Functionalized surface arrays for spatial targeting of immune cell signaling

Article abstract of DOI:10.1021/ja058701p

The effect of surface topography and chemistry on cellular response is of fundamental importance, especially where living systems encounter device surfaces as in medical implants, tissue engineering, and cell-based sensors. To understand these biological

Full text of DOI:10.1021/ja058701p

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Published on Web 04/06/2006
Functionalized Surface Arrays for Spatial Targeting of Immune Cell Signaling
Wageesha Senaratne,† Prabuddha Sengupta, Vladimir Jakubek, David Holowka,
Christopher K. Ober,* and Barbara Baird*
Department of Chemistry and Chemical Biology and Department of Materials Science, Cornell UniVersity,
Ithaca, New York 14853
Received December 22, 2005; E-mail: cober@ccmr.cornell.edu; bab13@cornell.edu
An important problem in cell biology with broad implications
is how cellular responses are affected by the chemistry and
topography of interfacing surfaces. Fundamental to normal physiol-
ogy, these interactions are also highly relevant for any situation
where living systems encounter device surfaces such as in medical
implants and cell-based sensors. Because of widespread interest in
understanding and regulating such cellular processes, significant
efforts are being made to tailor surface-active materials and develop
effective surface chemistry.1
Self-assembled monolayers (SAMs) formed by adsorption of
alkanethiols on a gold layer provide well-packed, chemically stable
surfaces.2 Surfaces patterned with modified SAMs can be used to
present specific ligands with controlled surface densities within
well-defined regions. Thus, for ligands that bind specifically to cell
surface receptors, this provides a tunable means for investigating
spatial control of cellular responses. For example, optical or electron
microscopy can be utilized for direct visualization and quantitative
analysis of subcellular reorganization that occurs in response to
these spatially defined stimuli. These observations can in turn
provide valuable mechanistic insights about the biochemical
pathways that regulate and target these processes. As described in
this report, we employed high-resolution lithography to pattern
SAMs with submicrometer feature size. We evaluated specific
binding by anti-DNP immunoglobulin E (IgE), both for the soluble
form of this antibody and when it is associated with its high affinity
receptor (FcꢀRI) on the surface of rat basophilic leukemia (RBL)
mast cells. These ligand-SAM substrates were also tested for their
capacity to investigate early signaling processes that are stimulated
by the engaged IgE-FcꢀRI.
Clustering of cell surface IgE-FcꢀRI by multivalent ligands
(antigens) initiates intracellular signaling pathways associated with
mast cell effector functions in allergic and inflammatory responses.3
Recently, micrometer-size patterned lipid bilayers containing 2,4-
dinitrophenyl-caproate (DNP-cap)-modified lipids were used to
control the location and size of IgE-FcꢀRI clusters, and intracellular
components were shown to redistribute selectively with the patterns
of receptors.4 To visualize the earliest molecular interactions
associated with receptor-mediated signaling requires even finer
resolution. Patterning of biological molecules at the submicrometer
scale has been achieved with various techniques, including dip-
pen, block copolymer micelles, and supramolecular stamping.5 A
useful alternative to these selective bottom-up approaches is top-
down, electron beam lithography, which creates ∼10 nm to 1 µm
features with high accuracy and reproducibility and offers easy
access to submicrometer resolution of cellular redistributions.6,7 Our
approach further incorporates SAMs with specific binding groups
and other desired, quantifiable properties. We used a lift-off process
to generate gold arrays of submicrometer features on a silicon chip
(Supporting Information, Figure S2). The gold/titanium posts (50
nm in height, 1 µm to 45 nm in width) were then selectively
chemically modified using ligand-SAMS that were designed to
interact specifically with antibodies and cells.
The DNP-cap-PEG4C16 molecules synthesized for these SAMs
contain a long alkyl chain, (CH2)n (n ) 16), with a disulfide terminal
group and an oligoethylene glycol (OEG) spacer terminated by a
DNP-cap (Scheme S1). The hydrocarbon segment (n > 10) served
to enhance molecular organization and packing,8 while the OEG
linker was incorporated into the system to introduce an interfacial
water layer that would orient the headgroup away from the surface
as well as minimize nonspecific interactions of the proteins with
the underlying surface.9 The terminal DNP-cap group binds
specifically to anti-DNP antibodies, including anti-DNP IgE.10
SAM-modified, patterned surfaces were incubated with Alexa-488-
labeled anti-DNP IgE at 37 °C for 1 h. Fluorescence images reveal
that this antibody binds to the patterned patches of DNP-cap-
PEG4C16-SAMs with high specificity (Figure 1a, 1 µm features;
Figure S3, 600 nm features). A conservative estimate of the specific/
nonspecific ratio is ∼5 (see Supporting Information). An immu-
noassay of these ligand-modified gold chips showed saturation
binding of anti-DNP antibodies (Figure S4) with coverage corre-
sponding to more than 1011 antibody molecules/cm2. These results
demonstrate that patterned surfaces of this type can provide well-
defined arrays of antibodies or other proteins with submicrometer
features for potential applications in biosensors.
To examine specifically stimulated cellular responses, the
patterned substrates were incubated with a suspension of RBL-
2H3 cells that had been sensitized with AlexaFluor-488-labeled anti-
DNP IgE bound to cell surface FcꢀRI receptors. The cells settled
on the silicon chip and became adherent within 2-3 min at room
temperature (RT). The fluorescently labeled IgE receptor complexes
that were originally dispersed over the cell surface became clustered
after binding to the DNP-cap-PEG4C16-SAM modified features
(Figure 1b,c). We confirmed that the gold structures do not interfere
with the fluorescence signal (Figures 1b, S3, and S5). Receptor
clustering is due to ligand binding because the cell-bound fluores-
cent IgE remains dispersed and does not concentrate on the features
when the chips are preincubated with soluble anti-DNP IgE or when
the chips are modified instead with methoxy-PEG4C16-SAMs
(Figure S5). Thus, DNP-cap PEG4C16-SAMs presented on sub-
micrometer surface features engage specific cell surface receptors
and cluster them in the same defined pattern.
We used these patterned surfaces to extend our investigation of
spatially regulated, transmembrane signaling events. It is known
that clustering of IgE-FcꢀRI causes intracellular Lyn kinase to co-
redistribute and phosphorylate tyrosine residues in the receptors’
cytoplasmic segments. RBL cells sensitized with unlabeled anti-
DNP IgE were incubated with the DNP-SAM chips for 15 min at
RT, then fixed and labeled with monoclonal anti-phosphotyrosine
† Current affiliation: Corning Inc., Thin Films and Surfaces, Corning, NY 14831.
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J. AM. CHEM. SOC. 2006, 128, 5594-5595
10.1021/ja058701p CCC: $33.50 © 2006 American Chemical Society

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C O M M U N I C A T I O N S
Figure 1. Confocal fluorescence images of surfaces patterned with DNP-cap-PEG4-C16 SAMs of 1 µm (a-c) or 600 nm (d, e) feature sizes. (a) Specific
binding of soluble anti-DNP IgE labeled with Alexa488. (b-e) Adherent RBL cells sensitized with Alexa488 labeled (b, c) or unlabeled (d, e) anti-DNP IgE
which binds to cell surface FcꢀRI receptors. (b, c) IgE-FcꢀRI clusters (green) on cell surface within 2-3 min in a pattern that coincides with the DNP-
SAM-modified gold (red). (d) Antibody 4G10 (green) marking Lyn-mediated tyrosine phosphorylation that co-clusters with IgE-FcꢀRI on DNP-SAM-
modified gold (red) after 15 min. (e) LAT-EGFP (green) co-clusters with IgE-FcꢀRI on DNP-SAM-modified gold after 15-20 min.
antibody (4G10) and Alexa 488-labeled secondary antibody. As
shown in Figure 1d, anti-phosphotyrosine concentrates on the
patterned features corresponding to the clustered IgE-FcꢀRI,
demonstrating that initiation of cell activation occurs with these
chips similarly to the patterned lipid bilayers used previously.4 In
a control experiment, the secondary antibody showed no concentra-
tion on the patterns in the absence of 4G10 (Figure S6).
containing amine or caproate groups, respectively. Biotin-avidin
sandwiches also could be readily incorporated within this scheme.
With fluorescence and electron microscopy visualization, patterned
SAMs provide a powerful tool for systematic examination of the
earliest receptor-mediated cellular signaling interactions that occur
with spatial resolution on the nano and longer length scales.
Acknowledgment. This work was supported by Grants NIH
AI18306 and NSF NIRT E74-8395 and drew from the resources
of Nanobiotechnology Center (NSF-ECS-9876771). The LAT-EFFP
construct was a generous gift from L. E. Samelson (NIH).
We then evaluated spatial redistribution of a subsequent signaling
component, not previously reported for patterned surfaces. Linker
for activation of T cells (LAT) is an important adaptor molecule
in immune cells. In mast cells, LAT recruits other protein
participants in activation pathways leading to release of chemical
mediators of inflammation.10 RBL cells were transfected with a
LAT-EGFP construct,11 and live cells expressing this endogenous
fluorescent protein were incubated with the DNP-SAM chips and
examined with microscopy in real time. Concentration of LAT-
EGFP over the patterned features was observed in more than 60%
of the adherent cells within 15-20 min of incubation with the
modified surfaces (Figure 1e). Interestingly, the LAT clusters appear
to become more diffuse over time, although they remain concen-
trated in the region of clustered receptors (data not shown). The
resolution of fluorescence microscopy cannot distinguish overlap-
ping from proximal clusters that are on the order of 100 nm. Similar
patterns with electron microscopy visualization will allow testing
of the hypothesis that LAT clusters separate from receptor clusters,
possibly representing secondary signaling domains.12 A major
advantage of the patterned presentation is that co-localization within
an array is readily determined and quantified using, for example,
Fourier transform analysis. Our results with cells confirm that
receptor-mediated signaling can be stimulated with submicrometer
spatial definition using this approach.
In summary, we demonstrate that E-beam lithography and
designed SAMs can be combined to create arrays for spatially
defined presentation of specific ligands (on feature sizes 1 µm to
600 nm), which in turn bind specifically to receptor proteins in
solution or on cells in the same pattern. In addition to their potential
for protein arrays for diagnostic or detector applications, these are
proving to be valuable for evaluating spatial regulation and targeting
of receptor-mediated cellular signaling. The ligand specificity can
be generalized; for example, succinimidated or hydroxylated PEG4-
C16-S-S- derivatives could be used to attach specific ligands
Supporting Information Available: Materials, syntheses, fabrica-
tion, and experimental procedures including control experiments. This
material is available free of charge via the Internet at http://pubs.acs.org.
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