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

ii

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

16 16 16 19

20 22 22 26 28 29 31

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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38 39 40 42

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Introduction

1

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

2

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

3

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

4

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

5

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

6

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

7

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

8

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

10

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).


12

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


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


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


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


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


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


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.


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]


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.


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


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]


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]


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]


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]


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.


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


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:


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


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.


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


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


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,


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


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


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.


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.


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


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.


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.


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 intramolecular 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.


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),


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


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


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.


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]


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.


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.


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


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


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.


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.


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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