BY SUM-FREQUENCY GENERATION SPECTROSCOPY...much like talking with Niels, Arend during some drinks...
Transcript of BY SUM-FREQUENCY GENERATION SPECTROSCOPY...much like talking with Niels, Arend during some drinks...
MOLECULAR VIEW OF DNA-LIPID INTERACTION
BY SUM-FREQUENCY GENERATION SPECTROSCOPY
Thesis for graduation as Master of Science in Physics
May 2009
NGÔ THị MINH THÙY
Supervised by Prof. Dr. Mischa Bonn
UNIVERSITY OF AMSTERDAM
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MOLECULAR VIEW OF DNA-LIPID INTERACTION
BY SUM-FREQUENCY GENERATION SPECTROSCOPY
By
NGO THI MINH THUY B.S. HANOI UNIVERSITY OF TECHNOLOGY 2007
DISSERTATION Submitted in partial satisfaction of the requirements for the degree of
MASTER OF SCIENCE in
PHYSICS-CONDENSED MATTER SCIENCE
of the
UNIVERSITY OF AMSTERDAM
Approved: Prof. Dr. Mischa Bonn Prof. Dr. Wybren-Jan Buma Dr. Kramer R. Campen Ms. Maria Sovago
Amsterdam, 31st May 2009
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For my love
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ABSTRACT Understanding and controlling the interaction between DNA and lipids is strongly desirable
to optimize the process of gene transfection to cells by mean of lipids. Ideally, one would like
to obtain quantitative information on the molecular interactions in a label-free manner. With
this goal in mind, we apply Vibrational Sum Frequency (VSFG) Spectroscopy and surface
pressure-area isotherms to non-invasively characterize the binding of DNA to self-assembled
monolayers of cationic and zwitterionic lipids. VSFG allows us to record the vibrational
spectrum of specifically the interfacial molecules in this system, the lipids and the water
molecules, and allows us to record the effect of the interaction of DNA on those molecules.
The presence of DNA at the lipid interface can be inferred from changes in the intensity
associated with oriented, interfacial water. The VSFG spectra reveal that the driving force for
the DNA-lipid complexes formation is coulombic: the poly-anion DNA binds readily to
cationic and slightly interacts with zwitterionic lipids. Interestingly, we found that VSFG
signal of water does not solely depend on the surface electric field but also on interfacial
water density and the dielectric constant at the water surface. Upon the formation of the
DNA-lipid surface complex, the decrease of these quantities cause the reduction of VSFG
signal of water.
In addition, we have elucidated details about the restructuring of lipid and water molecules
at the interface. The DNA-induced changes in Π-A isotherms curves and lipid molecular
order suggest that the lipids monolayer are more condensed in the presence of DNA.
Furthermore, changes of water hydrogen bonding network was observed. A weak H-bond
network of water molecules confined between the lipid monolayer and the DNA layer gives
new OD stretch peaks in the VSFG spectra.
From this study, we suggest that diC14-amidine is a possible vector for gene therapy.
Moreover, we have raised a few problems and proposed several approaches for next studies:
determining change of the medium properties at the surface upon the binding of DNA to the
lipid monolayer, directly probing the appearance of DNA at the surface and controlling the
DNA-lipid interaction by ions.
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ACKNOWLEDGEMENT This study is part of a Master (MSc) program in Physics-Condensed Matter Science at the University of Amsterdam. I have been awarded a Huygens Scholarships (HSP) by Dutch Ministry of Education, Culture and Science to follow this MSc program for two years. This Master’s thesis is the result of a research project in the Biosurface Spectroscopy Group led by Prof. Dr. Mischa Bonn at the FOM Institute for Atomic and Molecular Physics (AMOLF) in Amsterdam. The research project is part of the research program of the “Stichting voor Fundamenteel Onderzoek der Materie (FOM)”, which is financially supported by the Nederlandse organisatie voor Wetenschappelijk (NWO). Foremost, I would like to thank Mischa, my supervisor for giving me an opportunity to join the BaBo twin-group. Being part of this ambitious and warmly group, I have not only gained essential experiences for a scientist but also had a feeling of being in a family. I sincerely share my respect to the way that Mischa decorates his office with pictures drawn by his daughter. This convinces me that a scientist has enough time for his/her family, inspiring me to go further in a scientific career. Last but not least, I am definitely in debt to Mischa with his very sharp advices and quick help at any time needed. I highly appreciate Maria and Kramer, my daily supervisors for their infinite help. Maria gave me expertise in aligning VSFG setup and troubleshooting many difficulties in analysing the data. I very much enjoyed critical discussions with Kramer about both theoretical and practical problems. I am grateful to prof. Ruysschaert from the Université Libre de Bruxelles, Belgium, for providing the Amidine lipids. Many thanks for Avi, Han-Kwang, Susumu and Ellen for their useful instruction and discussion. I was very happy in the time doing experiments and joining courses together with Steven and Ruben. Joep, Ronald and Maaike, thanks for sharing the Ti:Saphire laser with me. I wish to thank the BaBo lunch club for joined lunches. Furthermore, I would like to thank every body with whom I shared experiences in life. I very much like talking with Niels, Arend during some drinks and meals. I would like to acknowledge thầy Huy, thầy Chiến, thầy Tụng, cô Liên, cô Hải for their encouragements. Thank my classmates, Adrian, Maria and Rosanne. Many thanks for the Vietnamese community in the Netherlands for making a home feeling and helping me during the time I lived abroad. Especially, I am thankful to every one who shared house with me: Shai, Kierann, chị Vân Anh and chị Thảo, Philipp and Edwin. Thank Dịu and my friends for our trips in Paris and Amsterdam. Last but not least, I am grateful to my family and my boy friend for their loving support. Grandparents, Mum, Dad, sisters and my honey, your loves are my strongest motivation and belief.
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CONTENTS 1. Introduction…………………………………………………………………………
1.1. Lipid based gene therapy……………………………………………………. 1.1.1. General description……………………………………………………. 1.1.2. Molecular structure of DNA lipids…………………………………... 1.1.3. DNA-lipid complexes…………………………………………………. 1.1.4. Biophysical studies……………………………………………………..
1.2. Langmuir monolayer………………………………………………………… 1.3. Vibrational sum-frequency generation spectroscopy……………………..
1.3.1. General description……………………………………………………. 1.3.1.1. Sum-frequency generation…………………………………… 1.3.1.2. Resonance with vibrational modes…………………………. 1.3.1.3. Broken inversion symmetry selective……………………….
1.3.2. Apply VSFG spectroscopy to probing DNA-lipid interaction……. 1.4. Objective of study…………………………………………………………….
2. Experimental approach……………………………………………………………. 2.1. Materials………………………………………………………………………. 2.2. VSFG setup……………………………………………………………………. 2.3. VSFG measuring procedure………………………………………………… 2.4. Pressure-area isotherm……………………………………………………….
3. Binding of DNA to cationic and zwitterionic lipid…………………………… 3.1. VSFG probing of the binding of DNA to cationic and zwitterionic lipids
3.1.1. Interaction of DNA with cationic lipid………………………………. 3.1.2. Interaction of DNA with zwitterionic lipid………………………….. 3.1.3. Comparing the interaction of DNA with cationic and zwitterionic
lipids……………………………………………………………….......... 3.2. Gouy-Chapmann model……………………………………………………..
3.2.1. Surface potential……………………………………………………….. 3.2.2. Debye length…………………………………………………………… 3.2.3. Disagreement of VSFG results with Gouy-Chapmann model…….
3.3. Tentative explanation of VSFG results…………………………………….. 3.4. Conclusion…………………………………………………………………….
4. Lipid monolayer condensation………………………………………………….. 4.1. Surface pressure………………………………………………………………
4.1.1. Surface tension………………………………………………………… 4.1.2. Changing of water surface tension in the present of a lipid
1 1 1 2 3 4 5 5 5 6 6 7 8 9
11 11 12 14 15
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32 32 32
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monolayer………………………………………………………………. 4.1.3. Surface pressure………………………………………………………..
4.2. Phase diagram………………………………………………………………… 4.2.1. Surface pressure-area isotherm of DPTAP and diC14-amidine…… 4.2.2. Influence of DNA………………………………………………….…...
4.3. C-H vibrational stretches……………………………………………………. 4.4. Order parameter………………………………………………………………
4.4.1. Probing the order of lipid layers by VSFG spectroscopy-order parameter………………………………………………………………..
4.4.2. Changing of diC14-amidine monolayer order upon compression.. 4.4.3. Condensation of diC14-amidine monolayer induced by DNA……
4.5. Conclusion……………………………………………………………………..
5. Interfacial water structure………………………………………………………… 5.1. Intermolecular coupling………………………………………………………
5.1.1. Hydrogen-bond network……………………………………………… 5.1.2. Spectra features of D2O-air interface…………………………………
5.2. Intramolecular coupling……………………………………………………… 5.2.1. Fermi resonance………………………………………………………… 5.2.2. SFG spectra of water and lipid at the interface with diC14-amidine
monolayer………………………………………………………………. 5.3. Weak H-bond network of interfacial water upon DNA-lipid surface
complex formation…………………………………………………………….
6. Conclusion and Outlook………………………………………………………….. 6.1. Conclusion…………………………………………………………………….. 6.2. Outlook………………………………………………………………………...
6.2.1. Finding the change of the interfacial water density upon the binding of DNA to lipid monolayer…………………………………..
6.2.2. Directly probing the appearance of the DNA layer at the surface… 6.2.3. Controlling the electrostatic interaction by ions…………………….
Appendix. Dependence of VSFG spectra on experimental conditions……………..
1. Polarization and incident angles………………………………………………. 2. Be careful with the impurity of solvent………………………………………..
Bibliography……………………………………………………………………………...
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Introduction
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CHAPTER 1
INTRODUCTION
We describe here general descriptions of lipid-based gene therapy and vibrational sum frequency generation (VSFG) spectroscopy. By that, we address the topic of the research:
study the interaction of DNA and lipids by VSFG technique.
1.1. Lipid based gene therapy
1.1.1. General description
Gene therapy is the insertion of nucleic acid (with a vector) to patient’s cell for some
therapeutic purpose. There are two major categories of vehicles for gene transfer: namely
viral and non-viral. However, viral vectors exhibit various disadvantages, such as induction
of strong immune response, virus particle associated toxicity, limited target cell specificity
[1]. Therefore, non-viral vectors are introduced as possible solutions for gene therapy. The
vectors used for non-viral protocol can be: the direct injection of purified plasmid, gene gun
and lipids. Lipid liposomes show a several advantages and potential for the delivery of gene
to cells [2]. First, DNA/lipid complexes are easy to prepare and there is no limit to the size of
genes that can be delivered. Second, they may evoke much less immunogenic responses
since carrier systems lack proteins. More importantly, the cationic lipid systems are
associated with a much reduced risk of generating the infectious form because genes
delivered have low integration frequency and cannot replicate or recombine.
From a physical point of view, we are interested in the DNA/lipid complex formation and
the delivery mechanism. The latter can occur through possible two mechanisms (figure 1.1):
endocytosis and fusion. Endocytosis process is the predominant one. The scope of this thesis
focuses on the former - the interaction of DNA and lipids to form a complex.
Introduction
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Figure 1.1. Mechanism of DNA delivery by mean of lipid liposome: Endocytosis (left) and Fusion (right)
1.1.2. Molecular structure of DNA and lipids
The molecular structure of DNA is depicted in figure 1.2. It consists of double nucleotide
strands connecting to each other by hydrogen bonds. Each nucleotide includes three
moieties: sugar, base and phosphate. The sugar and phosphate moieties are connected via
covalent bonds to form a sugar-phosphate backbone. As each phosphate group has one
negative charge, DNA is a poly-anion.
Nucleotide
Figure 1.2. Molecular description of DNA: DNA consists of double nucleotide strands connecting to each other by hydrogen bonds. Each nucleotide includes three moieties: sugar (S), base (A, T, G or C) and phosphate (P). Left panel: illustrating for how different part of DNA connect to each other. Middle panel: describing the 3D geometry of DNA. Right panel: zoom in the molecular structure of one nucleotide. Phosphate group has a negative charge, therefore DNA is a polianion.
Lipids molecules are amphiphilic, i.e. possessing both hydrophilic and hydrophobic
properties. They consist of two parts: the headgroup and the tails. The hydrophilic
headgroup of the lipid molecule is attracted by water molecules while the hydrophobic tails
Introduction
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repel water. In this thesis, we study lipids with saturated alkyl tails and positive or
zwitterionic headgroups.
Figure 1.3 shows molecular structure of N-t-butyl-N9-tetradecylamino-propionamidine
(diC14-amidine), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) and 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Each of these lipids contains two
saturated hydrocarbon tails. DPTAP and DPPC have of 16 carbon atoms in the alkyl chains
while diC14-amidine has 14 atoms. DiC14-amidine and DPTAP have monovalent positively
charge headgroup whereas DPPC is zwitterionic, i.e. electrically neutral but carrying formal
positive and negative charges on different atoms.
Hydrophobic tails Hydrophilic headgroup
Figure 1.3. Molecular structure of diC14-amidine (a), DPTAP (b) and DPPC (c)
1.1.3. DNA-lipid complexes
As DNA is negative, cationic lipid spontaneous forms complexes with DNA and thus
become prevalent non-viral vector. To improve transfection, most cationic lipid are
combined with at least one neutral lipid, the so-called helper lipid [3, 4]. There are three
structures of cationic liposome – DNA complexes that have been observed so far (figure 1.4):
lamellar liquid crystal (LaC), inverted hexagonal (HIIC) and hexagonal (HIC). Lamellar liquid
crystal structures are the alternatives of layers of lipid bilayers and DNA layers. This is the
most favorable structure for lipids because it confers the lowest hydrophobic energy caused
a
b
c
Introduction
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by the interaction between lipid tails. In the inverted hexagonal phase, lipids are organized
in inverse cylindrical micelles and DNA is localized inside these micelles. HIIC structure is
preference for lipid with cone-shaped molecular structure. Hexagonal lattice HIC consist of
lipid micelles with cylindrical shapes immersed in a DNA network. These structural
complexes were found for multivalent lipids, i.e. lipid with positive head group charge of 4+
to 16+ [5].
Figure 1.4. Structure of cationic lipd-DNA complexes for Gene therapy: (a) lamellar liquid crystal (LaC), (b) inverted hexagonal (HIIC) and (c) hexagonal (HIC) [5].
1.1.4. Biophysical studies
Extensive studies and several clinical trials in gene therapy are currently ongoing. The factor
that limits the success of lipid-based gene therapy is the low transfection efficiency [2, 6]. An
understanding of the mechanism of the fundamental biophysical interaction of DNA and
lipid may permit further optimization of deliver strategies [7]. Indeed, many questions need
to be answered: which lipid binds to DNA, how do they interact to form their complexes,
what is the role of helper lipids, ions and water in the binding process, and is there a change
of DNA and lipid conformation and the complex structure upon binding? Although
extensive studies have been conducted, the DNA/lipid interaction is still not completely
understood [8, 9]. Specifically, the driving force for the binding is a question of debate: what
is the contribution of electrostatic, hydrophobic and hydration forces in the interaction [9,
10].
Several approaches have been applied for investigation DNA/lipid interaction: isothermal
titration calorimetry (ITC) [9, 11, 12], fluorophore label probes techniques [10, 13, 14], light
scattering [12], Fourier Transform Infrared Spectroscopy (FTIR) [15], Atomic Force
Introduction
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Microscopy (AFM) [16, 17], X-ray diffraction [1], pressure-area isotherms [1, 18] and
vibrational sum frequency spectroscopy (VSFG) [19]. Among them, we choose VSFG in
conjunction with pressure-are isotherms to give a molecular view of the interaction of DNA
to cationic and zwitterionic lipids because VSFG is a label-free and surface-sensitive
technique (see 1.3.2).
1.2. Langmuir monolayer
A Langmuir monolayer is a monomolecular film formed at the air-water interface, usually
composed of amphiphilic molecules. Amphiphilic substances insoluble in water can be
spread on water surface to form an insoluble monolayer at water/air interface. Headgroups
of lipid molecules are attracted to the water phase and lipid tails intrude into the air phase as
illustrated in figure 1.5.
Langmuir lipid monolayers are appropriate models for investigation the interaction between
DNA and lipid as it is a good model for membrane and bilayer. Furthermore, Lipid/DNA
complexes primarily form a multilayered sandwich structure (LaC) with lipid bilayers
altering with DNA layers [8]. Each lipid bilayer consists of weakly contacted two monolayers
of lipid molecules. 1.3. Vibrational Sum Frequency Generation Spectroscopy
1.3.1. General description
Vibrational sum frequency generation (VSFG) spectroscopy is a second-order nonlinear
optical technique that provides a vibrational spectrum of the molecules in a medium with a
broken symmetry. In this technique, a visible (VIS) beam with fixed frequency and a tunable
infrared (IR) laser beam combine at the surface and generate a coherent signal beam at the
sum frequency of the two incoming beams [20].
Figure 1.5. Lipid monolayer on water sub-phase.
Introduction
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1.3.1.1. Sum-Frequency Generation:
If the VIS and IR beam overlap and have considerable intensity, the second-order
polarization (P(2)) is induced. This is the source of the sum frequency signal.
SFGfactorLs
IRs
VISfactorK
IRVIS EPEEEE )2()2(
,
IRVISeffSFG EELKNE )2( (1.1)
Where: EVIS, EIR are the electric fields of the VIS and IR beams, P(2) is the second-order
polarization, ESFG is the electric field of the sum-frequency signal generated by the second-
order polarization, χ(2) is the second-order susceptibility, K-factor and L-factor are Fresnel
factors, Neff is the effective number of molecules in the asymmetric region, in which the
ordering degree of molecules must be taken into account.
The intensity of SFG signal is proportional to the square of the generated electric field:
2)2(2
IRVISeffSFGSFG EELKNEI (1.2)
1.3.1.2. Resonance with vibrational modes:
In eq. 1.2, only the second-order susceptibility, χ(2), changes as function of the infrared
frequency and it is therefore responsible for the vibrational information obtained from a sum
frequency spectrum.
The second-order susceptibility, χ(2), consists of both a resonant (figure 1.6) and non-resonant
response. )2()2()2(
RNR (1.3)
Introduction
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Figure 1.6. Energy diagram of the sum frequency generation process on vibrational
resonance
The non-resonant second-order susceptibility is small for liquid surface [21]. The resonant
response can be expressed as a Lorentzian function:
n nIRn
nR i
A
)2( (1.4).
When the IR frequency (ωIR) is resonant with a molecular vibration (ωn), (ωIR- ωn) goes to
zero and the resonant second-order susceptibility and thus intensity of SFG reaches its
maximum amplitude. Therefore with SFG, we probe the vibrational modes of the molecules
at the interface, and each vibration will appear as a peak feature in the SFG spectrum.
1.3.1.3. Broken inversion symmetry selective:
The second-order non-linear susceptibility, χ(2), which describes the relationship between the
induced second-order polarization and two applied electric fields, is a third rank tensor with 27 elements )2(
ijk with i,j,k=x,y,z. The element )2(ijk is the SFG response component in the i
direction when the applied VIS and IR electric fields are polarized in j and k directions, respectively. Because changing the sign of the subscripts of )2(
ijk is equivalent to reversing
the applied direction of VIS or IR electric fields or SFG response component, )2(ijk must
reverse its sign:
)2()2(kjiijk (1.5)
IR
VIS SFG
Introduction
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If an environment is inversion symmetric, i.e. all directions are equivalent, the value of )2(ijk
for all opposite directions is identical:
)2()2(kjiijk (1.6)
From equation (1.5) and (1.6), it follows that )2(
ijk must be equal to zero. This means that in
an inversion symmetric environment, SFG is forbidden. SFG occur only in a medium where
inversion symmetry is broken. Surfaces and interfaces are examples.
In this study, we monitored sum-frequency signal generated from the water surface as well
as the lipid monolayer on water. This system is isotropic in the plane of the surface whose symmetry properties confer four independent non-zero element of )2(
ijk [22].
)2()2()2()2()2()2()2( ,,, zyyzxxyzyxzxyyzxxzzzz
With xy plane is the surface plane and z is the surface normal. The ssp polarization accessed
vibrational modes with the transition moments parallel to the surface normal [22]. We chose
this polarization combination for measurement, because we purpose to record the SFG signal
of interfacial water molecules whose symmetric stretch mode have the transition dipole
moment pointing out of the surface plane.
1.3.2. Apply VSFG spectroscopy to probing DNA-lipid interaction
We look at the DNA-lipid interaction indirectly by probing the water molecules at the
interfaces. Because VSFG can be used to probe the interfacial water molecules through their
vibrational modes, VSFG spectra contain information about the interaction of DNA with
lipid monolayer.
The electric field produced by lipid monolayer aligns interfacial water molecules. Due to the
orientation of the interfacial water molecules, the inversion symmetry is broken not only at
the water-lipid interface but also for the underneath region where water molecules is aligned
by the static electric field. Moreover, the electric field produces an additional contribution to
the nonlinear polarization. As a result, the water VSFG signal is strongly enhanced at the
water- lipid interface (figure 1.7.a).
Introduction
9
Figure 1.7. (a) The presence of a cationic lipid monolayer at the air-water interface aligns the first few water layers. This breaks the symmetry, giving rise to a large vibrational sum-frequency generation (VSFG) signal. (b) Due to the strong binding of DNA to the cationic lipids, the electric charges are screened and the orientation order of water is lost, leading to a sharp decrease of the water signal. [19].
The interaction of DNA at the lipid interface will result in the change of interfacial water
structure. For instance, if DNA binds to lipid monolayer, the interfacial charge is screened by
the negative charge of DNA molecules. As a result, interfacial water molecules are less
ordered than in the absence of DNA and thus O-H stretch oscillator strength is decreased in
VSFG spectra (figure 1.7.b).
Label-free: Using VSFG technique, we monitor the intrinsic molecular properties of water
and lipid molecules and thus no labelling is required. The interfacial water structure gives
detailed information on the lipid-DNA interaction. In other words, VSFG spectroscopy is a
label-free technique.
Surface-sensitive: VSFG spectroscopy probes only molecules at the interface where the
interaction occur. DNA-lipid interaction results in a significant change of molecules at
interface and a small relative change in the bulk. VSFG detects this significant absolute
change therefore VSFG is a sensitive technique for probing the DNA-lipid interaction.
1.4. Objective of study
The understanding of the DNA/lipid interaction is important for both fundamental scientific
interest as well as its applications in gene therapy. This subject has been extensively
investigated with a variety of other techniques employing (fluorescent) labels (see 1.1.4).
Here, we aim to characterize the nature of DNA/lipid interaction at a molecular level using
Introduction
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VSFG spectroscopy, a label-free and sensitive technique. We probe here the binding of DNA
to cationic and zwitterionic lipids. The relationship between DNA and lipid upon binding
were quantified by finding the associate/disassociation constant. Furthermore, we monitor
details about lipid conformation and the restructuring of interfacial water.
We choose cationic lipid diC14-amidine and zwitterionic lipid DPPC for investigation.
DPTAP, a cationic lipid, is used for comparison with diC14-amidine. In general, cationic
lipids are toxic for cells, however diC14-amidine and zwitterionic lipid DPPC are proved as
nontoxic [1, 23]. We examine the binding possibility of these nontoxic lipids and provide
detail information about this interaction. This is important for designing lipid-based gene
therapy processes.
Experimental approach
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CHAPTER 2 EXPERIMENTAL APPROACH
We study the interaction between DNA and lipid monolayers by monitoring the change of interfacial molecules (water and lipid) when changing the DNA concentration in the subphase of lipid monolayer. The techniques applied here are vibrational sum-frequency
generation spectroscopy and the surface pressure-area isotherm. In this chapter, I describe the details of these experimental approaches.
2.1. Materials
The chemicals used in this study are: water, heavy water (D2O), several different types of
lipids and lambda-DNA. Water was filtered using a Millipore filter unit and had a resistivity
of 18.2 MΩ. The heavy water (D2O) was obtained from Cambridge Isotope laboratories (MA,
USA) with the impurity of 99.96%. 1,2-dipalmitoyl-3-trimethylammonium-propane (chloride
salt) (DPTAP) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased
from Avanti Polar Lipids Inc (Birmignham, AL, USA). DiC14-amidine was synthesized by
the Ruysschaert group [24]. Lambda DNA was purchased from Fermentas (Germany). Calf-
thymus DNA was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Lambda-DNA which was purchased from Fermentas is stored in H2O. In our experiments,
DNA in D2O solution is required. On this purpose, DNA was extracted from H2O by QIAEX
II Gel Extraction Kit (QIAGEN, CA, USA), then dissolved in TRIS buffered D2O (10mM TRIS-
HCl, pD=7). We repeated this procedure 3 times to ensure low percentage of H2O in DNA
solution and ~80% of the initial DNA is recovered. After buffer exchange, the concentration
was determined by measuring the absorption at 260nm (A260).
Experimental approach
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2.2. VSFG set up
The SFG setup is schematically depicted in figure 2.1. The setup consist of four main parts:
generating 800nm pulsed laser, generating mid-IR beams, generating SFG signal and
detecting SFG signal.
As shown in figure 2.1, an 800 nm laser pulses are generated by a regeneratively amplified
Ti:sapphire laser (Legend, Coherent Inc., CA, USA). This laser operates by amplifying single
pulses from an oscillator. The oscillator (laser 1, Vitesse) provides low-energy pulses at high
repetition rates. To produce laser pulses centered at ~800nm of 3mJ at 1 kHz repetition rate
with pulse duration of ~100fs and ~12nm broad in wavelength, the Coherent Legend
amplifier is pumped by a nanosecond pulses laser (laser 2, Evolution, Coherent Inc., CA,
USA).
About 1.5 mJ of laser energy is used to run SFG experiments. 1mJ is used to generate tunable
mid-IR pulses by a tunable optical parametric amplifier (TOPAS, Light Conversion Ltd.,
Lithuania). The TOPAS unit converts the 800nm pulses to mid-IR pulses through in a two-
step process: optical parametric generation and amplification (OPA) followed by difference-
frequency generation (DFG). In the OPA process, a β-barium borate (BBO) is used to “split”
800 nm photons into the signal (1140-1600nm) and the idler (1600-2650nm) photons. In the
DFG step the signal and idler are mixed in a AgGaS2 crystal giving difference-frequency light
at infrared frequencies (IR):
idlersignalVIS
idlersignalIR
The frequencies of generated signal and idler and thus IR pulses depend on the phase
matching condition of the BBO crystal. Therefore, we used a program WIN TOPAS, software
written by Light Conversion, to allow easy tunability of the IR produced by this unit.
The remaining 0.5 mJ of 800 nm light is converted to a narrow beam with bandwidth of
25cm-1 using one etalon. The frequency-width of VIS laser controls the resolution of the SFG
spectra; therefore we use a Fabry-Perot etalon to narrow the VIS beam.
VIS and IR beams are focused onto the sample by lenses L1 (f=30 cm) and L2 (f=5 cm).
Because we wish to probe a certain combination of polarization of VIS and IR, polarizers and
λ/2 plates were used in order to “clean” the polarization of lasers and ”rotate” the
Experimental approach
13
polarizations of laser beams, respectively. VIS and IR beams are overlapped at the sample in
space and time. In order to achieve the time overlap condition, the optical path of VIS beam
is controlled by a delay stage on which mirror M7 and M8 are mounted. The VIS and IR
beams should strike on the sample with near-identical incident angles in order to increase
the overlap region between two beams (increase the coherence length). The incident angles
of VIS and IR were chosen 35o and 40o relative to the surface normal. Under these incident
angles, we obtain maximum VSFG intensity for water and lipids for ssp polarization
combination (s-SFG, s-VIS and p-IR).
The SFG signal is collimated by a lens and then pointed to a spectrograph through some
filters (the purpose of the filters being to remove any remaining 800 nm light). In the
spectrograph, the SFG photons are dispersed by a grating and detected by a Electron-
Multiplied CCD camera (EMCCD, Andor Technologies, USA)
Figure 2.1. A schematic representation of the SFG setup. A pulsed laser beam is generated by a regenerative amplifier Ti:sapphire laser. This laser operates by amplifying single pulses from an oscillator. The laser is a pulsed laser centered at 800nm of 3 mJ at 1kHz repetition rate with pulse duration of 100fs and 12nm broad in wavelength. Half of the energy of seed laser is used to generate tunable mid-IR pulses in TOPAS and a half is converted to a narrow visible (800nm) with band with of 25cm-1 using one etalon. The telescope is used to change the beam diameter. The optical path length of VIS beam is controlled by a delay stage on which M7 and M8 are mounted. Polarization of VIS and IR beams is controlled by polarizers and λ/2 plates. The VIS and IR beams are focus by lens L1 (f=30 cm) and L2 (f=5 cm) on the sample. The incident angles of VIS and IR are 35o and 40o , respectively. SFG signal is collimated by lens L3 (f=10cm), then pointed into a
Experimental approach
14
spectrograph (detected by CCD camera). Additional filters F2, F3 and F4 are used to block the VIS beam.
2.3. VSFG measuring procedure
Lipid monolayers were prepared on neat water and DNA solutions in a home-made trough.
We checked the density of lipid monolayer by measuring the surface pressure using a
tensiometer (Kibron Inc., Finland). Then SFG signal was collected from the monolayer. A
background is recorded in the same condition but IR blocked. Then the SFG signal is
corrected for the background, which is subtracted. To take into account for the dispersion of
IR generation, i.e. the dependence of IR intensity on generating frequencies (IR generation is
not equally efficient at all frequencies in the spectra) and the sensitivity of the camera, SFG
spectra of the sample were divided to SFG signal obtained from a z-cut quartz crystal. It is
noted that the height of the sample and the height of quartz surface is the same to make sure
that both were exposed to the same overlap condition of IR and VIS beams and signal were
recorded with the dispersion of the spectrograph.
150x103
100
50
0
VIS
inte
nist
y (a
.u.)
825800775Wavelength (nm)
100x103
80
60
40
20
0
SFG
Inte
nsity
(a.u
.)
320028002400
IR frequency (cm-1)
OD region CH region
Entire region
Figure 2.2. (a): the VIS beam center at 797.4nm with the FWHM of 12cm-1 in frequency. (b): SFG spectra on the z-cut quartz crystal with different regions of tunable IR laser. OD and CH regions cover frequencies from 2300cm-1 to 26cm-1 and from 2700cm-1 to 3000cm-1, respectively. Entire region cover both OD and CH stretches.
Figure 2.2.a illustrates the beam profile of the VIS beam which is center at ~797.3 nm and
having FWHM of 25 cm-1. SFG spectra from a z-cut quartz plate are shown in figure 2.2.b.
The region of frequencies where IR is tuned depends on which vibrational modes we are
interested in. If the interested modes are within 200cm-1, the bandwidth of the IR beam from
the TOPAS is sufficient to cover those modes. With a wider region (such as is the case for the
a b
Experimental approach
15
OD stretch), IR frequencies need to be scanned by tuning the angle of the BBO and AgGaS2
crystals. In our experiments, IR laser was scan in the region from 1050 cm-1 to 3250 cm-1.
2.4. Pressure-Area isotherm
A pressure-Area (π-A) isotherm is the measurement of the surface pressure of an insoluble
monolayer on water as a function of the area per insoluble molecule. The measurement is
carried out at a constant temperature.
Lipids were dissolved in Chloroform to be used as a stock solution. The monolayer was
prepared on the subphase in a trough by droplet method using a micro-syringe. Each drop
has volume of 0.5 µl. The trough was cleaned by immersion in Hellmanex 2% solution for
one hour then thoroughly rinsed with mili-Q water and ethanol.
In our study, we measure the Pressure-Area isotherm curves of DPTAP and diC14-amidine
monolayer on neat water and on DNA solutions. A π-A isotherm is recorded by compressing
the monolayer from very large to smaller area per molecule. The monolayer is prepared in a
Micro Trough X (Kibron Inc., Finland) and compressed by two Teflon barriers at a constant
rate. At the starting point of the compression process, the surface pressure is close to zero.
Upon compression, the surface pressure is measured by a tensiometer which is based on the
maximum pull on a rod method (Kibron Inc., Finland). We compressed until the Pressure-
Area isotherm appeared non-monotonic (the sign is that the solution flows over the edges of
the trough). Such a change in the manner in which surface pressure increases with
decreasing area is usually taken to suggest that the surface monolayer has fractured.
Binding of DNA to lipid monolayer
16
CHAPTER 3 BINDING OF DNA TO LIPID MONOLAYER
I present here the result and discussion of probing the interaction between DNA and various lipids using the VSFG technique. Cationic lipid diC14-amidine and DPTAP and zwitterionic lipid were chosen for study. Pronounced changes in spectral amplitudes and line shapes were
observed as a function of DNA concentration in the subphase. We present a tentative model to describe the system, which contains partly Gouy-Chapman model.
3.1. VSFG probing of the binding of DNA to cationic and zwitterionic lipids
We demonstrate here that the interaction of DNA and lipid can be probed by the vibrational
sum frequency generation of water molecules. As a result of its symmetry selection rules,
SFG at frequencies of the OD or OH stretch is generated only from interfacial water
molecules whose orientation and arrangement are affected by the presence of lipid and
DNA. Therefore, VSFG spectra contain detailed information about the interaction.
3.1.1. Interaction of DNA with cationic lipid
Monolayers of cationic lipids (diC14-amdine and DPTAP with area per molecule of 52Å2
(equivalent to surface pressure of 23mN/m) were spread on the surfaces of neat D2O and
with various concentrations of DNA. Then VSFG spectra of the samples were collected with
tunable IR laser in the frequency region from 2050 cm-1 to 3250 cm-1 which covers both OD
and CH stretches. As shown in figure 3.1, the spectra show two features: an intensive and
broad bands with two peaks appear in the region from 2200cm-1 to 2700cm-1 and narrow
resonances around 2900cm-1. The later is assigned to the lipid CH stretches modes and the
former is attributed to the OD stretch modes of hydrogen bonded water. The presence of two
OD peaks is due to the splitting by intramolecular coupling [25] while the broadening of the
band is caused by intermolecular coupling of interfacial water (see 5.1). Detail explanations
Binding of DNA to lipid monolayer
17
at a molecular view of these resonances are presented in chapter 4 and chapter 5. Here, we
have traced the change of VSFG signal of OD stretches to give information about the DNA-
lipid interaction.
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
30002800260024002200
IR frequency (cm-1)
DPTAP[lambda DNA]
0 pM 26 pM 47 pM 94 pM
water/air
D2O
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
30002800260024002200
IR frequency (cm-1)
diC14-amidine[lambda DNA]
0 pM 10 pM 18 pM 26 pM 56 pM 243 pM
water/air
D2O
Figure 3.1. VSFG spectra (thin lines) of water and lipids (top panel: DPTAP and bottom panel: diC14-amidine) at the cationic lipid/D2O interface for different concentration of DNA in water subphase, all at pD=7.0. The SFG spectrum of water/air interface (black line) is shown for comparison. Thick lines are fitting curves of VSFG data using multiple Lorentzian peaks from a global fitting procedure. With increasing DNA concentration, the SFG intensity of OD stretches decreases and a new mode of water with weak H-bond appears. These observations are indication of the strong binding of DNA to cationic lipid.
VSFG signal of water is strongly enhanced at the water-charged lipid interface when
compared with neutral interface (see water/air interface). As shown in figure 3.1, OD
stretches SFG intensity at interface with DPTAP and diC14-amidine is about 20 times higher
than at water/air interface. This enhancement can be ascribed to three reasons. Firstly, the
Binding of DNA to lipid monolayer
18
electric field created by the charge surface aligns water molecules underneath the surface
(figure 1.7.a); this leads to a significant increase in the depth of the broken symmetry region.
Secondly, these interfacial water molecules are more ordered by the large electric field.
Thirdly, the electric field produces an additional contribution to the nonlinear polarization
[22], given by the formula 3.0:
(2) (3)
0( )sfg eff VIS IR VIS IRP N E E E E E [3.0]
Because both the second-order and the third-order susceptibility contain non-resonant and
resonant component, an effective susceptibility can be presented by the equation 1.4. As a
result, VSFG generated from these water molecules is significantly enhanced.
40x10-3
30
20
10
0
I SFG
(a.u
.)
100806040200DNA concentration (pM)
diC14-amidine DPTAP
weak H-bond OD stretch
Figure 3.2. VSFG intensity of the weak hydrogen bonded OD stretch which is centered at ~2630 cm-1 as function of DNA concentration. VSFG intensity was calculted as the square of the ratio of the amplitude and the FWHM at the resonance of 2630 cm-1, with these values were taken from the fitting procedure. This VSFG signal is generated form water molecules confined between lipid monolayer (square: diC14-amidine and star: DPTAP) and DNA layer underneath.
In the presence of DNA in the subphase (see figure 3.1), the SFG signal of water is sharply
reduced. This reduction is an indicator for the binding of DNA to the lipid. The DNA
binding to the lipids results in several effects: screening the charge of the lipid monolayer
and causes the decline of interfacial water density as well as changes the dielectric constant.
The reduction of water signal is marked at a small DNA concentration of 10pM. And at
around 100pM, the signal is equivalent to that from neat water/air interface. This observation
Binding of DNA to lipid monolayer
19
is an indicator of the strong binding of DNA to cationic lipids, which is consistent with
previous measurement on DPTAP of Wurpel et.al. [19].
In addition, a broad band peak appears at around 2630cm-1 (figure 3.1) which increases in
intensity with increasing DNA concentration (figutr 3.2) This high frequency band implies
the appearance of a weak H-bond network of water molecules confined between the lipid
monolayer and DNA layer. This peak does not appear when Chloride ions bind to the lipid
monolayer instead of DNA [19] because there is no water confinement effect in this case. In
another measurement, we monitor the decrease of OD signal by diluted D2O with H2O, there
is also no appearance of a high frequency peak (see appendix), meaning that this peak
originated form water rather than lipids or DNA.
3.1.2. Interaction of DNA with zwitterionic lipid
A similar approach was applied to examine the interaction of DNA with zwitterionic lipid
DPPC. Monolayers of DPPC were prepared at 47Å/molecule on water and several DNA
solutions. Collected VSFG signal of water and lipid of these systems were depicted in figure
3.3.
1.0
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
30002800260024002200IR frequency (cm-1)
DPPC[lambda-DNA]
0 pM 52 pM 78 pM1800pM
water/air
D2O
Figure 3.3. VSFG spectra (thin lines) of water and zwitterionic lipid (DPPC) at the lipid/D2O interface for different concentration of DNA subphase solutions, all at pD=7.0. The SFG spectrum (black line) of water/air interface is shown for comparison. Thick lines are fitting curves of VSFG data using multiple Lorentzian peaks global fitting procedure. With increasing DNA concentration, there is no marked change of the SFG intensity of OD stretches and the shape of the spectra remains. These observations indicate that interfacial water properties are not affected by the presence of DNA because DNA has no or little coordination with zwitterionic lipid.
Binding of DNA to lipid monolayer
20
At the interface with zwitterionic DPPC lipid, the enhancement of the OD stretches in the
SFG signal is qualitatively the same as that at the interface with charged lipid but the
magnitude of the enhancement is smaller. This increase of water SFG signal intensity is due
to the water alignment by the electric filed of the surface dipole made by DPPC headgroup.
Our result is consistent with previous measurements of surface potential in these systems.
According to Shushkov et.al [26] and Shapovalov et.al [27], the surface potential of DPPC
monolayer is about 0.5V at 47Å/molecule. While the surface potential for cationic lipid
monolayer in the measurement of McLoughlin et.al [18] at about 50Å/molecule is 0.9V, nearly
double electric field produced by a zwitterionic lipid monolayer.
With the introduction of DNA in the subphase solution, there is slight variation in SFG signal
of water. The effect of DNA-DPPC interaction to the change of VSFG water intensity is not as
significant as observed for DNA-cationic lipid binding. At low concentrations of DNA
(<78pM), there is gradually fall of the VSFG water intensity, then this change reaches
saturation, i.e. there is no further drop of VSFG water intensity regardless of how much
DNA present in the solution (1800pM). This observation proves that DNA interacts weakly
with the zwitterionic lipid.
3.1.3. Comparing the interaction of DNA with cationic and zwitterionic lipids
In the previous sections, we show that DNA interacts differently with cationic and
zwitterionic lipids. Here, we quantify this difference. The VSFG data can be fitted to multiple
Lorentzian peaks spectra (equation 3.1) with a global a fitting procedure [19, 22]. This
approximation bases on a physical property that the SFG signal is strongly enhanced when
the frequency of IR beam is resonant with a vibrational mode which is SFG active. In the
fitting procedure, the non-resonance part and the resonance frequencies were held constant,
the amplitudes An were free and the width Γn can be varied but constraint. 2
2)2()2(2)2(
n nIRn
niNRRNRSFG i
AeAI NR
[3.1]
When IR frequency matches the resonant frequency of a vibrational mode, SFG intensity is 2
,n
nnSFG
AI
[3.2]
Binding of DNA to lipid monolayer
21
For sake of convenience, the OD stretch band which is spitted by an Evans hole (see chapter
5) is considered as two resonance peaks. The SFG intensity of water was calculated as the
average of SFG intensity of two OD stretch modes. This was normalized to the signal
obtained from the lipid monolayer on neat water without DNA and to the water air interface
as:
, , , /
, , _ , , /
SFG n SFG n water airSFG
SFG n without DNA SFG n water air
I INormalized I
I I
[3.3]
1.0
0.8
0.6
0.4
0.2
0.0
Nor
mal
ized
I SFG
120100806040200Concentration of DNA (pM)
DPPC DPTAP diC14-amidine
Figure 3.4. Normalized SFG intensity of OD stretch of water at the water/lipid interfaces (red: DPPC, green: DPTAP and blue: diC14-amidine) as a function of DNA concentration in the subphase. Marked dots are data points calculated from global Lorentzian fitting procedure. The solid lines are used to guide the eyes.
The normalized SFG intensity of water is plotted as function of DNA concentration in figure
3.4 for different lipid monolayers. The sudden drop of the SFG signal of water with
increasing DNA concentration occurs for both diC14-amidine and DPTAP. In molecular
structure (figure 1.3), both diC14-amidine and DPTAP have one charge per headgroup.
However the charge on diC14-amidine is delocalized but that on DPTAP is localized on
nitrogen atom. This implies that the behaviour of DNA with lipid only depends on the
charge of the lipid regardless of the difference in the charge distribution. Moreover, each tail
of diC14-amidine has 14 carbon atoms while DPTAP has 16. This difference does not affect
much on the interaction of lipid with DNA, showing that hydrophobic interaction of the tails
has minor contribution for driving the binding of DNA to lipid. In contrast with cationic
lipid, zwiterionic lipid slightly interacts with DNA. Given that argument, SFG intensity of
water is gradually and slightly reduce in the presence of DNA.
Binding of DNA to lipid monolayer
22
To conclude, the interaction of DNA to lipid depends mainly on the charge of the lipid. DNA
strongly binds to cationic lipid but slightly interact with zwitterionic lipid. Therefore, the
driving force for the binding is electrostatic.
3.2. Gouy-Chapmann model
3.2.1. Surface potential
We expect the SFG signal to be influenced by the surface potential in this manner (as shown
in equation 3.0). We can relate changes in surface potential to amount of DNA on the surface
using the Gouy-Chapman model [19, 22].
We approximate the surface charge of a cationic lipid monolayer as a continuous charged
surface on an electrolyte solution (pure water is considered as an electrolyte solution with
H3O+ and OH- concentration of 10-7M). The surface charge and ions in the solution create an
electric field. In the bulk, the field vanishes due to the random distribution of both negative
and positive ions. At the surface, the superimposed electric field is non zero characterized by
the surface potential.
The electric field at the surface is the superimposition of the fields generated by the surface
charge and electrolytes therefore the surface potential Ψ0 depends on both the surface charge
density and the distribution and concentration of electrolytes. The Gouy-Chapman model is
based on the Boltzmann distribution of ions in the solution and Poison equation [28]. The
physical assumptions that go into this model: ions are point charges, there is no ion pairing,
the solvent (water) is only represented in terms of its macroscopic dielectric constant.
Electrostatic binding model:
Boltzmann distribution of ions in the solution:
kTzee /
[3.4]
Where:
ρ is the density of ion at the distance x from the interface[ion/m3]
z is the charge of ion (including the sign of the charge)
ψ is the electric potential, which depends on the distance x from the interface
k is the Boltzmann's constant
T is the absolute temperature in Kelvins
Binding of DNA to lipid monolayer
23
NA is Avogadro's number
e is the elementary charge
Differentiating eq. towards x [3.4]:
dxdeze
kTdxde
kTze
dxd kT
zekT
ze
0
0 [3.5]
Where:
ε0 is the permittivity of free space
ε is the dielectric constant
Poisson equation:
2
2
0 dxdze
[3.6]
Or
02
2 zedxd
[3.7]
To combine the Boltzmann distribution and Poisson equation, substituting ρ from [3.4] to
[3.7] eq., we obtain kTze
ezedxd
0
2
2
[3.8]
Substitute [3.8] to [3.5] 2
02
20
0
0
2
dxd
dxd
kTdxd
dxd
kTdxdeze
kTdxd kT
ze
[3.9]
Integrating eq. [3.9], we obtain;
220
2 dxd
dxd
kT xx
[3.10]
Where ρx is the density at the distance x from the surface
In the bulk: dx
d=0
At the surface: ρx= ρ0, ψ= ψ0, 00
Edxd
, where Eo is the electric field at the surface
20
00 2
EkT
[3.11]
Binding of DNA to lipid monolayer
24
From the electro-neutrality condition, we have surface
bulk
dxze [3.12]
Where is the surface charge density.
Substituting [3.6] to [3.12], we have
00002
2
0 Edxd
dxd
dxddx
dxddxze
surfacebulksurface
surface
bulk
surface
bulk
[3.13]
00
E [3.14]
Substituting [3.14] to [3.11], we obtain kTkT 0
22
0
00 22
[3.15]
In the case where more than one type of ion is present in the solution:
kT0
2
0 2
[3.16]
Eq. [3.16] is the so-called Grahame equation. The physical meaning of this equation is that
the difference of the ion density at the surface and in the bulk is caused by the charge on the
surface.
Check unit of eq. [3.16]:
JmJCmC
m 112
22
3
.1
[3.17]
Dividing eq. [3.17] by 1000 and Avogadro’s numberNA=6.023x1023 we have
22
2 1 1
.1000 A
C mmoleMl C J m J N
[3.18]
Dividing the Grahame equation by 1000xNA, we obtain: 2
002 1000 A
C CkT N
[3.19]
To find the surface potential as a function of the concentration of ions, we find the
solution for Grahame equation. Assume that we have N ions in the solution with the
concentration C1, C2 …, CN. Then equation [3.19] is
0 2
02 1000
iz eN NkT
i ii i A
C e CkT N
[3.20]
Binding of DNA to lipid monolayer
25
In case the ionizable surface sites are partially neutralized by the binding of specific ions
from the solution to the surface, the surface charge density does not remain constant: 0 , where 0 is the surface charge density without binding (fully dissociated)
and α is the fraction of unbound charge on the surface.
In case of a 1:1 electrolyte in the solution and when the surface sites are fully dissociated
N=2, z1=1, z2=-1, C1=C2=C, equation [3.20] becomes:
0 0 2
0
22 1000
e ekT kT
A
C e ekT N
[3.21]
0 0 0 0
2 2 22 2 2 2
0
22 1000
e e e ekT kT kT kT
A
C e e e ekT N
[3.22]
0 0
2 22 2
02 1000
e ekT kT
A
C e ekT N
[3.23]
0 2
2 2
0
sinh8 1000
ekT
A
CkT N
[3.24]
In case of 1:1 electrolyte (NaCl for example) in the solution and surface sites are partial
neutralized due to the binding of ion to the surface:
0 [3.26]
Equation [1.20] becomes:
10
0
2 sinh8 1000 A
kTe kT N C
[3.25]
1 00
0
2 sinh8 1000 A
kTe kT N C
[3.25]
Binding of DNA to lipid monolayer
26
We can find α from the associate-disassociate equilibrium condition: ionsurfaceionsurface K
Where the association constant K is:
0ionsurface
ionsurfaceK
[3.28]
000
0 11ionion
K
[3.29]
In case of 1:1 electrolyte in the solution, surface sites are partially neutralized and the
cooperativity in the adsorption process is included:
nionK
0
1
[3.30]
Where n is the Hill constant
kTezion
eionion0
0
(Boltzmann distribution)
zion=-1 kTe
eionion0
0
[3.32]
[ion]=C
The formula [3.28] agrees with Marty et al. [15] and the formula [3.30] agrees with Luo et al.
[29].
For 1:1 electrolyte, we solve eq. 3.25, 3.31 and 3.32 to find the surface potential as function of
electrolyte concentration (for 1:1 electrolyte). For other case, we solve eq. 3.20, 3.26, 3.31. and
3.32.
3.2.2. Debye length
The electric field of a charged surface causes the difference in distribution between negative
and positive electrolytes in the solution. The Debye length 1/κ is the distance from the
surface over which significant charge separation can occur (figure 3.5) [28].
nionK 011
[3.31]
Binding of DNA to lipid monolayer
27
0 02 2
1
i i i ii i
kT kTe z e NA z C
[3.33]
where Ci is the concentration of electrolyte i in the solution.
Figure 3.5. Potential and ionic density profiles for 0.1M monovalent electrolyte such as NaCl near a surface of charge density -0.0621 Cm-2. [28] The Debye length 1/κ is the characteristic decay length of the potential.
The variation of the potential away from the surface for a 1:1 electrolyte is given by the so-
called Debye-Hückel equation 3.34:
0x
x e [3.34] Equation 3.34 shows that the Debye length 1/κ is the characteristic decay length of the
potential. The depth of the asymmetric region, where water is ordered, is on the order of the
Debye length.
Binding of DNA to lipid monolayer
28
3.2.3. Disagreement of VSFG results with Gouy-Chapmann model
The binding of DNA to lipid monolayer influences the organization of water molecules at the
surface [19]. Therefore, we monitor the SFG intensity associated with the interfacial water,
which is aligned by the surface electric field in order to quantify the DNA-lipid interaction.
The VSFG water signal is proportional to the square of the number of oriented water at the
surface [30]. Furthermore, the electric field, which is generated by the lipid monolayer,
produces an additional contribution to the nonlinear polarization [22]. Given that argument,
SFG intensity of water would be expected to be proportional to the square of the surface
potential.
1.0
0.8
0.6
0.4
0.2
Nom
anized ISFG
10-9 10-7 10-5 10-3 10-1 101
Concentration of nucleotide (M)
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
02
(V2 ) Nomanized ISFG
n=5
n=15
Figure 3.6. The normalized SFG intensity (closed circles) of water obtained from the Lorentzian fits of the VSFG date (see section 3.1.3) (right axis). The theoretical calculation of the square of surface potential from the Guoy-Chapmann model for a surface charge of 0.307Cm-2 and 1:1 electrolyte with association constant of ion to surface of Ka=105 and cooperative factor of 5 (black line) and 15 (gray line) are plotted against the left axis.
In figure 3.6, we compare the normalized SFG water signal with the theoretical calculation of
the square of surface potential from the Guoy-Chapmann model (see 3.2.1) for a surface
charge of 0.307Cm-2 (correspond to the lipid density of 52Å2/molecule) and 1:1 electrolyte
with association constant of ion to surface of Ka=105 and cooperative factor of 5 and 15.
We choose the value for the association constant as Ka=105 because the dissociation constant
of DNA and cationic lipid is about Kd=10-5 M-1 expressed in concentration of DNA nucleotide.
The dissociation constant (Kn) can be estimated within the regions where near-saturation
occurs [31]. As shown in figure 3.4, the interaction of DNA to cationic lipid reaches
saturation at the order of 100pM. Therefore the association constant of DNA and cationic
Binding of DNA to lipid monolayer
29
lipid is 10-10M expressed in DNA concentration or 10-5M expressed in concentration of
nucleotide. The binding constant Ka is the invert of dissociation constant, hereby Ka=105M-1.
The comparison gives an obvious disagreement of the SFG intensity with the square of
surface potential calculated by Gouy-Chapmann model. The SFG intensity plotted as
function of nucleotide concentration is much steeper than the square of surface potential.
With increasing the cooperative factor, the slope of the square of surface potential increases,
however its steepness is smaller than that of the SFG data. This indicates that the physics of
the studied system are not fully captured by this model.
3.3. Tentative interpretation of VSFG results
The disagreement of the SFG data with the Gouy-Chapmann calculation implies that the
VSFG water signal drops much faster than the surface potential when DNA binds to a lipid
monolayer. To further test this hypothesis, we performed a VSFG measurement on a system
with known surface potential. The result is illustrated in figure 3.7. McLoughlin et.al [18]
measured the surface potential of cationic lipid monolayer on neat water and on calf-thymus
DNA of 0.1mg/ml concentration as function of area per lipid molecule. They show that at
52Å2/molecule (equivalent to 0.307Cm-2 surface charge density), in the presence of DNA,
surface potential drops 70% in comparison to the absence of DNA. While in our
measurement, VSFG signal of water decrease by more than 99% (the normalized intensity is
0.06) in the presence of DNA. In previous measurement with lambda-DNA (see section 3.1),
at the concentration of 100pM (equivalent to 0.003mg/ml) which is two orders smaller than
concentration of 0.1mg/ml calf thymus DNA, the SFG signal is already reduce by more than
99% (the normalized intensity is 0.07). This comparison suggests that indeed the SFG signal
is not solely influenced by the surface potential.
We propose that the reduction of the VSFG water signal is due not only to the reduction of
surface potential by screening effect but also due to the reduction of the number of water in
the region where the potential applied. We note that:
2SFGI N [3.35]
where ISFG is the VSFG water signal and β is the effective fraction of oriented water which in
turn is proportional to the surface potential
0 [3.36]
N is the number of water in the region where the potential is present:
Binding of DNA to lipid monolayer
30
1N V A
[3.37]
with is the density of interfacial water, V is the volume of the asymmetric region where
sum-frequency signal of water vibrational is generated. This volume can be approximated by
the region within Debye length 1/κ [22] multiplied by A, the relevant area (e.g. focus spot of
IR beam on the sample surface).
From eq. 1.35, 3.36 and 3.37, we have; 2
01
SFGI
[3.38]
2.0
1.5
1.0
0.5
0.0
I SFG
/I ref (
a.u.
)
270026002500240023002200
IR frequency (cm-1)
diC14-amidine[calfthymus-DNA]
0 mg/ml 0.1 mg/ml
water/air
D2O
1.0
0.8
0.6
0.4
0.2
0.0
Nom
aniz
ed I S
FG
10-5 10-2 101 104
DNA concentration (mg/ml)
Norm
alized 0
2 (V2)
02
ISFG
Figure 3.7. (a): VSFG spectra (thin lines) of water and lipids at the cationic lipid/D2O interface for different concentration of calfthymus-DNA, all at pD=7.0. The SFG spectrum (black line) of water/air interface is shown for comparison. Thick lines are fitting curves of VSFG data using multiple Lorentzian peaks global fitting procedure. (b): Normalized VSFG intensity of water (star) and the square of the surface potential normalized to the potential in the absence of DNA (closed square, this data is taken from the result of McLoughlin et.al [18] Dot line: extrapolation of the normalized SFG intensity as function of DNA based on data of lambda-DNA with sigmoid fitting. Dash line: The expected square of surface potential according to the Gouy-Chapmann model.
Within the scope of this study, we have not quantified how much of the decrease of SFG
signal is due to the change of surface potential, interfacial water density and the depth of the
asymmetric region. Here, we qualitatively discuss these contributions. Upon the formation of
DNA/lipid surface complex, the surface potential decrease due to the screening of DNA
charge, the interfacial water density decline resulting from squeezing water out by DNA.
The Debye length might be subject to the change of dielectric constant and electrolyte
concentration by the presence of DNA/lipid complex at the surface. From the figure 3.7.b) we
a b
Binding of DNA to lipid monolayer
31
can estimate that more than 50% of the decrease of SFG water signal is due to the change of
the depth and water density of the asymmetric region.
3.4. Conclusion
DNA strongly binds to cationic lipid but slightly interacts with zwitterionic lipid due to the
electrostatic force. This interaction can be indirectly probed by monitoring the VSFG spectra
intensity associated with the lipid-bound water. Upon the binding, the change surface
potential, the asymmetric region depth and interfacial water density in this region cause the
reduction of VSFG water signal.
Lipid monolayer condensation
32
CHAPTER 4 LIPID MONOLAYER CONDENSATION
In this chapter we consider the change in the order of the lipid monolayers upon DNA binding. Firstly, we briefly explain the method to determine the monolayer order by surface
pressure measurement and vibrational sum frequency generation spectroscopy. Then we apply these methods to investigate the phase behavior of particular lipid diC14-amidine and DPTAP monolayers. Finally, we show that DNA induces the condensation of these lipid
monolayers when it binds to the monolayer and the DNA-lipid surface complex is formed.
4.1. Surface pressure
4.1.1. Surface tension
We are considering a liquid-gas interface. Intermolecular
forces at the surface of a liquid cause surface tension. In the
bulk of the liquid, the interaction of a molecule with other
molecules is balanced by equally attractive forces in all
direction, resulting in a net force of zero. At the surface,
intermolecular forces exert on a molecule are imbalance as
the symmetry is broken (figure 4.1.). Therefore, there is a
free energy existing at the surface or the so-called surface
energy.
To minimize the surface energy, there is a driving force to diminish the surface area. The
surface tension (γ) is the needed work to increase the surface area by 1 m2. The unit of
surface tension is J/m2 or N/m. By this definition, surface tension can be understood as the
excess energy at the surface.
Figure 4.1. Forces exert on molecules in the bulk and at the surface of liquid.
Lipid monolayer condensation
33
In another way, surface tension is defined as the force measured in a unit length along a line
on the surface. Therefore, surface tension can be measured by the NuNouy-Padday rod
method [32].
4.1.2. Changing of water surface tension in the present of a lipid monolayer
Water is a polar liquid with strongly intermolecular interactions. The surface tension of
water is around 74mM/m at 22oC.
In the presence of an insoluble lipid monolayer, because of its amphiphilic property, the
strong imbalance interaction at the water surface is reduced. The hydrophilic headgroup of
lipids is immersed in the water and the hydrophobic tails are pointing toward air (figure 1.5).
This results in the decrease of the excess surface energy and thus surface tension.
4.1.3. Surface pressure
The surface pressure (π) is the difference of the surface tension in the absence of a monolayer
(γ0) and with the monolayer presence (γ).
π = γ0- γ
Surface pressure is an important determination of the properties of an amphiphilic layer. The
value of surface pressure indicates how the surface energy changes in the presence of the
monolayer. The arrangement of lipid molecules on the surface affect the intermolecular
interaction at the surface and hence surface energy. Due to that, surface pressure can be used
as an indicator of the condensation of lipid monolayer. The lipid presence changes the
surface pressure and this depends on the lipid density.
4.2. Phase diagram
4.2.1.Surface pressure-Area isotherm of DPTAP and diC14-amdine
Surface pressure- Area isotherm (π-A) is curve describing the surface pressure as a function
of the surface area per lipid molecule at a constant temperature. This isotherm curve is
equivalent to the P-V curve of a gaseous-liquid phase transition of a three dimensional gas
such as water. When the area per lipid molecule is large, the lipid molecules are far apart
from each other and their interaction is weak. In this condition, the monolayer has a small
effect on the surface tension and hence the surface pressure is close to zero. This monolayer
can be regarded as a two dimensional gas. Upon compression, the area available for each
lipid molecule is reduced and therefore the interaction between lipid molecules becomes
Lipid monolayer condensation
34
stronger. In this process, the monolayer undergoes transitions from a gas phase to more
ordered phases.
40
30
20
10
0
(m
N/m
)
14012010080604020
Area/molecule (Å2)
DPTAP diC14-amidine
LC
LE+LC
LE
T=22 oC,
LE
Figure 4.2. Phase diagram of DPTAP (black line) and diC14-amidine (gray line) showing the gaseous phase, the gaseous-liquid expanded coexistence state (G), the liquid expanded state (LE), the liquid expanded-liquid condensed coexistence state (LE+LC) and the liquid condensed state (LC). These surface pressure isotherms are obtained at constant temperature T=22oC. Inset is a cartoon of lipid molecules arrangement at various phases.
Phase diagrams of DPTAP and diC14-amidine monolayers are depicted in figure 4.2. These
surface pressure–area isotherms are obtained at constant temperature of 22oC. With
decreasing surface area, several phases appear. For DPTAP: a liquid condensed phase (LC) at
a high compression (area per molecule A<70Å2), a coexistence liquid condensed and
expanded phase (LE+LC) between 70 and 90Å2 per molecule and a liquid expanded phase
(LE) at >110Å2/molecule. For diC14-amidine, only liquid expanded phase exists regardless of
how compressed the monolayer is.
The phase behavior of DPTAP and diC14-amidine are remarkably different at room
temperature. DPTAP undergoes a transition from liquid expanded to liquid condensed
phase while there is no phase transition for diC14-amidine. DPTAP has a liquid condensed
region with a very steep isotherm curve region. This steepness indicates that DPTAP has a
small compressibility. In the zone where both liquid expanded and liquid condensed phases
Lipid monolayer condensation
35
exist, there are two bounding condition: saturated liquid expanded state (90Å2) and
saturated liquid condensed state (70Å2). The existence of a phase transition region implies
that the temperature in the measuring condition (room temperature) is below the critical
temperature of DPTAP. The critical point in a π-A (or P-V) phase diagram is the temperature
which separates the π-A (P-V) into two regions: above that point, the π-A curve is
monotonic, i.e. there is no phase transition and below that temperature the π-A curve show a
transition region between two phases. In the transition region, the surface pressure π does
not change with reducing the area per lipid molecule. Figure 4.3 also shows that diC14-
amidine can be compressed to 40 Å2/molecule, a value lower than the 50Å2/molecule limit for
DPTAP. This clearly implies that diC14-amidine is more compressible than DPTAP. The
unique liquid expanded phase of diC14-amidine upon compression suggests its critical point
is lower than room temperature. This also infers that diC14-amidine monolayer can not be
compressed to a condensed phase in an isotherm process at room temperature.
The different phase behavior between diC14-amdine and DPTAP can be attributed to the
difference in the tail lengths and charge distributions of the headgroup. Although, both of
them have a double saturated alkyl tail, the number of carbon atoms in the back-bone tails of
DPTAP and diC14-amidine is 16 and 14, respectively. An increasing in the tail length results
in a stronger hydrophobic attraction between molecules.
4.2.2.Influence of DNA
With the presence of DNA in the subphase, the shape of the isotherm curves are similar as on
neat water subphase but shifted to higher area per molecule for both DPTAP and diC14-
amidine (figure 4.3). Firstly, this similarity indicates that DNA has no prominent effect on the
fudamental phase behaviour of the lipids. Secondly, the compression isotherm of both
DPTAP and diC14-amidine shifts to appreciably higher surface pressures due to the presence
of DNA. It can be described as the monolayers on a DNA solution subphase having a higher
surface pressure than that on neat water under the same area per molecule, meaning that the
monolayer is more ordered with DNA underneath. In other words, to reach the same surface
pressure, i.e. the same ordering level of the monolayer, each lipid molecule still can occupy a
more area on average.
The phase behavior of the monolayer depends on the cohesive and repulsive forces existing
between headgroups and the adhesive forces between lipid and water molecules. Therefore,
Lipid monolayer condensation
36
the charge and hydration of headgroup influences on molecular packing orientation. In
chapter 3, we point out that DNA binds to diC14-amidine and DPTAP and form surface
complexes. Due to the binding, hydrated water molecules around lipid headgroups is
squeezed out, reducing water intermolecular interaction. As a result, surface tension
decreases or surface pressure increases. Moreover, poly-anion DNA binds and neutralizes
charges of the headgroups, leading to decrease of the repulsive force. The net effect results in
a better orientation or more condense phase of the phospholipids monolayers when it binds
to DNA.
40
30
20
10
0
(m
N/m
)
140120100806040
Area/molecule (Å2)
(a) DPTAP water DNA 90pMLC
LE+LC
LE
40
30
20
10
0
(m
N/m
)
20015010050
Area/molecule (Å2)
(b) diC14-Amidine water DNA 150pM
LE
Figure 4.3. Pressure-area (π-A) isotherm of DPTAP (a) and diC14-amidine (b) monolayer on different sub-phases: neat water (black curve) with buffer TRIS-HCl 10mM pH=7 and 90pM and 150 pM lambda-DNA solution (gray curve). The gaseous phase (G), liquid condensed phase (LE), coexistence region of the two phases (G+LE) and (LE+LC) and liquid condensed phased phase (LC) are indicated along the π-A curves. ). These surface pressure isotherms are obtained at constant temperature T=22oC.
To conclude, from the compression isotherm investigation we elucidate the influence of
DNA on lipid monolayer and interfacial water molecules: DNA induces the condensation of
lipid monolayer and reduces intermolecular interaction between interfacial water molecules.
4.3. C-H vibrational stretches
The alkyl tails of lipid molecules contain C-C and C-H bonds. The C-C stretch vibrations are
usually weak. There are six C-H stretching vibrations of saturated hydrocarbon chains of
phospholipids commonly observed (figure 4.4). The vibrational frequency depends on the
bonding stiffness and thus the subgroup containing the bond. Each of the methylene methyl
groups on the back-bone of alkyl tails produce two stretching modes: the symmetric and
Lipid monolayer condensation
37
asymmetric. The symmetric stretch is split by Fermi resonance with an overtone of an
asymmetric bending mode.
Figure 4.4. Methylene and methyl stretching modes: CH2SS and CH2AS are the symmetric and asymmetric methylene stretches, CH3SS and CH3AS are the symmetric and asymmetric methyl stretches.
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
I SFG
/I ref
(a.u
.)
30002950290028502800
IR frequency (cm-1)
CH2SS
CH3SS
diC14-amidine26mN/m
DPTAP20mN/m
CH3FR
CH3AS
CH2AS
R=1.1
R=7.0CH2FR
Figure 4.5. SFG spectra (dot curves) of diC14-amdine at 26 mN/m (gray) and DPTAP (black) at 20mN/m. The vertical lines indicate the positions of methylene symmetric stretch (CH2SS), methyl symmetric stretch (CH3SS), methylene asymmetric stretch (CH2AS), methyl symmetric stretch-Fermi resonance (CH3FR) and methyl asymmetric stretch (CH3AS). All spectra are normalized to a reference SFG signal from z-cut quartz. The solid curves represent fits to the data using a Lorentzian model.
Vibrational stretch frequencies of C-H bonds on lipid alkyl chains of DPTAP and diC14-
amidine are determined by recording VSFG spectra of monolayers of these lipids on a water
subphase (TRIS-HCl buffer 10mM pH=7) at 20mN/m and 26mN/m, respectively. As shown
in the figure 4.5 five vibrational modes can be observed: the symmetric methylene stretch
(CH2SS), the symmetric methyl stretch (CH3SS), the asymmetric methylene stretch (CH2AS),
the symmetric methyl stretch Fermi resonance (CH3FR) and the asymmetric methyl stretch
CH3SS CH3AS CH2SS CH2AS
Methylene Methyl
Lipid monolayer condensation
38
(CH3AS) are found at 2846, 2871, 2908, 2935 and 2955 cm-1, respectively. The Fermi resonance
of CH2SS is too weak to be visible.
As elucidated in chapter 1, sum frequency photons only generated under broken symmetry
condition. Based on this principle, VSFG signal of C-H modes can be monitored to provide
information about the conformation order of lipid layers [33].
4.4. Order parameter
4.4.1.Probing the order of lipid layers by VSFG spectroscopy-order parameter
The ordering of the lipid monolayer can be further investigated at a molecular view by
vibrational sum-frequency generation (VSFG) spectroscopy. The SFG intensity at resonance
frequencies with the vibrational modes is related to the ordering level of molecules. Upon
compression, the monolayer is more ordered and the alkyl tails transform from cis to trans
configuration. Due this geometric transformation, the angular distribution of the terminal
methyl group is narrower. Under this condition the angle distribution of the transition dipole
moment of methyl symmetric stretch is small, leading to an increase in the SFG intensity at
CH3SS resonance. In contrast, the CH2SS resonance is decrease due to a local symmetry point
along the lipid tail.
Given the above arguments, the ratio R of the CH3 and CH2 symmetric stretch oscillator
strengths provides a sensitive measure for the order of the lipid monolayer [34]. The higher
R implies the more ordered of the monolayer.
As shown in figure 4.5, the ratio R of diC14-amidine monolayer at 26mN/m is 1.1 while that
of DPTAP at 20mN/m is 7.0. This result is consistent with the phase behavior determined by
compression isotherm measurements, which showed that at 20mN/m, DPTAP monolayer is
already in the liquid condensed phase, whereas diC14-amidine monolayer remains in the
liquid expanded phase at room temperature at all pressures. The ratio R of diC14-amidine is
about 7 times smaller than for DPTAP, meaning that the monolayer made by diC14-amidine
is much less ordered than by DPTAP even if both monolayers are compressed to the same
area and the surface pressure for diC14-amidine monolayer higher than DPTAP. This implies
that VSFG is not only a complementary technique with compression isotherm measurement
but also is a very powerful, sensitive probe of molecular orientation and phase behavior of
Langmuir monolayers.
Lipid monolayer condensation
39
4.4.2.Change of diC14-amidine monolayer order upon compression
To quantify the order changing of diC14-amidine monolayer upon compression, the VSFG
spectra were recorded in the C-H stretches frequency zone at different surface pressure or
area per molecule along the isotherm (figure 4.6, upper panel). In general, along the
isotherm, all C-H stretch modes for both methyl and methylene groups are visible. At low
surface pressures (π<18mN/m), the amplitude of the CH2SS modes are larger than the
CH3SS’s one. Even a high compressed conditions (π>26mN/m), there is no marked increase
of the CH3 symmetric stretch intensity. This is testimony to the disorder in the monolayer
regardless of how compressed the monolayer is. Interestingly, the obvious appearance of
asymmetric methylene oscillator confirms a clearly difference of diC14-amidine in
comparison with DPTAP (see 4.4.1) and DPPC [34]. This strongly evident for the liquid
expanded state since only with this condition, both asymmetric and symmetric transition
dipole moments are favorable for SFG active.
However, the increase in CH3 symmetric oscillator strength upon compression is not
negligible. As shown in figure 4.6 (lower panel), the oscillator strength of CH2SS is round
constant value while that of CH3SS increase with the surface pressure. The overall change in
the order of monolayer is quantified by the ratio R of SFG intensity at CH3SS to CH2SS
modes. A general trend of the change of R upon compression is an increase of the ratio R.
This ratio slowly increases at low surface pressure (π<15mN/m), increases faster from 15 to
30mN/m and then reaches saturation at compression. At a saturated level, the methylene
symmetric oscillator is slightly stronger than the methyl symmetric oscillator, evidenced by
the value of around 1.2 for ratio R. The saturation value of R for diC14-amdine monolayer is
ten times smaller than for DPPC which was found at 10.0 [34], confirming for the disorder
state of the diC14-amidine monolayer. This phase behavior found by VSFG measurement is
consistent with that determined by π-A isotherm diagram: the diC14-amidine monolayer is
more ordered upon compression but still in the liquid expanded phase.
Lipid monolayer condensation
40
1.5
1.0
0.5
0.0
I SFG
/I ref
(a.u
.)
30002950290028502800
IR frequency (cm-1)
diC14-amdine 4 mN/m 8 mN/m 18 mN/m 26 mN/m 36 mN/m
CH2SSCH3SS
0.40
0.35
0.30
0.25
0.20
0.15
0.10
ISFG
(a.u.)
353025201510Surface pressure (mN/m)
1.2
1.0
0.8
0.6
Rat
io R
Ratio R
CH2SS CH3SS
Figure 4.6. Upper panel: SFG spectra (dot curves) of the diC14-amidine monolayer on water (buffer TRIS-HCl 10mM buffer pH=7) at different surface pressures: 4mN/m, 8mN/m, 26mN/m and 36mN/m. The vertical lines indicate the positions of methylene symmetric stretch (CH2SS), methyl symmetric stretch (CH3SS). The solid lines represent fits to the data using a Lorentzian model. Lower panel: SFG intensity of CH2SS and CH3SS resonances (opened circle and star, right axis) and their ratio R (square dots, left axis) of diC14-amidine monolayer at different surface pressures. The solid line a guide to the eyes.
4.4.3.Condensation of diC14-amidine monolayer induced by DNA
We applied the VSFG measurement approach for a broad frequency band which covers both
OD and CH stretches zones and then extracted the C-H region to monitor the influence of
DNA on the order of diC14-amidine monolayer. Because we are interested in monitoring the
ordering of the monolayer, only the CH stretch region is presented in figure 4.7 (upper
panel). The diC14-amidine monolayer is prepared at the area of 52Å2/molecule on neat water
subphase and DNA solution at different concentrations. Overall, the SFG spectra upon
injection of DNA change in the same fashion with the change upon compression: with
Lipid monolayer condensation
41
increasing the DNA concentration, the SFG signal of the CH3 symmetric stretch, an indicator
for the ordering of monolayer, lifts up (illustrated in lower panel) and all stretch modes of
methyl and methylene groups remain visible. It is noteworthy that the CH2SS vibration
seems to increase in a similar way with CH3SS mode due to the interference with a lower
frequency broad band appearing from free OD stretch when DNA bind to diC14-amidine
(see chapter 3 and 5). In fact, from the fitting results, the SFG intensity of CH2SS shows a
slight decrease, to then remain constant (figure 4.7, lower panel).
0.6
0.5
0.4
0.3
0.2
0.1
0.0
I SFG
/I ref
(a.u
.)
30002950290028502800
IR frequency (cm-1)
diC14-amidine[lambda-DNA]
0 pM 10 pM 26 pM 56 pM
CH2SSCH3SS
2.0
1.8
1.6
1.4
1.2
1.0
0.8
Rat
io R
100806040200DNA concentration (pM)
100x10-3
90
80
70
60
50
40
ISFG (a.u.)
Ratio R
CH2SS CH3SS
Figure 4.7. Upper panel: SFG spectra (dot lines) of the diC14-amidine monolayer (52Å2/molecule) on neat water (red) and DNA solution with different concentrations. The vertical lines indicate the positions of methylene symmetric stretch (CH2SS), methyl symmetric stretch (CH3SS). The solid lines represent fits to the data using a Lorentzian model. Lower panel: SFG intensity of CH2SS and CH3SS resonances (opened circle and star, right axis) and their ratio R (square dots, left axis) of diC14-amidine monolayer on DNA solution with various concentrations. The solid line a guide to the eyes.
Lipid monolayer condensation
42
The ratio R resulting from an analyzing procedure with a Lorentzian multiple peaks fitting is
plotted in figure 4.7 (lower panel) as a function of DNA concentration. The starting point of
this curve corresponds to the monolayer on neat water with the value of 0.69. This value is
reasonable: the monolayer was compressed to 52Å2/molecule which is equivalent to surface
pressure of 20-25mN/m (figure 4.2). As shown in figure 4.6, diC14-amidine monolayer at this
condition (20-25mN/m) has the ratio R of ~0.8. The ratio R sharply increases proportionally
to DNA concentrations within small range (DNA concentration<30pM), and above 30pM is
nearly independent on DNA concentration. This trend is similar to the high surface pressure
part of figure 4.6. The steep increase of R in order at low concentration range, saturating at
higher concentrations is in good agreement with the results from monitoring the binding of
DNA to diC14-amidine (chapter 3) and the phase diagram observation (figure 4.3).
4.5. Conclusion
The observation in the change of the order of the diC14-amidine monolayer is complement
for the probing of DNA-lipid binding. DNA binds to diC14-amidine and change the
molecular arrangement at the surface, evident by the squeezing out of interfacial water (see
chapter 3 and 5). With increasing DNA concentration in the solution, the number of hydrated
water around lipid headgroups rapidly decreases and surface charge of the headgroups is
screened. This change is obvious in a small range of DNA concentration at about 10-60pM
then completely saturates above 100pM. Actually, within 10-100pM DNA, the diC14-amidine
transforms from a lipid monolayer to a surface complex together with DNA and then above
100pM, the surface complex will not change any more. Corresponding to this observation,
the ratio R or the order of lipid tails quickly raises upon increasing DNA concentration
(<40pM), then remains constant. This condensation inducing by DNA is in consistence with
the compression isotherms for diC14-amidine on water and DNA solutions (figure 4.3). From
these observations, we conclude that the binding of DNA to diC14-amidine induces the
order of lipid monolayer.
Interfacial water structure
43
CHAPTER 5 INTERFACIAL WATER STRUCTURE
In this chapter, I present our investigation of the water hydrogen bond network at the surface upon the binding of DNA to diC14-amdine. We first report the method used for investigation: monitoring H-bond via tracing fundamental hydrogen bonded OD stretch and a treatment to
simplify water spectrum which is caused by Fermi resonance. Tracing fundamental hydrogen bonded OD stretches shows that a weak hydrogen network of sparse interfacial water appears when the lipid-DNA complex is formed.
5.1. Intermolecular coupling
5.1.1. Hydrogen-bond network
Hydrogen bonding is of great important biological molecules, and certainly for water
molecules. Hydrogen bonding can be defined as the attraction which occurs between a
highly electronegative atom and a hydrogen atom. As the polar inherent property, water
molecules interact with each others through hydrogen bonding (figure 5.1). The oxygen atom
of one water molecule has two lone pairs of electrons, each of which can form a hydrogen
bond with hydrogen atoms on two other water molecules. This can repeat so that every
water molecule is able to form H-bonded with up to four other molecules.
As the frequencies of water modes are known to be highly sensitive to intermolecular
hydrogen bonding between water molecules [35], the vibrational frequency of the hydrogen
bonded O-H stretch is a good reporter of the local H-bonding network of water.
Interfacial water structure
44
Figure 5.1. Hydrogen bonding of water molecules
Physically, the dependence of O-H stretch frequency can be explained by considering the
vibration of a molecular bond as a harmonic oscillator. The molecular dipole moment
oscillates when two atoms of the bond move back and forth with the same frequency. Each
bond can be treated as a spring with the force constant k. With this approximation, the
frequency of the vibration relates to the reduce mass m of two atoms in the bond and the
stiffness of the bond k as
mk
21
Inter- and intra- molecular interaction affects the stiffness k of the bonds and hence alters the
vibration frequencies.
5.1.2. Spectra features of D2O-air interface
Here, we apply vibrational sum frequency spectroscopy (VSFG) to record vibrational bands
of OD stretch which provides a molecular view of interfacial water structure and hydrogen
bonding. Figure 5.2 show a VSFG spectrum of the heavy water-air interface. The sharp peak
at 2725cm-1 (corresponding to 3700cm-1 for H2O [35]) is attributed to the “free OD bond” of
water molecules that protrudes into the air phase and does not form hydrogen bond with
other water molecules. There is a broad band with a hole at frequencies (2300 to 2600 cm-1)
suggests the presence of a hydrogen network at water surface. The mean observations of this
band are: lower in energy, wider in band shape in comparison with the “free OD” peak and
band splits into two peaks. As participating in the hydrogen bonding interaction, the
weakening of OD oscillator results in a red-shift in energy. The broadening of spectral peak
is a common characteristic of a hydrogen bonded system [36] but its causes are still unclear.
Intermolecular collision is one contribution for peak broadening but whether it is a
homogeneous or inhomogeneous broadening might be answer if we can determine the
interfacial H-bond is a fast mode or slow mode. The splitting of the peak will be considered
in the section 5.2.a.
Interfacial water structure
45
100x10-3
80
60
40
20
0
I SFG
/I ref
(a.u
.)
30002800260024002200
Wave number of IR (cm-1)
23882518
2725water-air interface
Figure 5.2. VSFG spectrum of the heavy water-air interface (thin and gray line). The thick and black curve represent fits to the data using a Lorentzian model with multiple peaks (indicated by arrows) at frequencies of 2388cm-1, 2518cm-1 and 2725cm-1 with full width half maximum (FWHM) Γ of 115cm-1, 171 cm-1 and 40cm-1, respectively.
To sum up, the hydrogen bonded OD stretch band contains rich information about the
interfacial water network at a molecular view. Following, we report our investigation of the
change in interfacial water structure and the role of water upon the binding of DNA to lipids
by monitoring hydrogen bonded OD spectra bands.
5.2. Intramolecular coupling
5.2.1. Fermi resonance
The splitting of the interfacial hydrogen bonded OD band causes the complexity for tracing
the change of the H-bond water network species at the surface. Sovago et.al [25] have
suggested a convincing explanation for this splitting. Based on their argument, we employ
the single fundamental OD stretch of deuterated water instead of double peak of pure heavy
water. This choice is motivated in the following.
The hydrogen bonded OD spectra bands from 2300cm-1 to 2600 cm-1 in figure 5.2 is the
resonance at the symmetric stretching mode of O-D bond which is split by an anharmonic
intramolecular interaction. As indicated above, OD stretch of a hydrogen network is an
inherent broad band. The overtone of OD bending mode is a sharp, low intensive vibrational
transition centre at 2405 cm-1 which fall within the broad band of hydrogen bonded OD
stretch. As the overtone (2δOD) close to the hydrogen bonded asymmetric OD stretch (ODss),
Interfacial water structure
46
2δOD bending overtone 2δOD
bending overtone
2δOD bending overtone
ODss stretch
ODss stretch
ODss stretch
(A1) (A2) (A3)
Fermi resonance occur giving rise to the double peaked structure in the SFG spectrum. The
dip in the SFG spectrum between two peaks is named Evans window. Figure 5.3
schematically illustrated the formation of Evans window depending on the energy difference
between ODss and 2δOD vibrational frequencies. The Evans window close 2δOD energy and
slightly shifted to the ODss stretch direction. Qualitatively, as band overtone is far apart
from the ODss stretch band, the Evans hole becomes wide and shadow and then disappears.
Figure 5.3. Fermi resonance between the symmetric OD stretch (ODss) and the second overtone of OD bending mode (2δOD) cause the splitting of spectral peak and forming a Evans hole. A [37]: The position of the 2δOD changes resulting in the different in the coupling of ODss and 2δOD. B[25]: Energy level diagram for the OD vibrational modes of D2O and HDO.
5.2.2. SFG spectra of water and lipid at the interface with the diC14-amidine
monolayer
The SFG spectra of pure heavy water D2O and deuterated water HDO at the interface with
the diC14-amidine monolayer is shown in figure 5.4. There is two-peak feature for D2O but
one-peak for HDO.
For D2O, the spectra feature corresponds to a similar case as illustrated in figure 5.3.A2: the
2δOD state is higher in energy than that of ODss stretch state. The positions of two ODss
peaks of water lipid interface centered at 2379cm-1 and 2509 cm-1 are slightly red shifted from
that of water air interface (2388cm-1 and 2518cm-1). The interaction of water molecules with
Interfacial water structure
47
lipid headgroups is responsible for the shifting of associated ODss stretch frequency. The
lower energy peak is more intensive than the higher one because the center of associated
ODss stretches band places at lower wavenumber than the band overtone.
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
I SFG
/I ref
(a.u
.)
320030002800260024002200
IR frequency (cm-1)
2379
2509
2450
diC14-amidine D/(D+H)=1 D/(D+H)=0.33
Figure 5.4. VSFG spectra of the lipid (diC14-amidine)-water interfaces. For pure heavy water (gray line): there are two peaks (indicated by arrows at frequencies of 2379 cm-1 and 2509cm-1) or a broad band with an Evans hole in the O-D stretch region. For the mixture (black line) of heavy water with light water (H2O:D2O=2:1 or D/(D+H)=0.33): there is only one broad band which fit in well to Lorentzian peak at frequency of 2450cm-
1. Lorentzian spectral fit to data superimpose as thick solid lines.
For deuterated water HDO, the bend fundamental is located at ~1450 cm-1, so that the
overtone is at 2900cm-1, at much than higher frequency than the ODss mode [25]. As a result,
the Evans window does not appear and there is a broad band centre at the fundamental
frequency of OD stretch. For the interface between deuterated water and diC14-amidine, the
fundamental OD stretch frequency was found at 2450cm-1 (figure 5.4, black curve). In
addition, the broad band originated from the hydrogen bonded OH stretch which expected
to centre in the region from 3100cm-1 to 3400cm-1 also appears in the spectrum. A deep,
narrow dip locates at ~2960cm-1 due to the destructive interference of the hydrogen bonded
OH stretch mode of HDO water molecules with asymmetric methylene stretch mode of
diC14-amidine.
5.3. Weak interfacial H-bond network upon DNA-lipid surface complex formation
To effectively monitor the change of the interfacial H-bond network, we trace the
fundamental OD mode of deuterated water. Figure 5.5 (upper panel) shows the VSFG
Interfacial water structure
48
spectra of the diC14-amidine monolayer on neat water (gray) and lambda-DNA solution of
62pM (black). Water was deuterated by mixing D2O with H2O at the ratio of 1:2 or
D/(D+H)=0.33 since at this ratio SFG spectrum of water is dominated by fundamental OD
mode and D2O modes contributes only 10% [25]. In the presence of DNA, water signal at OD
as well as OH modes almost disappear. The significant decrease of water signal was
mentioned in chapter 3 already: interfacial water molecules are not only less ordered due to
the screening of lipid charges but also squeeze out from the interface by the formation of
DNA-diC14-amidine complex. It is noteworthy that SFG spectrum in the C-H vibrations
region look to be decrease due to the reduced constructive interference with OD and OH
resonances. Actually, the amplitudes of C-H stretches do not change, as can be concluded
from Lorentzian multiple peaks fitting procedures. Moreover, a marked appearance of a
band centre at 2540 cm-1 and a sign of band at 2630 cm-1 were observed.
The appearance of high frequency peaks indicates the presence of weak H-bond water
network. As DNA binds to diC14-amidine, charges of lipid headgroups were screened
resulting in a change of hydrophobic property of this complex in comparison with that of the
diC14-amidine monolayer. There is less water molecules hydrated at lipid headgroups
(figure 1.7), presumably leading to a weak hydrogen bonding network of sparse water
confined between lipid monolayer and DNA layer. If we consider the OD oscillator as a
spring, and each spring connects to adjacent springs of neighbouring water molecules by H-
bond, then the weakening of H-bond network results in strengthening of O-D bonds and
thus alters the OD vibration to higher energy.
It is notable that, there are two differences of the appearance of high frequency OD peaks in
the SFG spectra between using the subphase of D2O and HDO. Firstly, the presence of the
peak at 2630 cm-1 is obvious in the spectra for D2O but small for HDO. It is because of the
dilution of deuterium by proton. Secondly, the peak at 2540 cm-1 is not visible in the spectra
of D2O. The reason might be that this peak is buried by two strong H-bonded OD peaks. In
the scope of this study, we have not had an explanation for why SFG signal of strong H-
bonded OD stretch is still visible in D2O spectra but not in HDO spectrum when DNA-lipid
complex is formed.
Interfacial water structure
49
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
320030002800260024002200
IR frequency (cm-1)
diC14-Amidine[lambda DNA]
0 pM 62pM
D/(D+H)=0.33
2540
2461
2630
-0.8
-0.6
-0.4
-0.2
0.0
Im(2
) (a.u
.)
320030002800260024002200
IR frequency (cm-1)
2461
2540
2630
Figure 5.5. Upper panel: VSFG spectra (thin lines) of the diC14-amidine monolayer on neat water (gray) and DNA solution of 62pM (black). Lower panel: Imaginary component of the second order susceptibility χ(2) found by applying the Maximum entropy method to VSFG spectra. The arrows (upper panel) and vertical dash lines (lower panel) indicate the positions of OD stretches with strong hydrogen bond (at 2461cm-1) and weak hydrogen bond (at 2540cm-1). The thick lines in the upper panel represent fits to the data using a Lorentzian model with single peak at 2461cm-1 (without DNA-gray lines) and 2540cm-1 (with DNA-black lines).
To further investigate the orientation of interfacial water, we apply Maximum Entropy
Method (MEM). MEM allows us to retrieve the phase and then calculate the real and
imaginary components of the second susceptibility which is proportional to SFG intensity
[38]. 2
2)2()2(2)2(
n nIRn
niNRRNRSFG i
AeAI NR
[5.1]
Interfacial water structure
50
n nnIR
nn
n nIRn
n
n nIRn
nR
AiAi
A2222
)2(
)()( [5.2]
n nnIR
nnR
A22
)2(
)(Im
[5.3]
As shown in formula 5.3, the sign of imaginary component at resonance ωn is to the same as
the sign of the Amplitude at resonance An. The orientation of transition dipole moment and
thus orientation of vibration group can be inferred from the amplitude An [30].
The MEM procedure applied to SFG spectra of diC14-amidine monolayer on deuterated
water and DNA solution 62pM returns imaginary component of χ(2) shown in figure 5.5
(lower panel). These plots confirm the appearance of a higher OD frequency peak upon the
formation of DNA-diC14-amidien complex. The OD and CH are negative in the Imχ(2
spectrum. This result implies that the interfacial water molecules at diC14-amidine-water
interface are oriented with their oxygen towards the surface, as for DPTAP-water interface
[39]. Furthermore, there is no change in the sign of Imχ(2) indicating that orientation of
interfacial water in the presence of DNA remains as in the absence of DNA. The pointing
down of dipole moment of water molecule (direction of dipole moment point away from
oxygen atom) infers that interfacial water molecules are mainly hydrated underneath lipid
headgroups. When DNA binds to phospholipids, positive charges of lipid headgroups
attract oxygen toward the surface and DNA carrying negative charges attract the hydrogen
moiety of water molecule pointing downward, therefore, the orientation of interfacial water
remain unchanged (figure 1.7).
For a finer determination the change of the interfacial H-bond network upon the binding of
DNA to diC14-amidine monolayer, we record SFG spectrum of diC14-amidine on DNA
solution with different concentrations with deuterated water subphase at ratio H2O:D2O=3:1
or D/(D+H)=0.25. As shown in figure 5.6, with increasing H2O potion in water mixing, the
contribution of D2O to the fundamental OD vibration band decreases and therefore fitting of
SFG spectrum to a single Lorentzian peak more accurate. This experiment confirms the
appearance of the high OD frequency peak. However, there is no gradual shift to higher
energy of OD stretch. This result implies that another type of water network, a weak H-bond
one, appears when DNA-lipid complex formed. This weak hydrogen bonded water network
species is present when major part of interfacial water molecules are squeezed out and leave
a few molecules weakly connected to each other at the surface.
Interfacial water structure
51
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
260025002400230022002100
IR frequency (cm-1)
diC14-Amidine[lambda DNA]
0 pM15 pM 25 pM 34 pM 62 pM
D/(D+H)=0.252461
2540
Figure 5.6. VSFG spectra (thin lines) of the diC14-amidine monolayer on neat water (red) and DNA solution of 15pM, 25pM, 34pM, 62pM. The vertical dash lines (lower panel) indicate the positions of OD stretches with strong hydrogen bond (at 2461cm-1) and weak hydrogen bond (at 2540cm-1). The thick lines represent fits to the data using a Lorentzian model with single peak at 2461cm-1 with FWHM of 300cm-1 (for DNA concentration of 0pM, 15pM and 25pM) and 2540cm-1 with FWHM of 200cm-1 (for DNA concentration of 34pM and 62pM).
Conclusion and outlook
52
CHAPTER 6 CONCLUSION AND OUTLOOK
6.1. Conclusion
In conclusion, we have demonstrated that Vibrational Sum-Frequency Generation (VSFG)
Spectroscopy can be used to probe the DNA-lipid interaction. The vibrational response of
interfacial water provides detail for tracing the interaction of DNA to lipid monolayer. Using
this technique, the DNA-lipid interaction can be monitored in a label-free manner since
interfacial water is used as a natural label for the probing. Moreover, VSFG is sensitive for
probing DNA-lipid interaction because with this technique, we detect the absolute
reorganization of molecules at interfaces upon the binding.
DNA has strong coordination with cationic lipid with association constant of 1010 M-1
(expressed in DNA concentration) but slightly interacts with zwitterionic lipid. The driving
force is the electrostatic force. The interaction solely depends on the number of unit charge
per lipid headgroup regardless of the delocalization of the charge and the length of the lipid
tails. When DNA binds to the lipid monolayer, the surface potential decreases because DNA
screens charges of lipid. In the presence of the DNA-lipid complex at the surface, the
interfacial water density and/or the dielectric constant dramatically reduces. The reductions
of surface potential and water density/dielectric constant result in the sharply decrease of
VSFG water signal.
In addition, the VSFG spectra of lipid CH stretch modes in conjugation with the compression
isotherm provide information about the condensation of the lipid monolayer. DNA binds to
diC14-amidine and induces the monolayer to be more condensed.
Conclusion and outlook
53
Moreover, the appearance of an interfacial weak H-bond network when DNA binds to lipid
is observed. This water network is attributed to be confined between lipid monolayer and
DNA layer. The OD vibrational frequency of this water species appears at higher energy
(2630 cm-1 and 2540cm-1) in comparison with the absence of DNA condition (2450cm-1) due to
the weakness of hydrogen bonds of the sparse water network.
From this study, we suggest that diC14-amidine is a possible vector for gene therapy. It is
proved as non-toxic and does not require helper lipids[23]. We demonstrated here that this
lipid binds strongly to DNA via electrostatic force.
6.2. Outlook
6.2.1. Finding the change of the interfacial water density upon the binding of DNA
to lipid monolayer
The observation in §3.3, the suddenly decrease of VSFG water signal when DNA binds to
lipid monolayer is not fully understood. We point out that there are three possible reasons
for this sharply reduction: the screening effect, the dwindling of the Debye length due to the
diminishment of dielectric constant and the reduction of interfacial water density caused by
the hydrophobicity of DNA/lipid complex at the surface. From a fundamental scientific point
of view, it is desirable to distinguish and quantify these contributions to the change of VSFG
signal.
We propose an experiment approach that is a combination of the measurement of VSFG and
surface potential simultaneously and neutron reflectivity technique. The VSFG procedure is
described above. The surface potential can be measure by the vibrating capacitor method
[27]. The depth of the asymmetric region might be determined by neutron reflectivity [6].
6.2.2. Directly probing the appearance of the DNA layer at the surface
We might directly probe the appearance of the DNA layer at the surface via the C=O and PO2
vibrational modes of bases and phosphate moieties. The vibrational frequencies for PO2
asymmetric and symmetric stretches are 1222cm-1 and 1088 cm-1. The C=O frequencies for
guanine, thymine and adenine are 1717 cm-1, 1663 cm-1 and 1609 cm-1, respectively.
Physically, it is difficult to detect the vibrational sum-frequency generated from these modes
because of the local symmetry along the DNA backbones. With the limitation of our set up in
generating the IR laser at these frequencies, we have not performed experiments to test
Conclusion and outlook
54
whether these mode are IR active. However, it is worthy, in my view, to perform a directly
observation of the appearance of the DNA at the surface.
6.2.3. Controlling the electrostatic interaction by ions
In this study, we probed and characterized the interaction of DNA to lipids. The ultimately
goal is controlling the interaction. Furthermore, it is useful if we can enhance and control the
binding of DNA to zwitterionic lipid because this type of lipid is more biological friendly to
mammal’s cell (cell membranes of mammal consist of zwitterionic and negative
phospholipids).
In a previous study, Sovago et.al [34] addressed that Ca2+ can associate with zwitterionic
lipid (figure 6.1). This result suggests that we may use Ca2+ to control the interaction of DNA
to zwitterionic lipid. Furthermore, by infrared reflection absorption spectroscopy (IRRAS)
measurements, Gromelski et.al stated that the DNA-zwitterionic lipid interaction can be
mediated by divalent cations Ca2+.
Figure 6.1. Schematic of the mediation of Ca2+ for the DNA-lipid interaction. Ca2+ ions are attracted to the negative potions of DPPC headgroup. Therefore the positive components of DPPC effectively attract DNA.
Given above ideas, we conducted VSFG experiments to probe the interaction of DNA with
DPPC with the mediation of Ca2+. The results show that with the increasing of DNA
concentration, the SFG intensity of OD stretches changes with different trends for different
concentration of Ca2+. At 50mM (figure 6.2.a), VSFG of water gradually decrease with adding
more DNA in the subphase. At 5mM (figure 6.2.b), VSFG signal of water increases then
Conclusion and outlook
55
decreases with the raising of DNA concentration. These observations indicate that the effect
of Ca2+ on DNA-zwitterionic lipid interaction depends on Ca2+ concentration. It is necessary
to carry out more measurements and investigation to understand more about the mediation
of divalent cations to DNA-lipid interaction and further reach the goal of controlling the
interaction.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
I SFG
/I ref
(a.u
.)
30002800260024002200IR frequency (cm-1)
DPPC 50mM CaCl2[lambda-DNA]
0pM 26pM 41pM 404pM
D2O
2.0
1.5
1.0
0.5
0.0
I SFG
/I ref (
a.u.
)
30002800260024002200
IR frequency (cm-1)
DPPC5mM CaCl2[lambda-DNA]
0pM 200pM 1000pM 2333pM
D2O
Figure 6.2. VSFG spectra (thin lines) of water and zwitterionic lipid (DPPC) at the lipid/D2O interface on the subphase solutions containing CaCl2 and DNA with various concentrations, all at pD=7.0. Thick lines are fitting curves of VSFG data using multiple Lorentzian peaks global fitting procedure (a) At 50mM: VSFG of water gradually decrease with adding more DNA in the subphase. (b) At 5mM: VSFG of water increase then decrease with the increase of DNA concentration. These observations indicate the effect of Ca2+ on DNA-zwitterionic lipid interaction depends on CaCl2 concentration.
a
b
Dependence of VSFG spectra on experimental condition
56
APPENDIX
DEPENDENCE OF VSFG SPECTRA ON EXPERIMENTAL CONDITIONS
1. Polarization and incident angles
The intensity of VSFG and the ratio of SFG intensity of different vibration modes vary
depending on: the polarization and incident angles of IR and VIS. Here, we briefly report our
examination of these dependences.
0.8
0.6
0.4
0.2
0.0
SFG
Inte
nsity
(a.u
)
30002800260024002200
IR frequency (cm-1)
DPPC on D2O pH=7surface pressure 34mN/mpolarization
ssp ppp
Figure 1. VSFG spectra of water and lipid (DPPC) at the lipid/D2O with different combination of the incoming VIS and IR beams.
The polarization and incident angles of the coming laser define the direction of electric fields
of light. If the electric field is favourable for the transition dipole moment of mode, SFG of
this mode is enhanced. The evidence for this dependence is shown in figure 1. Under the ssp
polarization combination, it is favourable for OD stretch, and for symmetric methylene
stretch mode CH3SS than for its Fermi resonance.
Dependence of VSFG spectra on experimental condition
57
1.2
1.0
0.8
0.6
0.4
0.2
0.0
SFG
Inte
nsity
(a.u
)
30002800260024002200
IR frequency (cm-1)
DPPC on D2O pH=7surface pressure 34mN/mincident angles
ssp VIS 31o, IR 41o
ssp VIS 28o, IR 37o
Figure 2. VSFG spectra of water and lipid (DPPC) at the lipid/D2O with different incident angles of the incoming VIS and IR beams.
If the angle between the electric fields of IR and VIS and the transition dipole moment is
small, SFG intensity is large. Figure 2 illustrates the dependence of SFG spectra on the
incident angles.
2. Be careful with the impurity of solvent
With different degrees of isotopic dilutions, the feature of VSFG spectral of water gradual
change with the disappearance of the double-peak with decreasing D2O fraction. The
intensity of SFG intensity of OD stretch band from 2200cm-1 to 2600cm-1 is contributed from
the double peak of D2O and single peak of HDO. With increasing H2O potion in the D2O:H2O
mixture, SFG spectra of water is decrease in intensity and change in shape. It is shown in
figure 3 that a small percentage (6.2% impurity) of H2O contaminated in D2O results in a
substantial change (decrease by 50%) of SFG spectra. Therefore, when performing
experiment we should be aware of the impurity of D2O.
Dependence of VSFG spectra on experimental condition
58
1.2
1.0
0.8
0.6
0.4
0.2
0.0
SFG
Inte
nsity
(a.u
)
30002800260024002200
IR frequency (cm-1)
diC14-amidine% of H
0% 2.2% 6.2% 16.6% 28.5%
D2O
Figure 3. VSFG spectra of water and lipid (diC14-amidine) at the lipid/heavy water with different impurities of D2O by H2O.
59
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