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www.elsevier.com/locate/cap
Current Applied Physics 5 (2005) 92–97
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, Indiab 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.
� 2004 Elsevier B.V. All rights reserved.
PACS: 68.18; 68.47.p; 72.80; 87.80Keywords: Langmuir–Blodgett films; Electrical conductivity; Biological techniques; Biomedical engineering
1. Introduction
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. Inthis 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
1567-1739/$ - see front matter � 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.cap.2004.06.021
* Corresponding author.
E-mail address: [email protected] (B.D. Malhotra).
devices based on direct spatial coupling between a bio-
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 applicationsin medical diagnostics, environmental monitoring and
genetics, food processing industries and defense. Due
B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 93
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 ofimmobilized 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 developmentof 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 chargetransport 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-polymercharge 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 immersedon 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 transferredas the substrate is raised through the sub-phase and
these molecules stack in a head-to-head and tail-to-tail
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 ofmonolayers 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 amountof 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 galactoseoxidase (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 withpoly(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/GaOLB 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
0
150
300
450
600
750
900
0 1 2 3 4 5 6 7Galactose (g/dL)
Cur
rent
(nA)
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).
300
400
500
600
700
0 20 40 60 80 100No. of days
Cur
rent
(nA)
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.
94 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97
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 �Cwhen 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
100
300
500
700
900
5.5 6.0 6.5 7.0 7.5 8.0 8.5pH
Cur
rent
(nA)
Fig. 2. Effect of pH on the amperometric response of P3HT/SA/GaO
LB film electrode at 3 g/dl galactose in 0.1 M phosphate buffers of
different pH at 0.4 V.
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 10days. 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
20
40
60
80
100
120
140
0 0.1 0.2 0.3 0.4 0.5Concentration (gl-1)
Cur
rent
(nA)
Fig. 4. Amperometric response of P3HT/SA/GaO LB films at different
concentrations of galactose in 0.1 M phosphate buffer, pH 7.4 in blood
serum.
55
65
75
85
0 20 40 60 80No. of Days
Cur
rent
(nA)
Fig. 5. Stability of P3HT/SA/GaO LB films on storage at 4 �C.Stability was monitored at 10 days interval amperometrically using 0.2
g l�1 galactose in 0.1 M phosphate buffer, pH 7.4.
80
90
100
B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 95
35% loss in amperometric response (Fig. 5) was ob-
served in three months.
0
10
20
30
40
50
60
70
2 4 6 8 10 12Lactose (g/dL)
Cur
rent
(nA)
Fig. 6. Amperometric response of P3HT/SA/b-Gal/GaO LB films at
different concentration of lactose in phosphate buffer, pH 7.0 (�) and
in milk (m). Different concentrations of lactose were prepared in
lactose and galactose free milk and change in current was observed.
0
50
100
150
200
250
10 20 30 40 50 60 70Temperature (°C)
Cur
rent
(nA)
Fig. 7. Effect of temperature on the amperometric response of P3HT/
SA/b-Gal/GaO LB films in the presence of 3 g/dl lactose in phosphate
buffer, pH 7.0 at 0.4 V (bias voltage).
4. Lactose biosensor
Lactose is the major carbohydrate present in the
milk. Most people, who suffer from the deficiency lac-
tase (b-galactosidase b-Gal), are not able to metabolize
lactose, present in most dairy foods. The absence or de-
crease of lactase activity in human leads to the clinical
syndrome ‘‘lactose intolerance’’. Lactose intolerance
causes various physical symptoms such as excessive
intestinal gas, nausea, cramps and diarrhea.We have developed an amperometric biosensor sensi-
tive to lactose as well as galactose, by immobilizing
b-galactosidase (b-Gal) and galactose oxidase (GaO)
in Langmuir–Blodgett (LB) films of poly-3-hexyl thio-
phene (P3HT) mixed with stearic acid (SA). The mono-
layers of P3HT/SA were fabricated by dispensing a
solution (1:1) of P3HT (1 mM) and SA (2 mM) in chlo-
roform onto water sub-phase containing CdCl2 (0.2mM), using Joyce-Loebl LB trough. Such P3HT/SA
monolayers were transferred onto the ITO-coated glass
plates at a surface pressure of 30 mN/m at 30 �C by ver-
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)
were mixed in a solution of P3HT/SA in chloroform
and this solution was spread onto air-water interfaceof 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.
The b-Gal/GaO immobilized P3HT/SA LB films were
characterized using Fourier-Transform-Infra-Red spectr-
oscopy (FTIR) and scanning electron microscopy (SEM)
technique. Performance and characteristics of P3HT/
SA/b-Gal/GaO LB electrodes were studied with respect
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
are shown in Fig. 6. P3HT/SA/b-Gal/GaO LB electrode
shows linearity from 1 to 6 g/dl after which a limitingvalue of current was obtained. The thermal stability
and effect of pH on b-Gal/GaO immobilized P3HT/SA
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
obtained as a function of temperature by holding the
P3HT/SA/b-Gal/GaO film in a lactose solution (3 g/dl
in phosphate buffer, pH 7) at different temperatures. Itshows that b-Gal/GaO activity increases up to 40 �Cand then it decreases drastically.
30
50
70
90
110
130
5.5 6 6.5 7 7.5 8 8.5pH
Cur
rent
(nA)
Fig. 8. Effect of pH on the amperometric response of P3HT/SA/b-Gal/
GaO LB films in the presence of 3 g/dl lactose in phosphate buffer of
different pH at 0.4V (bias voltage).
0
0.2
0.4
0.6
0.8
100 200 300 400 500 600Glucose Concentration (mg/dL)
Abso
rban
ce (O
.D.)
Fig. 9. Absorbance of P3HT/SA/GOX LB electrode at 540 nm as a
function of glucose concentration.
96 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97
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.
ð1Þ
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,
NPL for his interest in this work. We are also thankful
B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 97
to Prof. Keiichi Kaneto, Kyushu Institute of Engineer-
ing and Technology, Iizuka, Fukuoka, Japan for his val-
uable suggestions.
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