See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/229218341

Phosphate biosensor based on polyelectrolyte-

stabilized pyruvate oxidase

Article in Analytica Chimica Acta January 2001

DOI: 10.1016/S0003-2670(00)01204-6

CITATIONS READS

50 48

2 authors, including:

Nikos Chaniotakis
University of Crete
109 PUBLICATIONS 3,796 CITATIONS

SEE PROFILE

All content following this page was uploaded by Nikos Chaniotakis on 01 September 2014.

The user has requested enhancement of the downloaded file.

 Analytica Chimica Acta 427 (2001) 271277

Phosphate biosensor based on polyelectrolyte-stabilized

pyruvate oxidase
Vasilis G. Gavalas, Nikolas A. Chaniotakis
Laboratory of Analytical Chemistry, Department of Chemistry, University of Crete,
71409 Iraklion, Crete, Greece
Received 27 June 2000; accepted 11 September 2000

Abstract
In this work the development of a pyruvate oxidase-based phosphate biosensor is illustrated. The use of polyelectrolyte
stabilized recombinant pyruvate oxidase in conjunction with a porous conductive carbon results in the development of a
simple, reproducible and stable phosphate biosensor. The polyelectrolyte diethylaminoethyl-dextran or DNA was used as
the enzyme stabilizer, and the resulting enzymepolyelectrolyte complexes were physically adsorbed into the transducer, a
highly porous and conductive carbon electrode, for the construction of the biosensor. The optimized biosensor exhibits high
operational (67% remaining activity after 220 h) and storage (49% remaining activity after 24 weeks) stability, and very good
sensor-to-sensor reproducibility. The optimized phosphate biosensor was used for the measurement of the phosphate ion
activity in serum. 2001 Elsevier Science B.V. All rights reserved.
Keywords: Recombinant pyruvate oxidase; Phosphate; Biosensor; Polyelectrolyte; Porous carbon electrode; Serum analysis

1. Introduction glucomutase and glucose-6-phosphate dehydrogenase

The simple and accurate measurement of the ac-
tivity of the orthophosphate anion is very important
in clinical, biological and environmental studies. A
sensor system (ISE or biosensor) for the sensitive and
selective measurement of this illusive anion is still a
challenge [1]. The development of biosensors sensitive
to phosphate has attracted many researches over the
past 25 years [2]. Amperometric biosensors have been
usually constructed based on multi-enzyme schemes:
alkaline phosphatase and glucose oxidase [2,3] or
polyphenol oxidase [4], nucleoside phosphorylase and
xanthine oxidase [58], phosphorylase A, phospho-
Corresponding author. Tel.: +30-81-393618;
fax: +30-81-393601.
E-mail address: nchan@chemistry.uch.gr (N.A. Chaniotakis).
[9,10], maltose phosphorylase and glucose oxidase
[11], co-immobilized with phosphatase and mura-
torase [12] have been employed with various degrees
of success. Camman et al. [12] have reported the low-
est detection limit (0.01 M) up until now, while the
best operational stability (70% remaining activity after
20 h) was reported by Coulet et al. [5] using nucleoside
phosphorylase and xanthine oxidase. Additionally the
best storage stability (66% remaining activity after 5
weeks dry storage at 20 C) was reported by Cosnier
et al. [4] using alkaline phosphatase and polyphenol
oxidase. The simplest and most efficient approach is
achieved with the use of pyruvate oxidase (POD), an
enzyme that catalyzes the following reaction
Pyruvate + H2 PO4 + O2
Acetylphosphate + CO2 + H2 O2 (1)

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 2 0 4 - 6
272 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277
POD requires the presence of thiamine pyrophos-
phate (TPP), flavine adenine dinucleotide (FAD) and
Mg(II) for the catalysis to take place. These cofactors
make the system not very selective, while POD-based
biosensors are shown to suffer form low operational
and storage stability [13,14]. The absence of phos-
phate anions from the working and storage buffer that
is crucial for the enzyme stability conduces to these
problems [13].
In this work, the construction of stable phosphate
biosensor based on recombinant pyruvate oxidase
stabilized with polyelectrolyte is presented. Pyruvate
oxidase from Lactobacillus plantarum contains the
cofactors TPP, FAD and Mg(II) bound in the active
center [15] and is genetically engineered to enhance
its stability [1618]. Bergmann et al. [19] have uti-
lized this enzyme in conjunction with horseradish
peroxidase for the construction of a bienzyme mod-
ified carbon paste biosensor for the measurement of
pyruvate.
In this work, the stabilized pyruvate oxidase is
immobilized by physical adsorption into a highly
porous and conductive carbon electrode. This mate-
rial has been used with great success for the con-
struction of highly stable and reproducible glucose
and lactate biosensors [2224]. The polyelectrolytes
diethylaminoethyl-dextran and DNA are employed
during the immobilization procedure to stabilize POD
via a cage formation around the POD. The use of
polyelectrolytes such as diethylaminoethyl-dextran
or polyethylenimine (positively charged macro-
molecules) have been proved to stabilize enzymes
[1927] in various environments. It has been shown
that these polyelectrolytes form a cage-like stable mi-
croenvironment around the enzyme via electrostatic
interactions. Recently, DNA (a negatively charged
macromolecule) was shown to enhance the stability of
certain biosensors [28]. Here, the effects of the type
and the amount of stabilizer used during the construc-
tion of the POD-based biosensor on the characteristics
of the sensor are evaluated, and an optimized sys-
tem is constructed. The optimized biosensor is then
incorporated in a flow system for the measurement
of phosphate levels in serum. The usually occurring
serum interferences from the easily electro-oxidazible
species are eliminated using an efficient preoxidizing
cell [29], allowing for the fast and accurate measure-
ments of phosphate in this complicated matrix.
2. Experimental
2.1. Materials-reagents
Recombinant pyruvate oxidase (POD) from Lac-
tobacillus plantarum (EC 1.2.3.3, 1.66 U/mg), avail-
able from Roche Diagnostics GmbH (Germany), was
kindly provided by Dr. Spohn (University of Halle).
Diethylaminoethyl-dextran (deae-dextran) (D-9885)
and DNA sodium salt (D-1626) were obtained from
Sigma. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfo-
nic acid (HEPES), biochemica grade was purchased
from Fluka.
The porous carbon was obtained from BF HIRM
(Austria). Vitreous carbon foam (bulk density
0.05 g/cm3 , porosity 96.5%), carbon fabric (weight/
m2 200 g, warp and weft yarn 200 Tex, plain
weave) and platinum mesh (wire diameter 0.06 mm,
wires/inch 82 82) were purchased from Goodfellow
Cambridge Ltd. In all experiments nano-pure water
(18 M, EASYpure model D7033, Barnstead) was
used. All other reagents used were of analytical grade.
2.2. Biosensor construction
A rod (2.1 mm diameter, 4.0 mm height) of the
porous carbon was cleaned in a sonicated ethanol bath
for 10 min and then in water bath for 10 min, followed
by oven drying at 150 C for 30 min. Pyruvate oxidase
was dissolved (to a final concentration 100 U/ml) in
10 mM HEPES buffer (pH 7.5) containing the desired
amount of the stabilizer. The enzyme was allowed to
react with the polyelectrolyte for 20 min at +4 C. Af-
ter this, the cleaned and dried carbon rod was placed
into the enzyme solution for 20 h at +4 C. The car-
bon rod was then removed from the solution, washed
thoroughly with buffer, and placed in a testing holder.
It is estimated, based on the amount of solution ad-
sorbed by the carbon, that 1 unit per carbon rod is im-
mobilized. The electrical contact was achieved from
the backside through a platinum wire. When not used
the biosensors were kept at +4 C in HEPES buffer.
2.3. Pre-oxidizing cell
The pre-oxidizing cell used for the blood measure-
ments was slightly modified from the one previously
described [29]. The cell consists of two separate parts,
 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277
the oxidation and the reduction part connected to a dc
power supply. The constant potential method at an op-
erational potential above that required to oxidize all
interfering species, but bellow that for the oxidation of
H2 O is chosen. Each part has separate inlet and outlet
of the flow sample. The inlet of the oxidation part is
connected to the injection valve so that the sample af-
ter the injection will pass only through this part of the
cell. The outlet of the oxidation part (anode) of the cell
is connected to the wall-jet flow cell where the biosen-
sor is placed. The inlet of the reducing part (cath-
ode) of the cell is connected to a reservoir containing
0.1 M KCl while the outlet is connected to the wastes.
The electrical contact of the two half-cells is
achieved through a cellulose acetate membrane (12
16 kDa cut-off) placed between the upper side of the
vitreous carbon foam and the lower side of the plat-
inum mesh. The reactor where the oxidation takes
place is the lower part of the cell, which contains the
vitreous carbon foam (6.3 cm 0.6 cm 0.5 cm). It
is composed of a polymethylmethacrylate (PMMA)
plate (8.3 cm 4.0 cm 1.0 cm) where a hole in the
dimensions of the foam is made. To assure homo-
geneous distribution of the potential throughout the
carbon without any IR drop, the electrical contact to
the power supply is achieved from the lower side of
the carbon plate using carbon fabric.
The cathode compartment (also made by PMMA
8.3 cm 4.0 cm 1.0 cm) contains the platinum
mesh (3.5 0.6). The outlet of this part of the cell
is designed so that the hydrogen bubbles generated
during the experiment will be quickly removed from
the platinum surface.
2.4. Electrochemical measurements
In all experiments a three electrode Metrohm 641
VA-Detector, a silver/silver chloride double junction
reference electrode (Model 90-02, ORION Res. Inc.),
and a platinum counter electrode (CORNING, cat. no.
476060) were used. The working potential was set at
+800 mV in all experiments, except from the oper-
ational stability where the sensors were polarized at
+600 mV versus Ag/AgCl. The signal was recorded
via a personal computer equipped with a 16-bit A/D
converter and controlled with software written in BA-
SIC. Temperature control at 24.00.1 C was achieved
with a circulating bath (Model 9105, PolyScience).
273
The flow injection system consisted of a wall-jet flow
cell, an injection valve with loop volume 400 l, while
the solvent delivery was done using a peristaltic pump
(No 7554-30, Cole-Palmer). The response is calcu-
lated as the peak height from the baseline.
3. Results and discussion
A detailed experiment in order to obtain the opti-
mum operational pH for the phosphate biosensor re-
veals that the pH profile of the sensor is bell shaped
(data not shown), with optimum value at around pH
7.5. This is slightly shifted to basic pH compared with
the POD pH profile that has been shown to be at pH
7.0 [30]. Further experiments were performed using
10 mM HEPES buffer adjusted to pH 7.5.
3.1. Effect of pyruvate concentration
From Eq. (1) it is clear that pyruvate, a co-substrate
in the enzymatic reaction, is required for the proper
operation of the system, and the measurement of
phosphate. The optimum amount of pyruvate was
determined by varying its concentration in the carrier
stream from 0.110 mM, while monitoring its effect
on the sensitivity and detection limit as determined
from the calibration curve obtained for each pyruvate
concentration. The results shown in Fig. 1 indicate
that an increase in the pyruvate concentration up to
1.0 mM results in an increase in the sensitivity (linear
Fig. 1. Effect of pyruvate concentration, incorporated in the HEPES
buffer, on the sensitivity () and the detection limit () of the
phosphate biosensor.
274 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277
range 0.051.0 mM phosphate). Further increase in
the pyruvate concentration results in lower sensitivi-
ties. Even though this decrease of the sensitivity was
also observed by Kubo et al. [14], the large negative
slope was not expected. Based on this observation, the
detection limit of the sensor might also be influenced
by the background pyruvate concentration. When the
pyruvate concentration is increased from 0.1 up to
0.5 mM, there is no influence on the lower detection
limit. When the concentration of pyruvate is increased
from 0.5 to 10 mM the lower detection limit also in-
crease linearly. The fact that the sensitivities obtained
with 5 or 10 mM pyruvate were similar indicates that
at these pyruvate concentrations the rate limiting sub-
strate is oxygen. From these results the 1 mM pyruvate
was chosen to be used for all further experiments.
3.2. Effect of polyelectrolyte
The effect of the amount and type of polyelectrolyte
on the phosphate biosensor was then investigated. Sen-
sors were prepared from solutions containing vari-
ous amounts of deae-dextran or DNA. Deae-dextran
was varied from 0.1 to 0.5%, w/v, while two different
amounts of DNA were tested (0.1 and 0.25%, w/v).
Higher amount of DNA resulted in the formation of a
suspension, preventing the construction of the sensor.
To monitor the effect of the amount and type of poly-
electrolyte, the sensors were periodically tested. Be-
tween the test times, all sensors were stored at 4 C in
buffer solution (10 mM HEPES, pH 7.5). Table 1 sum-
marizes the results of the sensitivity and the remaining
activity of the sensors, 1 month after their construction.
From this data it is clear that both polyelectrolytes in-
crease the stability of the biosensor. It is interesting to
note that when the amount of deae-dextran used is in-
Table 1
Effect of polyelectrolyte type and concentration on the sensitivity and remain activity of the phosphate biosensor
Polyelectrolyte Concentration (%, w/v)
1 5.56
2 Deae-dextran 0.10
3 Deae-dextran 0.25
4 Deae-dextran 0.50
5 DNA 0.10
6 DNA 0.25
a One month after construction.
creased beyond the level of 0.25%, w/v the stabilizing
effect is eliminated and the sensor loses its sensitivity
even faster than the control. The reduced biosensors
stability at higher amounts of deae-dextran has been
previously observed with other biosensors [2224] and
has been attributed to the reduction of enzyme activity
at high polyelectrolyte concentrations. Additionally, it
is shown that DNA has a smaller stabilizing effect on
the sensor. When 0.1%, w/v DNA is used the initial
sensors sensitivity is smaller than the control, and the
stability of the sensor is slightly reduced. Increasing
the concentration to 0.25%, w/v, the stability of the
sensor is improved over that of the control, but to a
lesser extent from the deae-dextran stabilized sensor.
Based on these results sensors 1, 2 and 6 were
chosen for further evaluation of their operational sta-
bility. For this experiment the sensors were incorpo-
rated in a flow system and were continuously po-
larized at +600 mV versus Ag/AgCl. The flow rate
was 0.65 ml/min, the temperature was held constant
at 24.0 0.1 C and the working solution was 10 mM
HEPES, 1 mM pyruvate and 1 mM phosphate adjusted
to pH 7.5. Under these conditions (buffer stream con-
taining the required substrates, pyruvate and phos-
phate), there is a continuous enzymatic catalysis. The
system was calibrated daily, and the sensitivity was
used as a measure to evaluate the activity of the sen-
sor. For the calibration procedure, the phosphate was
removed from the buffer stream and after equilibration
different concentrations of phosphate were injected.
Table 2 shows the remaining activity of the three
sensors under the continuous operation test. It is inter-
esting to note that the operational stability of all the
sensors monitored is better than any such sensors pre-
sented so far in the literature (70% remaining activity
after 20 h [5]). Moreover, the operational stability of
Sensitivitya (A/mM) Remaining activitya (%)
0.06 51.8
7.94 0.12 77.1
5.88 0.14 68.0
5.31 0.08 43.4
3.00 0.03 49.6
6.15 0.12 57.5
 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277 275

Table 2
sensitivity is 10.3 0.2 A/mM and the response
Operational stability of the 1, 2 and 6 phosphate biosensors
is linear to phosphate concentrations from 50 to
Hours polarized Remaining activity (%) 1250 M. The limit of detection for phosphate based
1 2 6 on a signal-to-noise ratio of three is 4.8 M. The re-
20 77 114 104 sponse time varies between 20 and 40 s, depending on
40 72 106 94 the concentration of the analyte. The sensor-to-sensor
60 72 97 89 reproducibility based on the sensitivities is 2.2%
80 69 86 84
100 65 79 79
RSD, while based on the response to 1 mM phos-
120 61 73 72 phate it is 7.6% RSD. These results indicate that the
140 59 71 69 method developed enables the construction of very
220 51 67 56 reproducible sensors. The storage stability of the
phosphate biosensor was then examined, using the
the sensor, 2 is exceptional, with 67% remaining ac- calibration curve of the fresh sensors as the reference
tivity after 220 h. DNA stabilized sensor, 6 has 56% curve. These studies indicate that there is a 84 5%
and the control sensor has 50% remaining activity. residual activity after 2 months of dry storage at 4 C,
while after 6 months the residual activity is reduced to
49 4%.
3.3. Characterization of the phosphate biosensor

A batch of the most stable sensors (2) was then
prepared. Three of these sensors were used to study
their analytical characteristics, and the remaining were
freeze dried and stored at 4 C for storage stability
measurements. At the end of each month one sensor
was rehydrated for 20 h and then its residual activity
was calculated from the sensitivity obtained from the
calibration curve.
In Fig. 2, the initial average calibration curve of
the three freshly prepared sensors is presented. The
Fig. 2. Calibration curve of the phosphate biosensor in 10 mM
HEPES, 1 mM pyruvate buffer adjusted at pH 7.5. The insert
presents the linear part of the curve. The working potential was
+800 mV vs. Ag/AgCl.
3.4. Application to serum analysis
A key factor for the application of an amperomet-
ric sensor in real life samples is its selectivity towards
the easily electro-oxidazible species. In the case of
serum, ascorbic acid, uric acid, acetaminophen and
many other compounds can interfere. The porous car-
bon used here for the construction of the phosphate
sensor has shown to be sensitive to these species (be-
tween 10 and 13 A/mM), preventing its application
to serum analysis. For this reason a pre-oxidizing cell
[29] was incorporated in the FIA system, to oxidize
these species before they reach the electrode surface.
Initial experiments indicated the need for the buffer
to be mixed with the sample after the pre-oxidation
step. When the buffer was introduced prior to the cell
the response of the sensor was not reproducible. The
final experimental set-up is shown in Fig. 3. The con-
centration of pyruvate in blood varies considerably
(70150 M) [31]. Unless there is a high and constant
concentration of pyruvate in the buffer stream, this
fluctuation will introduce an error to the biosensors
response. Therefore, the concentration of pyruvate in
the buffer stream was raised from 1 to 10 mM. At the
same time, the concentration of HEPES in the buffer
stream was also raised in order to increase its buffer
capacity. Thus, the buffer stream was 50 mM HEPES
and 10 mM pyruvate adjusted to pH 7.5 at a flow rate
of 0.65 ml/min and the flow rate of the sample was
also 0.65 ml/min.
276 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277
Fig. 3. Diagram of the flow injection system. The buffer consists of 50 mM HEPES and 10 mM pyruvate adjust to pH 7.5. The sample
passes through the pre-oxidizing cell where the interfering species are electrooxidized, then is mixed with the buffer and reach the biosensor
to complete the analysis.
The analytical characteristics of the phosphate
biosensor incorporated in the flow injection system
are as follows. The sensitivity is 445 9 nA/mM, the
response is linear from 0.5 to 10 mM, while the de-
tection limit is less than 0.3 mM. In Fig. 4, a typical
recording of phosphate injections is presented.
Prior to real sample analyses, the efficiency of the
cell in the elimination of interferents was then investi-
gated. With the cell current off, the phosphate biosen-
sor exhibits sensitivities 1.72, 2.27 and 1.63 A/mM
Fig. 4. Recording of the response of the biosensor incorporated in
the flow injection system shown in Fig. 3 to phosphate injections
(2.5, 5.0, 7.5 and 10 mM phosphate, respectively). The sensor is
polarized at +800 mV vs. Ag/AgCl. The flow rate is 0.65 ml/min
and the buffer consist of 50 mM HEPES and 10 mM pyruvate
adjusted to pH 7.5.
for ascorbate, acetaminophen and urate, respectively.
When the cell current is on, all sensitivities are re-
duced to 0.26, 0.30 and 0.51 A/mM, respectively.
With these sensitivities the detection limit of the sys-
tem for these species is 362, 853 and 816 M for
ascorbate, acetaminophen and urate, respectively, val-
ues that are much higher than their normal concentra-
tion in serum.
The above experimental set-up was then used to de-
termine the phosphate level in serum. Three samples
of serum were analyzed and the results were com-
pared with those obtained with ion-chromatography.
The samples were injected in the system without di-
lution and the calculated concentration was found to
be 1.03 0.06 mM. The results from the chromato-
graphic analysis were 0.99 0.02 mM, very close to
the results obtained with the biosensor.
4. Conclusions
The development and evaluation of a phosphate
biosensor based on stabilized with polyelectrolytes
recombinant pyruvate oxidase adsorbed into a porous
carbon has been presented. Both electrolytes used
were shown to have a positive effect on both the oper-
ational and storage stability of the resulting biosensor,
with 0.1%, w/v deae-dextran being the optimum. The
phosphate biosensor exhibits exceptional operational
and storage stability while the simple construction
 V.G. Gavalas, N.A. Chaniotakis / Analytica Chimica Acta 427 (2001) 271277
procedure results in a very good sensor-to-sensor
reproducibility. The use of the sensor in an FIA sys-
tem equipped with a pre-oxidizing cell allows for
the measurement of the activity of phosphate in dif-
ficult samples such as those of human serum with
results in good agreement with those obtained with
ion-chromatography.
Acknowledgements
We would like to thank, Dr. U. Spohn (University of
Hall) for supplying the enzyme, G. Kouvarakis (ECPL,
University of Crete) for the ion-chromatography mea-
surements and the Greek Secretary General of Re-
search and Development for financial support through
the YPER program (97 YPER-219).
References
[1] S.O. Engblom, Biosens. Bioelectron. 13 (1998) 981.
[2] G.G. Guilbault, M. Nanjo, Anal. Chim. Acta 78 (1975) 69.
[3] Y. Su, M. Mascini, Anal. Lett. 28 (1995) 1359.
[4] S. Cosnier, C. Gondran, J.-C. Watelet, W. De Giovani, R.P.M.
Furriel, F.A. Leone, Anal. Chem. 70 (1998) 3952.
[5] E.M. dUrso, P.R. Coulet, Anal. Chim. Acta 239 (1990) 1.
[6] E.M. dUrso, P.R. Coulet, Anal. Chim. Acta 281 (1993) 535.
[7] S.D. Haemmerli, A.A. Suleiman, G.G. Guilbault, Anal.
Biochem. 191 (1990) 106.
[8] U. Wollenberger, F. Schubert, F.W. Scheller, Sens. Act. B 7
(1992) 412.
[9] J.J. Fernandez, J.R. Lopez, X. Correig, I. Katakis, Sens. Act.
B 47 (1998) 13.
[10] J. Parellada, A. Narvaez, M.A. Lopez, E. Dominguez, J.J.
Fernandez, V. Pavlov, I. Katakis, Anal. Chim. Acta 362 (1998)
47.
View publication stats
277
[11] S. Huwel, L. Haalck, N. Conrath, F. Spener, Enz. Microb.
Technol. 21 (1997) 413.
[12] N. Conrath, B. Grundig, S. Huwel, K. Cammann, Anal. Chim.
Acta 309 (1995) 47.
[13] N. Gajovic, K. Habermuller, A. Warsinke, W. Schuhmann,
F.W. Scheller, Electroanalysis 11 (1999) 1377.
[14] I. Kubo, M. Inagawa, T. Sugawara, Y. Arikawa, I. Karube,
Anal. Lett. 24 (1991) 1711.
[15] Y. Muller, G. Schumacher, R. Rudolph, G. Schulz, J. Mol.
Biol. 237 (1994) 315.
[16] E. Kopetzki, K. Lehnert, P. Buckel, Clin. Chem. 40 (1994)
688.
[17] B. Risse, G. Stempfer, R. Rudolph, H. Mollering, R. Jaenicke,
Prot. Sci. 1 (1992) 1699.
[18] B. Risse, G. Stempfer, R. Rudolph, G. Schumacher, R.
Jaenicke, Prot. Sci. 1 (1992) 1710.
[19] W. Bergmann, R. Rudolph, U. Spohn, Anal. Chim. Acta 394
(1999) 233.
[20] A. Appleton, T.D. Gibson, J.R. Woodward, Sens. Act. B 43
(1997) 65.
[21] T.D. Gibson, J.R. Woodward, in: P.G. Eldman, J. Wang (Eds.),
Biosensors and Chemical Sensors, ACS Books, 1992, p. 40.
[22] V.G. Gavalas, N.A. Chaniotakis, T.D. Gibson, Biosens.
Bioelectron. 13 (1998) 1205.
[23] V.G. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 404 (2000)
67.
[24] V.G. Gavalas, N.A. Chaniotakis, Mikrochim. Acta, submitted
for publication.
[25] U. Spohn, D. Narasaiah, L. Gorton, Electroanalysis 8 (1996)
507.
[26] H. Liden, J. Volc, G. Marko-Varga, L. Gorton, Electroanalysis
10 (1998) 223.
[27] M. Tessema, E. Csoregi, T. Ruzgas, G. Kenausis, T. Solomon,
L. Gorton, Anal. Chem. 69 (1997) 4039.
[28] P. Dantoni, S.H.P. Serrano, A.M.O. Brett, I.G.R. Gutz, Anal.
Chim. Acta 366 (1998) 137.
[29] V.G. Gavalas, M.G. Fouskaki, N.A. Chaniotakis, Anal. Lett.
33 (2000) 2391.
[30] Roche Molecular Biochemicals 1999/2000.
[31] R.K. Murray, D.K. Granner, P.A. Mayes, V.W. Rodwell,
Harpers Biochemistry, Appleton & Lange, 1996, p. 829.