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.

<ol>
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The authors declare that they have no competing interests.

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 silanization. During the actual PEGylation, the substrate must not dry out. Furthermore, the Silane-PEG-Biotin must be stored properly to preserve its reactivity (section 1.2) and to avoid crosslinking. To assess the outcome of the PEGylation, substrates can be incubated with fluorescently labeled streptavidin and compared to non-PEGylated controls. Successfully PEGylated substrates should bind considerably more streptavidin than the controls.
Also, in later stages of the lithographic process, drying of the chip has adverse effects, resulting e.g. in the removal of already bound DNA or in a higher adsorption of DNA to unexposed regions on the substrate.
As mentioned above (section 3.2), the resolution of the lithographic process and the necessary exposure times depend on the experimental setup (objective, light source, etc.). Therefore, in a first step, a wide range of exposure times should be tested.
For gene expression experiments, the experimental setup should be customized to fit the available hardware (microscope, temperature control, etc.). We showed two such implementations, one for short experiments (4 h, section 5), where the system was encapsulated in a box to reduce the evaporation of the expression mix, and one for long observation (section 6), where a microfluidic device enabled a continuous supply of precursor molecules. In both cases, the alignment of DNA brushes on the substrate and compartments in the PDMS represented the trickiest step. We therefore recommend trying this step several times with a dummy substrate before using a patterned one (see also supplementary information, section 6). In the assembly of large systems of distinct gene brushes, long incubation times, precise angular alignment of chambers and brushes over long distances, and contamination by adsorption of DNA to non-patterned regions depending on the quality of passivation, may pose limitations to the size of practically feasible systems.
An alternative to this technique was demonstrated by Karzbrun et al.25, who created compartments in a chip by inductively coupled plasma reactive-ion etching (Bosch process), followed by plasma-enhanced vapor deposition (PECVD) of a silicon dioxide layer. After surface functionalization, patterning and placement of nanoliter DNA droplets by a pipetting robot, the compartments were closed with a PDMS-covered glass slide. This procedure has the advantage that the DNA can be directly immobilized inside the compartments without the danger of contaminating nearby chambers, but it requires additional fabrication steps and laboratory equipment.
The protocol presented here enables researchers working in cell-free synthetic biology to transfer their systems of study from solution-based setups to chip-based reaction containers. Utilizing DNA strand displacement as the central step during development of the resist enables labeling of exposed regions with unique DNA sequences, which provides a facile approach towards biocompatible multistep lithography. Combination with microfluidics allows the observation of gene expression processes over extended periods of time. Apart from the investigation of cell-free gene expression processes under open flow reactor conditions, the same methodology could be used to immobilize and spatially organize other biomolecules on a chip surface. This should prove useful for a wide variety of functional and biophysical studies, such as the fluorescence-based single-molecule experiments demonstrated here.

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><tbody><tr><td>Silicon wafer with 50 nm silicon dioxide (Bephore substrate)</td><td>Siegert Wafer</td><td></td><td>Thickness (&amp;micro;m): 525 &amp;plusmn;25, Diameter (mm): 100</td></tr><tr><td>Silicon wafer (for PDMS master mold)</td><td>Siegert Wafer</td><td></td><td>Thickness (&amp;micro;m): 525 &amp;plusmn;25, Diameter (mm): 76.2 (3&amp;rdquo;)</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;GmbH</td><td></td><td>Photoresist</td></tr><tr><td>mr-Dev 600</td><td>micro resist technology&amp;nbsp;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&amp;nbsp;</td><td></td></tr><tr><td>UV-protection yellow foil</td><td>Lithoprotect (via MicroChemicals)</td><td>Y520E212</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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 by partly bleaching the fluorescent protein and observing the recovery of the fluorescence signal, disregarding the immediate recovery, which does not result from protein expression. After the first bleaching at two hours of expression, the fluorescence intensity recovered quickly and rose above its value before the bleaching. After four and six hours, the fluorescence did not recover to its previous intensity, indicating that without the supply of fresh expression mix, the reaction terminated after approximately 3-4 h.
Coupling to microfluidics: Gene expression can be sustained over longer periods of time by supplying the expression compartments with additional precursor molecules via microfluidics. Figure 7 shows such a system, enabling the expression of YPet over 10 h.
TIRF observation: Bephore can also be applied to study the interaction of fluorescently labeled proteins with a DNA brush at the single molecule level. Especially in a noisy environment, lithography helps to distinguish between specific and unspecific interaction with the brush or the surface, respectively. Figure 8 gives such an example, with fluorescently labeled T7 RNA polymerase binding or adhering preferentially to the DNA brush compared to the surrounding surface.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58634/58634fig1.jpg" /> 
Figure 1: Bephore photolithography.&#160;A. A substrate with a surface of silicon dioxide (SiO2) is covered with a layer of biotinylated polyethylene glycol (PEG-bt), which is biocompatible and allows for the attachment of a photocleavable DNA hairpin via biotin-streptavidin interactions. Here, PC contains sequence domains abcb* and a photocleavable modification (purple star) and is hybridized to the strand PH with domain a*. B. Ultraviolet (UV) illumination cleaves PC, releasing a DNA fragment (cb*) into solution. C-D. The resulting single-stranded region on PC (b) aids as a toehold for the displacement of PH by a fluorescently labeled (green star) DIS strand. E. Also longer, double-stranded DNA (&#34;Template&#34;) can be attached to the patterned surface. Such DNA is prepared by PCR with a primer carrying a DIS sequence at its 5&#39; end, where primer and DIS are separated by a triethylene glycol spacer to keep DIS single-stranded during PCR (see also supplementary information, sections 2-4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58634/58634fig2.jpg" /> 
Figure 2: Sample preparation for photolithography. A. Place a Bephore chip with an alignment mark on a microscopy slide or another chip holder (e.g. the holder in Figure 3B). Apply two-component silicone glue as a hydrophobic barrier along the edges of the chip (steps 3.1.2-3). B. Place the mask onto a mask holder. Here, we removed the iris of the field stop and modified its holder so the mask can be held by small magnets. For precise (angular) alignment in multi-step lithography, ensure that alignment marks on the holder and the mask match (step 3.2.1). C. To navigate on the chip and to align the mask with the alignment marks on the chip, slide in the red filter. For the UV exposure, insert the UV filter (steps 3.2.2-5). Figure reproduced from the Supporting Information of previous work27. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58634/58634fig3.jpg" /> 
Figure 3: Assembly of a holder for the observation of compartmentalized gene expression. A. Parts of the holder (ruler unit: cm). B-C. Top and bottom part of the holder are assembled separately, with the bottom holder carrying a patterned Bephore chip (section 5.1) and the top holder carrying a PDMS chip with compartments (section 5.2). D-F. Chip and PDMS are carefully brought into tight contact, while simultaneously observing and aligning compartments and DNA brushes in a stereoscopic microscope (steps 5.3.1-6). G. Before transferring the holder to an inverted, temperature-controlled microscope, the whole system is encapsulated in an anti-evaporation box (steps 5.3.7-8). Figure reproduced from the Supporting Information of previous work27. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58634/58634fig4.jpg" /> 
Figure 4: Microfluidic setup and sample holder for the compartmentalized gene expression. A. Parts of the sample holder, the PDMS device, the temperature-controlled microscope stage and the Bephore glass slide (ruler unit: cm). B. A microscopy slide carrying the PDMS with compartments is glued to the PDMS holder and exposed to oxygen plasma together with the PDMS in a plasma cleaner. The PDMS is then inserted into the top holder (steps 6.2.2-5). C. The cell-free expression system tube is connected to a pressure controller and placed on ice. The tubing (red dashed line in the inset) for the cell-free expression system is cooled by rubber tubing (blue dashed line) through which ice water is pumped by a peristaltic pump (section 6.1). D. The tubing is connected to the inlet position on the PDMS device. Another piece of tubing is connected to the outlet (steps 6.2.6-7). E. The top holder is placed on the microscope stage and carefully lowered towards the Bephore slide. The PDMS holder can still be moved in the x-y-plane to align the compartments in the PDMS with the gene brush on the Bephore chip. The wingnuts are used to press the PDMS onto the Bephore chip and fix the top holder to the microscope stage (steps 6.4.1-3). F. Cell-free expression system is pumped through the micro-channels in the PDMS and gene expression from DNA brushes can be monitored in epifluorescence microscopy (step 6.4.4). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58634/58634fig5.jpg" /> 
Figure 5: Two-step photolithography. A-B. Fluorescently labeled oligonucleotides (green: ATTO 532; red: Alexa Fluor 647) were consecutively immobilized on a Bephore glass slide via mask projection lithography with exposure times of 45 s. C. Overlay of subfigures A and B, demonstrating the precise alignment of the single exposures. Scale bars: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58634/58634fig6v2.jpg" /> 
Figure 6: Compartmentalized gene expression. A. A DNA brush on a Bephore glass slide (UV exposure time: 2 min), coding for the fluorescent protein YPet was aligned with a compartment on a PDMS chip (see section 5 and Figure 3). B. Gene expression in the chamber with DNA yielded a strong fluorescence signal with a protein concentration gradient forming along a channel, which connected the chamber to the expression mix outside of the PDMS device. The control chamber without immobilized genes remained relatively dark. C. Fluorescence intensity profile over time for both chambers. Every two hours the fluorescent protein was partly bleached (black arrows) to check whether the expression was still active. After 4 h, the fluorescence did not recover to its previous intensity, indicating that expression had terminated. Scale bars: 300 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58634/58634fig7v2.jpg" /> 
Figure 7: Sustained compartmentalized gene expression. A. A DNA brush on a Bephore glass slide (UV exposure time: 2 min), coding for YPet was aligned with a compartment on a PDMS chip. The compartments are connected to a supply channel (white arrow) through which cell-free expression system is pumped (see section 6 and Figure 4). B. The compartment containing the gene brush shows a fluorescence signal from YPet expression (in green) by the cell-free gene expression system. The neighboring compartment without a gene brush remains relatively dark. Fresh components of the cell-free expression system flow through the supply channel and diffuse into the compartments, while waste products are transported away. C. Fluorescence intensity profile over time for both chambers. The fluorescent proteins were partly bleached at different points in time (black arrows) to check whether expression was still active. Due to flow in the supply channel, gene expression was maintained for at least 10 h. (The peak in the red trace marked by the red arrow was caused by an air bubble that temporarily drained the solution from the compartments). Scale bars: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58634/58634fig8.jpg" /> 
Figure 8: Single-molecule studies on Bephore glass slides in total internal reflection fluorescence microscopy (TIRFM). A. Objective-type TIRFM enables single-molecule imaging close to the glass-water interface. We immobilized fluorescently labeled genes (green, ATTO 532, UV exposure time: 2 min) with a T7 promoter along lithographically defined stripes and observed the behavior of T7 RNA polymerase (orange, labeled with Alexa Fluor 647) interacting with the surface. B. Fluorescence image showing two stripes of immobilized genes. C. T7 RNA polymerases attach specifically and non-specifically to the surface (single image, 50 ms exposure time). D. An average image obtained from all frames of a fluorescence video (5,000 frames like in subfigure C) exposes the specific interaction of the RNA polymerase with the DNA brush. Scale bars: 10 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/58634fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/SI_Lithography_masks.pdf" target="_blank">Please click here to download this file</a>.
Supplemental File 2.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58634/SI_Pardatscher_JoVE.docx" target="_blank">Please click here to download this file</a>.

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 ...

functional-surface-immobilization-genes-using-multistep-strand

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.4K Views.  Technical University of Munich. DNA-biochips zijn een aantrekkelijk onderzoeksplatform voor zelfretische biologie, oorspronkelijk ontwikkeld door de BASF-groep, dat de werking van in vitro gencircuits over langere tijd mogelijk maakt. Met dit protocol hopen we deze technologie beschikbaar te maken voor een breder scala aan onderzoekers. Onze methode, beforore lithografie genoemd, vertegenwoordigt een strategie voor de immobilisatie van DNA op basis van de DNA-strengverplaatsingsreactie.De kracht van deze techniek lig...

Video: Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

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.

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 ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

Biology

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Medicine

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

Developmental Biology

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

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 ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

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