One-step procedure for the preparation of functional polysaccharide/fatty acid multilayered coatings [original]

ARTICLE
One-step procedure for the preparation
of functional polysaccharide/fatty acid
multilayered coatings
Samantha Micciulla 1,2,3 , Dominic W. Hayward 1,2 , Yuri Gerelli 2 , Alain Panzarella 4 ,
Regine von Klitzing 1,5 , Michael Gradzielski 1 & Leonardo Chiappisi 1,2
Soft, strati ﬁ ed, amphiphilic systems are recurrent motifs in nature, e.g., in myelin sheaths or
thylakoid stacks, and synthetic analogues are increasingly being exploited in the areas of
biocatalysis, biosensing, and drug delivery. The synthesis of such complex multilayered
systems usually requires lengthy preparation protocols. Here, we demonstrate the formation
of multilayered fatty acid/polysaccharide thin ﬁ lms prepared via a single step protocol, which
exploits the spontaneous self-assembly of the components into vesicular systems in aqueous
solution. The solutions are characterized by light and neutron scattering experiments and the
thin ﬁ lms by neutron re ﬂ ectometry, optical ellipsometry, atomic force microscopy, and x -ray
diffraction. The thin ﬁ lms exhibit structural features with sub-10 nm dimensions, stemming
from the ordered sequence of hydrophilic and hydrophobic layers and respond strongly to
changes in ambient humidity. Using this approach, ﬁ lms with a total thickness varying from
tens to hundreds of nanometers can be easily prepared.
https://doi.org /10.1038/s42004-01 9-0155-y OPEN
1 Stranski Laboratorium für P hysikalische und Theo retische Chemie, Institut für Chemi e, Technische Universität B erlin, Strasse des 17. Juni 124, 10 623
Berlin, Germany. 2 Institut Max von Laue - Paul L angevin, 71 av enue des Martyrs, 38042 Grenoble, France. 3 Max Planck Institute o f Colloids and
Interfaces, Am Mühlenb erg 1, 14476 Potsdam, Germany. 4 The European Synchrotron, 71 avenue de s Martyrs, 38042 Grenoble, France. 5 Technische
Universität Darmstad t, Fachbereich Physik, Ala rich- Weiss-Strasse 10, 64287 Darms tadt, German y. Correspondence and reque sts for materials
should be addressed to L.C. (email: l [email protected] )
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S urface modi ﬁ cation is an excellent method to provide int erfaces
with a tailored functionali ty wh ile preserving the bulk prop-
erties of the substrate. Functio nal coatings are used across a
broad range of disciplines from biomedicine to nanotechnology with
countless applications in cluding: biomolecular sensing, mechani cal
actuation, ﬁ re protection, anticorrosion , a nd antifoul ing. Despit e
rapid progress in the ﬁ eld, the formation of well-de ﬁ ned, responsiv e,
nanostructured layers remains a ch allenging task. Within the domain
of soft -matter fu nction alizat ion, mon olayer ed or mul tilayer ed coat -
ings can be obtained by spin coating, drop casting, dip coa ting, or
sprayin g 1 – 10 . The different methods hav e been extensively employed
to modify the surface of a very diverse range of substrates, with only
few restriction s on the chemical comp osition of the thin ﬁ lms. In
contrast to protocols requiring subsequent synthetic steps and
comple x experiment al set-up, the clas sical layer -by-lay er technique
remai ns one of the mo st versa tile me thods to pr epare mult i-
functi onal, lay ered co atings. F irst empl oyed for t he success ive
adsorption of oppositely charged polyelectrolytes onto solid, planar
surfaces, the method has rapidly been extended to t he incorporation
of colloidal parti cles into thin ﬁ lms. Such ﬁ lms may also be formed
on soft and non-planar objects 11 – 16 .
A common aim for research in this ﬁ eld is the preparation of
hierarchical, compartmentalized ﬁ lms with a reversible response
to external stimuli and the ability to load active agents, stored in
speci ﬁ c reservoirs. To achieve this, intense work has been con-
ducted on the preparation of composite coatings where polymeric
materials are combined with low-molecular-weight block copo-
lymer or surfactant assemblies, i.e., micelles, vesicles, or bilay-
ers 15 – 20 . The strati ﬁ ed structure of alternating lipid or surfactant
and polyelectrolyte layers has shown highly promising results in
the ﬁ elds of bio-catalysis and bio-sensing and is generally pre-
pared by sequential adsorption of the different components 21 – 23 .
However, for the incorporation of micelles, intact vesicles, or
amphiphilic bilayers into multilayered ﬁ lms, additional steps
aimed at stabilizing intermediate structures are required 16 , 17 , 19 .
This results in complex preparation protocols, which are not
easily controlled and reproduced.
In contrast to the active deposition methods commonly
employed so far, in this work, we exploit the spontaneous self-
assembly of polymers and surfactants in bulk aqueous solution
into complex structures to prepare responsive, nanostructured
coatings via a simple, single-step spin-coating deposition proce-
dure. To prepare thin ﬁ lms of alternating polymer/surfactant
layers, mixtures of biopolycation chitosan and oppositely charged
oligo ethylene oxide (EO) alkyl ether carboxylic acids (AECs) of
general formula C
i
EO
j
CH
2
COOH are chosen (C
i
is the usual
nomenclature for fatty acids and j is the average number of EO
units). These mixtures are very versatile as they co-assemble into
a large variety of structures depending on the solution acidity, the
surfactant-to-polymer molar mixing ratio Z , and the number of
EO units of the surfactant 24 – 26 .
The approach presented in this work aims at the direct transfer
of the spontaneously formed aggregates from the aqueous solu-
tion onto a solid substrate via a simple spin-coating procedure.
The aim is to exploit the high degree of structural control, con-
ferred by the solution self-assembly process, for the functionali-
zation of solid substrates.
Results
Self-assembly in aqueous solutions . AECs with a lauric (C
12
)o r
oleic (C
18:1
) aliphatic chain and an average of 5 EO units form
vesicles in a narrow pH range of 4 – 4.5, close to the pKa value of
the fatty acid 27 – 29 . The representative chemical formula of the
surfactant and a schematic representation of the self-assembled
vesicles in aqueous solution are given in Fig. 1 a. In mixtures with
O
O
O
–
O
O
OH
5
5
H O
HO
O
CH 3
NH
OH
O HO
O O
OH
OH
NH 2
N*(1-DA)
N*(1-DA)
i
i
~7 nm
~4 nm
10 4
10 3
10 2
10 1
10 0
10 –1
0.01 0.1 1
I( q ) (cm –1 )
q (nm –1 )
x10
C 18:1 E 5 CH 2 COOH
C 18:1 E 5 CH 2 COOH/Chitosan
Chitosan
x2
a
b
c
d
O

O

O

O

Fig. 1 Description of the materials used and their solution self- and co-assembly behavior. a Representative chemical formula of an alkyl ether carboxylic
acid (AEC), with i = 18:1 or 12, at a pH value close to the pKa, and a representation of the bilayer and vesicle structure, formed in aqueous solution.
b Chemical formula of chitosan with a degree of polymerizat ion N and degree of deacetylation DA; c Schematic represen tation of the multilayered vesicles
found in aqueou s mixtures of polymer and AECs. d Small-angle neutron scattering patterns arising from a 0.6 wt% chitosan solution, 0.5 wt%
C
18:1
EO
5
CH
2
COOH solution, and their 1:1 mixture (0.3 wt% chitosan and 0.25 wt% fatty acid). Full lines represent curves calculated using models
describing the bilayer vesicle, the multilay er vesicle, and the linear polysaccharide depicted in a – c . The mathematical description of the corresponding
models is given in the Supplementary Note 3. Curves are scaled for improved readability. Error bars represent standard deviations of neutron counts
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the oppositely charged biopolymer chitosan (Fig. 1 b), well-
de ﬁ ned onion-like vesicles are formed 24 , represented schemati-
cally in Fig. 1 c. The formation of onion-like assemblies is com-
monly observed in mixtures of vesicle-forming surfactants and
oppositely charged macromolecules 30 – 32 . The structure of the
spontaneously formed assemblies in aqueous solutions can be
resolved in high detail using small-angle neutron scattering
(SANS) and dynamic light scattering (DLS). Representative
scattering patterns arising from aqueous solutions of chitosan,
C
18:1
EO
5
CH
2
COOH, and of their mixture are shown in Fig. 1 d.
The SANS data from the polysaccharide is described with a
generalized Gaussian coil model 33 and indicates that the acetic
acid/acetate buffer is a poor solvent for chitosan. In the surfactant
solution, we can identify the presence of large, polydisperse
vesicles, with the core formed by the aliphatic chains and the shell
by the hydrated EO units and carboxylic acid/carboxylate moi-
eties. The scattering pattern arising from the chitosan/AEC
mixture exhibit a clear Guinier region at low- q ( R
g
~ 40 nm),
indicating the well-de ﬁ ned size of the aggregates, and a pro-
nounced correlation peak at q ~ 0.9 nm − 1 , substantiating the
internal periodic structure of the aggregate. The scattering pattern
was successfully modeled with a periodic sequence of concentric
shells and a polydisperse core radius. Further details on the model
and analytical expressions used for the description of the SANS
data are provided in the Supplementary Note 3. To analyze the
data in absolute units, the molecular volumes and the scattering
length densities (SLD) of the components were obtained
according to Supplementary note 1 and Supplementary Figure 3.
The SANS pattern reveals the formation of multilayered vesicles,
formed by an average of three repeating chitosan/surfactant
layers of 7.9 nm thickness, and an average vesicle outer radius of
~30 nm. Multiangle DLS experiments show a diffusive behavior
for the system with a hydrodynamic radius of R
h
~ 70 nm (see
Supplementary Figures 6 and 7). This is in good agreement with
the expected value calculated from the structural parameters
obtained from the analysis of the SANS data (see Supplementary
Table 2). The apparent discrepancy between the vesicle sizes
obtained from the SANS and DLS lies in the polydispersity of the
sample, as demonstrated in Supplementary Notes 3 and 4. The
size of the vesicles, the layer thickness, and the number of
the layers can be controlled via the mixing conditions, i.e., the
surfactant-to-polymer mixing ratio, the pH, or the length of the
hydrophobic chain of the AEC (see Supplementary Fig. 5, Sup-
plementary Table 2, Supplementary Note 3 and ref. 24 ). In the
following, we exploit this spontaneous, controllable assembly
behavior for the preparation of thin ﬁ lms with similar internal
layered structures.
Multilayered thin ﬁ lms . When an aqueous solution, containing
the spontaneously assembled chitosan/AEC multilayered vesicles,
is spin-coated onto a silicon substrate, a macroscopically homo-
geneous ﬁ lm is formed (see Fig. 2 ). The ﬁ lm is highly sensitive to
variations in relative humidity (RH), as clearly demonstrated by
the change in color of the layer when gently blowing on the
surface (see Supplementary Movie 1), leading to a humidity
increase from approximately 30% to 60% RH.
Thin ﬁ lms were prepared from complexes formed by either
C
12
EO
5
CH
2
COOH or C
18:1
EO
5
CH
2
COOH and chitosan. These
chain lengths were chosen as they determine the thickness of the
layers in the multilayered vesicles: 6.3 nm for the C12-based
surfactant and 7.9 nm for the C
18:1
-based complexes. In addition
to the chain length variation, the effect of spin-coating conditions,
i.e., rotation speed, Ω , and the total concentration of polymer and
surfactant, c
tot
, on the optical ﬁ lm thickness, d
tot
, was system-
atically investigated by ellipsometry. The relationship between the
spin-coating parameters and the ﬁ lm thickness was found to be
d tot / c tot  Ω  0 : 6 (see Supplementary Figure 8), as predicted for
simple polymeric systems 34 (see Supplementary Note 5). It is
noteworthy that the same spin-coating procedure performed with
a pure surfactant solution (0.3 wt%) leads to the formation of
macroscopically highly heterogeneous ﬁ lms, while spin-coating
the pure chitosan (0.3 wt%) solution results in a ﬁ lm with a dry
thickness of <2 nm (details in Supplementary Note 6, Supplemen-
tary Figures 10 and 11, and Supplementary Table 3). In summary,
by simply varying the spin-coating conditions, thin ﬁ lms with
thickness varying from a few tens to hundreds of nanometers can
be prepared.
In or de r to eva lu ate th e in te rn al st ru ct ur e of the thin ﬁ lm s,
neut ro n re ﬂ ec to me t ry (NR ) ex pe ri me nt s w er e perf o rm ed un de r a
cont ro ll ed atm osph ere of hea vy water ( D
2
O) . The o bj ec ti ve of th e
swel lin g stud y wit h D
2
O was tw ofol d: ﬁ rst, i t allo wed the in ter na l
st ruct ure of th e ﬁ lm to be prob ed, by pr ovi ding an en han ced
cont rast be twee n hy dr ate d, hydr ophi li c regi ons — ch ito sa n and
surf act ant he ad gr ou ps — a nd th e hydr opho bic re gio ns — surf act ant
alky l chain s. Seco nd, it off ered si gni ﬁ ca nt insi ght s int o swel ling
beha vior of t he th in ﬁ lm, w hich is of pa ramo unt im port ance fo r
man y appl icati ons, e. g., ch em ical se nsi ng, upt ake /rel ease , and
me cha nica l actua tion , as the ph ysic o-c hemi ca l prop erti es of th e
coa ting a re expe cted to be st rong ly depe nden t on its wa ter cont ent.
A representative set of NR curves, recorded for the chitosan/
C
18:1
EO
5
CH
2
COOH coating at increasing RH, is shown in Fig. 3 .
The ﬁ lm was prepared by spin-coating a mixture containing 0.3
wt% chitosan and 0.3 wt% C
18:1
EO
5
CH
2
COOH ( Z = 0.3) at pH
4.3 at 500 RPM for 180 s. Further NR curves are shown in
Supplementary Fig. 12. The re ﬂ ectivity data exhibit a character-
istic evolution with increasing humidity. First, the apparent
critical edge shifts toward higher q values at increasing RH,
indicating an increase of the average SLD of the coating as it takes
up D2O (SLD of 6.33 × 10 − 4 nm − 2 ). Second, with the exception
of the curve at 0% RH, i.e., under an atmosphere of N
2
, the ﬁ rst
minimum moves to lower q values while the oscillation frequency
of the Kiessig fringes increases, characteristic of increasing layer
thickness. Finally, a broad Bragg peak appears at q ~1n m
-1 and
moves toward smaller q values with increasing RH, which is a
clear indication of the swelling of a multilayered system with
structural periodicity.
Th e quan tita tive da ta ana lysi s of th e NR patt erns w as perf orme d
by mo deli ng th e int erfa cial di stri but ion (v olum e frac tio ns) o f
30% RH 60% RH
a b c
Fig. 2 Photographs of representative samples investigated in this work. a Photograph of the aqueous solution with a composition of 0.3 wt% chitosan and
0.3 wt% C
18:1
EO
5
CH
2
COOH (mixing ratio, Z = [surfactant]/[chitosan units] = 0.3) at pH = 4.3, b of the silicon block coated with the same solution spin-
coated at 500 RPM for 180 s at ambient humidity of approximately 30% RH and c at approximately 60% RH
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all th e ch em ica l spec ies pr es en t. Th e volu me fra ct ion pr o ﬁ le s, ϕ
i
( z ),
are us ed to co mp ut e the in terf acia l SL D pro ﬁ le , whic h is, in tu rn,
us ed to gen era te th e corr espo ndin g re ﬂ ect ivit y cur ve, R ( q ), vi a the
Pa rratt re cursi ve algo rith m 35 . Co mp utin g the SLD pr o ﬁ le fr om th e
vo lume fr acti ons, and no t vice vers a, i s an e ffec tiv e way t o inclu de
mo lecula r cons tra ints a nd to ext ract ph ys ica lly mea ning ful
pa ramet ers fr om the ﬁ tting pr oce dure 36 – 38 .T h e ﬁ lm is mo dele d
as a se quen ce of al tern atin g poly mer a nd su rfact ant la yers , de ﬁ ned
by th eir thi ckne ss, roug hn es s, and t otal nu mber of la yer s. In the
fo ll owi ng, a laye r is de ﬁ ned as a one -comp onen t regi on of the ﬁ lm ,
ch ito san, su rfac tant hyd roph obi c chai ns, or surf acta nt hy drop hili c
he ad gr oup. As in the mo del of mu ltil ayer ve sicl es, on e repe ati ng
un it is fo rm ed by a sequ ence of fo ur laye rs comp osed of su rfa ctant
he ad gr oup, surf act ant ta il, su rfact ant he ad gr ou p, an d chit osan.
Th e who le ﬁ lm con tain s N repe ati ng un it s in a dditi on to a n ini ti al
ch ito san la yer. In term ixi ng be twe en the com pone nts is al lowed .
Th e inc oher ent su m o f the NR curv es from la ye rs fo rmed by N and
N + 1 re pe at ing un its a llow s the NR pr o ﬁ le of th in ﬁ lm fo rm ed by
a no n- in te ger a vera ge n umbe r of re peat ing unit s to be calc ul ate d.
Fo r ea ch hu midi ty, th e wate r cont ent in the hy dro phili c laye rs is
all owed to va ry. Wi th th ese a ssum pt io ns, th e vo lume fr act ion
pr o ﬁ le s ϕ
i
( z ) a re desc ribe d by a set of para mete rs com mon to the
who le sw elli ng se ries (n umb er of la ye rs , to tal amo unt o f ch itos an
and su rfac tan t, and su bstr ate ro ug hn ess ) and by a set of hu midi ty -
sp ec i ﬁ c para mete rs (w ater co nte nt of th e hydr ophi lic la yers , void
co nten t of the ﬁ lm in dry co ndit ions , roug hnes s of th e layer , and
in term ix ing of th e compo nen ts). Thes e para mete rs wer e var ied
un ti l the be st ag re emen t of the sim ulate d NR cu rve wi th the
ex pe ri me nta l on es wa s ac hi eved an d are re po rted in S uppl emen -
ta ry Ta bl es 4 and 5. A graph ica l repr esen tat ion of th e mode l is
gi ven in Fig . 3 d and full de tail s are gi ven in t he “ Me thod s ” sect ion.
The model was used to describe the NR patterns shown in
Fig. 3 a. In particular, the SLD pro ﬁ les (Fig. 3 b) were obtained
from the volume fraction pro ﬁ les in Fig. 3 c. This highly
constrained model is able to reproduce the most relevant features
of the experimental re ﬂ ectivity data: the shift in the critical edge;
the position, width, and intensity of the Bragg peak; and the
position of the Kiessig fringes. All of these features validate the
underlying structural model. However, some predicted fringes are
more pronounced than they appear in the experimental data. This
discrepancy likely arises from sample inhomogeneities over the
illuminated area of 2.5 × 3.0 cm 2 , a factor not taken into account
by the model. The structural model is further supported by a
qualitative analysis of the off-specular re ﬂ ectometry signal (see
Supplementary Note 10 and Supplementary Figures 15-17),
which con ﬁ rms the presence of Bragg sheets. Moreover, the
observed linear relation between the diffuse scattering and the
ﬁ lm SLD indicates that no strong structural changes, e.g., layer-
to-vesicle transition or lateral phase separation, take place during
the swelling process.
The volume fraction pro ﬁ les deduced from the NR experi-
ments (Fig. 3 c) clearly show that the spin-coating process of
polysaccharide/oleic acid-based surfactant, self-assembled, multi-
layered vesicles produces thin, multilayered ﬁ lms. The internal
periodicity results in the consistent alternation of hydrophilic
(chitosan and surfactant head group) and hydrophobic (surfac-
tant tail) layers, with the swelling water enriching only the
hydrophilic region. The size of the repeating unit is 4 – 7 nm,
depending on the RH value (see Supplementary Table 5), thus
reaching the limits of nano-patterning in thin ﬁ lms by
spontaneous phase separation required for new-generation
nano-lithography 39 – 41 . With the aim of achieving even smaller
RH = 00 %
RH = 0.0 %


i (z)
Si
SiO 2
Chitosan
1
0


i (z)
1
0


i (z)


i (z)
1
0


i (z)
1
0


i (z)
1
0
Alk. Chain Air Repeating unit
Si block
SiO 2
Chitosan
Chitosan
Chitosan Alkyl chain
SiO 2
Si
D 2 O
Chitosan
Chitosan
Humid air
Surfactant
Headgroup
Air
Alkyl chain
Alkyl chain
Alkyl chain
EO 5 CH 2 COOH
EO 5 CH 2 COOH
EO 5 CH 2 COOH
EO 5 CH 2 COOH
EO 5 CH 2 COOH
EO 5 CH 2 COOH
D 2 O
EO 5 COOH
37.0 %
47.0 %
53.0 %
65.0 %
Reflectivity
0.1
5
4
SLD (10 –4 nm –2 )
3
2
1
0 0.0 10.0 20.0 30.0 40.0 50.0 60.0
0 1 53 04 56 0
SLD(z)
Z
z (nm)
z (nm)
0.2 0.4
q (nm –1 )
0.8 1.6
37 %
47 %
53 %
65 %
RH = 00 %
37 %
47 %
53 %
65 %
ac d
b
Fig. 3 Characterization of the thin ﬁ lms by neutron re ﬂ ectometry (NR). a NR patterns measured at controlled D
2
O humidity of a chitosan/
C
18:1
EO
5
CH
2
COOH thin ﬁ lm. x axis is shown on a logarithmic scale and curves are scaled to enhance readability. Solid lines represent calculated re ﬂ ectivity
curves. b Scattering length density (SLD ) pro ﬁ les used to calculate the NR curves. c Volume fraction pro ﬁ les, ϕ
i
( z ), used to calculate SLD( z ). d Graphical
description of the model underpinning the NR data analysis including the representat ion of the layered model used to describe the NR curves and the
corresponding volume fraction and SLD pro ﬁ les
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domain sizes, additional thin ﬁ lms were prepared from mixtures
of chitosan and the lauric acid-based surfactant C
12
EO
5
CH
2
-
COOH, see Supplementary Note 7. The re ﬂ ectivity curves are
shown in Supplementary Fig. 12 and reveal a similar swelling
behavior, with the ﬁ lm being formed by fewer and thinner repeat
units. Interestingly, in both cases the thickness of the repeat unit
at the highest probed humidity of 65% RH (6.5 and 7.4 nm for the
C
12
and C
18:1
-based AECs, respectively) exactly equals the
repeating distance found in the multilayered vesicles, providing
further evidence of the direct link between assemblies in aqueous
solution and surface structure. The analysis also reveals that the
composition of the thin ﬁ lms is close to that of the multilayered
vesicles (see Supplementary Table 7).
It is worth noting tha t all NR curves rec orded at 0% RH , i.e.,
under N
2
atmosp here, could onl y be describ ed by assuming a large
fracti on (appro ximately 50 vol %) of voids in the lay er. The hi gh air
conte nt results in the, seemin gly paradoxic al, case of thin ﬁ lms
being thi cker in dry co nditio ns than in humid air , when water is
absor bed inside the layer . In order to valid ate the results from the
NR analys is with the compl ex multil ayer model , the low- q par t of
the dat a, whe re the sm all-sc ale, intern al structur al features of the
ﬁ lm can be negle cted, was describe d with a homogen eous ﬁ lm
model (see Suppl ementary Note 8). In the an alysis, onl y the total
amo unt of surfac tant, pol ymer, air, and D
2
O was allow ed to vary
(see Suppl ementary Not e 9, and Supplementa ry Figures 13 and
14). Aside fro m small varia tions, the analy sis leads to layers wi th
the sam e thickness and co mpositio n as found by th e multilaye r
model , as summariz ed in Supplemen tary Table 6. Th e presence of
voids in polye lectro lyte multil ayer thin ﬁ lms has previo usly been
repor ted in the litera ture, althou gh only to a maximum void
conte nt of 20 vol% 42 – 44 . We hy pothesiz e that the lar ge fracti on of
voids ari ses due to a dry, rigid str ucture with numer ous defec ts
and that water act s as pla sticiz er, tr iggeri ng the transi tion to a
softer and mo re compac t struct ure.
Film morphology and crystalline structure . The hypothesis of
crystalline-to-amorphous transition causing the anomalous
behavior in dry conditions was examined by atomic force
microscopy (AFM) and X-ray diffraction in grazing incidence
geometry (GI-XRD) experiments under controlled humidity
conditions. AFM surface height scans performed on an area of
400, 25, and 1 μ m 2 are given in Fig. 4 , and the corresponding
phase traces are provided in Supplementary Fig. 18. The results
show the formation of a polycrystalline ﬁ lm at 0% RH, with ﬂ at,
crystalline domains with a typical size of ~200 nm, and an almost
parallel orientation to the silicon substrate. Platelets, as shown in
Fig. 4 , have also been reported in other chitosan-based compo-
sites and chitosan single crystals 45 – 47 . In contrast, a homo-
geneous, softer ﬁ lm is formed at 40% RH. The surface roughness,
evaluated as the standard deviation of the height pro ﬁ le on the
20 × 20 μ m 2 scans, is approximately 7 nm for both humidity
conditions, in good agreement with the decay of the SLD pro ﬁ le
from the NR analysis.
This cry stalli ne-to- amorphou s transiti on is clear ly suppor ted by
the GI-XR D patterns rec orded at different hum idity con ditions
(see Fig. 4 ). Under dry co ndition s, three clear peaks are found at q *
values of 2.14, 4. 52, and 6.66 nm − 1 , correspo nding to lattic e
spac ings of 2.9 3, 1.39, an d 0.9 4 nm , respe ctively . Some resid ual
crysta llinity is fo und at 25% RH, as eviden ced by the large
should er at low q and by the correl ation peak at q * = 4.4 1 nm − 1 ^ ¼
1.42 nm. The signal at approxim ately 6.5 nm − 1 wea kens wit h
increa sing humidi ty, while a residu al, very wea k and broad signal
at appr oxi matel y 14 nm − 1 persis ts at all humidity condi tions.
Chitosa n exhibi ts a complex crysta llization beha vior and different
crysta lline struct ures are obtaine d depending on the cr ystalliza tion
condit ions, i.e ., th e temper ature of pre paratio n, the natur e of the
coun terion, the pre sence of wa ter in the crys tal, and the tim e
since pre paratio n of the crysta l 47 – 52 . The si gnal s at approx imate ly
6.5 nm − 1 an d approxim ately 14 nm − 1 can be direc tly ascrib ed to
the chitosa n chains 48 , 53 . Moreo ver, the GI-XRD data exc lude the
format ion of sodiu m acetat e crystal lites, whose dif fracti on signals
are found between 6.2 and 25 nm − 1 54 .
In summary, the XRD and AFM results show that the
adsorption of water in the polysaccharide/surfactant induces
conformational changes of the chitosan chains, with the presence
of crystalline domains at low humidity and a fully amorphous
ﬁ lm at high humidity values. Dynamic mechanical analysis on
chitosan ﬁ lms have shown a gradual decrease in the storage
modulus with increasing humidity 55 , corroborating the hypoth-
esis of water acting as plasticizer in the ﬁ lm, thereby triggering
the transition from a semi-crystalline ﬁ lm at low humidity to
an amorphous ﬁ lm at high humidity. Finally, the AFM scans
con ﬁ rm the full conversion of the vesicles into a
homogeneous ﬁ lm.
RH = 00% RH = 25% RH = 40% RH = 65%
40% RH
Intensity (Log) / a.u.
51 0 1 5
15
10
5
Height (nm)
–5
–10
–15
0
20
Intensity (a.u.)
123456
q (nm –1 )
q (nm –1 )
789 1 0
0% RH
5 µ m 1 µ m
1 µ m
5 µ m
0.2 µ m
0.2 µ m
a b c
d e f
g
Fig. 4 Morphology of the chitosan/C
18:1
EO
5
CH
2
COOH coatings as seen by atomic force microscopy (AFM) and X-ray diffraction in grazing incidence
geometry (GI-XRD ). On the left, AFM scans performed on different areas: 20 × 20 μ m 2 ( a , d ), 5 × 5 μ m 2 ( b , e ), 1 × 1 μ m 2 ( c , f ), dry conditions ( a – c ), and at
40% relative humidity of water ( d – f ). Squares indicate the magni ﬁ ed areas shown to the right of each frame. The GI-XRD patterns recorded with a grazing
incidence angle of 0.2° at different humidity values are shown in g . The shaded area represents the standard deviation resulting from at least ﬁ ve
measurements. Inset shows high- q data on a logarithmic scale
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Sample ageing and repeatability of ﬁ lm preparation .A ﬁ nal
aspect probed in this work is the evolution of the thin ﬁ lms with
time. Nine identical samples were prepared by spin-coating the
same chitosan/C
18:1
EO
5
CH
2
COOH solution ( Z = 0.3 and at pH
4.3) at 500 RPM for 180 s. The ﬁ lms were stored in closed
chambers in contact with silica gel (dry conditions), with a
saturated potassium acetate solution (23% RH), and with a
saturated sodium chloride solution (75% RH). The evolution of
the ellipsometric ﬁ lm thickness and refractive index of the ﬁ lms
was followed for 35 days and is shown in Fig. 5 . No change in the
ﬁ lm thickness and refractive index is found for samples stored in
dry conditions and at 23% RH. In contrast, a clear decrease of
thickness and increasing inhomogeneity, as evidenced by the
large error bars, is found for the samples stored at 75% RH. The
latter also exhibits small heterogeneities visible by naked eye.
Accordingly, optical images of the thin ﬁ lms stored under dif-
ferent conditions were taken with different level of magni ﬁ cation
and are shown in Fig. 6 . The images clearly demonstrate that the
ﬁ lms stored in dry conditions and at high humidity exhibit phase
separation, while no sign of demixing is found in the ﬁ lms stored
at 23% RH. In particular, the morphology of the phase-separated
aggregates strongly varies with humidity. After 35 days of storage
at high humidity, large (approximately 100 μ m), star-like aggre-
gates with few branches are formed. In contrast, after long storage
in dry conditions, smaller (10 – 20 μ m) fractal-like aggregates are
found. The AFM height traces, which allow for a stronger mag-
ni ﬁ cation, further con ﬁ rm the presence of fractal-like phase
separation for samples stored in dry conditions, the formation of
large, rigid star-like objects at 75% RH, and the presence of a
homogeneous ﬁ lm when the sample is stored at 23% RH.
Finally, the repeatability and the effect of the age of the
multilayered vesicle solution on the ﬁ lm thickness were probed.
In Fig. 5 , the dependence of the ellipsometric thickness of the thin
ﬁ lms is plotted as a function of the day of preparation for two
different solutions with different age. The data reveal a random
variability of the thickness with time of approximately 7%. We
ascribe the variability to ﬂ uctuations in the humidity of the
laboratory during the ﬁ lm preparation (see Supplementary Fig. 9),
which is a parameter affecting the spin-coating deposition from
aqueous solutions 56 , 57 . The variability of the thickness of samples
prepared on the same day from the same solution is 4%, while a
systematic deviation between the thicknesses from the two
solutions of 5% was found.
Discussion
The combination of the structural information from NR and
morphological features from AFM results in the following uni ﬁ ed
picture: thin polysaccharide/lipid ﬁ lms, with an ordered multi-
layer structure, can be prepared in a single and rapid step, by
spin-coating a solution of spontaneously co-assembled multilayer
vesicles onto a solid surface. The hydrophilic regions of the ﬁ lm,
namely, the chitosan chains and the fatty acid PEGylated head
groups, selectively and reversibly absorb water as a function of the
RH, resulting in a remarkable color change of the ﬁ lm, thereby
allowing for optical sensing of humidity and the design of
humidity-triggered optical ﬁ lters. Moreover, in dry conditions,
the ﬁ lm switches to a polycrystalline morphology, with partially
ordered platelets formed by crystalline chitosan/lipid domains. A
schematic representation of the process is given in Fig. 7 . A slow
phase separation in the ﬁ lm is observed over a time scale of
several weeks, which is strongly dependent on the humidity
conditions at which the ﬁ lms are stored.
In summary, we report the formation of well-de ﬁ ned poly-
electrolyte/lipid coatings, whose mesoscopic structure is the result
of a spontaneous co-assembly process of chitosan and oppositely
charged ethoxylated fatty acids into multilamellar vesicles in bulk
aqueous solution. The preparation is rapid, low-cost, repro-
ducible, and highly versatile, as it allows ﬁ lms to be prepared with
a controlled total thickness, number of layers, and periodicity. In
particular, the thickness of the repeating unit is dictated by the
length of the fatty acid, while the total ﬁ lm thickness is deter-
mined during the spin-coating process via the total concentration
and the spin speed. NR reveals a clear segregation between the
hydrophilic and hydrophobic regions, with a strong swelling
response of the hydrophilic regions to changes in RH. The pre-
paration procedure is, at this moment, restricted to relatively
small, rigid, and ﬂ at substrates, due to the intrinsic limitations of
the spin-coating deposition procedure. It is worth conducting
further studies, with the aim of exploring the potential of these
responsive, compartmentalized membranes as functional coatings
for food preservation, hierarchical nanoreactors, and transport
membranes and to extend the deposition process to different
substrates.
60
65
70
75
80
85
90
95
100
a
b
d tot / nm
d tot / nm
1.45
1.50
1.55
1.60
1.65
1.70
0 5 10 15 20 25 30 35 40
n
Age of sample/days
Dry conditions
23% RH
75% RH
60
65
70
75
80
85
90
Date of coating preparation
Solution prepared on
10. Dec. 2018
14. Jan. 2019
01/16/19
01/18/19
01/20/19
01/22/19
01/24/19
01/26/19
01/28/19
01/30/19
01/01/19
01/03/19
01/05/19
01/14/19
Fig. 5 Time-dependent evolution of the ellipsometric thickness and
refractive index for chitosan/C
18:1
EO
5
CH
2
COOH thin ﬁ lms. a The evolution
of samples stored under different conditions as a function of sample age.
b The ellipsometric thickness as a function of the day of preparation of the
coating from two solutions of different age; the broken lines represent the
average thickness and the shaded areas the standard deviations. For each
condition, three samples were prepared and investigated; error bars
represent standard de viation between different points on the same sample
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The method prese nted in this work is no t restricted to
polymer/surfactant mixtures , and we expect that similar
multilayered coat ings can be prepared applyin g an analogous
rapid, single-step procedure to a large variety of spontaneously
assembled, hybrid, layered systems in bulk solution,
such as: catanionic 58 , 59 , surfactant/polyoxometalates 60 ,f u l l -
erene/polyoxo metalates 61 , and DNA/lipid multilayer 62 , 63
vesicles.
Methods
Materials . Chitosan was obtained from TCI Europe. It is characterized by a
viscosity-average molecular weight of 100 kDa and a degree of acetylation of 0.15
determined by 1 H-nuclear magnetic resonance (NMR) and puri ﬁ ed before use as
described previously 25 . Two oligo ethylene oxide alkylether carboxylic aci ds were
obtained from Kao Chemicals and are available under the trade names AKYPO RO
50 VG and AKYPO RLM 45 CA. The surfa ctants are of technical grade and were
used without puri ﬁ cation. They were, however, characte rized by 1 H-NMR spec-
troscopy 27 , revealing that the hydrophobic part of RO 50 VG is a 3:1 mixture of
C
18:1
and C
16:0
aliphatic chains, while that of RLM 45 CA is a 2:1 mixtures of C
12
and C
14
chains. The degree of ethoxylation is 4.7 and 4.6 for RO 50 VG and RLM
45 CA, respectively. The degree of carboxymethy lation is for both surfactants ca.
0.9. RO 50 VG is referred to with the representative formula of C
18:1
EO
5
CH
2
-
COOH and RLM 45 CA with C
12
EO
5
CH
2
COOH. The 1 H-NMR spectra were
recorded on a Brucker Avance II spectrometer operating at 400 MHz and are
provided in Supplemen tary Figs. 1 and 2.
Bulk solution preparation . Solutions were prepared in an acetic acid buffer with a
total concentration of acetate/acetic acid (Roth, 99%) of 200 mM. pH was adjusted
using a concentra ted sodium hydroxide solution. Solutions were prepared at a
constant chitosan concentra tion of 0.3 wt% and at a mixing ratio of Z = 0.3,
de ﬁ ned as the ratio between the moles of surfactants and the moles of deacetyla ted
chitosan units (using an effective M
w
of 196.8 g mol − 1 ). Unless otherwise stated,
solutions were prepared in D
2
O.
Dry conditions
5 mm 5 mm 5 mm
500 µ m 500 µ m 500 µ m
5 mm 5 mm 5 mm
500 µ m 500 µ m 500 µ m
23 %RH 75 %RH
1-day storage 35-day storage
0 200 400 600 800 0 100
Height trace (nm) Height trace (nm) Height trace (nm)
ab c
de f
gh i
jk l
mn o
10 µ m 10 µ m 10 µ m
20 40 60 80 100 300 200
Fig. 6 Morphological investigation of identically prepared thin ﬁ lms stored under different humidity conditions. The ﬁ lms were prepared by spin-coating the
same chitosan/C
18:1
EO
5
CH
2
COOH solution ( Z = 0.3 and at pH 4.3) at 500 RPM for 180 s. The images show: optical microscopic images ( a – l ) of samples
stored for 1 day ( a – f ) and for 35 days ( g – l ) in dry conditions ( a , d , g , j ), at 23% relative humidity (RH) ( b , e , h , k ), and at 75% RH ( c , f , i , l ); atomic force
microscopic (AFM) height traces ( m – o ) of samples stored for 35 days in dry conditions ( m ), at 23% RH ( n ), and 75% RH ( o ). Note the different
magni ﬁ cations in the optical images and the different height scales in the AFM images
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Substrate cleaning . The substrates consisting of silicon mono-crystalline blocks
cleaved along the (111) direction (5 × 5 × 1 cm 3 ), were cleaned in an ultrasonic bath
in an ethanol/acetone 1:1 mixt ure, extensively washed with Milli-Q water (speci ﬁ c
resistivity 18.2 M Ω cm), and dried under nitrogen ﬂ ow. Surface activation was
performed by ozone etching (BioForce Nanosciences UV/ozon e ProcleanerTM
Plus) for 10 min. The thickness of the silicon oxide layer was checked prio r to the
re ﬂ ectivity experiments with optical ellipsometry.
Coating preparation . The chitosan – surfactant mixtures were spin-coated on
freshly cleaned silicon blocks, using an SÜSS MicroTec Delta 6 spin-coater.
Approximately 2 mL of solution were deposited onto the blocks to cover the entire
substrate surface. Where not otherwise stated, the spin-coating process wa s per-
formed with a rotation speed of 500 RPM for 3 min. The coated samples were
kept under saturated D
2
O atmosphere before NR experiments.
Small-angle neutron scattering . SANS curves were recorded on D11 at the
Institut Laue-Langevin (ILL) in Grenoble, France 64 . Three different con ﬁ gurations
were used, with a wavelength of λ = 0.6 nm; sample-to-detector distances (SD) of
1.5, 8, and 34 m; and collimation lengths of 8, 8, and 34 m, respectively, covering a
q -range of 0.02 – 4n m
− 1 , where
q ¼ 4 π sin ð θ = 2 Þ = λ ð 1 Þ
is the magnitude of the scattering vector and θ is the scattering angle. The
differential cross-sections (absolute scaling) were obtained by comparison with the
scattering from a water sample with 1 mm path length. The data were described
in absolute units using the SLD values and molecular volumes reported in Sup-
plementary Table 1. The SANS pattern arising from the chito san solution was
previously published 25 and recorded on D11 using three con ﬁ gurations with
sample-to-detector distan ces (SD) of 1.2, 8, and 34 m and collimations of 4, 8, and
34 m, respective ly, and wavelength of λ = 0.6 nm.
Light scattering . Static and dynamic light scattering experiments were simult a-
neously performed at 25 °C on a compact ALV/CGS-3 instrument, equipped with a
He – Ne laser with a wavelength of 632.8 nm. The absolute intensity was obtained
using toluene a standard , where a Rayleigh ratio of 1.34 × 10 − 5 cm − 1 was used 65 .
Experiments were performed at variab le scattering angles between 21° and 145°,
thus covering a q -range of 4.8 × 10 − 3 – 2.3 × 10 − 2 nm − 1 .T h e ﬁ eld autocorrelation
function was computed by the ALV 5000/E multiple- τ corr elator and was
described as 66 :
g ð 1 Þ ð q ; τ Þ¼ exp ð Γ ð q Þ τ Þ 1 þ μ 2 ð q Þ
2 τ 2
 ð 2 Þ
with Γ being the mean decay rate, τ the delay time, and μ
2
is related to the second
moment of the distributi on of decay rates. The diffusion coef ﬁ cient D
h
was
obtained from the relation Γ ( q ) = D
h
q 2 . The hydrodynamic radius is ﬁ nally
calculated using the well-known Stoke s – Einstein relation:
R h ¼ k B T
6 πη 1 D h ð 3 Þ
where k
B
, T , and η
1
are the Boltzmann constant, the absolute temperat ure, and the
viscosity of the solvent, respectively. The data were also analyzed using the con-
strained inverse Laplace transformat ion CONTIN 67 , provided with the ALV data
analysis software.
Optical ellipsometry . Single-wavelength (632 nm) multiple-angle ellipsometry was
used to determine the total ﬁ lm thickn ess. Experiments were performed on a
Picometer Light ellipsometer (Beaglehole Instruments, New Zealand) with incident
angles between 50° and 80° with step increment of 2°. The reported thickness es are
the average from three randomly chosen points on the surface.
Neutron re ﬂ ectivity . NR measurements were performed on the D17 re ﬂ ectometer
at the Institut Laue-Langevin (ILL, Grenoble, France) 68 (https://doi.org/ILL-
DATA.9?12?455/ILL-DATA.9-12 -455). The measurements were performed in
time-of- ﬂ ight (ToF) mode with instrumen tal resolution of Δ Q / Q between 3% and
6% and wavelengths ranging from 2 to 22 Å. The footprint length (in the beam
direction) and width (in the perpendicular direction) were L = 2.5 cm and W = 3
cm, respective ly.
The samples were placed inside a humidity chamber 69 . The chamber and the
sample were kept at constant temperature of a 25 °C via a thermostatic bath, while
the temperature of four, small reservoirs inside the chamber ﬁ lled with D
2
O was
controlled via piezo-elements to reach the desired humidity value following the
psychrometric charts. The humidity level was monitored by a commercia l humidity
sensor (Honeywell Inc., USA) placed inside the cell in close proximity to the
sample. NR experiments in dry conditions were performed by ﬂ ooding the sample
chamber with dry nitrogen. After temperature stabilization, the sample was allowed
to equilibrate to the new humidity levels for at least 15 min before starting the
acquisition of the re ﬂ ectivity curves. Further details on data reduction can be found
in the Supplementary Note 2.
Spontaneous
self-assembly
in solution
Swelling
F rom bulk to surf ace
by
spin-coating
60% RH 30% RH 0% RH
Cr ystallization
Film proper ties
OH
OH OH
OH
HO
NH 2
NH 2
NH 2
NH 2 O
O
O
O
O
O O O O
HO
HO
HO
T otal thickness from
spin-coating parameters
Repeating unit thickness from
f atty acid chain length
Fig. 7 Schematic representation of the spontaneous co-assembly of ethoxylated fatty acid and chitosan into multilayered vesicles and the response
behavior of the corresponding multilayered ﬁ lm
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Neutron re ﬂ ectivity data analysis . The SLD pro ﬁ le, ρ ( z ), used for the calculation
of the re ﬂ ectivity curve s, was obtained via a three-step procedure, schematically
represented in Fig. 3 d. In the ﬁ rst step, each coating is described by a sequence of
discrete layers, characte rized by their content in chitosan, hydrop hobic surfactant
chains, surfactant head groups, and D
2
O. The thickness and content of the layers
are de ﬁ ned by a set of parameters common to all curves and by a set of parameters
that are humidity dependent and de ﬁ ned hereafter as P ( h ). The sequence of layers
is equal for all investigated systems and built-up as follows: the ﬁ rst, semi-in ﬁ nite
layer is made of pure silicon, followed by a thin layer of silicone oxide. The
thickness of the silicon oxide layer was determined by optical ellipsometry. The
interface between the silicon oxide layer and the coating was set at z = 0 nm.
The organic layer was built-up as a sequence of layers composed of mainly
surfactant head group, surfactant alkyl chain, surfactant head group, and chitosan
layers. This sequence of laye rs is repeated Floor( N ) and Ceil( N ) times. Floor( x ) and
Ceil( x ) are the ﬂ oor and ceiling functions, which return the greatest integer less
than or equal to x and the least integer greater than or equal to x , respectively.
Mixing between surfactant tails and head group is quanti ﬁ ed by the ﬁ t parameter
Ξ ð h Þ . The amounts of surfactant head group and tails in the respective layers, given
in volume per surface area, were calculated as:
d 0
a ¼ d a
a ð h Þþ 2 d h
a ð h Þ ð 4 Þ
d 0
h ¼ d h
h ð h Þþ d a
h ð h Þ
2 ð 5 Þ
Ξ ð h Þ¼ d h
a ð h Þ
d h
a ð h Þþ d h
h ð h Þ ¼ d a
h ð h Þ
d a
h ð h Þþ d a
a ð h Þ ð 6 Þ
with d 0
a and d 0
h being the amount of surfactant head group and tail per repeating
unit. d a
a ð h Þ and d h
a ð h Þ are the amounts of surfactant tails in the layers composed of
mainly alkyl chains and head groups, respective ly. Similarly, d a
h and d h
h are the
amounts of surfactant head groups in the layers composed of mainly alkyl chains
and head groups, respectively. The total amount of surfactant head groups and tails
in the ﬁ lm is given by 2 Nd 0
h and Nd 0
a , respectively. The amounts are constraint via
the molecular volumes of the hydrop hilic v
h
and hydrophobic v
a
surfactant parts as
d 0
h ¼ d 0
a  v a = ð 2 v h Þ . Equations 4 – 6 can be solved leading to the amount of
surfactant head groups and tails in the re spective layers:
d a
a ð h Þ¼ d 0
a ð Ξ ð h Þ 2  2 Ξ ð h Þþ 1 Þþ d 0
h ð 2 Ξ ð h Þ 2  2 Ξ ð h ÞÞ
1  2 Ξ ð h Þ ð 7 Þ
d h
h ð h Þ¼ d 0
a ð Ξ ð h Þ 2  Ξ ð h ÞÞ þ d 0
h ð 2 Ξ ð h Þ 2  4 Ξ ð h Þþ 2 Þ
2  4 Ξ ð h Þ ð 8 Þ
d a
h ð h Þ¼ 2 d 0
h Ξ ð h Þ 2 þ d 0
a ð Ξ ð h Þ 2  Ξ ð h ÞÞ
2 Ξ ð h Þ 1 ð 9 Þ
d h
a ð h Þ¼ d 0
a Ξ ð h Þ 2 þ d 0
h ð 2 Ξ ð h Þ 2  2 Ξ ð h ÞÞ
4 Ξ ð h Þ 2 ð 10 Þ
Ξ ð h Þ is allowed to vary between 0 and d 0
h = ð d 0
h þ 0 : 5 d 0
a Þ and is a humidity-
dependent parameter. The surfactant was not allowed to diffuse into the chitosan
layer. In contrast, penetration of chitosan into the surfactant layers is allowed, and
the parameter φ ( h )d e ﬁ nes the ratio between the volumes of chitosan and
surfactant in the “ surfactant laye r ” as:
ϕ ð h Þ¼ d h
ch ð h Þ
d h
a ð h Þþ d h
h ð h Þ ¼ d a
ch ð h Þ
d a
a ð h Þþ d a
h ð h Þ ð 11 Þ
with d h
ch ð h Þ and d a
ch ð h Þ being the amounts of chitosan in the laye rs composed of
mainly alkyl chains and head groups, respective ly. The amount of chitosan in the
chitosan layer d ch
ch was then calculated as:
d ch
ch ð h Þ¼ d 0
ch  ϕ ð h Þð d 0
a þ 2 d 0
h Þ ð 12 Þ
with d 0
ch being the amount of chitosan per repeating sequence. φ ( h ) is humidity
dependent and varies with the distance from the substrate as:
ϕ ð j ; h Þ¼ erf ð j  j 0 ð h ÞÞ g 0 ð h Þ ½ þ 1
2 ð 13 Þ
erf is the error function, j is the numbe r of the repeating unit starting at 1
and ending at Floor( N ) or Ceil( N ), and j
0
( h ) and g
0
( h ) are the ﬁ tted humidity-
dependent parameters that descri be φ ( h ). The choice of the erro r function to
describe the intermixing between chitosan and surfactant, i.e., the extent of
segregation within the system, is arbitrary and is motivated by the property of
the error function to describe both relatively constant φ values (when g
0
( h )i s
close to zero), increasing and decreasing values according to the sign of g
0
( h ) and
strongly j -dependent values when g
0
( h ) is large. j
0
( h )d e ﬁ nes the position of the
in ﬂ ection point of the function. The maximum value of φ ( j , h ) is limited to
ϕ ð j ; h Þ <d 0
ch = ð d 0
a þ 2 d 0
h Þ . An additional chitosan layer is inserted between the
multilayer and the silicon oxide.
The water content of each layer is calculated under the assumption that
chitosan and the surfactant adsorb water to the same extent and is comput ed as:
d i
w ¼ð d i
ch þ d i
h Þ φ ð h Þ
1  φ ð h Þ ð 14 Þ
with ϕ ( h ) being the volume fraction of water in the ﬁ lm relative to the surfactant
head group and chitosan content.
Finally, the sum of the amounts of the different components, D
2
O, chitosan,
surfactant head group, and surfactant tails, in each layer resulted in the total layer
thickness. The volume fraction of the i th component is re adily calculated as the
ratio of its amount and the layer thickness. In addition to thickness and
composition, each layer is characte rized by its roughness. In particular, the
roughnesses of the silicon/silic on oxide and silicon oxide/coating interfaces, σ
Si
and
σ SiO 2 , are constant within each series.
The water content of the layer is set to 0 w hen the system is put under dry,
nitrogen atmosphere. For this part icular case, the presence of voids in the
system is accounted for. In particular , the following strategy was fol lowed: the
total number of repeating units for ming the ﬁ lm was recalculate d as N /(1 −
ϕ
void
) and the volume fractions of the com ponents multiplied by the factor (1
− ϕ
void
).
The second step consists of converting the discrete sequence of layers with
de ﬁ ned thickness and composition and roughness of their interfaces into
continuous volume fraction pro ﬁ les for each component. For the k th components,
it reads:
φ k ð z Þ¼ φ k
0 þ X
n
i ¼ 1
φ k
i  φ k
i  1
2 1 þ erf z  z i
ﬃﬃ ﬃ
2
p σ i
! "#
ð 15 Þ
with φ k
0 being the volu me fraction of k th component in the ﬁ rst layer, i.e., the
silicon block where all volume fractions, except that of silicon, are zero. z
i
is the
distance of the i th interface from the silicon oxide/co ating interface set at z = 0, and
n is the total number of layers in the ﬁ lm.
The third step consists of calculating of the SLD pro ﬁ le ρ ( z ) from the volume
fraction pro ﬁ les, as the volume weight average of the SLD of the single
components:
ρ ð z Þ¼ X
k
φ k ð z Þ SLD k : ð 16 Þ
The SLD values of the k th component (SLD
k
) and the molecular volumes used
for the calculations are given in Supplementary Table 1. Owing to technical
reasons, the humidity cell has to be brie ﬂ y opened before the measuremen t under
N
2
atmosphere can be performed, which exposes the sample to ambient air. In
order to take into account a possi ble exchange of the labile protons of chitosan, the
SLD of chitosan for the 0% RH dataset is a ﬁ tted paramet er, which is allowed to
vary between the two extreme values provided in Supplem entary Table 1. For the
experiments under D
2
O atmosphere, a value of 4.64 × 10 − 2 nm − 2 was used.
Finally, the SLD pro ﬁ le SLD ( z ) was discretized into thin and sharp slabs of 1 Å
thickness, and the re ﬂ ectivity curve was calculated by applying Fresnel ’ sr e ﬂ ection
laws at each slab interf ace using the iterative procedure proposed by Parratt 35 .
No effects in the re ﬂ ectivity curves were seen when the thicknes s of the slabs is
further reduced.
The last step is to perform the incoherent average of the re ﬂ ectivity patterns
calculated for the Ceil( N ) and Floor( N ) coatings. The weighted ave rage was
performed as:
R ð q ; N Þ¼ Ceil ð N Þ N ½ R ð q ; Floor ð N ÞÞ þ N  Floor ð N Þ ½ R ð q ; Ceil ð N ÞÞ ð 17 Þ
The analysis was performed using python script language making use of the
LM ﬁ t ﬁ tting routine based on the SciPy package. Data ﬁ tting with the simpler,
homogeneous layer model was performe d using the Levenberg – Marquardt
minimization routine, while when the more complex, multilayered model was
employed, the quasi-Newto n limited-memory
Broyden – Fletcher – Goldfa rb – Shanno minimization algorithm was used.
Atomic force microscopy . AFM me asurements were performed on a Cypher S
Scanning Probe microscope (As ylum Research, USA). T he instrument was oper-
ated in intermi ttent contact mode (AC-mode) in air under controlled RH. The
humidity was set by ﬂ ooding the AFM me asurement chamber with an atmosphere
of desired humidity, set by appropriate mixing of dry and water-saturate d air. To
reach dry condit ions, the experimental chamber was ﬂ ooded with dry air. Several
hours were needed to reach dry conditions. Microcanti levers AC240TS-R3 from
Asylum Research (Oxford Instruments) were used, with dimensions of (240 × 40 ×
2.3) μ m 3 , spring constant 2 N/m, and a resonant frequency of 70 kHz in air. A
tetrahedral silicon tip was mounted on the cantilever, which has a radius of 7(±2)
nm. The oscillation amplitude was set to 3.3 V (175 nm) and the integral gain was
varied between 30 and 100 depending on surface asperities to optimize the surface
scan quality. The size of the scan box was varied from (20 × 20) μ m 2 to (1 × 1) μ m 2 ,
with a scan rate varying between 1.5 and 4 Hz. Image treatment and analysis were
performed using the Gwyddion open-source software 70 , version 2.50. The data
were corrected for sample tilting by subtracting a linear ﬁ t and the height was
rescaled with respect to the average sample height. The roughness was calculated as
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COMMUNICATIONS CHEMISTRY | (2019) 2:61 | https://d oi.org /10.1038/s42004-019-015 5-y | www.nature.com/commschem 9

the root mean square, r
rms
r rms ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
1
n X
n
n ¼ 1 ð h i  h Þ 2
s ð 18 Þ
with h being the average ﬁ lm height.
X-ray diffraction . XRD experiments were performed with an Empyrean dif-
fractometer from PANanalytical (Brevannes, France) in grazing incidence geo-
metry, with an incident angle of 0.2°. The da ta were corrected for the signal
arising from a bare silico n wafer placed in the same sample hold er. The
monochromatic X-ray source was a Cu anode emitting K
α
radiation (wavelength
λ = 1.54 Å). On the incoming beam side, a divergence slit 1/8°, focusi ng mirror
for Cu source and anti-scatter slit of 1/4° were used. Between mirror and anti-
scatter slit, a mask of 2 mm and a Soller slit of 0.02° was used to reduce the
horizontal beam divergence. On the detector site, the diffracted beam op tics
consisted of a collimation slit of 0.18°, a Soller slit of 0.04°, and a PIXcel3D
scintillator detector. The experiments were performed using the sample c ell as
descri bed in Supplementary Fig. 4.
Binocular lenses . The morphological investigation of the ageing samples was
performed using an Olympus- type SZ61 Stereo Microscope equipped with a 5.1
Megapixel ToupCam UCMOS05100KPA sensor. Experiments were performed
under ambient conditions.
Data availability
The neutron re ﬂ ectometry raw data and reduced data are available at https://doi.org/ILL-
DATA.9?12?455/ILL-DATA.9 – 12 – 455. All other data that sup port this study are
available from the correspon ding author upon reasonable request. The code used to ﬁ t
the neutron re ﬂ ectometry data and to 3D-print the cell used for the XRD experiments are
available from the correspon ding author on reasonable request.
Received: 6 November 2018 Accepted: 12 April 2019
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Acknowledgements
We thank the ILL for the allocation of neutron beamtim e and the Partnership for
Soft Condensed Matter (PSCM) for pr oviding the laboratory infrastructu re for the
layer preparation and characterization and the humidity contro ller set-up used for
the experiments. D.W.H. was supporte d by BMBF grant 05K16KT1. The open access
fee was covered by FILL2030, a European Union project within the European
Commission's Horizon 2020 Research and Innovation programme under grant
agreement N°731096. The useful discussions and suggestions of Emanuel Schneck,
Philipp Gutfreund, and Sebastian Sc hön are gratefully acknowledged.
Author contributions
S.M., Y.G., A.P., and L.C. performe d experiments; S.M. and L.C. analyzed data; D.W.H.
designed and made the sample cell used for the XRD ex periments; L.C. conceived
experiments; S.M., D.W.H., Y.G., R.v.K., M.G., and L.C. wrote the manuscript. All
authors agree with this submissio n.
Additional information
Supplementary info rmation accompanies this paper at https://doi.org/10.103 8/s42004-
019-0155-y .
Competing intere sts: The authors declare no competing interests.
Reprints and permissio n information is available online at http://npg.n ature.com/
reprintsandpermissio ns/
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