Photochemical Micropatterning of Carbohydrates on a Surface

Photochemical Micropatterning of Carbohydrates on a Surface
Gregory T. Carroll,† Denong Wang,‡ Nicholas J. Turro,†,§ and Jeffrey T. Koberstein*,§
Department of Chemistry, Columbia UniVersity, 3000 Broadway, MC 3157, 10027, New York, New York,
Carbohydrate Microarray Laboratory, Departments of Genetics, Neurology, and Neurological Sciences,
Stanford UniVersity School of Medicine, Beckman Center B007, 94305, Stanford, California, and
Department of Chemical Engineering, Columbia UniVersity, 500 West 120th Street, 10027,
New York, New York 10027
ReceiVed NoVember 16, 2005. In Final Form: January 9, 2006
In this report, we demonstrate a versatile method for the immobilization and patterning of unmodified carbohydrates
onto glass substrates. The method employs a novel self-assembled monolayer to present photoactive phthalimide
chromophores at the air-monolayer interface. Upon exposure to UV radiation, the phthalimide end-groups graft to
surface-adsorbed carbohydrates, presumably by a hydrogen abstraction mechanism followed by radical recombination
to form a covalent bond. Immobilized carbohydrate thin films are evidenced by fluorescence, ellipsometry and contact-
angle measurements. Surface micropatterns of mono-, oligo-, and polysaccharides are generated by exposure through
a contact photomask and are visualized by condensing water onto the surface. The efficiency of covalent coupling
is dependent on the thermodynamic state of the surface. The amount of surface-grafted carbohydrate is enhanced when
carbohydrate surface interactions are increased by the incorporation of amine-terminated molecules into the monolayer.
Glass substrates modified with mixed monolayers of this nature are used to construct carbohydrate microarrays by
spotting the carbohydrates with a robot and subsequently illuminating them with UV light to covalently link the
carbohydrates.Surface-immobilizedpolysaccharidesdisplaywell-definedantigenicdeterminantsforantibodyrecognition.
We demonstrate, therefore, that this novel technology combines the ability to create carbohydrate microarrays using
the current state-of-the-art technology of robotic microspotting and the ability to control the shape of immobilized
carbohydrate patterns with a spatial resolution defined by the UV wavelength and a shape defined by a photomask.
Introduction
Carbohydrates, like nucleic acids and proteins, carry important
biological information. The development of high-throughput
technologies for generating DNA and protein microarrays has
been vigorously explored and has contributed greatly to the fields
of genomics and proteomics. A newer field that explores the
information content of carbohydrates, called glycomics, has
recently emerged and has been facilitated by the relatively recent
development of carbohydrate microarrays.1-10 Already, carbo-
hydrate microarrays have been used to investigate the SARS6
and HIV7 viruses. In addition, enzyme activity,3,11 glycome
sequencing,2 and carbohydrate interactions with cells,12,13
antibodies,1 and proteins4,14 have been studied with carbohydrate
microarrays.Immobilizingcarbohydratesonsurfaceshasbecome
a major preliminary challenge in the area. Most current methods
involve either a noncovalent immobilization that becomes less
stable as the molecular weight (MW) decreases, or synthetic
methods in which each carbohydrate to be spotted must first be
chemically modified. To develop a simple and universal approach
to carbohydrate microarray fabrication, it is very important to
devise methods that allow for covalent immobilization of
carbohydrates on a surface without prior chemical derivatization.
Only a few methods have been reported that demonstrate this
goal. Underivatized carbohydrates have been covalently attached
to monolayers bearing phenylboronic acid groups,15 polysac-
charide films bearing diazirine groups,16 and hydrazide-coated
glass slides.17 Only the latter two methods were used to construct
microarrays.
Carbohydrates also contain important physical and chemical
propertiesthatmayfindutilityinbiotechnologyandnoveldevices.
Surface-immobilized carbohydrates are potential components in
biological sensors,18 scaffolds for tissue engineering,19 templates
forstudyingcellbehaviorinaconfinedspace,20 suprabiomolecular
structures on surfaces,21 host-guest complexes on a surface,
* Corresponding author.
† Department of Chemistry, Columbia University.
‡ Stanford University School of Medicine.
§ Department of Chemical Engineering, Columbia University.
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10.1021/la0531042 CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/15/2006

and three-dimensional biochips.22 It is desirable to have spatial
control over surface immobilization when incorporating carbo-
hydrates into many of these devices. One widely accessible
approach involves pattern transfer via a photomask or stamp.
Although patterning biological materials on surfaces using these
versatile approaches has been demonstrated,23,24 there are
relatively few reports focusing on carbohydrates.25
Irradiating photoactive surfaces in the presence of a photo-
mask26 is a well-known technology that employs photons as
traceless reagents for pattern formation without the use of a
spotter, microstamps, or an atomic force microscope and has
been used to control the spatial deposition of various materials
including proteins,27 DNA,28 cells,29 and colloidal and nano-
particles.30 The resolution of such patterns is controlled by the
size of the illumination pattern and is not dependent on the size
of the drop placed on the surface by a spotter. Such features
allow greater control over the size and shape of a micron-sized
architecture. As the size of such patterns continues to decrease,
scaffolds of individual macromolecules could be produced,
allowing for interactions at the single-molecule or few-molecules
level to be interrogated. In addition, reducing the size of the
pattern reduces the amount of material consumed.
In this report, we demonstrate a versatile method for covalent
immobilization and patterning of unmodified mono-, oligo-, and
polysaccharides onto glass substrates. This technology involves
self-assembly of a new class of photoactive monolayers onto
glass substrates. The monolayers present phthalimide chromo-
phores31 at the surface that, upon exposure to light, graft surface
adsorbed carbohydrates by hydrogen abstraction followed by
radical recombination. Using a robotic spotter, we are able to
generate a microarray of carbohydrates and demonstrate high-
throughput characterization of antigen-antibody interactions.
The surface-immobilized carbohydrates retain their immunologi-
calproperties.Incomparisonwithnitrocellulose-coatedsubstrates,
an established technology for carbohydrate microarrays,1,2,6,32
this novel approach is much less dependent on the MW of the
spotted carbohydrates and shows a higher grafting efficiency for
lower MWs. The photochemical patterning method described
herein requires no chemical modification of the sugars prior to
deposition, is applicable for carbohydrates of different MWs,
requires no chemical reagents for covalent coupling of carbo-
hydrates on the surfaces, and uses existing microspotting devices
for high-throughput microarray construction. The methodology
we present has potential applications in materials, biological,
and medical research.
Results and Discussion
To covalently link carbohydrates to a surface without prior
derivatization, we created self-assembled monolayers (SAMs)
containing aromatic carbonyls that can react with C-H groups
upon absorption of a photon to form a covalent bond.33
Phthalimide derivatives can undergo all the major photochemical
reactions of aromatic carbonyls.31 Exposure to UV light produces
an excited n-π* state that can abstract a hydrogen atom from a
nearby molecule. The resulting radicals can then recombine,
forming a covalent bond as shown in Figure 1. Other secondary
processes are also possible, including disproportionation and
back-transfer.CarbohydratesubstratescanundergopH-dependent
and independent rearrangements, depending on the structure of
thecarbohydrate.34 Facileincorporationofpotassiumphthalimide
into bromine-terminated silanes allows for self-assembly on
silicon, glass, or quartz substrates. Terminal groups other than
silanes could readily be employed in the synthesis to create
phthalimides for self-assembly onto other substrates.
To create a novel surface suitable for immobilizing carbo-
hydrates, a phthalimide-derivatized silane was synthesized in
one step by reacting 11-bromoundecanetrimethoxy silane with
potassium phthalimide in dimethylformamide (DMF) to produce
11-phthalimidoundecanetrimethoxy silane (compound 1). Com-
pound 1 was self-assembled on silicon, glass, and quartz in
anhydrous toluene to produce SAM 1 as shown in Figure 2. The
self-assembly of compound 1 on the surface was verified by
UV/Visible (UV-vis) spectroscopy as shown in Figure 3. Under
the rough assumption that the extinction coefficient of the
chromophore on the surface is the same as that in solution, the
approximate surface coverage was calculated to be 5.5 molecules/
nm2.35 A rough calculation using Chem.3D suggests that about
4.9 aliphatic phthalimides can fit in a space of 1 nm2, a value
that is the same order of magnitude as the experimental value,
suggesting that SAM 1 is densely packed. In addition, an H2O
contact angle of 65 (1° and an ellipsometric thickness of 1.4
( 0.1 nm indicated the self-assembly of compound 1 on silicon.
To test the ability of surface-bound phthalimides to photo-
chemically immobilize sugars, 2000 kDa fluorescein isothio-
cyanate (FITC)-conjugated R(1,6)dextran polysaccharide films
were spin-coated onto SAM 1 from an aqueous solution and
irradiated for approximately 1 h with a 300 nm rayonet bulb in
an inert environment. Two controls were also prepared. In the
first, polysaccharides were spin-coated onto SAM 1 and left in
the dark. In the second, polysaccharides were spin-coated onto
an underivatized silicon wafer. All three samples were placed
in water-filled vials for 12 h. After removing the samples and
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Figure 1. Schematic of a phthalimide derivative undergoing a photochemical hydrogen abstraction reaction followed by recombination to
form a covalent bond.
2900 Langmuir, Vol. 22, No. 6, 2006 Carroll et al.

rinsing with water and methanol followed by blow-drying with
argon, the fluorescence spectrum of each sample was obtained,
as shown in Figure 4. Preferential retention of polysaccharides
on the irradiated sample relative to the two controls indicates the
photochemical immobilization of the polysaccharides on SAM
1.
The film thicknesses of the three samples were measured using
a Beaglehole ellipsometer in variable angle mode. A refractive
index value of 1.5 was used for the organic layer. The irradiated
sample retained 7.1 ( 0.3 nm of material after the rinse. The
thickness of the material on SAM 1 unexposed to light was 0.7
( 0.3 nm, and the thickness on the underivatized silicon wafer
was 0.4 ( 0.3 nm. The reported thicknesses do not include the
thickness of SAM 1. The surfaces were further investigated with
water contact-angle measurements. The hydrophilic nature of
the sugars reduced the water contact angle from 65 ( 1 to 28
( 1° on the irradiated SAM. Inefficient immobilization on the
dark control is evident from a post-rinse contact angle of 62 (
1°. The higher retention of material on the irradiated SAM
demonstrates that self-assembled phthalimide monolayers are
capable of photochemically bonding to an overlayer sugar film,
despite any spatial restrictions on the chromophore as a result
of placement in a constrained environment. We speculate that
the nature of the bonding is covalent and results from radical-
radical recombination following hydrogen abstraction.
The above experiments were also performed on SAMs
comprised of benzophenone chromophores, another class of
aromatic carbonyls that can photochemically abstract hydrogen
from C-H groups and have been shown to graft polymers to
surfaces.36 Although the benzophenone monolayers were able to
graftthesugars,theresultingsugarfilmthicknessandfluorescence
intensity were lower, and the contact angle was higher than that
of the films on SAM 1. The lower performance may be due to
the radical center in the benzophenone SAM residing further
from the surface than that in the phthalimides, self-quenching
of the excited state, or a higher interfacial tension between the
more hydrophobic benzophenone monolayer and the sugar film
compared to the phthalimide-sugar interaction. Benzophenone
SAMs have more hydrophobic character than phthalimide SAMs,
as evidenced by a higher water contact angle of about 85°.
Preliminary experiments with a microarray spotter have shown
thathydrophilicsurfacesaremoreeasilyspottedthanhydrophobic
substrates. We found that more material physisorbed onto SAM
1 in comparison to a benzophenone-terminated SAM. In any
case, other photoactive carbonyl groups capable of abstracting
hydrogen atoms can be substituted and may enhance or retard
the reaction because of the efficiency of self-assembly, steric,
and thermodynamic constraints.
In addition to covalently attaching underivatized sugars to a
substrate, we are also able to generate patterns of grafted sugars.
Our strategy for immobilizing carbohydrates on SAM 1 in a
spatially controlled fashion is presented in Figure 5. Spin-coated
polysaccharide films were covered with a photomask consisting
of a copper grid with spacings of 280 µm and irradiated for 2
h as described above. The photoreaction is restricted to the opaque
regions of the mask, leaving the pattern of the mask written to
the surface via attached carbohydrates. We removed ungrafted
sugars by sonicating films in water for 15 min, changing the
water and vial every 5 min.
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Figure 2. Synthesis of compound 1 and SAM 1.
Figure 3. UV/vis spectra of compound 1 in ethanol (dashed line)
and SAM 1 (solid line).
Figure 4. Fluorescence spectra of 2000 kDa FITC-conjugated R-
(1,6)dextran films under three conditions: irradiated SAM 1 (dashed
line), dark SAM 1 (dotted line), and underivatized silicon (solid
line). Each spectrum was obtained after washing the substrates for
12 h in H2O.
Photochemical Micropatterning of Carbohydrates Langmuir, Vol. 22, No. 6, 2006 2901

Condensing water onto the substrate provided a quick way to
visualize the hydrophilic patterns using optical microscopy.37
Figure 6 presents an optical microscope image of water
condensation onto patterned R(1,6)dextran polysaccharides with
a MW of 2000 kDa. The hydrophilic attraction between water
andthepolysaccharidesrelativetotheunmodifiedmaskedregions
of the monolayer causes water to preferentially reside on the
areas of the substrate containing polysaccharide. The results were
similar when 20 kDa R(1,6)dextrans were patterned.
Other strategies involving immobilization without prior
derivatization have been successful with carbohydrate-containing
macromolecules noncovalently adsorbed on nitrocellulose1 or
oxidized polystyrene (PS);5 however, the nitrocellulose study
showed that the immobilization efficiency decreases with the
MW of the polysaccharides.1 To show the versatility of our
method, we tested glucose and sucrosestwo simple sugars at the
low extreme of MW, containing both six- and five-membered
sugar moieties. The resulting water condensation images are
presented in Figure 7. The visible patterns clearly show that our
method extends to sugars of the lowest MWs.
To show that our method is applicable for the high-throughput
production of carbohydrate microarrays, we investigated whether
carbohydrates could be microspotted and subsequently photo-
immobilized using a robotic spotter. We applied the FITC-
conjugated polysaccharides as probes to monitor the spotting
(37) Lopez, G. P.; Biebuyck, H. A.; Frisbie, C. D.; Whitesides, G. M. Science
1993, 260 (5108), 647-649.
Figure 5. The strategy for direct chemical patterning of a surface with carbohydrates involves a photolithographic technique. Carbohydrates
are spin-coated onto SAM 1 and covered with a mask. Irradiation induces a photochemical reaction that covalently links the carbohydrates
to SAM 1.
Figure 6. Optical microscope images of water condensation on
photochemically generated patterns of polysaccharides on SAM 1.
Figure 7. Optical microscope images of breath condensation on
photochemically generated patterns of (a) sucrose and (b) glucose.

process. After irradiation for 1 h, extensive washing, and
“blocking” with bovine serum albumin (BSA), we introduced
specific antibodies and lectins to detect immobilized carbohy-
drates. Bound antibodies were revealed with a streptavidin-Cy3
conjugate. We found that the thermodynamic parameters of the
surface needed to be adjusted to transfer a detectable amount of
carbohydrates from the pin of the spotter to SAM 1. To make
the surface more attractive to carbohydrates, we made mixed
phthalimide-amine monolayers (PAM) from a solution contain-
ing a 5:1 ratio of aminopropyltrimethoxy silane to compound 1.
Presumably,thehydrophilicaminegroupinteractsmorefavorably
with the carbohydrates compared to the more hydrophobic phenyl
ring of compound 1, decreasing the interfacial tension between
the carbohydrate and the substrate, which allows a sufficient
amount of carbohydrates to be adsorbed to the surface for
subsequent photoimmobilization.
Figure 8a,b presents the results after spotting FITC-conjugated
R(1,6)dextrans with MWs of 20, 70, and 2000 kDa on PAM and
nitrocellulose-coated FAST slides. The FAST slide was treated
to provide a comparison of our new method with an established
platform. By examining the fluorescent signals of the spotted
slides before irradiation and washing (Figure 8a), we found that
theamountsofcarbohydratesadsorbedontoPAMaresignificantly
less than those spotted on the FAST slide. This may be attributed
to the two-dimensional nature of PAM, which allows less
polysaccharides to be delivered and adsorbed in comparison to
nitrocellulose surfaces with thicker three-dimensional coatings.
However, staining the slides with an anti-R(1,6)dextran antibody
(16.4.12E), which is specific for the terminal nonreducing end
epitopes displayed by all three dextran conjugates1 revealed that
the PAM surface retains a similar amount of polysaccharides
regardless of the MW of the polysaccharides spotted (Figure
8b). Neither an underivatized glass substrate nor PAM without
UVirradiationshowedadetectablesignalwithanti-R(1,6)dextran
antibodies under the same experimental conditions. These results
were reproduced in multiple microarray assays (data not shown).
Thus, not only is PAM suitable for use in the high-throughput
construction of polysaccharide microarrays, but the photoim-
mobilized carbohydrates also retain their immunological proper-
ties, as defined by a specific antibody, after immobilization.
We further examined a panel of mono- and oligosaccharide
arrays on PAM and FAST slides. The spotted arrays were probed
with a biotinylated lectin, Concanavalin A (Con A; Figure 8c),
which is Man- and/or Glc-specific and requires the C-3, C-4, and
C-5 hydroxyl groups of the Man or Glc ring for binding. We
found that oligosaccharides with three (IM3), five (IM5), and
seven glucoses (IM7) are reactive to Con A on the PAM slide
but not on the FAST slide. However, none of the spotted
monosaccharides were reactive to the lectin on these surfaces.
The method of photocoupling, which can target any CH- group
on the sugar rings with varying specificity depending on the
structure of the ring34,38,39 (Figure 5), may interfere significantly
with the lectin binding of monosaccharides, Man, or Glc. The
limited specificity of the reaction and the lesser amount of
saccharide epitopes present for smaller carbohydrates reduces
(38) Madden, K. P.; Fessenden, R. W. J. Am. Chem. Soc. 1982, 104 (9), 2578-
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Figure 8. Immobilization of mono-, oligo-, and polysaccharides on PAM. A FAST slide is included for comparison. (A) Fluorescence images
and intensity values of the spotted polysaccharides, the FITC-conjugated R(1,6)dextrans of 20-, 70-, and 2000 kD, before treatment with
light. The three-dimensional FAST slide adsorbs more material than does the two-dimensional PAM. (B) Fluorescence images and intensity
values after treatment with light, rinsing, and staining with a biotinylated anti-dextran antibody (16.4.12E), followed by staining with a
Streptavidin-Cy3 conjugate. Immobilization on PAM is not dependent on MW, and a greater amount of 20 kD polysaccharides are retained
even though much less material could initially be spotted. (C) Fluorescence intensity values of mono- and oligosaccharide arrays after
treatment with light, rinsing, and staining with a biotinylated lectin, Con A, followed by staining with a Streptavidin-Cy5 conjugate. IM3,
IM5, and IM7 refer to isomaltotriose, isomaltopentose, and isomaltoheptaose, respectively.

the probability that a biologically active epitope presents itself
at the air-monolayer interface.
These results show that PAM offers a plausible alternative to
nitrocellulosefordisplayingpolysaccharidesandoligosaccharides
on glass chips. Larger panels of carbohydrates with structural
and immunological diversities must be introduced to further
validate and explore the potential of this novel chip substrate for
microarray technologies.
Althoughthisreportfocusesontheapplicationofimmobilizing
andpatterningcarbohydrates,phthalimide-containingmonolayers
are suitable for patterning virtually any material containing C-H
groups. In addition to various carbohydrates, we are also able
to pattern a variety of polymers. PS, poly(methyl methacrylate)
(PMMA), and poly(vinyl alcohol) (PVA) were all immobilized
and patterned on SAM 1 (see Supporting Information for images).
Theversatilityofourmethodallowsmaterialswithvaryingsurface
tensions and chemical functionalities to be immobilized and
patterned on a surface, allowing for a fast and simple approach
to design organic scaffolds for novel materials and devices.
Summary
We have shown that a new class of SAMs containing
phthalimide chromophores is capable of photochemically im-
mobilizing carbohydrates on a flat substrate. The method requires
no chemical modification of the carbohydrates prior to deposition
and is not limited to carbohydrates of high MWs. Further,
immobilizedcarbohydrateantigensareshowntoretaintheirability
to interact with the corresponding antibody or lectin. The
photochemicalnatureofthetechniqueallowspatternstobecreated
and makes the method adaptable to the full potential of
photolithography, which is currently used in industry for the
high-throughput fabrication of computer chips and nanoscale
patterning. Multiple carbohydrate patterns can be immobilized
by repeating the reaction with a different carbohydrate in a
previously masked region. In conjunction with a microarray
spotter, large libraries of carbohydrates can be immobilized on
our surface without previous derivatization. The versatility and
ease of the method provides a platform for biologists, chemists,
and engineers to investigate and create new biological materials
aswellascharacterizecarbohydrateinteractionsinarapidmanner.
Methods
Synthesis of Compound 1. A 3.3 mmol portion of 11-
bromoundecanetrimethoxysilane (Gelest) was added to a solution
of an equimolar amount of potassium phthalimide (Aldrich) in 60
mL of anhydrous DMF (Aldrich). The solution was stirred overnight
at room temperature (RT) under argon. Chloroform (50 mL) was
added. The solution was transferred to a separatory flask containing
50 mL of H2O. The aqueous layer was separated and then extracted
with two 20 mL portions of chloroform. The combined chloroform
extract was washed with several 20 mL portions of H2O. The
chloroform was removed by rotoevaporation, and residual DMF
was removed on a high vacuum line to give a pale yellow liquid
(0.99 g, 72% yield). The compound was used without further
purification. Note that, for self-assembly experiments, residual DMF
was not removed. 1H NMR: (CDCl3) δ 7.82 (m, 2H), 7.69 (m, 2H),
3.66 (t, J ) 7 Hz, 2H), 3.55 (s, 9H), 1.44-1.15 (m, 18H), 0.71-0.51
(m, 2H). LRMS-FAB+ (m/z): (M - H) 420.2 (experimental), 420.2
(calculated); (M - OCH3) 390.1 (experimental), 390.2 (calculated).
Self-Assembly of SAM 1. The substrates consisted of glass
(ArrayIt), quartz (SPI), or silicon (wafer world). The substrates and
glassware were cleaned by being boiled in a “piranha” solution (7:3
sulfuric acid/H2O2) for 1 h followed by an extensive rinse with water
and methanol. Substrates were dried with a stream of argon and
immersed in a 1 mmol solution of compound 1 in anhydrous toluene
(Aldrich). The solution was kept under argon and left undisturbed
for 12 h. The resulting SAMs were rinsed with toluene and sonicated
three times for 2 min each in toluene, toluene/methanol (1:1), and
methanol. Substrates were kept in argon-purged vials until further
use.
Preparation of PAM. PAM was made in the same manner as
SAM 1, except that a 5× molar amount of aminopropyltrimethoxy
silane (Gelest) was simultaneously added with compound 1. The
contact angle of the resulting surface was 72 ( 1°.
Photochemical Grafting of Polysaccharide Films. FITC-
conjugated R(1,6)dextrans weighing 20 or 2000 kD (Dextran-FITC)
(Sigma) were spin-coated from a 10 mg/mL aqueous solution at
3000rpmfor90sandplacedinargon-purgedquartztubes.Irradiation
was carried out for 70 min with a Rayonet photochemical reactor
equipped with lamps that emit at 300 nm. For ellipsometry and
fluorescence experiments, the surface was rinsed by placing in H2O
for 12 h followed by rinsing with methanol. Substrates were blown
dry with argon.
Instrumental Measurements. UV-vis spectra were obtained
using a Shimadzu (UV-2401PC) UV-vis recording spectropho-
tometer. Contact-angle measurements were performed with a Rame-
Hart 100-00 contact-angle goniometer using Millipore Milli-Q water.
At least three droplets were measured on each sample and averaged.
ThicknessesweremeasuredwithaBeagleholeellipsometerinvariable
angle mode. A refractive index of 1.5 was used for all samples.
Measurements were performed three times in different locations on
the surface and averaged. Fluorescence spectra were obtained using
a Jobin Yvon Fluorolog 3 spectrofluorometer in front face mode.
The surface was placed at an angle of 20° to a line parallel to the
plane of the detector.
Photochemical Patterning of Carbohydrates. A 75-mesh
transmission electron microscopy (TEM) grid (Electron Microscopy)
was used as a photomask for all patterning experiments. Dextran-
FITC (2000 kD) and 20 kD polysaccharide films were prepared as
described above. Glucose (Aldrich) was spin-coated from a solution
of 26 mg in 1 mL of acetonitrile at 3000 rpm for 90 s. One drop
of a sucrose (Aldrich) solution containing 1.5 g in 1 mL H2O was
placed on the substrate using a pipet. Approximately three-fourths
of the drop was removed with a pipet. In all cases, the photomask
was placed on top of the sugar film or droplet and pressed down with
a quartz plate. Irradiation was carried out in an argon-filled glovebag
with a desktop lamp containing a 300 nm Rayonet bulb for
approximately 2 h. Samples were rinsed by sonication in H2O for
15min,withthewaterandvialbeingchangedevery5min.Sonication
was accompanied by extensive rinsing with water and methanol.
Samples were blown dry with argon.
Visualization of the Chemically Patterned Surface. Patterns
were visualized by condensing water onto the pattern and imaging
with a Nikon Eclipse optical microscope equipped with an INSIGHT
digital camera. Two methods were used to condense water onto the
surface. In the first, the surface was exposed to an extended breath.
In the second, the substrate was held over boiling water for
approximately 10 s.
Microarray Construction. Antigen preparations were dissolved
in saline (0.9% NaCl) at a given concentration and were spotted as
triplet replicate spots in parallel. The initial amount of antigen spotted
was approximately 0.35 ng/spot and was diluted by serial dilutions
of 1:5 thereafter (see also the microarray images inserted in Figure
8). A high-precision robot designed to produce cDNA microarrays
(PIXSYS 5500C, Cartesian Technologies, Irvine, CA) was utilized
to spot carbohydrate antigens onto chemically modified glass slides
as described.1,6 Both FAST slides (Schleicher & Schuell, Keene,
NH) and PAM slides were spotted. The printed FAST slides were
air-dried and stored at RT. The printed PAM slides were subjected
to UV irradiation to activate the photocoupling of carbohydrates to
the surface.
PhotocouplingofCarbohydratesontheChips.Aftermicroarray
spotting, the PAM slides were air-dried and placed in a quartz tube.
The sealed tube was subsequently purged with argon or nitrogen
before irradiation. UV irradiation was conducted by placing the
quartz tube under a desktop lamp containing a 300 nm Rayonet bulb

for 1 h. Precaution was made to avoid skin and eye contact with the
radiation during the irradiation process.
Microarray Staining, Scanning, and Data-Processing. Im-
mediately beforeuse,theprintedmicroarrayswererinsedandwashed
withphosphate-bufferedsaline(PBS)(pH7.4)andwith0.05%Tween
20 five times, with 5 min of incubation in each washing step. They
were then “blocked” by incubating the slides in 1% BSA in PBS
containing 0.05% NaN3 at RT for 30 min. Antibody staining was
conducted at RT for 1 h at given dilutions in 1% BSA/PBS containing
0.05% NaN3 and 0.05% Tween 20. Since a biotinylated anti-dextran
antibody (mAb 16. 4.12E, adapted from the late Professor Elvin A.
Kabat at Columbia University) and lectin Con A (EY Laboratories,
San Mateo, CA) were applied in this study, streptavidin-Cy3
conjugateorstreptavidin-Cy5conjugate(AmershamPharmasia)were
applied to reveal the bound anti-dextran antibodies or lectin Con A,
respectively. The stained slides were rinsed five times with PBS and
with 0.05% Tween 20 after each staining step. A ScanArray 5000A
standard biochip scanning system (Perkin Elmer, Torrance, CA),
equipped with multiple lasers, emission filters, and ScanArray
acquisition software, was used to scan the microarray. Fluorescence
intensityvaluesforeacharrayspotanditsbackgroundwerecalculated
using ScanArray Express (Perkin Elmer, Torrance, CA).
Acknowledgment. This material is based upon work sup-
ported by, or in part by, the U.S. Army Research Laboratory and
the U.S. Army Research Office under Contract/Grant No. DA
W911NF-04-1-0282, the National Science Foundation under
Grant Nos. DMR-02-14263, IGERT-02-21589, and CHE-04-
15516 to N.J.T. and J.T.K. at Columbia University, and the Phil
N. Allen Trust and the Herzenberg Trust to D.W. at Stanford
University. This work used the shared experimental facilities
that are supported primarily by the MRSEC Program of the
National Science Foundation under Award No. DMR-0213574
and the New York State Office of Science, Technology and
AcademicResearch(NYSTAR).G.T.C.acknowledgesanIGERT
fellowship. Any opinions, findings, and conclusions or recom-
mendations expressed in this material are those of the author(s)
and do not necessarily reflect the views of the National Science
Foundation.
SupportingInformationAvailable: Optical microscope images
of patterns of PVA, poly(tert-butyl acrylate) (PTBA), and PS. This
material is available free of charge via the Internet at http://pubs.acs.org.
LA0531042

Photochemical Micropatterning of Carbohydrates on a Surface

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Photochemical Micropatterning of Carbohydrates on a Surface