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Continued AFM Exploration of Attachment Strategies for DNA to Silicon By Presenter* , Koshala Sarveswaran, and Marya Lieberman School Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 Introduction DNA is self-assembling (meaning the strands will connect through base pairs) and can be manipulated in predictable ways to create two dimensional and three dimensional structures. For these reasons, nanostructures can be made using DNA. In this case, plasmid DNA. One such purpose is for DNA to act as scaffolding for circuitry in the field of nanotechnology. To be able to do this, however, a semiconductor is needed to act as a substrate for the DNA molecule. In my experiment, silicon is used to act as the semiconductor. The problem is that the silicon dioxide surface is negatively charged, as is the sugar-phosphate backbone of the DNA, so they repel each other. A positive adhesive is needed in order to attach the plasmid to the surface. In this case, the liquid solution called APTES (aminopropyl triethoxysilane) is normally used, but the APTES binds the DNA too strongly to the silicon surface, causing the DNA to appear too coiled and constricted when seen on the atomic force microscope (AFM). This was the topic of my research in 2006. In the summer of 2007, I tested other neutral molecules such as PEG (Polyethylene glycol) and OTS (octadecyltrichlorosilane) which are used to loosen the strong positivity of APTES. To help see the DNA better on surfaces, restriction enzymes were used to cleave the DNA plasmid, causing it to linearize. By using restriction enzymes to cut the DNA plasmid, the DNA would appear longer, allowing me to see the effects of the monolayer on the DNA in greater detail, and hopefully the linearization would help the DNA relax. Below are the molecular structures of APTES, PEG, and OTS. The AFM The AFM or atomic force microscope was the main tool in the process of imaging and analyzing my samples of DNA on the monolayer and silicon. A cantilever holder and tip is put in the microscope itself above the sample. The tip scans the sample back and forth, moving up and down in accordance with the surface texture. A laser is beamed on the tip, and the laser is reflected into a photodiode as seen in figure 6, which interprets the movement of the laser on to the computer which displays the AFM image. Figure 7 illustrates how the AFM looks as a whole. Procedure The first step of the project is cut 1 cm by 1 cm squares of silicon and clean the wafers by washing them in different solutions such as toluene after which they are rinsed in water and dried. After the cleaning process, different solutions are deposited on the silicon wafers for varying amounts of time. In the case of the 1% APTES, the solution is deposited on the wafer for 15 minutes, then washed with water and dried with nitrogen gas. 1 For the 1% PEG with dry toluene, two deposition times of 21 hours and 48 hours were used. 2 When the .2% OTS solution was used, I went to the dry box (an oxygen free box, as seen in figure 9) in order to deposit this solution. Here, I left the silicon wafers submerged in the OTS for at least 3.5 days. After taking the silicon out, I rinsed the samples in chloroform and ethanol, then dried the samples in nitrogen gas. 3 In all cases, the silicon wafers were completely submerged in the solutions. After the monolayers were deposited, I used contact angles to verify that the monolayers indeed grew on the silicon surface. First, I used the contact angle instrument, which measures the contact angles of water bubbles that are dropped on the surface of the silicon. Because OTS is known to be hydrophobic (the angle is greater than 90 º) while plain silicon, APTES, and PEG are hydrophobic (the contact angle is less than 90º), I took the angle measurements, in the manner shown in figure 10, of the monolayers to make sure that they indeed grew on the surface. As it turned out, the contact angles of OTS, APTES, and PEG were around 117 º, 61º, and 36º, respectively. For the other component of the project, I used linearized DNA. To do this, circular (1 microgram/microliter) DNA is mixed with Buffer solution, BSA, the kpn1 restriction enzyme and water. This mixture goes through a long process of time in the centrifuge and freezer ( Promega ). The final concentration after mixing it with TAE/Mg 2+ buffer is .025 micrograms/microliter. This portion of the procedure was completed by Dr. Koshala Sarveswaran. After the DNA deposition, the silicon wafer is again washed with water and dried with nitrogen gas. When this is completed the sample is ready to be brought to the atomic force microscope to be imaged and analyzed. Discussion and Conclusion ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Acknowledgements Dr. Thomas Loughran Christina Arisio Bo Gao Mr. Mark Mankowski University of Notre Dame, Department of Chemistry and Biochemistry Radiation Laboratory All images taken in “ tapping mode” Figure 8 displays how an image is seen on the computer using the Nanoscope ® program. Displayed is a section analysis. Figure 8: An example of an AFM section analysis Figure 9: The Dry Box Figure 10: Diagram of Contact Angling Figure 11: DNA deposition on APTES (Drawn by Dr. Koshala Sarveswaran) ,[object Object],After the monolayer is grown on the surface, DNA plasmids (.01 micrograms/microliter concentration) were deposited as diagramed in figure 11, for either 15 minutes, or 30 minutes, depending on the experiment done. Results References Figure 4: The Biology Corner . 11 Mar. 2008 <http://www.biologycorner.com/resources/DNA-colored.gif>. Figure 6: "AFM (Atomic Force Microscope)." Soft Matter. University of Greifswald. 11 Mar. 2008 <http://www3.physik.uni-greifswald.de/method/afm/eafm.htm>. Figure 7: "Schneider Research Group." Carnegie Mellon University. 11 Mar. 2008 <http://www.andrew.cmu.edu/user/jamess3/JWSfac.htm>. Figure 10: Quéré, David. "Surface Chemistry: Fakir Droplets." Nature Materials 1 (2002): 14-15. 11 Mar. 2008 <http://www.nature.com/nmat/journal/v1/n1/fig_tab/nmat715_F1.html>. 1. Sarveswaran, Koshala, Hu, Wenchuang, Huber, Paul W, Bernstein, Gary H, and Lieberman, Marya. "Deposition of DNA Rafts on Cationic SAMs on Silicon." Langmuir 26 (2006): 11279-11283. 2. Papra, Alexander, Nikolaj Gadegaard, and Niels Larsen. “Characterization of Ultrathin Poly(ethylene glycol) Monolayers on Silicon Substrates.” Langmuir 17 (2001): 1457-1460. 3. Wang, Yuliang, and Lieberman, Marya. "Growth of Ultrasmooth Octadecyltrichlorosilane." Langmuir 19 (2003): 1159-1167. N OEt Si OEt EtO H H Figure 1: APTES molecule drawn by Dr. Koshala Sarveswaran Figure 2: PEG molecule Repeats 6 to 9 times Me C 17 H 34 Si Cl Cl Cl Figure 3: OTS molecule The DNA As is shown in figure 1, the DNA double helix held together by bonding base pairs which are a part of a nucleotide of which DNA is made. As a part of my project, DNA plasmids (circular DNA) were used because they are easy to acquire and easy to use. For linearizing DNA, the plasmid was digested using restriction enzymes, in this case, the kpn1 enzyme. Figure 4: DNA structure Kpn1 enzyme restriction site Puc19 DNA plasmid Figure 5: Plasmid DNA with labeled enzyme restriction site Figure 6: AFM Diagram Figure 7: Atomic force microscope Plasmid DNA on OTS On Silicon = +/- 8.44 nm = +/- 56.3 nm = +/- 25.1 nm On Silicon (3.9) = +/- .1 plasmids = +/- .78 plasmids = +/- 1.6 plasmids Plasmid DNA on 48 hour PEG Plasmid DNA on 21 hour PEG Plasmid DNA on APTES Plasmid DNA on PEG and APTES Images Plain APTES with an rms of .14 nm Plain OTS with an rms of .15 nm Plain 21 hour PEG with rms of .244 Plain Sample Images Linearized DNA on APTES Linearized DNA for 30 min. on APTES Linearized DNA with Mg 2+ buffer on Mica On Silicon On Mica = +/- 2.3 plasmids = +/- 2.4 plasmids = +/- .9 plasmids Surface Density of Linearized DNA 11 11.52 0.655 0 2 4 6 8 10 12 14 APTES DNA (30 min.) on APTES Mg2+ Buffer Number of DNA per square micrometer On Silicon On Mica = +/- 72.4 nm = +/- 46.3 nm = +/- 101.2 nm Size of Linearized DNA 213.492 167.48 478.77 0 100 200 300 400 500 600 APTES DNA (30 min.) on APTES MG2+ buffer Length in nanometers Linearized DNA on APTES and with Mg 2+ buffer on Mica Images

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