Site-selective chemistry and the attachment of peptides to the surface of a microelectrode array

Article abstract of DOI:10.1021/ja308121d

Peptides have been site-selectively placed on microelectrode arrays with the use of both thiol-based conjugate additions and Cu(I)-coupling reactions between thiols and aryl halides. The conjugate addition reactions used both acrylate and maleimide Michae

Full text of DOI:10.1021/ja308121d

Article
pubs.acs.org/JACS
Site-Selective Chemistry and the Attachment of Peptides to the
Surface of a Microelectrode Array
Melissae Stuart Fellet, Jennifer L. Bartels, Bo Bi, and Kevin D. Moeller*
Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States
S
* Supporting Information
ABSTRACT: Peptides have been site-selectively placed on microelectrode arrays with the use of both thiol-based conjugate
additions and Cu(I)-coupling reactions between thiols and aryl halides. The conjugate addition reactions used both acrylate and
maleimide Michael acceptors. Of the two methods, the Cu(I)-coupling reactions proved far superior because of their
irreversibility. Surfaces constructed with the conjugate addition chemistry were not stable at neutral pHs, especially the surface
using the maleimide acceptor. Once a peptide was placed onto the array, it could be monitored in “real-time” for its interactions
with a biological receptor.
INTRODUCTION
■
Microelectrode arrays have great potential as tools for
monitoring the interactions of small molecules and biological
receptors in “real-time”.1−3 Typically, when microelectrode
arrays are used as bioanalytical sensors, the surface of the array
is functionalized with a receptor or antibody, and then the array
is used to identify ligands for the receptor in solution.4−6 We
have been working to reverse this approach so that the
electrochemical impedance experiments can be used to probe
the binding of small-molecule libraries to receptors as those
events occur. To accomplish this task, the molecules in the
molecular library must be built or placed onto the micro-
electrode array so that each unique member of the library is
located next to a unique, individually addressable micro-
electrode in the array.7 The microelectrodes are then used to
Figure 1. Planned impedance experiment.
monitor interactions that involve molecules in the library and a
receptor as illustrated in Figure 1.8
One of the most attractive features of this experiment is that
In this experiment, a current is established at each electrode
in the array by the addition of an iron redox couple to the
solution above the array. The iron(II) species in the couple is
oxidized at the electrodes in the array (the anode), and the
iron(III) species is reduced to iron(II) at a remote cathode.
When a receptor in solution (green ball) binds to one of the
molecules in the library on the array, it impedes the iron(II)
from reaching the array. The result is a drop in the current
measured at the associated electrode that both signals the
binding event and identifies the molecule involved.
it can be used to rapidly provide biological data for a newly
synthesized molecule without the need for a labeled receptor or
immunological assay. For example, if one has a library of
molecules that target a particular receptor already on an array,
then a new molecule can be added to the existing library, the
impedance experiment conducted with the receptor in
question, and the binding-data obtained for the new molecule
Received: August 15, 2012
Published: September 19, 2012
© 2012 American Chemical Society
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immediately compared with data gathered for the existing
library. These data can then be used to guide subsequent
synthetic efforts. Of course, key to this experiment is the ability
to place a newly synthesized molecule on the array so that it is
located exclusively on the electrodes used to monitor its
behavior.
in Scheme 2. In this case, the base-catalyzed esterification
reaction between the activated acrylate ester and an agarose
Scheme 2
With this in mind, we have been developing methods for site-
selectively adding peptides and peptidomimetics to the surface
of a microelectrode array.8b The focus on small peptides and
peptidomimetics is motivated by the overall utility of the
molecules as probes for interrogating a variety of different
biological targets.9
AN INITIAL APPROACH
■
The initial plan called for the use of a thiol-based conjugate
addition strategy to add the peptides to a polymer surface-
coating on the arrays (Scheme 1).8b Our hope was to convert a
Scheme 1
coating was used to place the “Michael acceptor” by every
microelectrode in a 1K-array (an array that has 1028
microelectrodes/cm2.11 The conjugate addition with the thiol
nucleophile was then conducted by the generation of base at
every other electrode in the array. The nucleophile was labeled
with a pyrene moiety so that its placement on the array could
be monitored with a fluorescence microscope. Following each
reaction, the array was washed with an excess of ethanol, water,
and then ethanol again. After the washing steps, the array was
allowed to dry. As shown in the image provided, the conjugate
addition reaction happened to a nearly equal extent at every
microelectrode in the array. There was no difference between
electrodes selected for the conjugate addition and those that
were not.
Fortunately, the base-catalyzed first step in the sequence can
be confined.12 The reaction works by reducing vitamin B12 to a
radical anion that in turn deprotonates alcohols on the agarose
surface. The alkoxides then react with the solution-phase-
activated ester, a reaction that consumes the alkoxide and
generates a solution that is not basic enough to catalyze
additional esterification reactions. Of course, the base being
generated at the selected electrodes can migrate to other
regions on the array if either the reduced vitamin B12 or the
alkoxides on the surface of the array deprotonate methanol.
Hence, to confine the esterification to selected electrodes on
the array requires a confining agent in solution that can
consume methoxide. To this end, the use of excess activated
ester is ideal (Scheme 3). In the experiment shown, a single
electrode in a 12K-array (an array that has 12,544 micro-
electrodes/cm2) was used as a cathode to conduct the
esterification reaction with the activated ester. The entire
array was then used to generate base and trigger the thiol-
derived conjugate addition reaction. Clearly, the addition
occurred at only the microelectrode used for the esterification.
The conjugate addition strategy can also be used to place
peptides on the arrays (Scheme 4).
well-known approach for functionalizing polymers10 into a site-
selective reaction on the array. Initially, two strategies were
followed. One placed a maleimide group onto the array, and the
second placed an acrylate moiety (as illustrated [Scheme 1])
onto the array. In either case, the surface of the array needed to
be coated with a porous reaction layer. This reaction layer
provides attachment sites for fixing substrates to the surface of
the electrodes. For the exploration of new array-based
reactions, agarose is an ideal surface. It is both stable to a
host of chemical reagents and easily removed from the surface
of the array following the completion of a reaction. This last
point is important because it allows for the arrays to be
recycled. In this way, a single microelectrode array can be used
to probe a variety of different reaction conditions.
To conduct the sequence outlined in Scheme 1 in a site-
selective fashion, the initial placement of the “Michael acceptor”
on the surface of the array needs to be confined. This is
required because thiol-derived conjugate addition reactions are
base-initiated chain reactions in that the product from the
conjugate addition reaction is a strong enough base to
deprotonate the thiol substrate and regenerate the nucleophile.
Hence, the thiolate is not consumed and can migrate to other
sites on the array. For example, consider the reaction illustrated
In the reaction shown, a fluorescently labeled RGD-peptide
(1) was placed by 10-electrodes in a checkerboard pattern on a
1K-array. The success of the reaction was monitored with a
fluorescence microscope. The RGD-peptide-functionalized
array allowed us to demonstrate the utility of the electro-
chemical impedance experiment suggested in Figure 1.8b
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Scheme 3
Scheme 5
Scheme 4
nucleophile was added to the solution during this second
incubation period. A fluorescence microscope was then used to
take a second image of the arrays (Figure 2).
Figure 2. (a) Maleimide as the Michael acceptor, pH = 7; (b)
maleimide, pH = 4; (c) acrylate as the Michael acceptor, pH = 7; (d)
acrylate, pH = 4.
PROBLEMS WITH REVERSIBILITY AND
■
CONVERSION
As can be seen in a and b of Figure 2, when maleimide was
used as the electron-poor olefin, the conjugate addition was
reversible at pH = 7. This conclusion was reached because the
only source of fluorescence during the second 45-min
incubation period of the experiment was the peptide originally
attached to the checkerboard pattern of electrodes at the
bottom of the image. Hence, the fluorescence observed at the
“dot in a box pattern” was derived from the originally
functionalized electrodes. Incubation at a pH = 4 reduced the
amount of the migration but did not stop it. The use of an
acrylate group as the “Michael acceptor” led to less of a
migration at a pH = 7, but it clearly still occurred (Figure 2c).
Fortunately, with the acrylate group the migration could be
minimized nicely with the use of pH = 4 conditions (Figure
2d). For this reason, an acrylate group was used as the “Michael
acceptor” on the array for all subsequent studies. With that said,
the instability of the maleimide-based system under all of the
conditions examined is worrisome in light of its popularity as an
attachment strategy for making bioconjugates.
Yet, while the thiol-Michael reaction worked and allowed us to
conduct an initial signaling study on the array, it was far from
ideal. Thiol-based conjugate addition reactions are reversible,
and on the array the reverse reaction proved difficult to stop.
This compromised the stability of the functionalized surface.
The two experiments shown in Scheme 5 highlight this
problem. In the first, an acrylate group (2) was used as the
electron-poor olefin for the conjugate addition. In the second, a
maleimide group was used. In both cases, an initial base-
catalyzed esterification was used to place the olefin onto a 12K-
array in a checkerboard pattern. A conjugate addition reaction
was then used to add the RGD-peptide derivative (1) to those
sites on the array. For the conjugate addition reaction, we
found that it was not necessary to pass any current through the
cell. Simply incubating the array with the peptide in a 5×PBS,
pH = 7 buffer, for 45 min was enough to accomplish the thio-
Michael reaction. A fluorescence image of the array was taken
to ensure that the reaction proceeded at the selected electrodes.
Next, a second set of electrodes was functionalized with the
electron-poor olefin. This time a “dot in a box pattern” of
electrodes was used. The arrays were then incubated for 45 min
in a 5×PBS buffer solution at either pH 4 or pH 7. No
Since more basic reaction conditions did seem to favor the
migration reaction, we worried that some migration might
occur during the based-catalyzed esterification reaction used to
place the “Michael acceptor” by a second set of electrodes on
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the array. Such a migration would occur if any of the base
generated at the selected electrodes for the second placement
reaction migrated to electrodes already functionalized with
peptide 1. While the experiment leading to Figure 2d suggested
that this was not a problem, a more careful experiment that
examined just the second acrylate placement reaction was
conducted. In this experiment, the chemistry shown in Scheme
5 was repeated on a 12K-array, and then the array was
examined after the second acrylate placement reaction but
before the incubation step. The first acrylate and hence peptide
1 was placed on the array in a “checkerboard within a box”
pattern. The fluorescence image presented in Figure 3a was
Scheme 6
conjugate addition was conducted at a pH of 4 so that
migration of thiol groups that were attached to the array would
not occur. Hence, any fluorescence that occurred at the first site
would be the result of unreacted acrylate at that site undergoing
a conjugate addition reaction during the second procedure. The
results of the experiment are shown in Figure 4. Figure 4a
Figure 3. (a) Initial placement of acrylate onto the array followed by a
Michael reaction with peptide 1. (b) Site of the second acrylate
placement on the array.
then acquired in order to assess the quality of this placement
reaction. The first step of the procedure was then repeated at a
second location on the array. This time, the pattern used was a
set of “parallel lines in a box”. The pattern was selected so that
there would be no confusion as to which reaction gave rise to
which pattern. An image of the array was then taken with a
fluorescence microscope to see if any of the fluorescently
labeled peptide migrated from the checkerboard pattern to the
parallel lines of acrylate placed on the array. Very faint
fluorescence did appear at the second site (Figure 3b). A
quantitative comparison of the fluorescence intensity at the two
locations on the array indicated that the line pattern had about
0.3 0.3% of the intensity of the checkerboard pattern. Clearly,
very little migration of the peptide occurred during the second
acrylate placement.
A third issue with the conjugate addition approach was the
“completeness” of the initial reaction. As illustrated in Scheme
2, thiol-based conjugate addition reactions are base-initiated
chain reactions. Once initiated at any electrode in an array, they
occur at every site on the array that has been previously
functionalized with a “Michael acceptor”. This can be
problematic with respect to the placement of multiple
molecules on an array. If a conjugate addition to place one
molecule onto an array does not go to completion, then
unreacted “Michael acceptor” will remain proximal to the
electrodes. A thiol-based conjugate addition conducted to place
a second molecule at a different site on the array will then place
some of the second molecule by the original electrodes as well.
The result would be a mixture of molecules by the original
electrode.
Figure 4. (a) Reaction of peptide 1 with acrylate at the second site on
an array.(b) Reaction of peptide 1 with unreacted acrylate at the first
site on an array following incubation with 2-mercaptoethanol.
shows the “dot in a box” pattern generated by the second
conjugated addition reaction with peptide 1. The reaction
proceeded nicely and was well confined with no migration to
the surrounding electrodes. However, a small amount of
fluorescence was observed at the initial checkerboard pattern of
electrodes (Figure 4b). The initial conjugate addition had not
gone to completion. A quantitative measure of the fluorescence
image from the two sites indicated that the reaction at the initial
site (checkerboard) proceeded to an extent of 3 2% of the
reaction at the second site. While the amount of fluorescence at
the initial site was small, its presence did indicate that a
capping-step would be needed at the site of the initial conjugate
addition reaction in order to consume any unreacted Michael
acceptor at that location.
The reversibility of the thiol-based conjugate addition
reaction was an even greater problem with the use of an
amine nucleophile (Scheme 7). In this case, the nucleophile
placed on the surface of the array was sensitive to the base
conditions used to add the electron-poor olefin to the array.
The experiment illustrated began with the placement of peptide
2 onto a 12K-array with the use of a lysine side chain and an
acrylate “Michael acceptor” on the array. The acrylate on the
array was placed down in a “checkerboard in a box” pattern in
the manner outlined above. The conjugate addition reaction
was then conducted with the pH = 4 conditions employed for
the previous thiol-based addition. The success of the reaction
In order to determine if this was a problem, one site on an
array was functionalized with a checkerboard pattern of the
acrylate moiety (Scheme 6). The array was then incubated with
a 10 mM solution of 2-mercaptoethanol in 5×PBS at a pH = 6
for 45 min. A second area of the array was then functionalized
with the acrylate in a “dot in a box” pattern. The electrodes
used in this second pattern were then employed to initiate a
conjugate addition of peptide 1 to the acrylate moiety in a
fashion identical to that illustrated in Scheme 4. This second
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or an unprotected N-terminus. For this reason, the reaction
needs to be conducted with either a peptide containing an
unactivated olefin,15 or performed by modifying the surface of
the array so that it contains the olefin and then modifying the
peptide with the necessary aryl halide. In a similar fashion, the
use of a Suzuki reaction would require either the addition of a
phenylboronic acid to the peptide or modification of the surface
to contain the phenylboronic acid and incorporation of an aryl
halide into either the polymer or the peptide.
Scheme 7
On the other hand, Cu(I)-catalyzed coupling reactions
require no modification of the peptide. The site-selective
additions of alcohol-, amine-, and thiol-based nucleophiles to
aryl bromide-functionalized diblock copolymers16 have all been
shown to work on both 1K- and 12K-arrays.14 Hence, with the
use of Cu(I) the same peptide substrates synthesized for the
conjugate addition chemistry can potentially be used to place
molecules on the array in an irreversible fashion. The method
offers additional advantages in that Cu(I) reactions show
ligand-dependent chemoselectivity of heteroatom-/aryl halide-
coupling reactions.17 In this way, the method has the potential
to selectively place peptides onto arrays with alcohol- and thiol-
containing side chains even if the N-terminus is not protected.
Preliminary results indicate that such selectivity is possible with
1,3-dicarbonyl ligands for Cu(I) leading to much faster
reactions between thiol nucleophiles and arylbromides on the
surface of an array relative to reactions using alcohol and amine
nucleophiles.14b
was verified with a fluorescence microscope. The acrylate
placement reaction was then repeated with a second set of
electrodes in a “parallel lines in a box” pattern. This reaction led
to faint fluorescence at this second site, fluorescence that could
only arise from migration of the peptide previously placed by
the electrodes used in the checkerboard pattern.
However, is the site-selective generation of Cu(I) on the
array compatible with more complex peptide substrates that can
serve to chelate the metal? In order to test this idea, peptide 3
was placed on a 12K-microelectrode array that had been
precoated with a diblock copolymer (Scheme 9). The diblock
A SOLUTION USING SITE-SELECTIVE Cu(I)
■
CATALYSIS
While the reversibility of the conjugate addition of a thiol
nucleophile can be stopped by using a pH = 4, this requirement
was not compatible with the more biologically relevant
conditions needed for signaling studies using intact proteins.
At neutral pHs, a library placed on an array would scramble.
What was needed was a nonreversible method for placing
peptides and peptidomimetics onto an array. To this end, a
transition metal-based option appeared ideal. Both Pd(0)- and
Cu(I)-coupling reactions have proven to be very useful
synthetic tools for site-selectively adding new molecules to
microelectrode arrays.13,14 Of these two approaches, the Cu(I)-
based method illustrated in Scheme 8 is particularly attractive.
Scheme 9
Scheme 8
copolymer was composed of a cinnamoyl-functionalized
methylmethacrylate block used to coat the array and then
provide stability to the surface after photochemical-cross-
linking, and a 4-bromostyrene was used to provide a coupling
partner for the Cu(I)-coupling reaction. The functionalized
array was submerged in a 7:2:1 acetonitrile/dimethylformade/
water solution that contained the peptide, copper sulfate, a 1,3-
dicarbonyl ligand for the copper, and tetrabutylammonium
bromide as an electrolyte. Selected electrodes in the array were
then used as cathodes by setting them to a potential of −1.7 V
While both Pd(0) Heck and Suzuki strategies work
beautifully on the arrays, they require modification of both
the peptide and the surface prior to the placement reaction. For
a Heck reaction, the peptide must be functionalized with an
olefin, and the surface, with an aryl bromide. Frequently, Heck
reactions employ activated olefins. However, the inclusion of an
activated olefin in the peptide can lead to its polymerization,
especially if the peptide contains either a nucleophilic side chain
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relative to a remote Pt anode. The electrodes were turned on
for two periods of 90 s each in order to give rise to the image
shown. Oxygen was used in the solution as a ‘confining-agent”
(oxidant for Cu(I)) in order to prevent migration of the Cu(I)-
catalyst to electrodes not selected for the reaction. Upon
completion of the reaction, the array was washed with an excess
of ethanol and DMF and then allowed to dry. Because the
coupling reaction is catalyzed by Cu(I) and not a “chain-
reaction” as for the conjugate addition, there was no reaction at
any site on the array that was not used for the generation of
Cu(I). For the experiment described, 10 blocks of 12 electrodes
each were used for the reduction. The fluorescence image
shown in Scheme 9 shows one of these blocks of electrodes
along with the surrounding electrodes. The very high level of
selectivity for the electrodes used is clearly evident.
Figure 6. Summary of cyclic voltammetry data.
SIGNALING
■
With the placement of the peptide on the array complete, two
questions immediately arose. Was the peptide available for
signaling studies, and was the new surface stable enough for us
to conduct the analytic experiment multiple times to establish a
relationship between binding on the surface of the array to the
concentration of receptor in solution? Answers to these
questions were probed with the use of an integrin receptor.
To this end, the array made in Scheme 9 was functionalized
with a second, non-RGD-peptide, 4. This second peptide was
also placed on 10 blocks of 12 electrodes each. The array was
then incubated with various concentrations (10−15 to 10−5 M)
of the integrin receptor αIIbβIII in a phosphate buffer solution
(1×PBS, pH ≈ 7.4) that also contained a 1:1 mixture of
K3[Fe(CN)6] and K4[Fe(CN)6]. For each concentration of the
receptor, a cyclic voltammogram was recorded for the iron
redox couple. On an array, there is no reference electrode.
Hence, the potentials measured represent the potential at the
electrode on the array relative to the Pt-counterelectrode
located 0.9 mm away.18 The data obtained for one of the blocks
of 12 electrodes functionalized with the RGD-peptide 3 are
shown in Figure 5. Note how the peak current at 30 mV for the
cyclic voltammogram decreases as the concentration of integrin
receptor in solution increases. This decrease in current reflects
the binding of integrin to the peptide on the polymer coating
the electrodes. The data for three blocks of the electrodes
functionalized with peptide 3 are summarized in Figure 6
(black line). Each data point on the line represents the average
current recorded for three of the blocks of electrodes at 30 mV.
The error bars indicate the spread in the data for the three
points.
Figure 6 also includes the summary for data obtained at the
electrodes functionalized with non-RGD-peptide 4 (green line)
and electrodes that were not functionalized at all (red line).
Little impedance was observed at the electrodes functionalized
with 4, indicating a low-level nonspecific binding to the
functionalized surface of the array. The steady climb in
impedance at the sites functionalized with 4 without any
leveling off of the curve suggested that the nonspecific binding
that was observed was a result of the functionalized surface and
not peptide 4. There is only 10−50 fmole of peptide bound to
the surface of the electrode, and thus binding to the peptide
itself would lead to a leveling off of the impedance with
increasing concentration of the receptor. This leveling off of the
impedance was observed with the binding of the receptor to
peptide 3. At the unfunctionalized electrodes, no binding was
observed, indicating that functionalization of the surface did
increase the level of nonspecific binding of the integrin receptor
to the polymer surface. The level of impedance observed for the
electrodes functionalized with 3 relative to those functionalized
with 4 clearly demonstrated that the integrin receptor
recognized the RGD-peptide on the surface.
Binding of the integrin receptor to the RGD-peptide on the
surface of the array was reversible. Incubating the chip used to
obtain the data shown in Figure 6 with a buffer solution led to
recovery of the current at the electrodes functionalized with the
RGD-peptide. Reintroducing the integrin receptor then caused
the current to drop again.
At this time, it is not known why the impedance
measurement made on the array is more sensitive than the
typical nanomolar binding constant associated with RGD−
integrin binding. Now that we have a method for placing
peptides irreversibly on an array, efforts to understand this
effect, to develop the use of the arrays for quantitative
measurements, and to explore the chemistry of libraries with
the arrays can commence.
CONCLUSIONS
■
We have found that peptides can be placed site-selectively on
microelectrode arrays with the use of either thiol-based Michael
chemistry or Cu(I)-catalyzed coupling reactions. Both reactions
take advantage of a cysteine in the peptide to provide the
Figure 5. CV data for one block of electrodes functionalized with
RGD-peptide 3.
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Richardson, A. VLSI Design 2008, 9 pages DOI: 10.1155/2008/
437879. (i) Zhang, Y.; Wnag, H.; Nie, J.; Zhang, Y.; Shen, G.; Yu, R.
Biosens. Bioelectron. 2009, 25, 34. (j) Maurer, K.; Yazvenko, N.;
Wilmoth, J.; Cooper, J.; Lyon, W.; Danley, D. Sensors 2010, 10, 7371.
(k) Li, X.; Tian, Y.; Xia, P.; Luo, Y.; Rui, Q. Anal. Chem. 2009, 81,
8249. (l) Chan, E. W. L.; Yousaf, M. N. ChemPhysChem 2007, 8, 1469.
nucleophile for the reaction. The conjugate-addition strategy
was confined by controlling the placement of the “Michael
acceptor” on the array. These reactions were problematic
because of their reversibility. When an acrylate acceptor was
used on the array, the retro-conjugate addition could be
stopped when the reactions were run at a pH = 4. For a
maleimide acceptor, the reversibility of the conjugate addition
could not be stopped. The use of a Cu(I)-catalyzed addition of
the thiol nucleophile to a arylbromide surface on the array did
not suffer from these issues. The reactions were confined with
the use of air as an oxidant for the Cu(I)-catalyst generated on
the array. With this chemistry, an RGD-peptide was site-
selectively placed on a 12K-array along with a second non-
RGD-peptide. Once there, binding of the peptide to its integrin
receptor was monitored in “real-time” with the use of an
electrochemical impedance experiment. The work demon-
strates the potential for microelectrode arrays as a platform for
analyzing peptide-based molecular libraries.
(4) For reviews see: (a) Gyurcsan
́ ́
yi, R. E.; Jagerszki, G.; Kiss, G.;
Toth, K. Bioelectrochemistry 2004, 63, 207. (b) Roberts, W. S.;
́
Lonsdale, D. J.; Griffiths, J.; Higson, S. P. J. Biosens. Bioelectron. 2007,
23, 301. (c) Scanning Electrochemical Microscopy, 2nd ed.; Bard, A. J.,
Mirkin, M. V., Eds.; Marcel Dekker, Inc.: New York, NY, 2001.
(5) For examples using macroscopic electrodes see: (a) Dykstra, P.
H.; Roy, V.; Byrd, C.; Bentley, W. E.; Ghodssi, R. Anal. Chem. 2011,
83, 5920. (b) Murata, M.; Gonda, H.; Yano, K.; Kuroki, S.; Suzutani,
T.; Katayama, Y. Bioorg. Med. Chem. Lett. 2004, 14, 137.
(6) For related references with ion-channel mimic sensors see:
(a) Endo, A.; Hyashita, T. J. Ion Exch. 2008, 19, 110. (b) Wirtz, M.;
Martin, C. R. Sensors Update 2002, 11, 35. (c) OdashimaK.Sugawar-
aM.UmezawaY. Channel Mimetic Sensing Membranes Based on
Host−Guest Molecular Recognition by Synthetic Receptors. ACS
Symposium Series 561; American Chemical Society: Washington, DC,
1994; p 123 (d) Yue, M.; Zhu, X.; Zheng, Y.; Hu, T.; Yang, L.; Wu, X.
Electrochim. Acta 2012, 73, 78. (e) Malecka, K.; Grabowska, I.;
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental and characterization data for all substrates
and products; copies of proton and carbon NMR spectra are
included. This material is available free of charge via the
Internet at http://pubs.acs.org.
́
Radecki, J.; Stachyra, A.; Gora-Sochacka, A.; Sirko, A.; Radecka, H.
Electroanalysis 2012, 24, 439. (f) Zhy, J.; Qin, Y.; Zhang, Y. Anal.
Chem. 2010, 82, 436. (g) Xu, Y.; Bakker, E. Langmuir 2009, 25, 568.
(h) Kurzatkowska, K.; Dolusic, E.; Dehaen, W.; Sieron
́
-Stoltny, K.;
Sieron, A.; Radecka, H. Anal. Chem. 2009, 81, 7397. (i) Komura, T.;
́
Yamaguchi, T.; Kura, K.; Tanabe, J. J. Electroanal. Chem. 2002, 523,
126. (j) Odashima, K.; Kotato, M.; Sugawara, M.; Umezawa, Y. Anal.
Chem. 1993, 65, 927.
(7) For recent examples and a list of references to earlier work see:
(a) Tanabe, T.; Bi, B.; Hu, L.; Maurer, K.; Moeller, K. D. Langmuir
2012, 28, 1689. (b) Bi, B.; Huang, R. Y. C.; Maurer, K.; Chen, C.;
Moeller, K. D. J. Org. Chem. 2011, 76, 9053.
AUTHOR INFORMATION
Corresponding Author
moeller@wustl.edu
■
Notes
The authors declare no competing financial interest.
(8) For examples see reference 7a as well as: (a) Tesfu, E.; Roth, K.;
Maurer, K.; Moeller, K. D. Org. Lett. 2006, 8, 709. (b) Stuart, M.;
Maurer, K.; Moeller, K. D. Bioconjugate Chem. 2008, 19, 1514.
(9) (a) Cain, M.; Kulkarni, V. V.; Hruby, J. J. Peptides and
Peptidomimetics. In Textbook of Drug Design and Discovery, 4th ed;
Krogsgaard-Larsen, P., Stroemgaard, K., Madsen, U., Eds.; CRC Press:
Boca Raton, 2010; p 123. (b) Vagner, J.; Qu, H.; Hruby, V. J. Curr.
Opin. Chem. Biol. 2008, 12, 292.
(10) For a review see: Lowe, A. B. Polym. Chem. 2010, 1, 17.
(11) For the use of base-catalyzed esterification reactions to place
molecules onto an array see: (a) Tesfu, E.; Roth, K.; Maurer, K.;
Moeller, K. D. Org. Lett. 2006, 8, 709. (b) Tesfu, E.; Maurer, K.;
Ragsdale, S. R.; Moeller, K. D. J. Am. Chem. Soc. 2004, 126, 6212.
(c) Tesfu, E.; Maurer, K.; McShae, A.; Moeller, K. D. J. Am. Chem. Soc.
2006, 128, 70.
(12) For a preliminary account of this work please see reference 8b.
(13) For the use of Pd(0) to place molecules onto an array see:
(a) Tian, J.; Maurer, K.; Tesfu, E.; Moeller, K. D.. J. Am. Chem. Soc.
2005, 127, 1392. (b) Tian, J.; Maurer, K.; Moeller, K. D. Tetrahedron
Lett. 2008, 49, 5664. (c) Hu, L.; Maurer, K.; Moeller, K. S. Org. Lett.
2009, 11, 1273. (d) Hu, L.; Stuart, M.; Tian, J.; Maurer, K.; Moeller, K.
D. J. Am. Chem. Soc. 2010, 132, 16610.
(14) For the use of Cu(I) to place molecules onto an array see:
(a) Bartels, J. L.; Lu, P.; Walker, A.; Maurer, K.; Moeller, K. D. Chem.
Commun. 2009, 5573. (b) Bartels, J.; Lu, P.; Maurer, K.; Walker, A. V.;
Moeller, K. D. Langmuir 2011, 27, 11199.
ACKNOWLEDGMENTS
■
We thank the National Science Foundation (CHE-0809142)
for their generous support of our work. We also gratefully
acknowledge the Washington University High Resolution
NMR facility, partially supported by NIH Grants RR02004,
RR05018, and RR07155, and the Washington University Mass
Spectrometry Resource Center, partially supported by
NIHRR00954, for their assistance.
REFERENCES
■
(1) For a description of the chips used here see: Dill, K.;
Montgomery, D. D.; Wang, W.; Tsai, J. C. Anal. Chim. Acta 2001,
444, 69 1k chips: electrode diameter = 92 μm; distance between the
Pt-electrodes (rectangular cells) = 245.3 and 337.3 μm; 12k slide:
diameter = 44 μm; distance between the Pt-electrodes (square cells) =
33 μm..
(2) Microelectrode arrays can be purchased from CustomArray, Inc.,
18916 North Creek Parkway, Suite 115, Bothell, WA 98011 (www.
CustomArrayInc.com).
(3) For alternative approaches see: (a) Sullivan, M. G.; Utomo, H.;
Fagan, P. J.; Ward, M. D. Anal. Chem. 1999, 71, 4369. (b) Zhang, S.;
Zhao, H.; John, R. Anal. Chim. Acta 2000, 421, 175. (c) Hintsche, R.;
Albers, J.; Bernt, H.; Eder, A. Electroanalysis 2000, 12, 660.
(d) Gardner, R. D.; Zhou, A.; Zufelt, N. A. Sens. Actuators, B 2009,
136, 177. (e) Beyer, M.; Nesterov, A.; Block, I.; Konig, K.;
̈
(15) Lambert, J. D.; Rice, J. E.; Hong, J.; Hou, Z.; Yang, C. S. Bioorg.
Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann,
M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling,
F. Science 2007, 318, 1888. (f) Devaraj, N. K.; Dinolfo, P. H.; Chidsey,
C. E. D.; Collman, J. P. J. Am. Chem. Soc. 2006, 128, 1794.
(g) Wassum, K. M.; Tolosa, V. M.; Wang, J.; Walker, E.;
Monbouquette, H. G.; Maidment, N. T. Sensors 2008, 8, 5023.
(h) Kerkoff, H. G.; Zhang, X.; Mailly, F.; Nouet, P.; Liu, H.;
Med. Chem. Lett. 2005, 15, 873−876.
(16) Hu, L.; Bartels, J. L.; Bartels, J. W.; Maurer, K.; Moeller, K. D. J.
Am. Chem. Soc. 2009, 131, 16638 The cross-linked polymer has pore
sizes on the order of 19 3 nm..
(17) Shafir, A.; Lichtor, P. A.; Buchwald, S. L. J. Am. Chem. Soc. 2007,
129, 3490.
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Journal of the American Chemical Society
Article
(18) A detailed description of the electrochemical apparatus used and
procedures for the experiments are provided in the Supporting
Information.
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