Ultrathin Covalently Bound Organic Layers on Mica: Formation of Atomically Flat Biofunctionalizable Surfaces

Article abstract of DOI:10.1002/anie.201701301

Mica is the substrate of choice for microscopic visualization of a wide variety of intricate nanostructures. Unfortunately, the lack of a facile strategy for its modification has prevented the on-mica assembly of nanostructures. Herein, we disclose a convenient catechol-based linker that enables various surface-bound metal-free click reactions, and an easy modification of mica with DNA nanostructures and a horseradish peroxidase mimicking hemin/G-quadruplex DNAzyme.

Full text of DOI:10.1002/anie.201701301

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International Edition: DOI: 10.1002/anie.201701301
German Edition: DOI: 10.1002/ange.201701301
Surface Chemistry
Hot Paper
Ultrathin Covalently Bound Organic Layers on Mica: Formation of
Atomically Flat Biofunctionalizable Surfaces
Rickdeb Sen+, Digvijay Gahtory+, Rui Rijo Carvalho, Bauke Albada, Floris L. van Delft, and
Han Zuilhof*
Abstract: Mica is the substrate of choice for microscopic
visualization of a wide variety of intricate nanostructures.
Unfortunately, the lack of a facile strategy for its modification
has prevented the on-mica assembly of nanostructures. Herein,
we disclose a convenient catechol-based linker that enables
various surface-bound metal-free click reactions, and an easy
modification of mica with DNA nanostructures and a horse-
radish peroxidase mimicking hemin/G-quadruplex DNAzyme.
three 2,3-dihydroxybenzoyl moieties and a lysine tail. In this
“two-punch” approach, the NH3 moiety displaced the K+
+
ions, followed by surface attachment of the catechol moiety.
Unfortunately, the six-step synthesis required for the scaffold
hampers its widespread application. Even more, post-attach-
ment functionalization of mica substrates with interesting
biomolecules such as DNA and proteins is still an uncharted
domain. Such an approach would require both facile and
strong adhesion, combined with the presence of a moiety that
can be routinely used for post-attachment functionalization.
The current approaches[7,8] do not provide such a handle, and
as such there is still need for a platform that allows various
surface modification strategies and a stepwise observation of
the assembly processes of biological nanostructures in
a controlled fashion.[10]
Herein, we address this unresolved problem by a dual
approach through the development of a simple molecule that
allows both 1) covalent modification of mica, and 2) post-
modification stepwise growth and study of molecular assem-
blies. To this end, we envisaged and synthesized a surface
anchor (1) that possesses the structural characteristics
required to combine optimal adhesion characteristics with
a handle for modular functionalization (Figure 1). We exten-
sively characterized the modified surfaces (M1) by static water
contact angle (SCA), X-ray photoelectron spectroscopy
(XPS), and AFM measurements. To illustrate the potential
for surface functionalization, we explored several metal-free
strain-promoted click reactions. Finally, to demonstrate bio-
functionalization we pursued the stepwise formation of
functional DNA constructs, such as G-quadruplex (GQ, G =
guanine) structures on covalently modified surfaces by AFM.
T
he atomically flat nature of mica has made it the substrate
of choice for microscopic visualization of dimensional param-
eters of various pre-fabricated nanomaterials such as DNA
origami[1] and protein conjugates,[2,3] among others. Mica is
a hydrophilic aluminosilicate, which in saline solution is
covered by a hydration layer with K+ ions that are tightly
bound to the anionic silicate. While widely used for atomic
force microscopy (AFM) studies,[4] the surface modification
and chemical functionalization of mica itself has received
surprising little attention.[5]
Lately, catechol-based coatings including mussel-adhesive
polydopamine proteins have found increasing attention for
mica surface modification.[6] Despite a general acceptance
that the catechol moiety in mussel proteins is central to their
adhesion ability,[7] its exact adhesion mechanism is still
unknown.[8] Butler et al. recently showed that a pending
amine functionality is central to binding.[9] To demonstrate
this, a symmetric trichrysobactin was synthesized that bears
[*] R. Sen,[+] D. Gahtory,[+] R. R. Carvalho, B. Albada, F. L. van Delft,
Prof. Dr. H. Zuilhof
Laboratory of Organic Chemistry
Wageningen University & Research
Stippeneng 4, 6708 WE Wageningen (The Netherlands)
E-mail: han.zuilhof@wur.nl
Prof. Dr. H. Zuilhof
Department of Chemical and Materials Engineering
King Abdulaziz University, Jeddah (Saudi Arabia)
and
School of Pharmaceutical Sciences and Technology
Tianjin University
92 Weijin Road, Nankai District, Tianjin, 92000 (P.R. China.)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201701301.
ꢀ 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co.
KGaA. This is an open access article under the terms of the Creative
Commons Attribution Non-Commercial NoDerivs License, which
permits use and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
Figure 1. Tentative mechanism of mica modification by surface anchor
1.
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As shown by Butler et al.[9] and Hwang et al.,[11] mica-
adhesive molecules should likely contain both catechol and
amino groups. However, in their case this resulted in a poorly
defined polymeric layer possibly due to intramolecular
Michael-type addition reactions between the catechol and
free amino groups.[12] Spencer et al. have tackled this issue of
polymerization by inclusion of a strongly electron-withdraw-
ing nitro group in the catechol motif to minimize auto-
oxidation.[13] Using this information, the design of our surface
anchor 1 was based on the retention of the key components,
that is a catechol moiety linked proximally by an electron-
withdrawing amide group to a protonated amine (Figure 1).
A first step forward was the synthesis of surface anchor 1:
2,3-dihydroxybenzoic acid (DHBA) and mono-Boc-protected
1,6-hexanediamine were linked by a conventional amide
coupling, followed by subsequent Boc-deprotection (Fig-
ure 2a) to afford 1 in an overall yield of 66% (0.78 g). A 1 mm
solution of 1 in PBS buffer (pH 7.4) allowed for the
simultaneous modification of large numbers of freshly
cleaved mica surfaces in 24 h. A constant low water contact
angle (558; Figure 2b), the emergence of N1s signal in XPS
(Figure 2c), and the XPS analysis of subsequent modifica-
tions on M1 confirmed modification of the mica surfaces with
surface anchor 1 (see the Supporting Information). Impor-
tantly, molecules similar to surface anchor 1, but lacking one
of the mentioned components, performed inferior than
anchor molecule 1 (Table S1).
XPS analysis of modified surfaces also showed the overall
chemical composition of M1 to fit the molecular composition
of 1 within experimental error (C/NXPS = 6.8; C/NTheor = 6.5;
see the Supporting Information, Section S4.2). As shown by
Hwang et al., the formation of polymeric layers on mica
surfaces leads to the disappearance of the Al2p, K2s, and Si2p
signals in the XPS spectrum.[11] Interestingly in our case, the
continued high intensity of these substrate-specific signals
hinted at formation of ultrathin layers (Figure 2c).
The deconvolution of the C1s narrow scan clearly
À
revealed carbon atoms in distinct environments, namely, C
À
À
=
C, C N, C O, and C O (Figure 2c, inset). Experimental XPS
binding energy results correlated nicely with simulated values
obtained from DFT calculations (Section 6.1).[14] AFM anal-
ysis of modified surfaces (M1) displayed a remarkably low
surface roughness of 0.4 nm with a layer thickness of 2.5 Æ
0.5 nm (Figure S1–S3). This confirms exclusive formation of
ultrathin layers (length of 1 in stretched-out fashion is ca.
1.3 nm) on mica surfaces. We note that the low (0.4 nm)
roughness makes our modified surfaces amenable for reliable
microscopic visualization of various covalently attached
nanostructured biomolecules.
In view of the importance of metal-free click chemistry in
bioconjugation chemistry, we performed various state-of-the-
art “metal-free” strain-promoted click reactions (Figure 3).
For this, amine-terminated layers M1 were converted to azide-
functionalized coatings M2 (see Supporting Information) by
a Cu-catalyzed azide transfer reaction.[15] The resulting azide-
terminated layers underwent a facile SPAAC reaction with
Figure 2. a) Synthesis of surface anchor 1. b) Increase of the water
SCA during the modification process. c) XPS wide-range spectra of
bare (M0) and modified (M1) mica (inset: narrow scan of the C1s
signal).
Figure 3. Functionalized mica as building block: General scheme
showing the cycloaddition adducts of various metal-free click reactions
on M1.
2
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a fluorinated labile BCN aromatic ester. This allowed easy
Having established prominent bioconjugation reactions
characterization of the resulting surfaces M2 by XPS and
direct analysis in real time high-resolution mass spectrometry
(DART-HRMS),[16] which showed the characteristic m/z peak
of the surficial 4-perfluorinated butyl benzoate anion
(339.0062) in the extracted ion chronogram (EIC; Figure S4).
A clear XPS F1s signal was evident at 686.0 eV, indicating
a successful reaction (50%) (Figure 4a). An inverse strategy
on mica, we switched our attention to our other objective: the
formation of AFM-observable covalently linked DNA nano-
structures. Building on the insights from Famulok et al., a 168-
bp DNA minicircle consisting of eight distinct DNA frag-
ments[18] was assembled in a step-wise fashion after one of the
components was tethered covalently onto mica surface M1.
Covalent attachment of strand D1 (Table S2) allowed its
hybridization to a preformed assembly of oligomers D2–D8
(Table S2), forming circular structures. As shown in Fig-
ure 5a, 3D height images of DNA-containing surfaces
Figure 4. a) Stacked XPS wide scans of final cycloaddition adducts of
SPAAC with azide on surface (M3; bottom), BCN on surface (M5;
middle), and SPOCQ (M7; top). The F1s and N1s peaks were used for
determining the yield of these reactions. b) XPS narrow scan C1s
region (left) and N1s region (right) of SPAAC cycloaddition adducts
(M5).
Figure 5. a) 3D height images of DNA circles assembled on mica.
b) 3D height and c) quadrature image of a single DNA circle.
revealed circular structures that possess an internal cavity
(Figures S8–S11). A large number of contiguous shapes with
circular features were obtained, with an internal diameter of
20 Æ 2 nm (Figure S12), in accordance with literature
(18.2 nm).[18] When significantly lower concentrations of
strands D2–D8 were applied (100-fold dilution, that is,
0.1 mm), we observed mostly open structures, and circles of
which the larger internal diameter hinted to the formation of
semi-closed dimeric and trimeric structures (Figures 5b,c, S13
and S14). Control experiments with bare mica surfaces, that is,
without the anchored DNA, did not show any circles or other
(linear) structures, proving the need for a covalently attached
anchor.
Lastly, we added another dimension to our method by the
attachment of functional DNA, namely the horseradish
peroxidase-mimicking hemin/G-quadruplex (hGQ) DNA-
zyme.[19] Such a hGQ DNAzyme can oxidize a variety of
organic substrates in the presence of H2O2.[20] To demonstrate
the feasibility of hGQ catalytic activity of DNAzymes on mica
for the surface-bound SPAAC reaction was also pursued with
BCN immobilized on the surface (M4) and a reaction with an
azide coupled to a fluorinated tail (Figure 3), to yield surface
M5. The XPS F1s peak at 686.0 eV was used to demonstrate
the extent of reaction (83%; Figure 4a and Section S4.10),
while also the C1s and N1s spectra confirm the success of this
reaction (Figure 4b).
The strain-promoted click reaction between BCN and
ortho-quinones (SPOCQ)[17] was investigated as a second
example on mica surfaces modified by our method. To this
end, 2,3-dihydroxybenzyl amine was attached to surface M1
using a DIC coupling reaction, and subsequently oxidized to
its corresponding 1,2-quinone (M6) using NaIO4. XPS analysis
confirmed the presence of a modified anchor on mica
(Section S4.13). The quinone underwent a facile SPOCQ
reaction producing in excellent yield (91%) surface M7 in 4 h,
as evidenced by the F/N ratio in XPS (Figure 4a and
Section S4.14).
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surfaces, we attached the guanine-rich sequence EAD2
Acknowledgements
(Table S2) covalently to M1 mica surfaces. Covalent attach-
ment of DNA was confirmed with XPS analysis by the
emergence of P2s and P2p signals in the XPS wide spectra at
189.0 eV and 133.0 eV, respectively (Figure S4.17). Further-
more, complexation of hemin [FeIII-protoporphyrin IX chlo-
ride] to the formed GQ under K+-rich conditions was
confirmed by the emergence of characteristic XPS Fe2p
peaks[20b] (710.0 and 721.0 eV) (Figure S4.17, inset).
The authors thank The Netherlands Organization for Scien-
tific Research (NWO) for funding via ECHO project number
712.012.006. We also thank Medea Kosian, Dr. Sidharam
Pujari, and Dr. Jorge Escorihuela for stimulating discussions.
Conflict of interest
After validation of the covalent attachment of DNA, we
assessed its HRP-mimicking ability using 2,2’-azino-bis(3-
ethylbenzothiazoline-6-sulfonate) (ABTS2À), which is con-
verted into ABTSCÀ in the presence of the hGQ DNAzyme
and H2O2.[20a] Indeed, upon addition of H2O2 to an ABTS-
solution that contained just a single hGQ-functionalized mica
slide (12 mm diameter), within 5 min a green-colored solution
with lmax = 414 nm (Figure 6) was formed. Control experi-
The authors declare no conflict of interest.
Keywords: click chemistry · DNA · enzymes · surface chemistry ·
ultrathin layers
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Manuscript received: February 6, 2017
Final Article published: && &&, &&&&
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Communications
Surface Chemistry
Catechol-based thin films: The facile and
covalent modification of mica is reported
using a catechol-based surface anchor
with a flexibly linked amino group.
Robustly bound, low-roughness, and
ultrathin amine-terminated layers are
obtained. This approach makes possible
highly flexible surface modifications
using metal-free click reactions.
R. Sen, D. Gahtory, R. R. Carvalho,
B. Albada, F. L. van Delft,
H. Zuilhof*
&&&& — &&&&
Ultrathin Covalently Bound Organic
Layers on Mica: Formation of Atomically
Flat Biofunctionalizable Surfaces
6
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Angew. Chem. Int. Ed. 2017, 56, 1 – 6
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