Sensors and Actuators B 148 (2010) 583589

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Two-enzyme lactose biosensor based on -galactosidase and glucose oxidase

deposited by AC-electrophoresis: Characteristics and performance for
lactose determination in milk
Malika Ammam , Jan Fransaer
Department of Metallurgy and Materials Engineering (MTM), KU Leuven, Kasteelpark Arenberg 44, B-3001 Heverlee, Belgium

a r t i c l e

i n f o

Article history:
Received 19 March 2010
Received in revised form 2 May 2010
Accepted 11 May 2010
Available online 24 May 2010
Keywords:
Lactose sensor
AC-EPD of enzymes
-Galactosidase
Glucose oxidase
Lactose determination in milk

a b s t r a c t
In this paper, we have investigated the deposition of two enzymes -galactosidase (-Gal) and glucose
oxidase (GOx) using alternating current electrophoretic deposition (AC-EPD) to manufacture a lactose
sensor. Using the optimal deposition parameters and optimal testing conditions, the sensor has a sensitivity up to 111 nA/mM mm2 . This sensitivity is high considering the low activity of the enzymes employed
in this study (9 units/mg for -Gal and 5.6 units/mg for GOx). The sensor has a large linear range up
to 14 mM lactose, fast response time (8 s) and reasonable stability without employing any stabilizers
or outer polymer membrane. In addition, it is easy and simple to manufacture, highly reproducible and
cheap because low activity enzymes are used. The sensor was tested to determine lactose in milk samples
and is demonstrated to be accurate.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Lactose is a disaccharide that consists of galactose and glucose fragments bonded through a -1 4 glycosidic linkage. It
is present most notably in milk and makes up 28% of milk (by
weight), although the amount varies among species and individuals. For example, the lactose percentage found in milk of healthy
humans might reach 8% [1], and the level of lactose in unprocessed milks from animals such as cow, goat, buffalo, yak and sheep
is respectively about 4.7%, 4.1%, 4.86%, 4.93%, and 4.6% [26]. In
industry, determination of lactose in milk and dairy products is
important since the lactose content is a basic indication for evaluating milk quality and detecting abnormal milk. In this regard, it has
been reported that milk from cows suffering from mastitis shows
lower lactose levels [2]. On the other hand, the precise control of
the amount of lactose in dairy food products is very important, as
many people are unable to digest the sugar. This medical condition is called lactose intolerance, which is related to the inability to
metabolize lactose into galactose and glucose, because of a lack of
the required enzyme lactase (-galactosidase) in the digestive system [7]. Therefore, the precise determination of lactose is important
for industry and public health. Various methods have been devel-

Corresponding author. Tel.: +32 16 321260; fax: +32 16 321991.

E-mail addresses: Malika.Ammam@mtm.kuleuven.be, m78ammam@yahoo.fr
(M. Ammam).
0925-4005/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2010.05.027

oped for lactose determination including physical methods such as

gas, liquid, and high-pressure liquid chromatography [810], gravimetric analysis [11], titrimetry by chloramine-T method [12] and
infrared spectroscopy [13]. However, many of these methods are
complex, expensive and time consuming.
Electrochemical methods are advantageous over the other
methods in terms of cost and time. In the last decade, the
immobilization of enzymes on electrodes for the design of amperometric biosensors for lactose determination has been an area
of intense research. Several types of enzymatic electrodes have
been developed for lactose, based on two immobilized enzymes,
-galactosidase and glucose oxidase. The enzymes were immobilized on a Clark-type oxygen electrode [1416], on a commercial
hydrogen peroxide sensor [17] or on Pt electrodes [18]. In
these types of biosensors, the electrochemical response is based
on the direct measurement of peroxide. In another type, the
enzymes are attached to electrodes and a mediator, such as benzoquinone, is present in the solution [19]. Enzymes have also
been immobilized onto electrodes together with a mediator such
as tetrathiafulval-inium tetracyanoquinodimethanide that reacts
with glucose oxidase [20,21]. The mediators increase the rate of
the enzymatic reaction and amplify the electrochemical signal. All
these modication methods have their advantages and disadvantages.
Previously, we described glucose and glutamate sensors based
on deposition of glucose oxidase and glutamate oxidase on the
transducer platinum electrode using asymmetrical alternating

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Fig. 1. Asymmetrical AC-signals tested for the electrophoretic deposition of the enzyme mixture (-Gal + GOx). (A) Triangular, (B) sine and (C) square waveform.

current electrophoretic deposition (AC-EPD), and have shown

that this method results in sensors with improved characteristics
[2224]. We have demonstrated that compared to other electrochemical deposition methods, thick and highly active layers of
enzyme can be deposited. In addition, compared to other immobilization methods such as encapsulation in polymer or gel matrices
and other hand operated techniques, the most obvious advantage of
AC-EPD is the automated manufacturing process. This leads on the
one hand, to the ease of the manufacturing process and, on the other
hand, to a high reproducibility. Thus, we believe that the process
warrants further investigations. In this paper, we have investigated
the possibility of the simultaneous deposition of two enzymes (galactosidase and glucose oxidase) using the process of AC-EPD to
manufacture a lactose sensor. The bi-enzyme lms are deposited
from a mixture of the two enzymes and from low conductivity solutions. The effect of nature of the asymmetrical AC-signal on the
amperometric response of the sensor was investigated for triangular, sine and square waves. The effect of pH and temperature on the
sensor performance towards the response to lactose was explored.
The ability of the sensor for lactose determination in real samples
was examined by the amperometric determination of lactose in
milk samples.
2. Experimental
2.1. Materials
Ultrapure water milliQ grade with a resistance of 18.2 M cm
was used for all the experiments. Glucose oxidase (GOx) crude
from Aspergillus niger 5.6 units/mg and -galactosidase or lactase
(-Gal) from Aspergillus oryzae 9 units/mg were purchased from
SigmaAldrich. d-Glucose 99% from Fisher Scientic, and the solution was prepared 24 h before use. d-Lactose 99% was purchased
from SigmaAldrich, citric acid and sodium phosphate analytical
grade were purchased from Acros Organic. The buffered saline solutions are prepared using 20 mM sodium phosphate and 10 mM

citric acid then adjusted to the desired pH. Platinum wire with a
diameter of 250 m is used for the sensor preparation. It is 99.99%
pure and was purchased from Goodfellow. The whole, skimmed
and semi-skimmed milk are products from SPAR (Everyday) and
the extra concentrated whole milk is a product from Nutroma.
2.2. Apparatus
The set up used for the AC-EPD of the enzymes was previously
described [2224]. Briey, it consisted of an arbitrary waveform
generator (ww5061, Tabor electronics) connected to a bipolar highvoltage operational amplier (BOP 1000M, Kepco). The applied
waveform was monitored using a digital oscilloscope (Explorer
III oscilloscope, Nicolet Instrument Corporation). The AC-signal
is composed of two triangular, sine or square waves of opposite
amplitude but with different heights and durations as shown in
Fig. 1. The half-cycle with the highest amplitude and the lowest
duration corresponds to the negative part of the AC-signal. However, the area of both waves is equal, so that the signal has no net DC
component, i.e. the integral of the AC-signal over one period is zero.
Before each experiment, the AC-signal was integrated to verify that
the integral of the applied signal over one period is equal to zero.
The outputs of the amplier are connected to an electrochemical
cell. This electrochemical cell contains two electrodes, a platinum
counter electrode and a platinum working electrode on which the
enzymes are deposited and that is later used as a biosensor. The
biosensor electrode is connected to the electrode whose polarity
with respect to the counter electrode is positive for a longer time
at low amplitude and becomes negative for a short duration at high
amplitude as previously demonstrated [2224].
2.3. Sensor preparation
A 4-mm long platinum wire (250 m diameter) was soldered
to the bare end of an insulated copper wire to provide electrical
connection. A 4-mm heat-shrink tube was used to insulate the

M. Ammam, J. Fransaer / Sensors and Actuators B 148 (2010) 583589

585

Fig. 2. Typical amperometric response of the bi-enzyme electrode (-Gal + GOx) to two injections of 1.4 mM lactose prepared using (A) triangular, (B) sine and (C) square waveforms. Sensor preparation: enzyme concentrations (10 mg GOx + 90 mg -Gal)/1 mL ultrapure water, deposition at 30 Hz, and 120 Vpp for 30 min. Test in phosphatecitrate
buffer solution pH 4.9 at 30 C. Polarization: +0.65 V vs. AgCl/Ag.

platinumcopper junction. The platinum end was then cut to a

length of 1 mm, and the tip was sealed with acrylic glue. The sensing area was 0.78 mm2 . The sensing area was cleaned by dipping
into a mixture of nitric acid (7%) and hydrogen peroxide (30%) for a
few seconds, and then rinsed abundantly with ultrapure water and
acetone.
The enzyme deposition was accomplished in the following way:
the sensor electrode is immersed in an enzyme solution containing (10 mg GOx + 90 mg -Gal) dissolved in 1 mL ultrapure water. A
platinum wire in the form of a spiral was used as a counter electrode
and positioned around the sensor electrode. This conguration permits the electrophoretic deposition of a homogeneous bi-enzyme
layer on the cylindrical sensor electrode. The asymmetrical ACsignal is then applied at 30 Hz and 120 Vpp for period of 30 min.
Next, the sensor is rinsed gently with ultrapure water and then
tested in 5 mL phosphatecitrate buffer solution at various pHs and
temperatures by injecting 10 L of lactose (1.4 mM).
A potentiostat (CMS 100, GAMRY) connected to a computer for
data acquisition was used for the testing of the sensors (amperometry). AgCl/Ag was used as a reference electrode, and the polarization
was set at +0.65 V vs. AgCl/Ag.
3. Results and discussion
3.1. Lactose sensor based on AC-EPD of -Gal and GOx
The goal of this paper is to demonstrate that two enzymes
such as -galactosidase and glucose oxidase can simultaneously
be deposited by means of AC-EPD to manufacture a lactose sensor.
Parameters such as nature of the AC-signal, enzyme proportions,
frequency, amplitude, pH and temperature have been shown to

affect the response of the sensor towards lactose. We found that

enzyme concentrations (10 mg GOx + 90 mg -Gal)/mL, amplitudes 120 Vpp , frequencies 30 Hz and deposition times of
20 min or longer to be optimal for a good sensor response. The
high ratio of -Gal used for enzyme mixture demonstrates that the
sensor response is specially -Gal rate dependent. This is understandable since our approach for determining lactose concentration
is based on the following coupled biochemical and electrochemical
reactions:
-Gal

lactose + H2 O glucose + galactose

GOx

glucose + O2 H2 O2 + gluconolactone
Pt

H2 O2 O2 + H2 O

(1)
(2)
(3)

The enzyme -galactosidase cleaves the disaccharide lactose,

producing glucose and galactose. The glucose reacts with the
immobilized GOx to produce H2 O2 , which in turn oxidizes at the
platinum electrode polarized at +0.65 V vs. AgCl/Ag resulting in an
amperometric signal proportional to the lactose concentration. The
high ratio of -Gal needed for enzyme mixture can also be related to
the low activity of -Gal enzyme used in this study. Compared to the
literature where high -Gal activities (>260 units/mg) were used
[2529], in this study the activity of -Gal is only 9 units/mg. On the
other hand, the low applied amplitude of 120 Vpp used for deposition compared to the optimal value of 160 Vpp reported previously
for GOx [22,23] is due to the presence of high amount of salts in Gal. These salts increase the conductivity of the bi-enzyme solution,
as a consequent increasing the electrolytic decomposition of water
(AC-electrolysis), which in turn resulted in a low deposition rate.

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Table 1
Estimated mass of (-Gal + GOx) deposited on the platinum substrate vs. nature of
the AC waveform.
AC waveform

Triangular

Sine

Square

Mass (g mm2 )

3.74

3.05

2.53

We have previously reported that the asymmetry in the triangular AC waveform is an important parameter for the electrophoretic
mobility as well as for the deposition of the enzyme on the substrate
[22]. In this work, we have investigated two other asymmetrical AC
waves (sine and square) shown in Fig. 1B and C on the amperometric response of the prepared electrodes. Fig. 2 shows a comparison
between the current response of the bi-enzyme layer deposited
on a platinum substrate at 30 Hz, 120 Vpp for 30 min using triangular, sine and square waves to the injection of 1.4 mM lactose. It
will be noted that under the same conditions, the amperometric
response of the bi-enzyme electrode manufactured using the triangular wave is higher than the one prepared using sine wave, and
the lowest sensitivity is obtained with the square wave. In order
to get a better understanding of the results, we have measured
the deposition rate for each electrode using a microbalance and
the results are gathered in Table 1. As can be seen, the amperometric responses observed in Fig. 2 are correlated to the amount of
deposited enzymes. In other words, the deposition rate using triangular wave is higher than the one obtained with sine wave, which
in turn is higher than the rate using square wave. The reason for
this has to do with the AC-electrolysis. It is observed that during
deposition, excessive gas bubbles are formed when sine or square
waves are applied. This electrolytic decomposition of water may
consequently decrease the deposition rate and denaturize some of
the deposited enzymes, leading to a low amperometric response.
In order to obtain the highest current response of the bi-enzyme
electrode, the asymmetrical triangular wave was used for the rest
of the study.
For each of the two enzymes present on the electrode, there
was an optimal pH and temperature: for -Gal it was pH 4.5 at
30 C and for GOx it was pH 5.1 at 35 C. To determine the optimal
pH and temperature for the combination of the two enzymes, the
pH of the testing solution was varied from 3.9 to 8.2, and the temperature was varied between 25 and 40 C. The current response of
the sensor manufactured under the optimal conditions vs. pH and
temperature of the testing buffer solution are shown respectively
in Fig. 3A and B. As can be seen at pH 4.55 and temperature of
3035 C, the highest current response is obtained. Therefore, pH
4.9 and 30 C were selected as optimal testing parameters.
The deposition of the two enzymes -Gal and GOx and testing of the sensors under the optimal conditions lead to a high
current response of the bi-enzyme electrode to lactose. Fig. 4A
shows the amperometric response of the sensor to several injections of 1.4 mM lactose. It will be noted that current responses to
lactose of 111 nA/mM mm2 can be obtained with these electrodes.
Compared to the sensitivities reported in the literature [2529],
this sensitivity is high considering the low activity of the enzymes
employed in this study (9 units/mg for -Gal and 5.6 units/mg for
GOx), compared to the literature where at least 260 units/mg is
used [2529]. For example, despite of the high employed enzymes
activity (910 units/mg for -Gal and 151 units/mg for GOx), the
sensitivities obtained by Xu et al. [28] are only few nA/mM lactose. As pointed out before, this sensitivity also demonstrates that
the rate determining reaction is the hydrolysis of lactose by Gal because, as we have previously reported using only GOx the
obtained current responses are much higher [22,23]. On the other
hand, this lower sensitivity improves the linearity of the biosensor.
As shown in Fig. 4B, the response of the sensor without employing any outer polymer layer to regulate the diffusion of lactose and

Fig. 3. Effect of pH (A) and temperature (B) on the current response to 1.4 mM lactose. Sensor preparation: enzyme concentrations (10 mg GOx + 90 mg -Gal)/1 mL
ultrapure water, deposition at 30 Hz, and 120 Vpp for 30 min using triangular waveform. Test in phosphatecitrate buffer solution at different pHs and temperatures.
Polarization: +0.65 V vs. AgCl/Ag.

oxygen is linear up to 14 mM lactose. This linearity is 34 times

higher than what has been reported in the literature [25,26,29,30].
This extended linearity is probably related to the reasonable sensitivity. Because the current response to 1.4 mM is not too high, the
available dissolved oxygen around the sensing part of the electrode
is consumed slowly. As displayed in Figs. 2A and 4A, the sensor also
has a fast response time, reaching 95% of the nal value in less than
10 s. The detection limit of the lactose sensor prepared by AC-EPD
is less than 0.1 mM (S/N = 3). However, it is important to note that
lactose, typically measured in milk and dairy products, is present
at high concentration (>100 mM) and therefore do not pose detection limit problems. Finally, in regard to selectivity of this biosensor,
since each diary product possesses different interferences, it would
be more appropriate to adjust the sensor fabrication for each product. For milk, this parameter will be discussed in detail in one of
the following paragraphs. The main characteristics of the biosensor
shown in Fig. 4A are gathered in Table 2.
Fig. 5A shows the current response of the biosensor to lactose
for a period of 3 weeks. The sensor was stored in air at room temperature and the response to lactose was checked every 25 days.
It is worth noting that these sensors have no stabilizing polymer or
outer membrane layers and therefore show how the activity of the

M. Ammam, J. Fransaer / Sensors and Actuators B 148 (2010) 583589

Fig. 4. (A) Typical amperometric response of the bi-enzyme electrode (-Gal + GOx)
to successive injections of 1.4 mM lactose. (B) Linear relationship between sensor
response and lactose concentration. Sensor preparation: enzyme concentrations
(10 mg GOx + 90 mg -Gal)/1 mL ultrapure water, deposition at 30 Hz, and 120 Vpp
for 30 min using triangular waveform. Test in phosphate citrate buffer solution pH
4.9 at 30 C. Polarization: +0.65 V vs. AgCl/Ag.

bi-enzyme layer varies with time. The response to lactose is quasistable during the rst week but shows a continuous decrease during
the two last weeks. This may be attributed to the dissolution of the
deposited bi-enzyme layer during each test in the buffer solution.
As shown in Fig. 5A, the stability of the sensor can be improved
by applying a thin layer of polyurethane (PU) prepared by the
procedure previously reported [23,24]. With a PU outer layer the
sensitivity of the sensor decreased from 111 to 98 nA/mM mm2 .
No PU outer layer is needed if the sensor is used for 1 or 2 tests
as it will be demonstrated in the following paragraphs for milk
samples, but the PU outer layer may be useful when the sensor
Table 2
Characteristics of the bi-enzyme electrode (-Gal + GOx) shown in
Fig. 4.
Area
Sensitivity
Linearity
Response time
Detection limit

0.78 mm2
111 2 nA/1 mM lactose
0.114 mM lactose
81s
<0.1 mM lactose

587

Fig. 5. (A) Stability of the bi-enzyme (-Gal + GOx) electrodes manufactured at

30 Hz, 120 Vpp for 30 min in the absence and presence of the outer membrane of
polyurethane (1 PU spray). (B) Curve showing the response of three different lactose
sensors manufactured using the same process to 1.4 mM lactose (reproducibility).
Sensor preparation: enzyme concentrations (10 mg GOx + 90 mg -Gal)/1 mL ultrapure water, deposition at 30 Hz, and 120 Vpp for 30 min using triangular waveform.
Test in phosphatecitrate buffer solution pH 4.9 at 30 C. Polarization: +0.65 V vs.
AgCl/Ag. Sensor stored in air at room temperature. The sensor with PU outer layer
is prepared using 1 PU spray, the procedure is previously reported [23,24].

is used for continuous measurements. Finally, the reproducibility

of the biosensor response can be estimated to 85% as shown in
Fig. 5B. This good reproducibility is attributed to the automated
manufacturing process.
3.2. Determination of lactose in milk samples
In order to evaluate the performance of the sensor for lactose
determination, it was tested in the presence of different milk samples. Since the sensor will be used only 1 time, no outer PU layer was
used. It is important to note that polarization of the Pt electrode at
+0.65 V vs. AgCl/Ag may generate a current response if electroactive species such as uric acid, ascorbic acid or others are present in
milk. We have therefore tested the milk samples on a polarized Pt
electrode rst. Under permanent stirring, a bare Pt electrode and a
AgCl/Ag reference electrode were immersed in 5 mL buffer solution
pH 4.9 at 30 C. The Pt electrode was polarized at +0.65 V vs. AgCl/Ag
and injection of 50 L milk from each sample was carried out once a

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M. Ammam, J. Fransaer / Sensors and Actuators B 148 (2010) 583589

Fig. 6. (A) Current response to 50 L whole (S1), skimmed (S2), semi-skimmed (S3) and extra concentrated whole milk (S4), on a bare unmodied Pt electrode. (B) Current
response to 50 L whole (S1), skimmed (S2), semi-skimmed (S3), extra concentrated whole milk (S4) and 0.05 mM glucose of GOx coated Pt. (C) Current response to
50 L whole (S1), skimmed (S2), semi-skimmed (S3), extra concentrated whole milk (S4) and 0.6 mM lactose of (-GAl + GOx) coated Pt. Sensors preparation: (B) 10 mg
GOx/1 mL ultrapure water and (C) (10 mg GOx + 90 mg -Gal)/1 mL ultrapure water. Deposition at 30 Hz, 120 Vpp for 30 min using triangular waveform (B and C). Test in
phosphatecitrate buffer solution pH 4.9 at 30 C. Polarization: +0.65 V vs. AgCl/Ag.

stable background current was obtained. Fig. 6A illustrates the current response to 50 L whole, skimmed, semi-skimmed and extra
concentrated whole milk, respectively on the bare polarized Pt electrode. As can be seen, a 12 nA current increase is observed for
each sample, meaning that some electroactive species are present
in the milk samples. It is clear that the extra concentrated milk
(S4) contains a higher amount of interferences compared to the
other samples. However, since the current response is very low
(less than 2 nA), the effect of interferences on lactose determination will be negligible because of the relatively high sensitivity
of the biosensor towards lactose (111 nA/mM mm2 ). Besides, we
have previously reported [23] that the enzyme layer deposited by
means of AC-EPD screens out a large part of the interferences, and
since the concentration of the interferences in the milk samples
is very low, it is possible that the bi-enzyme layer will entirely
screen them out. The second potential interference to examine is
the presence of glucose traces in milk samples and, because the
bi-enzyme layer contains GOx, it will respond to glucose. In order
to get a better idea about the glucose present in milk samples, we
have deposited GOx at the same concentration used for deposition of the two enzymes (10 mg/mL) and under the same applied

parameters of 30 Hz, 120 Vpp for 30 min. The Pt modied GOx was
then immersed in 5 mL buffer under the same conditions as previously described, and injections of 50 L milk samples were carried
out followed by an injection of 0.05 mM glucose for calibration.
The results are shown in Fig. 6B. It is worth noting that glucose
sensors manufactured using AC-EPD have previously been demonstrated to be linear up to 20 mM without using any outer layer of
polyurethane [23]. Therefore, at these low concentrations, linearity
does not pose problems and one calibration point is enough to estimate the glucose level in milk samples. As can be seen in Fig. 6B, the
amperometric response is low (<1 nA), meaning that the concentration of glucose in milk samples is low. Assuming total rejection
of the interferences by the bi-enzyme layer, the concentrations
of glucose estimated from Fig. 6B for the different milk samples
are gathered in Table 3. These concentrations are close to the glucose concentrations in milk samples reported elsewhere [31]. It
is clear from Table 3 that the low glucose concentrations will not
interfere with the lactose measurements. However, for more accurate determination of lactose, it is possible to subtract these values
from the concentrations estimated by the bi-enzyme (GOx + -Gal)
electrode. After examination of the current responses to the two

Table 3
Concentrations of glucose and lactose in the different milk samples purchased from SPAR and Nutroma and laboratory reference for lactose concentrations.
Milk sample

Glucose concentration determined

with glucose sensor (mM)

Lactose concentration determined

with lactose sensor (mM)

Laboratory reference for

lactose (mM)

Whole milk (S1)

Skimmed milk (S2)
Semi-skimmed milk (S3)
Extra concentrated whole milk (S4)

0.50
0.41
0.46
0.75

141.4
139.1
137.6
326.2

139.8
142.9
142.9
321.0

M. Ammam, J. Fransaer / Sensors and Actuators B 148 (2010) 583589

main interferences (acids and glucose), the concentration of lactose

can be accurately determined by the bi-enzyme modied Pt electrode. Fig. 6C displays the current response to respectively 50 L
whole (S1), skimmed (S2), semi-skimmed (S3) and extra concentrated whole milk (S4), followed by an injection of 0.6 mM lactose
as a calibration point. It can be seen that the current response to
the three rst injections are practically equal, indicating a similar level of lactose in these samples. However, the level of lactose
in the last sample (extra concentrated whole milk (S4)) is much
higher. Taking into account the estimated glucose concentrations,
the determined lactose concentrations from Fig. 6C are gathered
in Table 3. Compared to the laboratory lactose concentrations provided by the producers, it can be seen that the values are very close.
The good correlation between the data indicates the reliability of
the present sensor for lactose determination in real milk samples.
4. Conclusions
We have demonstrated that the enzymes -galactosidase and
glucose oxidase can be deposited together by means of asymmetrical AC-signal to form a stable bi-enzyme layer. The triangular
asymmetrical AC waveform is shown to provide the highest current
response compared to sine and square waves. Under the optimal
deposition conditions of 30 Hz, 120 Vpp , 30 min deposition time
and, the optimal testing conditions of pH 4.9 and 30 C, the sensor
has a sensitivity for lactose of up to 111 nA/mM mm2 . This sensitivity is high considering the low activity of enzymes employed in
this study (9 units/mg for -Gal and 5.6 units/mg for GOx), compared to the literature where high activity enzymes of at least
260 units/mg are used. The sensor has a large linear range up to
14 mM lactose, a fast response time (8 s) and a reasonable stability without employing any stabilizers or outer polymer layer.
Furthermore, the sensor is easy and simple to manufacture, highly
reproducible and cheap because low activity enzymes are used.
The applicability of the sensor to determine lactose in milk samples is demonstrated and the accordance between the data make
it a good candidate for lactose measurements in real milk samples.
For other dairy products where the concentration of the interferences may be signicant, adjustment in the sensor manufacturing
might be necessary in order to have an accurate estimation of lactose.
Acknowledgements
The authors acknowledge the support of the Research Fund KU
Leuven (GOA/08/007) and the Belgian Federal Science Policy Ofce
(BELSPO) through the IUAP project INANOMAT (contract P6/17).
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Biographies
Malika Ammam received her Ph.D. from the University of Paris Sud XI, France in
September 2005. From 2006 to 2007, she worked as a research fellow at the University of Kansas (USA) under the supervision of Dr. G.S. Wilson on the development
of glucose and glutamate sensors commercialized by Pinnacle Technology Inc. She
is currently a post-doctoral position at the University of KU Leuven, Belgium. Her
research interests are biosensors and chemical sensors, biofuel cells, electrochemistry of inorganic and organic molecules (polyoxometalates and thioethers).
Jan Fransaer is currently a professor at the University of KU Leuven, Belgium. His
research interests are electrochemistry, materials and mathematical modeling.