Current Applied Physics 5 (2005) 92–97

www.elsevier.com/locate/cap

Recent trends in biosensors

Bansi D. Malhotra a,*, Rahul Singhal a, Asha Chaubey b,
Sandeep K. Sharma c, Ashok Kumar c
a
Biomolecular Electronics & Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012, India
b
Regional Research Laboratory, Jammu, India
c
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India

Available online 17 July 2004

Abstract

Rapid advances in biosensors have recently been reported. This has been possible due to rapid growth in the development of new
biomaterials such as conducting polymers, copolymers and sol gels etc and the reported improvements in sensing techniques. Bio-
sensors are miniaturized devices employing biochemical molecular recognition as the basis for a selective analysis. The response gen-
erated as a result of biochemical reaction is detected by a transducer to give a signal (optical/electrical/thermal) that can be used with
or without amplification for the estimation of the concentration of an analyte in a given test sample. Among the various biosensors,
electrochemical sensors, especially amperometric biosensors presently hold a leading position.
Due to specificity, portability, simplicity, high sensitivity, potential ability for real-time and on-site analysis coupled with the
speed and low cost, biosensors have been projected to have applications in food analysis, environment control, clinical detection,
drug and agriculture industries etc. Besides this, biosensors offer exciting opportunities for numerous decentralized clinical applica-
tions, ranging from emergency room screening, home self testing and alternative site testing, continuous and real-time in vivo mon-
itoring. New generation of biosensors combining new bioreceptors with the ever-growing number of transducers is emerging. The
present paper highlights some of the recent advances in the area of biosensors contributed by our laboratory.
Ó 2004 Elsevier B.V. All rights reserved.

PACS: 68.18; 68.47.p; 72.80; 87.80

Keywords: Langmuir–Blodgett films; Electrical conductivity; Biological techniques; Biomedical engineering

1. Introduction devices based on direct spatial coupling between a bio-

In the recent past, there has been a tremendous de-
mand of modern techniques that have great potential
for industrial applications for a variety of analytes. In
this context, biosensors have the potential to overcome
most of the disadvantages of the conventional methods.
Though literature on the biosensor technology is well
documented [1–31], there is still a lack of proper utiliza-
tion of the knowledge of biosensor technology for com-
mercial applications. Biosensors are in general small
*
Corresponding author.
E-mail address: bansi@mail.nplindia.ernet.in (B.D. Malhotra).
logically active compound and a signal transducer
equipped with an electronic amplifier.
Biosensors are analytical devices incorporating bio-
logical materials such as enzymes, tissues, micro-organ-
isms, antibodies, cell receptors or biologically derived
materials or a biomimic component in intimate con-
tact with a physico-chemical transducer or transducing
microsystems. Transducers are the components that
convert a biochemical signal into a quantifiable electri-
cal signal. The transducing microsystem may be elec-
trochemical, thermometric, optical, piezoelectric or
magnetic. Biosensors have found immense applications
in medical diagnostics, environmental monitoring and
genetics, food processing industries and defense. Due

1567-1739/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.cap.2004.06.021
 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97
to their simplicity, high sensitivity and potential ability
for real-time and onsite analysis, biosensors have been
widely applied in various fields including industrial proc-
ess, clinical detection, environmental control etc. [4–7].
The most important part of biosensor is the immobi-
lization of a desired enzyme. However, the usefulness of
immobilized enzyme electrodes depends on factors such
as the immobilization method, the chemical and physi-
cal conditions (pH, temperature and contaminants),
thickness and stability of the membrane used to couple
the enzyme. Immobilization of enzyme in several matri-
ces has been used for the fabrication of biosensors for
estimation of glucose [8,9], urea [10,11], cholesterol
[12] etc. A number of papers relating to development
of conducting polymer sensors (chemical and electro-
chemical), self-assembled monolayer assembly, Lang-
muir–Blodgett (LB) film deposition etc. have recently
appeared in literature [13,14].
Among the conducting polymers, poly-alkyl-thiophe-
nes (PATs) have rapidly become the subject of consider-
able interest. From theoretical point of view, PATs have
often been considered as a model for the study of charge
transport in conducting polymers with non-degenerate
ground states. The high environmental stability of both
its doped and undoped states together with its structural
versatility has led to various applications such as elec-
trode materials, organic semiconductors etc. Besides
this, polyaniline can also be used for the sensor applica-
tion because it exhibits two redox couples in the conven-
ient potential range to facilitate an enzyme-polymer
charge transfer. Remarkable stability and solubility of
polyaniline in various solvents makes it an attractive
candidate for the technical development of a biosensor.
The present paper relates to some of the recent develop-
ments on application of Langmuir–Blodgett films to
biosensors carried out at our laboratories.
2. Langmuir–Blodgett films
Langmuir—Blodgett (LB) films are formed by first
dispensing a small quantity of an amphiphilic material
dissolved in a volatile organic solvent onto the surface
of purified water (sub-phase). As the solvent evaporates,
a monolayer is formed as dictated by the amphiphilic
nature of the molecules; the head group is immersed
on the water surface and the tail groups remain outside.
The molecules in their closest packed arrangement
(solid phase) are removed from the surface of water by
suitably dipping and raising a desired substrate through
air/water interface.
Three deposition types viz X, Y, Z deposition are pos-
sible depending on the nature of substrate. If the sub-
strate is hydrophilic, the first monolayer is transferred
as the substrate is raised through the sub-phase and
these molecules stack in a head-to-head and tail-to-tail
93
configuration. This deposition mode is referred to as
Y-type deposition. This results in an odd number of
monolayers being transferred onto the solid substrate.
However, if the solid substrate is hydrophobic, a mono-
layer will be deposited as it is first lowered into the sub-
phase, thus a Y-type film containing an even number of
monolayers can be fabricated. If a monolayer is depos-
ited on the substrate when the solid substrate enters
the sub-phase this deposition is called X-type deposition.
On the other hand, if a monolayer is deposited on the
substrate when it is withdrawn from the sub-phase, it
is called Z-type deposition.
3. Biosensors based on Langmuir–Blodgett films
There are several methods for immobilization of en-
zymes. Some of these suffer from drawbacks. For exam-
ple, the physical adsorption method is prone to leaching
and shows instability whereas the covalent linking re-
sults in reduced activity of the biomolecule. Most of
these methods do not provide the control on the amount
of enzyme to be immobilized. Langmuir–Blodgett tech-
nique can be used to obtain highly ordered and desired
orientation of the enzyme molecules, leading to faster
response [15,30]. LB technique is known to be an impor-
tant method for the immobilization of desired biomole-
cule. With a view to develop a desired biosensor, we
have used Langmui–Blodgett films of poly-3-hexylthio-
phene for the immobilization of developed galactose
oxidase (GaO), lactase (b-galactosidase, b-Gal) and glu-
cose oxidase (GOX), respectively.
3.1. Galactose biosensor
An enzymatic amperometric biosensor has been
developed for the estimation of galactose in milk and
blood serum. Galactose oxidase was immobilized with
poly(3-hexyl thiophene)/stearic acid (P3HT/SA) onto in-
dium tin-oxide (ITO) coated glass plates using Lang-
muir–Blodgett film deposition technique. The effect of
galactose concentration, pH, and stability of the immo-
bilized galactose oxidase in LB films were studied.
Fig. 1 shows the amperometric response of P3HT/SA/
GaO LB film at 25 °C with varying concentration of
galactose in phosphate buffer (pH 7.0). P3HT/SA/GaO
LB electrode shows linearity for 1–4 g/dl galactose in
0.1 M phosphate buffer and soya milk, after which a lim-
iting value of current was obtained. It was also observed
that the same electrodes could be repeatedly used for
about 10 times. Thereafter, a severe drop in the current
was noticed. The repeated polarization of the electrode
at 0.4 V might have perhaps caused the denaturation
of GaO.
Amperometric response of the P3HT/SA/GaO LB
electrodes was also taken at room temperature and at
94 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97

900
700
750

Current (nA)
600
600
Current (nA)

500
450

300 400

150
0
0 1 2 3 4 5
Galactose (g/dL)
Fig. 1. Amperometric response of P3HT/SA/GaO LB film electrode at
different concentrations of galactose in 0.1 M phosphate buffer, pH 7.0

( ) and in soya (lactose/galactose free) milk solution (n).
2 g/dl galactose solution using different pH of the solu-
tion. The current increases with the increase in pH.
The highest value of the current was obtained at pH
7.0 (Fig. 2), indicating that these P3HT/SA/GaO LB
electrodes can be used at pH 7.0 for the detection of
galactose.
P3HT/SA/GaO LB films were tested for stability
under the same operating conditions as those for amper-
ometric response measurements. The response of the
P3HT/SA/GaO LB electrode was measured once in 7
days. The enzyme LB electrodes were stored at 4 °C
when not in use. The response was measured on a fresh
electrode. Fig. 3 shows the amperometric response of the
P3HT/SA/GaO LB film electrode in galactose solution
(3 g/dl) in 0.1 M phosphate buffer (pH 7.0) as a function
of days. It can be seen that the response of these elec-
trodes is almost same for about 20 days after which
the electrodes show a gradual decrease in current re-
sponse. This may be due to partial decay in the enzyme
activity. The amperometric current decreases from 675
900
300
0 20 40 60 80 100
No. of days
6 7
Fig. 3. Stability of P3HT/SA/GaO LB film electrode on storage at 4
°C. Stability was monitored at 10 days intervals amperometrically
using 3 g/dl galactose in 0.1 M phosphate buffer, pH 7.0.
to 655 nA for 28 days and it decreases to 625 lA for
35 days. The half-life of these P3HT/SA/GaO electrodes
was determined to be 90 days.
Different concentrations of galactose were also pre-
pared in serum samples (containing galactose <0.05
g l
1). P3HT/SA/GaO LB electrode showed linearity
from 0.05 to 0.5 g l
1 in blood serum after which a lim-
iting value of current was obtained (Fig. 4). Separate
electrodes were used for different concentration of galac-
tose.
Further, no significant effect of the interferents (as-
corbic acid, calcium chloride and uric acid) was ob-
served at their physiological concentrations. Further, it
was found that P3HT/SA/GaO electrodes can be used
for galactose estimation in the temperature range 25–
40 °C.
The stability of P3HT/SA/GaO LB films was tested
under the same operating conditions as those for amper-
ometric response measurements. The response of the
P3HT/SA/GaO electrodes was measured once in 10
days. The response was measured on a fresh electrode.
The enzyme electrodes were stored at 4 °C, when not
in use. The amperometric current response was found
to decrease from 85 to 75 nA in 30 days and about

700 140
Current (nA)

120

500
Current (nA)

100

80

300 60

40

100 20
5.5 6.0 6.5 7.0 7.5 8.0 8.5 0 0.1 0.2 0.3 0.4 0.5
pH Concentration (gl-1)

Fig. 2. Effect of pH on the amperometric response of P3HT/SA/GaO Fig. 4. Amperometric response of P3HT/SA/GaO LB films at different
LB film electrode at 3 g/dl galactose in 0.1 M phosphate buffers of concentrations of galactose in 0.1 M phosphate buffer, pH 7.4 in blood
different pH at 0.4 V. serum.
 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 95

85 to varying concentrations of lactose, temperature and

pH.
The results of the amperometric response determined
at room temperature for P3HT/SA/b-Gal/GaO LB films
75 are shown in Fig. 6. P3HT/SA/b-Gal/GaO LB electrode
Current (nA)

shows linearity from 1 to 6 g/dl after which a limiting

value of current was obtained. The thermal stability
and effect of pH on b-Gal/GaO immobilized P3HT/SA
65
LB film was investigated at 3 g/dl lactose concentra-
tion by amperometric measurements. Fig. 7 shows the
results of enzyme (b-Gal/GaO) response measurements
55 obtained as a function of temperature by holding the
0 20 40 60 80 P3HT/SA/b-Gal/GaO film in a lactose solution (3 g/dl
No. of Days
in phosphate buffer, pH 7) at different temperatures. It
Fig. 5. Stability of P3HT/SA/GaO LB films on storage at 4 °C. shows that b-Gal/GaO activity increases up to 40 °C
Stability was monitored at 10 days interval amperometrically using 0.2 and then it decreases drastically.
g l
1 galactose in 0.1 M phosphate buffer, pH 7.4.
100
35% loss in amperometric response (Fig. 5) was ob- 90
served in three months. 80

70
Current (nA)
4. Lactose biosensor 60

50
Lactose is the major carbohydrate present in the 40
milk. Most people, who suffer from the deficiency lac-
30
tase (b-galactosidase b-Gal), are not able to metabolize
20
lactose, present in most dairy foods. The absence or de-
crease of lactase activity in human leads to the clinical 10

syndrome ‘‘lactose intolerance’’. Lactose intolerance 0

causes various physical symptoms such as excessive 2 4 6 8 10 12

intestinal gas, nausea, cramps and diarrhea. Lactose (g/dL)

We have developed an amperometric biosensor sensi- Fig. 6. Amperometric response of P3HT/SA/b-Gal/GaO LB films at
tive to lactose as well as galactose, by immobilizing different concentration of lactose in phosphate buffer, pH 7.0 ( ) and 
b-galactosidase (b-Gal) and galactose oxidase (GaO) in milk (m). Different concentrations of lactose were prepared in
in Langmuir–Blodgett (LB) films of poly-3-hexyl thio- lactose and galactose free milk and change in current was observed.
phene (P3HT) mixed with stearic acid (SA). The mono-
layers of P3HT/SA were fabricated by dispensing a
250
solution (1:1) of P3HT (1 mM) and SA (2 mM) in chlo-
roform onto water sub-phase containing CdCl2 (0.2
mM), using Joyce-Loebl LB trough. Such P3HT/SA 200
monolayers were transferred onto the ITO-coated glass
plates at a surface pressure of 30 mN/m at 30 °C by ver-
Current (nA)

150
tical dipping method. The dipping speed during up-
stroke and downstroke was maintained at 5 mm/min.
b-Galactosidase and galactose oxidase (2.5 mg each) 100
were mixed in a solution of P3HT/SA in chloroform
and this solution was spread onto air-water interface
50
of the LB trough. Thirty monolayers of P3HT/SA/
b-Gal/GaO were then transferred onto indium-tin-oxide
(ITO) coated glass plates by vertical dipping method. 0
The b-Gal/GaO immobilized P3HT/SA LB films were 10 20 30 40 50 60 70

characterized using Fourier-Transform-Infra-Red spectr- Temperature (°C)

oscopy (FTIR) and scanning electron microscopy (SEM) Fig. 7. Effect of temperature on the amperometric response of P3HT/
technique. Performance and characteristics of P3HT/ SA/b-Gal/GaO LB films in the presence of 3 g/dl lactose in phosphate
SA/b-Gal/GaO LB electrodes were studied with respect buffer, pH 7.0 at 0.4 V (bias voltage).
96 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97

130 0.8

110
0.6

Absorbance (O.D.)
Current (nA)

90
0.4

70

0.2
50

0
30
100 200 300 400 500 600
5.5 6 6.5 7 7.5 8 8.5
Glucose Concentration (mg/dL)
pH
Fig. 9. Absorbance of P3HT/SA/GOX LB electrode at 540 nm as a
Fig. 8. Effect of pH on the amperometric response of P3HT/SA/b-Gal/ function of glucose concentration.
GaO LB films in the presence of 3 g/dl lactose in phosphate buffer of
different pH at 0.4V (bias voltage).

Fig. 8 shows the results of P3HT/SA/b-Gal/GaO film
response measurements obtained as a function of varia-
tion of pH by holding the electrode in a lactose solution
(3 g/dl) made in different pH buffers. It shows the max-
imum activity at pH range 7.0–7.2. It can be concluded
from these studies that the P3HT/SA/b-Gal/GaO
electrodes can be used for lactose estimation in the tem-
perature and pH range 25–40 °C and 7.0–7.2, respec-
tively.
5. Glucose biosensor
The prevalence of diabetes in industrialized countries
amounts to approximately 4% and hence the demand
and the necessity for the determination of blood glucose.
The normal concentration of glucose in blood serum
ranges between 4.2 and 5.2 mmol/l. The determination
of glucose is one of the most frequently performed rou-
tine analyses in clinical chemistry as well as in the micro-
biological and food industries [29–31]. Keeping the
above in view, an attempt has been made towards the
preparation and characterization of LB films of poly-
3-hexyl thiophene mixed with stearic acid (SA). Further,
enzyme glucose oxidase (GOX) has been immobilized
onto the P3HT/SA LB films via LB technique. The
GOX immobilized P3HT/SA LB films have been sys-
tematically investigated.
The activity of the glucose oxidase (GOX) immobi-
lized onto P3HT/SA LB films were performed by color-
imetric method using UV-visible spectrophotometer
(Schimatzu 160A). The following reaction occurs and
gives a brown color dye.
ð2Þ
The dye produced adsorbs the light at 540 nm. Pho-
tometric response of glucose oxidase (GOX) immobi-
lized P3HT/SA LB film was also monitored with
varying concentration of glucose in phosphate buffer
(pH 7.0). The intensity of dye produced was found to
be directly proportional to the concentration of glucose
in the solution.
A plot between the absorbance at 540 nm with vary-
ing concentration of glucose is plotted and is shown in
Fig. 9. It is clear from the fig. that the absorbance in-
creases linearly as glucose concentration increases form
100 to 500 mg/dl. These results suggest that these P3HT/
SA/GOX LB electrodes may be used for the estimation
of glucose from 100 to 500 mg/dl glucose solution [26].
6. Conclusions
An attempt has been made to present some of the re-
cent work in the area of biosensors carried out at our
laboratories. Further, it has been shown that conducting
polymer based Langmuir–Blodgett films of poly3-hexyl-
thiophene can be used for immobilization of galactose
oxidase (GaO), lactase (b-galactocidase, b-Gal) and glu-
cose oxidase, respectively. These P3HT/SA/GaO, P3HT/
SA/b-Gal/GaO, P3HT/SA/GOX and P3DT/SA/GOX
LB electrodes can be used for the estimation of galac-
tose, lactose and glucose in desired test specimens,
respectively.
Acknowledgments

We are grateful to Dr. Vikram Kumar, Director,

ð1Þ NPL for his interest in this work. We are also thankful
 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97
to Prof. Keiichi Kaneto, Kyushu Institute of Engineer-
ing and Technology, Iizuka, Fukuoka, Japan for his val-
uable suggestions.
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