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Biological materials investigation by atomic force microscope (AFM)

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Biological materials investigation by atomic force microscope (AFM)

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Content BIOLOGICAL MATERIALS INVESTIGATION BY
ATOMIC FORCE MICROSCOPE (AFM)
by
Ankita Agarwal
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL,
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIO-MEDICAL ENGINEERING)
December 2002
Copyright 2002 Ankita Agarwal
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UMI Number: 1414829
UMI
UMI Microform 1414829
Copyright 2003 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
AKSKrrA JX y X R u iA L .__________________
under the direction o f h & s> i thesis committee, and
approved by all its members, has been presented to and
accepted by the D irector o f Graduate and Professional
Programs, in partial fulfillment o f the requirements fo r the
degree of
M .aste'5 ol Sc-*e-v*c£- wAexivco.1
Director
Thesis Committee
Chair
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Acknowledgements
ii
I would like to take the opportunity to recognize the guidance and support of many
people that made this dissertation into a reality. I am greatly fortuned to know and
work with Professor Aristedes A.G. Requicha, who has guided me to develop my
technical skills and his encouragement and advices throughout the years.
I would also like to thank Professor David Z. D’Argenio for his insightful
discussions that helped me to understand a lot of issues and for his valuable
comments on my dissertation and for serving on my guidance and dissertation
committee. I would also like to thank P rofessor Yamashiro for his interest in my
thesis problem and his feedbacks on my dissertation and for serving on my guidance
and dissertation committee.
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iii
Contents
Acknowledgements ii
List of Figures v
Abstract vii
1. Introduction 1
2. Atomic Force Microscope (AFM) 3
2.1. Background 3
2.2. Operating Principle of AFM 3
2.3. Prerequisites for the observation of native biological systems 6
2.4. Possible substrates for imaging biological material 7
2.4.1. Mica 7
2.4.2. Silicon 8
2.4.3. Glass 8
3. Sample Preparation Techniques 9
3.1. Background 9
3.2. Approach 9
3.2.1. Mica with Poly-L-lysine 10
3.2.1.1. Surface Preparation 10
3.2.1.2. DNA Deposition 10
3.2.1.2.1. Images (Set 1) 13
3.2.1.2.2. Discussion (Set 1) 13
3.2.1.3. Effect of finger pressure 14
3.2.1.3.1. Images (Set 2) 15
3.2.1.3.2. Discussion (Set 2) 16
3.2.1.4. Effect of Time 18
3.2.1.4.1. Images (Set 3) 18
3.2.1.4.2. Discussion (Set 3) 19
3.2.1.4.3. Images (Set 4) 19
3.2.1.4.4. Discussion (Set 4) 19
4. DNA Manipulation 20
4.1. Mechanical nano-manipulation of DNA 20
4.1.1. Discussion 21
4.2. Atomic Force Microscope observation of de-oxy ribose nucleic acid
with restriction enzyme 21
4.3. Digestion Procedure 22
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4.4. Discussion
5. Conclusions
References
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V
List of Figures
1. Operating principle of AFM 4
2. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine. 11
3. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine 12
4. Tapping mode AFM height image of a mica surface.
(a) X ssDNA on poly-L-lysine. 12
(b) Height profile along cross-section along Line 3. 12
(c) Measured heights along Lines 1, 2 and 3 12
5. Tapping mode AFM height image of a mica surface with X ssDNA over 14
poly-L-lysine. ssDNA was deposited while applying increased pressure.
6. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine. ssDNA was deposited while applying increased pressure. 14
7. Tapping mode AFM height image of a mica surface.
(a) Well-extended X ssDNA on poly-L-lysine. 15
(b) Height profile along cross-section along Line 2. 15
(c) Measurement of heights along lines 1 and 2. 15
8. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine. ssDNA was deposited while applying excessive pressure. 16
9. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine. ssDNA was deposited while applying excessive pressure on
the cover slip. 17
10. Tapping mode AFM height image of a mica surface with X ssDNA over
poly-L-lysine. This sample was 7 months old. 18
11. Tapping mode AFM height image of a mica surface.
(a) Well-extended X ssDNA on poly-L-lysine. The DNA was seven
month old. 19
(b) Height profile along cross-section along Line 19
(c) Measurement of height along lines 1. 19
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v i
12. (a) Tapping mode image of ssDNA on mica before pushing with AFM
tip. 20
(b) Tapping mode image of ssDNA on mica after pushing DNA strand
at two positions. 20
13. Tapping mode image of ssDNA after digestion with EcoRl on mica.
(a) Scan size is 1*1 micron 22
(b) Height profile for cross-section along line 2. 22
(c) Heights along lines 1 and 2. 22
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v ii
Abstract
The Atomic Force Microscope (AFM) has the potential to image biological systems
in their native state with molecular resolution. The limitations to the practical
application of the AFM in studying biological systems are sample preparation and
the adhesion of biological macromolecules to the scanning tip. The biomolecule
must be attached to the substrate surface to avoid movement caused by the lateral
forces generated during scanning else the drift experienced by the biomolecule limits
image resolution or precludes imaging altogether. We have analyzed and compared
two sample preparation techniques that ensure reliable deposition of Deoxyribose
Nucleic Acid (DNA) onto mica that is used as a substrate. DNA is an attractive
candidate f or s tudy b ecause o f i ts e xquisite s pecificity i n b ase p airing a nd ~ 1 n m
radius. The effects of pressure and time on DNA samples, mechanical nano
manipulation of DNA and the action of restriction enzymes on surface-bound DNA
have been analyzed experimentally.
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1
CHAPTER - 1
Introduction
Microscopy has laid the foundation for many revolutions in biology since
Leeuwenhoek first glimpsed “animules” through a glass lens. Feynman saw
microscopy of single molecules as the key to the problems of modem molecular
biology [1 ]. In the two decades since the invention of the scanning tunneling
microscope (STM), probe microscopy in general has made a dramatic impact in
fields as diverse as material science, semiconductor physics, biology,
electrochemistry, tribology, biochemistry and medical implant technology. The
reason for the instantaneous acceptance of scanning probe microscopy (SPM) is that
it provides three-dimensional, real-space images of surfaces at high spatial
resolution. These instruments visualize a surface by ‘feeling’ it with a sharp probe
while conventional (far field) microscopes image by collecting radiation transmitted
through, or reflected from the sample. Depending on the particular SPM, the images
can represent physical surface topography, electronic structure, electric or magnetic
fields, or a number of other local properties. In the case of some materials, such as
biological tissue and large organic molecules, SPM allows imaging at unprecedented
levels of resolution without destroying samples, in contrast to other characterization
techniques, including scanning electron microscopy and transmission electron
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2
microscopy, which require relatively high vacuum conditions. The atomic force
microscope (AFM) is now used routinely as a diagnostic probe of biomaterials.
This thesis concentrates on the use of the AFM to investigate biological material,
mainly Deoxyribose Nucleic Acid (DNA). DNA is an attractive candidate for study
because of its exquisite specificity in base pairing and ready availability. Methods for
extending and manipulating DNA for various purposes are reported. Usually,
biological samples must be strongly attached to an atomic flat matrix surface so that
they are immobile in a buffer solution and not swept away during imaging with
AFM. Chemically modified mica and silica have both shown good performance in
our experiments. The possibility of forming molecular patterns by manipulating
DNA molecules mechanically is briefly addressed. Finally, the action of restriction
enzymes on surface-bound DNA is studied.
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3
CHAPTER - 2
The Atomic Force Microscope (AFM)
2.1 Background
The first thing to consider when thinking about how an AFM works is that all
notions of conventional microscope design need to be disregarded, since it has no
lenses of any kind. The AFM images samples by ‘feeling’ rather than by ‘looking’.
This method of imaging can produce an exquisitely detailed picture, not just of the
topography of the picture being studied but also of its texture or material
characteristics, soft or hard, springy or compliant, sticky or slippery. Let us consider
how does an AFM provides topographical information.
2.2 Operating Principle of AFM
The most important part of the AFM is the tip. The tip consists of a micro -
fabricated, extremely sharp spike mounted on the end of a cantilever. The sharpness
of the spike (or tip) determines the resolving power of the instrument. The cantilever
on which it is mounted allows the tip to move up and down as it tracks the sample.
The cantilever typically has a very low force constant enabling the AFM to control
the force between the tip and the sample with great precision. The cantilever-tip
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assembly is generally made of silicon or silicon-nitride, these materials being hard,
wear-resistant and ideally suited to micro-fabrication.
Laser diode
j
.
End face of the photo
diode detector
Photo diode
detector
Piezo tube scanner
for xyz motion
Feedback
electronics
image
T
Deflection
vibration amplitude
phase difference
Figure 1: Operating Principle of AFM
The second important feature of AFM is the scanning mechanism. This mechanism
refers t o t he m eans o f p recisely p ositioning t he tip r elative t o t he s ample s urface.
This is done by means of a piezoelectric transducer. If a potential difference is
applied to the piezoelectric ceramic, it expands. This motion is incredibly
reproducible and sensitive. With a clean electric signal, the piezoelectric ceramic can
be made to move with an accuracy of atomic dimensions, providing AFM with the
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5
capability to accurately position the sample or the tip. In the AFM used in our
experiments, the sample is mounted atop the piezoelectric transducer thus controlling
the motion of the sample in three orthogonal directions, x, y and z. The sample can
now be positioned in extremely close proximity to the AFM tip using the z channel
and then line scanned using the x and y channels in order to build up an image of a
selected area on the sample surface.
The final important feature of the AFM is the detection mechanism that refers to
monitoring the motion of the tip as it traverses the sample. A laser beam is focused
onto the end of the cantilever, preferably directly over the tip, and then reflected off
onto a photodiode detector that has been split into four segments. During scanning,
as the tip moves in response to the sample topography the angle of reflection of the
laser changes. The laser spot falling on the photodiode detector moves producing
changes in intensity in each of its quadrants, which produces an electrical signal
quantifying the motion of the tip. When the sample is scanned the surface
topography of the sample causes the cantilever to deflect as the force between the tip
and sample changes. System electronics operate a feedback circuit that accesses
different signals to control tip oscillation and position depending on the imaging
mode. The x, y and z displacements of the piezoelectric scanner are recorded and
displayed to produce an image of the sample surface.
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2.3 Prerequisites for the observation of native biological systems with
the atomic force microscope
Atomic force microscopy is a powerful tool for imaging the structure of biological
specimen in their physiological environment. Sample preparation and an appropriate
imaging environment are crucial for imaging successfully using an atomic force
microscope. The most basic and important requirement of the sample preparation is
to fix the specimens firmly to a supporting surface so that the position of the probe
with respect to the specimen can be defined with high precision during image
acquisition. Also, the lateral resolution using atomic force microscope is intrinsically
dependent on the sample, the finite size and shape of the tip and the compression due
to probe force [7 ]. Chemically appropriate and atomically flat substrates are important
for getting sub-nanometer resolution through the atomic force microscope. The
atomically flat substrates are necessary as they allow the differentiation between the
topography of the adsorbed bio-macromolecules, especially single molecules, from
that of the solid support. Thus, the number of suitable substrates is limited due to the
requirements of flatness and biocompatibility.
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2.4 Possible substrates for imaging biological material using the atomic
force microscope
2.4.1 Mica
The most commonly used substrate for AFM imaging is mica. The advantages of
using mica are:
1. Layered crystal structure
2. Relatively inert chemically
3. Ease of getting atomic flat surfaces over several microns
We have used muscovite mica in our experiments. A central layer of Al(OH)2 links
tetrahedral sheets of alumino- silicate. A layer of hexagonally coordinated potassium
ions balances the net negative charge of the basal oxygen between these double
layers. This layer is disrupted by cleavage (by a scotch tape). The resultant basal
plane is highly negatively charged (0.015 electrons per surface unit cell)[5 ].
Mica was obtained from Ted Pella, Inc., Redding, CA. The thickness of mica sheets
was 0.23 mm and they were muscovite, die cut, ruby, flat, very clean and with
minimum of inclusions and air bubbles.
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2.4.2 Silicon
Silicon is also widely used as a substrate for the atomic force microscope studies.
Silicon chips are opaque, of low electrical resistance and are highly smooth. The
advantages of using silicon are
1. Highly smooth surface
2. Chemically inert
3. Good substrates for growing or mounting cells.
Silicon for our experiments was obtained from Ted Pella, Inc., Redding, CA. The 4-
inch silicon wafer was precut on 1-0-0 axis and the chips were separated as needed.
2.4.3 Glass
Glass cover slips and slides are useful as transparent specimen support for the atomic
force microscope imaging. The amorphous surface can be chemically or physically
modified to manipulate its adsorption or chemisorption properties.
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CHAPTER - 3
Sample preparation techniques
3.1 Background
Imaging applications by biological atomic force microscopy can be divided into two
categories namely, imaging of stationary biomolecules and imaging of biomolecules
in motion [2 ]. This section highlights some of the techniques of imaging stationary
biomolecules as applied to de-oxy ribose nucleic acid (DNA). DNA is an attractive
candidate for study because of its exquisite specificity in base pairing, ~ 1 nm radius
and ready availability. We have used both plasmid and phage lambda DNA in our
experiments. The super coiled plasmid DNA (pUC 19) was obtained from
Worthington Biochemical Corporation. The Lambda DNA was obtained from New
England Biolabs Inc., Beverly, MA. Lambda DNA is 48,502 base pairs in length.
Both pUC 19 and Lambda DNA are isolated from E.coli.
3.2 Approach
Since DNA is negatively charged for the proper electrostatic binding of DNA to the
substrate surface, it is necessary that the surface be positively charged. We thus try to
coat the substrate surface with some chemical so as to make the surface positively
charged.
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The various sample preparation techniques are listed next.
3.2.1. Mica with Poly-L-lysine
3.2.1.1 Surface Preparation
1-ppm solution of poly-L-lysine in water was made from a 0.1% (w/v) aqueous
solution containing preservatives (obtained from Sigma Aldrich). 0.5-inch diameter
circular mica was cleaved using a scotch tape. For cleaving, care was taken that the
peeled off layer (which comes out, sticking on the scotch tape) was free of holes,
cracks and flakes and was visually smooth. This ensures that no liquid will penetrate
the inner mica layers during sample preparation. If some liquid enters the inner mica
layers, there is a high chance that the upper layer will come off while drying with dry
compressed nitrogen. After cleavage the mica surface is rendered hydrophilic. 25
micro liter of 1-ppm poly-L-lysine was deposited onto freshly cleaved mica exactly
for five minutes. The sample was then rinsed in a stream of de-ionized water for
approximately 20 seconds and dried under a stream of dry compressed nitrogen. As a
result of poly-L-lysine deposition, the surface becomes hydrophobic.
3.2.1.2 DNA Deposition
Lambda DNA as obtained was double-stranded. It was diluted to 5 ng/pl in 10 mM
Tris, 1 mM EDTA, pH 8.0 (TE Buffer)(10X TAE Buffer was obtained from
Invitrogen Life Technologies). To make single stranded DNA (ssDNA) we heat the
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11
diluted DNA solution to 95° C for 4 minutes. This denatures double stranded DNA
(dsDNA) converting it to ssdNA. 1 |il drop of ssDNA was deposited on the treated
mica surface [8 ]. A glass cover slip (made of superior quality borosilicate glass,
obtained from Ted Pella Inc., Redding, CA) that had been sonicated for 10 minutes
each in acetone, isopropyl alcohol and methanol (anhydrous grade solvents) in order
to remove any organic contamination was put over the DNA droplet gently. Extra
pressure was applied slowly with a finger over the glass cover slip in a linear
direction for approximately 7 seconds. This made the DNA strands straighter (or in
other words extended) [4 ]. After 3 minutes, the cover slip was rinsed off using de
ionized water and the sample was dried with dry compressed nitrogen prior to
imaging.
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i m a g e o f a m i c a s u r f a c e w i t h X s s D N A
o v e r p o l y - L - l y s i n e .
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F i g u r e 3 . T a p p i n g m o d e A F M h e i g h t i m a g e
o f a m i c a s u r f a c e w i t h X s s D N A o v e r p o l y -
L - l y s i n e .
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1 me 1 403.4 nm $311 0% 0.82 m r $80.0% 0.6? r 3 8H S0 nm 0.5343 n r r j h 0 0007484 f ir
F i g u r e 4 . T a p p i n g m o d e A F M h e i g h t i m a g e o f a m i c a s u r f a c e , ( a )
X s s D N A o n p o l y - L - l y s i n e . ( b ) H e i g h t p r o f i l e a l o n g c r o s s - s e c t i o n
a l o n g L i n e 3 . ( c ) M e a s u r e d h e i g h t s a l o n g L i n e s 1 , 2 a n d 3
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13
3.2.1.2.1. Images (Set 1)
The above images were obtained in air using tapping mode Auto Probe CP atomic
probe microscope (Park Scientific Instruments). The scan rate was 1 Hz. Figures 2, 3
and 4 show the same area on the sample but with different scan sizes. Figure 4 also
shows a line scan and height measurements along DNA strands.
3.2.1.2.2. Discussion (Set 1)
Many DNA strands are clearly visible in Set 1 images. We could see many blobs and
holes on our images. The blobs are contributed by the secondary structure of DNA
and impurities present in the air. The observed height of strands is 0.7 nm as
compared to the literature value of 0.4 nm. This inconsistency may be attributed to
different imaging conditions and parameters. Also, we did not calibrate the
instrument for accurate, absolute z measurements. Figure 2 has spurious blotches all
over the image, presumably due to salt deposits. The higher features in figure 4a near
the crossover points are probably due to the super coiling of DNA.
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14
3.2.1.3. Effect of finger pressure
I
I
B ajHMBBB
h h h h IS f®
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1 1 1®
lim p
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Figure 5. Tapping mode AFM height image of a mica
surface with X ssDNA over poly-L-lysine. ssDNA was
deposited while applying increased pressure.
I ? » 2 s
^ I
illlll
H M
ML
0.00 1.00 2.00 pm
Figure 6. Tapping mode AFM height image of a mica
surface with X , ssDNA over poly-L-lysine. ssDNA was
deposited while applying increased pressure.
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15
MMHI
Ra: 0.8430 nm
Rq: 0.2224 nm
H eight Profile
Rp: 0,8266 nm
Rv: -0.2705 nm
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1.001 pm 330.0% 0.87 mr 380.0% 0.69 nrr ) 8266 nm O 7765 nrv h: 0.0008072 pij
Figure 7. Tapping mode AFM height image of a mica surface, (a)
Well-extended X ssDNA on poly-L-lysine. (b) Height profile
along cross-section along Line 2. (c) Measurement of heights
along lines 1 and 2.
3.2.1.3.1. Images (Set 2)
Figure 5 shows a representative image of a sample prepared using the same
technique as in Set 1 images but with increased finger pressure. Figures 5, 6 and 7 all
refer to the same area on the sample but different scan sizes. The scan rate used was
1 Hz. Figure 7 also shows a cross-section and height measurements along DNA
strands.
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16
3.2.1.3.2. Discussion (Set 2)
We can vaguely notice a general alignment of DNA strands in figure 5. This is due to
the increased linear pressure during DNA deposition which forces the DNA strands
to become well extended and oriented in the direction of applied pressure. In this set
of images, not only do we see blotches (presumably due to salt deposition) but also
some holes. The holes are due to inconsistencies in the uppermost mica surface and
poly-L-lysine layer. Also evident is the impurity on the sample (near the lower left
corner in figure 6). This impurity might be due to a variety of reasons. It could be
due to suspended particles in the air or precipitation of salt. In figure 7a we have
captured an almost linear segment of DNA. The height measurement of this strand is
0.3 nm, which matches with the theoretical value of 0.3 - 0.44 nm.
m m S m m
Figure 8. Tapping mode AFM height image of a mica
surface with X ssDNA over poly-L-lysine. ssDNA was
deposited while applying excessive pressure.
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17
I -------------------1 -------------------1 ---------
0 400 800 urn
Figure 9. Tapping mode AFM height image of a mica surface
with X ssDNA over poly-L-lysine. ssDNA was deposited while
applying excessive pressure on the cover slip.
We also show that applying excessive pressure can destroy the DNA strands, as it is
a soft bio-molecule. This is evident from figure 8 and figure 9. Figure 9 is a zoom on
the area of the sample shown in figure 8. It is very clear from figure 9 that DNA
strands have been damaged and fragmented due to excessive pressure. Since the fact
that DNA has been fragmented cannot be appreciated visually, we zoomed in on the
area to as low as 0.4*0.4 micron and it did show that the strand in figure 9 was made
up of small fragments of DNA.
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18
3.2.1.4. Effect of Time
We have tried to observe the stability of DNA over time both when stored at -4°C in
buffer after denaturing and when attached to the surface.
3.2.1.4.1. Images (Set 3)
Figure 10. Tapping mode AFM height image of
a mica surface with X ssDNA over poly-L-lysine.
This sample was 7 months old.
To judge the effect of time on surface-bound DNA, we stored our sample at room
temperature under ambient conditions and imaged it after 7 months. Figure 10 shows
the images obtained from the same sample as in Set 1 images.
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19
3.2.1.4.2. Discussion (Set 3)
By comparing Set 3 images to Set 1 images we don’t find any differences in general.
We can thus conclude that surface-bound DNA is fairly stable at room temperature
for extended periods of time.
3.2.1.4.1b. Images (Set 4)
To study the effect of time on denatured DNA stored at -4° C in buffer, we used
seven-month-old denatured DNA. Before depositing it on mica, we heated it to 65° C
for 10 minutes to linearize it. Figures 11 shows a sample prepared using old DNA.
■ H R
Ra. 0.9494 nm
Rq: 0.4350 cm
Rp: 1.544 nm
Rv: -0.81 $9 nm
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F i g u r e 1 1 . T a p p i n g m o d e A F M h e i g h t i m a g e o f a m i c a s u r f a c e , ( a ) W e l l - e x t e n d e d X
s s D N A o n p o l y - L - l y s i n e . T h e D N A w a s s e v e n m o n t h o l d . ( b ) H e i g h t p r o f i l e a l o n g
c r o s s - s e c t i o n a l o n g L i n e 1 . ( c ) M e a s u r e m e n t o f h e i g h t a l o n g l i n e s 1 .
3.2.1.4.2b. Discussion (Set 4)
As with the images in the previous section, there does not seem to be a difference in
the images in Set 4 and Set 1. Thus we can conclude that DNA (in buffer or surface-
bound) is very stable under certain temperature conditions (-4° C for DNA in buffer
and room temperature for surface-bound DNA).
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20
CHAPTER - 4
DNA Manipulation and Imaging
4.1. Mechanical nano-manipulation of DNA
In the previous chapter we demonstrated how ssDNA can be reliably deposited on
mica. We also performed sample manipulations on the surface. We tried to cut DNA
by pushing with the AFM tip. Figures 12 and 13 show a mica sample before and
after pushing.
Figure 12. (a) Tapping mode image of ssDNA on
mica before pushing with AFM tip. (b) Tapping
mode image of ssDNA on mica after pushing DNA
strand at two positions.
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21
4.1.1. Discussion
As is evident from the images above, it is easy to push and manipulate DNA
mechanically using AFM tip. In the above images when we tried to cut DNA strand
at the lower arrow position, we could not cut it because the force applied was
insufficient. We were able to make a clean cut at another location with slightly
higher force. Some spherical aggregation of material at the two ends of the broken
DNA is often observed. DNA folding during the cutting possibly causes these
clumps of shorter DNA segments at the end of DNA strand.
4.2. Atomic Force Microscope observation of de-oxy ribose nucleic acid
with restriction enzyme
Restriction enzymes have been used extensively in gene manipulation to define and
isolate specific DNA fragments. The enzyme recognizes specific sequence sites of
double stranded DNA and cleaves the phophodiester bonds [3 ]. AFM is useful for the
direct observation of biological materials as AFM images can give us direct
information from analysis by electrophoresis.
We tried to digest Lambda DNA using EcoRl.
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22
4.2.1. Digestion Procedure
EcoRl (obtained from New England Biolabs) has a concentration of 100,000 units
per milliliter. This enzyme is diluted to a concentration of 0.1 units per milliliter in
diluent buffer C. Take 44.5 micro liter of de-ionized water in a vial. Add to it 5
micro liter 10X NE buffer EcoRl.To the reaction mixture add 0.5 micro liter of
diluted enzyme. Put this 50 micro liter volume of reaction mixture on a DNA sample
prepared over poly-L-lysine on mica. Incubate the sample for an hour at 37 °C. Rinse
the sample with de-ionized water for approximately 20 seconds. Dry using
compressed nitrogen prior to imaging.
Figure 13 shows the image of DNA after incubation with EcoRl.
w B cB8 b « I |
1 1 1
*
R.a: 4.877 am
Rq: 2.097 nm
H eight P ro file
nm
9 000H
>
1 0 0 0 -
V
A
400
Rp: 5.066 nm
Rv: -4.197nm
v ^ v v
800 nm
I B B
'' 1 i
Bear.ng i M i |
r n t i i
i i B i p
i.Uf;. pm b.'1 ! i nm j ''g c tu j* j.uy mn | y.; wn ; "■■■c tut! tf.wouiv pin
1 mp ? 1.010 pm §30.0% 6.03 nrrliB0.0% 3 06 nn(5.066 nm |4 .1 3 7 nm h: O.OOMMOlu1
Figure 13. Tapping mode image of XDNA after digestion with EcoRl on mica,
(a) Scan size is 1*1 micron, (b) Height profile for cross-section along line 2. (c)
Heights along lines 1 and 2.
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2 3
4.2.2. Discussion
We can see from figure 13 that the DNA is no longer easily visible. Most of the area
on the sample is filled by enzymatic residue. A close visual inspection in the area
around Line 1 suggests that the enzyme has indeed cut DNA but it is not clearly
visible due to the ‘curtain’ of enzymatic residue.
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24
CHAPTER - 5
Conclusions
We analyzed and tested a sample preparation technique for imaging single stranded
DNA and studied the effects of pressure and time on surface-bound DNA and DNA
in solution. We also showed that it is possible to mechanically push and cut DNA
using an AFM probe. We tried to digest DNA with EcoRl to see if the restriction
enzyme would bind to DNA even when the DNA is surface bound. The resulting
images are difficult to interpret due to the presence of enzymatic residue. Future
work might involve trying to mechanically push away the residue from the sample
and confirming if EcoRl binds to DNA. That would be a step further in the direction
of site-recognition.
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2 5
References
1 . Feynman, R.P., J. Micro mechanical Systems 1(1992), 60, ‘There’s plenty of
room at the bottom’
2. Hansma, Helen G., J. Vac. Sci. Technol. B 14(2) (1996), 1390-1394,’Atomic
force microscopy of biomolecules’
3. Nakamura, T. et al., J. Vac. Sci. Technol. B 17(2)(1999), 288-293, ‘Atomic
force microscope observation of plasmid deoxyribose nucleic acid with
restriction enzyme’
4. Ouyang, Zhen-qian; Hu, Jun et al., J. Vac. Sci. Technol. B 15(4) (1997),
1385-1387,’Molecular patterns by manipulating DNA molecules’
5. Pashley, R. M., J. Colloid Interface Science 83(1981), 531-546,’ DLVO and
hydration forces between mica surfaces in Li+, Na+, K+ and Cs+ electrolyte
solutions: A correlation of double-layer and hydration forces with surface
cation exchange properties’
6. “Scanning Probe microscopy and spectroscopy, Theory, Techniques and
Applications”, Second edition, Edited by Dawn Bonnell
7. Wagner Peter, FEBS (Federation of European Biochemical Societies) Letters
430(1998), 112-115,’Immobilization strategies for biological scanning probe
microscopy’
8. Woolley, Adam T.; Kelly, Ryan T„ Nanoletters vol 1(7) (2001), 345-
348,’Deposition and characterization of extended single-stranded DNA
molecules on surfaces’
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.

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Creator Agarwal, Ankita (author)

Core Title Biological materials investigation by atomic force microscope (AFM)

School Graduate School

Degree Master of Science

Degree Program Biomedical Engineering

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Computer Science,engineering, biomedical,OAI-PMH Harvest

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Advisor D'Argenio, David (committee chair), Requicha, Aristides (committee member), Yamashiro, Stanley (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-297220

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