Protein patterning based on electrochemical activation of bioinactive surfaces with hydroquinone-caged biotin

Article abstract of DOI:10.1021/ja0459330

We report a protein attachment and patterning method based on a hydroquinone-caged biotin surface that generates bioactive biotin by mild electrochemical perturbation. The electrochemical activation proceeds under the buffered aqueous environment at neutr

Full text of DOI:10.1021/ja0459330

Published on Web 11/06/2004
Protein Patterning Based on Electrochemical Activation of Bioinactive
Surfaces with Hydroquinone-Caged Biotin
Kyuwon Kim,† Haesik Yang,*,‡ Sangyong Jon,¶ Eunkyung Kim,§ and Juhyoun Kwak*,§
Korea Research Institute of Standards and Science, Daejeon 305-600, Korea, Department of Chemistry,
Pusan National UniVersity, Pusan 609-735, Korea, Department of Life Science, Gwangju Institute of Science and
Technology, Gwangju 500-712, Korea, and Department of Chemistry, Korea AdVanced Institute of Science and
Technology, Daejeon 305-701, Korea
Received July 7, 2004; E-mail: Juhyoun_Kwak@kaist.ac.kr (J.K.); hyang@pusan.ac.kr (H.Y.)
An efficient attachment and patterning method of proteins on
surfaces is crucial to the construction of protein chips1 and
bioelectronic devices.2 In the attachment step, the site-specific
immobilization of proteins is required to retain their biological
activity. As a platform satisfying the requirement, a biotin-modified
surface has been widely used because either avidin or biotin-labeled
proteins can be selectively immobilized in right orientation through
avidin/biotin interaction with high affinity (Ka ≈ 1 × 1015 M-1).3
On the other hand, the serial patterning of proteins is essential to
Figure 1. (A) Cyclic voltammogram of I-based SAM on a gold electrode
the preparation of protein chips for multiple analysis.4 To prepare
in 0.1 M phosphate buffer (pH 7.2) at a scan rate of 50 mV/s. The solid
such patterns utilizing biotin/avidin interactions, two kinds of
strategies have been developed. First is site-selective immobilization
of biotin before protein attachment by using the soft lithography
technique5 or scanning probe microscopy.6 Although these proved
to be versatile protein patterning methods, several drawbacks restrict
their application. For example, time consuming in the complex and
serial patterning may lower the activity of previously attached
proteins under ambient condition. Electrochemical approaches based
on this strategy have been also reported.7 However, their reaction
conditions are not compatible with the previously presented protein.
They may suffer from ligand contamination bringing about cross
adsorption of the protein. The second strategy is a site-selective
bioactivation of initially deactivated biotin surface by photo
irradiation.8 In the approach, a whole surface is precoated with
deactivated biotin containing a photolabile protecting group, and
then only selected sites are activated by the photo irradiation through
the photo mask. When the surface is exposed to a solution of a
target protein labeled with avidin, the protein is immobilized on
the activated sites through avidin linkage. In some cases, however,
longer photo irradiation to achieve complete deprotection can cause
irreversible damage to the surface, which results in increased
nonspecific adsorption of the protein. This should be overcome for
the retention of protein activity as well as for higher contrast
patterning.
line is the first scan, and the dashed line is the second scan. Reference
electrode is Hg/Hg2SO4. (B) Grazing angle FT-IR spectra obtained for a
freshly prepared I-based SAM (top) and after the SAM was subjected to
electrochemical reaction (bottom).
Scheme 1. Schematic Representation of the Electrochemically
Oxidative Reaction for the Generation of Bioactive Biotin on the
HQ-Caged Biotin-Modified Gold Surface
mechanism for the generation of a bioactive biotin surface after
electrochemical oxidation. Once the hydroquinone (HQ) moiety is
oxidized, it is converted to a benzoquinonium cation. Subsequently,
benzoquinone (BQ) and CO2 are immediately released after
nucleophilic acyl substitution by H2O, and finally, the bioactive
biotin surface is generated. To demonstrate the surface reaction
proposed in Scheme 1, cyclic voltammetry (CV) was performed
with the self-assembled monolayers (SAMs) of compound I,
prepared by immersion of gold surfaces in 1 mM THF solution of
I for at least 12 h. Figure 1A shows an irreversible anodic peak at
0.20 V on the first scan and a dramatic disappearance of the peak
on the second cycle, representing very fast and nearly complete
release of the BQ moiety from the initial surface. For the second
cycle, no change in the charging current of the double-layer region,
compared to that of the first cycle, indicates that the oxidation
potential is mild enough to not destroy the remaining organic SAMs.
Figure 1B shows the grazing angle Fourier transform infrared
(FT-IR) spectra of chemical environments on SAM surfaces, which
support our view that the electrochemical bioactivation step is very
Therefore, it is of great benefit to develop a fast, mild, and
protein-friendly method for protein patterning. In this paper, we
report a method for protein patterning based on an electrochemically
active biotin derivative that generates a bioactive biotin surface by
mild electrochemical perturbation. The electrochemical activation
proceeds under the buffered aqueous environment at neutral pH. It
also allows site-selective generation of bioactive biotin for the
immobilization of the target protein by using prepatterned electrode
arrays.
Scheme 1 displays the chemical structure of the electrochemically
active biotin derivative (I) employed in this paper and a proposed
† Korea Research Institute of Standards and Science.
‡ Pusan National University.
¶ Gwangju Institute of Science and Technology.
§ Korea Advanced Institute of Science and Technology.
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10.1021/ja0459330 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
electrochemically treated at 0.23 V for 1 min. The resulting substrate
was immersed in phosphate-buffered saline solution containing
pHRP-SA. After pHRP-SA immobilization, the pattern was visual-
ized by a color change that resulted from catalytic precipitation of
4-chloro-1-naphthol by HRP. Figure 2B shows an optical micro-
scopic image of micropatterned pHRP-SA on IDA. Bright and dark
regions correspond to the electrodes before and after electrochemical
activation, respectively. The two electrodes highly contrast each
other, which indicates that the present approach is very efficient
for site-selective protein micropatterning.
In conclusion, we demonstrated that site-specific, as well as site-
selective, protein patterning with high contrast could be achieved
by means of an electrochemical bioactivation reaction of a rationally
designed HQ-caged biotin surface under mild electrochemical
conditions. Since the present patterning process, including surface
activation and protein attachment, can be conducted under neutral
buffer conditions in a short time period, it enables the serial
patterning of multiple proteins while retaining the activity of
previously attached proteins. Therefore, it might have applications
for fabricating protein chips and biomolecular electronic devices.
Figure 2. (A) SPR sensorgrams of SA adsorption on various mixed SAMs
for ΓI ) (a) 0.25, (b) 0.5, (c) 0.1, (d) 1.0, and (e) 0. Adsorption of SA (f)
preblocked with excess free biotin at ΓI ) 0.25. SPR sensorgrams for SAM
surfaces for ΓI ) (g) 0.5 and (h) 1.0 before electrochemical treatments. (B)
Optical microscope image of pHRP-SA-patterned IDA.
fast and complete. In the spectrum of I-based SAMs, we observed
two strong bands at 1743 and 1796 cm-1 corresponding to the Cd
O stretching of three esters and two urethane groups (a and b in
Scheme 1), respectively. The peak at 1511 cm-1 is characteristic
of aromatic CdC stretching of the HQ moiety. The C-H stretching
bands of the alkyl chain appear at 2929 and 2861 cm-1. The
spectrum of the monolayers after electrochemical reaction in a 0.1
M phosphate buffer solution showed distinct changes from original
SAMs.9 While the peak at 1743 cm-1 (CdO stretching in the ester
moiety) revealed no significant change, the aromatic CdC stretching
at 1511 cm-1 disappeared, and the peak intensity at 1796 cm-1
(CdO stretching in urethane) was reduced by half of the initial
intensity. These results indicate that the HQ moiety was completely
detached, and the CdO of “b” was also removed from the I-based
SAMs. Moreover, the negligible changes in intensity, as well as
the position of the C-H stretches, also imply that the other parts
of I are kept intact. This implication also supports the view that
the electrochemical reaction of HQ is mild.
Acknowledgment. J.K. gratefully acknowledges support from
the National R&D project for Nano Science and Technology, as
well as Brain Korea 21, MICROS, and IMT-2000 projects. H.Y.
is grateful for Grant 02-PJ3-PG6-EV05-0001.
Supporting Information Available: Detailed synthetic procedure
of compounds I and II. This material is available free of charge via
the Internet at http://pubs.acs.org.
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To evaluate biological activity of the activated biotin surface
after the electrochemical reaction, we tested a biospecific interaction
with streptavidin (SA). Tri(ethylene glycol) ester of thioctic acid
(II) was used as a diluent of I-based SAMs not only to adjust proper
density of surface biotin but also to prevent nonspecific adsorption
of protein.10 Under the condition, we optimized the surface density
of I to maximize SA binding. The density can be easily controlled
by changing the mixing ratio of I and II in adsorbent solutions.
The binding of streptavidin on mixed SAMs with various mole
fractions of I in the solution (ΓI) was monitored using SPR after
the electrochemical oxidation (Figure 2A).11 When ΓI was 0.25 and
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the absolute surface density from CV was 5 × 10-11 mol cm-2 12
,
(9) All electrochemical treatments were conducted by holding a SAM-
modified gold surface as a working electrode at 0.23 V for 1 min on the
CV scan.
the maximum attachment of SA was observed (175 ng cm-2, curve
“a”). This density is consistent with that of the previous reports on
the maximum binding condition of surface biotin.8c,13 Curve “e”
shows that nonspecific binding of SA was negligible in II-based
SAMs (2 ng cm-2). Curve “f” supports that these bindings are
biospecific. The biospecific interactions between SA and the mixed
SAMs before electrochemical bioactivation were negligible (curves
“g” and “h”). This SPR study confirms that bioinactive HQ-caged
biotin is completely converted into a bioactive biotin surface under
mild electrochemical reaction.
(10) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-
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(11) BIACORE X SPR and SIA Kit Au surfaces were used for SA binding
studies. The 1000 RU corresponds to an SA density of 1 ng mm-2
.
(12) The absolute surface density of I in the mixed monolayers can be easily
obtained from integrating the oxidation peak of the HQ of I on the CV.
(13) Due to the nonlinear structure of thioctic acid of compound I, ΓI, presenting
maximum binding of SA, is higher than the mole fraction of biotin/
alkanethiols (∼0.1) of the linear methylene chain in other investigations.
Nevertheless, the absolute surface density of biotin is similar to that
expected in them. (a) Spinke, J.; Liley, M.; Schmitt, F. J.; Guder, H. J.;
Angermaier, L.; Knoll, W. J. Chem. Phys. 1993, 99, 7012-7019. (b)
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Golledge, S. L.; Sasaki, T.; Castner, D. G.; Campbell, C. T.; Stayton, P.
S. Langmuir 2001, 17, 2807-2816.
We constructed I-based SAMs on an interdigitated microelec-
trode array (IDA) for protein micropatterning. To visualize the
pattern, we used poly-(horseradish peroxidase)-conjugated SA
(pHRP-SA).14 Using SPR, we determined ΓI, presenting maximum
pattern contrast between deactivated and activated biotin surfaces,
by comparing the amounts of pHRP-SA bound to the surfaces
before and after electrochemical activation.15 Maximum contrast
was observed at ΓI ) 0.5, and thus we employed this condition for
micropatterning. Only one electrode of an IDA substrate was
(14) HRP mediates the catalytic oxidation of 4-chloro-1-naphthol by H2O2.
Only the pHRP-SA bound region produces precipitates of the oxidized
product in aqueous solution, which paints the region in dark color.
Patolsky, F.; Katz, E.; Barden, A.; Willner, I. Langmuir 1999, 15, 3703-
3706.
(15) SA binding onto the surfaces before electrochemical activation is also
dependent on ΓI, although the binding constant of them must be much
lower than that of HQ-released surface after electrochemical activation.
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