Surface patterning with fluorescent molecules using click chemistry directed by scanning electrochemical microscopy

Article abstract of DOI:10.1021/ja078183d

Scanning electrochemical microscopy (SECM)-based surface patterning can be achieved to form useful μm size structures for various kinds of applications. Here we report a novel SECM-based surface patterning of fluorescent molecules onto a solid substrate b

Full text of DOI:10.1021/ja078183d

Published on Web 02/02/2008
Surface Patterning with Fluorescent Molecules Using Click Chemistry
Directed by Scanning Electrochemical Microscopy
Sung-Yu Ku, Ken-Tsung Wong,* and Allen J. Bard*
Center for Electrochemistry, Department of Chemistry and Biochemistry, UniVersity of Texas at Austin, Austin,
Texas 78712, and Department of Chemistry, National Taiwan UniVersity, Taipei 106, Taiwan
Received October 25, 2007; E-mail: ajbard@mail.utexas.edu; kenwong@ntu.edu.tw
We introduce a new approach to pattern fluorescent molecules,
which do not participate in an electrochemical reaction themselves,
onto an azido functionalized glass substrate using “click” chemistry
directed by scanning electrochemical microscopy (SECM). Our
technique extends the surface patterning ability of SECM to the
level at which any inert small molecule or macromolecule can be
grown on an insulating substrate through a click chemistry reaction
to form a covalent bond directly under a microelectrode.
SECM is a powerful tool for studying local electrochemical and
chemical reactions and the kinetics of a fast reaction by scanning
an ultramicroelectrode (UME) along a substrate while the currents
produced by the oxidation or reduction of the electrochemical
species at the electrodes are recorded.1 When the UME is held about
a radius distance from the substrate surface, electron transfer is
confined to a small area and local chemical or electrochemical
information can be obtained from the positive or negative feedback
current, providing local topographic or kinetic information. By
scanning the UME at a constant height, useful information about
the spatial distribution of local surface conductivity and electro-
chemical activities of reactive sites can be obtained.
SECM-based surface patterning can be used to form a variety
of µm-sized structures, for example, by electrochemical etching of
a semiconductor,2 metal deposition and etching,3 deposition of
conducting polymers,4,5 and inducing biomacromolecules to im-
mobilize on a self-assembled monolayer (SAM) which is patterned
using a UME.6 With SECM no preformed stamp or mask is needed
prior to patterning the substrate, since the pattern is formed by
scanning the UME. Surface patterning of different materials (metal,
polymer, semiconductor) at high density onto various kinds of
substrates at the µm, or even nm, scale is possible, and the surface
chemistry is controlled to deposit precise amounts of materials by
the UME at a given applied potential. These existing SECM
patterning methods either depend on patterning a surface through
destructive modification of a substrate (i.e., etching) or heavily rely
on the electrochemical behavior of the target materials that will be
deposited onto a substrate under an applied potential between the
tip and the substrate. Recently Collman, Chidsey and co-workers7
described the modification of a self-assembled monolayer coated
Au microelectrode surface (ferrocene attachment) by “click”
chemistry catalyzed by the electrochemical reduction of Cu(II)
catalyst. Here we describe a different approach, where the Cu(II)
reduction is carried out at a SECM tip to modify a glass surface
beneath.
The reaction is useful in linking molecules onto a solid surface
and can proceed in a variety of solvents, tolerate a wide range of
pH values, and perform well over a broad temperature range. For
example, azido-terminated SAMs can easily be connected with
alkynes by 1,2,3-triazole formation in the presence of a Cu(I) active
catalyst.10 In this report, an acetylene-functionalized fluorophore
was used in an electrochemical cell to react with an azido-terminated
monolayer self-assembled onto a glass substrate to form triazole
through a click reaction. As depicted in Scheme 1, a gold
microelectrode was used to synthesize Cu(I) locally in the small
gap between a tip and a substrate to catalyze the click reaction in
a small volume. The small gap between a UME and a substrate
can not only produce sufficient Cu(I) species to ensure immobiliza-
tion of acetylene fluorophore derivatives onto a small area of the
substrate, but can also maintain the stability of Cu(I), since it can
be oxidized easily in solution. The amount of deposited molecules
can be controlled by adjusting the tip-substrate distance, the
amount of Cu(I), and the reaction time. The maximum amount of
dye molecules attached to the SAM is determined by the total
number of azido groups functionalized on the glass substrate.
Benzothiodiazole acetylene 1 was designed and synthesized as
the fluorescent ink (see Supporting Information for the detailed
synthesis and optical properties of 1). The azido-terminated SAM
on a glass substrate was prepared by treating a bromo-terminated
monolayer with NaN3. The bromo-terminated SAM was obtained
by the immersion of a glass substrate in a solution of 11-
bromoundecyltrichlorosilane in dry dichloromethane (1%, v/v) (see
Supporting Information for detailed preparation conditions).
Since aquo-Cu(I) has poor stability and can disproportionate to
Cu(0) and Cu(II), we selected Cu(II) salen as the Cu-source and
DMF as the solvent to achieve stable Cu(I) species and solubility
of the other reagents. Stable Cu(I) was generated at an UME in
the SECM to catalyze the click reaction in the small gap between
the gold UME and the glass substrate.7 A typical cyclic voltam-
mogram (CV) of Cu(II) salen11 at a gold microelectrode in DMF,
shows Nernstian behavior at -1.0 V vs Ag QRE (see Supporting
Information). CVs at a larger electrode demonstrate that a stable
Cu(I) intermediate can be obtained. This electrochemical behavior
of Cu(II)/Cu(I) is attributed to stable tetrahedral intermediates of
the Cu(I) species in the presence of the salen ligand in DMF.11
To pattern the azido-functionalized glass substrate with the
fluorescent dye molecule, an approach curve to the azido-func-
tionalized glass substrate with the gold microelectrode (100 µm
diameter) at a potential of 0.8 V (vs silver quasi-reference electrode)
(QRE) and a solution of ferrocene in DMF/n-Bu4NBF4 was obtained
to allow the tip to be positioned at the desired height. The
microelectrode was then held at a constant height of 28 µm, where
the tip current decreased to 50% of the steady-state current at long
distance (see the Supporting Information). This small distance above
the glass substrate reduces the diffusion time of the Cu(I) species
The “Sharpless click” reaction8,9 refers to a Cu(I) catalyzed
Huisgen 1,3-dipolar cycloaddition of azide and alkynes to form
1,2,3-triazoles with high efficiency.
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C O M M U N I C A T I O N S
trode may play a role in the formation of the interesting ring
features. Such patterns could arise from the relative concentration
gradients of the three components of the click reaction, e.g., Cu(I),
azido monolayer and fluorescence dye derivative, and the spatial
distribution of the reaction rates. The formed rings resemble
“Liesegang rings”,12 which are formed in reaction-diffusion systems.
Each of the rings also shows a pronounced “tail” which is probably
formed because of convection caused by Ar gas bubbling. The
distance between two ring centers was about 700 µm, which was
controlled exactly by moving the microelectrode position with the
SECM stepping motor. Different patterns of the dye molecule could
be written on the azido-terminated SAMs in the same way by
programming the SECM tip movement, as shown in previous
SECM patterning.
This technique represents a novel method for attaching fluores-
cent molecules to a solid substrate by using SECM and click
chemistry. This approach can probably also be used to transfer other
molecules, and nanoparticles to a solid substrate. In addition, we
have found the pattern generated by SECM-mediated click chem-
istry with a Liesegang ring-type behavior. On the basis of previous
SECM work, the pattern feature size and shape should be
controllable by selecting the tip size and the relative distance from
tip to substrate. Larger scale and higher density patterning of protein
arrays and other biologically interesting substances should also be
possible.
Figure 1. Typical fluorescence microscopy image of a patterned ben-
zothiodiazole array on the glass substrate.
Scheme 1 Local Reduction of Cu(II) to Cu(I) at a Gold
Microelectrode (Left) and Immobilization of Acetylene Fluorophore
Derivatives onto a Glass Substrate through Click Chemistry.
Acknowledgment. We thank the financial support from the
National Science Council of Taiwan (NSC-96-2120-M-002-006)
and the Robert A. Welch Foundation. S.Y.K. thanks Dongping Zhan
and Shanlin Pan for their assistance with the SECM work and
Kwok-Fan Chow for obtaining the fluorescent images.
from tip to substrate and ensures a small reaction area. The ferrocene
solution for tip approach and alignment was then replaced by the
Cu(II) salen and 1 in DMF/n-Bu4NBF4 solution for surface
patterning. The solution was deaerated with argon to stabilize the
Cu(I) intermediate and prevent oxygen from participating in the
electrochemical reaction. The Cu(I) species generated at the gold
microelectrode at -1.08 V catalyzed the click reaction. After surface
patterning, the glass substrate was washed well several times with
acetone and EtOH to remove excess dye molecules nonspecifically
anchored to the substrate. The pattern formed was measured by
fluorescence microscopy. The fluorescence pattern on the glass
substrate indicates that acetylene fluorophores were covalently
linked to the substrate via 1,2,3-triazole formation.
Figure 1 shows a fluorescence microscopy image of a typical
array. The array contains four spots, where each spot shows inner
circular centers and outer rings. Since we used a 100 µm gold
electrode for patterning, the 300 µm diameter inner centers
correspond to the surface area of azido-functionalized glass directly
below the gold microelectrode, where the produced Cu(I) has the
highest concentration close to the glass substrate because of the
short diffusion distance. The outer rings were about 500∼600 µm
in diameter and presumably result from a concentration gradient
of Cu(I)-acetylene intermediates. The reaction rate of the click
reaction and the thick insulating layer around the gold microelec-
Supporting Information Available: Additional experimental de-
tails of synthesis and photophysical properties of 1, CV of Cu(II) salen,
and procedures for the preparation of azido-functionalized SAM and
SECM-aided patterning. This material is available free of charge via
the Internet at http://pubs.acs.org.
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