Towards the Hybrid Biosensors Based on Biocompatible
Conducting Polymers
1
A. Ramanavičienė , A. Finkelšteinas2, A. Ramanavičius2,3,4*
1. Laboratory of Ecological Immunology, Institute of
Immunology of Vilnius University, Moletų pl. 29, Vilnius,
Lithuania;
2. Department of Analytical and Environmental Chemistry,
Vilnius University, Naugarduko 24, Vilnius, Lithuania;
3. Sector of Immunoanalysis and informatics, Institute of
Immunology of Vilnius University, Moletų pl. 29, Vilnius,
Lithuania;
4. Laboratory of bioanalysis, Institute of Biochemistry,
Mokslininkų 12, Vilnius Lithuania.
*Corresponding author e-mails: arunas@imi.lt and/or arunas.r@bchi.lt.
The effective combination of biological and physical methods in
analytical device could provide the basis for direct detection of wide
range of analytes with great sensitivity and specificity. The most
powerful alternative to conventional analytical techniques,
harnessing the specificity and selectivity of biological systems in
small, low cost devices is biosensor technology.
A biosensor is described as compact analytical device,
incorporating a biological or biomimetic sensing element to, or
integrated within, a transducer system. The detection is based on
specific complementary binding or catalytic conversion of analyte of
interest by bio-recognition element immobilized on the suitable
signal transducer. The specific interaction of analyte with biorecognition element results in a change of one ore more
physicochemical properties (electron transfer, capacity, optical
properties etc.) those are detected and can by measured by the signal
transducer.
Analyte
Analitė
Analyte
Analitė
Analyte
Part of biological recognition
Signal transducer
Registration
device
Depending on the method of signal transduction, biosensors
are divided into different groups: electrochemical, optical,
thermometric, piezoelectric or magnetic. Electrochemical
biosensors are the most commonly reported class of biosensors.
The main advantages of electrochemical transduction systems
are low cost, simple operation and the use of disposable
electrodes. Limitations of electrochemical transducers include
interference from electro-active compounds and as usually low
sensitivity. Here mentioned problems can be solved if additional
alternative detection method is applied. The most powerful
alternative is optical signal transduction.
The conducting polymers can be considered as effective materials for creation of
immunosensing devices and especially for immobilization of Bio-molecules.
Or
gM
et
n
Polyacetylene
N
S
n
H
Polypyrrole
Polythiophene
H
S
n
ythiophene
O
Polyfurane
N
n
n
Polyaniline
n
O
Polyfuran
The main aim of here presented work is to construct affinity
biosensors based on dual detection (optical and
electrochemical) system. Current state of research and
future perspectives of conducting (π-π conjugated) polymer
polypyrrole application in hybrid biosensors will be
presented. Since according to our initial investigations
florescence of polypyrrole is undetectable, we believe that it
can be successfully exploited as the immobilization matrix
in fluorescence based biosensors.
Electrochemical synthesis of polypyrrole
N
H
e-
.
+N
H
2e
N
H
H
N
N
H
(2 n -1 )e
N
H
H
me
H
N
N
H
-
N
H
O2
NH
H
N
H
n
N
H
N
H
n
R
R
R
CH2O H
COH
COOH
The biological recognition parts of affinity biosensors were
based on electrochemically synthesized polypyrrole (Ppy)
doped by bovine leukemia virus (BLV) proteins. Bovine
leukemia virus was selected as a model system very similar to
human immunodeficit virus (HIV). BLV proteins were able
selectively bind specific antibodies indicating BLV infection.
Next biosensor was based on Ppy doped by single-stranded
DNA able specifically recognize and bind provirus DNA.
New real-time electrochemical detection methods were
elaborated for direct detection of protein-protein and DNADNA binding.
–NO 2
Elektrocheminė
Electrochemical
polimerizacija
polymerisation
PPy –NO 2
Pt electrode
+Pirolas
Pt electrode
Pt electrode
PPy
+Cu(NO3) 2
Attachment of
nitrogroups
–NO2
+ +H +e
Electrochemical
reduction
-H O
2
–NO2
–NO2
Pt electrode
Electrochemical
reduction
–NH 2
QHADH
Activation of HOOCby EDC
–NH 2 -H2 O
–NH 2
PPy
+EDC
–
OC
PPy–NH 2
Pt electrode
HO
–NH 2
–NH 2
–NH–CO–
–NH 2
–NH–CO–
–NH 2
QHADH
QHADH
Immobilization of
bio-molecules on the
surface of
electrochemically
synthesized
polypyrrole
Generation of immune response
un
m
m
I
n se
o
p
s
e re
Y
Blood
Y
Y
Y
Y
Y
Antibodies against infection
Y
Y
In
fe c
tio
n
Y
Y
Structure of Retroviruses
V
i
r
u
s
a
s
Reverse transcriptase
Lipid layer
Integrase
Human
immunodeficit virus
p24
gp51/gp30
RNR
p15
Proteins applied
in construction of
immunosensors
BLV
PPy–NO 2
Electrochemical
polymerization
Pt electrode
+Pyrrole
Pt electrode
PPy
Pt electrode
+Cu(NO3) 2
–NO2
+ +H +e
El. reduction
-H O
2
–NO2
–NO2
–NH 2
gp51
aktivation of
HOOC-
–NH 2
–NH 2
PPy
Pt elektrodas
El. reduction
–NH 2
–
OC
HO
–NH 2
PPy–NH 2
Pt elektrodas
Covalent
attachment
–NO 2
+EDC
-H2 O
gp51 – Glicoprotein from bovine
leukemia virus (antigen)
–NH–CO–
gp51
–NH 2
–NH–CO–
–NH 2
gp51
Y Y
Y
Reference
electrode
Incubation
Counter electrode
Reference
electrode
YY Y
Working electrode
YY Y
Counter electrode
Y Y
Y
R
C
Y Y
YY
Y
YY
Working electrode
General detection scheme used during application of affinity sensors
T
y
r
i
m
ų
s
h
e
m
a
dR
dC
Detection of protein-protein binding event by cyclic
voltammetry
200 mV/s
300
200
100
0
0
100
200
300
400
500
600
700
-100
-200
-300
pe
ri š n
i k u bav m
i ą
Before incubation
-400
pon
i k ub av m
i o
After incubation
800
In recent years, a key stimulus for the development of optical
biosensors has been the availability of high-quality and variety
LED’s, fibres and other optoelectronic components. The
electrochemical/optical biosensor format may involve direct
detection of analyte of interest or indirect detection though labelled
probes. The optical transducers may detect changes in the
fluorescence, luminescence, absorbance, polarisation, refractive
index etc. The advantages of optical transducers are their speed, the
immunity of signal to electrical or magnetic interference and the
potential for higher sensitivity and advanced information content
but several years ago the main drawback was high cost of some
optical instrumentation. Currently, costs of optical components
dramatically decreased and it offers a great opportunity for
construction of biosensors suitable for mass production.
We hope that multiple-transducers based biosensors offers
great advantages over conventional mono-transducers based
techniques. The multi-functionality of transdusing part of
biosensor offers the opportunity for development of highly
specific devices for real-time analysis in complex mixtures,
without the need for extensive sample pre-treatment or large
sample volumes. Since those biosensors promise to be highly
sensitive, rapid, reproducible, simple to-operate and
multipurpose analytical tools, they will find great application
not only in analytical fields but in scientific investigations as
well. Such systems are especially required in the fields of
proteomics and bioinformatics where wide spectrum of
information about interacting biological objects is desirable to
be collected in real-time.
Optical transducers based on fluorescence detection of
protein-protein and DNA-DNA binding events are under
development.
Detector
Optical
transducer
LED
Analyte
Analyte
Analitė
Analyte
Analitė
Part of biological recognition
Electrochemical transducer
Registration
device
Conclusions
„
„
„
„
„
„
From the analytical point of view Ppy has a few very attractive
characteristics:
(i) is biocompatible and, hence, causes minimal and reversible
disturbance to the working environment;
(ii) is capable of transducing the energy arising from
interaction between immune reagents into electrical signals
that are easily monitored;
(iii) protects electrodes from fouling and interfering materials
such as electroactive anions;
(iv) can be modified in situ in a controlled fashion.
Conducting polymers can be applied in design of intelligent
analytical signal transducers and amfilers.
Future trends:
Development of optical systems based on
conducting polymers.
Development of hybrid biosensors based on
dual detection system (optical +
electrochemical) for analytical and scientific
applications.
Advantages to be expected:
Operating in real-time, direct (label free)
detection systems will be developed.