Photoreactive polymer brushes for high-density patterned surface derivatization using a diels-alder photoclick reaction

Article abstract of DOI:10.1021/ja210350d

Reactive polymer brushes grown on silicon oxide surfaces were derivatized with photoreactive 3-(hydroxymethyl)naphthalene-2-ol (NQMP) moieties. Upon 300 or 350 nm irradiation, NQMP efficiently produces o-naphthoquinone methide (oNQM), which in turn undergoes very rapid Diels-Alder addition to vinyl ether groups attached to a substrate, resulting in the covalent immobilization of the latter. Any unreacted oNQM groups rapidly add water to regenerate NQMP. High-resolution surface patterning is achieved by irradiating NQMP-derivatized surfaces using photolithographic methods. The Diels-Alder photoclick reaction is orthogonal to azide-alkyne click chemistry, enabling sequential photoclick/azide-click derivatizations to generate complex surface functionalities.

Full text of DOI:10.1021/ja210350d

Communication
pubs.acs.org/JACS
Photoreactive Polymer Brushes for High-Density Patterned Surface
Derivatization Using a Diels−Alder Photoclick Reaction
Selvanathan Arumugam,† Sara V. Orski,†,‡ Jason Locklin,*,†,‡ and Vladimir V. Popik*,†
†Department of Chemistry and ‡Faculty of Engineering and the Center for Nanoscale Science and Engineering, University of Georgia,
Athens, Georgia 30602, United States
S
* Supporting Information
Scheme 1. Ligation of Two Substrates (S1 and S2) Using the
ABSTRACT: Reactive polymer brushes grown on silicon
Diels−Alder Photoclick Reaction between oNQM (2) and
oxide surfaces were derivatized with photoreactive 3-
(hydroxymethyl)naphthalene-2-ol (NQMP) moieties.
Upon 300 or 350 nm irradiation, NQMP efficiently
produces o-naphthoquinone methide (oNQM), which in
turn undergoes very rapid Diels−Alder addition to vinyl
ether groups attached to a substrate, resulting in the
covalent immobilization of the latter. Any unreacted
oNQM groups rapidly add water to regenerate NQMP.
High-resolution surface patterning is achieved by irradiat-
ing NQMP-derivatized surfaces using photolithographic
methods. The Diels−Alder photoclick reaction is
orthogonal to azide−alkyne click chemistry, enabling
sequential photoclick/azide-click derivatizations to gen-
erate complex surface functionalities.
Vinyl Ether
chemical precursor to oNQM, 3-(hydroxymethyl)naphthalene-
2-ol [naphthoquinone methide precursor (NQMP), 1].
NQMP-derivatized substrates require no special handling
because of the excellent stability of NQMP under ambient
conditions. Upon irradiation with 300 or 350 nm light,
photoconversion results in the efficient generation of oNQM
moieties (Scheme 1).
Here we report the functionalization of activated ester
polymer brushes with NQMP groups to allow selective
immobilization of vinyl ether-tagged substrates only upon
activation with light. A poly(N-hydroxysuccinimidyl 4-vinyl-
benzoate) [poly(NHS4VB)] brush coating was chosen as a
versatile surface platform because it is densely packed and
provides a facile template for postfunctionalization.15 The
polymer brush platform insures high mechanical and chemical
stability of the functionalized surfaces and allows for a much
higher surface density of the reactive groups than self-
assembled monolayers (SAMs).
Poly(NHS4VB) coatings (50 nm) were prepared using
surface-initiated atom-transfer radical polymerization.15 Over-
night incubation of the derivatized silicon wafers in an N,N-
dimethylformamide (DMF) solution of NQMP-TEG-amine
derivative 4 (TEG = triethylene glycol) resulted in immobiliza-
tion of the latter on the brush matrix (Scheme 2). The
conversion was quantitative, as evidenced by the complete
disappearance of the imide bands in the grazing-angle
attenuated total reflectance (GATR)-FTIR spectra (Figure
1a).16 Removal of acetal protection from immobilized 4 was
achieved by treating the wafers with 0.1 HCl in DMF.
The deprotection of surface-immobilized NQMP was
confirmed by conversion of the resulting diol group into
diacetate by overnight incubation in a dichloromethane
solution of acetic anhydride/pyridine. The change in polymer
layer thickness and hydrophobicity as well as the appearance of
hotochemical immobilization of carbohydrates,1 pro-
P
teins,2−4 DNA fragments,3,5 antibodies,4,6 and other
substrates7 allows for the formation of patterned or gradient
arrays on various surfaces. These techniques are widely used in
the development of novel high-throughput analytical methods.8
Several photoclick strategies suitable for surface patterning are
currently under development. Most of these methods rely on
the photochemical generation of reactive functional groups on
the surface, such as azide-reactive cyclooctynes,9 alkene-reactive
nitrile imines,10 or hydroquinone dienophiles.7 Photoreduction
of Cu(II) to catalytically active Cu(I) species also permits
spatial control over the alkyne−azide click reaction.11 While
photochemical activation in these techniques is virtually instant,
the actual “click” step takes from several seconds to hours,
significantly slowing down the overall process. Photoinitiated
thiol−ene12 and thiol−yne13 reactions proceed via the
generation of reactive radicals, allowing for very fast click
ligation. The high reactivity of the radical intermediates, on the
other hand, is reflected in low selectivity or poor “orthogon-
ality” of the method. In addition, technologies involving
photoreactive surfaces often require handling and functionaliza-
tion of substrates in a light-protected environment.
We have recently developed a photoligation strategy based
on very facile (k ≈ 4 × 104 M−1 s−1) hetero-Diels−Alder
addition of 2-napthoquinone-3-methides (oNQMs, 2) to vinyl
ethers (Scheme 1).14 In aqueous solutions, oNQMs are very
selective and undergo cycloaddition only to vinyl ethers to
produce a photochemically stable naphthopyran linker, 3.
Unreacted oNQMs rapidly hydrate to regenerate the photo-
Received: November 3, 2011
Published: December 14, 2011
© 2011 American Chemical Society
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Communication
To demonstrate the utility of the Diels−Alder photoclick
reaction for surface patterning, poly(NQMP-4VB)-coated
wafers were irradiated through a shadow mask to form
multicomponent surfaces with spatially resolved chemical
functionalities. Substrates were immersed in a 0.1 mM aqueous
solution of vinyl ether-derivatized fluorescein (EV−fluores-
cein)16 and irradiated at 300 nm for 1 min via square-patterned
transmission electron microscope (TEM) grids (250 and 12.5
μm pitch) using a hand-held lamp (Scheme 3). The
immobilization occurred only in the exposed areas, where
reactive oNQM moieties were formed upon irradiation (Figure
Scheme 2. NQMP Functionalization of a Brush Polymer
Figure 2. Fluorescence microscopy images of poly(NQMP-4VB)-
coated wafers (a, b) photopatterned in 0.1 mM EV−fluorescein
solution using (a) 250 and (b) 12 μm pitch TEM grids and (c) upon
sequential immobilization of rhodamine B and fluorescein.
2a,b). Photoimmobilization of EV−fluorescein was accompa-
nied by a sharp increase of the polymer layer thickness and
hydrophobicity.16
The photochemical stability of the naphthopyran linker
permitted multiple exposures of photoderivatized areas,
simplifying the patterning of multiple substrates. To illustrate
this capability of the photo-Diels−Alder immobilization
technique, a poly(NQMP-4VB)-coated wafer was irradiated
through a TEM grid mask in the presence of a vinyl ether−
rhodamine B conjugate (EV−rhodamine B). After the mask
was removed, the wafer was thoroughly washed with DMF and
flood-irradiated in a solution of EV−fluorescein. Figure 2c
shows a fluorescence image of the resulting two-color pattern
with negligible amounts of cross contamination between the
two dyes.
Diels−Alder photoclick chemistry is orthogonal to the
majority of other derivatization techniques, including the
well-developed alkyne−azide click chemistry. Concurrent or
sequential applications of photoclick and alkyne−azide click
ligations permit one-pot derivatization of substrates with
multiple moieties or for light-directed patterning of photo-
sensitive groups. In such sequential click immobilizations, an
azide or alkyne-containing vinyl ether was photopatterned onto
a poly(NQMP-4VB)-coated surface. The substrate of interest
was then immobilized using Cu(I)-catalyzed (CuAAC) or
strain-promoted azide−alkyne cycloaddition (SPAAC) chem-
istry (Scheme 4).
Figure 1. GATR-FTIR spectra and properties of the functionalized
polymer brush containing (a) NQMP, (b) NQMP diacetate, and (c)
NQMP−acetyl vinyl ether adduct.
ester bands in the IR spectrum (Figure 1b) confirmed the
formation of the diacetate.16
To test the efficiency of the new photoimmobilization
platform, the wafer coated with the NQMP-functionalized
polymer brush [poly(NQMP-4VB)] was immersed in an
aqueous solution of 2-(vinyloxy)ethyl acetate (0.1 mM) and
irradiated at 300 nm using a hand-held lamp (3.5 mW cm−2) for
2 min. Increases in the thickness and hydrophobicity of the
polymer layer as well as the appearance of an ester band at 1738
cm−1 in the IR spectrum (Figure 1c) clearly indicated formation
of naphthopyran 3 on the polymer brushes. Overnight
incubation of the irradiated wafer in acetic anhydride/pyridine
solution did not change the surface characteristics, confirming
complete conversion of the NQMP moieties to 3 (Scheme 3).
Scheme 3. Photo-Diels−Alder Functionalization of a
Poly(NQMP-4VB)-Coated Silicon Wafer
To test the feasibility of the sequential click surface
functionalization, poly(NQMP-4VB)-coated silicon wafers
were irradiated in aqueous solutions of the propargyl vinyl
ether of diethylene glycol or (2-azidoethoxy)ethyl vinyl ether
using a 12 μM pitch TEM grid (Scheme 4). The azide-
derivatized wafer was immersed in a 0.1 mM DMF solution of
ADIBO−rhodamine B conjugate17 for 4 h. A fluorescence
microscopy image of the resulting slide is shown in Scheme 4a.
The substrate containing the propargyl groups was subjected to
CuAAC coupling with azidofluorescein16 to give a clean pattern
(Scheme 4b). These images show that a sequential click
strategy permits clean and selective immobilization of azide- or
alkyne-tagged substrates.
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Journal of the American Chemical Society
Communication
zation14b of an epoxide SAM on a glass slide.16 Both slides were
immersed in a 0.1 mM aqueous solution of EV−fluorescein and
flood-irradiated using 350 nm light until full conversion was
achieved. The average surface emission of the fluorescein-
functionalized poly(NQMP-4VB) layer was 61 5 times more
intense than that of the NQMP-derivatized SAM (Figure 3B vs
Scheme 4. Sequential Click Functionalization: Photo-Diels−
Alder Surface Patterning Followed by Azide−Alkyne Click
Immobilization of Fluorescent Dyes via (a) SPAAC of
ADIBO−Rhodamine B on an Azide-Derivatized Surface and
(b) CuAAC of Azidofluorescein
Figure 3. Fluorescent emission of (A, B) EV−fluorescein immobilized
on (A) an NQMP-SAM on glass and (B) a poly(NQMP-4VB) layer
on a silicon wafer and (C, D) FITC−avidin immobilized on (C) an
NQMP-SAM and (D) poly(NQMP-4VB).
A, respectively). This experiment demonstrates the increased
surface density of solvent-accessible functional groups in the
polymer brush layers in comparison with SAMs.
NQMP-SAM and NQMP-polymer brush platforms were also
tested for the immobilization of much larger avidin molecules.
Both types of slides were photobiotinylated by exposure to 350
nm light in an EV−biotin solution and then stained with
FITC−avidin. The fluorescence intensity of the poly(NQMP-
4VB) slide was 15 2 times greater than that of the NQMP-
SAM analogue. The biotinylated polymer brush showed a
significant and uniform increase in hydrophilicity upon FITC−
avidin staining.16 This observation and GATR-FTIR data
indicate uniform protein immobilization on the poly(NQMP-
4VB)-coated surface. The appearance and position of amide I
and II bands in the FTIR spectra (Table S2 in the Supporting
Information) closely matched the absorbance of native avidin,
indicating that the protein was not denatured upon
immobilization.18 The changes in layer thickness, on the
other hand, were rather small,16 suggesting a relatively low
loading of the protein in the polymer matrix. This conclusion
agrees well with the muted fluorescence intensity enhancement
for the FITC−avidin-functionalized polymer brush platform
relative to the SAM (Figure 3D vs C, respectively).
The relatively low contact angle of the poly(NQMP-4VB)
substrate (55°) makes it a suitable platform for protein
immobilization. Thus, fluorescein isothiocyanate (FITC)−
avidin was photopatterned on the surface using a two-step
procedure. First, vinyl ether−biotin conjugate (EV−biotin) was
micropatterned on polymer brushes using the Diels−Alder
photoclick reaction (Scheme 5). The resulting patterned
Scheme 5. Diels−Alder Photoclick Patterning of FITC−
Avidin on a Poly(NQMP-4VB) Surface
We presume that the reduction in the loading level of avidin
compared to fluorescein is most likely due to the fact that the
entropic cost of having polymer brush chains extended from
the surface makes it difficult for large molecules to diffuse into
the brush.19 It is also possible that the bound avidin can cross-
link to the biotinylated polymer brushes, thereby preventing
further diffusion of protein into the matrix. However, it is
important to note that the loading level of protein on the
surface of the polymer brushes is still more than an order of
magnitude greater than that of SAMs.
In summary, we have demonstrated the utility of the light-
directed Diels−Alder click reaction for the patterned
derivatization of polymer-brush-coated surfaces. The very fast
o-naphthoquinone methide−vinyl ether cycloaddition allows
for temporal control of the immobilization process and can
enhance the efficiency of many biotechnological tools, such as
microprinting, microarray manufacturing, protein assays, etc.
Furthermore, the ability to attach reliably a larger amount of
protein than in monolayer methods enhances sensor readout
and potentially reduces the cost of biotechnology platforms.
This instantaneous photoclick reaction is orthogonal to the
majority of modern ligation techniques and can be used in
biotinylated slide was developed with FITC−avidin. Fluo-
rescence microscopy images demonstrated that avidin was
immobilized only in the exposed areas.
One of the most important advantages of the polymer brush
platform over SAM-based systems in sensor development is the
ability to achieve significantly higher functionalization and
therefore a stronger readout signal. We quantitatively compared
the binding capacity of the 65 nm poly(NQMP-4VB) layer with
a NQMP-TEG monolayer produced by quantitative derivati-
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NQMP group is readily and inexpensively prepared and can be
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based photoclick chemistry a promising tool for ligation and
immobilization procedures.
ASSOCIATED CONTENT
* Supporting Information
■
S
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(16) See the Supporting Information.
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AUTHOR INFORMATION
Corresponding Author
jlocklin@uga.edu; vpopik@uga.edu
■
ACKNOWLEDGMENTS
■
The authors thank the Georgia Cancer Coalition and the NSF
(CHE-0842590, ECCS 0901141, DMR 0953112) for support
of this project.
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