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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.
ProcedureThe 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 90º) 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 117º, 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/Mg2+ 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 ConclusionThe length of the plasmid on APTES is smaller than DNA on PEG for either deposition time. This shows how restricted the DNA is on APTES when compared to PEG, a neutral monolayer.
The increase in plasmid length on PEG for 21 hours versus the 48 hour deposition may be due to the fact that the optimum deposition time for PEG is 18-24 hours, so for that sample, the monolayer was at its sturdiest.
The DNA on PEG may be straight and directionalized because the plasmid might be imbedded into the surface of the monolayer. An average plasmid height of only around .96 nm supports this.
As for the average length of linearized DNA, the difference in deposition times for APTES (one is 15 minutes and one is 30 minutes) is negligible, as you can see the margins of error are quite similar.
The high plasmid count on APTES is due to the strong adhesion to APTES
The plasmid count on PEG solution is lower due to less adhesion
The higher plasmid DNA count versus the linearized DNA maybe because a higher concentration was used.
Acknowledgements Dr. Thomas Loughran
Christina ArisioBo Gao
Mr. Mark MankowskiUniversity of Notre Dame, Department of
Chemistry and BiochemistryRadiation Laboratory
All images taken in “ tapping mode”
ReferencesFigure 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
OEtEtO
HH
Figure 1: APTES molecule drawn by Dr. Koshala
Sarveswaran
Figure 2: PEG molecule
Repeats 6 to 9 times Me
C17H34
Si
Cl Cl Cl
Figure 3: OTS molecule
The DNAAs 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
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)
Plasmid DNA on OTS
The OTS solution causes the DNA to clump and overlap, making OTS a poor choice for a monolayer.
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
Size of DNA plasmids
257.813
424.81
95.183
0
100
200
300
400
500
PEG (48Hours)
PEG (21Hours)
APTES(15 min.)
Len
gth
in
nan
om
ete
rs
On Silicon
= +/- 8.44 nm
= +/- 56.3 nm
= +/- 25.1 nm
Surface Density of Plasmids
1.121.28
2.3
0
0.5
1
1.5
2
2.5
PEG (48Hours)
PEG (21Hours)
APTES
Nu
mb
er
of
pla
sm
ids p
er
sq
uare
mic
rom
ete
r
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 nmPlain 21 hour PEG with rms of .244
Plain Sample Images
Linearized DNA on APTESLinearized DNA for 30
min. on APTESLinearized DNA with Mg2+
buffer on Mica
On SiliconOn Mica
= +/- 2.3 plasmids
= +/- 2.4 plasmids
= +/- .9 plasmids
Surface Density of Linearized DNA
11 11.52
0.65502468
101214
APTES DNA (30min.) onAPTES
Mg2+Buffer
Nu
mb
er o
f D
NA
per
sq
uar
e m
icro
met
er
On SiliconOn Mica
= +/- 72.4 nm
= +/- 46.3 nm
= +/- 101.2 nm
Size of Linearized DNA
213.492 167.48
478.77
0100200300400500600
APTES DNA (30min.) onAPTES
MG2+buffer
Len
gth
in n
ano
met
ers
Linearized DNA on APTES and with Mg2+ buffer on Mica Images
