels of micron dik, kruisverwijzende films van een azlactone blok co-polymeer worden gemeld. Kritische experimentele stappen, representatieve resultaten en beperkingen van elke methode worden besproken. Deze methoden zijn handig voor het creëren van functionele interfaces met op maat gemaakte fysieke kenmerken en afstembare oppervlakte reactiviteit.

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

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

Chemistry

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Reactive Vapor Deposition of Conjugated Polymer Films on Arbitrary Substrates

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. Conductive polymers, poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3,4-propylenedioxythiophene) (PProDOT), and a semiconducting polymer, poly(thieno[3,2-b]thiophene) (PTT), were deposited on unconventional highly-disordered and textured substrates with high surface areas, such as paper, towels and fabrics. This reported deposition chamber is an improvement of previous vapor reactors because our system can accommodate both volatile and nonvolatile monomers, such as 3,4-propylenedioxythiophene and thieno[3,2-b]thiophene. Utilization of both solid and liquid oxidants are also demonstrated. One limitation of this method is that it lacks sophisticated in situ thickness monitors. Polymer coatings made by the commonly used solution-based coating methods, such as spin-coating and surface grafting, are often not uniform or susceptible to mechanical degradation. This reported vapor phase deposition method overcomes those drawbacks and is a strong alternative to common solution-based coating methods. Notably, polymer films coated by the reported method are uniform and conformal on rough surfaces, even at a micrometer scale. This feature allows for future application of vapor deposited polymers in electronics devices on flexible and highly textured substrates.

This paper presents a protocol for reactive vapor deposition of poly(3,4-ethylenedioxythiophene), poly(3,4-propylenedioxythiophene), and poly(thieno[3,2-b]thiophene) films on glass slides and rough substrates, such as textiles and paper.

We demonstrate a method of conformally coating conjugated polymers on arbitrary substrates using a custom-designed, low-pressure reaction chamber. ...

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing (R-&#181;CP) and Sequential Nucleophilic Substitution

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates presenting grafted poly(ethylene glycol) (PEG) brushes can be used to achieve this task by creating microscale, non-fouling and cell adhesion resistant regions as well as regions where cells participate in biospecific interactions with covalently tethered ligands. To engineer complex tissues using such substrates, it will be necessary to sequentially pattern multiple PEG brushes functionalized to confer differential bioactivities and aligned in microscale orientations that mimic in vivo niches. Microcontact printing (&#956;CP) is a versatile technique to pattern such grafted PEG brushes, but manual &#956;CP cannot be performed with microscale precision. Thus, we combined advanced robotics with soft-lithography techniques and emerging surface chemistry reactions to develop a robotic microcontact printing (R-&#956;CP)-assisted method for fabricating culture substrates with complex, microscale, and highly ordered patterns of PEG brushes presenting orthogonal &#8216;click&#8217; chemistries. Here, we describe in detail the workflow to manufacture such substrates.

Fabricating Complex Culture Substrates Using Robotic Microcontact Printing R- &#181;CP and Sequential Nucleophilic Substitution

Cell culture substrates functionalized with microscale patterns of biological ligands have immense utility in the field of tissue engineering. Here, we demonstrate the versatile and automated manufacture of tissue culture substrates with multiple, micropatterned poly(ethylene glycol) brushes presenting orthogonal chemistries that enable spatially precise and site-specific immobilization of biological ligands.

In tissue engineering, it is desirable to exhibit spatial control of tissue morphology and cell fate in culture on the micron scale. Culture substrates ...

Bioengineering

Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing challenge in the field is the fabrication of hybrid micro-nano structures using a facile, cost-effective, and high-throughput method. To overcome these challenges, this paper proposes a protocol to fabricate multiscale structures using only an imprint process with an anodic aluminum oxide (AAO) filter and an evaporative self-aggregation process of nanofibers. Unlike previous attempts that have aimed to straighten nanofibers, we demonstrate a unique fabrication method for multiscale aggregated nanofibers with high aspect ratios. Furthermore, the surface morphology and wettability of these structures on various liquids were investigated to facilitate their use in multifunctional surfaces.

Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing ...

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

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 ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

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

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