Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: https://www.tandfonline.com/loi/tbeq20

Enzyme based Biosensor for Heavy Metal Ions

Determination

R. Ilangovan, D. Daniel, A. Krastanov, C. Zachariah & R. Elizabeth

To cite this article: R. Ilangovan, D. Daniel, A. Krastanov, C. Zachariah & R. Elizabeth (2006)
Enzyme based Biosensor for Heavy Metal Ions Determination, Biotechnology & Biotechnological
Equipment, 20:1, 184-189, DOI: 10.1080/13102818.2006.10817330

To link to this article: https://doi.org/10.1080/13102818.2006.10817330

© 2006 Taylor and Francis Group, LLC

Published online: 15 Apr 2014.

Submit your article to this journal

Article views: 2138

View related articles

Citing articles: 13 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=tbeq20
ENZYME BASED BIOSENSOR FOR HEAVY METAL
IONS DETERMINATION
R. Ilangovan1, D. Daniel2, A. Krastanov3, C. Zachariah1, R. Elizabeth1
Vellore Institute of Technology, Department of Electronics and Instrumentation Engineering,
Vellore, Tamil Nadu, India1
Vellore Institute of Technology, Department of Chemical Engineering, Vellor, Tamil Nadu,
India2
University of Food Technologies, Department of Biotechnology, Plovdiv, Bulgaria3

ABSTRACT
Sol-gel-immobilized-urease conductometric biosensor on a thick film interdigitated elec-
trode was developed. The biosensor can be used for heavy metal ions determination in li-
quid samples. The biosensor exhibited good response to changes in urea concentration
within the range of 1mM to 15 mM. After standardizing the sensor for Urea, the biosensor
has been used to determine the heavy metal ions of different concentrations. The concen-
tration range of Urea that can be detected by using this sensor is 1mM to 15mM.The
heavy metals range is from 0.1mM to 10mM. Among the three metals used, the amount of
inhibition is found to be more in Cadmium, then Copper and then Lead. The sensitivity is
1 mM in spectrophotometric technique and 5 mM in electrical method. The described sen-
sor is tested for synthetic effluents in laboratory conditions. By further refinement, it can
be used to test real industrial samples.

Introduction rapidly on site at trace levels. The present

The accumulation of toxic substances in
the environment continuously increases due
to diverse pollutants from the industries.
Contamination of land and water due to
disposal of industrial effluents is the most
significant problem. Heavy metal ions are
regarded as one of the most toxic sub-
stances affecting the environment (1). The
presence of heavy metals in excess affects
air, water as well as soil. The plants grown
in such areas can accumulate heavy metals
like cadmium, zinc, lead and copper. These
metals have certain threshold levels for
essential functions of living organisms and
man, which turn into toxic actions if the
respective tolerance level for the respective
organism is exceeded. Due to the high
toxicity caused by the heavy metal ions
there is an obvious need to determine them
investigation aims at the development and
evaluation of a sol-gel based biosensor for
quantitative determination of heavy metals.
In recent years, biosensors are gaining
importance as suitable detectors for heavy
metal ions. They prove very promising for
environmental monitoring, since the system
is simple, rapid and selective. Several tech-
niques based on spectroscopy, ion-selective
electrodes, polarography and voltametry
have been described in the past (6).
Zhylyak et al. (9) developed a urease based
conductometric biosensor for the determi-
nation of heavy metal ions in wastewater.
The enzyme was immobilized by cross-
linking urease with bovine serum albumin,
which forms a biologically sensitive mem-
brane. An interdigitated gold electrode was
used as the transducer. The response of the

Biotechnol. & Biotechnol. Eq. 20/2006/1 184

sensor for varying concentrations of heavy
metal ions was evaluated by measuring the
urease activity after incubating the elec-
trodes in sample solutions of heavy metal
ions. Li et al. (7) used horseradish peroxi-
dase and developed an amperometric en-
zyme electrode for peroxide determination.
Urease isolated from pigeon pea was im-
mobilized in poly acrylamide gel and cal-
cium alginate beads analyzed for various
performance factors (4). Ho et al. (5) found
that an enzyme-catalyzed polymer trans-
formation when effectively combined with
a transducer could be used effectively as a
sensor. Srivastava et al. (8) immobilized
urease enzyme on gelatin beads via cross-
linking with glutaraldehyde. They analyzed
the immobilized enzyme for various per-
formance factors. Chern et al. (3) designed
a biosensor that contained two metacrylic-
acrylic membranes: One consists of proton
ionophore and the other contains the en-
zyme urease for the detection of urea.
When exposed to urea sample, the change
in pH at the membrane was detected by the
selective ionophore membrane and this was
converted into an electrical signal (mV) by
Ag/AgCl electrode.
Literature reports indicate that spectro-
scopic methods are expensive, as they re-
quire very sophisticated equipment, which
cannot be used for field monitoring. Both
polarographic and voltametric techniques
lack selectivity. Since ion selective elec-
trodes were based on the measurement of
the potential at an electrode surface caused
by a selective ion exchange reaction, the
design of ion-selective membrane was a
major difficulty in the development of this
type of sensor. Taking into consideration
the drawbacks mentioned above, there is a
need for the development of a cheap, sim-
ple and portable detector for heavy metal
ions.
In many instances, monitoring is not
continuous but requires a number of indi-
vidual measurements to be made at diffe-
rent times. In such cases, sensors should be
185
manufactured inexpensively so that they
may be disposed after a single reading.
Hence, the present work had the following
objectives: To develop an enzyme-based
biosensor, capable to detect heavy metal
ions in synthetic effluents and to evaluate
the performance of the sensor.
Materials and Methods
The determination of heavy metal ions
using the urease-immobilized biosensor is
based on the measurement of the urease
enzymatic activity, which is inhibited by
heavy-metal ions. The enzyme urease was
immobilized using the sol-gel process on a
screen-printed electrode and used as the
bio-recognition element in the biosensor.
The enzymatic reaction was converted into
an electrical signal using a transducer
whose impedance change was the measure
of the activity of the enzyme. The change
in impedance was converted into a voltage
signal by using a suitable circuit.
Construction of Biosensor
The sensor consisted of two electrodes
separated by a known distance was shown
(Fig. 1). The conductometric sensor con-
sidered for fabrication consists of two sets
of interdigitated fingers deposited on an
insulated substrate with each set having
four fingers. Each set of fingers is con-
nected to an electrical conductor that can
be constructed of gold, platinum, iridium,
carbon, copper or several other conductive
materials which are inert in the medium
and upon which coating containing urease
remain adherent. It is fabricated by using
PCB manufacturing technique.
Enzyme immobilization
A homogeneous stock sol-gel solution was
prepared by vigorously mixing 570 μl of
methanol, 50 μl of Tetra methoxy silicate
(TMOS), 10 μl of 3.8 % Cetyl trimethyl
ammonium bromide solution in a small test
tube at room temperature. This stock gel
solution was then cooled to 4 °C immedi-
ately after mixing. Enzyme stock solution
was prepared by dissolving a known quan-
Biotechnol. & Biotechnol. Eq. 20/2006/1
Fig. 1. Conductometric sensor strip used in the present work.
SENSOR
AC
Fig. 2. Measuring circuit.
tity of urease in 50 ml of 0.02 mM phos-
phate buffer (pH 7.0). Enzyme solution
was then stored at 4 °C in refrigerator. 50
μl of enzyme stock solution along with 5 μl
of glycerol was pipetted onto the surface of
the electrode and distributed gently over
the entire surface of the electrode with help
of a capillary tube. The electrode was al-
lowed to dry in ambient conditions for 1 h.
Equal amount of stock sol-gel solution was
Biotechnol. & Biotechnol. Eq. 20/2006/1
VIRTUAL
ZL INSTRUMENT
pipetted to cover the enzyme layer formed
over the surface of the electrode. The elec-
trode was allowed to polymerize and dried
for 1 h in ambient temperature. The en-
zyme electrode was immersed in a phos-
phate buffer and kept at 4 °C in a refri-
gerator overnight.
Measuring circuit
The impedance change was converted into
voltage using the circuit shown (Fig. 2).
186
 0 .6
0 .5 5
0 .5
Voltage(V)
0 .4 5
0 .4
0 .3 5
0 .3
0 .2 5
0 .2
0 5 10
C o n c e n tr a tio n o f U r e a
(m M )
Fig. 3. Concentration of Urea Vs Voltage.
The response of the sensor was measured
by applying a sinusoidal ac voltage of 1V
and 10 kHz frequency. The output voltage
was computed using the following equa-
tion:
Vo = [ZL/(Z+ZL)] Vi
The output voltage is acquired for further
analysis using LabView 7.1. A data logger
was designed using LabView to acquire
voltage and store it in the computer to
compute time response.
Urea and urease activity
Urea was determined by the diacetylmon-
oxime reaction as reported earlier (2). All
the measurements were carried out in 25 ml
beaker filled with 15-ml of test solution at
room temperature. Standard plot for vari-
ous concentrations of urea in the range of
1-15 mM were constructed. The immobi-
lized electrode was dipped in 25 ml beaker
which consisted of a known concentration
of urea for 10 minutes. The output of the
sensor was acquired by virtual instrumen-
tation. The sample was taken and analyzed
for urea concentration. The extent of urea
hydrolysis was determined by comparing
the obtained absorbance value with the
standard absorbance plot drawn for the
urea. The experiment was repeated to stan-
dardize the sensor for various concentra-
tions of urea. The response of the electrode
for 2 mM concentration was chosen for
further evaluation. The electrode was incu-
0.5
0.4
Voltage (V)
0.3
0.2
0.1
0
0 1 2 3 4
Time (min)
15 20
Fig. 4. Time response.
bated in a test solution containing heavy
metal ion for 10 minutes. The heavy metal
ions selected were copper, cadmium and
lead. After incubating in heavy metal solu-
tion, the electrode was then washed with
phosphate buffer and dipped in 2 mM of
urea solution in 25 ml beaker. The proce-
dure was repeated by changing the con-
centrations of heavy metal ions for differ-
ent metals. For each sample the output
voltage was acquired for further analysis
and the samples were subjected to Spectro-
photometric analysis to determine the %
inhibition caused by heavy metals.
Results and Discussion
The steady state response of the biosensor
as a function of urea concentration under
the specified conditions was examined. As
depicted in Fig. 3, the biosensor exhibited
good response to changes in urea concen-
tration within the range of 1 mM to 15
mM. The response was found to be linear.
The time response of the sensor was
studied by using acquired voltage at vari-
ous times for a known concentration of
urea. The response shows gradual increase
and reaches the steady state value after 3
min. The time response for 2 mM urea is
shown (Fig. 4).
Testing with metal ions
The rate of enzyme inhibition by an in-
hibitor (heavy metal ion in this study) is
187 Biotechnol. & Biotechnol. Eq. 20/2006/1
 ments. After standardizing the sensor for
49 Urea, the biosensor has been used to de-
termine the heavy metal ions of different
% Inhibition

44 concentrations. The concentration of heavy

metal ions is measured in terms of % Inhi-
39 bition and it is given by:
34 % Inhibition = [(Ao – A)/Ao x 100 %]
0 5 10 15 A - absorbance of urea obtained before
Concentration of Copper (mM)
incubation in metals
Ao - absorbance of urea obtained after in-
Fig. 5. Concentration of Copper Vs % Inhibition. cubation in heavy metals
The sequence of inhibition to the urease
activity is Cu2+>Cd2+>Pb2+, which is in
49 good agreement with the results observed
in previous reports (6). The plots for vari-
% Inhibition

44 ous concentrations of all the three metals

against % Inhibition are shown (Figs. 5, 6,
39 7). All the responses are for 2 mM of Urea.
34
Response curves for Metal ion
0 5 10 15
concentration Vs % Inhibition
Concentration of Cadmium (mM)
The Inhibition by Copper shows linear in-
crease in lower concentration range (0.1
Fig. 6. Concentration of Cadmium Vs % Inhibition. mM to 1 mM) and the increase in inhibi-
tion becomes less as the concentration in-
creases (1 mM to 10 mM).
The Inhibition by Cadmium shows al-
43
most linear increase in the concentration
% Inhibition

38 range (0.1 mM to 10 mM). The Inhibition

33
28
23
0 5 10
Concentration of Lead (mM)
Fig. 7. Concentration off Lead Vs % Inhibition.
rather slow. Therefore, the biosensor to be
tested should be pre-incubated for a certain
period of time in the test solution contain-
ing an inhibitor in order to obtain the
measurable inhibition. A measurable inhi-
bition is obtained within 5 to 10 minutes
pre-incubating period. Although it is
clearly dependent on the specific metal ion
and its concentration. An incubation time
of 10 minutes is shown for further experi-
by Lead shows rapid increase in lower con-
centration range (0.1 mM to 1 mM) and as
the concentration increases the increase in
inhibition becomes less but shows a linear
15 rise.
Performance factors
The storage time of the sensor is found to
be 6 to 7 days. It can be used 4 to 5 times
when it is used to measure Urea and only
one time if it is used to measure heavy me-
tals concentration. The concentration range
of Urea that can be detected by using this
sensor is 1 mM to 15 mM.The heavy me-
tals range is from 0.1 mM to
10mM.Among the three metals used, the
amount of inhibition is found to be more in
Cadmium, then Copper and then Lead. The
sensitivity is 1 mM in spectrophotometric
technique and 5 mM in electrical method.

Biotechnol. & Biotechnol. Eq. 20/2006/1 188

 The described sensor is tested for syn-
thetic effluents in laboratory conditions. By
further refinement, it can be used to test
real industrial samples. Future development
will include adopting suitable methods to
achieve selective detection of heavy metal
ions.
Conclusions
The studies conducted in this project shows
that the Sol-gel-immobilized-urease con-
ductometric biosensor on a thick film inter-
digitated electrode can be used as a reliable
sensor for heavy metal ion determination in
liquid samples. It has several advantages
like easy production of the sensor, low
cost, sensibility and ease of operation.
189
REFERENCES
1. Bontidean I., Ahlqvist J., Mulchandani A.,
Chen W., Bae W., Mehra R.K., Mortari A.,
Csoregi E. (2003) Biosensors and Bioelectronics, 18,
547-553.
2. Ceriotti G., Spandrio L. (1963) Clin. Chim.
Acta, 8, 295-299.
3. Chern L.H., Heng L.Y., Musa A. (2001) A
potentiometric biosensor based on urease enzyme.
Proc. NSF Workshop, Kuala Lumpur.
4. Das N., Kayastha A.M., Malhotra O.P. (1998)
Biotechnol. Appl. Biochem., 27, 25-29.
5. Ho W.O., Krause S., McNeil C.J., Pritchard
J.A., Armstrong R.D., Athey D., Rawson K. (1999)
Anal. Chem., 67, 3928-3935.
6. Lee S.M., Lee W.Y. (2002) Bull. Korean Chem.
Soc., 23, 1169-1172.
7. Li J., Tan S.N., Oh J.T. (1998) Journal of Elec-
troanalytical Chemistry, 448, 69-77.
8. Srivastava P.K., Kayastha A.M., Srinivasan
M. (2001) Biotechnol. Appl. Biochem., 34, 55-62.
9. Zhylyak G.A., Dzyadevich S.V., Korpan Y.I.,
Soldatkin A.P., Elskaya A.V. (1995) Sensors and
Actuators B., 24-25, 145-148.
Biotechnol. & Biotechnol. Eq. 20/2006/1