ReviewRecent advances in polyaniline based biosensors
Introduction
Biosensors are analytical devices that integrate biological sensing elements like enzymes, antibodies, nucleic acids, cells, etc. with electronic transducer equipped with an electronic amplifier. Biosensors represent conceptually novel approach to real-time, on-site, simultaneous detection of multiple hazardous agents. They provide speedy interactive information about the desired sample and found to have applications in various fields, e.g. clinical diagnostics, environmental monitoring, bioprocess monitoring, food and agricultural product processing, etc. Recently, IUPAC has given the specific definition of biosensor: it is a self-contained integral device that is capable of providing specific quantitative or semi-quantitative analytical information using a biological element (Thevenot et al., 1999). Biosensor has three major components: (i) a bio-recognition element such as deoxyribonucleic acid (DNA) or enzyme or antibody, etc. for the recognition of an analyte also called bioreceptor, (ii) an immobilization surface such as a conducting polymers (CP) (Gerard et al., 2002), nanomaterials (Luo et al., 2006), sol–gel films (Ansari et al., 2008), and self-assembled monolayers (Arya et al., 2009), etc., used for immobilization of biomolecule and (iii) a transducer unit for conversion of biochemical reaction product into a recognizable signal (Fig. 1). Bioreceptor and transducer together may be referred as biosensor membrane.
The history of biosensor began in 1962 with the development of first enzyme based glucose sensing device by Clark and Lyons (Clark and Lyons, 1962). This novel biosensing device relied on a thin layer of glucose oxidase (GOx) entrapped over an oxygen electrode via semi-permeable dialysis membrane. Measurements were based on the monitoring of the O2 consumed by the enzyme-catalyzed reaction as shown below
Though the current scenario of biosensors is much advanced than the Clark enzyme electrode, the researchers are facing new challenges and are struggling to find solutions to the instability of desired biomolecules. Until recently, the majority of biosensors are based on one or more biomolecules used in conjunction with an electrode. The redox reaction could be detected electrochemically by measuring the loss or formation of substrate or product, respectively, by the use of a small mediator species that shuttles between the biomolecule and the electrode, or less commonly by direct electron transfer (ET) between the biomolecule redox site and the electrode (Murphy, 2006). Direct ET can be difficult to achieve, since the redox site of a biomolecule is often hidden deep inside the biomolecule. Recent advances in achieving direct ET have been made by modification of biomolecules or electrode surfaces through the use of novel conducting materials as mediators and design of functional biointerfaces (Hartmann, 2005). Thus, highly conductive organic transducers are gradually emerging for the development of next generation biosensor design for highly reliable, stable and robust field-based diagnostic devices. There has been considerable interest towards the development of biosensor probes for detection of biologically significant molecules using CP. Hoa et al. (1992) have recently described a new generic concept based on the fact that the conductivity of this class of polymers is very sensitive to chemical potential of the microenvironment within a polymer matrix. And these polymers thus act as both immobilization matrices as well as the physicochemical transducer to convert a chemical signal into an electrical signal.
In general, CP are polymers with delocalized π-electron system with a wide band gap in their pristine state (Smilowitz et al., 1993) and can be made electrically conducting by doping. Doping in CP refers to the oxidation or reduction of π-electronic system, p-doping and n-doping, respectively, and can be effected chemically or electrochemically. To maintain electro-neutrality, doping requires incorporation of a counter ion. The doped and undoped states have different electronic, optical, physical, chemical, and electrochemical aspects. Thus, reversible interchange between redox states in CP gives rise to the changes in its properties including polymer conformation, doping level, conductivity, and colour. These make the CP suitable for applications to electrochromic devices (Beaujuge and Reynolds, 2010), energy storage devices (Cheng et al., 2009), actuators (Fuchiwaki et al., 2009), sensors (Hatchett and Josowicz, 2008), etc. Polyaniline (PANI), polypyrrole, polythiophene, and polyacetylene are some of the most explored and interesting CP and have been used for a variety of applications.
Among various conducting polymers, PANI has attracted much attention due to its unique and controllable chemical and electrical properties (Kang et al., 2004), its environmental (Pruneanu et al., 1999), thermal (Wang et al., 1995) and electrochemical stability (Chiang and MacDiarmid, 1986), and its interesting electrochemical, electronic, optical and electro-optical properties (Heeger, 2001). Moreover, PANI is known to have a broad range of tunable properties emanating from its structural flexibility leading to many applications in different fields such as anti-corrosive coatings, energy storage systems, gas sensing, as well as electrochromic and electrocatalytic devices. Chandrakanthl and Careem (2000) have claimed that PANI has the highest environmental stability and is recognised as the only conducting polymer that is stable in air (Sergeyeva et al., 1996). PANI has been found as an interesting material for sensor and biosensor interfaces since it acts as an effective mediator for electron transfer in redox or enzymatic reactions and can also be used as a suitable matrix for immobilization of biomolecules (Luo and Do, 2004). The former role is possible due to the inherent electroactivity of PANI. PANI has gained much popularity in biosensor applications, partially due to its favourable storage stability and simple synthetic procedures with good processibility. Advantages of PANI in the field of biosensor are indicated by impressive signal amplification and elimination of electrode fouling. Besides this, PANI exhibits two redox couples in the convenient potential range to facilitate an efficient enzyme–polymer charge transfer. Recent reports have elucidated the role of PANI as enzyme amplifier to provide signal amplification in the recognition process (Sergeyeva et al., 1996; Kim et al., 2000; Grennan et al., 2003, Morrin et al., 2003). The use of polyaniline as an enzyme switch, which yields “on” and “off” responses, has also been recently demonstrated (Iribe and Suzuki, 2002). From economic point of view, aniline monomer is less expensive than other monomers used for the synthesis of other conducting polymers.
The goal of this review is to describe at length the role of PANI for biosensor application in a comprehensive fashion. It covers PANI as active component mediating or transducing responses that indicate the presence of an analyte.
Section snippets
Polyaniline
PANI, a CP of semi-flexible polymer family, was discovered over about 150 years ago. Recently, PANI has captured attention of scientific community due to the discovery of its high conductivity and low cost. Therefore researchers are continuously exploring its applications including those in biosensors because of a number of useful features such as 1) direct and easy deposition on the sensor electrode, 2) control of thickness, 3) redox conductivity and polyelectrolyte characteristics, 4) high
Classification of enzymatic biosensors based on PANI as immobilization platform
Enzymatic biosensors utilize the bio-specificity of an enzymatic reaction on an electrode surface that can be detected and quantified using various transduction techniques. The concept of enzyme based sensor devices was presented first by Clark and was realized as a commercial clinical analyser (Clark and Lyons, 1962). Most enzyme-based biosensors employ a class of enzymes known as oxido-reductases mainly oxidases and dehydrogenases. Their general reaction sequences can be described by the
Classification of bioaffinity sensors based on PANI as immobilization platform
A bioaffinity sensor consists of a bioaffinity reagent in close proximity to the transducer. Measurement of a target analyte can be achieved by selectively converting molecular recognition occurring at analyte sensor interface from a nonelectrical domain to an electrical signal. Several bioaffinity reagents can be used to fabricate a sensor, including receptors, binding proteins, antibodies and nucleic acids immobilized on surface of transducer. The binding of analyte is detected through change
Other biosensors based on PANI
PANI has been utilized for detection of insecticides, pesticides and toxicants present in the environment and biological samples. A nucleic acid sensor based on PANI has been fabricated by covalently immobilizing dsCT onto perchlorate (ClO4–) doped PANI film for the insecticide (cypermethrin/trichlorfon) detection. This disposable dsCT-DNA-PANI-ClO4/ITO bioelectrode, stable for about 4 months, can be used to detect cypermethrin (0.005 ppm) and trichlorfon (0.01 ppm) in 30 and 60 s, respectively.
Commercialization and challenges
A large number of reports are available related to the fabrication of PANI based electrode for biosensor application, though its commercialization is still a challenge. This is due to the aging effect, low electrochemical stability of PANI and lack of well optimized deposition techniques. PANI consists of conducting grains of ES-I separated by thin insulating barriers. These grains are composed of crystallites surrounded by “paracrystalline” and “amorphous” ES-I (Stafstrom et al., 1987). During
Conclusions
This review has described how the synchronization of various remarkable characteristics of PANI like controllable conductivity, charge transfer capability, good processibility, environmental stability, mechanical flexibility, etc. makes this matrix a novel platform for fabrication of variety of biosensors. Efforts have also been made to unravel the implications of PANI nanostructures (nanospheres/nanotubes/nanofibers) for electric wiring of biomolecules with the electrode for direct ET and thus
Acknowledgements
We thank Prof. R.C. Budhani, Director, NPL, New Delhi, for providing facilities. Chetna Dhand and Maumita Das are thankful to CSIR, India, for the award of Senior Research Fellowship. We acknowledge the financial support received from the DST (GAP081132) and DBT, Govt. of India (GAP070832).
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