A multilayered approach to complex surface patterning

Article abstract of DOI:10.1021/la902966b

A method for developing complex nanopattems on surfaces has been developed by combining self-assembly, photolabile protecting groups, and multilayered films. An o-nitrobenzyl protecting group has been incorporated into molecular level films utilizing thiol-gold interactions. When the o-nitrobenzyl group is cleaved by ultraviolet light, a carboxylic acid terminated layer remains on the surface and is available for activation and further functionalization through amide bond, formation. Using this method, multilayered films have been constructed and characterized by contact angle goniometry, cyclic voltammetry, grazing incidence infrared spectroscopy, and X-ray photoelectron spectroscopy measurements. Complex surface patterns can be achieved by creating a surface array using a photomask and then further functionalizing the irradiated area through covalent coupling. Fluorophores were attached to the deprotected regions, providing visual evidence of surface patterning using fluorescence microscopy. This approach is universal to bind moieties containing free amine groups at defined regions across a surface, allowing for the development of films with complex chemical and physicochemical properties.

Full text of DOI:10.1021/la902966b

pubs.acs.org/Langmuir
© 2009 American Chemical Society
A Multilayered Approach to Complex Surface Patterning
Peter F. Driscoll, Eftim Milkani, Christopher R. Lambert,* and W. Grant McGimpsey*
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609
Received August 10, 2009. Revised Manuscript Received September 15, 2009
A method for developing complex nanopatterns on surfaces has been developed by combining self-assembly,
photolabile protecting groups, and multilayered films. An o-nitrobenzyl protecting group has been incorporated into
molecular level films utilizing thiol-gold interactions. When the o-nitrobenzyl group is cleaved by ultraviolet light, a
carboxylic acid terminated layer remains on the surface and is available for activation and further functionalization
through amide bond formation. Using this method, multilayered films have been constructed and characterized by
contact angle goniometry, cyclic voltammetry, grazing incidence infrared spectroscopy, and X-ray photoelectron
spectroscopy measurements. Complex surface patterns can be achieved by creating a surface array using a photomask
and then further functionalizing the irradiated area through covalent coupling. Fluorophores were attached to the
deprotected regions, providing visual evidence of surface patterning using fluorescence microscopy. This approach is
universal to bind moieties containing free amine groups at defined regions across a surface, allowing for the development
of films with complex chemical and physicochemical properties.
Introduction
dip-pen nanolithography,10 or removal of the monolayer from
specific regions using high energy sources such as lasers11 and
electron beams.12 Other methods have used photoreactive com-
pounds deposited as SAMs to generate surface patterns. Zhao et al.
have demonstrated an approach to directing flow patterns inside
silica microchannels using a self-assembled monolayer fabricated
from a compound with a photolabile protecting group.13-16 Several
groups have used nitroveratryloxycarbonyl (NVOC) protecting
groups to generate quinone, oxyamine, and amine reactive surface
sites that can then be further functionalized.17-20 Although these
approaches have all successfully patterned surfaces, some draw-
backs include; nonreactivity of the patterned substrates toward
further functionalization, deprotection steps necessitating solvent to
facilitate the photoreaction or to limit competing reactions, and the
inability to further pattern surfaces to increase complexity.
Recent reports have focused on chemical reactions on mono-
layer modified surfaces.21,22 These descriptions of “reactions in
two dimensions” have demonstrated that monolayers possessing
reactive sites can be further functionalized, in a similar way to
compounds in solution. Specifically, the functionalization of
carboxylic acidsurfacesbyamide bondformation has beenshown
to be a facile way to add additional functionality to substrates that
first involves activation of the acid using carbodiimide chemistry,
Chemical surface functionalization and patterning can be
achieved using a top-down or a bottom-up method.1 In a top-
down approach a functionalized surface is patterned using an
external technique, such as electronbeam lithography, to generate
small features on the substrate. A bottom-up method requires
control over surface functionalization in a step-by-step process
utilizing the tendency of molecules to self-assemble into larger
supramolecular structures. Self-assembled monolayers (SAMs)
offer one way to modify surfaces using the latter approach.
Alkane-thiols are widely known to have a high affinity for gold,
and SAMs of thiols on gold have been extensively studied.2-6
Thiols on gold form tightly packed, highly ordered monolayers
with uniform surface properties.2-4 Additionally, these mono-
layers have been shown to be stable under a variety of conditions
including irradiation by ultraviolet light at wavelengths as short
as 254 nm.7,8 Tailoring the chemical and physical properties of the
compound used to form the monolayer allows the surface proper-
ties of the resulting thin film to be controlled, and more complex,
multicomponent films can be created by SAM patterning.
In order to increase complexity and functionality, techniques
have been developed to create chemically modified surfaces with
complex patterns that exhibit different properties in specific areas.
Approaches to generating surface patterns include depositing a
monolayer in specific regions using microcontact printing9 and
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(12) Pallandre, A.; Glinel, K.; Jonas, A. M.; Nysten, B. Nano Lett. 2004, 4, 365–
371.
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Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321–335.
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1995, 99, 7663–7676.
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Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152–7167.
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Langmuir 2010, 26(5), 3731–3738
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DOI: 10.1021/la902966b 3731

Article
Driscoll et al.
followed by exposure to a compound with a free amine group. The
resulting multilayered films retain properties of the monolayer
such as stability and functionality.
the substrate and imaged the resulting patterns with fluorescence
microscopy. Each modified substrate has also been fully char-
acterized to confirm the step-by-step multilayered film assembly
process.
Previous work in our laboratory has focused on using chemical
surface modification for applications such as controllable switch-
able wettability, photovoltaics, and selective ion sensing.23-27 The
utility of these types of systems can be significantly expanded by
the ability to selectively pattern a substrate and attach desired
moieties to defined regions of the same surface. In the work
described here, we have combined photopatterning with covalent
surface coupling reactions to create complex multicomponent
films. Our approach to multilayered film assembly allows a
surface to be irradiated and patterned multiple times to increase
complexity and functionality. Advantages of this method over
other types of SAM patterninginclude the following: the ability to
achieve complete deprotection of the photolabile group in air in
the absence of solvent, that the surface activation step makes the
patterned areas reactive toward any compound with a free amine
group, and that the multilayered films created are stable to further
patterning.
Patterned multilayered thin films have potential use in applica-
tions such as biological sensing systems, directed cell adhesion,
antimicrobial surfaces, and micro- and nanofluidics. For exam-
ple, generating a multianalyte sensing platform by attaching a
selective sensor to one portion of a deprotected surface (through
amide bond formation) and then repeating the process in other
areas produces substrates with regions that respond to different
analytes. Chemically modified surfaces that control protein
adhesion as well as cell attachment and growth have significant
utility in biomedical applications.28 Patterned monolayers that
promote cell adhesion in specific regions of a substrate and those
that inhibit adhesion in others have increased utility and allow for
the development of complex cellular networks. Photolabile head
groups also have the potential to generate sufficient surface
separation for multilayer film assembly. We have previously
demonstrated the use of mixed monolayers to fabricate films with
switchable wettability, as the mixed SAM was effective in appro-
priately spacing the reactive sites for multilayer attachment.24
However, it is well know that mixed SAMs form islands of like
molecules on a surface which would result in a nonhomogeneous
multilayer and limit the degree to which the wettability can be
altered. An alternative approach to preparing multilayered films
using protecting groups and covalent coupling is likely to provide
superior reactive site separation. Following removal of a bulky
protecting group, the reactive site spacing is increased compared
to that of a conventionally prepared monolayer, which would
allow for the attachment of subsequent functionalities that under-
go a conformational change and alter the properties of the
surface, such as wettability.
Experimental Details
Materials and Methods. All chemicals and solvents were
reagent grade or better and used as received. 2-nitrobenzyl
alcohol, 11-mercaptoundecanoic acid, 1,3-dicyclohexylcarbodii-
mide (DCC), and potassium ferricyanide were all from Aldrich
(Milwaukee, WI). 4-(dimethlyamino)pyridine (DMAP) was ob-
tained from Avocado Research Chemicals (Heysham, U.K.).
1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochlor-
ide (EDC) and N-hydroxysuccinimide (NHS) were from Alfa
Aesar (Ward Hill, MA). Rhodamine 110 was purchased from
Acros Organics (Geel, Belgium). Cresyl violet 670 was from
Exciton (Dayton, OH). Ethanol (200 proof, absolute, 99.98%)
for all experiments was obtained from Pharmco Products
˚
(Brookfield, CT). Silica gel (40 μm, 60 A) for column chroma-
tography was obtained from J.T. Baker (Phillipsburg, NJ).
Deionized water was used from a Millipore (Billerica, MA)
Synergy UV deionization system that produces water with a
resistivity of 18 MΩ.
NMR spectra were obtained on a Bruker (Billerica, MA)
Avance 400 MHz spectrometer and referenced to tetramethylsi-
lane (TMS). Spectra were recorded at 400 MHz for 1H and 100
MHz for 13C, and all chemicalshifts(δ) are reportedinppm. Mass
spectrometry was performed on a Waters (Milford, MA) Micro-
mass model ZMD spectrometer using electrospray ionization and
a 50:50 acetonitrile:water solvent flow. Attenuated total reflec-
tance (ATR) infrared spectroscopy experiments were carried out
on a Thermo Scientific (Waltham, MA) Nicolet FT-IR model
6700 spectrometer using a liquid nitrogen cooled, mercury cad-
mium telluride (MCT) detector. UV/visible spectra were recorded
on a Perkin-Elmer (Wellesley, MA) Lambda 35 UV/vis double
beam spectrometer, and a baseline correction (solvent vs. solvent)
was performed prior to each experiment. Fluorescence measure-
ments were performed on a Perkin-Elmer model LS50B lumines-
cence spectrometer. Transmission (bright-field) optical micro-
scopy was performed on a Fisher (Pittsburgh, PA) Micromaster
optical microscope with an integrated digital camera coupled to a
computer running Micron (v. 1.01) software.
Preparation of Self-Assembled Monolayers. Gold surfaces
were obtained commercially from Evaporated Metal Films
(EMF) (Ithaca, NY). The float glass slides (25 mm ꢀ 75 mm ꢀ
˚
1 mm) are coated with 50 A of a chromium adhesion layer
˚
followed by 1000 A of gold. Prior to monolayer formation, the
slides were cut to the desired size and cleaned by immersion in a
piranha solution (70% concentrated sulfuric acid, 30% concen-
trated hydrogen peroxide) at 90 °C for 10 min (Caution! piranha
reacts violently with organic compounds and should not be stored in
closed containers). The slides were then washed thoroughly with
distilled water, followed by absolute ethanol, and then dried in a
stream of nitrogen. The cleaned slides were immediately placed in
the monolayer solution (5 mM in ethanol) overnight. After
deposition, and prior to any characterization, the films were
removed from solution, rinsed with ethanol, and dried with
nitrogen. Characterization was carried out on freshly prepared
films.
Overall, the utility of the system presented is universal to
selectively attaching moieties with free amine groups to site
specific areas of a surface. To demonstrate the functionality of
our patterning method we have attached multiple fluorophores to
(23) Driscoll, P. F.; Douglass, E. F., Jr; Phewluangdee, M.; Soto, E. R.; Cooper,
C. G. F.; MacDonald, J. C.; Lambert, C. R.; McGimpsey, W. G. Langmuir 2008,
24, 5140–5145.
(24) Driscoll, P. F.; Purohit, N.; Wanichacheva, N.; Lambert, C. R.; McGimp-
sey, W. G. Langmuir 2007, 23, 13181–13187.
(25) Cooper, C. G.; MacDonald, J. C.; Soto, E.; McGimpsey, W. G. J. Am.
Chem. Soc. 2004, 126, 1032–1033.
(26) Soto, E.; MacDonald, J. C.; Cooper, C. G.; McGimpsey, W. G. J. Am.
Chem. Soc. 2003, 125, 2838–2839.
(27) Wanichacheva, N.; Soto, E. R.; Lambert, C. R.; McGimpsey, W. G. Anal.
Chem. 2006, 78, 7132–7137.
Photodeprotection. Removal of the photolabile protecting
group was accomplished by exposing the slides to ultraviolet light
in a Rayonet reactor. The lamp used was a mercury arc lamp with
a maximum distribution of light centered at 300 nm (an emission
spectrumofthelampisprovidedasSupportingInformation). The
slides were placed in deionized water and irradiated for 2 h. For
photomask experiments the irradiation was accomplished by
tightly clamping the photomask to the substrate to limit the effect
of diffraction using a custom built clamping fixture. No solvent
was used for patterning experiments.
(28) Bush, K. A.; Driscoll, P. F.; Soto, E. R.; Lambert, C. R.; McGimpsey,
W. G.; Pins, G. D. J. Biomed. Mater. Res. A 2009, 90A, 999–1009.
3732 DOI: 10.1021/la902966b
Langmuir 2010, 26(5), 3731–3738

Driscoll et al.
Article
Photomasks. Custom fabricated photomasks were obtained
fromAdtek Photomask (Montreal, Canada). The specified design
and dimensions were printed on soda lime glass (300 ꢀ 300 ꢀ 0.0600)
using a chrome coating. The masks were rinsed thoroughly with
distilled water and ethanol after each use.
Fluorescence Microscopy. Patterned surfaces were examined
byfluorescencemicroscopy. Fluorescent imageswere obtainedon
a Nikon (Melville, NY) Eclipse model E600 fluorescence micro-
scope equipped with a Diagnostic Instruments (Sterling Heights,
MI) RT Color digital camera and Spot (v. 4.0) analysis software.
Samples were observed using a Nikon Plan Fluor 10X (N = 0.50)
Ph1 DLL lens. Illumination was provided by a 100 W mercury arc
lamp (Chiu Technical Corporation, Kings Park, NY) passed
through either a Nikon FITC-HYQ (excitation filter, 460-
500 nm; dichroic mirror cut on, 505 nm; barrier filter, 510-560
nm) filter cube for green fluorescence or a Nikon Texas Red HQ
(excitation filter, 532-587 nm; dichroic mirror cut on, 595 nm;
barrier filter, 608-683 nm) filter cube for red fluorescence.
Samples were observed in a darkened room to reduce stray light.
Patterned surfaces were prepared for microscopy analysis using a
method to remove the pattern from the surface and eliminate
electron transfer fluorescence quenching by the gold substrates.
Slides were coated with strips of Scotch brand Transparent tape
from 3M (St. Paul, MN) and incubated at 80 °C for 5 min to
facilitate pattern transfer. The tape was carefully removed from
the surface and adhered to a standard glass microscope slide for
analysis and imaging.
Surface Activation and Multilayered Film Assembly.
Irradiated substrates were exposed to a freshly prepared solu-
tion of 0.1 M EDC and 0.02 M NHS in deionized water for 30 min
while agitating with a Thermo Scientific (Waltham, MA)
Barnstead/Lab-line lab rotator on low speed to facilitate the
reaction. Following activation, the substrates were rinsed with
deionized water, dried with nitrogen, and placed in a 0.01 M
ethanolic solution of a fluorescent compound (rhodamine 110 or
cresyl violet 670) for 10 min (with agitation) to complete the
surface reaction. Longer exposure times resulted in nonspecific
attachmentofthe fluorophoretothesubstrate, which wasdifficult
to completely remove by rinsing with solvent. After amide bond
formation, the samples were thoroughly rinsed with ethanol and
dried with nitrogen. Patterned surfaces were shielded from light
during this process to protect the photolabile group.
Contact Angle Goniometry. Sessile drop contact angle mea-
surements were made using a Rame-Hart Model 300 Goniometer
(Netcong, NJ). Measurements were obtained using 1 μL drops of
deionized water deposited on the substrates using the Automated
Dispensing System accessory coupled to the goniometer. Images
were obtained by an integrated digital camera and the entire
system was under computer control using Rame-Hart’s DROP-
image Standard (v. 2.0.10) software package. The software auto-
matically provides contact angle measurements once the liquid is
dispensed. Five measurements were taken per substrate for five
different samples (25 total measurements for each type of modi-
fied surface) and the results were averaged.
Infrared Spectroscopy. Grazing incidence infrared spectra
were obtained using a Thermo Electron (Waltham, MA) Nicolet
FT-IR model 6700 spectrometer equipped with a Thermo Nico-
let grazing angle accessory and a liquid nitrogen cooled mercury
cadmium telluride (MCT) detector. The incident IR beam was at
75° to the gold substrates. Prior to measurement the optical path
was purged with a stream of nitrogen for 30 min, and purging was
continued during the experiments. A clean, bare gold slide was
used as the background, and a new background was collected
immediately prior to each sample run. The scan range was from
4000 to 600 cm-1, and 64 scans were collected for each sample.
The spectra were automatically corrected for H2O and CO2, and a
manual baseline correction was performed after each experiment.
Cyclic Voltammetry (CV). Electrochemistry experiments
were carried out using a Gamry Instruments (Warminster, PA)
Reference 600 potentiostat/galvanostat/ZRA. A typical three-
electrode cell was employed with the SAM functioning as the
working electrode, a platinum wire as the counter electrode, and
referenced against a standard saturated calomel electrode (SCE). An
alligator clip was used to attach the gold slide to the cell, and a surface
area of 1 cm2 was placed in the electrolyte solution. The electrolyte
was an aqueous 1 mM potassium ferricyanide solution with 0.1 M
potassium chloride as supporting electrolyte. Voltammograms were
obtained from -0.3 to þ0.7 V at a scan rate of 50 mV/s.
Synthetic Details. 2-Nitrobenzyl-11-mercaptoundecanoate
(Photolabile Compound). One equivalent of 2-nitrobenzyl
alcohol (2.17 g, 14.15 mmol) was combined with 1 equiv of 11-
mercaptoundecanoic acid (3.09 g, 14.15 mmol) and 0.1 equiv of
DMAP (0.173 g, 1.42 mmol) and dissolved in dichloromethane
(50 mL). To this mixture was slowly added a solution of DCC
(2.92 g, 14.15 mmol) in dichloromethane (30 mL) while stirring.
Upon addition, a white precipitate formed after a few minutes.
The mixture was stirred at room temperature overnight. Vacuum
filtration removed the white precipitate and the filtrate was
concentrated under vacuum. Silica gel column chromatography
was employed for purification using dichloromethane/methanol
(v/v 50:1) as eluent. The product was dried over sodium sulfate,
the solvent removed by rotary evaporation, and dried under
vacuum, producing a yellow solid. Yield: 3.57 g (71%). 1H
NMR (CDCl3), δ (ppm): 8.1 (d, 1H, Ar), 7.6 (t, 1H, Ar), 7.5
(d, 1H, Ar), 7.4 (t, 1H, Ar), 5.5 (s, 2H, O-CH2), 2.6 (t, 1H, SH) 2.5
(q, 2H, CH2-SH), 2.4 (t, 2H, CH2-CdO), 1.6 (m, 4H, CH2), 1.2
(m, 12H, CH2). 13C NMR (CDCl3), δ (ppm): 173.5, 147.0, 134.1,
132.7, 129.5, 129.2, 125.5, 63.2, 34.6, 34.4, 29.8, 29.7, 29.6, 29.5,
29.4, 28.8, 25.3, 25.1. MS (ESI): (M þ Na)þ = 376.19 (calcd
376.47).
Results and Discussion
An
alkanethiol,
2-nitrobenzyl-11-mercaptoundecanoate
(photolabile compound), with an o-nitrobenzyl protecting group
was synthesized and used as the base for multilayered film
assembly. o-Nitrobenzyl moieties are well-known as photolabile
protecting groups that readily undergo Norrish-type II reactions
when excited with ultraviolet light29,30 (absorption measurements
of the photolabile compound in solution before and after irradia-
tion are provided as Supporting Information). An accepted
mechanism for a Norrish-type II photocleavage involves the
excited nitro group abstracting a proton from the methylene
carbon on the aromatic ring forming a radical, the radical is
resonance stabilized by a five-membered ring intermediate which
rapidly decomposes to an aldehyde and a carboxylic acid.29 The
structure and expected photoproduct of the photolabile com-
pound on a surface is shown in Figure 1 (we note that this is an
idealized representation for clarity and not a depiction of the
molecular arrangement onthe surface). Following deprotection, a
carboxylic acid terminated layer will be exposed to the surface,
X-ray Photoelectron Spectroscopy (XPS). XPS analysis
was performed by Anderson Materials Evaluation, Inc.
(Columbia, MD). Spectra were collected on a Surface Science
Instruments SX-100 X-ray photoelectron spectrometer equipped
with a monochromatic Al KR X-ray source (1487 eV), a wide-
angle input lens, a hemispherical analyzer, and a multichannel
detector. AnellipticalX-ray beammeasuring 1.2 by0.6 mmonthe
major and minor axes was used. Spectra were collected from 0 to
1100eVata stepsizeof0.5 eVusing 32scans ofthe energyrange, a
neutralizer electron beam energy of 2.4 eV, and a dwell time of 100
ms at each step. The analysis chamber was at less than 9 ꢀ 10-10
Torr during data collection. The C 1s primary component binding
energy was set to 285.00 eV (that of unsaturated hydrocarbons)
and all other binding energies were adjusted accordingly.
(29) Bochet, C. G. J. Chem. Soc., Perkin Trans. 1 2002, 125–142.
(30) Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92,
6333–6335.
Langmuir 2010, 26(5), 3731–3738
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Article
Driscoll et al.
in solution.31-37 CVs of the protected and deprotected mono-
layers are provided as Supporting Information along with that of
a bare gold surface for comparison. When the bare gold surface is
used as the working electrode the expected redox of the ferricya-
nide in solution is readily observed. After SAM deposition the
monolayer blocks electron penetration to the surface and the
characteristic anodic and cathodic redox peaks of ferricyanide are
no longer seen. The attenuated current in the CV is indicative of a
well ordered, insulating monolayer on the surface. Following
irradiation the surface remains blocking (no current is seen for the
redox of ferricyanide), indicating that the exposure to ultraviolet
light had not destroyed or removed the monolayer.
XPS analysis of samples following monolayer deposition and
after irradiation with 300 nm light confirms successful SAM
formation of the photolabile compound and cleavage of the
protecting group after UV exposure. A summary of the elements
detected based on their electron orbital designation, binding
energy, and atomic percentages for each element in the SAM is
provided in Table 1 (complete XPS spectra are shown in the
Supporting Information). The elements observed are expected for
a SAM of the photolabile compound on gold with the exception
of trace amounts of Sn and I, which arise from the substrate
preparation process and are also seen in an XPS spectrum of the
bare substrate (see Supporting Information). From the XPS data,
a molecular formula for the monolayer of C18O4.8N1.2S0.6 (not
accounting for hydrogen) is obtained for the monolayer and is
consistent with the expected formula from the structure of the
photolabile compound of C18O4NS. The differences in the
measured and expected molecular formulas are because of the
depth of the individual elements in the film, electrons from deeper
in the film are more likely to have inelastic collisions and lose
energy prior to reaching the detector, consequently attenuating
their signals. XPS analysis indicates that the film is oxygen and
nitrogen rich and sulfur deficient, which is consistent with the
SAM being bonded to the gold through the sulfur and the rest of
the molecule oriented away from the surface, terminating with the
o-nitrobenzyl group. Additionally the binding energy for the
nitrogen 1s signal at 406.37 eV indicates that the nitrogen is
present as an organic nitro group.
After exposing the monolayer to UV light samples were re-
examined by XPS to confirm successful removal of the protecting
group. A summary of the elements detected, binding energy, and
atomic percentages for each element in the irradiated SAM is
provided in Table 2 (complete XPS spectra are shown in the
Supporting Information). It is evident from the XPS data that the
irradiation removed the o-nitrobenzyl protecting group from the
monolayer. A complete absence of nitrogen is observed in the
XPS spectrum of the irradiated SAM, and also the atomic
percentages of carbon and oxygen have decreased compared to
the monolayer, as expected due to the cleavage of the protecting
group as a nitrobenzyl aldehyde.
Figure 1. Structure and irradiation of the photolabile compound
on a surface.
providing a suitable reactive site for further functionalization.
Additionally, the surface can be deprotected in a specific pattern
leaving defined areas available for covalent coupling.
Successful SAM formation and photodeprotection was con-
firmed by contact angle, grazing incidence IR, cyclic voltammetry
and XPS measurements. It is expected that a portion of the benzyl
ring will be exposed to the surface and it will therefore be
hydrophobic. SAMs of the photolabile compound produced a
sessile water droplet contact angle of 72 ( 2.0°, as expected of a
hydrophobic terminal nitrobenzyl moiety. After irradiation and
removal of the protecting group, the contact angle of the
surface decreases to 16 ( 3.0°, consistent with an acid terminus
on the surface. Images of water droplets on a surface chemically
modified with the photolabile compound before and after irradia-
tion are shown in Figure 2. A significant increase in the wettability
of the surface is observed in Figure 2 (right) as compared to
Figure 2 (left).
Grazing incidence infrared spectra of the protected (top) and
deprotected (bottom) monolayer are shown in Figure 3. The
major IR absorption frequencies of the SAM are similar in
position to that of a solid sample of the photolabile compound
examined by ATR (see Supporting Information), indicating
successful monolayer deposition. Additionally the CH2 stretching
signals at 2920 and 2851 cm-1 are indicative of a crystalline
arrangement of alkyl chains.6 Removal of the protecting group
following irradiation is confirmed by examining the signal at 1534
cm-1, which is consistent with a nitro group. As shown in the
bottom spectrum of Figure 3, this signal disappears after irradia-
tion, indicating that the o-nitrobenzyl group has been cleaved.
The carbonyl stretching frequency shifts from 1744 to 1715 cm-1
after irradiation which is consistent with the original ester being
converted to a carboxylic acid. Stretching frequencies for the alkyl
chain are unchanged.
Following complete characterization of the chemically mod-
ified surfaces described above, SAMs of the photolabile com-
pound were irradiated through a photomask to afford substrates
that could be further functionalized in specific patterns. A SAM-
modified substrate was patterned by clamping a photomask (100
μm ꢀ 100 μm squares) to the surface, to limit diffraction, and
irradiating at 300 nm through the mask. The minimum feature
Cyclic voltammetry has been widely used to determine the
blocking or nonblocking nature of monolayers to a redox process
(35) Richardson, J. N.; Peck, S. R.; Curtin, L. S.; Tender, L. M.; Terrill, R. H.;
Carter, M. T.; Murray, R. W.; Rowe, G. K.; Creager, S. E. J. Phys. Chem. 1995, 99,
766–772.
(36) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E. D.; Linford, M. R.; Newton,
M. D.; Liu, Y.-P. J. Phys. Chem. 1995, 99, 13141–13149.
(37) Weber, K.; Creager, S. E. Anal. Chem. 1994, 66, 3164–3172.
(31) Alleman, K. S.; Weber, K.; Creager, S. E. J. Phys. Chem. 1996, 100, 17050–
17058.
(32) Cheng, Q.; Brajter-Toth, A. Anal. Chem. 1992, 64, 1998–2000.
(33) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682–691.
(34) Miller, C.; Cuendet, P.; Graetzel, M. J. Phys. Chem. 1991, 95, 877–886.
3734 DOI: 10.1021/la902966b
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Driscoll et al.
Article
Figure 2. Images of water droplets on a surface modified with the photolabile compound before (left) and after (right) irradiation and
removal of the protecting group.
Table 1. Summary of XPS Data for a SAM of the Photolabile
Compound
XPS line
BE (eV)
atom %
C 1s
Au 4f
S 2p
Sn 3d
O 1s
I 3d5
N 1s
285.00
84.12
60.69
16.49
2.02
163.20
487.08
532.97
618.07
406.37
0.41
16.21
0.15
4.03
Table 2. Summary of XPS Data for a SAM of the Photolabile
Compound Following Irradiation
XPS line
BE (eV)
atom %
C 1s
Au 4f
S 2p
Sn 3d
O 1s
I 3d5
285.00
84.31
162.81
487.00
532.87
619.10
56.59
28.00
2.76
0.43
12.05
0.17
Figure 3. Grazing incidence IR of the photolabile compound on a
surface before (top) and after (bottom) irradiation and removal of
the protecting group.
size on the mask is 100 μm to demonstrate the feasibility of the
technique and also to minimize distortion of the pattern due to
diffraction. Using a microscopic projection system or byclamping
a flexible mask to the substrate under vacuum, a significantly
higher resolution of micrometers or less would be expected.38,39
After irradiation the resulting surface pattern can be readily
observed as the protected and deprotected regions offer a drastic
difference in wettability that can be seen by applying droplets of
liquid to the surface. An optical micrograph of the pattern is
shown in Figure 4, the ethanol droplets are centered in the
irradiated areas of the surface and the distance between droplet
centers is approximately 200 μm, consistent with the dimensions
of the mask.
A primary reason for the choice of photolabile compound for
this study is that removal of the protecting group reveals a
carboxylic acid which provides a surface site for further functio-
nalization. SAMs with carboxylic acid terminal groups can be
activated with EDC/NHS toward amide bond formation.21,22
Following activation, exposure to a solution of a compound with
a free amino group will result in a covalent coupling on the
surface. The mechanism for amide bond formation using EDC/
NHS activation on a surface is shown in Figure 5. Following
substrate exposure EDC first reacts with the free carboxylate to
form an unstable amine-reactive intermediate which then under-
goes nucleophilic substitution with NHS to form a semistable
amine-reactive NHS ester. The NHS ester is then exposed to a
solution of a free amino compound (R-NH2) which displaces the
NHS moiety by nucleophilic substitution, forming the desired
amide bond. If the irradiation step was done through a photomask,
Figure 4. Optical micrograph of ethanol droplets on a surface
modified with a SAM of the photolabile compound after irradia-
tion at 300 nm through a 100 μm square photomask (shown in
reduced scale in inset). The scale bar represents 200 μm.
the resulting surface will have both reactive and nonreactive sites,
allowing a multilayer to be deposited in site-specific areas of the
substrate.
Successful surface activation was confirmed by characteriza-
tion on slides that had been irradiated completely (no photomask
used) and treated with EDC/NHS. The amine-reactive NHS ester
is stable in air for several hours, allowing for characterization by
the same methods used for the monolayer. EDC/NHS activated
surfaces demonstrated an expected increase in hydropho-
bicity, resulting in a contact angle of 44.8 ( 2.0°. This value is
(38) Jones, S. W. Photolithography; IC Knowledge LLC: Georgetown, MA, 2000.
(39) Rogers, J. A.; Paul, K. E.; Jackman, R. J.; Whitesides, G. M. Appl. Phys.
Lett. 1997, 70, 2658–2660.
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Driscoll et al.
Figure 5. Mechanism for surface amide bond formation.
completely (no photomask), activated with EDC/NHS, and
exposed to solutions of the fluorophores to form the desired
amide bond and multilayered film. Contact angles for rhodamine
110 functionalized surfaces were measured to be 48.4 ( 2.5°, and
contact angles for cresyl violet 670 were found to be 49.2 ( 3.0°.
Comparable values for each film are expected since the each
fluorophore has a similar structure, and the angles measured are
analogous to those measured for monolayers with an amino
terminated functionality (46 ( 2.0°). The formation of an amide
bond at one end of each fluorophore would result in the free
amino group at the other end being exposed to the surface.
Grazing incidence IR also confirms the presence of the fluor-
ophores onthemonolayer functionalized substrate. IRspectra for
the activated monolayer exposed to both rhodamine 110 and
cresyl violet 670 are provided as Supporting Infromation. Cyclic
voltammetry demonstrated that an insulating film is present on
the gold surface. Voltammograms of each functionalized surface
show blocking behavior, as expected if a well ordered layer is still
present (see Supporting Information).
Following irradiation of the monolayer with a photomask (100
μm ꢀ 100 μm squares or 100 μm lines separated by 150 μm), the
patterned surface was activated with EDC/NHS, followed by
exposure to one of the fluorophores. The free amino group of the
fluorophore reacts with the activated carboxylic acid and an
amide bond is formed on the surface in the desired pattern.
Metals, including gold, are know to quench fluorescence by an
electron transfer process,40,41 and direct observation of the
patterned surfaces by darkfield microscopy resulted in no fluor-
escence being detected on the patterned surfaces. A technique was
devised to remove the pattern from the gold surface so that
observation was possible and images could be obtained. Trans-
parent Scotch tape was carefully applied to the surface, allowed to
incubate at elevated temperature to facilitate transfer, and then
removed and adhered to a glass slide for observation. This lift-off
technique effectively transferred the fluorescent pattern from the
gold surface to eliminate the electron transfer fluorescence
quenching by the metal. Patterns were then observed with a
fluorescence microscope using the appropriate filters for each
fluorophore, and images of square and line arrays of each
fluorophore are shown in Figure 7.
Figure 6. Chemical structures of rhodamine 110 (left) and cresyl
violet 670 (right).
comparable to measurements taken on other carboxylic acid
terminated films activated with EDC/NHS and the decrease in
wettability is expected if the carboxylate terminated surface has
been replaced with a NHS ester functionality. Grazing incidence
IR shows the presence of NHS on the activated surface and also
confirms that treatment with EDC/NHS does not affect the
unirradiated surface, which is vital for further surface patterning.
IR spectra and analysis are provided as Supporting Infromation.
Cyclic voltammetry of NHS activated surfaces confirms that a
well ordered SAM remains on the substrate after EDC/NHS
exposure; the substrates remain blocking to a redox process in
solution (see Supporting Information).
In order to demonstrate the ability of our method to easily
generate substrates with defined areas of functionalization,
fluorescent compounds were attached to the activated surface
sites to provide visual evidence of multilayered pattern formation.
Two commercially available fluorophores were chosen to attach
to the patterned surfaces, rhodamine 110 (6-amino-9-(2-carbo-
xyphenyl)-3H-xanthen-3-iminium chloride), and cresyl violet 670
(9-amino-5H-benzo[a]phenoxazin-5-iminium perchlorate). These
fluorophores were selected because they both have free amino
groups for the necessary surface reaction. Chemical structures of
rhodamine 110 and cresyl violet 670 are shown in Figure 6; both
fluorophores are similar in size and offer two possible sites for
amide bond formation. Additionally, they are fluorescent in
different areas of the spectrum (rhodamine 110 in the green,
and cresyl violet 670 in the red), and can be used to provide
distinct visual evidence of multilayered film assembly in the
irradiated pattern. Absorption and fluorescence emission spectra
for the two selected fluorophores in solution (EtOH) are provided
as Supporting Information. It is noted that the maximum emis-
sion intensity differs by more than 100 nm and the majority of the
emission for each fluorophore lies within the appropriate band-pass
filter, also the absorbance of each fluorophore matches correctly
with the excitation profile of each of the respective filter cubes.
Successful amide bond formation and fluorescent ligand at-
tachment was confirmed on substrates that had been irradiated
It is noted that control experiments including; the initial SAM,
the irradiated SAM, EDC/NHS activated surfaces, and surfaces
exposed to fluorophores with no EDC/NHS activation, imaged
(40) Li, L.; Ruzgas, T.; Gaigalas, A. K. Langmuir 1999, 15, 6358–6363.
(41) Waldeck, D. H.; Alivisatos, A. P.; Harris, C. B. Surf. Sci. 1985, 158, 103–
125.
3736 DOI: 10.1021/la902966b
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Driscoll et al.
Article
Figure 7. Fluorescence microscopy images of surfaces patterned with 100 μm squares (top) and 100 μm lines separated by 150 μm spaces
(bottom), images on the left are of rhodamine 110 (green) and images on the right are cresyl violet 670 (red). Inset: reduced scale images of the
photomasks used to pattern the surfaces.
Further surface patterning can be carried out on these sub-
strates. By taking the initially patterned surface, as described
above, and reirradiating after rotating the photomask 45° to the
original, a second array of squares can be added to the surface.
The surface is once again activated with EDC/NHS and exposed
to the fluorophore. Imaging using the same method as described
previously reveals a second array of squares at 45° to the original.
Additional patterning experiments also demonstrated the ability
to put multiple fluorophores on the same substrate. In this case a
SAM was deposited, deprotected with a square array mask,
activated and exposed to rhodamine 110, then the substrate was
reirradiated (with the photomask at 45° to the original), activated
and exposed to cresyl violet 670. Fluorescence microscopy de-
monstrated that a two-colored dual array was created on the
surface. Observation with a green emission filter reveals the initial
pattern of the rhodamine dye, and observation in the same
location with a red emission filter shows a diagonal pattern of
the cresyl violet dye, a combined red/green image of the patterned
surface is shown in Figure 8. This dual patterned array demon-
strates the ability of this multilayered approach for site selective
surface patterning.
Figure 8. Combined red/green image of a dual patterned surface.
Conclusions
using the same methods, result in no patterns being observed,
indicating the fluorophore is coupled to the surface through the
generated amide bond between the carboxyl group of the film and
the free amino group of the fluorophore. Additionally, patterned
surfaces were treated by sonnication in ethanol, washing
with dilute acid and base (0.01 M HCl and 0.01 M NaOH), salt
(0.1 M AlCl3), and detergent (0.1 M sodium dodecyl sulfate).
Patterns are still observed after these surface treatments, support-
ing the formation of a covalently coupled, stable multilayered
surface.
A method for fabricating complex patterned surfaces has been
developed by combining photopatterning and covalent interac-
tions. The removal of a photolabile portion of a self-assembled
monolayer exposes a suitable reactive site which allows for the
addition of subsequent layers through the use of amide bond
formation. We have demonstrated this method of multilayered
surface patterning by incorporating fluorophores into the assem-
bly structure, allowing for pattern observation and imaging using
fluorescence microscopy. Further patterning has shown that the
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Driscoll et al.
initial pattern is stable and that the process can be repeated in
other regions of the surface. The multilayered assembly process
described has the ability to generate complex, multicomponent
surfaces with site-specific areas of chemical functionality
with potential utility in biological sensors, directed cell adhesion,
switchable surface wettability, and other nanoscale surface
applications.
No. W81XWH-07-2-0106. The views, opinions, and/or findings
contained in this report are those of the author(s) and should not
be construed as an official Department of the Army position,
policy, or decision unless so designated by other documentation.
Supporting Information Available: Text and figures de-
scribing the complete characterization data including an
emission profile of the lamp used for patterning experiments,
solution absorbance and emission studies of the photolabile
compound and the fluorophores, complete XPS spectra, and
additional IR and CV data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Acknowledgment. The authors gratefully acknowledge
the financial support of the Telemedicine and Advanced Tech-
nology Research Center (TATRC) at the U.S. Army Medical
Research and Materiel Command (USAMRMC) under Award
3738 DOI: 10.1021/la902966b
Langmuir 2010, 26(5), 3731–3738