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: leif.jonsson@kau.se (L.J. Jonsson).

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www.elsevier.com/locate/bios

0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

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.

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