Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on...
Transcript of Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on...
Rapid and convenient determination of oxalic acid employing a noveloxalate biosensor based on oxalate oxidase and SIRE technology
Feng Hong a,b, Nils-Olof Nilvebrant c, Leif J. Jonsson b,*a Department of Applied Microbiology, Lund University/Lund Institute of Technology, P.O. Box 124, SE-22100 Lund, Sweden
b Biochemistry, Division for Chemistry, Karlstad University, SE-65188 Karlstad, Swedenc STFI, Swedish Pulp and Paper Research Institute, P.O. Box 5604, SE-11486 Stockholm, Sweden
Received 4 February 2002; received in revised form 23 October 2002; accepted 4 November 2002
Abstract
A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE
(sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with
other present detection systems for oxalate. The total analysis time for each assay was 2�/9 min. A linear range was observed
between 0 and 5 mM when the reaction conditions were 30 8C and 60 s. The linear range and upper limit for concentration
determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20
mM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction
time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the
determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated
well (R�/0.952) with the colorimetric method.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Oxalic acid; Oxalate oxidase; Biosensor; SIRE technology
1. Introduction
Oxalic acid is widely distributed as calcium and
magnesium salts in plant cells and cell walls (Pundir
and Verma, 1993). In human, precipitation of calcium
oxalate may lead to the formation of kidney stones. The
determination of oxalic acid is therefore of considerable
significance, especially in clinical diagnosis. Nowadays,
oxalic acid is determined in the diagnosis and therapeu-
tic monitoring of primary hyperoxaluria type 1 (PH1;
Wilson and Liedtke, 1991), in preparation of low-
oxalate diets for hyperoxaluria patients (Lathika et al.,
1995; Savage et al., 2000), and in food industry such as
beer production (Haas and Fleischman, 1961). More
recently, there has been increasing demands on measure-
ment and control of oxalic acid in the pulp and paper
industry since high concentrations of oxalate are formed
during bleaching with strong oxidizing agents like
ozone, chlorine dioxide, oxygen, and hydrogen perox-
ide. The oxalic acid can easily precipitate in the form of
calcium oxalate crystals, which cause clog problems in
pipework, washing filters, and heat exchangers (Elsan-
der et al., 2000; Nilvebrant et al., 2002).
Currently, a large number of various methods for the
assay of oxalate are available such as high-performance
liquid chromatography (Holloway et al., 1989; Fry and
Starkey, 1991; Manoharan and Schwille, 1994), gas
chromatography (Gelot et al., 1980; Yanagawa et al.,
1983), ion chromatography (Schwille et al., 1989;
Petrarulo et al., 1993; Peldszus et al., 1998), spectro-
photometric methods based on oxalate oxidase (Kohl-
becker and Butz, 1981; Ichiyama et al., 1985; Salinas et
al., 1989; Petrarulo et al., 1994) or oxalate decarboxylase
(Hatch et al., 1977; Beutler et al., 1980), pH-electrode
determination coupled with an enzymic reaction (Boer
et al., 1984; Canizares and Luque de Castro, 1997), and
chemiluminescence detection (Balion and Thibert, 1994;
Gaulier et al., 1997a,b, 1998). Some of these methods
* Corresponding author. Tel.: �/46-54-7001-801; fax: �/46-54-7001-
457.
E-mail address: [email protected] (L.J. Jonsson).
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www.elsevier.com/locate/bios
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PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 2 5 0 - 6
provide high sensitivity and specificity but also have
drawbacks such as high costs for equipment and assays,
time-consuming and complicated operation as well as
being unsuitable for applications where mobility of theanalytical equipment is of advantage.
The development and application of biosensors have
attracted attention in many areas due to good selectiv-
ity, sensitivity, rapidity, stability, and reliability. Oxalate
biosensors based on immobilized oxalate oxidase (Nabi
Rahni et al., 1986; Dinckaya and Telefoncu, 1993;
Milardovic et al., 2000) and plant tissues (Glazier and
Rechnitz, 1989) have been constructed. A drawback ofthese traditional biosensors is that the life span is only
1�/3 months due to the instability of the enzyme and
they are relatively expensive. Therefore, such biosensors
are suited more for short-term processing of many
samples rather than occasional measurements under an
extended period of time. Here, we describe a new
method for the determination of oxalic acid based on
the use of oxalate oxidase in the SIRE (sensors based oninjection of the recognition element) technology.
The method is based on the injection of the recogni-
tion element (in this case oxalate oxidase) in a buffer
solution into an internal chamber (integration of an
electrochemical transducer and enzyme solution) of the
SIRE biosensor. The SIRE biosensor technology has
been described previously (Kriz and Johansson, 1996).
When the enzyme is introduced into the reactionchamber by flow-injection, it will be in close proximity
to an electrochemical transducer. A small amount of
enzyme is used for one measurement and is then
discarded. Compared to traditional biosensors, the
SIRE-based biosensor has several advantages (Kriz et
al., 2001): (1) it circumvents or avoids the problem
associated with the instability of the biological recogni-
tion element since a new and freshly prepared enzymesolution is used for each measurement; (2) a differential
measuring technique is employed where the sample is
measured both in the presence and absence of enzyme,
thus allowing the matrix signal to be estimated, which
increases the accuracy of the assay; (3) the automatic
temperature compensation that is used provides the
possibility to place the biosensor in a process line with
large temperature variations; and (4) the SIRE biosen-sor also can resist conditions of thermal stress due to the
fact that the recognition element is not immobilized on
the sensor probe, while a traditional biosensor will
permanently lose its response due to deactivation of
the enzyme if it is operated once at high temperature.
Oxalate oxidase (oxalate: oxygen oxidoreductase, EC
1.2.3.4, OXO) is capable of oxidizing oxalic acid to
carbon dioxide with the simultaneous production ofhydrogen peroxide which is given by the following
equation (Chiriboga, 1966):
HOOC�COOH�O2 0OXO
2CO2�H2O2: (1)
The hydrogen peroxide that is generated gives rise to an
electrochemical signal by its oxidation at the electrode in
the SIRE biosensor probe. The oxalic acid concentra-
tion in a sample can be determined via a calibrationcurve made by exposing the sensor probe to standard
solutions of oxalic acid. The effect of interfering
substances is minimized by the differential measuring
procedure and, as a consequence, the biosensor gives
more accurate results.
The characteristics of the oxalate SIRE biosensor
with regard to linear range, detection limit, sensitivity,
and precision were investigated. In addition, the influ-ence of factors including enzyme concentration, reaction
time, and temperature was evaluated. The performance
of the method was also investigated with regard to an
important potential application: determination of oxalic
acid in urine samples.
2. Materials and methods
2.1. Reagents and enzyme
All chemicals were of spectral or analytical grade.
Unless otherwise stated, all chemicals used were ob-
tained from Sigma Chemical Co. (St. Louis, MO). This
includes oxalate oxidase from barley seedlings (Cat. No.
O 4127), activated charcoal (washed with hydrochloric
acid, C 4386), the Sigma urinary oxalate assay kit (591),oxalate standard set (591-11), and oxalate urine controls
(O 6627). The oxalate oxidase was partially purified and
provided as a lyophilized powder (0.71 U/mg solid). The
oxalate standard set was used only in the assays of urine
specimens with the Sigma oxalate assay kit. Oxalic acid
(disodium salt) and ethylenediamine tetraacetic acid
disodium salt dihydrate (EDTA) were purchased from
Merck (Darmstadt, Germany). Distilled/deionized waterwas utilized throughout all the experiments.
2.2. Buffer and test solutions
A succinate-based stock solution (250 mM) was made
by dissolving disodium succinate hexahydrate in dis-
tilled water and adjusting the pH to 4.0 with succinic
acid. A succinate-based buffer (50 mM, pH 4.0) wasmade by diluting the succinate-based stock solution with
distilled water and adding Tween 20 to a final concen-
tration of 2 ml/l. The succinate-based buffer was used as
the carrier stream in the biosensor for the preparation of
oxalic acid solutions and for the preparation of oxalate
oxidase suspensions (prepared daily).
A succinic acid-based stock solution (250 mM) was
prepared from succinic acid and by adjustment of thepH to 4.0 with sodium hydroxide. This stock solution
was used for the preparation of a succinic acid-based
buffer (50 mM, pH 4.0) containing 0.2% (v/v) Tween 20,
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/11811174
which was used for preparing solutions of oxalic acid
and suspensions of oxalate oxidase in the determinations
of urinary oxalate. Dilution of urine samples was made
using 50 mM succinic acid and the pH was adjusted to5.0 with sodium hydroxide.
2.3. Biosensor and measuring procedure
The SIRE Biosensor P100 was obtained from Chemel
AB (Lund, Sweden). The biosensor was equilibrated
with the succinate buffer with a flow of 0.1 ml/min until
the current was stable at the applied potential (�/650
mV vs. silver wire reference electrode). The reactionchamber (approximately 1.3 ml) is covered by a dialysis
membrane and contains an amperometric transducer,
which has a potentiostatic three-electrode configuration.
The working and auxiliary electrodes consist of plati-
num wires while the reference electrode consists of a
silver wire (Kriz et al., 2001).
The biosensor probe was immersed into the sample
solution (30�/50 ml), which was constantly agitated bymagnetic stirring (300 rpm) and its temperature was
stabilized using a waterbath. To initiate a measurement,
0.1 ml of the oxalate oxidase suspension was injected
into the system. When the oxalate oxidase reached the
reaction chamber, the buffer flow stopped and the
reaction started. The oxalic acid, which passed from
the sample through the dialysis membrane into the
reaction chamber, served as substrate for oxalateoxidase, and hydrogen peroxide was generated. Hydro-
gen peroxide was then oxidized at the anode, giving rise
to the electrochemical signal. This signal reflected the
hydrogen peroxide plus potential interfering compounds
present in the matrix. Thereafter, the reaction chamber
was immediately washed with buffer to regenerate the
system, and then refilled with buffer (without enzyme).
Then, a second signal was obtained that reflected onlythe interfering compounds in the matrix. Subtraction of
the signal obtained in the absence of enzyme from the
signal obtained in the presence of enzyme provided a
differential measurement corresponding directly to the
concentration of oxalic acid in the sample.
The instructions in the manual were followed with the
exception of some minor modifications as stated below.
The succinate buffer that was used as the carrier streamwas degassed before use in order to avoid the formation
of air bubbles, which interfere with the response values.
A heating waterbath and a magnetic stirrer were applied
for achieving high sensitivity and stability. The tem-
perature and other conditions, such as stirring speed, stir
bar size, and reaction time, were kept constant for both
samples and standard solutions in order to maintain the
necessary accuracy. The interval between two measure-ments was maintained at least 30 s. The biosensor
system required washing once or twice per month with
the supplied washing solution, in particular, since the
commercial oxalate oxidase preparation had poor
solubility. For the same reason, the oxalate oxidase
suspension was mixed by gentle inversion to reach a
homogeneous state before each assay injection. Everysample should preferably be assayed at least in triplicate
to obtain a precise mean value.
2.4. Experimental
2.4.1. Enzyme concentrations
Oxalate oxidase was added to the succinate buffer (50mM, pH 4.0) with 0.2% (v/v) Tween 20 to give final
concentrations of 0.1 mg/ml (71 U/l), 0.2 mg/ml (142 U/l),
and 0.4 mg/ml (284 U/l), respectively. A suspension was
formed by vortexing gently. Sodium oxalate was dis-
solved in the succinate buffer to achieve a final oxalate
concentration ranging from 0 to 40 mM.
2.4.2. Temperature dependence
A 0.4 mM oxalate solution was incubated in a
waterbath. The temperature was varied from 25 to
50 8C. The assay was carried out with the biosensor
using 0.4 mg/ml oxalate oxidase. Other procedures were
performed as described in Section 2.4.1.
2.4.3. Urine preparation and determination of oxalate in
charcoal-treated urine
A series of urine samples from healthy volunteers
were collected in bottles containing EDTA to give a
final EDTA concentration of 10 mM (Chalmers et al.,
1985). The pH of the samples was then adjusted to 5.0
with succinic acid and stored at 4 8C. Before the assay,
the acidified urine was diluted with an equal volume ofsample diluent (Barlow and Harrison, 1990), prepared
from succinic acid and adjusted to pH 5.0 with NaOH.
The pH of the diluted urine was checked by using a pH-
meter. Thereafter, the diluted urine was mixed with 200
g/l activated charcoal for 5 min by using a rotating mixer
(Ichiyama et al., 1985; Li and Madappally, 1989). Then,
the mixture was filtered through a Buchner funnel with a
filter paper (1F quality, Munktell Filter AB, Grycksbo,Sweden) under vacuum. Oxalate standard solutions
were prepared and treated in the same way as the urine
samples. The filtrates were analyzed with the biosensor
at 38 8C using 0.2 mg/ml enzyme in the succinic acid-
based buffer. The concentration of oxalic acid in the
samples was determined from a calibration curve pre-
pared using several different oxalic acid standard
solutions. Both samples with and without charcoalpretreatment were assayed for oxalate content using
the Sigma urinary oxalate assay kit as the reference
method.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/1181 1175
2.4.4. Enzymatic colorimetric determination of urinary
oxalate
For the determination of oxalate in urine, the Sigma
oxalate assay kit was applied. The determination isbased on the oxidation of oxalate to carbon dioxide and
hydrogen peroxide by oxalate oxidase. The hydrogen
peroxide serves as a substrate for a peroxidase, which
catalyzes the formation of an indamine dye (with an
absorbance maximum at 590 nm) from 3-methyl-2-
benzothiazolinone hydrazone (MBTH) and 3-(dimethy-
lamino)-benzoic acid (DMAB). A Hitachi U-2000
spectrophotometer (Tokyo, Japan) was used for thecolorimetric determination method.
3. Results and discussion
3.1. Enzyme concentrations and linear range of biosensor
Oxalate oxidase in combination with an SIRE bio-
sensor was applied to develop a fast and selective
analysis method for oxalic acid. First, the amount of
enzyme that should be injected for an analysis wasdetermined. Three concentrations of oxalate oxidase,
0.1, 0.2, and 0.4 mg/ml, were employed. The response
signals of the biosensor using different oxalate oxidase
and oxalate concentrations are shown in Fig. 1. Only a
small difference in signal between 0.2 and 0.4 mg/ml was
found, whereas the signal obtained with 0.1 mg/ml
oxalate oxidase was markedly lower. The response
increased linearly with an oxalate concentration up to5 mM, with a curvature at higher concentrations.
However, the curvature observed prior to 10 mM (Fig.
1) decreased when higher enzyme concentrations were
used (0.2 and 0.4 mg/ml). This indicates that the enzyme
concentration was not the limiting factor when the
sample had an oxalate concentration of approximately
10 mM or more. Since the reaction catalyzed by oxalate
oxidase also requires molecular oxygen, this should at
some point become rate limiting. However, there was no
significant difference in signal between experiments inwhich degassed or not degassed carrier was used. This
indicates that the concentration of dissolved oxygen was
sufficient even when degassed solution was used.
Fig. 1 also indicates that the response was linear up to
approximately 5 mM and reached an upper limit at 15,
10, and 10 mM with 0.1, 0.2, and 0.4 mg/ml oxalate
oxidase, respectively. However, further studies indicated
that the linear range could be modified by decreasing thereaction time from 60 to 25 s when 0.1 mg/ml enzyme was
used, as shown in Fig. 2. When the reaction time was 25
s, a linear relationship was found between the signal and
the concentration of oxalate below 25 mM.
Around 100 ml of enzyme reagent was consumed for
each measurement and the amount of enzyme required
for each assay would therefore be in the range 10�/40 mg
when 0.1�/0.4 mg/ml is used. Due to the enzyme cost, 0.1mg/ml would, if possible, be preferred, and the concen-
tration of 0.1 mg/ml was therefore included in further
experiments, although the experiments described above
suggest that a higher enzyme concentration would be
preferable.
3.2. Regression analysis
Regression analysis of the results obtained with
injection of 0.1 mg/ml enzyme and oxalate concentrations
up to 5 mM (Fig. 1) gave the equation y�/890.08x�/
141.98 (R2�/0.9973). When 0.2 and 0.4 mg/ml enzymes
were used to measure oxalate concentrations up to 5
mM, the equations y�/1134.9x�/129.87 (R2�/0.9987)
and y�/1127.7x�/40.569 (R2�/0.9996) were obtained,respectively. Fig. 3 shows the difference of the slopes in
the concentration range up to 1 mM oxalic acid. The
Fig. 1. Typical response curves of the biosensor for different oxalate
concentrations using three different amounts of oxalate oxidase: (')
0.1 mg/ml, (j) 0.2 mg/ml, and (") 0.4 mg/ml. Measuring conditions: 50
mM succinate buffer, pH 4.0, 30 8C, and 60 s.
Fig. 2. Response of the biosensor to oxalate concentrations ranging
from 1 to 40 mM after decreasing the reaction time to 25 s. Measuring
conditions: 0.1 mg/ml oxalate oxidase, 50 mM succinate buffer, pH 4.0,
and 30 8C.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/11811176
regression equations were y�/670x�/33.333 (R2�/
0.9887), y�/951.79x�/40.476 (R2�/0.9890), and y�/
1065.7x�/12.857 (R2�/0.9917) with 0.1, 0.2, and 0.4
mg/ml oxalate oxidase, respectively. When 0.1 mg/mlenzyme was used for the determination of oxalic acid
with a reaction time of 25 s (Fig. 2), the equation y�/
99.371x�/22.968 (R2�/0.9999) was obtained.
3.3. Effect of reaction time and enzyme dose
The effects of reaction time on the response signal of
the biosensor were studied by measurements of oxalateconcentrations ranging from 0.2 to 1.0 mM (Fig. 4). The
response increased with increased reaction time, as
would be expected since more oxalic acid and more
oxygen would reach the enzyme by passing through the
membrane and since the enzyme would be given more
time to convert the substrates. The response signal was
not high even for high oxalate concentrations unless the
reaction time was prolonged, as shown in Fig. 4. This
result showed that a reaction time above 1 min was more
suitable for the detection of oxalate at low concentra-
tions (B/1 mM). Also, a higher signal could be reached
by adding more enzymes to accelerate the reaction, as
shown in Fig. 5. The signal increased almost linearly
with the increase in reaction time but also with the
amount of oxalate oxidase. Thus, the sensitivity can be
enhanced by taking advantage of the features mentioned
above. Our recommendations for oxalate determination
Fig. 3. Response signals and linear regression analysis for oxalate
concentrations up to 1.0 mM using different amounts of oxalate
oxidase: (') 0.1 mg/ml, (j) 0.2 mg/ml, and (") 0.4 mg/ml. Measuring
conditions: pH 4.0, 30 8C, and 60 s.
Fig. 4. Response of the biosensor with increased reaction time. Measuring conditions: 0.1 mg/ml oxalate oxidase, 50 mM succinate buffer, pH 4.0, and
30 8C.
Fig. 5. Effect of enzyme dosage on the sensitivity of the analysis. The
figure shows the response with increasing amount of oxalate oxidase
for a standard solution with 0.2 mM. Measuring conditions: 50 mM
succinate buffer, pH 4.0, 30 8C, and reaction time (") 60 s, (j) 120 s,
(m) 180 s, and (*) 240 s.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/1181 1177
with 0.1 mg/ml oxalate oxidase using the SIRE biosensor
are summarized in Table 1.
3.4. Improvement of the sensitivity by increased reaction
temperature
Although the sensitivity was enhanced by increased
reaction time at 30 8C, the signal associated with 0.2
mM oxalate was only about 650 arbitrary units (Fig. 4)
after 4 min, the maximum time that can be pre-set using
the SIRE biosensor. For clinical analyses, a lower
detection level is desirable. The sensitivity of theanalyses therefore needed to be further increased. For
achieving this, the effect of the reaction temperature was
investigated in spite of the fact that the optimum
temperature of oxalate oxidase has been reported to be
around 35 8C, a result obtained by investigating the
maximum activity at various temperatures for 30 min
(Sugiura et al., 1979). The possibility that the enzyme
would be stable enough for a 4 min reaction even at atemperature higher than 35 8C was considered. The
effect of the reaction temperature on the response of the
biosensor is displayed in Fig. 6. The response signal was
highly dependent on the reaction temperature. Above
30 8C, the increase was around 130 arbitrary units per
degree.
Theoretically, even a higher temperature than 50 8Ccould be utilized to improve the sensitivity of the
analysis since it has been reported that oxalate oxidase
from barley seedlings is relatively heat stable. Forinstance, 100% activity remained after incubation at
75 8C for 10 min (Dumas et al., 1993) and more than
80% of the activity remained after incubation at 75 8Cfor 30 min (Sugiura et al., 1979). However, too high
temperature would result in changes in the oxalate
concentration due to the evaporation of water. In
addition, the structure of the membrane of the biosensor
probe might be damaged over 60 8C. A temperaturerange between 30 and 50 8C is therefore desirable.
A comparison of the response signal between 30 and
45 8C is shown in Table 2. The results indicated that the
lower detection limit was 40 mM when the determination
was carried out at 30 8C for 4 min using 0.1 mg/ml
oxalate oxidase. However, the detection limit 20 mM
could be reached at 45 8C (Table 2).
3.5. Precision of measurements
The repeatability and intermediate precision were
studied for five different concentrations of oxalic acid(0.4, 0.6, 0.8, 1.0, and 3.0 mM) and the relative standard
deviation (r.s.d.) was calculated. Three measurements of
each of the five different concentrations of oxalic acid
were performed. The repeatability was defined as the
precision under the same operating conditions over a
short interval of time, while the intermediate precision
was defined as the within-laboratory variation observed
in measurements performed on different days and usingdifferent analysts. The repeatability was investigated by
using the different concentrations of oxalate measured
for 1 min at 30 8C during the same day. The SIRE
biosensor showed a repeatability of 6.5% (n�/15). The
intermediate precision was investigated in the same
manner except that the enzyme suspension, the buffer,
and the samples were changed and the measurements
were carried out on different days. The intermediateprecision was found to be 12% (n�/15). This could
probably be improved further if a completely soluble
enzyme preparation was used.
Table 1
Recommended reaction time based on different concentrations of
oxalate at 30 8C with 0.1 mg/ml enzyme
Oxalate concentration range (mM) Reaction time (s)
B/1 60�/240
1�/5 25�/60
5�/25 �/25
Fig. 6. Temperature dependence of the response signal of the
biosensor with 0.4 mg/ml oxalate oxidase and a standard solution
containing 0.4 mM oxalate. Measuring conditions: 50 mM succinate
buffer, pH 4.0, and 60 s.
Table 2
A comparison between 30 and 45 8C by the determination of 20�/100
mM oxalate with 0.1 mg/ml enzyme for 4 min
Oxalate (mM) Response signal of biosensor (arbitrary units)
30 8C 45 8C
20 0 55
40 20 170
60 50 335
80 95 440
100 135 620
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/11811178
3.6. Determination of oxalate in charcoal-treated human
urine
Twelve urine samples were used in order to assess the
applicability of the method for clinical analyses. A pre-
treatment with activated charcoal is normally carried
out following the dilution of the urine, since oxalate
oxidase otherwise becomes inhibited. Urine is reported
to contain inhibitors of oxalate oxidase such as chloride
ions (Potezny et al., 1983; Pundir, 1993; Pundir and
Verma, 1993). The differential measurement technique
will provide accurate results if there are no enzyme
inhibitors and if the concentrations of electrochemically
active compounds are low enough to allow the measure-
ments to be performed. Since enzyme inhibitors are
present in urine samples, a treatment prior to the
measurements is advisable so that the signals from the
samples are not lower than those from the standard
solutions.A conventional method for oxalate determination is
represented by the Sigma urinary oxalate assay kit,
which is based on a colorimetric measurement (Li and
Madappally, 1989) involving the toxic chemical MBTH.
This method was used for comparison, as shown in Fig.
7. The results obtained by using the biosensor agreed
well with the results obtained with the colorimetric
method (R�/0.952). It is probable that the correlation
coefficient will be higher if more samples are analyzed
and if higher enzyme concentrations are injected.
In a comparison of different methods used for the
determination of oxalic acid in biological samples,
Sharma et al. (1993) found that the scatter of results
among different laboratories was very wide for all
current methods with a coefficient of variation exceed-
ing 20%. Sharma et al. also indicated that most of the
laboratories used the Sigma kit procedure, and noted
that this appears to remain the most popular method for
clinical laboratories. Isotope dilution and quantitative
gas chromatography�/mass spectrometry was suggested
as a reference method for oxalate determinations by
Koolstra et al. (1987). However, because of the require-ments of time, labor, and instrumentation, the use of
that method remains limited (Sharma et al., 1993).
4. Conclusions
The SIRE biosensor method yielded satisfactory
results, and a low detection limit (20 mM) could be
achieved when the enzymic reaction was carried outunder optimized conditions. The sensitivity of the
response could be improved by increasing reaction
temperature, reaction time, or the dose of enzyme.
These three parameters can be changed simultaneously
to reach a higher sensitivity. Consequently, oxalate
analysis with biosensor can be considered for several
important applications. These include assays of oxalic
acid in food, such as fruits and leafy vegetables, as wellas clinical assays of the oxalic acid concentration in
urine. Efforts in our laboratory are proceeding towards
the determination of oxalic acid in bleaching filtrates
from pulp and paper mills. The evaluation of urinary
oxalate assay using the biosensor demonstrated a good
agreement with the conventional method. The biosensor
method can be used in a broad concentration range and
may become a valuable analytical tool for industrialapplications, clinical diagnosis, and scientific research.
Although immobilized oxalate oxidase has previously
been used successfully to construct oxalate biosensors
for detection of oxalate in clinical samples, it is the first
time that the enzyme is combined with SIRE biosensor
technology, which is commercially available. This
method has several advantages. (1) It is a cheap method.
The cost for carrying out the spectrophotometricprocedure is still high for some clinical laboratories
(Sharma et al., 1993). The biosensor does not require
any additional expensive equipment and only consumes
10�/40 mg of enzyme for each assay and some activated
charcoal. Moreover, a biosensor apparatus is less
expensive than a spectrophotometer. (2) The technology
can easily and conveniently be employed without special
expertise and training. (3) The SIRE biosensor technol-ogy is of particular advantage compared with a tradi-
tional biosensor when only few analyses are carried out
infrequently. (4) The method is selective for oxalic acid.
(5) The assay can be carried out rapidly. Total analysis
time for each assay was approximately 2�/9 min. (6) The
SIRE technology circumvents the problems normally
associated with the instability of oxalate oxidase-based
biosensors since new and freshly prepared enzymepreparations are used. (7) The differential measuring
technique employed allows the biosensor to be less
sensitive to interfering substances in crude samples.
Fig. 7. A comparison of oxalate concentrations in 12 normal urine
samples obtained by a spectrophotometric method and the biosensor.
The calculated regression line is shown.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/1181 1179
The drawbacks of the method include that the
membrane and tubing of the biosensor system are
blocked easily by partially insoluble oxalate oxidase.
In the beginning of the investigation, we observed thatthe enzyme suspension, especially if the enzyme con-
centration was 1.0 mg/ml or more, could precipitate on
the wall of the biosensor tubing to clog the system.
Through adding Tween 20, the clog problem in the
tubing was resolved but the problem with the membrane
being partially clogged remained. The response slowly
decreased initially when a new membrane was utilized.
The life span of a membrane should be shorter if anenzyme suspension is used rather than a completely
soluble enzyme preparation, and the precision would
also be affected negatively by the use of a suspension.
Measures to improve the performance by increasing the
solubility of oxalate oxidase or using only solubilized
oxalate oxidase would simplify the operation, provide
more precise results, and facilitate the maintenance of
the biosensor.
Acknowledgements
The financial support of Vinnova, the Swedish
Agency for Innovation Systems, is gratefully acknowl-edged.
References
Balion, C.M., Thibert, R.J., 1994. Determination of oxalate by luminol
chemiluminescence. Clin. Chem. 40, 1096�/1097.
Barlow, I.M., Harrison, S.P., 1990. Improved urinary oxalate kit. Clin.
Chem. 36, 1523.
Beutler, H.O., Becker, J., Michal, G., Walter, E., 1980. Rapid method
for the determination of oxalate. Fresen. Z. Anal. Chem. 301, 186�/
187.
Boer, P., van Leersum, L., Endeman, H.J., 1984. Determination of
plasma oxalate with oxalate oxidase. Clin. Chim. Acta 137, 53�/60.
Canizares, P., Luque de Castro, M.D., 1997. Enzymatic interference-
free assay for oxalate in urine. Fresen. J. Anal. Chem. 357, 777�/
781.
Chalmers, A.H., Cowley, D.M., McWhinney, B.C., 1985. Stability of
ascorbate in urine: relevance to analyses for ascorbate and oxalate.
Clin. Chem. 31, 1703�/1705.
Chiriboga, J., 1966. Purification and properties of oxalic acid oxidase.
Arch. Biochem. Biophys. 116, 516�/523.
Dinckaya, E., Telefoncu, A., 1993. Enzyme electrode based on oxalate
oxidase immobilized in gelatin for specific determination of
oxalate. Indian J. Biochem. Biophys. 30, 282�/284.
Dumas, B., Sailland, A., Cheviet, J.P., Freyssinet, G., Pallett, K., 1993.
Identification of barley oxalate oxidase as a germine-like protein.
CR Acad. Sci. 316, 793�/798.
Elsander, A., Ek, M., Gellerstedt, G., 2000. Oxalic acid formation
during ECF and TCF bleaching of kraft pulp. Tappi J. 83, 73�/77.
Fry, D.R., Starkey, B.J., 1991. The determination of oxalate in urine
and plasma by high performance liquid chromatography. Ann.
Clin. Biochem. 28, 581�/587.
Gaulier, J.M., Cochat, P., Lardet, G., Vallon, J.J., 1997a. Serum
oxalate microassay using chemiluminescence. Kidney Int. 52,
1700�/1703.
Gaulier, J.M., Steghens, J.P., Lardet, G., Vallon, J.J., Cochat, P.,
1997b. Chemiluminescent measurement of oxalate in serum by
detection of hydrogen peroxide generated through oxalate oxidase.
J. Biolumin. Chemilumin. 12, 295�/298.
Gaulier, J.M., Lardet, G., Cochat, P., Vallon, J.J., 1998. A serum
oxalate assay using chemiluminescence detection, adapted to a
paediatric population. J. Nephrol. 11 (Suppl. 1), 73�/74.
Gelot, M.A., Lavoue, G., Belleville, F., Nabet, P., 1980. Determina-
tion of oxalates in plasma and urine using gas chromatography.
Clin. Clim. Acta 106, 279�/285.
Glazier, S.A., Rechnitz, G.A., 1989. Construction and characterization
of a beet stem based biosensor for oxalate. Anal. Lett. 22, 2929�/
2948.
Haas, G.J., Fleischman, A.I., 1961. The rapid enzymatic determination
of oxalate in wort and beer. J. Agric. Food Chem. 9, 451�/452.
Hatch, M., Bourke, E., Costello, J., 1977. New enzymic method for
serum oxalate determination. Clin. Chem. 23, 76�/78.
Holloway, W.D., Argall, M.E., Jealous, W.T., Lee, J.A., Bradbury,
J.H., 1989. Organic acids and calcium oxalate in tropical root
crops. J. Agric. Food Chem. 37, 337�/341.
Ichiyama, A., Nakai, E., Funai, T., Oda, T., Katafuchi, R., 1985.
Spectrophotometric determination of oxalate in urine and plasma
with oxalate oxidase. J. Biochem. (Tokyo) 98, 1375�/1385.
Kohlbecker, G., Butz, M., 1981. Direct spectrophotometric determi-
nation of serum and urinary oxalate with oxalate oxidase. J. Clin.
Chem. Clin. Biochem. 19, 1103�/1106.
Koolstra, W., Wolthers, B.G., Hayer, M., Elzinga, H., 1987. Devel-
opment of a reference method for determining urinary oxalate by
means of isotope dilution-mass spectrometry (ID-MS) and its
usefulness in testing existing assays for urinary oxalate. Clin. Chim.
Acta 170, 227�/236.
Kriz, D., Johansson, A., 1996. A preliminary study of a biosensor
based on flow injection of the recognition element. Biosens.
Bioelectron. 11, 1259�/1265.
Kriz, K., Anderlund, M., Kriz, D., 2001. Real-time detection of L-
ascorbic acid and hydrogen peroxide in crude food samples
employing a reversed sequential differential measuring technique
of the SIRE technology-based biosensor. Biosens. Bioelectron. 16,
363�/369.
Lathika, K.M., Sharma, S., Inamdar, K.V., Raghavan, K.G., 1995.
Oxalate depletion from leafy vegetables using alginate entrapped
banana oxalate oxidase. Biotech. Lett. 17, 407�/410.
Li, M.G., Madappally, M.M., 1989. Rapid enzymatic determination of
urinary oxalate. Clin. Chem. 35, 2330�/2333.
Manoharan, M., Schwille, P.O., 1994. Measurement of ascorbic acid in
human plasma and urine by high-performance liquid chromato-
graphy: results in healthy subjects and patients with idiopathic
calcium urolithiasis. J. Chromatogr. B 654, 134�/139.
Milardovic, S., Grabaric, Z., Tkalcec, M., Rumenjak, V., 2000.
Determination of oxalate in urine, using an amperometric biosen-
sor with oxalate oxidase immobilized on the surface of a chromium
hexacyanoferrate-modified graphite electrode. J. AOAC Int. 83,
1212�/1217.
Nabi Rahni, M.A., Guilbault, G.G., de Olivera, N.G., 1986. Im-
mobilized enzyme electrode for the determination of oxalate in
urine. Anal. Chem. 58, 523�/526.
Nilvebrant, N.-O., Reimann, A., de Sousa, F., Cassland, P., Larsson,
S., Hong, F., Jonsson, L.J., 2002. Enzymatic degradation of oxalic
acid for prevention of scaling. Prog. Biotechnol. 21, 231�/238.
Peldszus, S., Huck, P.M., Andrews, S.A., 1998. Quantitative determi-
nation of oxalate and other organic acids in drinking water at low
microgram/l concentrations. J. Chromatogr. A 793, 198�/203.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/11811180
Petrarulo, M., Cerrelli, E., Marangella, M., Maglienti, F., Linari, F.,
1993. Ion-chromatographic determination of plasma oxalate reex-
amined. Clin. Chem. 39, 537�/539.
Petrarulo, M., Cerrelli, E., Marangella, M., Cosseddu, D., Vitale, C.,
Linari, F., 1994. Assay of plasma oxalate with soluble oxalate
oxidase. Clin. Chem. 40, 2030�/2034.
Potezny, N., Bais, R., O’Loughlin, P.D., Edwards, J.B., Rofe, A.M.,
Conyers, R.A., 1983. Urinary oxalate determination by use of
immobilized oxalate oxidase in a continuous-flow system. Clin.
Chem. 29, 16�/20.
Pundir, C.S., 1993. Purification and properties of an oxalate oxidase
from leaves of grain sorghum hybrid CSH-5. Biochim. Biophys.
Acta 1161, 1�/5.
Pundir, C.S., Verma, U., 1993. Isolation, purification, immobilization
of oxalate oxidase and its clinical applications. Hindustan Antibiot.
Bull. 35, 173�/182.
Salinas, F., Martinez-Vidal, J.L., Gonzalez-Murcia, V., 1989. Extrac-
tion-spectrophotometric determination of oxalate in urine and
blood serum. Analyst 114, 1685�/1687.
Savage, G.P., Vanhanen, L., Mason, S.M., Ross, A.B., 2000. Effect of
cooking on the soluble and insoluble oxalate content of some New
Zealand foods. J. Food Comp. Anal. 13, 201�/206.
Schwille, P.O., Manoharan, M., Rumenapf, G., Wolfel, G., Berens,
H., 1989. Oxalate measurement in the picomole range by ion
chromatography: values in fasting plasma and urine of controls
and patients with idiopathic calcium urolithiasis. J. Clin. Chem.
Clin. Biochem. 27, 87�/96.
Sharma, S., Nath, R., Thind, S.K., 1993. Recent advances in
measurement of oxalate in biological materials. Scanning Microsc.
7, 431�/441.
Sugiura, M., Yamamura, H., Hirano, K., Sasaki, M., Morikawa, M.,
Tsuboi, M., 1979. Purification and properties of oxalate oxidase
from barley seedlings. Chem. Pharm. Bull. 27, 2003�/2007.
Wilson, D.M., Liedtke, R.R., 1991. Modified enzyme-based colori-
metric assay of urinary and plasma oxalate with improved
sensitivity and no ascorbate interference: reference values and
sample handling procedures. Clin. Chem. 37, 1229�/1235.
Yanagawa, M., Ohkawa, H., Tada, S., 1983. The determination of
urinary oxalate by gas chromatography. J. Urol. 129, 1163�/1165.
F. Hong et al. / Biosensors and Bioelectronics 18 (2003) 1173�/1181 1181