Rapid preparation of multifunctional surfaces for orthogonal ligation by microcontact chemistry

Article abstract of DOI:10.1002/chem.201103422

Microcontact chemistry has been applied to patterned glass and silicon substrates by successive reaction of unprotected and monoprotected heterobifunctional linkers with alkene-terminated self-assembled monolayers (SAMs) to produce bi-, tri-, and tetrafunctional surfaces. Photochemical microcontact printing of an azide thiol linker followed by immobilization of an acid thiol linker on an undecenyl-terminated SAM results in a well-defined, micropatterned surface with terminal azide, acid, and alkene groups. Biologically relevant molecules (biotin, carbohydrates) have been selectively attached to the surface by means of orthogonal ligation chemistry, and the resulting microarrays display selective binding to fluorescently labeled proteins. An orthogonally addressable, tetrafunctional surface (azide, acid, alkene, and amine) can be prepared by an additional printing step of a tert-butyloxycarbonyl (Boc)-protected alkyne amine linker on the azide structures by using the copper(I)-catalyzed azide-alkyne Huisgen cycloaddition and subsequent removal of the protective group. Copyright

Full text of DOI:10.1002/chem.201103422

DOI: 10.1002/chem.201103422
Rapid Preparation of Multifunctional Surfaces for Orthogonal Ligation by
Microcontact Chemistry
Christian Wendeln,[a] Stefan Rinnen,[b] Christian Schulz,[a] Tobias Kaufmann,[a]
Heinrich F. Arlinghaus,[b] and Bart Jan Ravoo*[a]
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FULL PAPER
Abstract: Microcontact chemistry has
been applied to patterned glass and sil-
icon substrates by successive reaction
of unprotected and monoprotected het-
erobifunctional linkers with alkene-ter-
minated self-assembled monolayers
(SAMs) to produce bi-, tri-, and tetra-
functional surfaces. Photochemical mi-
crocontact printing of an azide thiol
linker followed by immobilization of
an acid thiol linker on an undecenyl-
terminated SAM results in a well-de-
fined, micropatterned surface with ter-
minal azide, acid, and alkene groups.
Biologically relevant molecules (biotin,
carbohydrates) have been selectively
attached to the surface by means of or-
thogonal ligation chemistry, and the re-
sulting microarrays display selective
binding to fluorescently labeled pro-
teins. An orthogonally addressable, tet-
rafunctional surface (azide, acid,
alkene, and amine) can be prepared by
an additional printing step of a tert-bu-
tyloxycarbonyl (Boc)-protected alkyne
amine linker on the azide structures by
using the copper(I)-catalyzed azide–
alkyne Huisgen cycloaddition and sub-
sequent removal of the protective
group.
Keywords: click chemistry · micro-
arrays
· microcontact printing ·
monolayers · self-assembly
Introduction
terning procedure itself. Several examples have been report-
ed in the last few years that demonstrate the potential of or-
thogonal functionalized surfaces for the attachment of vari-
ous (bio)molecules and nanoparticles. Del Campo et al. pre-
pared mixed monolayers of silanes, terminated with amine
and carboxylic acid groups, with photocleavable protecting
groups, and they used subsequent UV irradiation through
a photomask at different wavelengths for selective deprotec-
tion.[9] The generated pattern could be visualized by the as-
sembly of carboxylated poly(butylmethacrylate) colloids.
Slocic et al. examined plasma-enhanced chemical vapor dep-
osition (PECVD) of poly(allylamine) on a self-assembled
monolayer (SAM) of mercaptopropylsilane for the prepara-
tion of patterned thiol/amine-functionalized substrates.[10]
The surfaces were orthogonally modified by reaction with
thiol-decorated quantum dots and green fluorescent protein
(GFP). Similarly, PECVD of poly(allylamine) and poly(pro-
pargyl methacrylate) in combination with capillary force
lithography could be used for the preparation of alternating
amine/alkyne patterns that were chemically addressed by re-
action with fluorescent dyes.[8] High-throughput microarrays
with different reactive functional groups were prepared by
the incorporation of alkene-modified functional linkers in
a poly(ethylene glycol)-based matrix by using the photoin-
duced thiol–ene polymerization reaction.[11] Orthogonal liga-
tion chemistry was demonstrated by sequential reaction with
fluorescent dyes by hydrazone, active-ester, and thiol–ene
chemistry. The indirect immobilization of proteins on surfa-
ces based on orthogonal ligation was shown recently by Li
et al.[12] In this work, the Langmuir–Blodgett technique was
applied to generate a patterned azide/alkene-terminated
self-assembled monolayer. Orthogonal ligation reaction with
a biotin alkyne or a nitrilotriacetic acid alkyne derivative,
followed by the binding of streptavidin or histidine (His)-
tag-labeled GFP resulted in nanosized protein patterns. Fur-
thermore, bio-orthogonal ligation of azides on patterned
ethynyl/propiolate- and on dibenzocyclooctyne-modified
polymer surfaces by sequential immobilization has been
demonstrated.[13,14] In addition, tip-based electrochemical
oxidation[15] and microfluidic oxidation[16] of alkene and hy-
droxyl-terminated monolayers were applied to the prepara-
The controlled attachment of biomolecules to surfaces with
predetermined density, spacing, and orientation is funda-
mental to the development of biosensors and microarrays,
since natural recognition events are strongly influenced by
the accessibility and conformation of target molecules. Site-
specific immobilization strategies—especially of DNA,[1]
proteins,[2] and carbohydrates[3]—on substrate surfaces have
been intensively developed over the last decades to over-
come these challenges. Furthermore, various surface-pat-
terning approaches including photolithography,[4] soft lithog-
raphy,[5] electron-beam lithography,[6] and dip-pen nanoli-
thography[7] have successfully been applied for the site-spe-
cific attachment of biomolecules to surfaces. Nevertheless,
in most cases, the immobilization process is affected by the
patterning procedure, which can limit the integrity of the
molecules and therefore reduce the suitability of the
method. For instance, photolithography has the disadvant-
age that strong UV irradiation can induce biochemical
damage. Soft lithography and also dip-pen nanolithography
may lead to the dehydration of biomolecules, which poten-
tially causes reduction or loss of activity.[8]
Orthogonal ligation chemistry allows the development of
multifunctional pre-patterned surfaces that can be used for
specific immobilization within discrete areas under mild
conditions and without detrimental influences of the pat-
[a] C. Wendeln, C. Schulz, T. Kaufmann, Prof. Dr. B. J. Ravoo
Organisch Chemisches Institut and
Center for Nanotechnology (CeNTech)
Westfꢁlische Wilhelms-Universitꢁt Mꢂnster
Corrensstrasse 40, 48149 Mꢂnster (Germany)
Fax : (+49)251-83-33287
E-mail: b.j.ravoo@uni-muenster.de
[b] S. Rinnen, Prof. Dr. H. F. Arlinghaus
Physikalisches Institut
Westfꢁlische Wilhelms–Universitꢁt Mꢂnster
Wilhelm-Klemm-Strasse 10, 48149 Mꢂnster (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103422.
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B. J. Ravoo et al.
tion of orthogonally reactive alkene/acid- and alcohol/alde-
hyde/acid-functionalized surfaces.
Microcontact printing (mCP) is a versatile surface pattern-
ing method that has been exploited for many different pur-
poses.[17–19] mCP has successfully been applied to immobilize
various molecules on chemically modified surfaces by induc-
ing different types of reactions, for example, amide[20,21] and
imine[22] formation, Diels–Alder chemistry,[23] or epoxide-
ring opening with amines.[24] mCP also allows chemical sur-
face patterning at elevated temperatures and has been ap-
plied to the printing of fluorescent dyes on trifunctional
polymer surfaces by thermal azide–alkyne cycloaddition and
acyl ketene amine chemistry.[25] Moreover, mCP has advanta-
geous properties including high resolution, very short reac-
tion times, simple large-area patterning, and low cost. A
short while ago, we demonstrated the suitability of photo-
chemical mCP by thiol–ene and thiol–yne chemistry for the
patterning of alkene- and alkyne-terminated monolayers.[26]
In contrast to photomask-based approaches, this method
allows the very fast patterning of transparent substrate ma-
terials (e.g., glass) that are relevant to microarray applica-
tions. The general applicability of mCP for the preparation
of patterned alkyne/cyclodextrin-modified surfaces, suitable
for orthogonal covalent and noncovalent functionalization,
has been reported recently.[27]
In this article, we show that mCP can be used for the
chemical patterning of silane SAMs on silicon oxide[28,29] by
successive reactions with heterobifunctional linkers to gener-
ate patterned, orthogonal, multifunctional surfaces. It is
shown that in this way micropatterns of two, three, and four
orthogonal functional groups can be readily prepared on
a single substrate SAM. It is also shown that each functional
group can be independently ligated to a biologically relevant
ligand, which in turn displays the expected orthogonal bio-
molecular recognition.
Figure 1. Preparation of functional surfaces by photochemical mCP using
thiol–ene chemistry: A) Preparation of a patterned bifunctional alkene/
azide surface by mCP of azide thiol 1 and B) preparation of an alkene/
acid surface by printing of acid thiol 2. C) A trifunctional alkene/azide/
acid-modified surface can be prepared by initial mCP of azide thiol 1 fol-
lowed by mCP of acid thiol 2 in a perpendicular direction. Light-micros-
copy images of water condensed on the D) bifunctional alkene/azide,
E) on the bifunctional alkene/acid, and F) on the trifunctional alkene/
azide/acid-modified glass surface indicate successful immobilization.
densation of water droplets on the hydrophilic pattern (ad-
vancing contact angle (46Æ3)8, receding contact angle (25Æ
5)8) of the immobilized linker molecule on glass (Fig-
ure 1D). Similarly, mCP of carboxylic acid thiol 2 in 5 mm
broad lines spaced by 20 mm on an undecenyl-SAM led to
the formation of a patterned bifunctional alkene/acid sub-
strate (Figure 1B). Wettability experiments again directly
verified the successful immobilization of the hydrophilic
molecule (advancing contact angle (45Æ3)8, receding con-
tact angle (15Æ5)8; Figure 1E). The printing of both mole-
cules was further proven by X-ray photoelectron spectrosco-
py (XPS; see Figure S1 in the Supporting Information). The
measurements verified the presence of sulfur, and in the
case of the printed azide thiol 1 also the presence of nitro-
gen on the surface (see Figure S1B,C,E in the Supporting In-
formation). Furthermore, analyses of the C 1s signals
proved the immobilization due to the obvious appearance of
Results and Discussion
For the preparation of micropatterned functional surfaces,
glass and silicon substrates were modified with an alkene-
terminated monolayer of 10-undecenyltrichlorosilane. A
single mCP step of a thiol-functionalized heterobifunctional
linker by thiol–ene chemistry allowed the simple and effec-
tive preparation of bifunctional surfaces. Moreover, thiol–
ene chemistry is known to be compatible with a variety of
functional groups, thus making it very attractive for linker
attachment to surfaces. To demonstrate this, azide thiol
1 was printed on an alkene-terminated glass slide in 5 mm
broad lines that were spaced by 15 mm, thereby leading to
the formation of a bifunctional, patterned alkene/azide sur-
face (Figure 1A). The use of 80 mm ethanolic thiol solutions
for stamp inking in combination with a high-power UV-
light-emitting diode (LED) allowed highly effective attach-
ment of thiol molecules to the surface within 2 min of pho-
tochemical reaction time. The successful modification with
azide thiol 1 could simply be visualized by the selective con-
À
carbon atoms in a C O and carbonyl (acid linker) surround-
À
ing or of C O and amide (azide linker) carbon atoms (see
Figure S1A and D in the Supporting Information). Finally,
the presence of azide and carboxylic acid functional groups
on the surface was also verified by the detection of charac-
teristic azide (2104 cmÀ1) and acid (1717 cmÀ1) absorption
bands in the attenuated total reflectance (ATR)-FTIR spec-
tra (see Figure S2 in the Supporting Information). For both
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Multifunctional Surfaces
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XPS and ATR-FTIR measurements, alkene-terminated
monolayers were homogeneously modified by mCP with
a flat poly(dimethylsiloxane) (PDMS) stamp. To prepare
a trifunctional pre-patterned alkene/azide/acid surface, se-
quential mCP of azide thiol 1 (5 mm lines spaced by 15 mm)
followed by mCP of acid thiol 2 in a perpendicular direction
(5 mm lines spaced by 20 mm) was carried out (Figure 1C). In
this case selective wetting also indicated the reaction of
both molecules (Figure 1F).
A more detailed analysis of the alkene/azide/acid-pat-
terned layer on silicon was carried out by atomic force mi-
croscopy (AFM) and by time-of-flight secondary ion mass
spectrometry (ToF-SIMS)[30] (Figure 2). AFM shows the
presence of both immobilized linkers on the underlying un-
decenyl monolayer that together form the expected lattice
structure in the height image (Figure 2A) and also in the
phase image (Figure 2B). To obtain a higher microscopic
resolution, the surface was rotated approximately 458 clock-
wise. A large area scan attested to a good uniformity of the
pattern (see Figure S3 in the Supporting Information). Addi-
tionally, ToF-SIMS analysis was carried out. Successful im-
mobilization of both linkers was verified by the fact that the
rectangular pattern is clearly visible in the negative total ion
image (Figure 2C) and also in the positive total ion image
(Figure 2D). Detailed consideration showed that nitrogen-
containing, negatively charged fragments such as CNÀ and
CNOÀ (as well as C2H2OÀ) are exclusively formed in areas
in which the azide thiol linker 1 was immobilized, thereby
resulting in visible horizontal lines (Figure 2C). Fragments
that can be generated by both linkers (such as HSÀ and
C2HOÀ) show the complete lattice structure (Figure 2C).
The fragments C2H3OÀ, C3H3O2 (and C2H3SÀ), and
À
À
C2H3O2 are preferably formed in areas in which the acid-
modified linker 2 was printed (Figure 2C). The successful
immobilization of azide thiol 1 in horizontal lines is also
verified by the nitrogen-containing, positively charged frag-
ments CH4N+, C2H4N+ (and C2H2O+), and C2H6N+ (and
C2H4O+) in the positive-ion mode (Figure 2D). The sulfur-
containing fragment C2H5S+ again shows the presence of
both linkers with similar intensity (Figure 2D). The fragment
+
C3H3 is preferably formed by the terminal alkene groups
and therefore illustrates the unsaturated carbon chains in
the rectangular (15 mmꢃ20 mm) interspace (Figure 2D). In-
terestingly, the different modified areas could also be differ-
entiated by the fact that small amounts of Na+ ions re-
mained on the structures of both immobilized hydrophilic
linkers, whereas Mg+ ions were preferably generated in the
areas modified by carboxylic acid (Figure 2D).
After extensive analytical characterization of the trifunc-
tional surface, its suitability for orthogonal ligation chemis-
try was investigated. Azides often play a key role in bioor-
thogonal reactions due to their stability and ability to under-
go highly specific reactions that can proceed in the presence
of other biologically relevant functional groups. Examples
are the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddi-
tion with terminal alkynes (CuAAC),[31] strain-promoted cy-
cloaddition with cyclooctynes,[32] and the Staudinger liga-
tion.[31,33] To investigate the accessibility of azide groups on
the trifunctional orthogonal surface for specific immobiliza-
tion of (bio)molecules, modified glass substrates were ex-
posed to a 5 mm solution of biotin alkyne 3 in dimethylfor-
mamide (DMF) and reacted by means of CuAAC. The re-
maining carboxylic acid groups were afterwards blocked by
attachment of EG4 amine 6 by using conditions for inverse
solid-phase peptide synthesis (ISPPS). Alkenes were subse-
quently treated with EG4 thiol (7) by thiol–ene chemistry
under UV-light irradiation. The decoration of the surface
with diethylene glycol chains in combination with the hydro-
philic design of the heterobifunctional linkers 1 and 2
should allow the immobilization of proteins on the surface
with low nonspecific binding. To prove this assumption and
verify the successful attachment of biotin residues, the sur-
face was probed by exposure to a solution of DyLight 405-
labeled streptavidin in phosphate-buffered saline (PBS) so-
Figure 2. Investigation of a trifunctional alkene/azide/acid-modified sili-
con substrate by AFM and ToF-SIMS. A) AFM height and B) AFM
phase image of the surface. Analysis of the trifunctional patterned sur-
face by means of ToF-SIMS in the C) negative-ion mode and D) in the
positive-ion mode.
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B. J. Ravoo et al.
lution. Streptavidin is a tetrameric protein that shows ex-
tremely high affinity toward biotin (association constant
Ka =1015 mÀ1).[34] Fluorescence microscopy revealed that
biotin was exclusively attached to azide-modified areas
(5 mm broad horizontal lines spaced by 15 mm) and that neg-
ligible nonspecific protein binding did indeed take place
(Figure 3A).
ed (Figure 3B). The visible vertical lines (5 mm spaced by
20 mm) are broken by 5 mm gaps (at 15 mm distances) that
represent azide-containing areas. This observation is due to
the fact that alkene groups were already consumed by reac-
tion with azide thiol 1 in the first printing step and therefore
not available for reaction with acid thiol 2.
The third ligation reaction that can be used for orthogonal
immobilization on the trifunctional pre-patterned surface is
the addition of radicals to alkene groups. The radical thiol–
ene reaction is especially attractive in this context, as it is
biocompatible and can take place under mild conditions,
thus making it highly suitable for the immobilization of bio-
molecules on surfaces.[36–38] By using thiol–ene chemistry for
orthogonal attachment, mannose thiol 5 was reacted with
the surface by covering the alkene/azide/acid-modified glass
slide with a solution of the carbohydrate and 2,2-dimethoxy-
2-phenylacetophenone (DMPA) in diethylene glycol, fol-
lowed by irradiation with UV light for 10 min. The remain-
ing acid groups were blocked by reaction with EG4 amine 6
under conditions for ISPPS and azide groups were in the
last reaction step clicked to EG4 alkyne 8 by means of
CuAAC. Protein screening with fluorescein-labeled conca-
navalin A (ConA) resulted in a periodic, fluorescent pattern
(Figure 3C). This result proves that reaction occurred specif-
ically in areas with alkene groups (20 mmꢃ15 mm square in-
terspaces) because ConA is known for its a-d-mannose
binding affinity. Furthermore, this shows that ligation by the
thiol–ene reaction can specifically be induced in the pres-
ence of azide and carboxylic acid groups.
In addition to site-specific, orthogonal immobilization of
single biomolecules on the alkene/azide/acid-modified sur-
face, sequential immobilization of molecules 3, 4, and 5 was
carried out. In a first step, the surface was activated for pep-
tide coupling by treatment with DCC/NHS and subsequent-
ly treated with a solution of lactose amine 4 in DMF (Fig-
ure 4A). Then biotin alkyne 3 was immobilized on the sur-
face from solution by means of CuAAC. Finally, mannose
thiol 5 was immobilized by light-induced thiol–ene reaction.
For protein binding, the modified surface was exposed to
a mixture of Dylight 405-labeled streptavidin, rhodamine-la-
beled PNA, and fluorescein-labeled ConA in buffer solu-
tion. Fluorescence microscopy revealed that 1) lactose was
immobilized in acid-modified areas and showed selective
Figure 3. Fluorescence microscopy images of selective protein immobili-
zation on the alkene/azide/acid-functionalized surface after specific at-
tachment of biologically relevant binders. A) Attachment of DyLight
405-labeled streptavidin to biotin 3, immobilized by CuAAC. B) Binding
of rhodamine-labeled peanut agglutinin (PNA) to lactose 4, immobilized
by peptide-bond formation. C) Attachment of fluorescein-labeled conca-
navalin A (ConA) to mannose 5, immobilized by thiol–ene chemistry.
Besides azide-based ligation reactions, peptide-bond for-
mation represents a commonly used method for the immobi-
lization of biomolecules on surfaces. For example, microar-
rays of synthetic heparin have successfully been prepared by
microspotting amine-modified heparin oligosaccharides on
active ester surfaces.[35] Similarly, the trifunctional patterned
surface was activated by reaction with dicyclohexylcarbodi-
imide (DCC) and N-hydroxysuccinimide (NHS) to convert
carboxylic acid groups into NHS esters. The surface was
subsequently exposed to a 10 mm solution of lactose amine
4 in DMF for orthogonal immobilization. After modification
of the remaining azide groups by treatment with EG4 alkyne
8 and subsequent attachment of EG4 thiol 7 to the remain-
ing alkenes, the substrate was exposed to a solution of rhod-
amine-labeled peanut agglutinin (PNA). PNA is a tetrameric
carbohydrate binding protein (lectin) that recognizes termi-
nal b-d-galactose units. Selective protein immobilization
(and therefore also lactose immobilization) was exclusively
observed in areas in which carboxylic acid thiol 2 was print-
Figure 4. A) Schematic description of the sequential attachment of lac-
tose amine 4, biotin alkyne 3, and mannose thiol 5 on the alkene/azide/
acid-patterned surface and successive protein screening. B) Overlay of
fluorescence microscopy images of immobilized Dylight 405-labeled
streptavidin (blue), rhodamin-labeled PNA (red), and fluorescein-labeled
ConA (green).
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Multifunctional Surfaces
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binding to PNA (see Figure S4A in the Supporting Informa-
tion), 2) biotin was immobilized in azide-containing areas
and was recognized by binding to streptavidin (see Fig-
ure S4B in the Supporting Information), and that 3) man-
nose was immobilized in alkene-modified areas and shows
binding to ConA (see Figure S4C in the Supporting Infor-
mation). An overlay of the three fluorescence microscopy
images is shown in Figure 4B, thereby proving the area-spe-
cific, orthogonal immobilization of molecules 3, 4, and 5 and
their selective binding to proteins.
Microcontact chemistry also allows the rapid preparation
of tetrafunctional surfaces. To this end, additional printing
steps are required. Microcontact CuAAC[39–41] was used as
an effective method for further functional derivatization of
the trifunctional surface because it is fast, allows high con-
version, and shows (as already discussed) orthogonal reac-
tivity to alkene and carboxylic acid functions. The surface
modification with amine groups opens a path for the addi-
tional immobilization of electrophiles, for example, active
esters, acid halides, or isothiocynates. Therefore, tert-buty-
loxycarbonyl (Boc)-protected heterobifunctional linker 9
was synthesized and printed for 15 min perpendicularly
(5 mm lines spaced by 20 mm) on the azide structures. The
reaction occured exclusively in the contact area with the
azide pattern, thereby leading to Boc-protected amine
squares (5 mmꢃ5 mm) that are horizontally spaced by 20 mm
broad azide-functionalized gaps (Figure 5A). Surface treat-
ment with 90% trifluoroacetic acid (TFA) for 20 min result-
ed in the completely deprotected, functionally patterned
substrate. It was found that also mCP of the TFA salt of the
corresponding alkyne–amine linker on azide-functionalized
surfaces is possible; nevertheless, we preferred to introduce
the additional deprotection step, as this allows longtime stor-
age of the Boc-protected, tetrafunctional surface without
a loss of reactivity. To exemplify the immobilization by reac-
tion with terminal amines, the surface was exposed to a solu-
tion of biotin NHS ester 10 in DMF. Rhodamine streptavi-
din binding after further surface modification by reaction
with 1) EG4 alkyne 8, 2) EG4 amine 6, and 3) EG4 thiol 7
enabled us to visualize the successful formation and orthog-
onal modification of amines (5ꢃ5 mm2 squares) (Figure 5B).
To prove the complete orthogonal reactivity of the surface,
the deprotected substrate was also modified by means of
CuAAC of biotin alkyne 3 from solution. The remaining
amines were blocked by reaction with succinimidyl ace-
tate.[42] Acid groups were then decorated with EG4 amine 6
and alkenes modified with EG4 thiol 7 in the last reaction
step. Fluorescence microscopy analysis of the surface after
exposure to rhodamine-labeled streptavidin showed selec-
tive protein immobilization in the initially azide-modified
areas (Figure 5C). The nonfluorescent black gaps (5ꢃ5 mm2)
indicate near-quantitative attachment of the amine-terminat-
ed alkyne linker during mCP, since biotin residues were not
detectable in these areas.
Figure 5. A) Preparation of
a
tetrafunctional alkene/azide/acid/amine-
modified substrate surface by microcontact CuAAC of Boc-protected
aminoalkyne 9 and successive deprotection. Fluorescence microscopy
images of rhodamine streptavidin immobilized on biotin modified surfa-
ces, prepared by reaction with B) biotin NHS ester 10 and C) with biotin
alkyne 3. D) Binding of rhodamine-labeled PNA to the substrate surface
after orthogonal modification with lactose amine 4. E) Binding of rhoda-
mine-labeled ConA after attachment of mannose thiol 5.
Amines were subsequently capped by reaction with succini-
midyl acetate. Azide groups were treated with EG4 alkyne
8, and alkenes in the last step were decorated with EG4
thiol 7. Rhodamine-labeled PNA binding verified the specif-
ic ligation of the lactose residues to acid groups (Figure 5D).
Finally, orthogonal alkene modification was demonstrated
on the tetrafunctional surface. To this end, mannose thiol 5
was immobilized on the substrate by means of photoinduced
thiol–ene chemistry and the remaining reactive groups were
modified by subsequent reaction with 1) succinimidyl ace-
tate, 2) EG4 amine 6, and with 3) EG4 alkyne 8 before pro-
tein binding. ConA (rhodamine-labeled) screening showed
the same binding result as observed on the trifunctional sur-
face, thus proving complete chemically orthogonal addressa-
bility of the surface (Figure 5E).
Conclusion
Furthermore, orthogonal acid modification on the tetra-
functional surface was demonstrated by attachment of lac-
tose amine 4 after surface activation with DCC/NHS.
In this article, we have described the preparation of multi-
functional surfaces by successive immobilization of heterobi-
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B. J. Ravoo et al.
mCP of Boc-amine alkyne linker 9 by means of CuAAC: Oxidized PDMS
stamps were covered with a solution (60 mL) of Boc-amine alkyne linker
9 (40 mm) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA,
1 mm) in ethanol. In each case, an aqueous CuSO4 solution (5 mL, c=
10 mg CuSO4·5H2O/1 mL H2O) and an aqueous sodium ascorbate solu-
tion (5 mL, c=40 mg sodium ascorbate/1 mL H2O) was added to the
stamp surface and the liquids were mixed for 10 s. The stamps were
blow-dried in a stream of Ar and carefully placed on the azide-patterned
substrates. Printing was carried out in an oven at 508C for 15 min. PDMS
stamps were removed, and the surfaces were washed with Milli-Q water
and ethanol. Finally, the substrates were sonicated in ethanol (1 min) and
dried in a stream of Ar.
functional linkers on chemically modified substrates. Micro-
contact chemistry was exploited as a straightforward way to
locally induce immobilization reactions in a very fast and ef-
fective manner, thereby resulting in well-defined micropat-
terned arrays of orthogonally addressable functional groups.
These surfaces were highly suitable for the site-specific at-
tachment of biologically relevant molecules, since each of
the selected immobilization reactions could be induced in
a highly selective and mild way. The results presented here
attest to the versatility of click chemistry for the functionali-
zation of organosilane SAMs. In addition, these results dem-
onstrate the power of mCP for the preparation of chemical
and biological microarrays.
Synthesis of tetrafunctional alkene/azide/acid/amine-modified surfaces by
deprotection with TFA: Boc-protected amine surfaces were immersed in
a TFA solution (90vol% in CH2Cl2) for 20 min at room temperature.
The substrate surfaces were then washed with Milli-Q water, ethanol,
and ethanolic NEt3 solution (5vol%) and dried in a stream of Ar.
Orthogonal ligation
Azide modification by reaction with biotin alkyne 3 by means of CuAAC:
Azide-modified surfaces were immersed into a solution of biotin alkyne
3 (5 mm) in DMF under Ar, and aqueous CuI solution (1% vol DMF)
was added. The CuI solution was freshly prepared by dissolving
CuSO4·5H2O (c=5 mgmLÀ1) and sodium ascorbate (c=20 mgmLÀ1) in
water. The reaction mixture was heated for 8 h to 708C. Substrates were
extensively washed with Milli-Q water and ethanol and dried.
Experimental Section
General procedures and materials: 11-Undecenyltrichlorosilane was pur-
chased from ABCR. Silicon wafers (B-doped, 1-0-0 orientation, 20–30 W)
were kindly donated by Siltronic AG (Burghausen, Germany). Glass sub-
strates were prepared by cutting IDL (Interessengemeinschaft der Labor-
fachhꢁndler) microscope slides into pieces of about 2.6ꢃ1.4 cm2. Milli-Q
water used for surface cleaning was prepared from distilled water by
a PureLab UHQ deionization system (Elga). Fluorescein isothiocyanate
(FITC)-labeled concanavalin A (ConA) and bovine serum albumin
(BSA) were obtained from Sigma Aldrich. Tetramethylrhodamine iso-
thiocyanate (TRITC)-labeled peanut agglutinin (PNA) and tetramethylr-
hodamine isothiocyanate (TRITC)-labeled concanavalin A (ConA) were
purchased from Vector Laboratories. DyLight 405-conjugated and tetra-
methylrhodamine isothiocynate (TRITC)-conjugated streptavidin was de-
livered by Thermo Fisher Scientific. Surface irradiation for photochemi-
cal thiol–ene reaction was carried out using a high power UV-LED
(P8D236, Seoul Semiconductor, 365 nm peak wavelength, 18 nm spec-
trum half with, 90 mW optical power output) supplied by Conrad Elec-
tronics.
Acid modification by immobilization of lactose amine 4 by means of
active ester peptide chemistry: The procedure for the activation of acid-
terminated SAMs by reaction with dicyclohexylcarbodiimide and N-hy-
droxysuccinimide was adopted from Benters et al.[43] Dicyclohexylcarbo-
diimide (1.00m, 6.19 g, 30 mmol) and N-hydroxysuccinimide (1.00m,
3.45 g, 30 mmol) were dissolved in dry DMF (peptide synthesis grade,
30 mL) under Ar. Acid-modified substrates were immersed in the solu-
tion and the mixture was stirred at room temperature for 1 h under Ar.
The substrates were removed, thoroughly washed with CH2Cl2, and dried
in a stream of Ar. Substrate surfaces were covered with a solution of lac-
tose amine 4 (10 mm) and NEt3 (20 mm) in DMF and incubated for 5 h at
RT. Finally, the substrates were cleaned by washing with Milli-Q water
and ethanol and dried in a stream of Ar.
Alkene modification by immobilization of mannose thiol 5 by means of
thiol–ene chemistry: Mannose thiol
5 (5.0 mg, 15 mmol) was put on
Surface Preparation
a clean microscopy glass slide and mixed into a solution of 2,2-dime-
thoxy-2-phenylacetophenone (DMPA) in diethylene glycol (20 mg solu-
tion, 2wt% DMPA). The alkene-modified glass surface was put upside-
down on the liquid-coated microscopy glass slide, and the system was ir-
radiated at room temperature for 10 min with a 365 nm high power UV-
LED. The LED was positioned approximately 2 cm above the contact
area between both glass surfaces. Finally, the carbohydrate-modified sub-
strates were thoroughly washed with Milli-Q water and ethanol and dried
in a stream of Ar.
Preparation of poly(dimethylsiloxane) (PDMS) stamps: PDMS stamps
were prepared by casting a 10:1 (v/v) mixture of Sylgard 184 silicone
elastomer base and curling agent (Dow Corning) on a patterned silicon
master. The PDMS was curled at 808C overnight. Patterned areas were
cut out with a knife and oxidized using an UV-ozonizer (PSD-UV, No-
vascan Technologies Inc.) for 55 min prior to use. Flat PDMS stamps
were prepared analogously by using a flat silicon master.
Preparation of alkene-modified glass and silicon substrates: Glass and sili-
con plates were cut into pieces of 2.6ꢃ1.4 cm2 and cleaned by sonication
in pentane, acetone, and Milli-Q water. Substrates were immersed into
a freshly prepared piranha solution (H2SO4/H2O2 (30%) 3:1; CAUTION:
Piranha is a very strong oxidant and reacts violently with organic materi-
al). After 30 min, the substrates were thoroughly washed with Milli-Q
water, dried, and directly put in a stirred solution of 11-undecenyltri-
chlorosilane in toluene (0.1% vol) for 40 min.[26] Finally, the substrates
were cleaned by washing with ethanol and Milli-Q water and dried.
Amine modification by reaction with biotin sucinimidyl ester 10: Amine-
modified substrates were covered with a freshly prepared solution of
biotin succinimidyl ester 10 (40 mm) and diisopropylamine (20 mm) in dry
DMF and incubated for 1 h at RT. The substrates were washed with
CH2Cl2, Milli-Q water, and ethanol and dried in a stream of Ar.
Protein binding
Blocking of carboxylic acid groups by peptide coupling with EG4 amine
6: Acid-modified surfaces were covered with a freshly prepared solution
of EG4 amine 6 (0.2m), diisopropylethylamine (DIEA, 0.2m), and O-
mCP of azide thiol 1 and acid thiol 2 by photochemical thiol–ene reaction:
Oxidized PDMS stamps were covered with 1–2 drops of a solution of
thiol (80 mm) and 2,2-dimethoxy-2-phenylacetophenone (DMPA, 40 mm)
in ethanol. After an incubation time of 1 min, the stamps were blow-
dried in a stream of Ar and carefully placed on the alkene-modified sub-
strates. The substrates were irradiated at room temperature for 2 min
with a 365 nm high power UV-LED, which was positioned in each case
approximately 2 cm above the contact area between stamp and substrate.
After irradiation, the stamps were removed and the substrates cleaned
by extensive washing with ethanol and sonication in ethanol (1 min). The
surfaces were dried in a stream of Ar.
(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium
tetrafluoroborate
(TBTU, 0.16m) in dry DMF at room temperature. After 2 h, the surfaces
were washed with CH2Cl2, Milli-Q water, and ethanol and dried in
a stream of Ar.
Blocking of alkene groups by photoinduced thiol–ene reaction with EG4
thiol 7: 2,2-Dimethoxy-2-phenylacetophenone (DMPA) was dissolved in
EG4 thiol 7 (3wt%), and a drop of the solution was topically applied to
the alkene-modified substrate surfaces. The liquids were covered with mi-
croscopy cover slides and irradiated at room temperature for 10 min with
5886
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 5880 – 5888

Multifunctional Surfaces
FULL PAPER
a 365 nm high-power UV-LED. The LED was positioned approximately
2 cm above the contact area between both glass surfaces. The substrates
were thoroughly washed with Milli-Q water and ethanol and dried in
a stream of Ar.
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Blocking of azide groups by CuAAC with EG4 alkyne 8: Azide-modified
surfaces were immersed into a solution of EG4 alkyne 8 (50 mm) in DMF
under Ar and aqueous CuI solution (1vol% DMF) was added. The CuI
solution was freshly prepared by dissolving CuSO4·5H2O (c=5 mgmLÀ1
)
and sodium ascorbate (c=20 mgmLÀ1) in water. The reaction mixture
was heated for 4 h to 708C. Substrates were extensively washed with
Milli-Q water and ethanol and dried in a stream of Ar.
Blocking of amine groups by reaction with succinimidyl acetate: Amine-
modified surfaces were covered with a freshly prepared solution of succi-
nimidyl acetate (0.1m) and diisopropylethylamine (DIEA, 50 mm) in dry
DMF and incubated for 30 min at room temperature. The surfaces were
washed with CH2Cl2, Milli-Q water, and ethanol and dried in a stream of
Ar. In the case of carbohydrate-decorated surfaces, amine groups were
acetylated by coverage of the surfaces with aqueous solutions of succcini-
midyl acetate (10 mm) in sodium phosphate buffer (50 mm, pH 8.5) for
2 h at RT. The surfaces were cleaned by washing with Milli-Q water and
ethanol and dried.
PNA and ConA binding: Prior to protein binding, substrates were incu-
bated for 30 min in a solution of bovine serum albumin (BSA, 3wt%) in
phosphate-buffered saline (1ꢃPBS, pH 7.5) solution and then washed
with PBS buffer without BSA (2ꢃ5 min). The substrates were then cov-
ered with a solution of fluorescence-labeled lectin (FITC-labeled ConA,
TRITC-labeled ConA, or TRITC-labled PNA; c=20 mgmLÀ1) in 4-(2-hy-
droxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (20 mm
HEPES, pH 7.5, 0.15m NaCl, 1 mm CaCl2). In the case of ConA, MnCl2
was added to a concentration of 1 mm. After 30 min at room tempera-
ture, the substrates were washed with the same buffer without lectin,
rinsed with distilled water, carefully dried with a tissue, and analyzed by
fluorescence microscopy.
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The substrates were covered with a solution of streptavidine (TRITC-la-
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were washed with the same buffer without protein, rinsed with distilled
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copy.
Simultaneous binding of PNA, ConA, and streptavidin: Prior to protein
binding, substrates were incubated for 30 min in a solution of BSA
(3wt%) in PBS (1ꢃPBS, pH 7.5) and then washed with PBS buffer with-
out BSA (2ꢃ5 min). The substrates were covered with a solution of
TRITC-labeled PNA, FITC-labeled ConA, and Dylight 405-labeled
streptavidin in buffer for 30 min at room temperature. The protein solu-
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c=50 mgmLÀ1 in 20 mm HEPES, pH 7.5, 0.15m NaCl, 1 mm CaCl2) with
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washed with HEPES buffer (20 mm HEPES, pH 7.5, 0.15m NaCl, 1 mm
CaCl2, 1 mm MnCl2), rinsed with distilled water, carefully dried with
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Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft DFG
through grant Ra 1732/2.
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Received: October 31, 2011
Published online: April 16, 2012
5888
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 5880 – 5888