Immobilization of ligands with precise control of density to electroactive surfaces

Article abstract of DOI:10.1021/ja065828l

We report a broadly applicable surface chemistry methodology to immobilize ligands, proteins, and cells to an electroactive substrate with precise control of ligand density. This strategy is based on the coupling of soluble aminooxy terminated ligands wit

Full text of DOI:10.1021/ja065828l

Published on Web 11/10/2006
Immobilization of Ligands with Precise Control of Density to
Electroactive Surfaces
Eugene W. L. Chan and Muhammad N. Yousaf*
Contribution from the Department of Chemistry and Carolina Center for Genome Science,
UniVersity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290
Received August 24, 2006; E-mail: mnyousaf@email.unc.edu
Abstract: We report a broadly applicable surface chemistry methodology to immobilize ligands, proteins,
and cells to an electroactive substrate with precise control of ligand density. This strategy is based on the
coupling of soluble aminooxy terminated ligands with an electroactive quinone terminated monolayer. The
surface chemistry product oxime is also redox active but at a different potential and therefore allows for
real-time monitoring of the immobilization reaction. Only the quinone form of the immobilized redox pair
is reactive with soluble aminooxy groups, which allows for the determination of the yield of reaction, the
ability to immobilize multiple ligands at controlled densities, and the in-situ modulation of ligand activity.
We demonstrate this methodology by using cyclic voltammetry to characterize the kinetics of a model
interfacial reaction with aminooxy acetic acid. We also demonstrate the synthetic flexibility and utility of
this method for biospecific interactions by installing aminooxy terminated FLAG peptides and characterizing
their binding to soluble anti-FLAG with surface plasmon resonance spectroscopy. We further show this
methodology is compatible with microarray technology by printing rhodamine-oxyamine in various size
spots and characterizing the yield within the spots by cyclic voltammetry. We also show this methodology
is compatible with cell culture conditions and fluorescent microscopy technology for cell biological studies.
Arraying RGD-oxyamine peptides on these substrates allows for bio-specific adhesion of Swiss 3T3
Fibroblasts.
Michael addition,16,17 and other chemoselective ligation strate-
gies.18 However, to extend the utility of these surfaces to
Introduction
Strategies to immobilize biomolecules onto solid supports is
important for a wide variety of applications ranging from the
development of small molecule and protein microarrays to
model substrates for mechanistic studies of cell behavior.1-5
There have been several immobilization methods developed to
tailor surfaces for a variety of diagnostic and high-throughput
assays. The most common conjugation methods employ radical,
mediatedphotoimmobilization,6acid-basechemistry,7-9Staudinger
ligation,10-12 Click chemistry,13,14 Diels-Alder reaction,15
generate new types of biosensors and to study complex
biological processes, it is necessary to develop substrates with
more sophisticated and flexible surface properties. Surfaces for
these applications would require the precise control of ligand
density, the ability to immobilize multiple ligands, and the in-
situ modulation of ligand activity. For cell biological applica-
tions, the ability to precisely control low density of ligands is
particularly important. The key criteria to successfully preparing
these types of substrates rely on the surface to resist nonspecific
protein adsorption, the selective chemistry for ligand im-
mobilization, and the nature of the underlying substrate for
spectroscopic characterization of its interactions with biomol-
ecules.
(1) Tomizaki, K. Y.; Usui, K.; Mihara, H. Chem. Bio. Chem. 2005, 6, 783-
799.
(2) Schutkowski, M.; Reineke, U.; Reimer, U. Chem. Bio. Chem. 2005, 6, 513-
521.
(3) Min, D. H.; Mrksich, M. Curr. Op. Chem. Biol. 2004, 8, 554-558.
(4) Mrksich, M. Chem. Soc. ReV. 2000, 29, 267-273.
(5) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763.
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L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano, M. D.; Hu,
W.-S. Chem. Biol. 1997, 4, 731-737.
Our strategy utilizes the unique chemical properties of an
immobilized p-benzoquinone on a conductive surface. This
redox-active molecule provides many desirable features for
bioconjugation of molecules to surfaces. For example, the
quinone molecule reacts in high yield with a number of
(7) Peelen, D.; Smith, L. M. Langmuir 2005, 21, 266-271.
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J. AM. CHEM. SOC. 2006, 128, 15542-15546
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Immobilization of Ligands
A R T I C L E S
Figure 1. Redox-active hydroquinone monolayer undergoes electrochemical oxidation to the benzoquinone. The resulting quinone then reacts chemoselectively
with aminooxy acetic acid to give the corresponding oxime.
functional groups in solution, such as thiol,19 cyclopentadiene,20
hydrazide,21 and hydroxyamine.22 The reversible redox coupling
of quinone also provides a sensitive probe to monitor the extent
of interfacial reaction in-situ by cyclic voltammetry.23,24 Fur-
thermore, only the quinone form of the reversible redox reaction
is reactive; this important feature permits modulation of the
ligand activity by an electrochemical potential.25,26 Previous
work has shown that a quinone monolayer reacts with a
cyclopentadiene (cp) via a Diels-Alder reaction to immobilize
a variety of biologically active ligands.27,28 Although this
strategy provides a rapid and selective immobilization of
biomolecules, the syntheses of cyclopentadiene tethered mol-
ecules are cumbersome. In particular, the use of cyclopentadiene
for common solid-phase peptide synthesis is not feasible because
the cp readily decomposes under cleavage conditions. Alterna-
tively, the use of sulfhydryl groups to conjugate biomolecules
onto quinone surfaces via Michael addition provides a general
and straightforward route. Unfortunately, the resulting quinone-
thiol adduct has a redox-active signal similar to that of the
quinone starting material.29,30 Because there is no diagnostic
peak that distinguishes between the quinone and the product, it
is not possible to determine the surface density of immobilized
ligands electrochemically with this coupling strategy for a
variety of ligands. To extend the utility of immobilizing
molecules to electroactive substrates, it would be desirable to
develop other coupling chemistry for the quinone molecules that
allows for the determination of the yield of coupling and
therefore the ligand density on the surface. In this article, we
report a novel chemical approach to immobilize ligands with
precise control of density onto an electroactive self-assembled
monolayer (SAM) of alkanethiolates on gold. This approach is
based on the coupling between the ketone-bearing quinone
monolayer and soluble aminooxy terminated ligands. We further
Figure 2. (A) Cyclic voltammograms recorded in 5-min intervals at a scan
rate of 50 mV‚s-1 showed the extent of the interfacial reaction between
soluble aminooxy acetic acid (150 mM) and quinone monolayer. The peaks
at 580 and -30 mV correspond to the redox coupling of the quinone,
whereas the peaks at 450 and 371 mV correspond to the redox peaks of the
product oxime. (B) A plot of the peak currents versus time compares the
relative rates between the decrease of quinone monolayer and the formation
of oxime on the surface.
demonstrate the utility of this methodology to immobilize
ligands for selective protein binding and cell attachment on inert
surfaces.
Results and Discussion
Our interest in using oxyamine for the bioconjugation is
motivated by several considerations: (1) Oxyamine readily
reacts with quinone in high yield at physiological pH and room
temperature. (2) The two partners react covalently to form a
chemically stable oxime linkage.31,32 (3) The aminooxy groups
can be easily introduced into most biomolecules through
straightforward solution or solid-phase synthesis.33
We first demonstrate that oxyamine can undergo selective
immobilization onto quinone monolayer in high yield. In this
model study, we used the coupling between aminooxy acetic
acid and a monolayer presenting quinone groups (Figure 1).
Characterization of the redox reaction by cyclic voltammetry
(19) Li, W.-W.; Heinze, J.; Haehnel, W. J. Am. Chem. Soc. 2005, 127, 6140-
6141.
(20) Kwon, Y.; Mrksich, M. J. Am. Chem. Soc. 2002, 124, 806-812.
(21) Smith, L. I.; Irwin, W. B. J. Am. Chem. Soc. 1941, 63, 1036-1043.
(22) Baldwin, J. E.; Norris, R. k. J. Org. Chem. 1981, 46, 697-703.
(23) Yousaf, M. N.; Chan, E. W. L.; Mrksich, M. Angew. Chem., Int. Ed. 2000,
30, 1943-1946.
(24) Chan, E. W. L.; Yousaf, M. N. Mrksich, M. J. Phys Chem. A 2000, 104,
9315-9320.
(25) Yeo, W. S.; Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 2003, 125,
14994-14995.
(26) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Angew. Chem., Int. Ed.
2001, 40, 1093-1096.
(27) Houseman, B. T.; Mrksich, M. Chem. Bio. 2002, 9, 443-454.
(28) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286-4287.
(29) Curreli, M.; Li, C.; Sun, Y. H.; Lei, B.; Gundersen, M. A.; Thompson, M.
E.; Zhou, C. W. J. Am. Chem. Soc. 2005, 127, 6922-6923.
(30) Brown, E. R.; Finley, K. T.; Reeves, R. L. J. Org. Chem. 1971, 36, 2849-
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(31) Chan, E. W. L.; Lee, D. C.; Ng, M. K.; Wu, G. H.; Lee, K. Y. C.; Yu, L.
P J. Am. Chem. Soc. 2002, 124, 12238-12243.
(32) Chan, E. W. L.; Yu, L. P. Langmuir 2002, 18, 311-313.
(33) Horn, T.; Lee, B. B.; Dill, K. A.; Zuckermann, R. N. Bioconjugate Chem.
2004, 15, 428-435.
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A R T I C L E S
Chan and Yousaf
fitted the data for the product oxime peak currents to an
integrated first-order rate law (eq 2). The fit was excellent and
gave a pseudo first-order rate constant of 0.055 min-1. The rate
constants for the decay of the hydroquinone-quinone signal
and the formation of the product oxime are well within
experimental error. This result shows that the quinone monolayer
reacts chemoselectively with aminooxy acetic acid to give the
corresponding oxime on the surface.
Figure 3. Surface plasmon resonance characterization for the selective
immobilization of anti-FLAG onto mixed monolayer presenting tetra-
(ethylene glycol) groups and chemoselectively immobilized FLAG peptides.
The results showed that the antibody associated only to the monolayer
presenting the FLAG peptide ligands (top), whereas the use of a scrambled
peptide ligand (middle) and the addition of soluble FLAG peptide (bottom)
completely inhibited binding.
showed the monolayer presenting hydroquinone groups (1)
underwent a reversible oxidation at 580 mV and reduction at
-30 mV in 1 M perchloric acid. Addition of soluble aminooxy
acetic acid resulted in the loss of peak currents for the quinone
monolayer, and an increase in peak currents corresponding to
the oxidation (450 mV) and reduction (371 mV) of the oxime
product (Figure 2A). To confirm that the product redox-active
peaks are due to the reaction of aminooxy acetic acid with the
quinone monolayer, we independently synthesized the aminooxy
acetic acid tethered quinone alkanethiol. Cyclic voltammograms
of the monolayer for the control molecule showed identical peak
currents as the immobilization product. Furthermore, we used
X-ray photoelectron spectroscopy to show the characteristic
nitrogen peak at 401 eV after surface immobilization of the
aminooxy acetic acid to the quinone monolayer.
Figure 2B shows a plot of the peak currents versus time for
the cyclic voltammograms shown in 2A. We expected the rate
for the loss in peak currents of the quinone monolayer to follow
pseudo first-order kinetics because the aminooxy acetic acid
was present in large excess relative to the immobilized
quinone. The data were fit to an exponential decay (eq 1) to
give a pseudo first-order rate constant (k′) of 0.056 min-1, where
It is the peak current at time t, I0 is the initial peak current and
If is the residual
To demonstrate the utility of this methodology for biological
ligand immobilization, we used the association of anti-FLAG
antibody to a monolayer presenting immobilized FLAG peptide.
We prepared a mixed monolayer containing 1% hydroquinone
groups (2) and 99% tetra(ethylene glycol) groups (3). The
hydroquinone monolayer was electrochemically oxidized at 750
mV for 10 s in phosphate buffer saline (PBS) to give the
corresponding quinone. To demonstrate the reaction was
chemoselective in the presence of complex protein mixtures the
substrates were treated with a 0.1 M solution of an aminooxy
functionalized FLAG peptide (4) in HeLa cell lysates for 4 h
to ensure completion of the ligand immobilization. We char-
acterized the antibody immobilization by surface plasmon
resonance (SPR) spectroscopy. Figure 3 shows SPR sensorgrams
for the selective binding of anti-FLAG (0.1 mg/mL) onto the
monolayer presenting immobilized FLAG peptides. When a
It ) If + (I0 - If) exp-k′t
It ) I0 ‚ (1 - exp-k′t
(1)
(2)
)
nonfaradaic current. To compare the difference in the rates
between the loss of quinone and the formation of oxime, we
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Immobilization of Ligands
A R T I C L E S
of yield of immobilization of the rhodamine-oxyamine within
the printed spots. To show the flexibility of this methodology
a second substrate was printed with rhodamine-oxyamine to
generate larger-sized (250 µm in diameter) spots (Figure 4C).
Both large and small arrayed spots were printed on the same
size substrate with the same number of spots. The only
difference between the substrates is the size of the printed spots.
The larger spots (250 µm) are approximately 4 times greater in
surface area than the smaller (120 µm) printed spots. Compari-
son of the cyclic voltammograms for the smaller (Figure 4B)
and larger (Figure 4C) printed spots shows the difference in
yield to be approximately 4 times larger upon integration of
the peaks and is consistent with the difference in surface area
reacted. Figure 4D shows that upon addition of rhodamine-
oxyamine to the entire surface after printing the surface is
completely converted to the product oxime and characterized
by cyclic voltammetry. These results indicate the use of cyclic
voltammetry to precisely determine the yield of immobilization
and therefore density of ligand on an arrayed substrate. This
strategy can be used with microelectrode arrays to immobilize
multiple ligands with the exact density of each ligand determined
by electrochemistry.
Finally, we extend this immobilization strategy by combining
microarray technology, cell culture and high-throughput mi-
croscopy for cell biological studies and applications. We
prepared a surface composed of a mixed monolayer of 2%
hydroquinone (2) and 98% tetra(ethylene glycol) alkanethiol
(3). To this surface we printed an RGD-oxyamine peptide (7)
(30 mM in PBS, 1 h) and a control scramble peptide GRD-
oxyamine (8) in alternating spots. The RGD peptide is found
in the central binding domain of the extracellular matrix protein
fibronectin and is known to facilitate adhesion through cell
surface integrin receptors.34 Addition of Swiss 3T3 fibroblasts
to the entire surface resulted in cell attachment only to the RGD
spots and not to the control scramble peptide GRD spots (Figure
5). To show the interaction between cell and substrate is
biospecific, i.e., the only interaction between cell and substrate
is a ligand-receptor interaction, soluble RGD peptide was added
to the media (1 mM in PBS) as a competitive inhibitor of the
immobilized peptide to detach the fibroblasts (data not shown).
As a further characterization, fast scan electrochemistry was used
to determine the density of immobilized RGD peptide (data not
shown) and may provide in combination with microarray
technology a new method to interrogate how cells behave on
surfaces where the density of immobilized ligands can be
precisely controlled.
Figure 4. Fluorescent microscopy images of a quinone surface reacted
with rhodamine-oxyamine and characterized by cyclic voltammetry. (A) A
substrate presenting a mixed monolayer of hydroquinone and tetra(ethylene
glycol) groups (1:1). Rhodamine oxyamine was spotted using a microarray
on a quinone monolayer with an average spot diameter size of 120 µm (B)
and 250 µm (C). (D) Complete conversion of the surface to the oxime
product after rhodamine-oxyamine was reacted to the entire quinone
surface.
peptide oxyamine with a scrambled non-binding sequence (5)
was immobilized onto the quinone monolayer, no protein
association to the surface was observed. Furthermore, presatu-
ration of the antibody with soluble FLAG peptide (1 mM)
completely prevented binding of the antibody to the monolayer.
These results confirmed the association of the anti-FLAG to
the immobilized FLAG peptide ligands was biospecific.
We next show the immobilization and electrochemical
characterization strategy is compatible with microarray technol-
ogy. We prepared a mixed monolayer containing 50% hydro-
quinone groups (2) and 50% tetra(ethylene glycol) groups (3).
Cyclic voltammetry showed an oxidation peak at 539 mV and
reduction peak at 322 mV corresponding to the hydroquinone-
quinone redox couple (Figure 4A). A SpotBot microarrayer was
then used to print rhodamine-oxyamine (6) (50 mM in
methanol, 30 min) onto the substrate in 120 µm diameter spots
(Figure 4B). Subsequent cyclic voltammetry of the entire surface
showed characteristic redox active peaks for both the unreacted
surface bearing the hydroquinone-quinone groups (539 and 322
mV) and the printed regions containing electroactive oxime
product (624 and 484 mV). Integration and comparison of the
peaks of the cyclic voltammogram allows for the determination
Conclusion
We have demonstrated a straightforward approach to im-
mobilize ligands onto an electroactive quinone monolayer with
precise control of ligand density using high throughput mi-
croarray technology. We have further applied this methodology
to immobilize peptide ligands for selective protein binding and
cell attachment to inert surfaces. The use of quinone and
oxyamine provides a general route for the preparation of
substrates presenting a variety of biological ligands for biospe-
cific interactions. The redox activity between the quinone and
oxime groups permits the determination of the yield of reaction
and therefore the density of immobilized ligands in real-time
(34) Ruoslahti, E. Annu. ReV. Cell DeV. Biol. 1996, 12, 697-748.
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A R T I C L E S
Chan and Yousaf
Microscopy. Adherent cells were fixed in 3.7% paraformaldehyde
in phosphate buffer saline (PBS) for 10 min and then permeabilized
with 0.1% Triton X in PBS (PBST) for 10 min. Cells were then stained
with anti-tubulin (1:1000) in PBS containing 10% goat serum for 1 h,
followed by Alexa 488-conjugated goat anti-mouse IgG (1:100 in
PBST) and Dapi (1:300 in PBST) for 1 h. Substrates were rinsed with
deionized water before mounted onto glass cover slips for microscopy.
All optical and fluorescent micrographs were imaged using a Nikon
inverted microscope (model TE2000-E). All images were captured
and processed by MetaMorph.
Preparation of Monolayers. All gold substrates were prepared by
electron-beam deposition of titanium (5 nm) and then gold (15 nm for
microarray, 50 nm for electrochemical and spr measurements) on glass
cover slips (75 cm × 25 cm). All gold coated glass substrates were cut
into 1 cm2 pieces and washed with absolute ethanol. The substrates
were immersed in an ethanolic solution containing the alkanethiolates
(1 mM) for 12 h and then cleaned with ethanol prior to each experiment.
Electrochemical Measurements. All electrochemical experiments
were performed using a Bioanalytical Systems CV-100W potentiostat.
Electrochemistry on SAMs was performed in 1 M HClO4, using a
platinum wire as the counter electrode, Ag/AgCl as reference, and the
gold/SAM substrate as the working electrode. All cyclic voltammo-
grams were scanned at 50 mV/s.
Figure 5. A fluorescent image of attached cells on a surface arrayed with
immobilized RGD and GRD peptides. Cells were stained for microtubules
and nuclei. Both peptides (30 mM in PBS) were printed by a microarray in
alternating spots. The dotted circles represent the position where the control
GRD peptide was immobilized. Cells attached only to the spots presenting
the RGD peptide. A fluorescent image of attached cells on a RGD spot
indicated by a dotted square (inset) is shown at higher magnification.
Kinetic Measurements. The kinetic experiment was performed in
a glass cylindrical cell with stirring in the electrolyte and fitted with a
gold substrate presenting hydroquinone terminated alkanethiol. Hyd-
roquinone was electrochemically oxidized to the corresponding quinone.
Aminooxy acetic acid hydrochloride (150 mM) was then added to the
electrolyte to initiate the kinetic experiment. Cyclic voltammograms
were recorded in 5 min intervals to monitor the extent of the reaction
in-situ.
by cyclic voltammetry. The selective coupling between the
aminooxy groups to the oxidized quinone monolayer makes
possible the modulation of ligand activity by an electrochemical
potential. The immobilization strategy shown here, in combina-
tion with inert surfaces, may be used with microelectrode arrays
and microfluidic technology to generate complex biosensors with
massively parallel analysis. We believe this methodology will
provide a broad range of tailored substrates for new studies in
attached cell culture and applications in biotechnology.
Surface Plasmon Resonance Spectroscopy. All SPR measurements
were conducted using a Biacore 2000 instrument. Monolayer substrates
were incorporated into the Biacore cassettes by removing of the
manufacturer’s substrate and then fastened onto the cassettes with
double side tape (3 M). Measurements were performed with Dulbecco’s
phosphate-buffered saline (pH 7.4) as the running buffer and reported
as changes in resonance unit (R.U.).
Experimental Section
All the solvents for the synthesis were HPLC grade. THF was
distilled from sodium benzophenone under nitrogen before use. Absolute
ethanol was purchased from Aaper Alcohol Chemical Company. Flash
chromatography was carried out using silica gel (230-400 mesh). All
amino acids and resin were purchased from Anaspec, Inc. (La Jolla,
CA). All reagents were purchased from Aldrich and used as received.
All reagents used in cell culture were obtained from Gibco BRL.
X-Ray Photoelectron Spectroscopy. XPS characterization was
carried out on an AXIS HIS 165 and ULTRA Spectrometer, using Al
KR radiation (energy 1486.6 eV) in a vacuum of 5 × 10-9 Torr.
Cell Culture. Swiss Albino 3T3 cells (ATCC) were cultured in
Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10%
calf bovine serum and penicillin/streptomycin. Cells were removed with
a solution of 0.05% trypsin/0.53 mM EDTA, resuspended in serum-
free culture medium (10,000 cells/mL), and plated onto the SAM
substrates. After 2 h, the substrates were placed in serum containing
media and maintained at 37 °C in a humidified 5% CO2 atmosphere.
Synthesis of Alkanethiolates. Alkanethiolates terminated in the
hydroquinone groups (1, 2) and the tetra(ethylene glycol) group (3)
were prepared as previously described.20,35
Synthesis of Rhodamine Oxyamine. Rhodamine oxyamine was
synthesized in four steps. Rhodamine sulfonyl chloride was coupled
to N-Boc diaminobutane. Removal of the Boc group by trifluoroacetic
acid (TFA) gives the corresponding rhodamine butylamine. The
resulting amine was then reacted with N-Boc aminooxy acetic acid in
the presence of dicyclohexylcarbodiimide/N-hydroxysuccinimide. Boc
group deprotection by TFA gives the corresponding rhodamine
oxyamine conjugate.
Microarray. All molecules were printed onto monolayer substrates
using a SpotBot 2 microarrayer.
Acknowledgment. This work was supported by the Carolina
Center for Cancer Nanotechnology Excellence and grants from
the NIH to MNY and the Burroughs Wellcome Foundation
(Interface Career Award).
Solid-Phase Peptide Synthesis. All aminooxy terminated peptides
were synthesized using a peptide synthesizer (CS Bio) at 2 mM. To
cleave the peptide, the resin was placed in a solution of TFA containing
20% water and 10% phenol for 2 h, and filtered. The filtrate was
concentrated in vacuo to give a yellow oil, and then precipitate in cold
ether to give a white solid.
Supporting Information Available: Cyclic voltammogram
of the monolayer presenting the control molecule; XPS char-
acterization for the surface immobilization. This material is
available free of charge via the Internet at http://pubs.acs.org.
(35) Dillmore, W. S.; Yousaf, M. N.; Mrksich, M. Langmuir 2004, 20, 7224-
7231.
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