We acknowledge the financial support of the NSF award CMMI-1000724.

1A. Primary Monolayer Formation on Silicon
<ol>
	<li>Cut silicon wafer into 1cm2 substrates, dust and rinse with water and filtered ethanol.</li>
	<li>Remove organic contamination by submerging the silicon substrates in a glass dish containing Nano strip at 75&ordm;C. After 15 minutes, rinse each substrate with deionized, filtered water.</li>
	<li>Place each substrate in a 5&#37; HF solution (Warning: HF is an extremely dangerous material) to remove the native oxide layer. After 5 minutes dry the oxide-free silicon with nitrogen</li>
	<li>To produce a chlorinated substrate, immediately submerge each oxide-free silicon piece in a scintillation vial containing 2 ml of saturated PCl5 in chlorobenzene. This solution should be filtered to 0.2 &mu;m. </li>
	<li>Assemble a vial condenser on top of each vial and place them in a heatblock set to 112&deg;C for one hour. </li>
	<li>After reaction is complete, let vials cool and rinse each surface with chlorobenzene and dry under filtered nitrogen.</li>
	<li>To form a propenyl-terminates substrate, place each chlorinated silicon surface in a pressure vial containing 4 ml of propenyl magnesium chloride. Place each pressure vial in a heatblock at 130 &deg;C for 24 hours. </li>
	<li>Take each pressure vial out of the heatblock and let cool.</li>
	<li>Rinse each surface quickly with DCM and ethanol and dry under filtered nitrogen.</li>
</ol>
1B. Primary Monolayer Formation on Germanium
<ol>
	<li>Cut germanium wafer into 1cm2 substrates, dust and rinse with water and filtered ethanol.</li>
	<li>Remove organic contamination by submerging the surfaces in a glass dish containing acetone for 20 minutes</li>
	<li>Place each surface in a 10&#37; HCl solution for 15 minutes. This process simultaneously removes the native oxide layer and chlorinates the surface. After 5 minutes dry the substrates with nitrogen.</li>
	<li>To form an octyl-terminated substrate, place each chlorinated germanium surface in a pressure vial containing 4 ml of octyl magnesium chloride (2 mM). Place each pressure vial in a heatblock at 130 &deg;C for 48 hours. </li>
	<li>Take each pressure vial out of the heatblock and let cool to room temperature.</li>
	<li>Rinse each surface quickly with DCM and ethanol and dry under filtered nitrogen.</li>

</ol>
2. NHS Substrate Functionalization on Silicon and Germanium
<ol>
	<li>Prepare a filtered 0.1 M NHS-diazirine solution in carbon tetrachloride. Warning: Keep light exposure to a minimum. </li>
	<li>Pipet a few drops of the solution onto the methyl terminated surfaces. Allow solution to spread across the entire surface.</li>
	<li>Place the surfaces under a UV lamp (&#9744;=254 nm, 4400/cm2 at 0.74 inches). Allow the surfaces to react under UV light for 30 minutes, then add more NHS-diazirine to the surface and let the reaction proceed for an additional 30 minutes. </li>
	<li>Rinse the NHS modified surfaces with DCM and ethanol and dry under filtered nitrogen.</li>
</ol>
3. Small Molecule Functionalization
<ol>
	<li>React NHS-modified substrates in a 20 mM tert-butyl carbamoyl (Boc-) ethylenediamine solution in dichloromethane (DCM) for two hours at room temperature. </li>
	<li>After the reaction, rinse the Boc-modified substrate with DCM and ethanol. </li>
	<li>Deprotect the Boc modified substrate using 25&#37; trifluoroacetic acid (TFA) in DCM for one hour at room temperature. </li>
	<li>Rinse the resulting surface with DCM, ethanol and 10&#37; (w/v) potassium bicarbonate in water and dry under filtered nitrogen. </li>
	<li>Analyze all surfaces by XPS to determine the elemental composition. </li>
</ol>
4. Acidic Polyurethane Acrylate Stamp (PUA) Preparation
<ol>
	<li>Dilute acrylate A by 30&#37; with trimethylolpropane ethoxylate triacrylate B to reduce viscosity. Add photoinitiators C and D to the reaction mixture (Figure 6).</li>
	<li>Add sodium 2-mercaptoethanesulfonate (0.2 g, 1.22 mmol) to a 4N HCl solution in dioxane (10 ml) and stir at room temperature for 2 minutes.</li>
	<li>Filter off the sodium chloride first through a fine glass filter and then through a 0.2 &mu;m PTFE membrane syringe filter to afford a clear solution of 2-mercaptoethanesulfonic acid in dioxane. </li>
	<li>Evaporate dioxane under reduced pressure </li>
	<li>React the resulting sulfonic acid with 2 ml of the polyurethane-acrylate prepolymeric mixture at room temperature and then under vacuum at 50 &deg;C. Be sure to completely free the mixture from trapped air bubbles.</li>
	<li>Cool the resulting solution to room temperature and polymerize between two glass microscope slides or a glass slide and a master by exposure to UV light for 2 hours at room temperature. </li>
	<li>After polymerization, carefully peel the stamp off the master and wash the stamp with ethanol and water and dry with filtered nitrogen. </li>
</ol>
5. Catalytic Printing and SEM/AFM Analysis
<ol>
	<li>Place the corresponding polyurethane-acrylate stamp on top of the NHS-modified substrate at room temperature for one minute with no external load to hold them together. </li>
	<li>After the reaction, separate the stamp and substrate.</li>
	<li>Rinse the substrate with ethanol, water, and ethanol then dry with filtered nitrogen. </li>
	<li>Rinse the stamp with ethanol, water, and ethanol then dry with filtered nitrogen.</li>
	<li>Keep stamps at room temperature before the next application.</li>
	<li>Analyze the produced pattern using contact mode lateral atomic force microsopy (AFM) and scanning electron microscopy (SEM)</li>
</ol>
6. Protein Patterning and Fluorescent Microscopy
<ol>
	<li>Submerge the NHS-patterned bifunctional substrate in Lysine-N,N-diacetic acid (20 mM) and Et3N (100 mM) in DMF:H20 (1:1) at room temperature for 1 hr and then rinsed with water and ethanol. </li>
	<li>Incubate the substrates in a 50 mM NiSO4 solution for 5 min at room temperature. </li>
	<li>Rinse the chelated substrates excessively with water and binding buffer (20 mM NaP, 250 mM NaCl, 10mM imidazole, pH 7.5) and submerge in a filtered GFP solution (&tilde;40 &mu;M) for 1 hr at 0&deg;C. </li>
	<li>Immediately rinse the substrates with binding buffer followed by PBS (pH 7.4). </li>
	<li>Keep substrates hydrated in PBS at 0&deg;C until they were ready for fluorescence microscopy analysis.</li>
</ol>
7. Protein Patterning and Fluorescent Microscopy
<ol>
	<li>Submerge the NHS-patterned bifunctional substrate in Lysine-N,N-diacetic acid (20 mM) and Et3N (100 mM) in DMF:H20 (1:1) at room temperature for 1 hr and then rinsed with water and ethanol. </li>
	<li>Incubate the substrates in a 50 mM NiSO4 solution for 5 min at room temperature. </li>
	<li>Rinse the chelated substrates excessively with water and binding buffer (20 mM NaP, 250 mM NaCl, 10mM imidazole, pH 7.5) and submerge in a filtered GFP solution (&tilde;40 &mu;M) for 1 hr at 0&deg;C. </li>
	<li>Immediately rinse the substrates with binding buffer followed by PBS (pH 7.4). </li>
	<li>Keep substrates hydrated in PBS at 0&deg;C until they were ready for fluorescence microscopy analysis.</li>
</ol>
8. Representative Results: 
An example of soft-lithographic catalytic nano patterning is shown in Figure 7. The approach creates chemoselective patterns on oxide-free silicon and germanium, which can be orthogonally functionalized with dissimilar chemical and biological moieties. The reaction between the NHS-functioanlized substrate and the catalytic patterned stamp leads to the hydrolysis of NHS moieties in areas of conformal contact, yielding a patterned bifunctional substrate bearing regions of NHS activated and free carboxylic acids. Due to the diffusion free nature of our method, we achieve resolution close to that of photolithography. For example, Figure 7 shows 125 nm features, which were uniformly reproduced across the entire silicon substrate surface. Remarkably, the catalytic stamp can be reused multiple times without losing efficiency.
Chemoselective functionalization of patterned semiconductors with biomolecules opens up the prospect of integrating traditional electronic materials with highly selective biological substrates for applications in sensing, diagnostic, and analytical areas of research. An example of such functionalization is shown in Figure 8, where NHS-patterned silicon was selectively functionalized with protein molecules. By exploiting the differential reactivities of activated and free carboxylic acids, we first affixed nitrilotriacetic acid-terminated (NTA) heterobifunctional linkers to the NHS-functionalized regions, and then used the resulting NTA-patterned surface as a template for the selective attachment of hexa-histidine-tagged GFP. Figure 8b clearly shows differential fluorescence intensity between GFP-modified and hydrolyzed free carboxylic acid regions. The size and shape of the replicated features are consistent between both NHS patterned surface (Figure 8a) and GFP-modified surface (Figure 8b), confirming the remarkable stability of carbon-passivated surfaces and the selectivity of the stamping approach. The protocol is not limited to His-tagged protein, and can be used to pattern other biomolecules including DNA and antibodies.
<img src="/files/ftp_upload/3478/3478fig1.jpg" alt="Figure 1"/> Figure 1.&nbsp;General scheme representing catalytic microcontact printing
<img src="/files/ftp_upload/3478/3478fig2.jpg" alt="Figure 2"/> Figure 2.&nbsp;Structure of bi-layered molecular system on Ge and Si. Primary alkyl monolayer forms stable Ge-C or Si-C bonds with the substrate and provides a chemically inert and close packed system that protects the underlying surface from degradation.(b) Secondary overlayer forms stable C-C bonds with primary protective layer and provides terminal functional groups
<img src="/files/ftp_upload/3478/3478fig3.jpg" alt="Figure 3"/> Figure 3.&nbsp;Reaction schemes representing formation of primary protective monolayers on Si (A) and Ge (B)
<img src="/files/ftp_upload/3478/3478fig4.jpg" alt="Figure 4"/> Figure 4.&nbsp;Chemical functionalization of the primary protective monolayer with a heterobifunctional carbene donor
<img src="/files/ftp_upload/3478/3478fig5.jpg" alt="Figure 5"/> Figure 5.&nbsp;Reaction scheme demonstrating small molecule modifications of NHS-functionalized substrates and the corresponding XPS spectra
<img src="/files/ftp_upload/3478/3478fig6.jpg" alt="Figure 6"/> Figure 6.&nbsp;Composition of the catalytic pre-polymeric mixture, polymerization conditions, and SEM images of the patterned sulfonic acid-modified stamp and the corresponding PMMA-Si master
<img src="/files/ftp_upload/3478/3478fig7.jpg" alt="Figure 7"/> Figure 7.&nbsp;SEM and AFM friction images of patterned SAMs on Si and Ge with an acidic stamp
<img src="/files/ftp_upload/3478/3478fig8.jpg" alt="Figure 8"/> Figure 8.&nbsp;Soft-lithographic patterning and functionalization of passivated silicon with organic and biological molecules. a: SEM image of the patterned NHS-modified substrate. b: Fluorescent micrograph of GFP modified substrate.

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B., Biebuyck, H., Delamarche, E., Michel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+in+microcontact+printed+self-assembled+monolayers.">Order in microcontact printed self-assembled monolayers.</a> J Am Chem Soc. 119 (13), 3017-3026 (1997).</li><li>Michel, B., Bernard, A., Bietsch, A., Delamarche, E., Geissler, M., Juncker, D., Kind, H., Renault, J. P., Rothuizen, H., Schmid, H., Schmidt-Winkel, P., Stutz, R., Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+meets+lithography:+Soft+approaches+to+high-resolution+printing.">Printing meets lithography: Soft approaches to high-resolution printing.</a> IBM Journal of Research and Development. 45 (5), 697-719 (2001).</li><li>Libioulle, L., Bietsch, A., Schmid, H., Michel, B., Delamarche, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact-Inking+Stamps+for+Microcontact+Printing+of+Alkanethiols+on+Gold.">Contact-Inking Stamps for Microcontact Printing of Alkanethiols on Gold.</a> Langmuir. 15 (2), 300-304 (1999).</li><li>Sharpe, R. B. A., Burdinski, D., Huskens, J., Zandvliet, H. J. W., Reinhoudt, D. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+Microcontact+Printing+without+Ink.">Catalytic Microcontact Printing without Ink.</a> Nano Lett. 3 (10), 1449-1453 (2003).</li><li>Shestopalov, A. A., Clark, R. L., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkless+Microcontact+Printing+on+Self-Assembled+Monolayers+of+Fmoc-Protected+Aminothiols.">Inkless Microcontact Printing on Self-Assembled Monolayers of Fmoc-Protected Aminothiols.</a> J. Am. Chem. Soc. 129 (145), 13818-13819 (2007).</li><li>Shestopalov, A. A., Clark, R. L., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+Microcontact+Printing+on+Chemically+Functionalized+H-Terminated+Silicon.">Catalytic Microcontact Printing on Chemically Functionalized H-Terminated Silicon.</a> Langmuir. 26 (3), 1449-1451 (2010).</li><li>Shestopalov, A. A., Clark, R. L., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inkless+Microcontact+Printing+on+SAMs+of+Boc-+and+TBS-Protected+Thiols.">Inkless Microcontact Printing on SAMs of Boc- and TBS-Protected Thiols.</a> Nano Lett. 10 (1), 43-46 (2010).</li><li>Snyder, P. W., Johannes, M. S., Vogen, B. N., Clark, R. L., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalytic+Microcontact+Printing.">Biocatalytic Microcontact Printing.</a> J. Org. Chem. 72 (19), 7459-7461 (2007).</li><li>Morris, C. J., Shestopalov, A. A., Gold, B. H., Clark, R. L., Toone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+NHS-Terminated+SAMs+on+Germanium.">Patterning NHS-Terminated SAMs on Germanium.</a> Langmuir. 27 (10), 6486-6489 (2011).</li><li>Shestopalov, A. A., Morris, C. J., Vogen, B. N., Hoertz, A., Clark, R. L., Toone, E. 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We have nothing to disclose

The presented protocol is a form or of inkless microcontact printing that can be universally applied to any substrate capable of supporting simple well-ordered monolayers. In this method, a stamp-immobilized catalyst transfers a pattern to a surface bearing corresponding functional groups. Because the process does not rely on ink transfer from stamp to surface the diffusive resolution limitation of traditional and reactive &mu;CP is obviated, permitting routine manufacturing of nanoscale objects. The incorporation of a primary highly-ordered molecular system provides complete protection of the underlying semiconductor from oxidation damage. At the same time, the method supports immobilization of bulky reactive groups by utilizing a secondary reactive overlayer; together the system achieves both protection and functionalization.
The technique begins with the formation of stable carbon-surface bonds allowing for chemically inert primary monolayer which serves as an effective barrier to oxide formation. Formation of a secondary reactive overlayer provides terminal NHS functional groups that serve as attachment points for a variety of chemical and biological moieties. This stable bilayered molecular system is subsequently patterned using our catalytic &mu;CP approach. The approach presented in this study offers a general method for patterning semiconductor substrates with a broad range of organic and biological materials. The ability to create patterned organic-semiconductor interfaces without expensive, complex instrumentation offers numerous opportunities in fields such as electronics, nanotechnology, biochemistry and biophysics.

<table border="1">
<tbody>
<tr>
	<th>Name of the reagent</th>
	<th>Company/model</th>
</tr>
<tr>
	<td>XPS spectrometer</td>
	<td>Kratos Axis Ultra</td>
</tr>
<tr>
	<td>Atomic force microscope</td>
	<td>Veeco D3100</td>
</tr>
<tr>
	<td>SEM-FEG microscope</td>
	<td>FEI XL30</td>
</tr>
<tr>
	<td>Fluorescent microscope</td>
	<td>Zeiss Axio Imager</td>
</tr>
<tr>
	<td>Heatblock</td>
	<td>VWR</td>
</tr>
<tr>
	<td>Vacuum pump</td>
	<td>Boc Edwards</td>
</tr>
<tr>
	<td>Water purification system</td>
	<td>Millipore</td>
</tr>
<tr>
	<td>TESP silicon probes</td>
	<td>Veeco</td>
</tr>
<tr>
	<td>Silicon</td>
	<td></td>
</tr>
<tr>
	<td>Pressure Vials</td>
	<td>Chemglass</td>
</tr>
<tr>
	<td>Vacuum manifold</td>
	<td>Chemglass</td>
</tr>
<tr>
	<td>UV Lamp</td>
	<td>UVP</td>
</tr>
<tr>
	<td>Stamp Material</td>
	<td>See references 20 and 18</td>
</tr>
<tr>
	<td>PFTE syringe filters</td>
	<td>VWR</td>
</tr>
<tr>
	<td>Nano Strip</td>
	<td>Cyantek</td>
</tr>
<tr>
	<td>HCl</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Ethanol</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Acetone</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>HF</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Chlorobenzene</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>PCl5</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Propenyl Magnesium Chloride</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Octyl Magnesium Chloride</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Carbon TetraChloride</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Boc protected ethylenediamine</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>TFA</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Sodium 2-mercaptoethanesulfonate </td>
	<td>Sigma</td>
</tr>
<tr>
	<td>4N HCl solution in dioxane</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Lysine-N,N-diacetic acid</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>Et3N</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>DMF</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>NiSO4</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>NaP</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>NaCl</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>imidazole</td>
	<td>Sigma</td>
</tr>
<tr>
	<td>PBS</td>
	<td>Sigma</td>
</tr>
</tbody>
</table>

soft lithographic functionalization and patterning oxide-free silicon and germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Watch this Scientific Journal Video about Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium at JoVE.com

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

soft-lithographic-functionalization-patterning-oxide-free-silicon

Research

JoVE Journal

Bioengineering

14.6K Views.  Duke University. Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

Video: Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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 ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...