Facile method for development of ligand-patterned substrates induced by a chemical reaction

Article abstract of DOI:10.1002/chem.201100084

Choose a pattern: A simple and efficient method for ligand patterning on a surface is reported. An organic chemical reaction induces various patterns of an amine functional group, which is further conjugated to a cell adhesion ligand to result in cell patterning (see figure). Copyright

Full text of DOI:10.1002/chem.201100084

DOI: 10.1002/chem.201100084
Facile Method for Development of Ligand-Patterned Substrates Induced by a
Chemical Reaction
Hyunjung Seo,[a] Inseong Choi,[a] Jeongwook Lee,[a] Sohyun Kim,[a] Dong-Eun Kim,[a]
Sang Kyung Kim,[b] and Woon-Seok Yeo*[a]
Patterned substrates have been widely used in various ap-
plications, including arrays of biomolecules and cells, high-
throughput assays, and direct target sensing.[1] In practice,
those demands have been achieved by either of or a combi-
nation of two strategies: 1) direct incorporation of biomole-
cules or functional-group-containing molecules into desired
patterns and 2) generation of functional-group-presenting
patterns by way of chemical conversions on the surface. The
former encompasses microcontact printing (mCP),[2] dip-pen
nanolithography (DPN),[3] polymer-pen lithography (PPL),[4]
microfluidic networks (mFNs),[5] and microarrays.[6] The
latter utilizes the “turning-on” strategy, in which inactive
substrates are switched to an active state to reveal organic
functional groups, in most cases by electrochemical or pho-
tochemical conversions.[7] Patterned functional groups in
both strategies are further used as chemical handles for im-
mobilization of biomolecules, such as cell-adhesion ligands,
enzyme substrates, proteins, oligosaccharides, and oligonu-
cleotides, to afford patterned substrates. As a typical recent
example, Rozkiewicz et al. reported on modified mCP for
the preparation of oligonucleotide micropatterns.[8] In their
report, oxidized PDMS stamps were first coated with posi-
tively charged dendrimers followed by negatively charged
oligonucleotides in a layer-by-layer arrangement, and were
transferred to a solid support for the generation of microar-
rays. Smith and co-workers introduced a photo-labile pro-
tecting group to a thiol functionality.[9] Various patterns of
small molecules and proteins were prepared by using a pho-
tolithographic method in combination with thiol-specific
conjugation chemistry. Yousaf et al. showed that ligand den-
sity and composition influence the rate of stem-cell differen-
tiation by using hydroquinone-based electroactive sub-
strates, which were patterned with a variety of ligands by
using microarray technology.[10] Although these two strat-
egies are reliable, well established, and, therefore, widely
used, each of the strategies offers limitations on practical
use as a general platform for ligand-patterned substrates.
For instance, direct contact printing methods, such as mCP,
cannot control ligand density on the surface, which can pro-
vide important quantitative information for use in experi-
mental design. A concern with regard to the turning-on
strategy is that in some cases activated functional groups re-
quire specified conjugation chemistry and, therefore, neces-
sitate preparatory steps (tagging steps) to make the ligands
compatible with the conjugation reaction.
Herein, we describe a simple, efficient, and straightfor-
ward method for ligand patterning on a surface, induced by
a non-invasive organic chemical reaction—which we have
termed a chemical-reaction-induced patterning (CRIP)—
and equipped with the capability for control of ligand densi-
ty. In addition, our method is compatible with common pat-
terning tools and conjugation chemistry. Herein, we demon-
strate our strategy by using two popular patterning tools,
mCP and microarray, and verify the fidelity of the prepara-
tion of ligand-patterned substrates by patterning cell-adhe-
sion ligands and aptamers.
Our strategy for preparation of ligand-patterned sub-
strates relies on conversion of a substrate from a dormant
(inactive) state to an active state by way of a chemical reac-
tion grafted with a patterning method (Figure 1). The result-
ing patterned area presents a chemically reactive functional
group, that is, a primary amine, which can be harnessed for
the immobilization of biomolecules of interest. Our ap-
proach utilizes self-assembled monolayers (SAMs) on gold
terminated with masked functional groups. Figure 1B shows
the structure of the monolayer and the chemical reactions
taking place on the surface for the preparation of the
amine-functionalized substrate (for the synthesis of the qui-
none-terminated alkanethiol, see the Supporting informa-
tion). The quinone-presenting monolayer[11] was treated with
a reducing agent by using patterning tools. Upon reduction
of the quinone to the corresponding hydroquinone, a cycli-
zation reaction ensues to afford an amine group. The result-
ing amine can react with linkers or ligands through amine-
specific bioconjugation chemistry. The surrounding tri(ethy-
lene glycol) groups provide inertness towards the nonspecif-
ic adsorption of proteins and cells, which is the most de-
manding feature of substrates in biological/biochemical stud-
ies at interfaces.[12] Herein, we demonstrate this CRIP strat-
egy by preparing various patterns of RGD (Arg-Gly-Asp)
[a] H. Seo, I. Choi, J. Lee, Dr. S. Kim, Prof. D.-E. Kim, Prof. W.-S. Yeo
Department of Bioscience and Biotechnology
Konkuk University, Seoul, 143-701 (Korea)
Fax : (+82)2-2030-7890
E-mail: wsyeo@konkuk.ac.kr
[b] S. K. Kim
Nano-Bio Research Center
Korea Institute of Science and Technology, Seoul, 136-791 (Korea)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201100084.
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Chem. Eur. J. 2011, 17, 5804 – 5807

COMMUNICATION
DMSO). MS analysis showed
that the peaks at m/z 803 and
825 were absent and showed a
new peak at m/z 976 [M+Na]+,
which was assigned to the male-
imide functionality on the mon-
olayer (Figure 2C). Treatment
of the maleimide-presenting
monolayer with CGRGDS pep-
tide (10 mL, 1 mm in phosphate
buffer solution (PBS), pH 7.4)
gave peaks at m/z 1212 [M+
H]+ and 1546 [M+H]+, corre-
sponding to peptide-conjugated
products in a free thiol form
and a disulfide form, respective-
ly (Figure 2D). Note that the
peak at m/z 693 [M+Na]+ cor-
responds to
a
tri(ethylene
Figure 1. A) Strategy of the chemical reaction induced patterning method. B) Structure of the monolayer-pre-
senting quinone functional group used in this study for preparation of amine-functionalized surfaces. Using
patterning tools, the quinone-presenting monolayer is treated with a reducing agent. Upon reduction of the
quinone to the corresponding hydroquinone, a cyclization reaction takes place to afford an amine group that
can be harnessed for immobilization of biomolecules through amine-specific bioconjugation chemistry.
glycol)-terminated disulfide and
the peak at m/z 1178 in Fig-
ure 2D corresponds to the mo-
lecular ion [M+HÀSH2]+,
tripeptide, a cell-adhesion ligand.[13] The ratio of the quinone
moiety to the tri(ethylene glycol) group was maintained at
<0.01 (1%) to ensure that cell adhesion to the patterned
monolayer was mediated by specific interactions between
receptors of cells and ligands on the patterns. As another
demonstration, the peptide aptamer DUP-1 (FRPNRAQ-
DYNTN), specific to the PC3 cell (one of the prostate
cancer cell lines), was patterned on the monolayer and cap-
tured PC3 cells specifically.[14]
SAMs of alkanethiolates on gold are well suited for analy-
sis with matrix-assisted laser desorption ionization time-of-
flight (MALDI-TOF) mass spectrometry (MS), termed
SAMDI MS.[15] SAMDI analysis affords the molecular
weight of constituents of monolayers (alkanethiolates) and
thus provides not only structural information but also quan-
titative information with regard to the composition of mono-
layers. In addition, because molecular weight is an intrinsic
property of a molecule, it allows us to discriminate between
signals from analytes and deleterious background noise.
Therefore, SAMDI is one of the most reliable techniques
for the investigation of chemical/biochemical transformation
of SAMs on gold.[16] We used SAMDI analysis to establish
chemical conversions on the monolayer, as depicted in Fig-
ure 1B. First, we prepared a quinone presenting monolayer
at a density of 20%. MS analysis of the monolayer gave a
major peak at m/z 1057 [M+Na]+, corresponding to the
quinone-containing disulfide (Figure 2A). This quinone
monolayer was treated with NaBH4 (10 mL, 50 mm in deion-
ized water) for 30 min, and then analyzed by MS. New
peaks corresponding to the amine product were observed at
m/z 803 [M+H]+ and m/z 825 [M+Na]+ (Figure 2B). To
verify that the resulting amine could serve to immobilize li-
gands, the amine-presenting monolayer was treated with a
bifunctional crosslinker, maleimide-NHS (10 mL, 50 mm in
Figure 2. Verification of surface chemistry by MALDI-TOF MS: qui-
none-presenting monolayer (A), after reduction (B), after treatment with
maleimide-NHS bifunctional cross-linker (C), and after CGRGDS conju-
gation (D). For details, see the main text.
which is often observed in the MALDI-TOF analysis of
SAMs on gold and results from the abstraction of SH2.
Taken together, these results establish the fact that the
chemical conversions proceeded as proposed in high yield.
Furthermore, SAMDI analyses ensured that we would ex-
clude undesired effects, which can be attributed to unreact-
ed or unidentified species on the surface, from our experi-
mental results.
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W.-S. Yeo et al.
The fidelity of our strategy was verified by fluorescence
by using two common patterning methods, mCP and micro-
array, on glass substrates. PDMS stamps with various fea-
tures, such as circles, squares, stripes, and mixtures (100 mm
size), were inked with a solution of NaBH4 in THF
(100 mm) and applied to the quinone-presenting glass sub-
strate for 30 min. Next, we patterned the quinone-presenting
glass substrate into circular regions by using a microarrayer
with an array pin (inner diameter of 100 mm) and NaBH4 so-
lution (50 mm, 10% DMSO in water). After thorough wash-
ing, the resulting substrates were treated with FITC, an
amine-specific fluorescent dye (0.25 mm in PBS, pH 7.4). We
examined the immobilization of FITC on the patterned sur-
faces by using a microarray scanner. Figure 3 shows fluores-
cence images from the substrates patterned by mCP (Fig-
ure 3A, B, C, D) and microarray (Figure 3E), which dis-
Figure 4. Application of the CRIP to cell patterning. Using mCP and microar-
ray, the quinone-presenting monolayer underwent a series of chemical conver-
sions to afford RGD patterns. After HeLa cell seeding and incubation for 12 h,
optical micrography clearly showed cell patterns as designed. Scale bar:
100 mm.
regions. As a control, we repeated this experiment with a
tri(ethylene glycol)-terminated monolayer. Cells did not
attach to the monolayer, which provided additional evidence
that cell adhesion to the substrate is biospecific and that
the patterning methods used in the study did not compro-
mise the inertness of the substrate. As another demon-
stration of biologically relevant applications of the strat-
egy, we tested aptamer-based cell capture on the pat-
terned substrate. The peptide aptamer DUP-1, which is
specific to the PC3 cell, was patterned by the same
method described above by using a microarrayer. As
shown in Figure 5, PC3 cells were captured on the apta-
mer-patterned substrate and was further treated with the
rhodamine-labeled aptamer (Rho-DUP-1) to give the flu-
orescent image in accordance with the cell pattern. Con-
Figure 3. Verification of CRIP by using fluorescence. Using mCP and microar-
ray, the quinone-presenting glass substrate was patterned into various features,
which were proved by fluorescence. Scale bar: 100 mm.
played clear green fluorescence exclusively from the pat-
terns. As a control, the quinone-presenting glass substrate
was treated by using the same process, except that the sub-
strate was incubated with acetic anhydride (100 mm in THF)
before FITC. As expected, no distinctive fluorescence was
observed (data not shown). This result verifies that amine
patterns were generated as desired and that FITC was selec-
tively conjugated to this patterned amine.
To demonstrate biologically relevant applications of the
CRIP strategy, Arg-Gly-Asp (RGD) peptide was conjugated
for the preparation of cell patterns. The RGD peptide is a
ligand that mediates cell adhesion through a specific interac-
tion with integrin receptors on the cell surface. As described
above, we prepared quinone-presenting monolayers and pat-
terned them by using mCP and microarray. The resulting
amine-patterned monolayer was reacted with the malei-
mide-NHS bifunctional linker (50 mm in DMSO) to afford
maleimide groups, and was subsequently treated with the
CGRGDS peptide (1 mm in PBS, pH 7.4). The RGD was
anchored to the monolayer by a Michael addition reaction
between the thiol in cysteine and the maleimide presented
on the surface.[17] This RGD-presenting monolayer was
treated with HeLa cells and incubated for 2 h. After brief
washing with sterilized PBS, the monolayer was transferred
to fresh media in a 12-well plate, incubated further for 10 h,
and photographed. Optical micrographs in Figure 4 display
cells that were patterned in accordance with the features of
the stamps and microarray, indicating that the RGD peptide
promoted selective cell adhesion to the chemically induced
Figure 5. The DUP-1 peptide aptamer-patterned substrate was seeded
with PC3 cells (A) and further treated with Rho-DUP-1 to give a fluores-
cent image in accordance with the cell pattern. Scale bar: 100 mm.
trol experiments with a different prostate cancer cell line
(LNCaP cell) and with an RGD-patterned substrate did not
afford distinctive cell patterns, which indicates that the PC3
cells were captured on the substrate through specific interac-
tions to DUP-1 aptamers on the monolayer (data not
shown).
Important aspects of the CRIP method described herein
include compatibility with many existing patterning methods
and versatility of the resulting reaction product, that is,
amines. Because our approach relies on chemical reduction
of terminal groups on the surface, any patterning tools that
deliver reducing agents to the surface can be harnessed. In
fact, Mirkin et al. reported on a nanopatterning method by
delivery of the oxidant to a hydroquinone-presenting surface
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Ligand-Patterned Substrates
COMMUNICATION
by using DPN.[18] Nucleophilic addition of amine-containing
molecules to the localized benzoquinone resulted in nano-
patterns. However, conjugation chemistry to the resulting
benzoquinone is not as versatile as popular organic function-
al groups, such as amines, carboxylic acids, aldehydes, and
thiols, which are the most commonly used functional groups
not only in chemistry but also in biology. Moreover, the sta-
bility of hydroquinone against oxidation under ambient con-
ditions sometimes causes unexpected results. Amine func-
tionalities, unlike other common functional groups, such as
carboxylic acids, aldehydes, and thiols, are quite stable
under physiological conditions, are tolerable to reduce or
oxidize, and maintain chemical reactivity under aqueous
conditions over a wide pH range. Furthermore, along with
development and commercialization of various amine-spe-
cific crosslinkers, almost every biomolecule can be immobi-
lized on the amine-presenting surface. Another significant
aspect of this work is the use of SAMs of alkanethiolates on
gold. For interrogation of specific interactions between li-
gands on the surface and biomolecules of interest in a quan-
titative manner, the ligand density on the surface should be
controllable and the surface should be inert to nonspecific
adsorption. In this context, mixed SAMs prepared from a
solution of ligand-terminated thiol and oligo(ethylene
glycol)-terminated thiol in various ratios can perfectly
supply those demands.
In conclusion, this report describes a simple ligand-pat-
terning strategy induced by a chemical reaction that gener-
ates a pattern of amine functional groups. We proved the fi-
delity of our strategy by demonstrating the RGD patterning
and subsequent cell patterns and the capture of prostate
cancer cells to a specific aptamer-patterned substrate. Due
to the chemical flexibility of the resulting amine functional
group and the compatibility of this strategy with many exist-
ing patterning methods, we believe that this method will be
a useful platform technology for the preparation of ligand-
patterned substrates. For example, we are currently carrying
out nano-patterning of multiple ligands by using grafting
dip-pen nanolithography for investigation of cellular behav-
ior on multi-ligand-presenting substrates.
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Acknowledgements
This research was supported by a Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (grant 2009-0064280).
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Keywords: aptamers · cell adhesion · mass spectrometry ·
monolayers · patterning
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Received: January 10, 2011
Published online: April 18, 2011
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