1001

Full Paper

A Homogenous Assay of FAD Using a Binding Between

Apo-Glucose Oxidase and FAD Labeled with an Electroactive
Compound
Kazuharu Sugawara,*a Naoto Kamiya,a George Hirabayashi,a Hideki Kuramitzb
a

Faculty of Education, Gunma University, Maebashi, Gunma, 371-8510 Japan

*e-mail: kzsuga@edu.gunma-u.ac.jp
b
Department of Environmental Biology and Chemistry, Faculty of Science, University of Toyama, Toyama 930-8555 Japan
Received: January 04, 2006
Accepted: March 07, 2006
Abstract
A homogenous assay of FAD using a binding between glucose oxidase (apo-GOD) and FAD labeled with an
electroactive compound was developed. Because daunomycin is sensitively detected with voltammetry, daunomycin
was connected to FAD with a cross-linker. The peak current decreased due to the apo-GOD-labeled FAD binding.
Competitive reaction to the apo-GOD between FAD and the labeled FAD produces the increase of peak current.
Accordingly, FAD is detected on the basis of the reaction. The merit of this method is that the influence from FMN
and riboflavin in the measurement of FAD can be suppressed by the high selective binding.
Keywords: FAD, Label, Glucose oxidase, Voltammetry
DOI: 10.1002/elan.200603491

1. Introduction
Flavin adenine dinucleotide (FAD), which is one of vitamin
B2 derivatives, exists in several beverages and foods. FAD is
related to their quality control and nutrition [1]. On the
other hand, FAD has a role as a medicament for angular
stomatitis, rash, pimple and conjunctivitis. It is known that
FAD has a fast-acting property in comparison with vitamin
B2. Therefore, many procedures to detect FAD have been
proposed. For instance, native fluorescence detection of
flavin derivatives by microchip capillary electrophoresis
with laser-induced fluorescence was reported [2, 3]. Determination of FAD was also attempted with high performance
liquid chromatography [4 6].
Flavin enzyme concerns intermediary metabolism of
sugar, amino acid and fatty acid in living bodies. In addition,
it has an important role for oxidation reduction reactions
such as the oxidative phosphorylation. FAD is a prosthetic
group for glucose oxidase (GOD), amino acid dehydrogenase and acetyl-CoA dehydrogenase. GOD from Asperigillus
niger which is a subunit with a molecular weight of 80 kDa
consists of a dimmer with equal subunits [7]. GOD has one
bound FAD per monomer, and the binding constant
between apo-GOD and FAD is about 1
1010 M1. Because
FAD does not combine with GOD covalently, FAD is
released from the holo-GOD without denaturation of the
protein. The prepared apo-GOD is not biocatalytically
active. When FAD is hold in the apo-GOD, the activity of
enzyme is restored. This concept can be applied to biological
molecule detection and in monitoring of biomolecule
Electroanalysis 18, 2006, No. 10, 1001 1006

interaction. Dosch et al. proposed a homogeneous immunoassay for the detection of trinitrotoluene using the reactivation of apo-GOD by a novel FAD-trinitrotoluene conjugate [8]. Spectrophotometric enzyme-amplified immunoassay for thyroid stimulating hormone was also investigated
[9]. In order to estimate the concentration of glucose, the
fluorescence emission of the apo-GOD has been used [10].
Since FAD is an electroactivity, the behavior of FAD in a
solution was investigated with an electrochemical procedure. Scheller et al. has reported reduction at D. M. E of the
prosthetic group in GOD and riboflavin flavoprotein [11].
The detection of FAD is carried out with an electrochemical
chromatography [12]. The detection limit was 108 M level.
This method was successfully applied to determine FAD in a
living sample only with pretreatment, such as filtration and
extraction. Furthermore, the evaluation of GOD-FAD
binding was attempted [13, 14]. Zayats et al. reported that
pyrroloquinoline quinone (PQQ) functioned as a mediator
when apo-GOD combined with PQQ-FAD conjugate
immobilized on an Au electrode [15]. The application of
redox enzymes for probing the antigen antibody association was also suggested [16].
In this study, we developed a sensitive and selective
detection of FAD based on a competitive reaction to apoGOD between FAD and FAD labeled with an electroactive
daunomycin. If the apo-GOD and labeled FAD are mixed in
a solution, the labeled FAD is hold in the binding site of apoGOD. The electrode response of labeled FAD decreases
due to the covering of electroactive moiety by the apoGOD. The decrease of diffusion constant of GOD with the
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K. Sugawara et al.

trode was a glassy carbon electrode (Model No. 11-2012,

BAS). In each measurement, the electrode was polished
with 1.0, 0.3, and 0.05 mm alumina (Baikoski International
Corp., Charlotte, NC). After polishing with alumina, an
ultrasonic bath was employed for cleaning the electrode
surface of alumina particles. An Ag/AgCl electrode (Model
No. 11-2210, BAS) was used as a reference electrode, and a
platinum wire was a counter electrode. All potentials were
measured against the Ag/AgCl electrode. The pH of a
solution was measured with a Horiba D-21 pH meter.
Spectrophotometric measurements were carried out with a
UV 1240 mini spectrophotometer (Shimadzu Tokyo, Japan).

2.2. Reagents
Fig. 1. Holding of labeled FAD in the binding sites of apo-GOD.

labeled FAD in a solution may be related to the decrease of

peak current. When FAD, labeled FAD and apo-GOD are
incubated in the solution, the competitive reaction to the
binding sites of apo-GOD between FAD and the labeled
FAD occurs (Fig. 1). Because FAD occupies the binding
sites of apo-GOD, the amount of labeled FAD increases.
That is, the electrode response of labeled FAD increases
with increasing the concentration of FAD. The binding
constant of labeled FAD-apo-GOD would be smaller than
that of FAD-apo-GOD due to the steric hindrance of
labeled FAD. The labeling using daunomycin has several
merits such as the probe monitoring the binding [17]. For
instance, this method can be detected without the need of a
more complicated enzymatic protocol. Labeled FAD
strongly adsorbs to a glassy carbon electrode because of
the property of daunomycin itself and the introduction of
hydrophobic spacer connecting daunomycin with FAD. High
sensitivity for FAD can be gained by the property of labeled
FAD. On the other hand, the detection of FAD is achieved
without a separation procedure of a free labeled FAD from a
bound one. Furthermore, the influence of flavin derivatives in
the measurement of FAD is reduced because of the selective
binding between apo-GOD and labeled FAD. The electrochemical measurement of FAD is generally influenced by the
electrode response of flavin derivatives. The peak potentials
of riboflavin and flavin adenine mononucleotide(FMN) are
similar to the peak potential of FAD. This method can be
utilized as a powerful tool especially to measure sample that
contains complex matrix, because the electrochemical property of daunomycin as a labeling reagent has different
potential from interference compounds.

2. Experimental
2.1. Apparatus
CV-50 W analyzer (Bioanalytical Systems Inc. (BAS)) was
used to all voltammetric measurements. A working elecElectroanalysis 18, 2006, No. 10, 1001 1006

FAD and flavin mononucleotide were purchased from

Sigma-Aldrich. Ethylene glycol bis[sulfosuccinimidylsuccinate](Sulfo-EGS) as a cross-linker was supplied from
Pierce. Daunomycin and riboflavin were from Wako Pure
Industries, Japan. FAD derivative solution was prepared
daily. Phosphate buffer which consisted of 0.1 M KH2PO4
and 0.1 M NaOH was used as incubation solution and as a
supporting electrolyte in electrochemical measurement.
High quality nitrogen gas was used for deaeration. All
chemicals used were analytical grade.

2.3. Procedure
Labeled FAD was prepared by mixing 4 mM daunomycin,
4 mM FAD and 2 mM Sulfo-EGS for overnight at 4 8C in
0.1 M phosphate buffer solution (pH 8.5) (Fig. 2). The
solution was spotted on a sheet of thin-layer chromatography (silica gel alumina sheet, MERCK) in chloroform:
methyl alcohol 4 : 1 (v/v)%. After developing, the labeled
FAD was stripped from the sheet and collected. The reagent
was dissolved in ethyl alcohol, and the solution was
centrifuged to exclude silica gel. The concentration of
labeled FAD was estimated from the mole absorbance
efficiency of daunomycin (490 nm).
GOD was dissolved in saturated (NH4)2SO4 solution, and
the pH of solution was adjusted to 1.4 with conc. H2SO4 [18].
The solution was mixed with stirring overnight at 4 8C and
was moved to centrifuge tube. The yellow supernatant was
removed after centrifugation. After the precipitate was
washed with saturated (NH4)2SO4 solution, the supernatant
was removed. The operation was repeated twice and the
precipitate was dissolved in 0.1 M phosphate buffer
(pH 7.0). The apo-GOD solution was stored with 20 %
(w/v) glycerol at 4 8C. The concentration of apo-GOD was
estimated by comparing the absorbance at 278 nm with the
absorbance of standard solution of GOD solution.
Apo-GOD and labeled FAD were mixed for 1 h in 0.1 M
phosphate buffer (pH 7.0). Deaeration with nitrogen was
carried out before each electrochemical measurement.
Next, a potential at  1.0 V for 5 min was applied to a

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Homogenous Assay of FAD

Fig. 2. Preparation of labeled FAD.

glassy carbon electrode with stirring of the solution. After a

rest period of 15 s, the potential between  1.0 and 0.0 V was
scanned with differential pulse voltammetry (scan rate:
5 mV s1, pulse amplitude: 50 mV, sample width: 2 ms, pulse
width: 50 ms, pulse period: 200 ms) in order to obtain an
oxidation response of labeled FAD. To detect FAD, the
competitive reaction between the labeled FAD and FAD to
the limited binding sites of apo-GOD was used. FAD,
labeled FAD and apo-GOD were mixed, and voltammetric
measurement was carried out under the same conditions
mentioned above.

3. Results and Discussions

3.1. Voltammograms of FAD Labeled FAD at a Glassy
Carbon Electrode
Voltammograms of FAD and labeled FAD were measured
with differential pulse voltammetry (DPV) at a glassy
carbon electrode in 0.1 M phosphate buffer (pH 7.0). When
the potential was scanned in the range of  1.0 V and 0.0 V
after the accumulation for 5 min at  1.0 V to the electrode
in the solution with 1
106 M FAD, an oxidation peak from
FADH2 to FAD was observed at  0.45 V(not shown).
Although the detection limit of FAD estimated from three
times the standard deviation (3s) is 8
108 M, the determination of FAD is carried out in 107 M order. Therefore, a
development of more sensitive method to detect FAD is
desired. To achieve the method proposed in this study, FAD
with an electroactive compound was prepared. Daunomycin
was connected to FAD with cross-linker, because it contains
of a property that strongly adsorbs to the electrode surface
and this can induce excellent sensitivity. The labeled FAD is
Electroanalysis 18, 2006, No. 10, 1001 1006

used as a probe for sensitive detection of FAD based on

labeled FAD-apo-GOD-binding. After applying a potential
of  1.0 V for 5 min to the electrode in the solution with 5

107 M daunomycin, an oxidation peak was obtained at
 0.60 V by scanning the potential. The peak was due to
reoxidation of quinone portion in daunomycin that was
reduced at  1.0 V. Although other oxidation peak appeared at more positive potential, the oxidation peak at
negative side was used because of high sensitivity [16]. The
peak of 5
107 M labeled FAD appeared at  0.60 V, and
the peak shift was not observed (Fig. 3). The reason for this
is that electroactive moiety and sugar moiety has amino
group that are far apart. It is anticipated that these functions
do not interfere each other.

3.2. Relation Between the Accumulation Time and the

Peak Current
The adsorption ability to a glassy carbon electrode of
daunomycin or labeled FAD was investigated. The measurements were carried out with DPV after a potential at
 1.0 V for 5 min was applied to the electrode in 0.1 M
phosphate buffer (pH 7.0) with 5.0
107 M daunomycin.
The peak current of daunomycin increased linearly up to
6 min with increasing the accumulation time and was
constant over 10 min. This is because daunomycin adsorbs
to a glassy carbon electrode and the saturation of the
electrode surface by the reagent occurs. The increase of
peak current for 5.0
107 M labeled FAD was linear up to
5 min and became a constant value over 9 min. This result
shows that the adsorption ability to the electrode of labeled
FAD is stronger than that of daunomycin. The reasons were
that the labeled FAD had an alkyl chain and the hydrophilic

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K. Sugawara et al.

Fig. 4. Dependence of the incubation time on peak current. a)

5.0
107 M Daunomycin 5.0
107 M Apo-GOD, b) 5.0

107 M Labeled FAD 5.0
107 M Apo-GOD. The incubation
was carried out in 0.1 M phosphate buffer (pH 7.0). After a
potential at  1.0 V for 5 min was applied to the electrode,
measurements were carried out with DPV.

Fig. 3. Voltammograms of labeled FAD and GOD at a glassy

carbon electrode. A) Holo-GOD: a) 0 M, b) 5
107 M. B) ApoGOD: c) 0 M, d) 5
108 M, e) 1
107 M, f) 5
107 M. GOD
and 5
107 M labeled FAD were incubated with stirring for 1 h
in 0.1 M phosphate buffer (pH 7.0). After a potential at  1.0 V
for 5 min was applied to the electrode, measurements were carried
out with DPV.

amino group was changed into a hydrophobic imino group.

On the other hand, the accumulation of native FAD to the
electrode was attempted under the same conditions. The
peak current of 5.0
107 M FAD without the accumulation
was similar to that after accumulation for 5 min. It is known
that FAD strongly adsorbs to a mercury electrode [11]. In
contrast, this result shows that the adsorption ability of FAD
to the glassy carbon electrode is low.

3.3. Voltammograms of Labeled FAD and GOD Added

Voltammograms of 5
107 M labeled FAD in the presence
of holo-GOD or apo-GOD were shown in Figure 3. When
5
107 M holo-GOD was added in a solution, the peak
current hardly decreased at the glassy carbon electrode. In a
solution with only holo-GOD of 5
107 M, an oxidation
peak did not appear. That is, the peak on the basis of
prosthetic group in the enzyme was not observed. In
contrast, the peak current of 5
107 M daunomycin did
not decreased in the solution with 5
107 M apo-GOD (not
shown). Therefore, the electrode reaction of daunomycin is
not influenced in the presence of 5.0
107 M apo-GOD.
When the concentration of apo-GOD was 7.0
106 M, the
peak current of daunomycin decreased to 90 % in comparison with that of 5
107 M daunomycin with 5
107 M
Electroanalysis 18, 2006, No. 10, 1001 1006

apo-GOD (not shown). This decrease of peak current is due

to the adsorption of apo-GOD to the electrode. On the other
hand, the peak current of 5
107 M labeled FAD decreased with increasing the concentration of apo-GOD. The
peak current of labeled FAD linearly decreased in the range
of 3
109  1
107 M. Consequently, it is expected that
daunomycin moiety of the labeled FAD is held in the
binding sites of apo-GOD to FAD and it lost electroactivity.
Figure 4 shows the relations between the peak current and
the incubation time. When 5
107 M daunomycin and
5.0
107 M apo-GOD were mixed for 120 min, the peak
current of daunomycin hardly changed. On the other hand,
5
107 M labeled FAD and 5.0
107 M apo-GOD were
incubated in 0.1 M phosphate buffer (pH 7.0). The peak
current of labeled FAD drastically decreased with increasing the incubation time and became a constant value over
45 min. This result shows that the labeled FAD combines
with apo-GOD as mentioned above.

3.4. Influence of Interfering Substances

Influence of interfering substances to a measurement of 5

107 M labeled FAD in 0.1 M phosphate buffer with 5

107 M apo-GOD was investigated. Table 1 indicates the
concentration limits at which interfering substance gives a
relative error of < 10% in the peak current. Coexisting
amino acids, such as cysteine, glycine, histidine, methionine
and phenylalanine did not interfere in concentrations up to
100 times that of labeled FAD. Several coexisting vitamins
and vitamin derivatives were also examined. The substances
were ascorbic acid, biotin, calciferol, flavin mononucleotide
(FMN), riboflavin and tocopherol. Biotin, calciferol and
tocopherol of 5
105 M did not influence the peak current.
However, the coexisting of 3
106 M ascorbic acid interfered the peak current. This is because an oxidation peak of
ascorbic acid which raises a background current appears at
0.10 V. Consequently, FAD could not be measured precisely
in coexisting with ascorbic acid. When a direct measurement
of FAD without the labeled FAD-apo-GOD binding is

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Homogenous Assay of FAD

Table 1. Concentrations in which other substances can be present
without causing interference. After 5
107 M apo-GOD, 5

107 M labeled FAD and a substance were mixed for 1 h in
0.1 M phosphate buffer (pH 7.0), measurements were carried out
at a glassy carbon electrode.

of the labeled FAD inhibits the binding between apo-GOD

and FAD.

Substance

Concentration (106 M )

3.6. Detection of FAD

Cysteine
Glycine
Histidine
Methionine
Phenylalanine
Ascorbic acid
Biotin
Calciferol
Flavin mononucleotide
Riboflavin
Tocopherol
Avidin
BSA

50
50
50
50
50
3.0
50
50
5.0
7.0
50
5.0
1.0

Detection of FAD was achieved with the competitive

reaction to the limited binding sites of apo-GOD between
labeled FAD and FAD. When the concentration of FAD
changed from 1
1010 to 5
106 M in 0.1 M phosphate
buffer (pH 7.0) with 5
107 M labeled FAD and 5
107 M
apo-GOD, the peak current of labeled FAD increased
(Fig. 5). This is because FAD occupies the binding sites of
apo-GOD. The relative standard deviation at 1
107 M
FAD was 5.3% (n 5). The limit of detection of FAD was
1
109 M. The definition was the same as mentioned
above. The calibration curve becomes a dose curve and not
linear. Detection of a simpler method was 8
108 M
mentioned above. The sensitivity of this method was
improved 80 times in comparison with that that of method
without labeled FAD. When 5
106 M FAD was added in
the solution, the peak of labeled FAD at  0.60 V was
influenced by that of FAD at  0.45 V. Therefore, the
investigation which exceeded 5
106 M FAD was not
carried out.
The proposed method was applied towards the detection
of FAD in commercially available eye drop because it has a
complicating matrix thus making it an appropriate example
to prove the applicability of the proposed method. The
constituents of eye drop are chondroitin sulphate sodium
salt, chlorphenylamine maleate, neostigmine methylsulfate
and tocopherol acetate. Boric acid, menthol, edetate
sodium, polyoxyethylene castor oil and polysorbatebenzalkonium chloride were used as additives. The sample (20 mL)
was diluted to two thousand folds with 0.1 M phosphate
buffer (pH 7.0). When the solution was directly measured
with voltammetry without the labeled FAD-apo-GOD
binding, an oxidation peak of FAD was not observed. It
was expected that co-existing substances of eye drop
inhibited the electrode reaction of FAD. On the other

attempted, the coexisting of FAD derivative even a onetenth concentration against the concentration of FAD
diminished the detection of FAD. The reason is that
oxidation potentials of FMN and riboflavin are similar to
that of FAD. This method on the basis of the selective
binding between labeled FAD and apo-GOD suppresses the
influence of flavin derivative. The allowed concentration of
flavin mononucleotide and riboflavin was 5
106 M and
7
106 M, respectively. The peak of flavin derivative at
about  0.45 Vaffects to that of labeled FAD at  0.60 V. To
examine inhibition of protein to the electrode response,
avidin and bovine serum albumin (BSA) were selected.
Coexisting of 5
106 M avidin and 1
106 M BSA was not
influenced to the peak current of labeled FAD. When the
concentration of protein exceeds the concentration mentioned above, the peak current decreases due to the
adsorption of the protein to the electrode surface. Thus,
this method has high selectivity to the detection of FAD and
is usable in complicating matrix.

3.5. The Binding Constant Between Apo-GOD and

Labeled FAD
The binding constant between apo-GOD and labeled FAD
under equilibrium conditions was measured. The concentration ratio (Abound/free) of bound and free labeled FAD to
apo-GOD was calculated from the peak current of free
FAD. For the estimation, we supposed that the labeled FAD
with apo-GOD became completely electroinactive. The
ratio was plotted against the amount of labeled FAD(bound) (Scatchard plot). A straight line was obtained
(Abound/free  1.1
108 Bbound 5.42), and the binding constant estimated from the measuring the slope of the line was
1.1
108 M1. The binding constant of the apo-GODlabeled FAD was smaller than that of the apo-GOD-FAD
binding Ka 1010 M1). This decrease of binding constant is
due to labeling of daunomycin. That is, daunomycin moiety
Electroanalysis 18, 2006, No. 10, 1001 1006

Fig. 5. Detection of FAD based on the competitive reaction to

the binding sites of apo-GOD between FAD and labeled FAD.
FAD, 5.0
107 M labeled FAD and 5
107 M apo-GOD were
incubated with stirring for 1 h in 0.1 M phosphate buffer (pH 7.0).
After a potential at  1.0 V for 5 min was applied to the electrode,
measurements were carried out with DPV.

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Table 2. Determination of FAD in eye drop. Number of

determinations 5; 1 : 2000 dilution with 0.1 M phosphate buffer
(pH 7.0).
Sample

Eye drop A
Eye drop B

Concentration of FAD (104 M )

Present
method

ManufacturerMs
value

2.3  0.1
5.7  0.3

2.4
6.0

hand, the detection of FAD was attempted with the

proposed method. The measurements were carried out by
a calibration curve, and the measurement time was about
1 h. Pretreatment of electrode was needed for each measurement. As a result, the peak of labeled FAD could be
obtained. This reason is that the adsorption of daunomycin
moiety in the labeled FAD to the electrode is stronger than
that of co-existing substance. The concentration of FAD
agreed to the certified value of the maker (Table 2).
Therefore, this method would be applied to the determination of FAD in pharmaceutical samples.

4. Conclusions
In this study, a sensitive and selective method to detect FAD
was established on the basis of a competitive reaction to the
limited binding sites of apo-GOD between labeled FAD and
FAD. Because the electrode response of FAD with daunomycin was more sensitive than that of FAD, the detection
limit of FAD became 109 M level. The influence of flavin
derivative to the measurement of FAD was suppressed by
using the selective binding between apo-GOD and labeled
FAD. This concept could be used to evaluate biomolecule
interaction.

Electroanalysis 18, 2006, No. 10, 1001 1006

5. Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research (No. 16550068) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan.

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