atomic force microscopy studies of generation 4 poly(amidoamine) (pamam) dendrimers on...

Surface Science 558 (2004) 99–110

www.elsevier.com/locate/susc

Atomic force microscopy studies of generation4 poly(amidoamine) (PAMAM) dendrimers

on functionalized surfaces

Hosam G. Abdelhady, Stephanie Allen *, Martyn C. Davies, Clive J. Roberts,Saul J.B. Tendler, Philip M. Williams

Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, University Park,

Nottingham NG7 2RD, UK

Received 18 December 2003; accepted for publication 23 March 2004

Available online 13 April 2004

Abstract

The aim of this work was to understand the factors effecting the interactions of generation 4 amino terminated

PAMAM (G4 PAMAM) dendrimers with chemically functionalized surfaces. To this end, the adsorption of G4 PA-

MAM dendrimers onto model alkanethiol self-assembled monolayer surfaces terminated in methyl, carboxyl and amine

functional groups was studied using the atomic force microscope (AFM). AFM images were obtained of all surfaces in

air and in liquid, before and following dendrimer adsorption. The similarity of the features observed at both of these

stages however, caused difficulties in their discrimination using AFM height images only. In situ imaging and force

measurement (force–distance and amplitude–phase–distance) experiments were therefore performed to determine

whether the imaged features originated from the PAMAM dendrimer molecules or the underlying functionalized

substrates. Interestingly, force–distance and amplitude–phase–distance measurements obtained with carboxyl func-

tionalized AFM probes suggested differences in the dendrimer conformation on the different alkanethiol surfaces.

� 2004 Elsevier B.V. All rights reserved.

Keywords: Self-assembly; Atomic force microscopy; Gold; Molecule–solid reactions

1. Introduction

Poly(amidoamine) (PAMAM) dendrimers are a

relatively new class of materials with a cascade,treelike architecture and a diameter of up to tens of

* Corresponding author. Tel.: +44-115-9515050; fax: +44-

115-9515110.

E-mail address: [email protected] (S. Al-

len).

0039-6028/$ - see front matter � 2004 Elsevier B.V. All rights reserv

doi:10.1016/j.susc.2004.03.049

nanometers [1–4], which are capable of interacting

with other molecules, such as DNA, to form a

range of different supra-macromolecular structures

[5–7]. The ‘dendrimer’ name reflects their ordered,branching treelike structure [1]. These water-solu-

ble macromolecules are constructed from various

initiator cores on which each complete itera-

tive reaction sequence results in a new dendri-

mer ‘generation’ with a larger molecular diameter,

twice the number of reactive surface sites, and ap-

proximately twice the molecular weight of the

ed.

mail to: [email protected]

100 H.G. Abdelhady et al. / Surface Science 558 (2004) 99–110

preceding generation. The most widespread water-

soluble dendrimers contain amine-terminated end

groups and tertiary amines in their repeat units.

These molecules can therefore be charged by

varying the pH of the solution and can be viewed

as polyelectrolyte networks of nanoscopic dimen-sions [8]. The large number of surface functional

groups of PAMAM dendrimers has enabled these

polymers to be employed for the multivalent rec-

ognition of viruses and cell surfaces [9]. Further-

more, they may neutralize the charge of the

phosphate groups of oligonucleotides, thereby

facilitating the delivery of genes and anti-sense

drugs to cells [5–7,10]. Complexing DNA withdendrimers results in condensation and aggrega-

tion of DNA, and increases DNA survival upon

delivery in vitro and in vivo [10,11]. PAMAM

dendrimers are approximately the same size as

many protein molecules, they are however, much

smaller than other biological targets such as viruses

[12].

The aim of this work was to understand the fac-tors effecting the interactions of generation 4 amino

terminated PAMAM (G4 PAMAM) dendrimers

with substrates of defined surface chemistry. An

understanding of these interfacial interactions is

important to the biomedical applications of these

polymers. This aim was achieved by using the

atomic force microscope (AFM) as an imaging

and force-measuring tool to visualize and charac-terize the adsorption of G4 PAMAM dendrimers

on three different model surfaces, namely, methyl,

carboxyl, and amino terminated alkanethiol self-

assembled monolayers (from hereon referred to as

SAM-CH3, SAM-COOH and SAM-NH2 surfaces

respectively). The formation of self-assembled

monolayers (SAMs) of alkanethiols on gold sur-

faces provides an excellent way to control bothsurface properties and chemical functionality [13,

14]. The self-assembly process is initiated by strong

chemical interactions between the sulfur end-

group and the gold surface, which is believed to

result in the chemisorption of the molecules as

thiolates, forcing the molecules to pack in registry

with the gold crystal lattice [15].

Due to their nanometer scale dimensions, vari-ous groups have begun to characterize a range of

dendrimeric polymers, and in particular their

interaction with different surfaces [16–20]. Indeed,

Hierlemann et al. [16], have shown that amine-

terminated PAMAM dendrimers spontaneously

form close-packed, monolayer-thick films when

exposed to gold surfaces. Lackowski et al. [18],

have also described a dynamic phase-segregationprocess involving dendrimer/n-alkanethiol mixed-

monolayers confined to Au(1 1 1) surfaces after

the sequential immersion of such substrates into

ethanolic solutions of, first, dendrimers and

then n-alkanethiols. For the smallest dendrimers

(G4 PAMAM dendrimers) used in their studies,

adsorption of the hexadecanethiol was found to

completely displace the dendrimers from the goldsurface after 96 h. All of these and several other

investigations, despite providing considerable in-

sights into the structure and surface behaviours of

dendrimers, were however restricted through their

analysis of air-dried samples. To further build on

such studies, M€uller and colleagues thus imaged

generation nine and five PAMAM dendrimers in

liquid on hydrophilic (mica) and hydrophobic(graphite) surfaces in aqueous media [21]. Within

the present study, we therefore extend the existing

AFM studies of dendrimeric polymers at interfaces

through the investigation of the adsorption

behaviour of G4 PAMAM dendrimers on alka-

nethiol surfaces with different chemical function-

alities. Such information is valuable for the

development of current and future applications ofthese materials, and in particular those within the

biomedical field.

2. Materials and methods

All materials, unless otherwise stated were

purchased from Sigma–Aldrich (Poole, England)and used without further purification. Solvents

utilized were of HPLC quality and obtained from

Fisher (Loughborough, Leicestershire, UK), and

deionized water obtained from ELGA MAXIMA

system (Lane End, High Wycombe, Bucks, UK)

with water of resistivity of about 18.2 MX cm.

G4 PAMAM dendrimers based on an ethylene-

diamine core (calculated formula weight 14,215)containing 64 surface primary amino groups, were

used in this study (Dendritech, Midland, MI).

H.G. Abdelhady et al. / Surface Science 558 (2004) 99–110 101

Samples were obtained as methanolic solutions.

To generate aqueous solutions, a portion of the

methanolic stock solution was dried under a

stream of argon and subsequently placed in a

vacuum (pressure� 0.013 mbar) for several hours.

The dried material was re-dissolved in deionizedwater to yield 20 lg/ml aqueous stock solu-

tions, which were stored at 4 �C for a maximum

of a few days. This method follows previous re-

ported work which avoids hydrolytic degradation

of PAMAM dendrimers at room temperature

[22].

Model surfaces were prepared using octadecyl

mercaptane, 16-mercaptohexadecanoic acid, and11-amino-1-undecanethiol hydrochloride (Doj-

indo Laboratory, Japan). These alkanethiols have

alkyl chain lengths of ðn > 10Þ and differ only in

the functionality of the terminal surface carbon, to

yield self-assembled monolayers with comparable

mechanical properties [13,23].

3. Sample preparation

3.1. Epitaxially grown gold substrates

Gold substrates were prepared using the vapor

deposition methods of DeRose et al. [24] and

Hegner et al. [25]. Gold (99.9%, Birmingham

Metals, UK) was first cleaned in acetone andplaced into a tungsten crucible, 30 mm beneath a

heating stage, in a vacuum coating system. Freshly

cleaved mica (Agar Scientific, Essex, UK) was

placed side down facing the gold filled crucible. A

vacuum of 10�6 mbar was reached before heating

the mica to 320 �C for 6 h to remove contaminants

from the mica surface. The gold was resistively

heated and evaporated at a rate of 0.1 nm/s ontomica at 315 �C. The formed gold film was then

annealed by further heating at 390 �C for 24 h.

Generally, thermal annealing of gold films causes

the grain boundaries to diffuse across the grains so

that they can merge to produce larger grains. The

specimen was allowed to cool at its natural cooling

rate. Returning the vacuum chamber to normal

atmospheric pressure allowed the retrieval of gold-coated surfaces. All gold surfaces were stored un-

der argon prior to use.

3.2. Preparation of alkanethiol self-assembled mono-

layers

1 mM solutions of octadecyl mercaptane,

16-mercaptohexadecanoic acid, and 11-amino-1-undecanethiol hydrochloride were separately

prepared in ethanol shortly before each experi-

ment. Prior to functionalization, gold substrates

(1 cm · 1 cm) were cleaned by passing �11 times

through hydrogen flame. Cleaned gold substrates

were then immediately immersed in the alkanethiol

solutions (2.5 ml) for 24 h. Alkanethiol-coated

substrates were then removed from the alkanethiolsolutions, and rinsed copiously with absolute eth-

anol to remove droplets of non-covalently bound

material, before drying in a stream of nitrogen.

3.3. Preparation of PAMAM dendrimer-coated

substrates

Solutions of G4 PAMAM dendrimers wereprepared at a concentration of 0.1 lg/ml in de-

ionized water (pH 6.5–6.9), immediately prior to

use. To produce dendrimer-coated substrates, a 40

ll drop of dendrimer solution was placed on each

nitrogen dried alkanethiol substrate and left for 15

min. PAMAM dendrimer-coated substrates were

then rinsed copiously with deionized water to re-

move loosely adsorbed G4 PAMAM dendrimermolecules before again being dried in a stream of

nitrogen.

3.4. Functionalization of AFM tips

Single beam tapping-mode etched silicon

probe (TESP) tips (Veeco, Santa Barbra, CA),

and V-shaped contact mode silicon nitride tips(Veeco, Santa Barbra, CA) were cleaned in ace-

tone, dried in nitrogen and placed into a gold

coater (Blazers Sputter Coater, SCD 030, Blazers

Union, SWISS). Both sides of these tips were

gold coated at 0.15 mbar, and a current of 30

mA for 45 s. Gold-coated tips were then im-

mersed in a 1 mM ethanolic solution of 16-

mercaptohexadecanoic acid for 24 h. The tipswere rinsed in ethanol, and allowed to dry in

nitrogen prior to use.


3.5. AFM experiments

All images and force measurements were ob-

tained using a multimode atomic force micro-

scope, with a Nanoscope IIIa controller (Veeco,Santa Barbara, CA, USA) utilizing a vertical en-

gage E scanner (10 · 10 · 2.5 mm). Tapping mode

imaging was utilized for the investigation of

functionalized surfaces in both air and liquid, and

all AFM experiments were performed within 1 day

of sample preparation.

During imaging, to achieve an efficient coupling

between the drive amplitude and cantilever ampli-tude response, the cantilever was ‘tuned’ within a

distance of �50 nm of the sample. Then to mini-

mize sample deformation and contamination of

the AFM tip, the image scan size and offset were

set to 0, before engaging the final tip approach

procedure. For tip approach, the piezo drive ampli-

tude was set to 300 mV, resulting in a 1 V ampli-

tude (corresponding to 50 nm, with sensitivity of 1nm/20 mV) of the cantilever. The noise of both the

topography and the phase signals were minimized

through optimization of the integral and propor-

tional gains and scan speed. Set points were chosen

as close as possible to the free oscillation ampli-

tude to minimize forces exerted on the interfacial

species. Typically, the difference between the set

point of jump in and the adjusted set point re-quired to achieve high-resolution topographs was

0.02–0.05 V, corresponding to a damping of 10–

25 nm of the free amplitude.

Functionalized gold substrates and dendrimer-

coated samples were imaged in air utilizing single

beam TESP silicon cantilevers with integrated tips

and with resonant frequencies between 278 and

300 kHz, and with �50 N/m force constant. Forliquid imaging, samples were prepared as de-

scribed above, but after washing were not allowed

to dry and were imaged in deionized water using

silicon nitride probes (100 lm long V-shaped

cantilevers, with �0.32 N/m spring constant, and

resonant frequencies in water between 9 and 12

kHz). As imaging in liquids eliminates the capil-

lary interaction between the AFM tip and thesample surface, imaging forces of less than 0.1 nN

could be achieved in order to minimize any sample

deformation by the probe [26,27]. It should also be

noted that while imaging the SAM-COOH sur-

faces, 50 ll of dendrimer solution (0.1 lg/ml in

deionized water) was injected into the liquid cell,

and adsorption visualized in situ. All images were

taken at a scanning rate of 2.77 Hz.

Force–distance measurements [28,29] were ob-tained in deionized water using SAM-COOH

functionalized probes to determine the relative

strength of the interaction with dendrimer-coated

surfaces. Before performing such measurements,

curves were recorded on the SAM surfaces so that

the background level of the interaction could be

determined, and as a check of surface and tip

functionalization. The slope of the contact regionof the force–distance measurement, recorded on a

non-functionalized gold surface, was used to con-

vert the measured cantilever deflection from the

relative units (V) to the absolute units (nm). Since

a small hysteresis typically exists between the ap-

proach and retract curves, the average of these two

curves was employed in calibration. Using the

cantilever spring constant (kc) in (N/m), the can-tilever deflection (dc) in (nm) was converted to

force (F ) in (nN) using Hooke’s law [28,29]. The

spring constant of the cantilever was determined

using the thermal noise method [30]. To facilitate

the comparison of data obtained in one experi-

ment, i.e. with one AFM probe, contact forces

were maintained at a constant value (typically 300

pN) so as to minimize any variation in probe–sample contact area between samples. A low level

of contact force was also chosen so to minimize

any deformation or damage of the functionalized

probes and/or surfaces during each experiment.

Amplitude–phase–distance (a–p–d) measure-

ments were obtained in air between SAM-COOH

functionalized TESP probes and dendrimer-coated

surfaces. For control measurements, a–p–d curveswere obtained on all SAM surfaces, again to

determine the background level of the interaction.

In this type of measurement, it is difficult to

determine the exact sample surface position. Ex-

perimentally, however, a significant decrease in

amplitude and an increase in phase from their free-

status values indicate a start point of tip–sample

interactions during tip approach. This can offer areference position for all a–p–d curves measured

on different samples [31]. This portion of the


measurement, recorded on non-functionalized

gold surfaces, was also used to convert the mea-

sured cantilever oscillation amplitudes (RMS val-

ues) to the absolute units (nm), assuming that

there was no deformation of gold surfaces and also

no deflection of the cantilever during tapping.Again, since a small hysteresis typically exists be-

tween the approach and retract curves, the average

of these two curves was used in calibration. In

each experiment we also aimed to maintain probe–

sample contact forces, and thus probe–sample

contact areas, at a constant value with the set-

point amplitude typically 10 nm below the free

oscillation value. For both force–distance and a–p–d measurements the number of data points

collected in one approaching–retracing cycle was

100, with scan windows of �100 nm.

4. Results and discussion

4.1. Gold substrate analysis

Fig. 1 shows a 1 lm · 1 lm AFM height image

of gold-coated mica prepared by the epitaxial

growth method. This method was found to pro-

duce films containing atomically flat gold islands

measuring up to 1000 nm in diameter. In these

Fig. 1. A representative 1 lm · 1 lm topography image of an

epitaxially grown gold film (z-scale 65 nm). The surface

topography consists of large flat gold islands of up to 1000 nm

in diameter (contact mode image obtained in air).

experiments, we found that gold films prepared at

a temperature of 315 �C and annealed at 390 �Cfor 24 h produced the smoothest surface mor-

phologies. Average surface roughness ðRaÞ and

root mean roughness ðRmsÞ values for the gold

films were found to be 52.4 ± 4 and 85.8 ± 2 �A, per1 lm2, respectively and to be 1.88 ± 0.3 and

2.79± 0.2 �A, per one island (215 nm · 215 nm),

respectively.

4.2. Images of SAM functionalized surfaces in air

Fig. 2 displays 1 lm · 1 lm images of (a) SAM-

NH2, (b) SAM-COOH, and (c) SAM-CH3 sub-strates, obtained at a set point of 98% of the free

oscillation amplitude. All the images show the is-

land features associated with the gold surface, with

an overlying patchy topography that we attribute

to the presence of the surface bound alkylthiols.

Such features were never observed on non-func-

tionalized gold surfaces. Here it should be noted

that the conditions and timescales employed forour SAM formation, are commensurate with those

well documented for the formation of ordered

monolayer films [23,32]. The brighter parts in these

images thus most likely correspond to small raised

islands of alkanethiols within a SAM monolayer.

The diameter of these features was about 20–60

nm, but sometimes they appeared to be aggregated

in clusters of up to 160–200 nm in diameter. Theheight of the islands above the background is

uniform and equal to 1.93 ± 0.24 nm, approxi-

mately the length for one alkylthiol molecule.

Similar islands have been observed in previous

ultra-high vacuum scanning tunneling microscopy

studies of alkylthiol functionalized surfaces [33].

4.3. G4 PAMAM dendrimer functionalized surfaces

Fig. 3 shows typical images of G4 PAMAM

dendrimers adsorbed onto (a) SAM-NH2, (b)

SAM-COOH, and (c) SAM-CH3 substrates. In

these images, spherical features can be observed

over the island features of the gold surface. The

observed diameters of these features were (a)

22.1 ± 2.5 nm, (b) 20.0 ± 6.5 nm, and (c) 30.5 ± 1.3nm, with respective heights of (a) 1.5 ± 0.4 nm, (b)

1.8 ± 0.4 nm, and (c) 2.1 ± 0.3 nm. These values of

Fig. 2. Representative topography images of the SAM surfaces formed through deposition of the alkanethiols (a) 11-amino-1-un-

decanethiol hydrochloride, (b) 16-mercaptohexadecanoic acid, and (c) octadecyl mercaptane onto epitaxially grown gold films (images

obtained in air; scale bars¼ 100 nm and z-scales¼ 8 nm).

Fig. 3. Representative topography images of the different functionalized SAM surfaces following their exposure to a solution of

dendrimers (0.1 lg/ml). (Images recorded in air. Scale bars¼ 200 nm, and z-scales¼ 70, 20 and 10 nm respectively.)


diameter are significantly larger than the predicted

diameter value of 4.5 nm for a spherical G4 PA-

MAM dendrimer molecule, while the values of

height are significantly smaller [19]. We attribute

these discrepancies to the following: Firstly, low-

generation members of a dendrimer homologousseries, such as those employed here, exist as open

plate like molecules in which the dendrimer bran-

ches of neighboring molecules can inter-penetrate

each other [16,19]. The observed features therefore

most likely represent dendrimer aggregates, as

opposed to single molecules. Secondly, drying of

the dendrimer samples may cause some degree of

collapse and spreading of the dendrimers, as pre-viously reported in several other AFM imaging

studies [20]. The observed features may also ap-

pear broadened due to the convolution effects of

the AFM tip [19,34,35], and deformation due to

the high frequency and vertical velocity by which it

is oscillated during tapping-mode imaging.

Fig. 4(a) depicts an image of a SAM-COOH

surface imaged in deionized water, also showing

islands of 20–50 nm in diameter and similar tothose observed in the air studies (see Fig. 3). Fig.

4(b) depicts an image of a SAM-COOH surface

upon which G4 PAMAM dendrimers have been

adsorbed, obtained also in deionized water. Fea-

tures consistent with the dimensions of individual

dendrimer molecules are not seen within this im-

age, but instead larger patchy features of 40–65 nm

in diameter are observed. The height of these fea-tures is 6.4 ± 1.6 nm, and they are most likely also

attributed to small aggregates of dendrimer mole-

cules.

Fig. 4. (a) A typical height image of SAM-COOH surface (recorded in deionized water, z-scale¼ 30 nm), showing raised islands of

diameter 20–150 nm. (b) A typical height image of the same surface following exposure to a 0.1 lg/ml dendrimer solution (recorded in

deionized water, z-scale¼ 12 nm). Here dendrimers can be seen to aggregate to give a patchy film with features of 40–65 nm in diameter

(scale bar¼ 100 nm).


While these images are consistent with previ-

ously published data [19] they however have fea-

tures, which are similar to those observed on the

SAM substrates, making the discrimination of

features related to either types of surface difficult.

Two approaches were thus subsequently employed

to solve this problem, namely; the in situ imagingof dendrimer adsorption, and by recording force

measurements on the functionalized substrates

before and after dendrimer adsorption.

Fig. 5 depicts a series of images recorded at one

position on a SAM-COOH surface in deionized

water over a 48 min time period, immediately be-

fore (Fig. 5(a)) and following the injection of a

dendrimer solution (Fig. 5(b)–(f)). Five minutesafter the injection of the dendrimer solution, pat-

ches (20–90 nm in diameter) were found to appear

in the AFM images (Fig. 5(b)). Again these fea-

tures are most likely to be dendrimer aggregates,

as they were observed to grow in size with time to

form a tightly packed layer in which individual

molecules and/or aggregates could not be dis-

criminated (Fig. 5(c)). Globular features, which weattribute to the presence of additional dendrimer

molecules and/or aggregates not able to be incor-

porated in the previous layer, were then seen to

appear over this layer, as can be seen in Fig. 5(d)

(highlighted with arrows). As the exposure time

increased, these globular features were also found

to grow in size, and coalesce to form another

densely packed layer (Fig. 5(e)). Finally in Fig.

5(f), obtained at t ¼ 48 min, features of diameter

�10 nm can be seen to yet again appear over this

newly formed layer.

This series of images clearly reveal a time-

dependent change in the sample topography, thus

confirming that dendrimers are able to adsorb to

SAM-COOH surfaces under the conditions em-ployed in our experiments. The images also indi-

cate that under our experimental conditions the

dendrimer molecules first tend to form a densely

packed film. Such a phenomenon has also been

observed in previous studies [19], and arises as the

dendrimers arrange themselves at the surface as to

maintain their lowest energy conformation. At a

certain point however, within this packing geo-metry additional dendrimer molecules will no

longer be able to be incorporated within this layer,

and will begin to form additional layers as evident

in images Fig. 5(d) and (f). Although the forma-

tion of multilayers of similarly charged molecules

may at first seem counterintuitive, we postulate

that such a condition can arise through the

hydrogen bonding between and/or inter-penetra-tion of dendrimer molecules.

4.4. Force–distance and a–p–d measurements

Adhesion force–distance measurements were re-

corded in deionized water (pH 6.5) between SAM-

COOH-coated tips and all SAM-coated surfaces,

prior to and following dendrimer adsorption.

Fig. 5. In situ imaging of the adsorption of dendrimers (from a 0.1 lg/ml solution) onto a SAM-COOH surface (scale bar¼ 35 nm and

z-scale¼ 30 nm). Arrows indicate dendrimer molecules and/or aggregates appearing over the underlying closely packed layer.


Previous studies performed by ourselves and othergroups have highlighted the sensitivity of adhesion

force measurements [36–38] and a–p–d curves [39]

to differences in the chemical nature of the probe–

sample interface, and in this study we wished to

investigate whether such measurements could be

exploited for the differentiation between SAM and

dendrimer-coated SAM substrates.

In all experiments to assess the reliability ofprobe and surface functionalization process, force

measurements were first recorded between SAM-

COOH functionalized probes and the three dif-

ferent SAM surfaces. Only those probes that

demonstrated adhesive behaviour consistent with

previous studies were then utilized in subsequent

measurements [36,37]. It should also be noted that

in these and subsequent dendrimer experiments wewere unable to determine the radius of curvature

of the functionalized probes, and thus also esti-

mations of probe–sample contact areas. Therefore,

in our experiments direct comparisons of forces

were only made between those obtained within one

experiment recorded with the same probe, andin which the probe–sample contact force was

maintained at a constant value. Only trends in

the obtained adhesive forces were compared be-

tween experiments obtained with different AFM

probes.

Fig. 6(a)–(f) shows a representative series of

force–distance measurements obtained for each

tip–substrate combination. Within these series ofmeasurements, it can be seen that the largest

adhesion forces were recorded on the SAM-NH2

surface (7.80 ± 0.13 nN) (Fig. 6(c)). In the other

two combinations (on the SAM-COOH surface,

Fig. 6(b) and on the SAM-CH3 surface, Fig. 6(a)),

smaller adhesive forces of 1.3 ± 0.2 and 2.4 ± 0.6

nN were observed respectively.

Several groups have indicated that the pKa

values of acid and amine functionalities in closely

packed self-assembled monolayers are markedly

different from their values when free in solu-

tion [38,40–42], attributed to intra-monolayer

hydrogen bonding between adjacent groups in the

c

a

b

d

e

f

a-SAM-COOH

b-SAM-CH3

f-SAM-NH2- G4

3nN

Forc

e (n

N)

Z-displacement

c-SAM-NH2

d-SAM-COOH-G4

e-SAM-CH3-G4

400nm

Fig. 6. Force–distance measurements recorded between a

SAM-COOH modified tip and the different SAM substrates.

All measurements were recorded in deionized water, and den-

drimer-coated SAM surface indicated in the legend by a ‘G4’

suffix.


respective monolayers. Indeed, in recent chemical

force titration experiments between carboxylicacid terminated SAMs [43] the pK1=2 (the solution

pH at which half the surface groups are ionized)

was found to be around 8, under conditions of

low ionic strength. This value was found to be in

good agreement with surface pKa values (also

approximately 8) derived from gas phase and

cyclic voltammetry experiments. Similar measure-

ments recorded between amine terminated SAMsrevealed that the behaviours of such surfaces were

more complex [44], although also consistent with a

model incorporating intra-monolayer hydrogen

bonding (at intermediate pH values and conditions

of low ionic strength).

Under the conditions employed in our ex-

periments (i.e. deionized water, pH� 6.5) it is

therefore likely that both the SAM-COOH and

SAM-NH2 surfaces would be composed of both

non-dissociated and ionized functional groups. We

thus attribute the larger adhesion forces, recorded

between SAM-COOH tips and SAM-NH2 sur-

faces, to electrostatic attractive interactions be-

tween any ionized carboxyl and amine groups andalso to inter-surface hydrogen bonds induced upon

probe–sample contact and which can occur by

virtue of the non-dissociated terminal functional-

ities [43]. The detection of adhesion forces when

recorded on a SAM-COOH surface is also con-

sistent with a model in which hydrogen bonds can

form during probe–sample contact, although here

such interactions will be opposed by repulsiveinteractions between any ionized carboxyl groups.

The intermediatary ranking of the forces recorded

on the hydrophobic SAM-CH3 surfaces, which is

both unable to interact with the SAM-COOH tip

electrostatically or via hydrogen bonding, is con-

sistent with previous observations [36].

Fig. 6(d)–(f), show representative force mea-

surements recorded on the same SAM substratesfollowing their exposure to an aqueous solution of

dendrimers. The probe utilized in these measure-

ments was also the same as that employed to re-

cord, Fig. 6(a)–(c). The mean adhesion force

obtained on the SAM-COOH surface was 2.4 ± 0.9

nN. Adhesion forces recorded with the same probe

on dendrimer treated SAM-CH3 and SAM-NH2

surfaces were found to be 5.2 ± 1.7 nN (Fig. 6(e)),and 2.7 ± 0.5 nN (Fig. 6(f)), respectively.

From these measurements it can be seen that

both the magnitude and order of ranking of the

adhesive forces differ markedly to those recorded

with the same probe (at the same contact force) on

the SAM surfaces prior to dendrimer adsorption.

This observation thus indicates the ability of

adhesion force measurements to detect the pres-ence of dendrimers on the surface. It should also

be noted that although the data displayed in Fig. 6

was obtained using only one probe, several repeats

of the experiment were actually performed. In all

experiments, the trends in the recorded adhesive

forces were the same as that displayed in Fig. 6,

with differences only in the absolute recorded force

values, i.e. due to variation in probe geometry.We postulate that the differences in the forces

recorded on the dendrimer-coated surfaces may

15 nm

10 n

m

Ampl

itude

Zdisplacement (nm)

Phas

e

15 nm

25 d

egre

es

a

b

c

d

e

a

b

c

d

e

a-SAM-NH2

b-SAM-CH3

c-SAM-COOH d-SAM-COOH-G4e-SAM-CH3-G4

Fig. 7. A–p–d curves recorded between a SAM-COOH modi-

fied TESP tip and the same range of surfaces as tested in Fig. 6.

All measurements were recorded in air, and again dendrimer-

coated SAM surfaces are indicated in the legend by a ‘G4’

suffix.


arise due to the high flexibility of the employed G4

dendrimer molecules and their subsequent ability

to change their conformation upon surface

adsorption [19]. Under our experimental condi-

tions, the surface amine groups of the dendrimers

would be protonated [8], and the polar solventwould cause their structures to be swollen. Within

a dendrimer molecule the interaction between

polymer branches is thus weak, leaving the surface

functional groups free to interact with the different

SAM substrates. When dendrimers are adsorbed

onto SAM-COOH substrates, we propose that the

initial layer of dendrimers may change their con-

formation by exposing some of their functionalgroups towards the small number of ionized car-

boxylic acid groups present on the surface, due to

the electrostatic attraction.

Contrary to this, in the case of the adsorption of

the dendrimers onto SAM-NH2 substrates, they

may change their conformation by exposing the

majority of their functional groups away from the

surface due to the repulsive electrostatic forcesimposed by the small number of protonated amine

groups present. This electrostatic repulsion will

also most likely reduce the number of dendrimer

molecules on the substrate, as compared with the

previous example. Assuming however, that these

molecules will also arrange themselves on the

surface as to minimize such repulsion, achievable

by maximizing their contact area with the surfacethrough exposure of their interior hydrophobic

branches, the molecules will be relatively elongated

and the surface amine groups distributed over a

large area of the elongated molecule relative to the

SAM-NH2 substrate.

Finally, if the substrate has no charge on its

surface (i.e. as for SAM-CH3), any change in

dendrimer conformation upon surface adsorptionwill largely be driven through the hydrophobic

interactions between the internal core of the den-

drimers and the underlying surface. As on the

SAM-NH2 substrate, the dendrimers would ar-

range themselves on the surface in order to maxi-

mize the hydrophobic interactions with the

substrate. However, unlike the case for that sub-

strate, electrostatic repulsive forces will not hinderdendrimer adsorption, and thus the number of

dendrimer molecules should be greater than on the

SAM-NH2 substrate. The resultant closer packing

of the dendrimer molecules on the hydrophobic

surface will result in a surface amine group density

greater than that for the dendrimers on the

oppositely charged SAM, but which is still lower

than the density obtained due to the SAM-NH2

substrate. To conclude, we thus suggest that the

observed differences in adhesion forces recorded

on the different dendrimer-coated SAM surfaces

are reflective of differences in the dendrimer sur-

face conformation and the subsequent change in

the density of protonated amine groups available

for binding to the SAM-COOH tip.

Representative a–p–d curves obtained using aSAM-COOH functionalized TESP AFM probe

and the same range of substrate surfaces, are

presented in Fig. 7. For clarity, only the approach

curves are shown. It should also be noted that


these measurements were recorded in air, unlike

the force–distance measurements presented in

Fig. 6. In this type of measurement, during tip–

sample approach prior to any significant tip–

sample interactions, both amplitude and phase

signals were constant for all the samples and thusthey appear as horizontal lines in the figures. At a

certain distance the cantilever becomes close en-

ough to the surface to experience both attractive

and repulsive forces, including capillary force

interactions, and the amplitude starts to decrease

while the phase lag starts to increase (observed as a

negative phase shift), indicating the onset of tap-

ping [31,39]. This change can be seen to continueduring approach until a point where the phase

shift suddenly switches from negative to positive,

indicating a change in tip–sample interactions

from attractive-dominated to repulsive-dominated

as shown in Fig. 7(b). Simultaneously, a disconti-

nuity can be seen in the amplitude curve. We also

found that in some cases, the transition of phase

shift from negative to positive did not always ap-pear as such a sudden jump, but a continuous

evolution, as shown in Fig. 7(c) and (d). Further-

more, in Fig. 7(a) and (e) there is no phase tran-

sition due to the strong attraction between the tip

and SAM-NH2 and the dendrimer-coated SAM-

CH3 substrates respectively. Accordingly, the

amplitude curves in these measurements also show

a smooth and usually non-linear decrease.Comparing profiles of the a–p–d curves ob-

tained from Fig. 7(a)–(e), it is clear that the a–p–d

measurements appear to indicate a difference in the

surface chemical functionality of these different

substrates. These results support the data obtained

in our force–distance measurements, in that the

carboxyl functionalized probe experiences different

forces and behaves differently when interactingwith each of the different substrates. The observed

trends in the a–p–d data recorded on the dendri-

mer-coated substrates are also consistent with the

force–distance measurements, further supporting

our hypothesis that the dendrimers are able to

change their conformation on the different alka-

nethiol surfaces due to their flexibility. These re-

sults are in line with the work of Danesh et al. whoused a–p–d curves to differentiate between differ-

ent functionalized surfaces [36,39].

5. Conclusion

AFM images in air and in liquid were found

to provide topological information about SAM

functionalized gold surfaces prior to, and follow-ing their exposure to aqueous solutions of G4

PAMAM dendrimers. However, due to the simi-

larity of the spherical features on both substrates,

it was difficult to discriminate between them

depending on data obtained from AFM height

images only. Therefore, utilizing carboxyl func-

tionalized AFM probes, and applying the AFM in

its force measurement modes, we have demon-strated that SAM functionalized probes can be

employed both to discriminate between features

related to the surface adsorbed dendrimers, from

those related to the underlying SAM substrate.

Interestingly, our results also suggest that the

surface conformation of such dendrimer molecules

maybe dependent upon the nature of the different

functionalized substrates, highlighting the impor-tance of understanding the interaction of such

molecules with surfaces of differing chemistry.

Acknowledgements

The authors acknowledge the support of Pro-

fessor X. Chen, funding from the Egyptian Gov-ernment, the University of Nottingham for HG’s

studentship and SA thanks Pfizer Global Research

and Development funding of her lectureship.

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