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An SECM Detection Scheme with Improved Sensitivity and Lateral Resolution Detection of Galactosidase Activity with Signal Amplification by Glucose Dehydrogenase.

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Communications
Scanning Electrochemical Microscopy
An SECM Detection Scheme with Improved
Sensitivity and Lateral Resolution: Detection of
Galactosidase Activity with Signal Amplification
by Glucose Dehydrogenase**
Chuan Zhao and Gunther Wittstock*
Scanning electrochemical microscopy (SECM) has grown to
be a powerful technique for the study of heterogeneous
electron-transfer reactions, charge-transfer at liquid–liquid
interfaces, and molecular transport across membranes.[1]
Since the inception of SECM, a major application has been
the study of biological systems, for example, the metabolic
activity of cells, whole organisms, subcellular particles, and
enzymes.[2] In particular, much attention has been devoted to
imaging the activity of immobilized enzymes on patterned
interfaces because of the practical significance of these
structures for the prototyping of integrated and miniaturized
biosensors and chip-based assays.[3] SECM can be used in the
feedback (FB) and generation collection (GC) modes, and
both modes have been applied successfully for imaging
enzyme activity at interfaces.
In the FB mode, an ultramicroelectrode (UME) probe is
used to oxidize or reduce a reversible electron mediator. The
feedback process occurs when the mediator diffuses to the
sample and is restored to its original oxidation state through
an electrochemical, chemical, or enzymatic reaction. The FB
mode provides better lateral resolution than GC imaging
because the regeneration of the mediator only occurs in close
[*] Dr. C. Zhao, Prof. Dr. G. Wittstock
Carl von Ossietzky Universitt Oldenburg
Faculty of Mathematics and Natural Sciences
26111 Oldenburg (Germany)
Fax: (+ 49) 441-798-3684
E-mail: gunther.wittstock@uni-oldenburg.de
[**] This work was supported by the Deutsche Forschungsgemeinschaft
(DFG, grant Wi1617/1-4). SECM = scanning electrochemical
microscopy.
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
proximity to the UME. The sensitivity of the feedback mode
is, however, very limited because the flux generated by the
enzymatic reaction at the interface must be detected against
the strong background signal resulting from the hindered
diffusion of the mediator from the bulk phase to the UME.
When imaging enzymatic activity the FB mode can only be
applied to oxidoreductases, as the immobilized enzyme
requires the mediator as a redox-active cofactor.
In the GC mode, the UME detects the concentration of a
species generated at the surface of the sample. Ideally, the
UME acts only as a passive sensor to produce concentration
maps of a particular chemical species near the sample surface.
The GC mode is more sensitive because the background
signal is very weak. However, lateral resolution in the GC
mode is rather limited as a result of the diffusion of detected
species from their source at the sample surface. GC imaging
can also be used for enzymes other than oxidoreductases if
the enzyme converts a substrate into a redox-active product
that is detected at the UME.[4]
Herein we introduce a new detection scheme which
combines the advantages of the FB and GC modes and allows
high sensitivity and lateral resolution in SECM imaging. The
method is demonstrated with a multienzyme system composed of immobilized galactosidase (Gal) and PQQ-dependent glucose dehydrogenase (GDH) (PQQ = pyrroloquinoline
quinone). Gal, one of most commonly used labeling
enzymes,[5] is not an oxidoreductase and can not be imaged
in the conventional FB mode. GDH is an extremely useful
quinoprotein for biosensor applications owing to its insensitivity to dissolved oxygen and its very high activity.[6] Both
enzymes have been studied individually by SECM in the GC
mode and GDH has also been studied in the FB mode.[7] In
conventional GC imaging of Gal, p-aminophenyl-b-d-galactopyranoside (PAPG) is added to the solution; p-aminophenol (PAP) is then generated from PAPG (Figure 1 a). The
potential of the UME is set to ET = 400 mV (Ag j AgCl), so
that PAP, but not PAPG, can be oxidized under diffusioncontrolled conditions at the UME to p-quinoneimine
(PQI).[7a]
In the new detection mode, the electrochemically produced PQI, which is an efficient cofactor of GDH,[8] diffuses
to the microspot, where it is reconverted into PAP by
immobilized GDH in the presence of d-glucose (Figure 1 b).
PAP then diffuses back to the UME and thus closes the
SECM feedback loop. The UME current, iT, therefore arises
from the oxidation of PAP formed at Gal-modified surfaces
from PAPG (GC mode) and at GDH-modified surfaces from
PQI (FB mode). Unlike in the conventional GC mode, the
signal amplification by the FB contribution only occurs in a
small region defined by the location of the GDH coupled to
the sample.
Gal and GDH were immobilized on separate batches of
paramagnetic microbeads. A mound of a 1:1 mixture of Galand GDH-modified beads was deposited on a hydrophobic
surface by a procedure developed in our laboratory.[4] The
optical microphotograph in Figure 2 a shows the bead spot
with a diameter of 100 mm. In the corresponding SECM image
from the combined detection scheme a well-defined, sharp
peak is observed above the microspot with the immobilized
DOI: 10.1002/anie.200454261
Angew. Chem. Int. Ed. 2004, 43, 4170 –4172
Angewandte
Chemie
Gal–GDH system (Figure 2 b). The peak is
characterized by an extremely small background current and a very steep increase in
the signal above the spot as a result of the
enzymatic feedback amplification. Such
sharply defined signals are not observed
in conventional GC experiments.
To differentiate between the contributions of Gal and GDH to the observed
electrode current, separate line scans were
performed across the center of the spot
with activated and deactivated GDH.
GDH catalysis was switched off by using
a d-glucose-free assay solution (peak a in
Figure 3). The peak corresponds to the
detection of PAP released through the Galcatalyzed reaction in the conventional GC
mode (Figure 1 a). After the addition of
glucose to the assay solution, a clear
Figure 1. Schematic representation of the combined detection principle for SECM in the
increase in the current strength was
immobilized Gal–GDH multienzyme system: a) conventional GC mode for Gal in the
observed above the spot (peak b in
absence of glucose; b) signal amplification in the presence of glucose. PAPG = p-aminoFigure 3) as a result of the additional
phenyl-b-d-galactopyranoside, PAP = p-aminophenol, PQI = p-quinoneimine. The diagram is
GDH-mediated feedback loop. Thus, in
not drawn to scale.
the combined mode iT is caused by the GC
contribution from Gal and the feedback
amplification by GDH. The peak current observed in the
combined mode is about 1.8 times stronger than that observed
in the conventional GC mode. The enhancement factor is
distance-dependent and increases with decreasing operating
distance. A further decrease in the operating distance was not
attempted because of the risk of displacing the bead
agglomerate mechanically with the UME.
The full width at half maximum (FWHM) of the signal is
about 140 mm for peak a (Figure 3) but only about 100 mm for
peak b. Evidently, the lateral resolution is also superior in the
combined detection scheme. The FWHM of peak b in
Figure 3 is in perfect agreement with the diameter of the
agglomerate in the optical microphotograph in Figure 2 a. The
combined mode enables the detection of Gal with a resolution
Figure 2. a) Optical microscopic and b) SECM images of the bead
microspot. The unsharp lines in (a) are from a microscopic rule and
have a periodicity of 100 mm. SECM conditions: HEPES buffer
(20 mm) containing KCl (30 mm), CaCl2 (1.0 mm), MgCl2 (1.0 mm),
PAPG (2.0 mm), and glucose (50 mm); rUME = 25 mm, rspot = 50 mm,
ET = 400 mV, d = 40 mm, translation speed = 10 mm s1.
Angew. Chem. Int. Ed. 2004, 43, 4170 –4172
Figure 3. SECM line scans across the center of the microspot: a) conventional GC mode; b) combined mode. Curve (a) was obtained for
the assay solution without glucose, and curve (b) was obtained after
adding glucose (50 mm) to the solution. The conditions were otherwise the same as those described for Figure 2.
www.angewandte.org
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
usually possible only in FB experiments, which can not be
used for Gal. This high resolution is a major advantage of the
new method. The principle can be applied directly to all
enzymes that produce PAP. The activity of alkaline phosphatase, which is another widely used labeling enzyme, has
already been imaged in the conventional GC mode through
the detection of PAP.[9] In this case the sensitivity was also
improved and a very low background current maintained by
using the new detection scheme.
In conclusion, a new combined detection scheme for
SECM was proposed and tested in the multienzyme system
Gal–GDH. The approach is a new SECM method for the
investigation and control of interactions between microstructured enzyme layers. The new detection scheme can be used
for the design and characterization of enzyme-based biochips
and biosensors and of immunoassays, for the detection of
enzyme labels, and for other SECM imaging experiments.
Further work is in progress towards the quantitative description of the detection scheme by digital simulations and its
development into a general detection principle for SECM.
[3] a) T. Wilhelm, G. Wittstock, Langmuir 2002, 18, 9485 – 9493;
b) T. Wilhelm, G. Wittstock, Angew. Chem. 2003, 115, 2350 –
2353; Angew. Chem. Int. Ed. Engl. 2003, 42, 2247 – 2250.
[4] C. A. Wijayawardhana, G. Wittstock, H. B. Halsall, W. R.
Heineman, Anal. Chem. 2000, 72, 333 – 338.
[5] E. Burestedt, C. Nistor, U. SchagerlHf, J. Emneus, Anal. Chem.
2000, 72, 4171 – 4177.
[6] a) J. A. Duine, J. Biosci. Bioeng. 1999, 88, 231 – 236; b) C.
Anthony, Biochem. J. 1996, 320, 697 – 711; c) A. Oubrie, H. J.
Rozeboom, K. H. Kalk, A. J. J. Olsthoorn, J. A. Duine, B. W.
Dijkstra, EMBO J. 1999, 18, 5187 – 5194; d) L. Ye, M. HImmerle, A. J. J. Olsthoorn, W. Schuhmann, H. L. Schmidt, J. A.
Duine, A. Heller, Anal. Chem. 1993, 65, 238 – 241.
[7] a) C. Zhao, J. K. Sinha, C. A. Wijayawardhana, G. Wittstock, J.
Electroanal. Chem. 2004, 561, 83 – 91; b) C. Zhao, G. Wittstock,
Anal. Chem. 2004, 76, 3145 – 3154.
[8] A. Rose, F. W. Scheller, U. Wollenberger, D. Pfeiffer, Fresenius J.
Anal. Chem. 2001, 369, 145 – 152.
[9] G. Wittstock, K. Yu, H. B. Halsall, T. H. Ridgway, W. R.
Heineman, Anal. Chem. 1995, 67, 3578 – 3582.
[10] a) D. J. Hnatowich, F. Virzi, J. Nucl. Med. 1987, 28, 1294 – 1302;
b) O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J.
Biol. Chem. 1951, 193, 265 – 275.
[11] C. Kranz, M. Ludwig, H. E. Gaub, W. Schuhmann, Adv. Mater.
1995, 7, 38 – 40.
Experimental Section
Biotinylated Gal was bound to streptavidin-coated microbeads as
described previously.[7] Apo-GDH (PQQ-dependent, from Escherichia coli, EC 1.1.99.17) was biotinylated, and then a Lowry protein
assay was performed to determine the concentration of the enzyme.[10]
Biotinylated GDH was treated similarly.[7] To reconstitute the holoGDH from apo-GDH, PQQ (0.5 mg mL1, 50 mL) and CaCl2 (0.1m,
10 mL) were added to a suspension of the beads (40 mL), and the
mixture was incubated at room temperature for 30 min. The Galcoated microbeads were then mixed with the GDH-coated microbeads in a 1:1 ratio, and the mixture was resuspended in HEPES
buffer (20 mm, 100 mL, pH 7.5; HEPES = (N-(2-hydroxylethyl)piperazine-N’-(2-ethanesulfonic acid)). A microspot of Gal–GDH-coated
beads was deposited on parafilm-coated glass slides (Figure 2 a)
according to a procedure described previously,[4] and then bathed in a
buffered assay solution of HEPES (20 mm) with KCl (30 mm), CaCl2
(1.0 mm), MgCl2 (1.0 mm), d-glucose (50 mm), and PAPG (2.0 mm).
The SECM measurements were performed with a self-built
instrument described previously.[3] An UME with a diameter of 50 mm
(RG 5) was made according to the method developed by Kranz
et al.[11] and was used throughout the SECM measurements at an
operating distance d = 40 mm.
Received: March 14, 2004 [Z54261]
.
Keywords: electrochemistry · enzyme catalysis · interfaces ·
scanning probe microscopy · SECM
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4172
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 4170 –4172
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