*Corresponding author.

Phone: 0046 0406657362; Fax: 0046 0406657030

e-mail address: alejandro.barrantes@mah.se, barrantes_ale@yahoo.es

1Present addres: Alfa Laval Lund AB, Rudeboksvägen 1, 22100 Lund, Sweden

Influence of pH on the Build-up of Poly-L-

Lysine/Heparin Multilayers

Journal of Colloid and Interface Science 388 (2012) 191

Alejandro Barrantes*, Olga Santos1, Javier Sotres and Thomas Arnebrant

Biomedical Science, Faculty of Health and Society, Malmoe University

Skåne University Hospital, Jan Waldenströms gata 25, 20506 Malmoe, Sweden

2

Abstract

The effect of pH on the build-up of polyelectrolyte multilayers, PEMs, composed by poly-

L-lysine and heparin onto two different substrates, silica and gold, has been studied by means

of ellipsometry and quartz crystal microbalance with dissipation, QCM-D. Ellipsometry

results indicate that the dry mass grows exponentially with the number of layers, and that this

amount is larger as the pH values are raised. From QCM-D data the viscoelastic properties of

the multilayered structure have been obtained. These data reflect that PEMs become more

viscoelastic as the pH values are increased for silica substrates, while for gold the highest

viscoelastic behavior is obtained at neutral pH and the elastic behavior becomes dominant as

the pH is further increased or decreased. By combining these two surface techniques it has

been also possible to determine the solvent content in the multilayers and reach a deeper

understanding of the internal structure.

KEYWORDS: Poly-L-Lysine; Heparin; Polyelectrolyte multilayer; Ellipsometry; QCM-D;

Viscoelasticity.

3

1. Introduction

The layer-by-layer assembly of polyelectrolyte multilayers, PEMs, has been extensively

used in the past years as a simple method to modify surfaces [1-5]. Basically, it consists in

alternately exposing a charged surface to oppositely charged polyelectrolytes [6, 7]. The

structure and properties of the PEM can be controlled by modifying the assembly conditions

such as pH or ionic strength [1, 5, 8, 9]. PEMs can be divided into two main groups

depending on their build-up behavior: the first kind exhibits a linear growth of the adsorbed

amount, , with the number of deposited layers, whereas the second type shows an

exponential growth regime [2, 7, 10].

Poly-L-lysine (PLL) is a biocompatible polycation (isoelectric point ~ 10) with a large

amount of active amino groups. In solution the polymer can adopt different secondary

structures (random coil, -sheet, or -helix) depending on the pH of the solution. PLL has

been used for many different purposes, such as the study of DNA-Protein interactions [11,

12], the immobilization of enzymes for biosensing [13, 14], cell labeling [15], and drug

delivery [16, 17]. Heparin (HEP) is a linear glycosaminoglycan composed of alternating units

of highly sulfated D-gluronic acid and D-glucosamine-N-sulfate. As a consequence of the

high degree of sulfated substitutions it is the biomolecule with the highest known negative

charge density. HEP is well known for its anticoagulant properties but, apart from this ability,

heparin is involved in many biological processes such as inflammatory processes, cell

differentiation, lipid transport, and cell-matrix interactions [18]. These characteristics make

the study of the formation of multilayers composed by both biomolecules very attractive, as

the fields of applicability are widely varied.

A sandwich type structure formed by PLL and HEP has been previously presented for the

analysis of biomolecular interactions on gold [19] and its validity for the study of DNA-

protein interactions [11] and protein aggregation [12, 20] has been demonstrated. Also, the

comparison between the hydration of PEMS formed by PLL combined with different

polysaccharides at pH 7, where HEP is included, has been reported [21]. However, to the

authors knowledge there is no previous report related to the effect of the pH and the surface

properties on the build-up and viscoelastic properties of PLL-HEP multilayers.

For these reasons this work is devoted to analyzing the effects of the pH on the build-up

and viscoelastic properties of the multilayered structure formed by PLL and HEP. The effect

4

of the substrate on the assembly process is also analyzed. For this purpose the combination of

two surface techniques, ellipsometry and QCM-D have been employed. This combination

does not only allow the detection of the effects of the pH in the thickness and mass adsorbed

onto the substrate, but also gives information about the level of hydration [3, 22] of the

multilayered structure and its rheological properties [5, 22].

2. Materials and Methods

2.1 Chemicals

Poly-L-Lysine hydrobromide, MW = 30000 – 70000 g/mol (cat. num. P-2636) and

Heparin sodium salt from intestinal mucosa (cat. num. H-3393) were purchased from Sigma.

H3BO3 and H3PO4 (85%) were purchased from Merk, and CH3COOH (>99%) from Fluka.

All experiments were run in Universal buffer composed of H3BO3, CH3COOH and H3PO4 (50

mM each) and adjusted to the desired pH with 1M NaOH solution. Water was treated by a

purifying unit (ELGA UHQ PS, Elga Ltd. UK).

2.2 Methods

Ellipsometry experiments were performed on two different substrates: on silicon wafers

with a silica layer of approximately 300 Å purchased from Semiconductor Wafer Inc.

(Taiwan), and on silicon wafers with a 200 nm thickness gold layer deposited onto a titanium

(25 Å) adhesion layer (Laboratory of Applied Physics, Linköping University, Sweden).

Hydrophilic silica substrates were cleaned following the procedure developed at RCA

laboratories [23]. The silica substrates were boiled for 5 minutes in an alkaline solution;

rinsed extensively with water; boiled again for 5 minutes in an acidic solution; and finally

rinsed with water and ethanol. The components of the alkaline solution were: NH4OH (25%),

H2O2 (30%), and water with a volume proportion of 1:1:5 respectively. The components of

the acidic solution were: HCl (37%), H2O2 (30%), and water with a volume proportion of

1:1:5 respectively. At the end of the cleaning procedure the surfaces were stored in ethanol.

Prior to use, the surfaces were plasma cleaned for 5 minutes in low pressure residual air using

a glow discharge unit (PDC-32 G, Harrick Scientific Corp., USA), as has been previously

suggested [24]. Gold substrates were cleaned by immersing the slides into ethanol (20

minutes) followed by NaOH 20 mM (20 minutes) and gently rinsing with water. After drying

with nitrogen the surfaces were plasma cleaned for 10 minutes. QCM-D measurements were

performed on AT-cut 5 MHz quartz crystals purchased from Q-Sense AB (Göteborg,

5

Sweden) and had silica or gold as outermost layers. Silica substrates were cleaned by

following the next procedure: 1) 10 minutes plasma treatment; 2) immersion into a

Hellmanex II solution (1% volume) for 20 minutes; 3) extensive rinsing with water; 4) a

second 10 minutes plasma treatment. Gold substrates were cleaned following a similar

procedure as the one performed for ellipsometry measurements. All cleaning procedures

yielded hydrophilic surfaces with water contact angles less than 5 degrees as measured with a

drop shape analyzer, DSA100 (Krüss, GmbH, Hamburg, Germany). All experiments were

performed twice and the results had a deviation smaller than 10%.

2.2.1 Ellipsometry

The assembly of PLL and HEP multilayers onto hydrophilic silica and gold surfaces was

monitored in situ by ellipsometry. The theoretical principles of the technique can be found

elsewhere [25]. The experimental setup is based on null ellipsometry according to the

principles of Cuypers [26]. The instrument used was a Rudolph thin film ellipsometer (type

43603-200E, Rudolph Research, USA) automated according to the concept of Landgren and

Jönsson [27]. A xenon arc lamp was used as the light source, and light was detected at 442.9

nm using an interference filter with UV and infrared blocking (Melles Griot, The

Netherlands). The trapezoid cuvette made of optical glass (Hellma, Germany) was equipped

with a magnetic stirrer and thermostated to 25 ºC. PLL and HEP stock solutions, 10 mg/ml

in UHQ water, were alternatively added to the cuvette containing the surface immersed in 5

ml of buffer solution to a final concentration of 20 g/ml. The adsorption of the

polyelectrolytes was monitored in situ in solution until a plateau was reached. A 3 minutes

long rinsing step with a polyelectrolyte-free buffer solution was introduced between each

polyelectrolyte addition. As the adsorption time until the plateau was reached varied with the

layer number, information related to this point is supplied in the tables presented in the

supplementary information.

The determination of the silicon complex refractive index, and of the thickness and

refractive index of the silicon oxide layer, was performed using air and water as ambient

media [27] and the determination of the gold complex refractive index was calculated only in

water. Four zone measurements were conducted to minimize systematic errors. Once the

optical properties of the substrate have been determined an average value for the adsorbed

film thickness and refractive index can be obtained by numerical iteration from the changes

in the ellipsometric angles and . The adsorbed amount, , was calculated by using de

6

Feijter’s equation [29] (Eq. 1), where nf is the refractive index and d the thickness of the

mixed polyelectrolyte multilayer. Although the dn/dc value for heparin is 0.13 ml/g [30], the

value for PLL, dn/dc = 0.15 ml/g [3], has been used for the whole multilayer. This procedure

will introduce an underestimation for the adsorbed amount of heparin [31].

dcdn

nnd

Bufferf (1)

Figure 1. General trend in ellipsometric adsorbed amount, (a) and (c), and thickness, (b) and (d),

obtained for the build-up of PLL-HEP multilayers on hydrophilic silica, for (a) and (b), and gold

surfaces, for (c) and (d). Polyelectrolyte concentration used was 20 g/ml, and in this particular

example adsorption was performed at pH 7.

2.2.2 QCM-D

The QCM-D measurements were performed by using an E4 system from Q-Sense AB,

Sweden. Both polyelectrolytes were supplied in solution at a concentration of 20 g/ml by

means of a peristaltic pump at a flow rate of 100 l/min until plateau values were obtained

7

with a rinsing step of 5 minutes between every addition. As was observed in the ellipsometry

measurements, the adsorption times that both polyelectrolytes needed to reach the plateau

increased with the number of layers. This information is supplied in the supplementary

material. QCM-D technique is able to simultaneously detect changes in the resonance

frequency, f, and in the dissipation factor, D, of the quartz crystal [32].

St

Dis

E

ED

2 (2)

In equation 2 the relation between D and the dissipation energy, Edis, is represented. Edis

represents the energy dissipated during a single oscillation after switching off the voltage and

ESt represents the initial energy of the chip. A detailed description of the technique and its

basic principles can be found elsewhere [32]. Briefly, an alternating-current voltage is applied

through a gold-coated quartz chip to stimulate the shear mode oscillation of the quartz

crystal. When a certain amount of mass, m, is adsorbed onto the sensor chip, a proportional

decrease in the resonance frequency, f, will be detected as stated in equation 3, known as

Sauerbrey’s equation [33].

C

mnf s

(3)

In the above equation (Eq. 3) n stands for the overtone number (n= 1, 3, 5 …), C is the mass-

sensitivity constant (C = 0.177 mg m-2

), and the subscript ‘s’ means Sauerbrey. From this

relation, a rough estimate of the mass can be made when the film deposited onto the chip can

be considered rigid.

For viscoelastic films, where high D values are obtained, this relation is no longer valid

and more trustable values are obtained by applying the Voigt model. By a numerical fit of the

frequency and the dissipation values obtained at three different overtones (n = 3, 5, and 7) not

only the mass can be obtained, but also the viscoelastic properties of the film, like the shear

elastic modulus,, and the shear viscosity, . The Q-Tools (Q-Sense AB) software was

employed for this purpose and the fixed values used were 1100 kg m-2

for the film density,

10-3

kg m-1

s-1

for the buffer viscosity, and 998 kg m-3

for the buffer density. Different values

for the density in the range between 1050 kg/m3 and 1200 kg/m

3 were tested and no effects

could be observed in the mass neither in the viscoelastic parameters, and .

8

Both parameters, and , can be related to the complex elastic modulus of the film G*

through equation 4:

fiiGGG 2'''* (4)

where G’ and G’’ are the storage modulus and loss modulus of the film respectively, and f is

the sensing frequency. From the ratio G’’/G’ a quantitative analysis of the viscoelastic

properties of the polyelectrolyte structure is performed, while a qualitative study of these

properties can be done simply by plotting D vs f [35, 36].

2.2.3 Atomic Force Microscopy

The PEM built on the same substrates used for ellipsometry experiments were examined with

a commercial Atomic Force Microscoscope (AFM) equipped with a liquid cell (MultiMode 8

SPM with a NanoScope V control unit, Bruker AXS). After preparation, samples were

immediately placed on the AFM not allowing them to dry at any moment. The surfaces were

visualized in liquid, at room temperature, by operating the AFM in the PeakForce Tapping ®

mode. Triangular silicon nitride cantilevers with nominal spring constant 0.7 N∙m-1

were

employed (ScanAsyst-Fluid, Bruker AXS). Analysis and processing of AFM images was

performed with the WSxM software [37].

3. Results

3.1 Ellipsometry

For both substrates the assembly of the multilayered system showed a similar trend in

thickness and adsorbed amount indicating that every time PLL or HEP were added they

bound to the oppositely charged surface (Figure 1). In this figure the alternate addition of

PLL and HEP solutions were performed at pH 7 with a rinsing step between them, as

described in the Methods section. However, substantial differences were detected depending

on both substrate and pH. In Figure 2 and d values obtained for both substrates under

different conditions are presented. It can be seen that the adsorbed amount increased in an

exponential way at all the experimental conditions studied for SiO2, showing a faster increase

as the pH values were raised. The behavior for the assembly of the multilayers was similar

when gold substrates were used. For both substrates, the semi-logarithmic representation of

the data could be fitted with a straight line, insets in Figure 2 (a) and (b), where it was found

that the higher the pH value the bigger the slope. It is worth mentioning that at pH 5.5 and 8.5

9

the adsorbed amount for the overall multilayered structure was highest for silica substrates,

whereas at pH 7 the largest adsorbed amount was obtained for gold substrates.

When adsorption was performed onto SiO2 substrates an obvious data scattering in the

thickness values was obtained for the first polyelectrolyte bilayer at all pH conditions.

However, the scattering was substantially diminished as the number of layers was increased.

Figure 2. Ellipsometric values for the adsorbed amount and thickness obtained under three different

pH values. Adsorbed amounts obtained for the consecutive adsorption of PLL (odd layer numbers)

and HEP (even layer numbers) onto (a) silica and (b) gold substrates are presented. The insets

represent the same data in a semi-logarithmic scale. In (c) and (d) the thickness evolution for the same

experiments, silica and gold respectively, is shown. Open symbols correspond to adsorption onto SiO2

substrates, while filled ones correspond to adsorption onto Au substrates. Experiments were

performed at pH 5.5 (circles); pH 7 (triangles); and pH 8.5 (squares).

10

This behavior agrees well with the one observed for the refractive index (Supplementary

material: Figure S1), where the scattering is also decreased as the amount and thickness of the

polyelectrolytes layers on the surface is increased. When Au substrates where used the

situation was completely different: only the first bilayer assembled at pH 8.5 showed high

scattering in d and also much higher values than the ones obtained for pH 7 and 5.5 (data not

shown). There are two possible explanations for the scattering observed for the ellipsometric

thickness: a) taking a look at the theoretical - plots (Supplementary information: Figure

S2) it can be seen that for very thin layers adsorbed the curves for different refractive indexes

are in the same order of magnitude as the resolution of the instrument, yielding scattered

values in thickness. b) Another possible explanation for this behavior may come from a low

surface coverage. The ellipsometric thickness and refractive index are obtained numerically

by assuming a homogenous layer model. Thus, a low surface coverage will not yield reliable

data for these parameters. In order to determine which one of the explanations best fits the

real situation, AFM topography measurements for the first polyelectrolyte bilayer have been

performed (Supplementary Information: Figures S3-1 and S3-2). From the analysis of the

images obtained it can be concluded that a) is the most likely explanation.

The behavior of the refractive index of the multilayers is affected by both the substrate and

the pH of the buffer solution (Figure 3). When adsorbed onto silica substrates the structures

assembled at pH 5.5 and 7 yielded very high refractive index values for the first PLL-HEP

bilayer. After the addition of the second PLL layer for pH 7 and of the second bilayer for pH

5.5 this value drops down to a minimum value from where it grows for every new bilayer

added. For the highest pH, 8.5, the value obtained for the refractive index of the first PLL

layer is the lowest (nPLL1=1.382), and increases with the addition of every new bilayer. The

refractive index values obtained after the final layer has been added, nML, are very close to

each other and are within the experimental error. If the substrate used is gold a similar trend

is observed: Again, the lowest value for the first PLL layer is obtained for the most alkaline

solution (nPLL1=1.371). After this, the value for the refractive index will again increase with

increasing number of bilayers; very high values were obtained for the initial layers when

working at neutral and acidic pH. However, some differences can be noticed for this

substrate, refractive index values remain very high up to the second and fourth bilayer, pH 7

and 5.5 respectively, and after the addition of new bilayers it stabilizes. The general trend

shown by nML is that the higher the pH the lower the values obtained.

11

3.2 QCM-D

Additional information for the PLL-HEP multilayer build-up was obtained by means of

QCM-D. Apart from measuring the mass of such films (including the mass of the trapped

solvent ), the viscoelastic properties were determined in two different ways: 1) the relation

between dissipation and mass was obtained by plotting D vs f; and 2) the ratio G’’/G’

could be calculated from the values obtained for the shear viscosity, , and the shear elastic

modulus, , when using the Voigt viscoelastic model [34].

Figure 3. Evolution of the refractive index with the layer number obtained by means of ellipsometry.

Results obtained for the build-up onto both substrates are presented (a) silica, and (b) gold

respectively. Different symbols represent different pH values: pH 5.5 (circles); pH 7 (triangles); and

pH 8.5 (squares).

3.2.1D vs f plots

12

From a qualitative analysis of the raw data very useful information was obtained. For both

substrates the total frequency change after the last polyelectrolyte layer was added, fT,

increased with the pH of the solution: fT (pH5.5)<fT (pH7)<fT (pH8.5). However,

for the dissipation the behavior depended on the substrate. For silica substrates the dissipation

increased with the pH: DT(pH5.5)<DT(pH7)<DT(pH8.5); whereas for gold substrates the

dissipation showed a maximum value for pH 7 and a minimum value for pH 5.5:

DT(pH5.5)<DT(pH8.5)<DT(pH7). These differences can clearly be observed in the

supplementary information (Figure S4).

13

Figure 4. Raw QCM-D data for the third overtone is presented as D vs f plots for consecutive PLL-

HEP bilayers adsorbed onto SiO2 (a; c; e) and Au (b; d; f) substrates at different pH values: pH 5.5 (a)

and (b); pH 7 (c) and (d); and pH 8.5 (e) and (f). Each color corresponds to a different bilayer number:

first bilayer (Black); second bilayer (Red); third bilayer (Green); fourth bilayer (Dark blue); and fifth

bilayer (Light blue). The graphs have been scaled so the slopes can be directly compared for all

conditions.

When taking a deeper look into the assembly bilayer-by-bilayer (Figure 4), it can be

observed that for the higher pH values the contribution to the dissipation is higher when PLL

is added than when the polyelectrolyte is HEP. This behavior is substrate independent.

However, at pH 5.5 this fact is no longer true for SiO2 substrates and it is HEP that produces

higher D while it is still true for Au substrates. Once the first PLL-HEP bilayer has been

adsorbed onto the surface, the addition of PLL causes an initial decrease in dissipation for all

cases (Figure S5). This effect is also observed for HEP, but only under certain circumstances:

Au substrates for pH 5.5 and 8.5; for SiO2 substrates for pH 7 and for the last two bilayers for

pH 5.5. When rinsing between the adsorption of both polyelectrolytes a decrease in f and

D is usually observed except for pH 7 on Au substrate. For this exceptional case while f

still decreases D is initially kept constant or is increased (depending on the layer number)

every time polyelectrolyte free buffer is supplied.

3.2.2 Sauerbrey vs Voigt model

The mass can be calculated both by using the Sauerbrey equation, which is a good

approximation for rigid layers or by means of the Voigt model, more appropriate for

viscoelastic films. The use of both models enables control of the validity of the

approximations assumed for the different conditions studied here, if the multilayered

structure is rigid or not. This may be done by comparing the mass obtained by using both

methods Sauerbrey, mS, and the Voigt model, mVoigt, as shown in Figure 5. In the plots it can

be seen that, for both substrates, the values obtained with the Sauerbrey approximation match

the ones obtained from the Voigt model under the more acidic conditions (pH 5.5). This is in

good agreement with the low values obtained for the ratio G’’/G’, which is a good way to

determine the viscoelastic character of the adsorbed film [5, 38, 39] (Figure 6). For pH 7 and

silica substrates, the difference between both modeled masses, although appreciable, is small

enough to consider the use of Sauerbrey approximation valid. On the other hand, for gold, the

14

difference is big enough to assume a viscoelastic system, and it justifies the use of the more

appropriate Voigt model. Again, these assumptions agree with the values obtained for the

ratio G’’/G’ obtained.

Figure 5. Comparison between masses calculated by means of Sauerbrey (open symbols) or Voigt

(filled symbols) model for the consecutive adsorption of PLL and HEP onto silica (a) and gold (b)

substrates. When both values are similar the former model can be assumed to be valid, while when

they are different the Voigt model was used. pH values used are: pH 5.5 (circles); pH 7 (triangles);

and pH 8.5 (squares).

Finally, for the more alkaline solution, pH 8.5, the situation is reversed and the PEMs

assembled onto the gold substrate become more rigid, as can be appreciated from both the

difference between Sauerbrey and Voigt masses and from the G’’/G’ ratio. For SiO2

15

substrates the multilayered structure exhibits a highly viscoelastic behavior that can be

deduced from the big difference between the modeled masses and the high values for G’’/G’.

Figure 6. Viscoelastic behavior of the multilayered structure represented as the ratio between the loss

(G’’) and the elastic modulus (G’) for both substrates: (a) Silica and (b) Gold. Circles represent data

obtained at pH 5.5; triangles at pH 7; and squares at pH 8.5.

As a general rule it can be stated that for both substrates tested with QCM-D in this work

as the pH is raised the more mass, mT, is adsorbed at the end of the assembly: mT (pH5.5)<

mT (pH7)< mT (pH8.5). Nevertheless, the ratio G’’/G’ that reflects the viscoelastic

properties exhibits a substrate dependent behavior, and for silica substrates the highest ratio

(more liquid-like multilayer) was found for the highest pH: G’’/G’ (pH5.5)< G’’/G’ (pH7)<

G’’/G’ (pH8.5); while for gold substrates it was the structure assembled at pH 7: G’’/G’

(pH5.5) G’’/G’ (pH8.5)< G’’/G’ (pH7).

Table 1. Mean values and standard deviations obtained from three independent measurements for:

Ellipsometric mass, wet mass (QCM-D), and solvent content for the complete multilayer.

pH 5.5 pH 7.0 pH 8.5

SiO2 Au SiO2 Au SiO2 Au

Ellip (mg/m2) 9±2 7.6±0.3 24.4±0.8 29±2 40±4 33±2

MassQCM (mg/m2) 19±1 15±1 34±3 53±2 160±10 65±3

Solvent Content 0.5±0.1 0.4±0.1 0.3±0.1 0.5±0.1 0.8±0.1 0.5±0.1

16

3.3 Ellipsometry and QCM-D combination

The QCM-D technique enables the measuring the mass of the layers deposited onto the

sensor surface. But as has been mentioned before, instead of the dry mass obtained by optical

means, like ellipsometry, the mass that QCM-D yields includes the solvent trapped between

the molecules forming the layer. Then, by combining both techniques, obtaining the solvent

content of the multilayered structure is a straightforward task, as has been proposed

previously [3, 22]. However, as some of the structures are highly viscoelastic the use of mVoigt

instead of mS will be applied in those situations. In Table 1 the mean values, obtained from

two independent measurements, for the ellipsometric and QCM-D adsorbed amount, as well

as for the solvent content of the overall multilayer are presented, while in Figure 7 the

dependence of the solvent content with the layer number is shown. In the plot it can be

observed that both the most and the least hydrated structures are obtained when the substrate

used is silica. The values range from nearly 80% for pH 8.5 down to approximately 30% for

neutral pH. When using gold as substrate the final values for the structure do not differ so

much for the different conditions. The most hydrated structures where obtained for the higher

pH values 8.5 and 7, with a solvent content of around 50% (very similar to the one obtained

for pH 8.5). The least hydrated is the multilayer obtained at pH 5.5 with an approximate

value of 40%.

17

Figure 7. Solvent content as a function of the layer number for PEMs built onto SiO2 (a) and Au (b)

surfaces at different pH values. Squares symbolize pH 8.5, triangles pH 7, and circles pH 5.5.

3.4 Atomic Force microscopy

AFM topography images of the complete five bilayers systems both on SiO2 and Au

substrates are shown for all the different experimental conditions (pH 5.5, pH 7 and pH 8.5)

in Fig. 8 along with images of the clean substrates. Representative cross-sectional profiles of

the surfaces are included as insets in all the figures. It is inferred by the changes they induce

in surface roughness that the PEM cover completely both types of substrates in all the

conditions tested. In fact, a complete coverage is achieved already after deposition of the first

bilayer (Supplemental Figures S3-1 and S3-2). Additionally, the AFM images show that the

roughness is considerably higher for the PEMs built at pH 8.5.

Figure 8. Topography images of a) a clean SiO2 surface immersed in water, and of five bilayers on a

SiO2 surface built and immersed in Universal buffer at b) pH 5.5, c) pH 7 and d) pH 8.5. Similar

images were obtained for Au surfaces: e) clean Au, and five bilayers on an Au surface built and

immersed in Universal buffer at f) pH 5.5, g) pH 7 and h) pH 8.5. Scan area of all images: 500nm x

500nm. Color scale goes from 0nm (black) to 30nm (white). The images include as an inset a cross-

sectional profile of the surfaces (locations indicated by blue lines in both images).

4. Discussion

18

4.1 Adsorption of the first Poly-L-Lysine layer

The behaviour observed for the build-up of the first PLL layer is dependent on the

experimental conditions, pH and substrate. This is of central relevance, as its formation will

affect the growth of the complete multilayered structure as can be deduced from the different

trends observed for G’’/G’, the solvent content, and the D vs f plots as also is concluded in

the work by Guillaume-Gentil et al.[40].

At pH 5.5 SiO2 substrates are strongly negatively charged while Au surfaces, with a pI

around 5 [41, 42], are only slightly negatively charged. At this pH, far below its pI (~10),

PLL is fully ionized and, in solution, it adopts an expanded random-coil configuration [43] as

result of the electrostatic repulsion between the lysine residues. Although both the dry and the

wet mass are very similar for both substrates, the dissipation values, which are much lower

for SiO2 substrates than for Au, implies different configurations for PLL on the two

substrates: a very flat one for silica as a result of the stronger electrostatic attraction, whereas

for gold PLL chains will be extended towards the bulk.

At pH 7 both substrates are strongly negatively charged. One of the most relevant

differences observed for the adsorption between both substrates is that for SiO2 D vs f

plots indicate a single process, whereas for Au a two-step process can be seen. The single

step process observed for SiO2 surfaces indicates the formation of a homogenously hydrated

polymer layer, in line with the random-coil structure of PLL in solution under these

conditions. Whereas, the two step process suggests the presence of two fractions of bound

molecules, the inner one formed by molecules strongly attached to the surface and a second

fraction of loosely attached molecules [44] having less contact points with the substrate The

behaviour observed is in good agreement with the values obtained for the solvent content and

for G’’/G’, and might be a consequence of the stronger attraction exerted on PLL by gold

compared to the one exerted by silica substrates. It has to be kept in mind that gold has a very

high dielectric constant offering possibilities for image charges and strong dispersion

interaction [45]. This fact has been demonstrated by Guillaume-Gentil et al.[40] to be of

extreme relevance in the interaction between polyelectrolytes and metallic surfaces. Also, for

gold substrates the highly mobile electrons allow the negative charge to come in close

proximity to the adsorbed positive charges [46] contributing to the stronger electrostatic

interaction.

19

At pH 8.5 PLL has lost some of its positive charges, and both substrates are strongly

negatively charged. D vs f plots suggest a two-step binding process where more mass is

adsorbed on silica. The reason might be found in the configuration in which PLL binds to

both surfaces: the contribution of other unspecific attractive interactions will make PLL adopt

a flatter conformation onto gold, and in this way, the surface is covered faster. This

explanation agrees with the lower solvent content (Figure 7) and a lower G’’/G’ values

obtained for gold.

The results in the present study are in good agreement with the ones previously reported

where PLL is attached to surfaces in a planar or in an extended configuration, depending on

the ambient conditions [19, 47, 48].

4.2 Build-up of the PLL-HEP multilayer (2nd

to 5th

bilayers)

It has been found that the adsorbed amount obtained by means of ellipsometry grows in an

exponential fashion under all the experimental conditions, a behavior very often observed for

multilayers where PLL is involved [2, 3, 49]. This indicates that at least one of the two

components of the PEM is diffusing within the layers as suggested by Picart et al. [50].

A detailed analysis of the viscoelastic properties represented in figures 4 and 6 sheds light

on the conformation of the polyelectrolytes in the multilayer. As a general trend, it can be

stated that everytime a polyelectrolyte solution is added the frequency decreases indicating

that mass has been adsorbed to the surface in an amount dependent on the pH for both

surfaces. The behavior of the dissipation also depends on the ambient pH, as has been already

indicated by Bieker et al [51]. Going into a detailed analysis it can be observed that for all

conditions a decrease in D and -f could be observed when PLL was added. This indicates

that the structure was initially compacted and solvent was expelled from the multilayer as a

result of the strong electrostatic interaction between PLL and HEP. After this, differences can

be observed depending on the substrate used as will be discussed below. On the other hand,

when HEP is added an initial decrease in the dissipation factor associated with no change in

mass indicates that HEP molecules replace the solvent within the polycation layer [52]

yielding a less hydrated film. After the initial compaction the anionic layer growth

represented in the D vs f plots consists of a multistep process suggesting a reorganization

of the structure or conformational changes induced by the interaction between the

polyelectrolytes.

20

It can be observed that for gold substrates, when PLL is added, D mainly exhibits a

linear increase with the decrease in frequency (homogenous hydration) where the slope is

nearly independent of the layer number but dependent on the pH:

(dD/df)pH5.5<(dD/df)pH8.5<(dD/df)pH7. This indicates that at neutral pH the multilayer has the

most viscous behavior, in good agreement with the trend observed for the G’’/G’ ratio,

although some exceptions were found. When the substrate used is SiO2, in general, PLL

shows a multistep behavior except for the second layer at pH 8.5, where a one-step regime is

observed.

The decreases observed in dD/df for the multistep processes in Figures 4d and 4e indicate

structural changes in the multilayer, and two possible explanations are proposed. First, it

might be a consequence of a conformational change in PLL, such as the formation of rigid -

helices induced by the presence of HEP, as has already been described for the interaction

between these polyelectrolytes in solution [53, 54]. It has previously been suggested that

bimolecular complexes have similar secondary structure in solution and when forming part of

a multilayer [55, 56]. Furthermore, the formation of -helices would also imply an expulsion

of the solvent, as observed in Figure 7. Second, the decrease in the slope might reflect the

diffusion of PLL facilitated by the highly hydrated structure. This effect is more pronounced

at the highest pH, where the charge density of PLL is lower, and then, it is easier for the

polyelectrolyte to diffuse [57] because the structure is more porous [58] and the inter- and

intra-molecular electrostatic repulsion is lower. The latter explanation would be in agreement

with the exponential growth for observed in Figure 2, as the inwards diffusion of PLL will

leave free sites at the outermost layer allowing the binding of more PLL molecules. Probably

a combination of both mechanisms is the most reasonable explanation for the observed

behavior; however, further efforts should be put on resolving the origin of this observation in

the future.

The peaks observed for G’’/G’ (Figure 6) at pH 7 and 8.5 for Au and at pH 8.5 for SiO2

reflect the point at which the structure of the multilayered film is becoming independent of

the substrate interaction. As the surface chemical properties have a short-range influence [40]

this is an effect of the high adsorbed amount of polyelectrolytes and layer thickness [56]

(Figure 2)., The high viscoelasticity of the films indicated by the high G’’/G’ ratio in the

peaks also contributes to this effect. These peaks are also present in Figure 7, pointing in the

same direction as stated above, and the down slope after the peaks indicate the formation of

21

more rigid structures. This last point again suggests the possibility that the presence of HEP

might be promoting the formation of PLL -helices.

4.3 Refractive index and solvent content.

The general trend observed for the refractive index as the PEM is built showed the

expected behavior under most experimental conditions, that is, it increased as the layer

number was increased [3, 56]. This indicates that the PEM becomes denser as more PLL or

HEP is incorporated. This trend is also observed through the solvent content although some

discrepancies can be observed between both parameters. Such disagreement may have two

different sources: one would be that the multilayer does not cover the surface completely, an

explanation that can be discarded after the performance and analyses of the topography

measurements (Fig. 8). The second and most probable reason are effects of surface

roughness. While the ellipsometer measures only the dry adsorbed mass, QCM-D measures

both the solvent trapped within polyelectrolyte layers but also by the hydrodynamically

coupled solvent [34].

5. Conclusions

We have investigated the effect of the ambient solution pH on the build-up of PLL-HEP

multilayers by means of ellipsometry and QCM-D. The study was made for two different

substrates: silica and gold, finding that the assembly of these polyelectrolytes is dependent on

both the buffer solution pH and the substrate.

The dry mass, obtained by means of ellipsometry, was found to have an exponential growth

under all experimental conditions when plotted against the layer number. On the other hand,

the wet mass, obtained by QCM-D, exhibited a linear or an exponential behavior depending

on the pH of the solution.

From the analysis of the viscoelastic properties, it can be concluded that when the

multilayer is build-up in an acidic medium, pH 5.5, the system is dominated by the elastic

term for both substrates as reflected by the low G’’/G’ ratio. Under these conditions a flat

conformation for the polyelectrolytes is proposed. When the viscous component becomes

predominant, at pH 8.5 for silica substrates, and at pH 7 for gold, the structure of the

22

multilayer becomes less influenced by the substrate interaction and PLL seems to adopt a -

helical structure.

The different growth behavior and layer properties observed for the substrates reflect

different interaction mechanisms with the polyelectrolytes.

Acknowledgment

The Gustaf Th. Ohlsson foundation and Malmö University (Biofilms-Research Center for

Biointerfaces) are gratefully acknowledged for financial support. We also thank Bo Thunér at

Linköping University for providing gold surfaces for ellipsometry.

Appendix A. Supplementary information

Additional plots where it is presented: The time evolution of the refractive index obtained by

means of ellipsometry; theoretical Psi-Delta plots; AFM topography and lateral force images

of the first polyelectrolyte bilayer and its interpretation; D vs f plots for the whole

multilayer and for a single bilayer with marks indicating where each polyelectrolyte addition

is made. A table with the times for each PLL and HEP step is also included. All presented for

both surfaces used. This information is available free of charge via the Internet at

http://www.sciencedirect.com

23

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