Name: functional surface-immobilization of genes using multistep strand displacement lithography
Uploaded: 2018-10-25T15:00:00
Description: Watch this Scientific Journal Video about Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography at JoVE.com

We gratefully acknowledge financial support for this project by the Volkswagen Stiftung (grant no. 89 883) and the European Research Council (grant agreement no. 694410 - AEDNA). M.S.-S. acknowledges support by the DFG through GRK 2062.

NOTE: A time schedule for the steps in the different sections is given in the supplementary information (section 1).
1. Chip Fabrication
NOTE: As substrates, use silicon wafers (100 mm diameter, 0.525 mm thickness) with a 50 nm thick layer of silicon dioxide or glass slides (24 mm x 24 mm, no. 1.5; 22 mm x 50 mm, no. 4). Depending on the application, other sizes and thicknesses may be more suitable.
<ol>
	<li>Cleaning the substrates via an RCA clean proce...

<ol>
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P., Schwartz, R., Leduc, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+crowding+shapes+gene+expression+in+synthetic+cellular+nanosystems.">Molecular crowding shapes gene expression in synthetic cellular nanosystems.</a> Nature Nanotechnology. 8 (8), 602-608 (2013).</li><li>van Nies, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-replication+of+DNA+by+its+encoded+proteins+in+liposome-based+synthetic+cells.">Self-replication of DNA by its encoded proteins in liposome-based synthetic cells.</a> Nature Communications. 9 (1), 1583 (2018).</li><li>Buxboim, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-step+photolithographic+interface+for+cell-free+gene+expression+and+active+biochips.">A single-step photolithographic interface for cell-free gene expression and active biochips.</a> Small. 3 (3), 500-510 (2007).</li><li>Bracha, D., Karzbrun, E., Daube, S. S., Bar-Ziv, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergent+properties+of+dense+DNA+phases+toward+artificial+biosystems+on+a+surface.">Emergent properties of dense DNA phases toward artificial biosystems on a surface.</a> Accounts Of Chemical Research. 47 (6), 1912-1921 (2014).</li><li>Bracha, D., Bar-Ziv, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+and+nanowire+assemblies+of+condensed+DNA+polymer+brushes.">Dendritic and nanowire assemblies of condensed DNA polymer brushes.</a> Journal of the American Chemical Society. 136 (13), 4945-4953 (2014).</li><li>Daube, S. S., Bracha, D., Buxboim, A., Bar-Ziv, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+by+directional+gene+expression.">Compartmentalization by directional gene expression.</a> Proceedings Of The National Academy Of Sciences Of The United States Of America. 107 (7), 2836-2841 (2010).</li><li>Karzbrun, E., Tayar, A. M., Noireaux, V., Bar-Ziv, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+on-chip+DNA+compartments+as+artificial+cells.">Programmable on-chip DNA compartments as artificial cells.</a> Science. 345 (6198), 829-832 (2014).</li><li>Tayar, A. M., Karzbrun, E., Noireaux, V., Bar-Ziv, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagating+gene+expression+fronts+in+a+one-dimensional+coupled+system+of+artificial+cells.">Propagating gene expression fronts in a one-dimensional coupled system of artificial cells.</a> Nature Physics. 11 (12), 1037-1041 (2015).</li><li>Pardatscher, G., Schwarz-Schilling, M., Daube, S. S., Bar-Ziv, R. H., Simmel, F. 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Bephore lithography is a robust and versatile technique for the patterned immobilization of DNA or RNA. Yet, the procedure includes several steps, which - if changed - may be a source for failure or reduced performance of the system.
A crucial step in the fabrication of Bephore chips is the PEGylation of the substrate, which provides the biocompatibility of the surface. Here, the cleaning step with an RCA procedure is important, since it also activates the surface for the subsequent silanizati...

Cell-free gene expression reactions are of great interest for various applications in biochemistry, biotechnology, and synthetic biology. Cell-free expression of proteins was instrumental for the preparation of pure protein samples, which were the basis for numerous studies in structural biology. For instance, cell-free systems were successfully used for the expression of protein complexes1 or membrane proteins2, which are difficult to produce using cell-based expression. Notably, cell-free gene expression reactions were also used to elucidate the structure of the genetic code, starting with the groundbreaking experiments by Nirenberg and Matthaei in 19613.
Recently, there has been a renewed interest in cell-free methods in biotechnology and synthetic biology4,5,6. Cell-free systems can be augmented with non-biological compounds, and components of diverse biological origin can be combined more easily7. Even though cell-free systems have the apparent disadvantage that they do not &#34;grow and divide&#34;, it is conceivable to prepare open cell-free bioreactors with basic metabolic functions and let them synthesize metabolites when provided with simple carbon and energy inputs8. Within the emerging field of synthetic biology, cell-free systems promise to be a more predictable &#34;chassis&#34; for the implementation of synthetic biological functions.
Currently, cell-free gene expression reactions are carried out either using cell extracts (from different sources such as bacteria, yeast, insects), or transcription/translation systems which were optimized for different applications (e.g., prokaryotic vs. eukaryotic gene expression, production of membrane proteins, etc.). A popular protocol for the preparation of bacterial cell extract (commonly termed TXTL) was provided recently by V. Noireaux and coworkers9. Its biophysical properties have been thoroughly characterized10, and the TXTL system has been already used successfully to perform a series of complex biochemical tasks: e.g., the assembly of functional bacteriophages via cell-free expression of the phage genome11, the synthesis of bacterial protein filaments12, or the implementation of cell-free gene circuits13,14.
Another system popular in cell-free synthetic biology is the PURE system, which is reconstituted from purified components15,16. Compared to the TXTL system, it does not contain nucleases or protein degradation machinery. While degradation of linear DNA, RNA molecules or proteins is less of an issue in the PURE system, decay pathways are actually important for the implementation of dynamical functions. In order to reduce the effect of exonuclease degradation of linear gene templates in the TXTL system (through RecBCD), the end-protecting GamS protein has to be added. Both the TXTL and the PURE system are commercially available.
A topic closely related to cell-free biology concerns the study of the effect of compartmentalization on biochemical reactions, and further the creation of artificial cell-like structures or protocells17,18,19,20. Research on artificial cells typically involves the encapsulation of a biochemical reaction system inside of vesicular compartments made from phospholipids or other amphiphiles. While such systems help to explore fundamental aspects of compartmentalization, or the emergence of cellularity and self-replicating structures, they face the typical problems of closed systems: in the absence of a functioning metabolism and appropriate membrane transport mechanisms, it is difficult to keep compartmentalized reactions running for extended periods of time - fuel molecules are used up and waste products accumulate.
An interesting alternative to compartmentalization inside of such cell-mimicking compartments is the spatial organization of genetic material using photolithographic methods. Immobilization of &#34;genes on a chip&#34; was pioneered by the Bar-Ziv group at the Weizmann Institute more than ten years ago21. Among the major issues that had to be resolved were the non-specific adsorption of DNA and the potential denaturation of proteins on the chip surface. Bar-Ziv et al. developed a dedicated photolithography resist termed &#34;Daisy&#34;, which was composed of a reactive terminal silane for immobilization of the resist molecules on silicon dioxide surfaces, a long polyethylene glycol (PEG) spacer that assured biocompatibility, and a photocleavable headgroup, which was converted into a reactive amine upon irradiation with ultraviolet (UV) light. It has been shown that Daisy can be used to immobilize gene-length DNA molecules (with lengths of several kilo base-pairs (kbp)) on a chip surface. From a polymer physics point of view, the systems represented polymer brushes grafted onto a solid substrate. Due to the polyelectrolyte nature of DNA, the conformation of these brushes is strongly affected by osmotic and other ion-specific effects22,23.
Most importantly, it has been shown that substrate-immobilized genes are still functional and can be transcribed and translated into RNA and protein. Gene brushes are accessible for RNA polymerases from solution24, and the complex macromolecular mixture of the transcription/translation is not denatured at the surface. One of the advantages of immobilization of genetic components on a substrate is that they can be operated in an open microfluidic reactor system that is continuously supplied with small precursor molecules and from which waste products can be removed25,26.
We recently developed a variant of this method termed Bephore (for Biocompatible electron-beam and photoresist)27, which was based exclusively on commercially available components and utilized sequence-specific DNA strand invasion reactions for the realization of a simple-to-implement multistep lithography procedure for the creation of chip-based artificial cells. A schematic overview of the procedure is shown in Figure 1. It is based on DNA hairpin molecules containing a photocleavable group, which are immobilized on a biocompatible PEG&#160;layer. Photocleavage of the hairpin exposes a single-stranded toehold sequence, through which DNA molecules of interest (containing the &#34;displacing&#34; DIS sequence) can be attached via toehold-mediated strand invasion.
While Bephore is potentially simpler to implement, Daisy allows the realization of very dense and clean &#34;gene brushes&#34;, which has advantages in certain applications. In principle, however, Daisy and Bephore lithography could be easily combined. A related lithography method utilizing DNA strand displacement for structuring DNA brushes on gold was previously developed by Huang et al., but was not utilized in the context of cell-free gene expression28,29.
In the following protocol we provide a detailed description of the production of DNA brushes for cell-free gene expression using the Bephore method. We describe how the gene chips are fabricated and demonstrate the use of multi-step photo-lithography for the spatially structured immobilization of genes on a chip. We also discuss the fabrication of reaction chambers and the application of microfluidics for the performance of on-chip gene expression reactions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Silicon wafer with 50 nm silicon dioxide (Bephore substrate)</td>
			<td>Siegert Wafer</td>
			<td></td>
			<td>Thickness (&#181;m): 525 &#177;25, Diameter (mm): 100</td>
		</tr>
		<tr>
			<td>Silicon wafer (for PDMS master mold)</td>
			<td>Siegert Wafer</td>
			<td></td>
			<td>Thickness (&#181;m): 525 &#177;25, Diameter (mm): 76.2 (3&#8221;)</td>
		</tr>
		<tr>
			<td>Glass slides no. 4</td>
			<td>Menzel</td>
			<td></td>
			<td>22 mm x 50 mm</td>
		</tr>
		<tr>
			<td>Glass slides no. 1.5</td>
			<td>Assistent</td>
			<td></td>
			<td>24 mm x 24 mm</td>
		</tr>
		<tr>
			<td>Biotin-PEG-Silane</td>
			<td>Laysan Bio</td>
			<td></td>
			<td>MW 5,000</td>
		</tr>
		<tr>
			<td>Anhydrous toluene</td>
			<td>Sigma Aldrich (Merck)</td>
			<td>244511</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Thermo-Fisher Scientific</td>
			<td>S888</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs</td>
			<td>M0531S</td>
			<td>PCR kit</td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td>Spin-column PCR clean-up kit</td>
		</tr>
		<tr>
			<td>PURExpress</td>
			<td>New England Biolabs</td>
			<td>E6800S</td>
			<td>Cell-free expression system</td>
		</tr>
		<tr>
			<td>PDMS&#160;</td>
			<td>Dow Corning</td>
			<td>Slygard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoSpheres</td>
			<td>Thermo-Fisher Scientific</td>
			<td>F8771</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE tubing (ID: 0.8mm, OD: 1.6 mm)</td>
			<td>Bola</td>
			<td>S 1810-10</td>
			<td></td>
		</tr>
		<tr>
			<td>EpoCore 20</td>
			<td>micro resist technology&#160;GmbH</td>
			<td></td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>mr-Dev 600</td>
			<td>micro resist technology&#160;GmbH</td>
			<td></td>
			<td>Photoresist developer</td>
		</tr>
		<tr>
			<td>Ti-Prime</td>
			<td>MicroChemicals</td>
			<td></td>
			<td>Adhesion promoter</td>
		</tr>
		<tr>
			<td>Two-component silicon glue</td>
			<td>Picodent</td>
			<td>Twinsil&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-protection yellow foil</td>
			<td>Lithoprotect (via MicroChemicals)</td>
			<td>Y520E212</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masks for photolithography</td>
			<td>Zitzmann GmbH</td>
			<td></td>
			<td>64.000 dpi, 180x240 mm</td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51</td>
			<td>Photolithography and fluorescence imaging</td>
		</tr>
		<tr>
			<td>60x water immersion objective</td>
			<td>Olympus</td>
			<td>LumPlanFl</td>
			<td>Used with Olympus BX51, NA 0.9</td>
		</tr>
		<tr>
			<td>20x water immersion objective</td>
			<td>Olympus</td>
			<td>LumPlanFl</td>
			<td>Used with Olympus BX51, NA 0.5</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Photometrics</td>
			<td>Coolsnap HQ</td>
			<td>Used with Olympus BX51</td>
		</tr>
		<tr>
			<td>Ligtht source</td>
			<td>EXFO</td>
			<td>X-Cite 120Q</td>
			<td>Used with Olympus BX51</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Ti2-E</td>
			<td>Fluorescence imaging of gene expression</td>
		</tr>
		<tr>
			<td>4x objective</td>
			<td>Nikon</td>
			<td>CFI P-Apo 4x Lambda</td>
			<td>Used with Nikon Ti2-E</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Andor</td>
			<td>Neo5.5</td>
			<td>Used with Nikon Ti2-E</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Lumencor</td>
			<td>SOLA SM II</td>
			<td>Used with Nikon Ti2-E</td>
		</tr>
		<tr>
			<td>Cage incubator</td>
			<td>Okolab</td>
			<td>bold line</td>
			<td>Used with Nikon Ti2-E</td>
		</tr>
		<tr>
			<td>Pressure Controller</td>
			<td>Elveflow</td>
			<td>OB1 MK3</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoPhotometer</td>
			<td>Implen</td>
			<td></td>
			<td>DNA concentration measurement</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Diener</td>
			<td>Femto</td>
			<td>200 W, operated at 0.8 mbar with the sample in a Faraday cage</td>
		</tr>
	</tbody>
</table>

functional surface-immobilization of genes using multistep strand displacement lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

Two-step lithography: Figure 5 shows the result of a two-step lithographic process on a glass slide with overlapping patterns of fluorescently labeled DIS strands.
Expression of a fluorescent protein from a gene brush: Figure 6 demonstrates the expression of the fluorescent protein YPet from immobilized DNA. At several points in time we assessed the gene expression rate...

Watch this Scientific Journal Video about Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography at JoVE.com

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

DNA biochips are an attractive research platform for self re-synthetic biology, originally developed by the BASF group, which allows for the operation of in vitro gene circuits over extended periods of time. With this protocol, we hope to make this technology available to a wider range of researchers. Our method, termed bephore lithography, represents a strategy for the immobilization of DNA based on the DNA strand displacement reaction.The strength of this technique lie in its multi-step lithographic capabilities, its simple reproducibility, and the commercial availability of all the components. Similar to other biochip solutions, bephore can be combined with different technologies. For example, functional gene brushes can be assembled within micro-compartments for protein expression.To begin, prepare an RCA mixture by mixing five parts water with one part of a 30%ammonia solution in a glass beaker. Heat the mixture to 70 degrees Celsius on a hot plate while stirring. Once it reaches 70 degrees Celsius, add one part 30%hydrogen peroxide.Then place a 100 millimeter diameter silicon wafer into the solution, to remove organic material and particles from it. After 30 minutes, take out the substrate, rinse it thoroughly with water from a wash bottle, and dry it with a nitrogen gun. Immediately proceed with the PEGylation of the substrate.To accomplish this, first dilute biotin-PEG-Silane in dry toluene to 5 milligrams per milliliter, and vortex the solution until it is well mixed. With the substrate placed in a glass Petri dish, slowly pipette the solution onto the substrate, covering the whole surface, but avoid allowing the solution to flow over the edge. Once the surface is covered, close the Petri dish, and then add an additional cover.After 30 minutes, remove the cover, and add approximately 40 milliliters of isopropyl alcohol to the Petri dish. Then take out the substrate, rinse it again thoroughly with isopropyl alcohol, and dry it with a nitrogen gun. When dry, store the substrate in the dark until it is needed.Prepare a substrate by using a glass cutter or a scalpel to form one centimeter by one centimeter pieces. Then, add a simple alignment mark by making a short scratch from the center towards an edge of the substrate. Blow any small particles off the chip using a nitrogen gun.Next, mix two droplets of a two-component silicone glue, and apply the glue to the surface of the chip, leaving the area around the tip of the scratch blank. There the glue provides a hydrophobic barrier, keeping aqueous solutions on the chip. This facilitates subsequent washing steps, and reduces the amount of DNA required for incubations.In a later step, the glue can be easily peeled off. In the following steps, photocleavable DNA is handled only in a yellow light environment. Cover the chip with about 10 microliters of a bephore mixture at room temperature, and place the chip in a box partially filled with water to reduce evaporation.After one hour, wash the chip several times with PBS to remove any unbound bephore mix. Next, add 10 microliters of the passivation mix to the chip. After two hours, wash the chip several times with PBS to remove the unbound passivation agents.Cut a printed photo mask to the appropriate size in order to fit a suitable mask holder. Insert it at the position of the field stop, and use the alignment marks to align the mask in the holder. With the substrate placed on the microscopy stage, insert a red filter into the illumination path, and focus onto the substrate surface.Then, navigate to the region of the substrate which will be exposed. Next, insert the mask holder, block the illumination, and change to the UV filter. At a low light intensity, open the shutter, and use the camera to quickly focus the mask on the substrate.With the substrate now aligned and the mask in focus, block the light path to the camera, and illuminate the substrate at a high light intensity for the desired exposure time. After exposure, remove the sample from the microscope, and carefully remove as much buffer as possible from the substrate without drying it. Then, add 10 to 20 microliters of DNA with the sequence for attachment to the surface.Place the substrate in a wet box to keep it from drying, and incubate the DNA on the substrate for two hours at room temperature. In order to observe the compartmentalized gene expression on an inverted microscope, first separately prepare the two parts of the sample holder. Start by gluing a bephore chip with an immobilized gene brush onto the chip holder, using double-sided adhesive tape.Next, add some vacuum grease around the central hole of the bottom part of the holder, and insert the chip holder. Now assemble the top part of the holder. Cut a thin PDMS chip with compartments as small as possible, leaving a channel open to one side to later allow for the exchange of waste and precursor molecules by diffusion Next, plasma-treat a glass cover slip in the back side of the PDMS chip with oxygen plasma.Immediately after treatment, place the PDMS chip at the center of the glass slide, with the compartments pointing upwards. Then, bake the glass with the PDMS for one hour at 70 degrees Celsius. Shortly before assembling the entire sample holder, plasma-treat the glass slide with the PDMS chip as before.Then, add some vacuum grease around the large hole of the top holder, and place the glass slide with the PDMS onto it. Gently press the glass onto the grease. Carefully remove the buffer from the chip, and wash it once with 10 microliters of self re-expression system.Next, add 60 microliters of self re-expression system onto the chip, and remove the two-component silicone glue from the edges. Now, add 20 microliters of the expression system onto the PDMS. After placing the droplet onto the PDMS, quickly check in the stereoscopic microscope that the compartments are well wet and without air bubbles.If there are air bubbles, try to wash them away. To assemble the two pieces of the holder, and to align chambers and DNA brushes, work under a stereoscopic microscope. Immobilize the bottom holder with a gripper arm so the two screws and wingnuts will be easily accessible with both hands.Insert the top holder into the bottom holder, and lower it until the cell-free expression system droplets fuse. Then, check to make sure the compartments and the alignment mark are in a similar region in the XY plane. From the bottom, screw up the wingnuts until they touch the bottom side of the holder.While aligning the compartments and the chip in the XY plane, gently tighten the wingnuts. This step requires some experience, and depends on the construction of the sample holder. The PDMS should be pressed onto the chip only with gentle force.Next, spray a sponge in the anti-evaporation enclosure with water, and insert the holder into the box. Then, fill a five milliliter syringe with two-component silicone glue, and use it to seal the box. Finally, transfer the box to a temperature-controlled microscope, and image the DNA brush and the reaction using fluorescence microscopy.A two-step lithographic process, here performed on a bephore glass slide, yields overlapping patterns of fluorescently labeled DNA strands. In this demonstration of on-chip gene expression, DNA is immobilized in the chamber on the left. When exposed to the expression mix, the yellow fluorescent protein YPet is synthesized from the immobilized DNA.To assess the activity of the gene expression system, the recovery of the fluorescence is observed after a bleaching step. At two hours, the fluorescence intensity recovered quickly. However, after four and six hours, it did not recover, indicating that without a fresh supply of the expression mix, the reaction terminated around hour four.Although gene expression is limited in a closed system, it can be sustained over longer periods of time by supplying the expression compartments with additional precursor molecules via micro-fluidics. The bephore method is used to immobilize oligonucleotides or gene length DNA, which can be applied to construct systems of interacting DNA brushes for self re-gene expression. The technique may also be used for other applications in biophysics, such as single molecule fluorescence studies.After watching this video, you should be able to fabricate bephore biochips from wafers or glass slides, and integrate them into your research projects.

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Research

JoVE Journal

Bioengineering

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 ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Robotic Mirror Therapy System for Functional Recovery of Hemiplegic Arms

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is conducted by eliciting an illusion through use of a mirror as if the hemiplegic arm is moving in real-time while moving the healthy arm. It can facilitate brain neuroplasticity through activation of the sensorimotor cortex. However, conventional mirror therapy has a critical limitation in that the hemiplegic arm is not actually moving. Thus, we developed a real-time 2-axis mirror robot system as a simple add-on module for conventional mirror therapy using a closed feedback mechanism, which enables real-time movement of the hemiplegic arm. We used 3 Attitude and Heading Reference System sensors, 2 brushless DC motors for elbow and wrist joints, and exoskeletal frames. In a feasibility study on 6 healthy subjects, robotic mirror therapy was safe and feasible. We further selected tasks useful for activities of daily living training through feedback from rehabilitation doctors. A chronic stroke patient showed improvement in the Fugl-Meyer assessment scale and elbow flexor spasticity after a 2-week application of the mirror robot system. Robotic mirror therapy may enhance proprioceptive input to the sensory cortex, which is considered to be important in neuroplasticity and functional recovery of hemiplegic arms. The mirror robot system presented herein can be easily developed and utilized effectively to advance occupational therapy.

We developed a real-time mirror robot system for functional recovery of hemiplegic arms using automatic control technology, conducted a clinical study on healthy subjects, and determined tasks through feedback from rehabilitation doctors. This simple mirror robot can be applied effectively to occupational therapy in stroke patients with a hemiplegic arm.

Mirror therapy has been performed as effective occupational therapy in a clinical setting for functional recovery of a hemiplegic arm after stroke. It is ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...