Journal of Biomaterials and Nanobiotechnology, 2011, 2, 91-97
doi:10.4236/jbnb.2011.21012 Published Online January 2011 (http://www.SciRP.org/journal/jbnb)
Copyright © 2011 SciRes. JBNB
Assembly of Oligoglycine Layers on Mica Surface*
Svetlana V. Tsygankova1, Alexander A. Chinarev1, Alexander B. Tuzikov1, Ilya S. Zaitsev1,
Nikolai Severin2, Alexey A. Kalachev3, Jurgen P. Rabe2, Nicolai V. Bovin1*
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; 2Department of Physics, Humboldt University Berlin,
Berlin, Germany; 3Plasmachem GmbH, Berlin, Germany.
Email: bovin@carb.ibch.ru
Received October 7th, 2010; revised November 10th, 2010; accepted December 20th, 2010.
ABSTRACT
Assembly of [Gly7-NHCH2-]4C, [Gly7-NHCH2-]3C CH3 and [Gly4NH(CH2)5-]2 peptides on mica surface in aqueous so-
lution was studied. The peptides are capable of forming atomically smooth (2.65-4.3 nm in height) layers assembled as
polyglycine II. Monomers in the layers are situated normally to the surface. Formation of analogous flat 2D structures
also takes place in solution but much more slowly than on mica surface, i.e. negatively charged surface plays an active
role promoting the assembly.
Keywords: Self-Assemblin g, Oligoglycine, Scanning Force Microscopy, Monolayers
1. Introduction
Self-assembly of small molecules on solid surface is an
attractive way for fabrication of advanced materials and
nano-devices [1]. Basically, self-assembled monolayers
(SAMs) constructed on gold from functionalized
long-chain hydrocarbon thiols [2] are used for this pur-
pose. Such SAMs, held primarily due to Van der Waals
interaction, are rather soft architectures precisely repro-
ducing imperfect curvature of the surface-template. In
contrast, peptides are capable of forming rigid architec-
tures due to multiple and highly narrowed hydrogen
bonds [3]. This property is intrinsic i.e. generally it does
not depend on the template. In polyglycine II structure,
parallel peptide chains are arranged as 31 helix forming a
net of hydrogen bonds (all CO and NH groups are in-
volved) [4-6]; this package is rigid and thus especially
attractive for design of new flat layers and for smooth-
ening of other rough surfaces. Canonical polyglycine II is
formed by a long-chain polymer Glyn in a solid phase [4];
earlier, we have demonstrated that short oligoglycines
with n = 7 are capable of forming the polyglycine II ar-
chitecture if four chains are organized in a star-like
manner [6]. Such symmetrical tetraantennary peptide
[Gly7-NHCH2]4C forms platelet-like 2D supramolecular
assemblies (hereinafter referred as tectomers), which are
proved to be stable not only in solid phase but also in
aqueous solutions. In the both cases Raman spectra of
tectomers have a band pattern consistent to that of crys-
talline polyglycine II. We analyzed conceivable ways of
[Gly7-NHCH2]4C packaging in tectomers and choose the
model, in which [Gly7-NHCH2]4C monomers with paired
antennae in polyglycine II conformation are situated
normally to the tectomer plane. Thickness of this model
structure is in the best agreement to thickness of tectom-
ers measured by scanning force microscopy (SFM), 4.5
nm [6]. Later we showed that tectomers can be formed
on a surface more readily than in solution volume, par-
ticularly on mica due to the opposite charges of terminal
NH2 groups and mica surface [7]. The current paper de-
scribes the assembly of tetra-, tri-, and biantennary oli-
goglycine peptides (Scheme 1) on mica surface more
closely and demonstrates that the assembly leads to
atomically smooth and durable mono- or bi-layers
(Scheme 2). Also we describe the application of scan-
ning force microscopy for investigation of sur-
face-promoted assembling of the antennary designed
oligoglycines. Tectomers are attractive material for na-
notechnologies, because they can serve as a platform for
fabrication of nano devices, as well as for smoothing,
strengthening, and functionalization of rough surfaces,
and for encapsulation of micro-objects [8].
2. Experimental
Peptides [Gly7-NHCH2-]4C, [Gly7-NHCH2-]3CCH3, and
[Gly4-NH(CH2)5-]2 (Scheme 1) were synthesized by ac-
tivated esters method in solution, by stepwise elongation
* The work was supported by the Program “Molecular and cell biol-
ogy”, from the Presidium of the Russian Academy of Sciences.
Assembly of Oligoglycine Layers on Mica Surface
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92
N
H
N
HN
H
N
HN
H
N
HN
H
NH2
O
O
O
O
O
O
O
N
H
N
H
N
H
N
H
N
H
N
H
N
H
NH2
O
O
O
O
O
O
O
NH2
N
H
N
H
N
H
N
HO
N
H
N
H
O
O
O
O
O
N
H
O
NH2N
H
N
H
N
HN
H
O
N
H
N
H
O
O
O
O
ON
H
O
N
H
N
H
N
H
N
H
N
H
N
H
N
H
NH2
O
O
O
O
O
O
O
NH2N
HN
H
N
HN
H
O
N
H
N
H
O
O
O
O
ON
H
O
NH2N
HN
H
N
HN
H
O
N
H
N
H
O
O
O
O
ON
H
O
CH3
NH2
N
H
N
H
N
H
N
HO
O
O
O
NH2N
H
N
HN
H
N
H
O
O
O
O
Scheme 1. Structures of bi-, tri- and tetraantennary oligoglycines.
Scheme 2. Association of oligoglycines on mica surface. (a) The tetraantennary peptide form monolayer (“2+2” packaging the
monomer); (b) The triantennary peptide form mono- or bilayer (“3+0” packaging); (c) Biantennary molecules capable of
forming mono- or bilayer (“2+0” packaging). To form durable layers with polyglycine II structure, that stabilized by exten-
sive network of H-bonds, the antennae of peptide molecules should have strict parallel orientation regarding each other, and
thus should be normally oriented toward surface. Obviously, the peptide molecules should be directed to mica surface having
negative zeta-potential by their amino groups, which may be positively charged; the suggested conformations of the peptides
allow maximum cooperation upon interaction with mica.
of the chain by one or two glycine residues [6,9], and
stored as HCl or CF3COOH salts. SFM images were re-
corded using a Nanoscope IIIa (Digital Instrument Inc.,
CA) in air in tapping mode using microcantilevers with a
typical resonance frequency 300 kHz and spring constant
42 N/m, and in aqueous solutions in contact mode using
Si3N4 cantilevers with force constants 0.06 N/m, 0.12
N/m, 0.32 N/m.
(a)
(b)
(c)
Assembly of Oligoglycine Layers on Mica Surface
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93
The samples for the investigation in air were prepared
as followed. Solution of NaHCO3 or Na2CO3 (0.1 M)
was added to the peptide salt solution, thoroughly mixed
and left to stay for 0-120 min. The mixture was placed on
freshly cleaved mica and was incubated (10 sec – 10 min)
at room temperature and normal pressure. The solution
was removed from mica surface by spin coating.
For SFM experiments in aqueous solution freshly
cleaved mica was placed to liquid cell followed by addi-
tion of water. Solution of NaHCO3 or Na2CO3 (0.1 M)
was added to peptide salt solution (0.1 or 1 mg/ml), tho-
roughly mixed for 10 sec and immediately placed to the
cell.
The obtained images were filtered with a flatten filter
from Nanoscope III software.
3. Results and Discussion
Protonated forms of oligoglycines (Scheme 1) are inca-
pable of self assembling due to the charge on the amino
group, thus they exist in solution as monomers [6]. To
initiate the assembling, an equivalent amount of base is
added to aqueous solution of peptide salt. As this takes
place the peptides adopt polyglycine II conformation
(Raman spectroscopy, data not shown) gradually evolv-
ing from solution into separate phase.
Thus, we can speculate that the peptide layers assem-
bled on mica would be 2D crystals of polyglycine II.
Analyzing possible ways of the peptides packing into
such crystalline layers we have fixed our attention on the
variants presented in Scheme 2. Thus, for the tetraanten-
nary peptide, “2+2” packaging, in which two coplanar
pairs of peptide antennae are oppositely directed, seems
to be favorable as also is for solution-formed tectomer
platelets studied earlier [6]. The triantennary molecule
are presumably packed in tectomer layer as a trident
(“3+0” packaging). As regards the biantennary peptide,
we suggest “2+0” shape of its molecules within layer;
molecular dynamic simulations of the peptide adsorption
on mica confirms this assumption [7]. The SFM results
obtained for each of the three peptides supports our pro-
posal, and considered below.
3.1. Tetraantennary Oligoglycine
[Gly7-NHCH2-]4C
Earlier we unambiguously showed, that platelet-like tec-
tomers with polyglycine II structure were formed in solu-
tion, and the assembling takes a few hours [6]. Indeed,
aqueous solutions with the deprotonated form of
[Gly7-NHCH2-]4C were allowed to stay for 60-120 min
before the tectomers visualized by STM of the samples
on of mica or graphite prepared from aliquots taken from
the solutions, i.e. the tectomers were already formed be-
fore application onto support surface. These observations
were confirmed by light scattering, which also showed
that spontaneous assembly of tectomer (in absence of
mica) proceeded slowly, tectomers reached maximal
sizes in about 1-2 h range, and then their precipitation
from solution is started. Other conditions were used in
this study, namely, the solution of the peptide was ap-
plied onto the mica surface immediately after adding
base. It can be seen on Figure 1 that tectomer formation
proceeds on mica surface much faster, over several min-
utes. Small islets are formed at first followed by gradual
increase in lateral dimensions and complete uniform
covering of the surface (layer T1); the layer roughness
does not exceed 0.2 nm. Sporadic appearing of the sec-
ond layer, T2, (Figure 1b) is observed before the com-
plete formation of the first one; the effect is more pro-
nounced at higher concentrations of the peptide solutions
(data not shown).
The thickness of the layer, about 4.3 nm, corresponded
to the length of the [Gly7-NHCH2-]4C molecule in “2+2”
conformation; this also correlating well with the data
obtained before for platelet-like tectomer formed in solu-
tion [6]. Morphological similarity between platelet-like
tectomers and tectomer islets on mica may point that the
both types of objects has the same nature, i.e. they are 2D
crystals of polyglycine II. Notworphy, angles between
tectomer edges are close to 120o (Figures 1(a,b)), which
justifies their polyglycine II structure. The same is fair
for the monolayer tectomer. Interestingly, the layer as-
sembly proceeded not evenly but with acceleration.
Contact mode scanning of 1000 x 1000 nm region of
the already formed layer led to its power-driven destruc-
tion (Figure 2), however during five minutes the layer
become restored due to peptides molecules being in
excess in solution. By this example we can suggest that
mica surface does not merely promote the assembly of
oligoglycine peptides but also takes part in restoration of
the tectomer layer.
Similar results were obtained when scanned in air in
tapping mode (Figure 3). Thus, formation of islet type
tectomer monolayer with the same height 4.3 nm, as in
case of the measurement in the liquid cell, took place.
However, here, continuous peptide layer on mica was not
formed; moreover, often the second, the third and etc.
layers were simultaneously appeared. Planar size of the
tectomer islets reaches several micrometers. Even though
the time of exposure of the peptide solution at mica when
scanned in air was relatively high (~20 min), it was still
less than time required for the tectomer formation with-
out mica (~60 min). Thus we concluded that peptide as-
sembling proceeded rather on surface than in solution.
However, a scenario when small primary (still easily
Assembly of Oligoglycine Layers on Mica Surface
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94
Figure 1. Growth of the tetraantennary peptide on mica surface; SFM contact mode in liquid cell. Т1, the first tectomer layer;
Т2, the second tectomer layer; M, uncovered mica regions. (a), 2 min after starting the experiment; (b), 3 min; (c), 4 min.
Figure 2. Destruction and restoring of the tectomer layer formed from [Gly7-NHCH2]4C; SFM contact mode in liquid cell. T,
tectomer layer. (а) Partial destruction of the tectomer during the scanning of a region with the surface 1 μm2 (square frame);
(b) Partial restoring of the damaged layer; (с) The layer is completely restored 5 min after its destruction.
Figure 3. Assembling of the tetraantennary peptide on mica; SFM taping mode in air. Thickness of the primary, secondary
and etc. tectomer layers is 4.3 nm.
diffusing) germs are formed in solution first followed by
their adsorption on the surface and final rapid overgrow-
ing of the uncovered regions cannot be completely ex-
cluded. Certainly, if the exposure time is over 60 min the
assembling takes place both in solution and on mica sur-
face.
(a) (b) (c)
Assembly of Oligoglycine Layers on Mica Surface
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95
3.2. Triantennary Oligoglycine
[Gly7- NHCH2-]3CCH3
In contrast to the tetraantennary analog, we failed to ob-
serve growth dynamics of the triantennary peptide layer
on mica surface (contact mode, liquid cell). Already in
the initial experiments (the exposure time ~2min) the
surface was completely covered with the peptide layer.
Moreover, the exhausting formation of the second layer
with the thickness of 2.8 nm proceeded for five minutes.
Such a drastic difference in the assembly of the tetra- and
triantennary peptides can be explained by the difference
of their packaging in tectomer layers. According to the
layer thickness of 2.8 nm, triantennary peptide is pack-
aged in tectomer as a trident (“3+0” conformation,
Scheme 2b). Thus, three amino groups per peptide mo-
lecule contact the mica surface. Tetraantennary peptide
assembled in monolayer in “2+2” conformation, i.e. only
two amino groups per molecule appear in contact with
mica surface. This leads to more quick assembling of the
tectomer monolayer by the triantennary molecules. In the
latter case the outer interface of the formed monolayer is
settled with hydrophobic methyl groups, which promotes
more slow assembly of the secondary layer, in “head to
head” manner. As the result, like in case of tetraanten-
nary peptide the final surface is settled with amino
groups, but the tectomer is bilayered here. The adherence
of methyl group-tethered layers to each other (Scheme
2b) should be low. Indirect prove: the second layer has
not been formed in the region where continuous scanning
was carried out (Figure 4).
At the same time, the second layer can be formed in
the regions where the scanning was not carried out.
Peeling of a large region of the second layer took place
sporadically when scanning (Figure 5) resulting in ex-
posure of atomically smooth hydrophobic surface. It
should be noted that edge of shear and angles of 120o
correspond to crystalline polyglycine II package [4,5].
The triantennary peptide covers the whole of the mica
surface, no islet-like structures were found in contrast to
the tetraantennary; the observed height of 2.8 nm corre-
sponding to “3+0” conformation monolayer (in air scan-
ning, data not shown).
3.3. Biantennary Oligoglycine [Gly4-NH(CH2)5-]2
Already at the end of the second scanning, i.e. in 2 min,
the surface was uniformly covered with the even de-
fect-free tectomer layer (Figure 6). The layer height ob-
served in liquid cell was 2.65 nm. This may be explained
with the model of the biantennary peptide in “2+0” con-
formation, capable of bilayer formation (Scheme 2c). As
in the case with the triantennary molecules, the primary
layer is assembled directly on mica (expected thickness
2.0 nm), and on its outer interface the secondary layer is
formed at due to hydrophobic interactions between hy-
drophobic -(CH2)10- moieties. Noteworthy, molecular
dynamic simulation of the peptide behavior in adsorption
layer on mica confirms possibility of polyglycine II
structure formation by the biantennary peptide in “2+0”
conformation; the calculated thickness of monolayer was
close to 2.0 nm [7]. In liquid cell experiments the secon-
dary layer was not observed, believed to be easily
scraped off by cantilever.
Also we measured the layer thickness of 3.7 nm (in air
experiments, tapping mode), which strictly corresponds
to the predicted thickness of the bilayer (Figure 7). To
prove that the upper tectomer layer is rather secondary
product, the sample firstly scanned in air was placed to
liquid cell into buffer solution. Morphology of the pri-
Figure 4. Assembling of [Gly7-NHCH2-]3CCH3 on mica. The second layer has not been formed in the region of continuous
scanning, whereas continued forming of it around this region took place. Т1, the first layer; Т2, the second layer. SFM contact
mode in liquid cell.
Assembly of Oligoglycine Layers on Mica Surface
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96
1 µm
T
2
T
1
Figure 5. Peeling of [Gly7-NHCH2-]3CCH3 upper layer;
SFM contact mode in liquid cell. Т1, the first layer; Т2, the
second layer.
mary layer remained practically unchanged, whereas
islets of secondary layers were gradually dissolved (data
not shown). Thus, simple washing seems to be a way to
tectomer surface “purification” from secondary aggre-
gates.
4. Conclusion
The assembling of three oligoglycine peptides, differing
by antennary and hydrophobicity, has been studied on
mica surface. All these peptides are capable of forming
flat 2D crystalline layers on mica, where monomers are
situated normally to the surface. The cases of lamellar
disposition were not observed. Tetraantennary peptides
form a monolayer whereas bi- and triantennary peptides
having a hydrophobic motif in the molecule’s central
region form monolayer or bilayer. Interlayer adherence
(between the hydrophobic motifs) is low; the upper layer
can be removed with cantilever. The velocity of surface
settlement increases by decrease of peptide antennary.
Though tectomer assembly can also take place in solu-
tion, the assembly process proceeds two orders of mag-
nitude more rapidly on mica surface, i.e. negatively
charged surface plays an active role in promoting the
assembly. We believe, oligoglycine tectomers can serve
as a platform for nanodevices design and for smoothing
and (due to amino groups) functionalization of rough
T
1
T
1
M
2 µm 2 µm
Figure 6. Growth dynamics of layer formed by [Gly4-NH(CH2)5-]2 (0.1 mg/ml) on mica surface, SFM contact mode in liquid
cell. The first scan, islets are observed immediately. The second scan, defect-free layer is formed. Т1, formed tectomer; M, yet
uncovered mica regions.
Figure 7. Tectomer layer formed by [Gly4-NH(CH2)5-]2 (0.1 mg/ml) on mica surface (1 min incubation time on mica surface),
SFM tapping mode in air. Т, bilayer; M, uncovered mica.
Assembly of Oligoglycine Layers on Mica Surface
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97
surfaces and seem to be а promising material for other
nanotechnology applications [8].
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