Aryl azide based, photochemical patterning of cyclic olefin copolymer surfaces with non-biofouling poly[(3-(methacryloylamino)propyl)dimethyl(3- sulfopropyl)ammonium hydroxide]

Article abstract of DOI:10.1002/asia.201000569

Micropatterns of cells were generated on a cyclic olefin copolymer (COC) by a combination of aryl-azide-based photopatterning of a polymerization initiator and surface-initiated atom-transfer radical polymerization of non-biofouling 3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium hydroxide (MPDSAH). The optical transparency of the COC made it possible to generate the patterns indirectly through a COC slab. Copyright

Full text of DOI:10.1002/asia.201000569

DOI: 10.1002/asia.201000569
Aryl Azide Based, Photochemical Patterning of Cyclic Olefin Copolymer
Surfaces with Non-Biofouling Poly[(3-
(methacryloylamino)propyl)dimethyl(3-sulfopropyl)ammonium hydroxide]
Jinkyu Kim,[a] Daewha Hong,[a] Seungpyo Jeong,[a] Bokyung Kong,[a] Sung Min Kang,[a]
Yang-Gyun Kim,*[b] and Insung S. Choi*[a]
Dedicated to Professor Eiichi Nakamura on the occasion of his 60th birthday
Copolymers of ethylene and a cyclic olefin, such as nor-
bornene or cyclopentene, are a new class of thermoplastics
commonly known as cyclic olefin copolymers (COCs). They
possess properties desirable for the fabrication of microflui-
dic devices, biosensors, medical devices, and diagnostic dis-
posables, including optical clarity, low autofluorescence, low
birefringence, moldability, high glass-transition temperature
(70 to 1608C), low water uptake, and resistance to common
organic solvents.[1–3] In spite of these favorable properties,
the use of COC as a material for the aforementioned appli-
cations has been limited owing to its hydrophobicity and the
lack of functionalizability. In this respect, some methods for
modifying/functionalizing the COC surface have recently
been reported.[4–6] Especially, functionalization of the inner
surface of channels has been attempted to minimize the hy-
drophobic interactions between the hydrophobic COC sur-
face and biomolecules, leading to the undesired adsorption
of biomolecules inside of the channels, and to improve the
efficiency of COC-based microfluidic devices.[4,6a]
Although most polymeric materials, such as poly-
carbonate, poly(methyl methacrylate), and polydimethylsil-
oxane, have been functionalized by simple chemical reac-
tions,[7] these methods are not applicable directly to the
functionalization of COCs because of its saturated hydrocar-
À
À
bon structure which has only C C and C H bonds; hence,
physical grafting[4] and chemical oxidation[5] have generally
been used. The chemical oxidation, such as ozone[5a] and
oxygen plasma treatments,[5b–e] generates oxygen-containing
functional groups, such as epoxide, ketone, alcohol, and car-
boxylic acid, which could be subsequently used for the at-
tachment of molecules of interest. On the other hand, pho-
tografting[6] is an attractive alternative to physical grafting
and chemical oxidation because it makes the strong covalent
attachment of molecules onto surfaces and allows for easy
fabrication of polymeric surfaces. In addition to the possibil-
ity of pattern generation by photografting,[6a] the UV trans-
parency of COCs would, in principle, make it possible to se-
lectively functionalize the inner surface of the COC-based
channels.
Nonspecific adsorption of bioentities occurs commonly in
contact with artificial surfaces and causes the deteriorated
performance of microfluidic channels as well as many unde-
sirable biological responses, such as thrombus formation and
bacterial infection.[8] The minimization of biofouling in mi-
crofluidic devices has been investigated based on polymer
coating. Ethylene glycol based compounds have generally
been used for non-biofouling coating, and zwitterionic poly-
mers, such as phosphorylcholine, sulfobetaine, and carboxy-
betaine, have recently been suggested as a new type of non-
biofouling materials.[9] For example, our group reported that
poly[(3-(methacryloylamino)propyl)dimethyl(3-sulfopropyl)-
[a] J. Kim,+ D. Hong,+ S. Jeong, Dr. B. Kong, Dr. S. M. Kang,
Prof. Dr. I. S. Choi
Molecular-Level Interface Research Center
Department of Chemistry
KAIST
Daejeon 305-701 (Korea)
Fax : (+82)42-350-2810
E-mail: ischoi@kaist.ac.kr
[b] Prof. Dr. Y.-G. Kim
Department of Chemistry
Sungkyunkwan University
Suwon 440-746 (Korea)
Fax : (+82)31-290-4574
E-mail: ygkimmit@skku.edu
ammonium hydroxide] [polyACTHNUTRGNEUGN(MPDSAH)] was highly effi-
[+] These authors contributed equally to this work.
cient in minimizing the nonspecific adsorption of proteins
(Figure 1a).[9a]
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201000569.
Chem. Asian J. 2011, 6, 363 – 366
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363

COMMUNICATION
After photoreaction, the
water contact angle of the sub-
strate was changed to 46.88
from 94.58, indicative of the
successful attachment of the ini-
tiator, 1, onto the COC surface,
by UV irradiation through the
COC slab (Figure 2a). The SI-
ATRP of MPDSAH further de-
creased the contact angle to
5.58.[9a] The attachment was fur-
ther confirmed by X-ray photo-
electron spectroscopy (XPS,
Figure 2b). In the XPS spec-
trum of the initiator-attached
substrate we observed charac-
teristic peaks of the initiator at
Figure 1. a) Structure of MPDSAH. b) Synthesis of aryl azide based, atom-transfer radical polymerization
(ATRP) initiator (1).
Aryl azide compounds—widely used in biology—have
been applied for photografting of carbon-based materials,
such as polymers[10a–c] and carbon nanotubes.[10d] Photolysis
of aryl azide yields a highly reactive nitrene, which inserts
À
À
into C H and C C bonds as one of the possible reaction
pathways.[11] An aryl azide containing aldehyde or quaterna-
ry amine has recently been patterned on the COC surface.[12]
In this work, we designed and synthesized an aryl azide con-
taining atom-transfer radical polymerization (ATRP) initia-
tor from methyl pentafluorobenzoate and used it as a photo-
grafting reagent for patterned, surface-initiated ATRP (SI-
ATRP)[13] of MPDSAH on the COC surface (Figure 1b; see
the Experimental Section for the synthesis). We found that
the photografting successfully occurred with a COC slab as
a physical barrier because of the optical transparency of
COC, which showed the possibility in functionalizations of
the inner surface of COC channels. Patterns of cells were
also generated on the COC surface by using the non-bio-
Figure 2. a) Static water contact angles of COC substrates: intact, initia-
fouling property of polyACTHUNTGRENNG(U MPDSAH).
tor-attached, and poly
substrates.
ACHTUNGTRENUN(NG MPDSAH)-coated COC. b) XPS spectra of COC
To investigate the photochemical attachment of the initia-
tor (1) onto a COC surface, we first performed the photore-
action in the absence of a photomask. The initiator was
spin-coated on a COC surface, followed by 100 s UV irradi-
ation (254 nm). Another COC slab was placed on the COC
substrate after spin-coating, for the confirmation of the pos-
sibility of indirect patterning of the initiator. The SI-ATRP
of MPDSAH was subsequently performed by following our
reported procedures:[9a] Briefly, copper(II) bromide, 2,2’-di-
pyridyl, MPDSAH, water, and methanol were added to the
Schlenk flask. The resulting solution was deoxygenated by
passing a continuous stream of dry argon, and to the solu-
tion was added copper(I) bromide. The initiator-coated
COC substrate was placed in another Schlenk flask, and
then the Schlenk flask was degassed under vacuum and
purged with dry argon. The reactant solution was trans-
ferred to the Schlenk flask containing the initiator-coated
COC substrate, and the polymerization was carried out for
14 h at room temperature.
687.66 (F 1s), 532.63 (O 1s), 70.23 (Br 3d), and 393.93 eV
(N 1s), while the spectrum of the intact COC was composed
of only the carbon peak. After the formation of poly-
AHCTUNGTREGUN(NN MPDSAH) films, the XPS spectrum showed peaks at
530.72 (O 1s), 399.5 (N 1s), 284.92 (C 1s), and 167.41 eV
(S 2p). In particular, the S peak at 167.41 eV appeared in
the spectrum owing to polyACTHNUTRGENUGN(MPDSAH). Taken together,
these results clearly showed that the aryl azide based ATRP
initiator was attached onto the COC substrate by photoreac-
tion, and the photoreaction could be achieved indirectly
with a COC slab with a barrier. In addition, as one of poten-
tial ATRP monomers, the non-biofouling MPDSAH was
successfully polymerized from the initiator-attached COC
surface.
Photoreactions have advantages for the pattern genera-
tion over other surface-based reactions, because they can be
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Chem. Asian J. 2011, 6, 363 – 366

Photochemical Patterning of Cyclic Olefin Copolymer Surfaces
seamlessly combined with conventional photolithographic
techniques.[14] In this work, we generated the micropatterns
were then transferred to a dish containing the COC sub-
strate and incubated at 378C for 24 h. The optical micro-
graph showed a grid pattern of cells (Figure 4b). As a con-
trol, we also incubated the cells on an intact, nonpatterned
COC substrate, and did not observe any patterns but did ob-
serve the nonspecific adsorption of the cells (see the Sup-
porting Information). Further experiments on the stability
of poly
and cell patterns by utilizing the non-biofouling property of
poly(MPDSAH). The procedure for the pattern generation
of the poly(MPDSAH) film is depicted in Figure 3. The ini-
ACHTUNGTRENNUNG(MPDSAH) and used them as a platform for protein
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
of the formed poly
ACHTUNGTRNE(NUNG MPDSAH) films showed that the expo-
sure of the poly(MPDSAH) films to the air did not deterio-
AHCTUNGTRENNUNG
rate their non-biofouling property for at least three days
(see the Supporting Information).
In summary, we suggested a photoreaction-based func-
tionalization of COC, based on aryl azide chemistry. The
possibility of functionalizing the inner surface of COC-
based channels was investigated by indirect UV irradiation,
and, as one of the applications, the non-biofouling coating
of poly
tern generation of NIH 3T3 fibroblast cells was achieved by
using the non-biofouling property of poly(MPDSAH). We
ACHTUNGTREN(NUNG MPDSAH) was demonstrated. In addition, the pat-
AHCTUNGTRENNUNG
believe that the patternability of the inner surface of COC-
based microchannels would widen the applications of COC
in microfluidic devices and related areas.
Figure 3. Procedure for pattern generation of polyACHTUNTRGNEU(GN MPDSAH) and NIH
3T3 fibroblast cells on COC surfaces by indirect photopatterning.
Experimental Section
tiator, 1, was spin-coated on a COC surface, followed by
placing a TEM grid as a model photomask. The sample was
then covered with another COC slab, and the UV light was
irradiated through the upper COC slab for 1 h. After 14 h
Experimental details, including the pattern generation of TRITC-SA and
cells, are described in the Supporting Information.
Synthesis of the ATRP initiator 1:
a) 4-Azido-2,3,5,6-tetrafluorobenzoic acid (2): To a stirred solution of
methyl pentafluorobenzoate (500 mg, 2.21 mmol, 1 equiv) in acetone
(30 mL) and water (10 mL) was added sodium azide (138 mg, 2.12 mmol,
0.96 equiv) at ambient temperature, and the reaction mixture was heated
at reflux for 8 h. The crude mixture was cooled down to room tempera-
ture, diluted with water (20 mL), and extracted with ethyl acetate (3ꢁ
50 mL). The combined organic layers were dried over MgSO4, filtered,
and concentrated to give 650 mg of yellow oil (4-azido-2,3,5,6-tetrafluoro-
benzoic acid methyl ester). To a stirred solution of 4-azido-2,3,5,6-tetra-
fluorobenzoic acid methyl ester (650 mg, 2.61 mmol) in methanol
(10 mL) and water (1 mL) was added aqueous NaOH solution (~1 mL,
20%), and the reaction mixture was stirred at ambient temperature over-
night. The reaction mixture was evaporated under reduced pressure and
then acidified with diluted aqueous HCl solution. The crude product was
extracted with dichloromethane (3ꢁ50 mL), dried over MgSO4, filtered,
and concentrated to give 554 mg of yellow solid in 90% yield. MS: calcd
m/z for [M+H]: 236.01; found [M+H]: 236.0.
SI-ATRP, the polyACHTUNGTRENNUNG(MPDSAH) pattern was visualized by
TRITC-streptavidin (Figure 4a): The relatively bright red
Figure 4. Pattern generation on COC surfaces: a) TRITC-streptavidin
and b) NIH 3T3 fibroblast cells. The scale bar is 100 mm.
b) 4-Azido-2,3,5,6-tetrafluorobenzoic acid 2,5-dioxo-pyrrolidin-1-yl ester
(3): To a stirred solution of compound 2 (554 mg, 2.36 mmol) in 10 mL
dichloromethane were added N-hydroxysuccinimide (NHS; 272 mg,
2.36 mmol, 1 equiv) and dicyclohexylcarbodiimide (497 mg, 2.41 mmol,
1.02 equiv) at ambient temperature. The reaction mixture was stirred at
ambient temperature overnight, and the resulting suspension was filtered
and washed with dichloromethane. The filtrate was evaporated under re-
duced pressure and purified by flash column chromatography (hexane/
ethyl acetate=5:1) to give 645 mg of white solid in 82% yield. 1H NMR
(500 MHz, CDCl3): d=2.92 ppm (s, 4H).
color was observed as a grid pattern that corresponded to
the area unexposed to the UV light. In other words, strepta-
vidin was adsorbed only onto the nonfunctionalized COC
surface, and polyACHTUNGTRENNUNG(MPDSAH) precluded the adsorption of
streptavidin effectively. After confirming the feasibility of
pattern generation by using the indirect photopatterning of
the compound 1 and SI-ATRP of MPDSAH, we initiated
the generation of cell patterns by the site-selective adsorp-
tion of fibronectin, which is known to enhance the attach-
c) 4-Azido-2,3,5,6-tetrafluoro-N-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-
benzamide (4): To a stirred solution of compound 3 (200 mg, 0.602 mmol,
1 equiv) and 2-(2-(2-aminoethoxy)ethoxy)ethanol (108 mg, 0.723 mmol,
1.2 equiv) in acetonitrile (30 mL) was added triethylamine (91 mg,
0.903 mmol, 1.5 equiv) at ambient temperature, and the reaction mixture
was stirred for 3 days. The crude mixture was diluted with water (30 mL)
and extracted with dichloromethane (2ꢁ50 mL). The combined organic
ment of cells, on the polyACTHNUTRGNE(UNG MPDSAH)-patterned COC sub-
strate. The protein, fibronectin, was adsorbed only onto the
areas of intact COC. NIH 3T3 fibroblast cells (5ꢁ105 cells)
Chem. Asian J. 2011, 6, 363 – 366
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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365

Y.-G. Kim, I. S. Choi et al.
COMMUNICATION
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quantitative). 1H NMR (500 MHz, CDCl3): d=3.59–3.68 (m, 12H),
2.01 ppm (s, 1H).
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Keywords: azides · atom-transfer radical polymerization ·
cyclic olefin copolymers · MPDSAH · photoreactions
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Received: August 14, 2010
Published online: October 4, 2010
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Chem. Asian J. 2011, 6, 363 – 366