Biomod/2014/AFM

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<h1> Team Nanoscooter Braunschweig </h1>

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<td> <font size="+2pt"><u>AFM</u></font> <br><br> <p align="justify"; style="line-height:2em"><font size="3pt"> The atomic force microscopy (AFM) uses a small (almost atomic size) metal tip to sense a surface to determine attributes of the surface like the topography. In these work two different modes, the AC-Mode and the Hyperdrive, of the AFM are used to verify that the DNA origami is folded as expected. <br><br> At the AC-Mode the tip, which is also known as cantilever, is constantly moved over the mica-surface. The cantilever starts gauging the surface at the left bottom of scan-region and ends at the right top. On the back of the cantilever a laser is focused. The distracted laser is registered by a diode (Figure 1). At the Hyperdrive an additional vibration of the cantilever leads to a higher resolution. </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/thumb/0/03/AFM.png/800px-AFM.png" width="50%" height="50%" > <br> </div> <br> <i><font size="3 "><div align="center">Figure 1: Laser distraction of an AFM.</font></i></div> <br><br>


<font size="+1pt">Mica Surface</font> <br> <p align="justify"; style="line-height:2em"><font size="3pt"> Mica is a sheet silicate mineral with a wide range of structural characteristics. On the basis of these structural differences, mica can be divided in classes for example called muscovit, lepidolite or biotit. As a natural occurring mineral mica can be found in sedimentary rock or graniticpegmatites and is mostly mined in the USA, Russia, Finland and China. <br><br> Mica is used for the brilliance in cosmetics and car finish or as electric isolator in heated filaments. Because of its nearly perfect cleavage, mica sheets as ultraflat surfaces can also be used for sample preparation in atomic force microscopy. <br><br>

<div align="center"><img src="http://openwetware.org/images/thumb/c/cd/Mica-Pl%C3%A4ttchen.jpg/734px-Mica-Pl%C3%A4ttchen.jpg" width="30%" height="30%" ></div> <br> <i><font size="3 "><div align="center">Figure 2: Mica plate for sample preparation in atomic force microscopy.</font></i></div> <br><br>

<p align="justify"; style="line-height:2em"><font size="3pt"> The ultraflat surface also allows the movement of DNA origami structures, through diffusion processes. Positive ions like Mg<sup>2+</sup> act as a salt bridge between the negativly charged mica surface and the negatively charged DNA origami. Electrostatic interactions lead to the adsorption of the DNA origami on the mica surface. The intensity of this adsorption can be controlled by the dosage of Mg<sup>2+</sup> ions in the buffer. The addition of monovalent ions like Na<sup>+</sup> can reduce these electrostatic interactions by replacing the salt bridge or the Mg<sup>2+</sup> ions, respectively. This enables a (controllable) movement of the DNA origami on the mica surface. </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/a/a9/Muskovit.png" width="" height="" ></div> <br> <i><font size="3 "><div align="center">Figure 3: Nearly transparent sheets of muscovit.<sup>[1]</sup></font></i></div> <br><br>


<font size="+1pt">Sample preparations</font> <br> <p align="justify"; style="line-height:2em"><font size="3pt"> For AFM imaging we used a NanoWizard<sup>®</sup> 3 ultra AFM (JPK Instruments AG, Berlin, Germany). As the mica surface (Quality V1, Plano GmbH, Wetzlar, Germany) has to be completely (atomically) flat in order to achieve best results, the first step in an AFM experiment is the cleavage of the surface. The negatively charged mica sheet gets then loaded with Mg<sup>2+</sup>-ions by incubating with a solution of 10 mM MgCl<sub>2</sub> for 2 minutes. <br><br>

After washing with MilliQ-water, the surface is dried with air and incubated with the DNA origami solution (diluted or as received after purification) for 2 minutes. The magnesium ions create a bridge between the negatively charged surface and the negatively charged DNA origami. The preparation is completed by washing with TAE buffer (40 mM Tris, 2 mM EDTA, 12.5 mM MgCl<sub>2</sub>). <br> For measurement in AC-Mode BL-AC40TS-C2 cantilevers and in Hyperdrive high dense carbon ultra-short cantilevers (NanoWorld, 330 kHz, and 0.3 N/m) were used.<br><br>For the list of the master mixtures used and staple sequences see <a href="http://openwetware.org/wiki/Biomod/2014/CACGTAATTGACGCCAGCTTTGAAT">here</a>.

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<font size="+1pt">Results</font> <br> <p align="justify"; style="line-height:2em"><font size="3"> By AFM, nanoscale structures can be made visible. This makes it an ideal tool to verify the correct folding of our Nanoscooter. In the following images, 6 µL of the DNA origami solution as received from purification are mixed with 2 µL of buffer and incubated as described above. </font></p> <br><br> <div align="center"><img src="http://openwetware.org/images/thumb/1/17/AFM-Scooter_neu.png/800px-AFM-Scooter_neu.png" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 4: AFM image of the Nanoscooter DNA origami. a) 3D image of several Nanoscooters. b) AFM image of a single Nanoscooter. c) Sketch of Nanoscooter in similar orientation as 1b).</font></i></div> <br>

<p align="justify"; style="line-height:2em"><font size="3pt"> Our AFM scans clearly verify the correct folding: We measure a length of about 70 nm, a width of 40 nm and a height of 10 nm in the front and 20 nm in the back of the DNA origami. This is in very good agreement with the structure designed so we are positive about successful creation of the first DNA origami Nanoscooter! <br><br> Consequently, our next step was to make sure our fuel (H<sub>2</sub>O<sub>2</sub>) does not destroy the DNA origami. For this we used a simpler DNA origami with rectangular shape (70 x 100 nm). </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/a/a3/AFM-NROl.png" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 5: Rectangular DNA origami. a) Sketch of the rectangular DNA origami: It has a size of 70 x 100 nm and consists of 24 helices. b) AFM image of the rectangular DNA origamis.</font></i></div> <br>


<p align="justify"; style="line-height:2em"><font size="3pt"> We took AFM images of the rectangular DNA origami before and after 4 hours incubation in a 30% H<sub>2</sub>O<sub>2</sub> solution. Sample preparation was carried out as described before: 2 µL of the purified DNA origami sample were diluted with 8 µL of buffer (TAE with 12.5 mM MgCl<sub>2</sub>) and incubated for 2 minutes. </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/7/7f/AFM-H2O2.png" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 6: AFM images of rectangular DNA origamis. a) Under normal conditions. b) After 4 hours of incubation in H<sub>2</sub>O<sub>2</sub>.</font></i></div> <br>


<p align="justify"; style="line-height:2em"><font size="3pt"> The comparison between the two AFM images clearly shows that during the incubation time of 4 hours, the DNA origamis stayed intact. We therefore state that our proposed fuel is compatible with our Nanoscooter. <br><br> Before we tried to detect moving Nanoscooter, we had to make sure that our AFM is stable over the period of time needed. We therefore bound our Nanoscooter to the mica surface and took multiple scans over about 40 minutes: </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/7/7b/AFM-Steady.gif" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 7: A time series of 5 AFM images (intevals of 10 min) shows that the Nanoscooters are firmly bound to the mica surface. Anyhow the AFM shows a visible drift to lower left corner.</font></i></div> <br>


<p align="justify"; style="line-height:2em"><font size="3pt"> As can clearly be seen, the Nanoscooters are firmly bound to the surface. Anyhow, this experiment demonstrates a movement to the lower left corner, which is caused by the drift of the AFM. <br><br> In the next step, we tried to visualize the movement of DNA origami on a mica surface.<sup>[2-3]</sup> We used the rectangular DNA origami and changed our buffer from Mg<sup>2+</sup> only to a mixture of Mg<sup>2+</sup> and Na<sup>+</sup>. As sodium is a monovalent ion, it does not bridge the negative charges from the mica and the DNA as effectively as Mg<sup>2+</sup> does. Anyhow, it shields the negatively charged mica surface, so repulsion is prevented: The DNA origami is not firmly bound anymore but does not unbind from the surface – it can freely diffuse on the surface now. <br><br> To proof this concept, we added 250 mM NaCl to our buffer and took consecutive AFM scans: </font></p> <br><br>


<div align="center"><img src="http://openwetware.org/images/thumb/6/68/AFM-Floaten.png/651px-AFM-Floaten.png" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 8: AFM-scans with rectangular DNA origami of the same region within 30 minutes. a) Picture without NaCl-solution. b) Image after 15 minutes of incubation with NaCl. c) After additional 15 minutes.</font></i></div> <br><br>


<p align="justify"; style="line-height:2em"><font size="3pt"> The series shows a movement of the rectangular DNA origamis. Unfortunately, we cannot follow the pathways of single DNA origamis here because of the slow frame rate of about 15 minutes. However, it shows clearly that upon changing the buffer, the DNA origamis can now freely diffuse on the surface. <br><br> Our solution to the limited time resolution is the use of a fluorescently labeled DNA origami and a camera based fluorescence microscope. Such microscopes have a frame rate of a few hundred ms and should therefore be ideal to track the pathways of our Nanoscooter. We also tried to actuate our engine by adding H<sub>2</sub>O<sub>2</sub> to a sample of Nanoscooters functionalized with Pt-nanoparticles. This unfortunately resulted in the visible development of gas bubbles (Figure 9) which destroyed the AFM cantilever and made AFM experiments simply impossible. But nevertheless, the development of gas indicated successful catalysis of our fuel into oxygen and water – as needed for successful propulsion! </font></p><br><br>


<div align="center"><img src="http://openwetware.org/images/0/08/Gasblasen.png" width="" height="" ></div> <br> <i><font size="3"><div align="center">Figure 9: Emerging oxygen gas of the H<sub>2</sub>O<sub>2</sub> decomposition.</font></i></div> <br><br>

<hr style="background-color:#be1e3c;"> <table> <tr><td><font size="2pt"><p align="justify";>[1]<br></font></td> <td><font size="2pt"><i>Freie Universität Berlin</i>, http://www.cms.fu-berlin.de/geo/fb/e-learning/petrograph/magmatite/pruefen/, final request: 23.10.14.</font></td></tr>

<tr><td><font size="2pt"><p align="justify"> [2]<br><br></font></td> <td><font size="2pt">A. Aghebat Rafat, T. Pirzer, M. Scheible, A. Kostina, F. C. Simmel <i>Surface-Assisted Large-Scale Ordering of DNA Origami Tiles</i>, Angewandte Chemie Int. Ed.,<b> 2014</b>, <i>53</i>, 7665-7668.</font></td></tr>

<tr><td><font size="2pt"><p align="justify"> [3]<br><br></font></td> <td><font size="2pt">S. Woo, P. Rothemund: <i>Self-assembly of two-dimensional DNA origami lattices using cation-controlled surface diffusion.</i>, Nature Cmmunications,<b> 2014</b>, <i>5</i>, 4889.</font></td></tr>

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