Effect of deoxyribonucleic acid interaction on the interfacial properties of a fluid functionalized lipidic matrix

Thin Solid Films 483 (2005) 319 – 329 www.elsevier.com/locate/tsf

Effect of deoxyribonucleic acid interaction on the interfacial properties of a fluid functionalized lipidic matrix Daphne´ L. Thomas, LoRc J. Blum, Agne`s P. Girard-EgrotT Laboratoire de Ge´nie Enzymatique et Biomole´culaire, UMR 5013/EMB2-CNRS/UCBL, Universite´ Claude Bernard Lyon 1, 43 bd du 11 novembre 1918, F-69622 Villeurbanne Cedex, France Received 21 July 2004; accepted in revised form 17 January 2005 Available online 19 February 2005

Abstract This report describes the effects of deoxyribonucleic acid (DNA) on the interfacial properties of an active lipidic matrix designed for polynucleotide immobilization. The synthetic lipid DiOctadecylamidoGlycylSpermine (DOGS) was spread at the air–water interface to form a functionalized film capable of capturing DNA molecules present in the subphase, as confirmed by Attenuated Total Reflection-Fourier Transform InfraRed spectroscopy. The predominance of electrostatic interactions in the DNA adsorption process onto the DOGS monolayer was demonstrated by changing the subphase ionic strength. In the presence of various DNA concentrations in the subphase and with increasing incubation times the compression isotherms of the DOGS monolayer were progressively modified, which suggested a change in monolayer fluidity. This finding was corroborated by the direct observation of the mixed monolayer morphology by Brewster Angle Microscopy. Taken together, these results suggest a reorganization of lipids at the interface following DNA adsorption. D 2005 Elsevier B.V. All rights reserved. PACS: 68.10.J Keywords: Langmuir monolayers; Surface pressure; Fourier transform infrared spectroscopy (FTIR); Brewster angle microscopy (BAM)

1. Introduction DNA molecules have unique recognition capabilities that can facilitate the construction of elaborate functional nanostructures and nanodevices for various nanotechnological applications. Beyond the now well-developed microarrays for genomics, the elaboration of DNA-modified surfaces has received considerable attention, as demonstrated with the variety of solid materials including glass, [1–4] silicon, [5– 9], metal surfaces [10,11] or synthetic polymers [12] used for the immobilization of DNA molecules. Besides that aspect of nanobiotechnology, there is current interest in using biomolecules as elementary structures to control the self-assembly of superstructures of predefined geometry. The potential of these two-dimensional molecular selfassemblies is clearly illustrated by Langmuir monolayers of T Corresponding author. Tel.: +33 472 448 532; fax: +33 472 447 970. E-mail address: [email protected] (A.P. Girard-Egrot). 0040-6090/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.tsf.2005.01.017

lipid molecules, which have been extensively used as models to understand the role and organization of biological membranes, and to acquire knowledge about the molecular recognition process [13–16]. The various advantages offered by this interfacial technology have since extended its initial use to nanoscale applications requiring a controlled immobilization of biomolecules onto a highly organized molecular film, through the fine control of the superstructure [17]. Recently, the study of spontaneous electrostatic interactions of biomolecules with charged Langmuir monolayers at the air–water interface has demonstrated interesting applications in various contexts such as the study of therapeutic molecules [18,19] or the immobilization of biomacromolecules such as DNA on a fluid medium [20– 25]. Immobilizing polynucleotides on a thin film at the air– water interface preserves the geometry and integrity of DNA molecules, as demonstrated by X-ray and neutron reflectometry techniques applied to liquid systems [26], when compared to deposited mixed monolayers including poly-

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nucleotides onto solid supports. A number of recent studies at the interface have indeed shown that DNA forms a compact layer adsorbed on cationic lipid monolayers. The average thickness of such a compact layer of DNA is 25 2, which is close to the diameter of 25–28 2 for a DNA molecule, compared to the value of 9–11 2 obtained for DNA kept in a dried state when transferred onto a solid support [27–29]. Additionally, particular polyvalent cations known to interact strongly with DNA, such as spermine, spermidine, Mn2+ and Co3+(NH3)6, have been shown to condense DNA molecules in such a way that DNA molecules have high degrees of freedom, as demonstrated by Atomic Force Microscopy studies [30]. From these results, our goal is to design a functionalized lipid monolayer allowing strong interactions of DNA at the air–water interface, and exploiting the efficiency of these well-characterized lipid–DNA electrostatic interactions, combined to the advantages of an interfacial study. In this case, the monolayer behaves as an active matrix playing the role of a soft support to immobilize DNA molecules, thereby allowing the investigation of these lipid–DNA interactions through the accurate control of its molecular organization. The focus on the two-dimensional state of this interfacial film upon interaction with DNA molecules will thus provide direct evidence for the presence of these polynucleotides. In the system used in this paper, the monolayer is formed by a cationic lipid, DiOctadecylamidoGlycylSpermine (DOGS), which is known to present a high affinity for both types of single-stranded and doublestranded DNA (105–107 M
1) due to a functionalized spermine headgroup able to anchor in the minor groove of DNA [31]. This lipid, initially used for cellular transfection [32–34], is spread at the air–water interface to form a monomolecular film able to specifically capture DNA strands by means of electrostatic interactions. In the present work, particular physico-chemical conditions are investigated in order to study the effect of DNA interaction on the interfacial properties of the DOGS monolayer, which can then be explored to demonstrate the effective immobilization of these macromolecules. When this film is allowed to interact with DNA molecules present in the subphase, the progressive adsorption of DNA strands onto the monolayer can be followed owing to the minute monitoring of surface pressure–area (p–A) isotherms, lateral compressibility traces and surface pressure–time (p–t) isotherms. The adsorption of DNA molecules onto the monolayer is then assessed by studying the effect of increasing ionic strength on the compression isotherms. This mixed DNA–lipid film is further characterized by Attenuated Total Reflection-Fourier Transform InfraRed (ATR-FTIR) spectroscopy after transfer onto a solid substrate at different pressures (Langmuir– Blodgett deposition). Finally, this study is completed with a direct observation at the air–water interface by Brewster Angle Microscopy (BAM), thus providing morphological information on the monolayer interfacial behavior upon interaction with DNA.

2. Experimental section 2.1. Materials DiOctadecylamidoGlycylSpermine (DOGS, N98% purity, Fig. 1) from Promega was solubilized in pure chloroform at about 1 mM. Calf thymus DNA (ctDNA, N98% purity) was purchased from Sigma and diluted in water to reach a final concentration of 10-6 M of DNA strands. All the solutions and subphases containing various DNA concentrations were prepared from this stock solution using ultrapure water having a resistivity of 18.2 MV cm (Milli-Q, Millipore). 2.2. Monolayer technique and surface pressure–area measurements The surface pressure–area experiments were performed on a computer-controlled KSV LB-3000 Langmuir trough enclosed in a cabinet permanently maintained under purified dry air flow (KSV Instruments, Finland). The surface pressure–area (p–A) isotherms were recorded by the Wilhelmy method, using a platinum plate as a surface pressure sensor. All experiments were performed at constant temperature using a cryostat. DOGS molecules were spread at the air–water interface (pH 5.6) to reach a final quantity of 50 nmol of lipids. The solvent evaporated within 15 min, leaving a monomolecular lipid layer on the subphase surface. This monolayer was then submitted to a symmetrical compression with barriers moving at a constant speed of 0.067 nm2 molecule
1 min
1 to produce a p–A isotherm diagram. In order to characterize the interfacial properties of a DOGS monolayer, a series of experiments was carried out by varying the temperature of a pure water subphase in the range of 12–35 8C. For all other experiments, the subphase temperature was maintained at a constant value of 20.5F0.5 8C. The effect of DNA on the interfacial properties of the functionalized monolayer was then investigated by spreading the DOGS solution on different subphases containing DNA concentrations rising from 10
10 to 10
6 M. The incubation time varied from 1 to 20 h. The monolayer was first (t 0+1 h) compressed up to a +

+

O N C

NH3

NH

C

O

NH2

+

+

NH2

NH3

Fig. 1. Structure of DiOctadecylamidoGlycylSpermine (DOGS).

D.L. Thomas et al. / Thin Solid Films 483 (2005) 319–329

limiting surface pressure of 45 mN/m (below the collapse point), then allowed to expand up to a fully expanded state (max trough area) and rest up to the next compression (t 0+5 h). The same protocol was repeated at t 0+10, 15, and 20 h. The analysis of the isotherms focused on two parameters showing important modifications in the presence of DNA in the subphase. These parameters were: (i) the variation in the mean molecular area compared to the control isotherm carried out on a pure water subphase, and (ii) the lateral compressibility. The two-dimensional surface compressibility values were directly calculated from the pressure–area data obtained from the monolayer compressions using the following equation [35] Cs ¼
1=Að dA=dpÞt where A is the molecular area at the corresponding surface pressure p. High C s values correspond to a high interfacial fluidity among packed lipids forming a monolayer [36]. This finding suggests that the lower the C s value of a monolayer, the more difficult to deform it, the minimum C s value representing a highly rigid state. As a control, the same experiments were carried out with a non-functionalized monolayer of neutral lipids DSPE (DiStearylPhosphatidylEthanolamine) to check the specificity of DNA binding. The series of experiments investigating the effect of ionic strength on DNA interaction were performed on aqueous subphases containing NaCl concentrations varying from 10
5 to 1 M, supplemented or not with ctDNA 10
8 M. All experiments were reproduced 4 to 5 times, the isotherm curves presented here being characteristic of a mean of 5 runs.

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transfer technique. After 1 h of incubation on a water subphase supplemented (or not) with ctDNA 10
8 M, the DOGS monolayer was compressed at a rate of 0.067 nm2 molecule
1 min
1, up to a target surface pressure value of 20 mN/m. The pressure was then allowed to stabilize for an appropriate relaxation time, prior to substrate withdrawal from the subphase (0.067 nm2 molecule
1 min
1), leading to the deposition of one single monolayer onto the solid substrate. Each transfer is characterized by a transfer ratio, calculated from the film surface removed and the substrate area immersed. To evaluate the effects of ionic strength on DOGS–DNA interaction, the same experiments were performed by supplementing the subphase with either 5.102 M or 1 M NaCl. Control experiments for specific adsorption were carried out on a DSPE monolayer according to the same protocol. The ctDNA spectrum was obtained by depositing a drop of DNA 10
8 M on each side of the crystal. After deposition, the crystal was dried under a filtered dry airflow and placed in the spectrometer equipped with a DGTS room temperature detector continuously purged with dry air from a Balston air purifier. The transmission–absorption spectra were obtained at normal incidence and recorded with a 510 M FTIR spectrophotometer (Nicolet instruments, France) operating at a 4 cm
1 resolution. The background signal, measured afterwards with the cleaned germanium plate, was then substracted. The Ge crystal had a refractive index of 4, and allowed for 8 internal reflections. Each spectrum resulted in the accumulation of 150 scans, collected in the single beam mode. 2.5. Brewster Angle Microscopy

2.3. Surface pressure–time measurements This series of experiments was carried out on a circular teflon trough having an area of 28 cm2 and a volume of 7 mL. The surface pressure was recorded with a filter paper Wilhelmy plate. In order to obtain an interfacial film with a definite surface pressure, lipids were spread drop by drop at the air–water interface until the desired pressure was reached. In the second step, after the monolayer stabilization, various volumes of DNA solutions were injected into the subphase and allowed to interact with the functionalized monolayer under mechanical stirring (100 rpm). The surface pressure increase (Dp) was recorded during 5 h for each DNA concentration, and plotted as a function of time. Control experiments were performed similarly with a DSPE monolayer, and without any monolayer at the interface. 2.4. Langmuir–Blodgett technique and ATR-FTIR spectroscopy Attenuated Total Reflection-Fourier Transform InfraRed spectroscopy measurements were performed after transfer of the monolayer onto a germanium ATR crystal by vertical dipping, according to the Langmuir–Blodgett

Brewster Angle Microscopy was used for microscopic observation of the functionalized monolayer morphology at the air–water interface, during the compression isotherms of the DOGS monolayer on different subphases. This microscopy technique uses the zero reflectance of an air–water surface for parallel polarized light at the Brewster angle of incidence (538 for the air–water interface). The condensed phase of a monolayer leads to a measurable change in reflectivity, thus allowing the visualization of the monolayer morphology. The physical principle of the BAM is described in detail in many papers and reviews [37,38]. In our study, the use of combined p–A isotherms and BAM measurements provides complementary information on the structural behavior of the functionalized lipids upon interaction of DNA molecules in the subphase. A Brewster Angle Microscope (BAM2Plus, Gfttingen, Germany) was mounted on a film balance (R&K Wiesbaden, Germany) equipped with a Wilhelmy filter paper. The spatial resolution of the microscope was 2 Am and the images (572768 Am) were taken with a CCD camera at various surface pressures during monolayer compression. The shutter speed of the CCD camera was adjusted to a value of 1/50 s for control measurements, and had to be increased

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to 1/120 s for the monolayer in the presence of DNA because of the steep increase in reflected light intensity.

3. Results and discussion 3.1. Characterization of DOGS monolayer interfacial properties As our study is proposing to investigate the effects of DNA interaction on the in-plane-elasticity behavior of a charged monolayer, it was first necessary to thoroughly determine the interfacial properties of this DOGS monolayer spread at the air–water interface. These preliminary control experiments carried out at different temperatures gave rise to a series of isotherm diagrams shown in Fig. 2A. The clear dependency of the isotherm shape as a function of temperature points out the existence of a phase transition separating two distinct physical states of the lipidic film, the liquid-expanded state (LE) and the liquid-condensed state (LC). This LE–LC phase transition exhibits a typical behavior of numerous complex lipids, since the use of

70

π collapse (mN/m)

Surface pressure (mN/m)

A

60

h g

50 40

f e

d c

30

b

20

80 70 60 50 40 30 0

10 20 30 40 T (ºC)

↑T

3.2. Modifications in p–A isotherm diagrams and monolayer compressibility upon DNA interaction

a

10 0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Mean molecular area (nm2/molecule) Surface pressure (mN/m)

B

50

e

40

h f

30

g d

c

↑T

b a

20 10 0 0

20

higher temperatures increases the initial surface pressure and reduces the initial molecular area values corresponding to the transition plateau [39]. Moreover, by extrapolating the liquid-condensed part of the isotherm diagram to zero pressure, it can be deduced that the mean limiting molecular area occupied by one DOGS molecule is about 0.55 nm2 when organized in an ordered structure. This value is in accordance with the predicted value taking into account the steric hindrance of polar headgroups. At 20 8C, the DOGS monolayer collapsed at a surface pressure of 58 mN/m. The decrease in collapse pressure according to the temperature increase (inset Fig. 2A) is in good agreement with the typical phospholipid behavior. A fine analysis of the twodimensional compressibility parameter was then used to more carefully characterize the LE–LC phase transition (Fig. 2B). The overall shape of the p–C s control curve reveals a progressive decrease in monolayer compressibility upon compression, preceding an abrupt increase in the C s value. As the magnitude of this peak varies with temperature and surface pressure in a manner that is similar to phospholipid phase transition, this maximum C s is attributed to the phase transition of the DOGS monolayer. Furthermore, it can be stressed that the analysis of the LE–LC phase transition by compressibility is more accurate and sensitive than the direct analysis on the p–A isotherm, particularly when the phase transition is shortened (highest temperatures) [40]. Lateral compressibility will thus provide a sensitive tool for analyzing the phase transition, and will be further used in this study to characterize the interactions of charged biomolecules with a functionalized monolayer.

40

60

80

100x10

-3

Compressibility Cs (m/mN) Fig. 2. Surface pressure–area (p–A) isotherms (A) and surface pressure– compressibility (p–C s) curves (B) of a DOGS monolayer on a water subphase, as a function of subphase temperature: (a) 12 8C, (b) 15 8C, (c) 18 8C, (d) 20 8C, (e) 22 8C, (f) 25 8C, (g) 30 8C, (h) 35 8C. Inset: p collapse versus temperature.

In order to demonstrate the effects of DNA interaction on the functionalized monolayer properties, our attention focused on p–A and p–C s isotherms in combination to perform the careful analysis of interfacial parameters. On one hand, surface pressure–area isotherms provide general information on the monolayer behavior upon compression, and on the other hand, compressibility studies allow a significant increase in the phase transition detectability and give additional information on the monolayer fluidity [41,42]. Fig. 3A shows some typical p–A isotherm curves recorded during the compression of DOGS monolayer, after various incubation times in the presence of different DNA concentrations in the subphase. After a short period of incubation (1 h), the main modifications occurring in the monolayer in the presence of a high DNA concentration (line 3), consist of an enlargement in the mean molecular area per molecule and a clear fading of the phase transition. After a long-term incubation (10–20 h), these characteristic changes are emphasized, as shown with the complete disappearance of the phase transition. The use of increasing incubation times also allows the detection of smaller quantities of polynucleotides (line 1). The time-dependent

D.L. Thomas et al. / Thin Solid Films 483 (2005) 319–329

Surface pressure (mN/m)

A

50

12

50

3

12

t0 + 1 h

40 30

t0+ 10 h

50

40

40

30

30

0 20

20

10

10

0

3

0

0.4

0.8

1.2

1.6

2.0

2.4

Mean molecular area (nm2/molecule)

∆Molecular area (nm2)

B

0,8 0.8 0,7 0.7

t0+ 20 h

3

0

10 0 0

0.4

0.8

1.2

1.6

2.0

2.4

Mean molecular area (nm2/molecule) 0.8 0,8 0.7 0,7

π = 20 mN/m

0,6 0.6 0,5 0.5

0.6 0,6 0.5 0,5

0,4 0.4 0,3 0.3 0,2 0.2

0.4 0,4 0.3 0,3

0,1 0.1 00

1 2

20

0 0

323

0

0.4

0.8

1.2

1.6

2.0

2.4

Mean molecular area (nm2/molecule)

π = 40 mN/m t0 + 1 h t0 + 10 h t0 + 20 h

0.2 0,2 0.1 0,1 10M 1010-10

x -10 5.10

10x-9

x -9 5.10

x -8 10

DNA concentration (M)

00

1010-10 M 10

x -10 5.10

10x -9

x -9 5.10

x -8 10

DNA concentration (M)

Fig. 3. (A) Surface pressure–area (p–A) isotherm of DOGS monolayer as a function of incubation time and DNA concentration. The compression isotherms were recorded after different incubation times varying from 1 to 20 h (t 0+1, 10, 20 h), on a subphase containing (0) pure water, supplemented with (1) ctDNA 10
10 M, (2) 10
9 M and (3) 10
8 M, at a constant temperature of 20 8C. (B) Variation of molecular areas at two different surface pressures, 20 and 40 mN/m, analyzed from the p–A isotherms of DOGS monolayer spread on a water subphase containing various DNA concentrations for different incubation times.

expansion of the film observed for the intermediate concentration of 10
9 M (line 2) gives a clear indication on the kinetic of nucleotide immobilization process onto the lipidic matrix. Fig. 3B focuses on the effect of time and DNA concentration on the film expansion amplitude for two different organization states corresponding to surface pressures of 20 and 40 mN/m (LE and LC respectively). This diagram suggests the progressive adsorption of DNA molecules onto the functionalized monolayer, until a probable saturation of the spermine sites occurs for high DNA concentrations. However, it has to be pointed out that the surface pressure of 20 mN/m shows enhanced variations due to the fluid state of DOGS molecules in the monolayer at 20 8C. This less compacted organization coupled to the use of a long incubation time thus enhances DNA immobilization. As a control, experiments carried out on a non-functionalized monolayer of DSPE did not show any modification in the p–A isotherm curves (data not shown). To better characterize the manifest change in the lateral packing properties of the DOGS monolayer upon DNA interaction, the two-dimensional compressibility was investigated in details. Fig. 4 shows the compressibility (C s) curves as a function of surface pressure (p) calculated from the p–A isotherms obtained for various DNA concentrations, after an incubation time of 1 and 20 h. In the presence of DNA in the subphase, the compressibility profile is markedly modified at two specific levels in a manner that is dependent of both DNA concentration and incubation time.

After a 1 h-long incubation with a saturating DNA concentration (line 3), the DOGS monolayer compressibility is reduced for low surface pressure values (0–5 mN/m), and the peak corresponding to the phase transition is clearly shifted towards higher surface pressure values (42 mN/m). These opposite effects seen in the compressibility profile suggest an ambivalent behavior of the lipids upon interaction with DNA, consisting of a condensing effect occurring at low surface pressures (in the fluid phase), and a fluidizing effect occuring at higher surface pressures (in the condensed phase). After an incubation period of 20 h, the condensing effect is enhanced for the lowest concentration (line 1), and the large peak corresponding to the phase transition has completely disappeared for DNA concentrations ranging from 10
9 to 10
8 M (lines 2 and 3, Fig. 4). These findings suggest that the interaction of DNA strands induces a progressive rearrangement of the DOGS molecules in the interfacial film, and finally the monolayer remains in the LE state throughout compression (e.g. DOGS molecules stay in a fluid conformation during the whole compression), as suspected from the p–A isotherms analysis. Both the condensing effect at low surface pressures and the fluidizing effect at high surface pressures can be linked to the polyanionic nature of DNA, which can, by direct interaction with the DOGS molecules, induce a modification of the lateral packing of the monolayer in a manner dependent on the state (fluid or condensed) of the monolayer. Indeed, the electrostatic

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Surface pressure (mN/m)

50

t0+ 1 h

40 30 20 10

2 3

Condensing effect at low surface pressures

1 0

0 0

5

10

15

20

25

30

35

40

45 50x10 -3

Compressibility Cs (m/mN)

distance between the lipid headgroup, thus inducing a condensation effect. On the opposite, the fluidizing effect observed at high surface pressures can be explained by an enlargement of the intermolecular distance between the spermine groups, due to their anchoring in the DNA groove. It thus hinders the close contact between the lipid molecules and consequently prevents the hydrocarbon chain crystallization, which normally induces the transition towards the LC phase. Indeed, the fact that individual DOGS molecules dispose of a wider area enhances the alkylated chains mobility and thus cause a global increase in monolayer fluidity. Hence, DNA immobilization has a strong impact on the in-plane-elastic properties of DOGS monolayer.

Surface pressure (mN/m)

50 t0+ 20 h

40

Fluidizing effect at high surface pressures

30 20

3

2 1

10

0

0 0

5

10

15

20

25

30

35

40

-3 45 50x10

Compressibility Cs (m/mN) Fig. 4. Compressibility profiles calculated from the p–A isotherms after 1 and 20 h of incubation in the presence of (0) pure water, (1) ctDNA 10
10 M, (2) 10
9 M and (3) 10
8 M.

interactions which can establish between the nucleotidic polyelectrolytes and the polycationic lipid headgroups acting as anchoring points for DNA, are suspected to bridge one DOGS molecule to another. In this way, the bridge effect can affect the molecule distribution in the low compressed monolayer and reduce the intermolecular

3.3. Direct evidence for DNA immobilization by ATR-FTIR spectroscopy measurements In order to demonstrate the presence of DNA interacting with the functionalized film, an IR characterization was performed after transfer onto a solid ATR substrate. Attenuated Total Reflection-Fourier Transform InfraRed spectroscopy indeed represents a powerful and highly sensitive method to provide information about the structure properties of a material on a submolecular level and particularly to study structural aspects of lipids–DNA complexes [43,44]. The IR spectra analysis focused on a specific spectral region, 1800–800 cm
1, shown to contain several characteristic peaks of strong intensity present in the ctDNA spectrum, essentially due to the phosphate backbone of DNA [45]. As seen in Fig. 5, the IR spectrum of the DOGS monolayer incubated with ctDNA (full line) reveals additional bands not observed in the IR spectrum of a single DOGS monolayer (dashed line). These bands were found in the ctDNA spectrum recorded previously (data not shown) and were mainly attributed to the phosphate groups of ctDNA. The main band of strong intensity located at 1690

B

A 0.0005

0.0005

0

Absorbance

Absorbance

0

1700.0

1650.0

Wavenumber (cm-1)

1200.0

1100.0

Wavenumber (cm-1)

Fig. 5. ATR-FTIR transmission spectra in the regions (A) 1750–1600 cm
1 and (B) 1275–975 cm
1 of a DOGS monolayer transferred at a surface pressure of 20 mN/m on a germanium crystal. The experiments were performed (a) on a pure water subphase (control), (b) supplemented with ctDNA 10
8 M after an incubation period of 1 h.

D.L. Thomas et al. / Thin Solid Films 483 (2005) 319–329

cm
1 (Fig. 5A) can be either assigned to CjO stretching (main contribution) or PjO stretching of the phosphate backbone of DNA; the bands of strong and medium intensities in the region 1225–1000 cm
1 (Fig. 5B), were assigned to ctDNA as followed: 1225 cm
1 band was attributed to PO2
asymmetric stretch, 1065 cm
1 to PO2
symmetric stretch, and 1013 cm
1 was either assigned to CO or phosphodiester stretch of DNA backbone. Additionally, the LB transfer of the DOGS monolayer onto the solid substrate gave rise to a mean transfer ratio of 1.09F0.03 in the presence of DNA in the subphase, accounting for a good transfer efficiency of the lipidic film interacting with DNA. All these results attest for the presence of DNA molecules on the monolayer, interacting with a sufficient strength with the lipidic matrix so as to keep binding during the compression at the air–water interface, and during the transfer onto a solid support. 3.4. Investigations of DNA–monolayer interactions: adsorption kinetics measurements and effect of ionic strength The electrostatic nature of DNA–DOGS interactions has been assessed respectively by following adsorption kinetics at the interface and investigating the effect of the subphase ionic strength. First, the adsorption phenomenon was studied by recording the lateral pressure variation (Dp) of the initially compressed DOGS monolayer (10–40 mN/m) after DNA injections in the aqueous subphase (Fig. 6). A control p–t measurement was carried out by injecting DNA molecules in the absence of the DOGS monolayer at the interface. In that case, regardless of the concentration, the smooth, flat trace corresponding to a Dp closed to zero demonstrated the complete lack of affinity of DNA for the air–water interface (data not shown). Conversely, in the presence of the functionalized lipid interface, the surface

Surface pressure (mN/m)

60

d

50 40

c

30

b

20

a

10 0 0

40

80

120

160

200

240

Time (min) Fig. 6. Kinetics of DNA interaction with the DOGS monolayer: variation of the surface pressure after injection of DNA into the subphase at t=0 min. The lipidic solution was deposited on a water subphase containing DNA 10
8 M (22 8C) so as to reach an initial surface pressure p i of (a) 10 mN/m, (b) 20 mN/m, (c) 30 mN/m, (d) 40 mN/m. Control experiments performed on a neutral monolayer of DSPE showed no change in surface pressure after injection of DNA.

325

pressure increases significantly upon DNA injection, showing a characteristic adsorption kinetics profile. One interesting point lies in the fact that, for a given DNA concentration, the extent of surface pressure variation is constant whatever the initial surface pressure of the lipid film. Indeed, as previously shown for proteins, the penetration of molecules into a lipidic monolayer at the interface is known to induce a shift in lateral pressure, which decreases significantly when the initial pressure value is increased [46]. This is due to the fact that the increasing condensation of the molecular arrangement upon compression prevents molecules from penetrating into the monolayer. Here, however, the shift in surface pressure upon injection of DNA in the subphase is equivalent (Dpc10 mN/m) in the presence of a loosely packed (low surface pressures) or a highly condensed (high surface pressures) lipid matrix. This finding strongly suggests that, whatever the organization state of the monolayer, these p–t isotherm modifications cannot be attributed to the insertion of nucleotides into the lipid film, and do rather result in a change in lipid organization upon adsorption of the charged polynucleotides with the ionized headgroups. After the initial shift, the surface pressure is constant, suggesting the great stability of these DOGS–DNA interactions all over the time, except for the higher surface pressure (40 mN/m) for which the monolayer is less stable and consequently less favorable for long-range DOGS–DNA interactions. In order to evaluate the electrostatic contribution in DOGS–DNA interactions, compression isotherms of DOGS monolayer were recorded on pure water subphases containing (or not) ctDNA, supplemented with various NaCl contents (Fig. 7). In the absence of DNA, the use of increasing ionic strengths was shown to induce a small shift of the p–A isotherm towards larger molecular areas, in an extent directly correlated to NaCl concentration. A similar dependence of the isotherm shapes on the subphase electrolyte concentration was observed earlier for other types of lipids [47–49]. In the first approximation, this tends to indicate that the subphase ions have an expanding effect on the DOGS monolayer. Such an expanding effect due to salts has been previously shown with a phosphatidylcholine monolayer, and was attributed in this case to a different orientation of the positive ammonium groups of choline headgroups upon electrostatic interactions [50]. In the presence of DNA however, two kinds of effects can be distinguished, depending on the importance of ionic strength. For low ionic strengths and for a given DNA concentration (10
8 M), the compression isotherms get drastically modified, whereby the increase in NaCl content up to V5.10
2 M causes a noticeable shift in the mean molecular area as well as a displacement of the phase transition towards higher pressure values. The use of the optimal concentrations of [NaCl]=10
2 and 5.10
2 M gives rise to an isotherm characteristic of the shape of isotherms recorded after longer incubation times in ctDNA 10
8 M (20 h, i.e. Fig. 3), illustrating the kinetic effect of low salt

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A Surface pressure (mN/m)

70 60

h g f

50 40

e 30

d c

20

b

a

10 0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Mean molecular area (nm2/molecule)

Surface pressure (mN/m)

B

70

h

60

g

f e

50 40

d

c b

30

a

20 10 0 0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Mean molecular area (nm2/molecule)

1.6

Fig. 7. Effect of ionic strength on surface pressure–area (p–A) isotherms of DOGS monolayer as a function of [NaCl]: (a) 0, (b) 10
5 M, (c) 10
3 M, (d) 10
2 M, (e) 5.10
2 M, (f) 10
1 M, (g) 5.10
1 M, (h) 1 M. The compression isotherms were recorded 1 h after lipid deposition, on a subphase containing either pure water (A), or water supplemented with ctDNA 10
8 M (B), at a constant temperature of 20 8C.

concentrations on DNA binding. This suggests that the expanding effect upon DNA binding is enhanced at low ionic strength. Higher ionic strengths however have a completely opposite effect on DNA interaction, since the use of NaCl concentrations from 10
1 to 1 M significantly decreases the mean molecular area, in such a way that the isotherms tend to adopt a global shape similar to the isotherms obtained without any DNA molecules in the subphase. This suggests that high ionic strengths probably cause the majority of DNA molecules to desorb from the monolayer. It is likely that the rest of the molecules are still being associated to the membrane through other types of interactions, as demonstrated by the incomplete reversibility of the isotherms for the highest salt concentrations. In order to confirm the effects of ionic strength on DOGS–DNA interaction, additional experiments were performed using ATR-FTIR spectroscopy. DOGS monolayers were incubated with ctDNA 10
8 M, in the presence of two specific NaCl concentrations, 5.10
2 M or 1 M, and transferred afterwards onto a solid ATR substrate. On the spectra

recorded, the focus was drawn on the 1690 cm
1 band, which was found to reveal the presence of ctDNA (Table 1). Upon addition of NaCl 5.10-2 M the intensity of this band is markedly increased (R=+1.21), suggesting a larger quantity of DNA was immobilized in this case. Conversely, this intensity is reduced in the presence of NaCl 1 M (R=
0.43), accounting for a significant decrease in DNA immobilization efficiency at high ionic strength. In accordance with the Gouy and Chapman theory, largely investigated for the case of anionic monolayers in presence of monovalent cations, some hypotheses can be proposed to explain such results [51,52]. Indeed, the presence of salts in aqueous phases has been shown to induce a local change in pH in the direct vicinity of the monolayer. When applying this theory to the case of a cationic layer, one can devise a model where, as a consequence of the massive positive charge accumulation at the interface, Cl
ions concentrate in the double diffuse layer. In a pure water subphase, the spermine headgroups of DOGS molecules are expected to oscillate between +3 and +4 positive charges, as seen with the pKa of the last primary amine, around 6.5 for the spermine headgroup. The massive Cl
concentration would thus modify the interfacial pH in such a way that the spermine group definitely acquires the additional positive charge. This excess in global positive charge of the monolayer would thus be responsible for a higher interaction between the DNA minor groove and spermine groups, with an increased efficiency and stability. Morevover, the electrostatic repulsion due to additional protonated amines explains the monolayer expansion occuring on NaCl aqueous subphases, thus favoring DNA interaction. It cannot be excluded though, that the presence of salts in the bulk of the subphase might have a stabilizing effect on DNA conformation in water. At high salt concentrations, the electrostatic interactions were significantly decreased, similar to traditional observations for ionic interactions. These findings, taken together with the adsorption kinetics performed at various p i, clearly revealed a relevant occurrence of electrostatic interactions during the DNA adsorption process onto the spermine groups of the DOGS monolayer.

Table 1 Quantitative effect of the subphase ionic strength on the DOGS–DNA interactions

DOGS/ctDNAc-pure water DOGS/ctDNAc-NaCl 5.10-2 M DOGS/ctDNAc-NaCl 1 M a

Intensity at 1690 cm
1a (Given in absorbance unit *10
3)

Relative evolution ratio (R)b

12.7 28.1 7.3

0 +1.21
0.43

m CjO and m PjO of phosphate backbone. Related evolution of the band intensity located at 1690 cm
1 (R=(I 1
I 0)/I 0) calculated using the intensity of 12.7 obtained on pure water as the reference value (I 0). c [ctDNA]=10
8 M. b

3.5. Morphological characterization of the mixed DNA/ DOGS monolayer at the air–water interface by Brewster Angle Microscopy As it allows the direct visualization of the two-dimensional organization of a lipid film at the interface without affecting it with probes or labels, the Brewster Angle Microscopy technique is well suited for investigating adsorption phenomena at the interface. As a control, the injection of DNA in the absence of the monolayer gave homogenous black pictures demonstrating the complete lack of affinity of DNA for the interface (data not shown). Fig. 8 shows the BAM images of a DOGS monolayer at the interface during compression, at selected surface pressures of 20 and 40 mN/m. When deposited on a pure water surface, the DOGS monolayer morphology consists of a heterogeneous surface with contrasted domains of high luminosity in a dark background, the shape and size of which increase as a function of the compression. By analogy with a typical phospholipid monolayer, these bright domains could be attributed to liquid condensed domains floating in a liquid expanded phase [53]. The domain shape, which is mainly determined by the anisotropies line tension and electrostatic repulsion between lipids, is specific of the type of lipid (e.g. chain length, headgroup structure and charges) and is known to be extremely sensitive to changes in the subphase composition and monolayer environment [54]. When DNA was allowed to interact with the lipidic matrix, the monolayer morphology was drastically modified, and displayed a relatively homogenous phase of very high luminosity. For monolayers exposed to DNA during a 20 h-long incubation, the appearance of brighter areas (arrows, Fig. 8D) suggests that DNA coupling to this

Grey level (arb. units)

D.L. Thomas et al. / Thin Solid Films 483 (2005) 319–329

250 2 200 150

1

100 50

Shutter speed: (1) 1/50 s and (2) 1/120 s

0

10

20

30

40

Time (min) Fig. 9. Plot of relative reflectivity (gray level) versus time recorded during slow dynamic compression of a DOGS monolayer incubated for 1 h on a water subphase (1) or on a subphase containing DNA 10
8 M (2).

membrane surface concentrates it locally. The steep increase in integrated reflectivity, directly correlated to light intensity, is shown in Fig. 9. It can be noticed that, due to the higher reflectivity observed in the presence of DNA, the shutter speed was reduced to 1/120 s compared to the DOGS monolayer alone. Generally, an increase in light intensity can be ascribed to a change in the average thickness of the molecular film. Here, this significant increase in the relative thickness of the interfacial film is explained by the presence of DNA molecules adsorbed onto the monolayer forming a network or layer covering the lipidic matrix. Though, it is not clear at this point whether the whole polynucleotidic chain is stretched underneath the monolayer or whether it forms loops at high surface pressures. Additionally, the change in lipidic domains morphology in the presence of DNA suggests that the lipidic distribution at the interface is ruled by these local DOGS–DNA selfassemblies, with DOGS molecules acting as anchor points

t0+ 1h

π = 20 mN/m

327

t0+ 20h

A

B

C

D π = 40 mN/m

B

A

D

C π = 20 mN/m

π = 40 mN/m

Fig. 8. BAM images of a DOGS monolayer recorded during slow dynamic compression. The images selected were taken at definite surface pressure values of 20 mN/m (A,C) and 40 mN/m (B,D) during compression of the lipid film either on a water subphase (A,B) or on a subphase containing DNA 10
8 M (C,D). Combined compression/BAM measurements were carried out 1 or 20 h after the monolayer was spread on the subphase surface. Image size: 572768 Am. Shutter speed: 1/50 s (A,B) and 1/120 s (C,D).

328

D.L. Thomas et al. / Thin Solid Films 483 (2005) 319–329

for DNA molecules. Thus, the interaction of nucleotides with individual lipid molecules induces a small change in their molecular structure, as shown previously for other types of lipids [55–57]. This molecular change has a direct incidence on the whole morphological feature and the phase behavior of the monolayer (e.g. the global organization of the DOGS monolayer). Indeed, the presence of polyelectrolytes in the direct vicinity of the monolayer induces electrostatic forces that greatly affect the inter-lipid interactions, and, consequently, the two-dimensional domain shape. This direct observation, along with isotherm expansion and compressibility profile, suggests energetically favorable ordering, which allows the positively charged headgroups to maximize their distance, so as to minimize the Coulombic repulsion. As a conclusion, this change in monolayer morphology and visco-elastic behavior attests drastic changes in the surface film properties upon interaction with DNA.

4. Conclusion In this study, the effects of DNA interaction with a monolayer of synthetic lipid chelating DNA at the air–water interface were investigated by means of several methods. The analysis of the interfacial behavior of this lipidic film was achieved by means of compression isotherms, which pointed out a change in the p–A isotherm shape in the presence of various DNA concentrations in the subphase, in a time and concentration-dependent manner. The main modifications, corresponding to a large shift towards higher molecular areas and a clear fading of the phase transition, were corroborated by the fine analysis of the monolayer compressibility profile, thus suggesting a characteristic change in the monolayer fluidity as a function of both time and DNA concentration. Adsorption kinetics measurements clearly demonstrated the progressive adsorption of DNA onto the monolayer, which was shown to be mainly dominated by electrostatic interactions through investigations of the effect of the subphase ionic strength. The mixed monolayer was analyzed by ATR-FTIR spectroscopy after transfer onto a solid support, which provided evidence for the presence of DNA strands interacting with the lipidic matrix. The BAM observation of the mixed monolayer morphology strongly suggested that DNA adsorption induces a reorganization of lipids at the interface, as evidenced by the change in the condensed lipidic domains morphology in the presence of DNA in the subphase. The different repartition of lipids molecules at the air–water interface could be correlated to the change in monolayer fluidity revealed by compression isotherms. Thus, DNA interaction was shown to induce significant modifications to occur within the functionalized monolayer, characterized by a dynamic change in the in-plane-elasticity of the DOGS monolayer. This system will be further applied to the study of the interaction of single-stranded polynucleotides and

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