Vous êtes sur la page 1sur 15

Biosensors & Bioelectronics 17 (2002) 345 359 www.elsevier.

com/locate/bios

Review

Application of conducting polymers to biosensors


Manju Gerard a, Asha Chaubey b,*, B.D. Malhotra b
b a Chemistry Department, Allahabad Agricultural Institute (Deemed Uni6ersity), Allahabad, India Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012, India

Received 14 August 2001; accepted 31 October 2001

Abstract Recently, conducting polymers have attracted much interest in the development of biosensors. The electrically conducting polymers are known to possess numerous features, which allow them to act as excellent materials for immobilization of biomolecules and rapid electron transfer for the fabrication of efcient biosensors. In the present review an attempt has been made to describe the salient features of conducting polymers and their wide applications in health care, food industries, environmental monitoring etc. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Conduction polymer; Biosensor; Immobilization; Electron transfer; Tranducer

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Conducting polymers. . . . . . . . . . . . . . . . . . . . . . . . 1.2. Historical background of electronically conducting polymers. 1.3. Conduction mechanism . . . . . . . . . . . . . . . . . . . . . . 1.4. Synthesis of conducting polymers . . . . . . . . . . . . . . . . 1.5. Applications of conducting polymers . . . . . . . . . . . . . . 2. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Transducer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biocomponents . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Importance of conducting polymers to biosensors . . . . . . . . . . 4. Immobilization of enzymes on conducting polymers . . . . . . . . 5. Types of biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Amperometric biosensors . . . . . . . . . . . . . . . . . . . . . 5.2. Potentiometric biosensors . . . . . . . . . . . . . . . . . . . . . 5.3. Conductometric biosensors . . . . . . . . . . . . . . . . . . . . 5.4. Optical biosensors . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Calorimetric biosensors . . . . . . . . . . . . . . . . . . . . . . 5.6. Peizoelectric biosensors . . . . . . . . . . . . . . . . . . . . . . 6. Applications of conducting polymers . . . . . . . . . . . . . . . . . 6.1. Biosensors for health care . . . . . . . . . . . . . . . . . . . . . 6.1.1. Glucose biosensors . . . . . . . . . . . . . . . . . . . . . 6.1.2. Urea biosensors . . . . . . . . . . . . . . . . . . . . . . . 6.1.3. Lactate biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 346 346 347 347 347 348 348 349 349 350 351 351 351 352 353 353 353 353 353 353 353 354

* Corresponding author. Fax: + 91-11-585-2678. E -mail address: achaubey@csnpl.ren.nic.in (A. Chaubey). 0956-5663/02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 3 1 2 - 8

346

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 354 354 354 355 355 355 355

6.1.4. Cholesterol biosensors . . . . . . . 6.2. Immunosensors . . . . . . . . . . . . . . . 6.3. DNA Biosensors . . . . . . . . . . . . . . 6.4. Biosensors for environmental monitoring 6.5. Biosensors for food industry . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . .

1. Introduction Polymers are being discarded for their traditional roles as electric insulators to literally take charge as conductors with a range of novel applications. Scientists from many disciplines are now combining expertise to study organic solids that exhibit remarkable conducting properties. A large number of organic compounds, which effectively transport charge are roughly divided into three groups i.e. charge transfer complexes/ ion radical salts, organometallic species and conjugated organic polymers. A new class of polymers known as intrinsically conducting polymers or electroactive conjugated polymers has recently emerged. Such materials exhibit interesting electrical and optical properties previously found only in inorganic systems. Electronically conducting polymers differ from all the familiar inorganic crystalline semiconductors e.g. silicon in two important features that polymers are molecular in nature and lack long range order (Duke and Schein, 1980). A key requirement for a polymer to become intrinsically electrically conducting is that there should be an overlap of molecular orbitals to allow the formation of delocalized molecular wave function. Besides this, molecular orbitals must be partially lled so that there is a free movement of electrons throughout the lattice (Bloor and Movaghar, 1983).

brane separations and chromatography. They also create new technological possibilities in design of chemical and biochemical sensors (Trojanowicz and vel Krawczyk, 1995; Bidan, 1992; Bartlett and Cooper, 1993).

1.2. Historical background of electronically conducting polymers


Research on conducting polymers intensied soon after the discovery of poly(sulphur nitride) [(SN)x ] in 1975 which becomes superconducting at low temperatures (Greene et al., 1975). Although, conducting polymer complexes in the form of tetracyano and tetraoxalato-platinates, the Krogman salts charge transfer complexes (Minot and Peristein, 1971) had been known earlier, the signicance lies in the rediscovery of PA in 1977 (initially discovered by Shirakawa et al., 1977 using a Ziegler Natta type polymerization catalyst) by MacDiarmid and Heeger, University of Pennsylvania. They were able to enhance the electrical conductivity of PA (10 9 S cm 1) by several orders i.e. 105 S cm 1 by simple doping with oxidizing agents e.g. I2, AsF5, NOPF6 (p-doping) or reducing agents (n-doping) e.g. sodium napthalide. This has generated renewed interest of the scientic community towards the study and discovery of new conducting polymeric systems. Poly-paraphenylene was synthesized by Ivory et al. (1979). It forms highly conducting charge transfer complexes with both n and p type dopants. Doping with AsF5 increases its conductivity to its values from 10 5 to 500 cm 1. Theoretical models and electron spin resonance measurements indicate that the charge transport in PPP is a polaron/bipolaron. PPS was the rst non-rigid, but not fully carbon backbone linked conducting polymer. Its discovery was particularly exciting, since its property of solution processability opened the door for potentially obtaining commercially viable conducting plastics (Rabolt et al., 1980). Amongst polyheterocyclines, polypyrrole (PPY) has been investigated the most. The electrochemical oxidation of pyrrole in aqueous H2SO4 can be carried out on platinum electrode. The product is a conducting polymer known as Pyrrole Black Kanazawa et al. (1979) produced coherent lms of PPY with a conductivity of 100 Scm 1 and exhibited excellent air stability. But the

1.1. Conducting polymers


Conducting polymers contain (p-electron backbone responsible for their unusual electronic properties such as electrical conductivity, low energy optical transitions, low ionization potential and high electron afnity. This extended (p-conjugated system of the conducting polymers have single and double bonds alternating along the polymer chain. The higher values of the electrical conductivity obtained in such organic polymers have led to the name synthetic metals. Many applications of conducting polymers including analytical chemistry and biosensing devices have been reviewed by various researchers (Trojanowicz and vel Krawczyk, 1995; Situmorang et al., 1998; Schuhmann, 1995; Wring and Hart, 1992; Guiseppi-Elie et al., 1997). They have widened the possibility of modication of surface of conventional electrodes providing new and interesting properties. They were applied in electrocatalysis, mem-

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

347

main hindrance of its processability is in its insolubility in any organic solvents. PTH shows remarkable stability of both oxidized (p-doped) conducting form and its neutral (undoped) insulating form in both air and water. It shows high doping level upto 50% which may be attributed to its partially crystalline nature that has been conrmed by X-ray photoelectron spectroscopy studies (Tourillon and Garnier, 1983). Many other conducting polymers such as polyfuran, polyindole, polycarbazole, polyaniline etc. have also been synthesized. Structures of some typical conducting polymers have been shown in Fig. 1

polymers is inuenced by a variety of factors including polaron length, the conjugation length, the overall chain length and by the charge transfer to adjacent molecules (Kroschwitz, 1988). These are explained by large number of models based on intersoliton hopping, hopping between localized states assisted by lattice vibrations, intra-chain hopping of bipolarons, variable range hopping in 3-dimensions and charging energy limited tunneling between conducting domains.

1.4. Synthesis of conducting polymers


Various methods are available for the synthesis of conducting polymers. However, the most widely used technique is the oxidative coupling involving the oxidation of monomers to form a cation radical followed by coupling to form di-cations and the repetition leads to the polymer. Electrochemical synthesis is rapidly becoming the preferred general method for preparing electrically conducting polymers because of its simplicity and reproducibility. The advantage of electrochemical polymerization is that the reactions can be carried out at room temperature. By varying either the potential or current with time the thickness of the lm can be controlled. Electrochemical polymerization of conducting polymers is generally employed by: (1) constant current or galvanostatic; (2) constant potential or potentiostatic; (3) potential scanning/cycling or sweeping methods. Standard electrochemical technique which employs a divided cell containing a working electrode, a counter electrode and a reference electrode generally produces the best lms. The commonly used anodes are chromium, gold, nickel, palladium, titanium, platinum and indium-tin oxide coated glass plates. Semi-conducting materials such as n-doped silicon (Nou et al., 1981), gallium arsenide (Nou et al., 1980), cadmium sulphide and semi-metal graphite (Bull et al., 1983) are also used for the growth of polymer lms. Electrochemical synthesis can be used to prepare free standing, homogeneous and self doped lms. Besides this, it is possible to obtain copolymers and graft copolymers. Polyazulene, polythiophene, polyaniline, polycarbazole and several other polymers have been synthesized using this approach.

1.3. Conduction mechanism


The mechanism of conduction in such polymers is very complex since such a material exhibits conductivity across a range of about fteen orders of magnitude and many involve different mechanisms within different regimes. Conducting polymers show enhanced electrical conductivity by several orders of magnitude of doping. The concept of solitons, polarons and bipolarons has been used to explain the electronic phenomena in these systems (Heeger, 1986). Conductivity in conducting

1.5. Applications of conducting polymers


The increasing number of academic, governmental and industrial laboratories throughout the world involved in basic research and assessment of possible applications of conducting polymers show that this area is interdisciplinary in nature. These conducting organic molecular electronic materials have attracted much attention largely because of their many projected applications in solar cells, light weight batteries,

Fig. 1. Structures of some conducting polymers commonly used in biosensors.

348

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

electrochromic devices, sensors and molecular electronic devices. Polymeric heterojunctions, solar cells have been fabricated by electrochemical deposition of PPY on n-silicon (Audebert and Bidan, 1985). Many conducting polymers such as polyacetylene, polythiophene, polyindole, polypyrrole, polyaniline etc. have been reported as electrode materials for rechargeable batteries (Saraswathi et al., 1999; Kawai et al., 1990; Santhanam and Gupta, 1993). It has been reported (Gazard et al., 1986) that the conducting polyheterocycles are good candidates for electrochromic displays and thermal smart windows. Scientists have used PPY lms in a neurotransmitter as a drug release system into the brain (Zinger and Miller, 1984). The potential for conducting polymers in the area of electronics and photonics (nonlinear optics) is enormous and has been used to fabricate diodes, capacitors, eld-effect transistors (FET) and printed circuit boards. Polyaniline PANI is being used by Hitachi-Maxell for anti-static coating of 4 MB barium ferrite oppy disk (Friend, 1993). In analytical chemistry the problem of selectivity, particularly at the low analyte concentrations and in the presence of interfering substances is of paramount importance. The development of sensors, which are highly selective and easy to handle opens the door to the problem in analysis. Conducting polymers have enough scope for the development of various sensors. A chemical or biosensor based on conducting polymers, also rely on sensible changes in the optical and electrical properties of these materials. The industrial applications of biochemical and morphological processes in elds such as production of pharmaceuticals, food manufacturing, waste water treatment and energy production is on increase. This has led to the development of biosensors. Biosensors have found promising applications in various elds such as biotechnology, food and agriculture product processing, health care, medicine and pollution monitoring.

Fig. 2. Schematic of a simple biosensor.

Biosensor instruments are specic, rapid, simple to operate, can be easily fabricated with minimal sample pretreatment involved. The apparently alien marriage of two contrasting disciplines combines the specicity and sensitivity of biological systems with the computing power of microprocessor. A general schematic of a biosensor is shown in Fig. 2. Depending on the level of integration, biosensors can be divided into three generations. In the rst generation, the biocatalyst is either bound to or entrapped in a membrane, which in turn is xed on the surface of the transducer. The second generation biosensors involve the adsorption of covalent xation of the biologically active component to the transducer surface and permits the elimination of semi-permeable membrane. In the case of third generation biosensors, it is the direct binding of the biocatalyst to an electronic device that transduces and amplies the signal. Conducting polymer-based biosensors come in this category.

2.1. Transducer
The biochemical transducer or biocomponent imparts to the biosensor, selectivity or specicity. A transducer converts the biochemical signal to an electronic signal. The transducer of an electrical device responds in a way that a signal can be electronically amplied, stored and displayed. Suitable transducing system can be adapted in a sensor assembly depending on the nature of the biochemical interaction with the species of interest. The physical transducers vary from electrochemical, spectroscopic, thermal, piezoelectric and surface acoustic wave technology (Svorc et al., 1997; Urban et al., 1990). The most common electrochemical transducers being utilized are amperometric and potentiometric. An amperometric biosensor measures the current produced during the oxidation or reduction of a product or reactant usually at a constant applied potential. Such sensors have fast response times and good sensitivity. However, the excellent specicity of the biological component can be compromised by the partial selectivity of the electrode. This lack of specicity requires sample preparation, separation or some compensation for interfacing signals. Potentiometric biosensors relate electrical potentials to the concentration of analyte by using an ion-selective electrode or a gassensing electrode as the physical transducer (Lewenstam et al., 1994; Domansky et al., 1998). These are

2. Biosensors The unprecedented interest in the development and exploitation of analytical devices for detection, quantication and monitoring of specic chemical species has led to the emergence of biosensors. The estimation of metabolites such as glucose, urea, cholesterol and lactate in whole blood is of central importance in clinical diagnostics. Biosensors represent a new trend emerging in the diagnostic technology. A biosensor is a device having a biological sensing element either intimately connected to or integrated within a transducer. The aim is to produce a digital electronic signal, which is proportional to the concentration of a specic chemical or set of chemicals.

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

349

selective, have large dynamic ranges and are nondestructive.

3. Importance of conducting polymers to biosensors Conducting polymers have attracted much interest as a suitable matrix of enzymes Conducting polymers are used to enhance speed, sensitivity and versatility of biosensors in diagnostics to measure vital analytes. Conducting polymers are thus nding ever increasing use in diagnostic medical reagents (Heller, 1990). In this context several reviews on the use of conducting polymers in fabrication of efcient biosensors have been published (Cosnier, 1999; Lewis et al., 1999; Kranz et al., 1998; Santhanam, 1998; Trojanowicz et al., 1997; Adeloju and Wallace, 1996; Trojanowicz and vel Krawczyk, 1995; Alva and Phadke, 1994; Contractor et al., 1994; Bartlett and Birkin, 1993; Schuhmann et al., 1993; Deshpande and Amalnerkar, 1993; Boyle et al., 1989; Bidan et al., 1988). Conducting polymers have attracted much interest as a suitable matrix for the entrapment of enzymes (Adeloju and Wallace, 1996; Bartlett and Cooper, 1993; Sung and Bae, 2000). The techniques of incorporating enzymes into electro-depositable conducting polymeric lms permit the localization of biologically active molecules on electrodes of any size or geometry and is particularly appropriate for the fabrication of multi-analyte micro-amperometric biosensors (Unwin and Bard, 1992). Electrically conducting polymers have considerable exibility in the available chemical structure, which can be modied as required. By chemical modeling and synthesis, it is possible to modulate the required electronic and mechanical properties. Moreover, the polymer itself can be modied to bind protein molecules (Lu et al., 1995; Mulchandani and Wang, 1996; Situmorang et al., 2000). Another advantage offered by conducting polymers is that the electrochemical synthesis allows the direct deposition of the polymer on the

2.2. Biocomponents
Biocomponents, which function as biochemical transducers can be enzymes, tissues, bacteria, yeast, antibodies/antigens, liposomes, organelles (Sadik and Wallace, 1993; Foulds and Lowe, 1988; Schmid, 1987). Within a biosensor the recognition biomolecule incorporated possesses an exquisite level of selectivity but is vulnerable to extreme conditions such as temperature, pH and ionic strength (Karyakin et al., 1999). Most of the biological molecules such as enzymes, receptors, antibodies, cells etc. have very short lifetime in solution phase. Thus they have to be xed in a suitable matrix. The immobilization of the biological component against the environmental conditions results in decreased enzyme activity (Schuhmann et al., 1992; Evtugyn et al., 1998). The activity of immobilized molecules depends upon surface area, porosity, hydrophillic character of immobilizing matrix, reaction conditions and the methodology chosen for immobilization. A number of techniques such as physical adsorption, cross-linking, gel entrapment, covalent coupling etc. have been used to immobilize biological molecules in carrier materials as shown in Table 1. Various matrices have been used for the immobilization of enzymes such as membranes, gels, carbon, graphite, silica, polymeric lms etc. (Trojanowicz and vel Krawczyk, 1995; Pandey, 1992; Ikeda et al., 1985; Gun and Lev, 1996). There is thus a great need to design the electrodes that are compatible with the biological component that can lead to rapid electron transfer at the electrode surface. Conducting polymers are attractive as possible materials for such applications.
Table 1 Conventional immobilization procedures Method Physical adsorption Advantages No modication of biocatalyst. Matrix can be regenerated. Low cost Disadvantages

Example Adsorption of glucose oxidase on conducting polymers for glucose detection Entrapment of urease and glutamate dehydrogenase in polypyrrole/polyvinyl sulphonate lms for urea detection Glutaraldehyde mediated linking of lactate dehydrogenase for lactate estimation GOD binding via poly(o -amino benzoic acid) for glucose detection

Reference Ramanathan 1995; Ramanathan et al., 1996a,b

Entrapment

Binding forces are susceptible to change in pH, temperature and ionic strength Only physical connement of High diffusion barrier biocatalyst near transducer. Low cost

Gambhir et al., 2001b

Cross-linking

Loss of biocatalyst is minimum. Moderate cost

Harsh treatment of biocatalyst by toxic chemicals Harsh treatment by toxic chemicals. Matrix not regenerable

Chaubey et al., 2000b

Covalent bonding Low diffusional resistance Stable under adverse conditions

Ramanathan et al., 2000

350

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

electrode surface, while simultaneously trapping the protein molecules (Bartlett and Whitaker, 1988; Gambhir et al., 2001a). It is thus possible to control the spatial distribution of the immobilized enzymes, the lm thickness and modulate the enzyme activity by changing the state of the polymer. The development of any kind of technology in this eld heavily depends on the understanding of the interaction at the molecular level, between the biologically active protein, either as a simple composite or through chemical grafting. For the proper relay of the electrons from the surface of the electrode to the enzyme active site, the concept of electrical wiring has been reported (Heller, 1990; Gregg and Heller, 1990). Conducting polymers are likely to provide a 3-dimensional electrically conducting structure for this purpose. Conducting polymers are also known to be compatible with biological molecules in neutral aqueous solutions. They can be reversibly doped and undoped electrochemically accompanied by signicant changes in conductivity and spectroscopic properties of the lms that can be used as a signal for the biochemical reaction (Wrighton, 1986; Kittlesen et al., 1984). The electronic conductivity of conducting polymers changes over several orders of magnitude in response to changes in pH and redox potential of their environment (Paul et al., 1985). Conducting polymers have the ability to efciently transfer electric charge produced by the biochemical reaction to electronic circuit (De Taxis du Poet et al., 1990). Moreover conducting polymers can be deposited over dened areas of electrodes. This unique property of conducting polymers along with the possibility to entrap enzymes during electrochemical polymerization has been exploited for the fabrication of amperometric biosensors (Umana and Waller, 1986; Foulds and Lowe, 1986; Iwakura et al., 1988; Bartlett and Whitaker, 1988; Pandey, 1988). Besides this, conducting polymers exhibit exchange and size exclusion properties due to which they are highly sensitive and specic towards substrate (Price et al., 1994; Mirmohseni et al., 1993; Teasdale and Wallace, 1993; Trojanowicz and vel Krawczyk, 1995). Numerous published papers reveal that the electrodeposition of conducting polymers serve as good matrices for the immobilization of enzymes (Trojanowicz and vel Krawczyk, 1995; Kranz et al., 1998; Cooper and Hall, 1992). They provide good detectability and fast response as the redox reaction of the substrate, catalyzed by an appropriate enzyme, takes place in the bulk of the polymer layer. Fig. 3 shows the pathway suggested for electron transfer in the conducting polymer based amperometric biosensors.

Fig. 3. Pathway suggested for electron transfer in conducting polymer based biosensors.

4. Immobilization of enzymes on conducting polymers In recent years, numerous papers have been published indicating organic conducting polymers as a convenient component, forming an appropriate environment for the immobilization of enzyme at the electrode surface and its interaction with metallic or carbon electrode surfaces. Stable immobilization of macromolecular biomolecules on conducting microsurfaces with complete retention of their biological recognition properties is a crucial problem for the commercial development of miniaturized biosensor. Most of the conventional procedure for biomolecule immobilization such as cross-linking, covalent binding and entrapment in gels or membrane suffer from a low reproducibility and a poor spatially controlled deposition. The other electrochemically prepared conducting polymers used for the biomolecule immobilization are polyacetylene, polythiophene, polyindole, polyaniline etc. (Bartlett and Whitaker, 1987, 1988; Pandey, 1988; Cooper and Hall, 1992). Also a few biosensors based on insulating electropolymerized lms polyphenol, poly(o -phenylenediamine), polydicholorophenolenedophenol and overoxidised polypyrrole have also been elaborated (Malitesta et al., 1990; Bartlett and Caruana, 1992; Groom and Luong, 1993). Ramanathan (1995a) have immobilized glucose oxidase (GOX) on different conducting matrices such as polypyrrole (PPY), polyaniline (PANI), polyaminobenzoic acid (PAB) and have studied the response characteristics, lifetime in detail. Ramanathan et al. (1996b) have also studied the changes in the dielectric constant in PPY immobilized GOD. Among the conducting polymers PPY and its derivatives play a leading role due to their versatile applicability and the wide variety of molecular (redox) species covalently linked to a pyrrole group (Bidan, 1992). Many recent reports have been published on the immobilization of different enzymes into the polyaniline lm (Ramanathan et al., 1995b; Yang and Shaolin, 1997; Chaubey et al., 2000a). Fig. 4 shows a conducting polymer based screen printed electrode for fabrication of amperometric biosensor. A number of enzymes or biomolecules have been immobilized physically on a number of conducting

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

351

polymers by (Ramanathan, 1995a; Ramanathan et al., 1996b; Verghese et al., 1998; Gambhir et al., 2001b; Chaubey et al., 2000a,b). This is the simplest method of enzyme immobilization. The binding forces involved are hydrogen bonds, multiple salt linkages, Vander waals forces etc. Many enzymes may be incorporated into conducting polymer lms during electrochemical deposition on appropriate electrodes. The entrapment of enzymes in conducting polymers provides a facile means for ensuring proximity between the active site of the enzyme and the conducting surface of the electrode with considerable potential for biosensor construction. This method provides a facile and controllable method for the deposition of biologically active molecules to dened areas on the electrodes. For electrochemical entrapment, a solution of the electro-polymerizable monomer and the aqueous buffer containing the enzyme is used for the deposition process. The polymer lm can be deposited electrochemically using potentiostatic or galvanostatic mode. It has been established that glucose oxidase can be successfully entrapped in polypyrrole lms (Umana and Waller, 1986; Foulds and Lowe, 1986) and the morphology of the lm alters to a more brillar form. During electropolymerization mediators can also be immobilized simultaneously with enzyme (Iwakura et al., 1988; Sun and Tachikawa, 1992; Bartlett and Whitaker, 1987) but they can also be conjugated with the monomer before polymerization. This well controlled procedure of enzyme immobilization by electropolymerization is of great signicance in fabrication of microsensors in the preparation of multilayer devices (Sun and Tachikawa, 1992; Strike and de Rooij, 1993) and multienzyme biosensor (Kajiya et al., 1991; Yon-Hin et al., 1990; Yon-Hin and Lowe, 1992; Tatsuma et al., 1993a,b; Garguilo et al., 1993). Many theoretical models have also been coupled with the electrochemical entrapment of enzyme for the evaluation of the role of the thickness of polymeric layers, enzyme location, enzyme loading etc. on the biosensor functioning (Bartlett and Whitaker, 1987; Gros and Bergel, 1995; Gooding et al., 1998).

5. Types of biosensors

5.1. Amperometric biosensors


Amperometric biosensors measure the current produced during the oxidation or reduction of a product or reactant usually at a constant applied potential. The most important factor affecting the functioning of amperometric biosensors is the electron transfer between catalytic molecule, usually oxidase or dehydrogenase, and the electrode surface most often involving a mediation or conducting polymer. Although the role of the electrodeposited conducting polymer lms is not fully understood and explained, various polymers used for enzyme immobilization may affect signicantly the response of biosensors sensitive to given species. Much work has been done in recent years on the application of electrochemically grown conducting polymer layers in amperometric biosensors. Redox enzymes have been either entrapped within conducting polymer layers or covalently bound to functional groups (Bartlett and Whitaker, 1987; Umana and Waller, 1986; Foulds and Lowe, 1986; Fortier and Belanger, 1991; Hammerle et al., 1992). Ramanathan et al. (1995c) have reported the immobilization of GOD via manipulation of pore size in PPY to improve loading parameters of enzyme leading to ve fold increase in its current response. Verghese et al. (1998) have conducted similar experiments in polyaniline/GOD lms. Table 2 shows the different biosensors developed for various substrates.

5.2. Potentiometric biosensors


Potentiometry is a rarely used detection method in biosensor with enzymes immobilized in an electrodeposited polymer layer, although certain advantages over amperometric detection for a PPY based electrode with immobilized GOX have been demonstrated. For biosensors having very slow response, the rate of potential change rather than steady state potential values, should be considered as the analytical signal for quantication of the substrate (Trojanowicz and vel Krawczyk, 1995). Potentiometric biosensors with conducting polymers can be produced using pH sensitivity of polymers (Ratcliffe, 1990). Polypyrrole sensitivity to NH3 was used to produce such biosensors (Pandey and Mishra, 1988; Trojanowicz et al., 1993). Conducting PPY molecular interfaces have also been implemented to modulate biological function of enzymes and living cells at the electrode surface by adjustment of electrode potentials. Often additionally obtained discrimination of electrochemical interfaces by using conducting polymers as matrices for enzymes allows the use of such biosensors for analysis of natural samples, e.g. ow injection determination of lactate in whole blood. Besides this, the

Fig. 4. A conducting polymer-screen printed electrode.

352

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

Table 2 Biosensors based on conducting polymers Substrates or species to be determined Glucose Enzyme Polymer Detection

Glucose oxidase

Polypyrrole

D-Alanine Atrazine Cholesterol Choline Glutamate Fructose Hemoglobin L-Lactate

Lipids Phenols Urea

Poly(N -methylpyrrole) Polyaniline Polyindole Glucose dehydrogenase Polypyrrole Polypyrrole D-amino acid oxidase Tyrosinase Polypyrrole Cholesterol oxidase and Cholesterol esterase Polypyrrole Choline oxidase Substituted Polypyrrole Glutamate dehydrogenase Polypyrrole Fructose dehydrogenase Polypyrrole Pepsin Polyaniline Lactate oxidase Polyphenyline di-amine Lactate dehydrogenase Polyaniline Polypyrrolepolyvinyl sulphonate Lipase Polyaniline Tyrosinase Substituted polypyrrole Urease Polypyrrole

Amperometry Potentiometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Conductometry Amperometry Amperometry Amperometry Conductometry Amperometry Amperometry Potentiometry Conductometry Capacitance Measurement Admittance measurement Amperometry Conductometry

Uric acid Triglycerides

Uricase Lipase

Polyaniline Polyaniline

advantage of well-dened formation of the polymer layer, this methodology yields some difculties, associated with a signicant chemical and electrochemical activity of the polymer matrix due to the similarity of these materials towards ion-exchange processes and redox equilibria. The interaction of NH3 with PPY was utilized for a design of a potentiometric biosensor of urea with urease immobilized in an electrodeposited PPY layer. Potentiometric sensitivity was only 17 mV/ decade with strong interference in the presence of potassium and sodium. The creatinine electrode was made by co-immobilization of creatininase, creatinase and sarcosine oxidase in a PPY matrix (Yamato et al., 1995). Difculties concerning the immobilization of enzymes in the electrodeposited polymer layer or the mechanism of the entrapment and dynamics effects on biosensors have been widely discussed and till now no conclusive reports on the comparison of different polymer matrices for immobilization of the same enzyme have been related.

5.3. Conductometric biosensors


Conductometric biosensors measure the changes in the conductance of the biological component arising between a pair of metal electrodes. Contractor et al. (1994) have constructed biosensors for estimation of glucose, urea neutral lipid/lipase and hemoglobin/ pepsin by monitoring the change in the electronic conductivity arising as a change in redox potential and/or pH of the microenvironment in the polymer matrix. Ramanathan et al. (1995b) have studied the application of polyaniline LB lms as a glucose biosensor. They have also investigated the dielectric spectroscopic measurements of PPY/glucose biosensor (Ramanathan et al., 1996b). Conductivity biosensors based on conducting polymers were developed for penicillin (Nishizawa et al., 1992) and also for glucose, urea, lipids and hemoglobin (Contractor et al., 1994). Microelectronic devices have been fabricated with a sensitivity to urea concentration in millimolar range in measurements of conductance, capacitance and admittance using urease

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

353

immobilized in a PPY layer on gold lm electrodes. A ow injection system was developed with covalently immobilized penicillinase on polypyrrole lms and changes in conductivity due to pH change was measured.

5.4. Optical biosensors


Optical biosensors are based on the measurement of light absorbed or emitted as consequence of a biochemical reaction. In such type of biosensors, light waves are guided by means of optical bers to suitable detectors. These type of biosensors have been used for the detection of pH, O2 and CO2 etc. Gerard et al. (1999) have measured the pyruvate concentration optically by immobilizing LDH onto polyaniline electrode. These electrodes were stable for 15 days and had a response time of 90 s. Chaubey et al. (1998) have immobilized LDH on PPY PVS electrodes on ITO plates and detected the lactate concentration by optical measurements. Singhal et al. (2000) immobilized glucose oxidase on polyhexyl thiophene by LB technique and fabricated optical glucose sensor using UV visible spectroscopy.

Health care: In medical diagnosis (glucose, fructose, lactate, ethanol, cholesterol, urea etc.) Immunosensors: Can be used in medical diagnostics and environmental sensors DNA sensors: In the detection of various genetic disorders. Environmental monitoring: For control of pollution and detection of hazardous chemicals in biosensors (polyphenols, sultes, peroxides, formaldehyde etc.) Food analysis: For detection of glucose, fructose, ethanol, sucrose, lactate, malate, galactose, citrate, lactose, urea, starch etc. in food industries.

6.1. Biosensors for health care


Selective determination of various blood analytes viz. glucose, urea, lactate, uric acid, cholesterol etc. is of utmost importance for the screening and treatment of a number of diseases.

5.5. Calorimetric biosensors


The basic principle of such biosensors is that all biochemical reactions involve a change in enthalpy Such a change in enthalpy is detected by calorimetric biosensors. Mosbach and Danielsson (1981) developed an enzyme thermister where the temperature change in an enzyme reaction was measured by a thermister device. A number of substrates, enzymes and antigens have been estimated using thermister biosensors. Xie et al. (1993) investigated a ferrocene mediated thermal biosensor that combines electrochemistry and calorimetry. For detection, thermal signal generated by the redox reaction was measured as opposed to measuring the electrochemical signal.

5.6. Peizoelectric biosensors


These biosensors operate on the principle of generation of electric dipoles on subjecting an anisotropic natural crystal to mechanical stress. Due to the adsorption of an analyte, the mass of the crystal is increased resulting in altered frequency of oscillation. Such type of biosensors have been utilized for the measurement of ammonia, hydrogen, methane, carbon monoxide, nitrous oxide and other organophosphorous compounds.

6.1.1. Glucose biosensors A number of reports on immobilization of glucose oxidase in conducting polymers for glucose estimation are available in literature (Fortier and Belanger, 1991; Foulds and Lowe, 1986; Ramanathan, 1995a; Ramanathan et al., 1995b,c, 1996b; Umana and Waller, 1986). It has been investigated that the polymers containing para - and ortho -quinone groups as electrontransfer relay systems for oxido-reductases can effectively catalyze the electro-oxidation of glucose. Ramanathan et al. (1995c) have immobilized GOD after manipulation of the pore size in polypyrrole to improve the loading of the enzyme. An exchange of para -toluene sulphonate and ferricyanide ions with a smaller ion like chloride in solution has been applied for making polypyrrole more porous enabling enhanced loading of GOD. An attempt has been made to covalently couple glucose oxidase (GOX) to poly(o -amino benzoic acid) (PAB), a carboxy group functionalized polyaniline (Ramanathan et al., 2000). Mediated (with ferrocene carboxylic acid and tetrathiafulvalene) and unmediated systems have been utilized for glucose concentrations. 6.1.2. Urea biosensors Most of the urea biosensors available in literature are based on detection of NH+ 4 or HCO3 sensitive electrodes (Koncki et al., 2000; Hirose et al., 1983; Cho and Huang, 1998; Pandey and Mishra, 1988; Gambhir et al., 2001a). Osaka et al. (1999) constructed a highly sensitive and rapid ow injection system for urea analysis with a composite lm of electropolymerized inactive polypyrrole and a polyion complex. Gambhir et al. (2001a) have recently co-immobilized urease and glutamate dehydrogenase on electrochemically prepared polypyrrole/polyvinyl sulphonate for the fabrication of urea biosensor.

6. Applications of conducting polymers Conducting polymers have been used in the fabrication of biosensors in various elds such as:

354

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

6.1.3. Lactate biosensors Lactate biosensors are based on enzymes like lactate oxidase and lactate dehydrogenase (Hart and Turner, 1996; Yang et al., 1999; Chaubey et al., 2000a,b Gerard et al., 1999). Regeneration of lactate by substrate recycling has been utilized by co-immobilization of lactate oxidase and lactate dehydrogenase by Chaubey et al. (2000a). Recently lactate biosensor based on polypyrrole/polyvinyl sulphonate composite lms has been reported by Chaubey et al. (2000b). 6.1.4. Cholesterol biosensors Being a very important parameter in clinical diagnostics, cholesterol biosensors have attracted much attention. Amperometric cholesterol biosensors are based on the enzyme cholesterol oxidase. Kajiya et al. (1991) immobilized cholesterol oxidase and ferrocene carboxylate in polypyrrole electrochemically to describe the sensitivity of the resulting lms. Kumar et al. (2001) have utilized dodecylbenzene sulphonate doped polypyrrole lms for immobilization of cholesterol oxidase by physical adsorption. These lms were utilized for cholesterol estimation via ferricyanide mediation. 6.2. Immunosensors
An effective combination of immunochemistry and electrochemistry in an analytical device could provide the basis of direct electrical detection of a wide range of analytes with great sensitivity and specicity. In this context, a number of immunosensors based on conducting polymers have been developed. A unique strategy for reversible immunosensors using conducting polymers based on pulsed electrochemical detection has been developed by Sargents group. The strategy has been utilized for the detection of organochlorine pesticides including PCBs, atrazines and chlorinated phenols. Porter (2000) investigated electroplated conducting polymers as antibody receptor in immunosensor. They have shown that antibodies against conducting polymers (carbazole as a hapten) may react to modulate the polymer electrochemistry. The reaction of the antiserum was found to inuence the polymer electrochemistry by an amperometric response and therefore can be utilized as a sensor for a direct immunoacid. AAI-Abtech product, lead by Anthony Guiseppi-Elie is sensitive to H2O2 and therefore may be used for enzyme, immunosensor, receptor and DNA probes and direct redox biosensor. The company is marketing a polymer based system to monitor sulphate-reducing desulphovibrio bacteria, implicated in corrosion and biofouling. In this system, which utilizes a disposable biosensor cartridge, antibodies specic to the bacterium are incorporated into an electroconductive polymer, which is deposited onto an array of micro-electrodes on a silicon chip substrate. When the electrodes are incubated with water samples, any

desulphovibrio bacteria present, may adhere to the antibodies and result in a measurable change in conductivity. A company, Ohmicron Corporation, in Pennsylvania has also developed an immunosensor which is a portable instrument for atrazine estimation. The instrument permits a total test time of less than 15 min and can detect the pesticide at the level of parts per billion.

6.3. DNA Biosensors


DNA biosensors have an enormous application in clinical diagnostics of inherited diseases, rapid detection of pathogenic infections, and screening of cDNA colonies required in molecular biology. Present methods of genetic analysis require pre- and post-treatments to modify DNAs with probes as proteins. Recently, DNA integrated electroactive polymers (thin lms or LB monolayers) have provided intelligent materials which possess superior intelligent material properties of self-assembly, self-multiplication, self-repair, self- degradation, redundancy, and self-diagnosis (Minehan et al., 1994; Garnier et al., 1999). A few reports of interaction of DNA with conducting polymers are available (Saoudi et al., 1997; Chehimi et al., 1996; Bruno et al., 1994). Livache et al. (1995) reported one-step electrodeposition of PPY lms functionalized by a covalently linked oligonucleotide. Another possibility to dope DNA probes within electropolymersised polypyrrole lms and monitoring of the current changes incurred by the hybridization (Wang et al., 1999). Gambhir et al. (2001b) have reported the results of the studies relating to the characteristics of physically adsorbed DNA (Calf thymus) on conducting PPY/PVS lms. Immobilization of DNA on a conducting polymer matrix facilitates the detection of a signal (amperometric or potentiometric) generated as a result of interaction of proteins or drugs with DNA.

6.4. Biosensors for en6ironmental monitoring


Biosensors show a potential to complement both laboratory based and eld analytical methods for environmental monitoring (Marco and Barcelo, 1996; Gabor, 2001; Lopez-Avila and Hill, 1997; Rogers, 1998). Although a wide range of biosensors have been reported for potential environmental applications, very few have progressed into commercial markets. Biosensors with potential for environmental applications have been the subject of a number of recent reviews. The conducting polymer based gas sensor for environmental monitoring is based on the fact that upon exposure to vapor, the polymers show rapid conductivity changes, which are generally reversible at room temperature. The change in conductivity probably results from a reversible reduction of the polymer on exposure to the organic vapor as well as change in the moisture content of the lm. (Harsanyi et al., 1999; Bartlett and LingChung, 1989a,b; Krutovertsev et al., 1992).

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359

355

A thin lm of conducting polymer based on microelectronic ammonia gas sensor device has been developed which is the rst application of conducting polymer in gas sensor devices for commercial applications. The device is a resistor element which shows large variations when ammonia is present in the environment in very low concentration and enables the necessary action or alarm activation (www.ett.bme.hu). Use of conducting polymers in impedance type sensors provide a possibility to build up low cost, highly sensitive and selective room temperature gas sensor (Bartlett et al., 1989c; Charlesworth et al., 1993). They described a number of sensors for organic sensors for organic vapours based on PPY, poly-N -methyl pyrrole, poly(5-carboxy indole) and polyaniline. These materials are however advantageous due to lack of specicity i.e. they show responses to a wide range of different gases and vapors. But their selectivity is found to be better than that of the inorganic materials based gas sensitive resistors. AAI-Abtech, a small biotechnology company in Pennsylvania, has developed a novel sensor based on conducting PPY on an array of micoelectrodes. It has been shown that a simple system using Mo(V)-catalyzed conversion of iodide to iodine can detect hydrogen peroxide. The polymer incorporates molybdenum together with iodide, hydrogen peroxide converts the iodide to iodine, which oxidizes the PPY, resulting in a measurable change in conductivity. Thomas Fare and coworkers have developed an immunosensor based on conducting polymer for screening of pesticide atrazine. Adeloju and Yuan (1999) have described a new approach for the ow amperometric biosensing of formate with single, double and triple layered arrangements of the ferrocyanide mediated PPY based biosensor. The detection of formate concentration has been found to be important in atmosphere, natural waters and sediments. This layered structure comprised of formate dehydrogenase, NAD and mediator and has been demonstrated for the ow amperometric biosensing of formate in aqueous media, with the detection limit of 2.5 mM. A cost effective polyaniline base ammonia gas sensor has been described by Lepsenyi et al. (1999). They deposited sensing lm of polyaniline, electrochemically doped by cyclic voltammetry on thin and thick lm electrodes where the isolation gap is formed by laser engraving. In this work, they investigated the sensitivity, selectivity, temperature, dependency and long term behavior of the sensor.

tion of biosensor technique may be an alternative to the present inefcient and expensive methods. Extensive work on development of analytical devices for estimation of glucose, sucrose, lactate, citric acid, ascorbic acid etc. in the food products has been pursued during the last few years (Pal et al., 1994; Stephens et al., 1998; Stredansky et al., 1999). A few reports on the conducting polymers based biosensors for application to food industries are available (Umana and Waller, 1986; Pal et al., 1994; Chaubey et al., 2000a). Ghosh (Hazra) et al. (1998) have developed a biosensor system for qualitative measurement of sh freshness. The biosensor is based on amperometric method using three ferrocene carboxylic acid mediator incorporated conducting polypyrrole enzyme electrodes with immobilized xanthine oxidase, nucleoside phosphorylase and nucleotidase enzymes for quantitative measurement of hypoxanthine, inosine and inosine monophosphate metabolites in sh tissue.

7. Conclusion Biochemical sensors have been shown to provide complementary and additional information to that contributed by the well-established bioanalytical techniques. Particular advantages of biochemical sensors concern the following: the possibility of miniaturizing the setup, in principle down to the molecular scale, the use of well-established microsystem technologies during manufacture, integration of signal preprocessing steps on a chip, and the building of arrays for more complex pattern recognition analysis. By combining the use of electronically conducting polymers with immobilized enzymes and by making use of the particular properties of conducting polymers, it is possible to develop novel enzyme-based bioelectronic devices.

Acknowledgements Authors are thankful to Dr. Krishon Lal, Director of NPL for his interest in this eld. Asha Chaubey is grateful to CSIR, India for the award of senior research fellowship.

References
Adeloju, S.B., Yuan, Y.J., 1999. Polypyrrole-based ow amperometric biosensor for formate, Electrochem. News, 4, online (http:// www.uws.edu.au). Adeloju, S.B., Wallace, G.G, 1996. Conducting polymers and the bioanalytical sciences. Analyst 121, 699 703. Alva, S., Phadke, R.S., 1994. Conducting polymers in the fabrication of efcient biosensors. Ind. J. Chem. 33A, 561 564.

6.5. Biosensors for food industry


Food industries need rapid and affordable methods to determine compounds in the products for quality control in food processing. In this context, the applica-

356

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359 Contractor, A.Q., Sureshkumar, T.N., Narayanan, R, Sukeerthi, S., Lal, R., Srinivasa, R.S., 1994. Conducting polymars based biosensors. Electrochim. Acta 39, 1321 1324. Cooper, J.C., Hall, E.A.H., 1992. Electrochemical response of an enzyme-loaded polyaniline lm. Biosens. Bioelectron. 7, 473 485. Cosnier, S., 1999. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized lms. A review. Biosens. Bioelectron 14, 443 456. De Taxis du Poet, P., Miyamoto, S., Murakami, T., Kimura, J., Karube, I., 1990. Direct electron transfer with glucose oxidase immobilized in an electropolymerized poly-N-methylpyrrole lm on a gold microelectrode. Anal. Chim. Acta 235, 255 264. Deshpande, M.V., Amalnerkar, D.P., 1993. Biosensors prepared from electrochemically synthesized conducting polymers. Prog. Polym. Sci 18, 623 649. Domansky, K., Baldwin, D.L., Grate, J.W., Hall, T.B., Li, J., Josowicz, M., Janata, J., 1998. Development and calibration of eld effect transistor-based sensor array for measurement of hydrogen and ammonia gas mixtures in humid air. Anal. Chem. 70, 473 481. Duke, C.B., Schein, L.B., 1980. Organic solids: is energy-based theory enough? Phys. Today 33, 42 48. Evtugyn, G.A, Budnikov, H.C., Nikolskaya, E.B., 1998. Sensitivity and selectivity of electrochemical enzyme sensors for inhibitor determination. Talanta 46, 465 484. Fortier, G., Belanger, D., 1991. Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole lm. Biotechnol. Bioengg. 37, 854 858. Foulds, N.C., Lowe, C.R., 1986. Enzyme entrapment in electrically conducting polymers. J. Chem. Soc. Faraday Trans. I 82, 1259 1264. Foulds, N.C., Lowe, C.R., 1988. Immobilization of glucose oxidase in ferrocene modied pyrrole polymers. Anal. Chem 60, 2473 2478. Friend, R.H. (Ed.) 1993. Conductive Polymer II, Rapra Review Report, 6 (3), 23. Gabor, H., (2001). Highly sensitive and selective ammonia sensor for monitoring and workplace safety applications, (www.ett.bme.hu). Gambhir, A. Gerard, M., Jain, S.K., Malhotra, B.D., 2001a. Characterization of DNA immobilized on electrochemically prepared conducting polypyrrole-polyvinyl sulphonate lms. Appl. Biochem. Biotech. (in press). Gambhir, A., Gerard, M., Mulchandani, A., Malhotra, B.D., 2001b. Co-immobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulphonate lms, Appl. Biochem. Biotechnol. (in press). Garguilo, M.G., Huynh, N., Proctor, A., Michael, A.C., 1993. Amperometric sensors for peroxide, choline and acetylcholine based on electron transfer between horseradish peroxidase and a redox polymer. Anal. Chem. 65, 523 528. Garnier, F., Korri-Youssou, H., Srivastava, P., Mandrand, B., Delair, T., 1999. Toward intelligent polymers: DNA sensors based on oligonucleotide-functionalized polypyrroles. Synthetic Metals 100, 89 94. Gazard, M., 1986. In: Skotheim, T.A. (Ed.), Handbook of Conducting Polymers, vol. 1. Marcel Dekker, USA, p. 673. Gerard, M., Ramanathan, K., Chaubey, A., Malhotra, B.D., 1999. Immobilization of lactate dehydrogenase on electrochemically prepared polyaniline lms. Electroanalysis 12, 450 452. Ghosh (Hazra), S., Sarker, D., Misra, T.N., 1998. Development of an amperometric enzyme electrode biosensor for sh freshness detection. Sens. and Actat. B 53, 58 62. Gooding, J.J., Hall, E.A.H, Hibbert, D.B., 1998. Thick lm to monolayer recognition layers in enzyme elcetrodes. Electroanalysis 10, 1130 1136. Greene, R.L., Street, G.B., Suter, L.J., 1975. Superconductivity in polysulfur nitride (SN)x. Phys. Rev. Lett. 34, 577 579.

Audebert, P., Bidan, G., 1985. Polyhalopyrroles: Electrochemical synthesis and some characteristics. J. Electroanal. Chem. 190, 129 139. Bartlett, P.N., Birkin, P.R., 1993. The application of conducting polymers in biosensors. Synth. Met. 61, 15 21. Bartlett, P.N., Whitaker, R.G., 1988. Strategies for the development of amperometric enzyme electrodes. Biosensors 3, 359 379. Bartlett, P.N., Cooper, J.M., 1993. A review of the immobilization of enzymes in electropolymerized lms. J. Electroanal. Chem. 362, 1 12. Bartlett, P.N., Ling-Chung, S.K., 1989a. Conducting polymer gas sensors part II: response of polypyrrole to methanol vapour. Sens. Actat. B 19, 141 150. Bartlett, P.N., Ling-Chung, S.K., 1989b. Conducting polymer gas sensors Part II: results for 4 different polymers and 5 different vapours. Sens. Actuat B. 20, 287 292. Bartlett, P.N., Archer, PBM, Ling-Chung, S.K., 1989c. Conducting polymer gas sensors part I: fabrication and characterization. Sens. Actuat. 19, 125 140. Bartlett, P.N., Caruana, D.J., 1992. Electrochemical immobilizaion of enzymes. Part V. Microelectrodes for the detection of glucose based on glucose oxidase immobilized in a poly(phenol) lm. Analyst 117, 1287 1292. Bartlett, P.N., Whitaker, R.G., 1987. Electrochemical immobilization of enzymes. Part II. Glucose oxidase immobilized in poly-Nmethyl pyrrole. J. Electroanal. Chem. 224, 37 48. Bidan, G., 1992. Electroconducting conjugated polymers. New sensitive matrices to build up chemical or electrochemical sensors. A Review. Sens. Actuat. B 6, 45 56. Bidan, G., Ehui, B., Lapkowski, M., 1988. Conductive polymers with immobilized dopants: ionomer composites and auto-doped polymers a review and recent advances. J. Apys. D. Appl. Phys. 21, 1043 1054. Bloor, D., Movaghar, B., 1983. Conducting polymers. IEEE Proceedings 130, 225 232. Boyle, A., Genies, E.M., Lapkowski, M., 1989. Application of electronic conducting polymers as sensors: polyaniline in the solid state for detection of solvent vapours and polypyrrole for detection of biological ions in solutions. Synth. Metals 28, C769 C774. Bruno, F.F., Marx, K.A., Tripathi, S.K., Akkara, J.A., Samuelson, L.A., Kaplan, D.L., 1994. Enzyme mediated polymerization of phenol and aniline derivatives on a langmuir trough to form ordered 2-D polymer lms. J. Int. Mat. Sys. Struct. 5, 631 634. Bull, R.A., Fan, F.R., Bard, A.J., 1983. Polymer lms on electrodes. J. Electrochem. Soc. 130, 1636 1638. Charlesworth, J.M., Partridge, A.C., Garrard, N., 1993. Mechanistic studies in the interactions between polypyrrole and organic vapours. J. Phys. Chem 97, 5418 5423. Chaubey, A., Pande, K.K., Singh, V.S., Malhotra, B.D., 2000a. Co-immobilization of lactate oxidase and lactate dehydrogenase on conducting polyaniline lms. Anal. Chim. Acta 407, 97 103. Chaubey, A., Gerard, M., Singhal, R., Singh, V.S., Malhotra, B.D., 2000b. Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrole-polyvinyl sulphonate composite lms for application to lactate biosensors. Electrochim. Acta 46, 723 729. Chaubey, A., Singhal, R., Gerard, M., Malhotra, B.D., 1998. Lactate biosensor based on polypyrrole-polyvinylsulphonate composites. Proceedings of the 6th Asian Conference on Solid State Ionics: Science and Technology, World Scientic Publishing Co. Ltd, Singapore, pp. 479 483. Chehimi, M.M., Abel, M.L., Saoudi, B., Delamar, M., Jammul, N., Watts, J.F., Zhdan, P.A., 1996. Adsorption of macromolecules onto conducting polymers. Polymery 41, 75 84. Cho, W.J., Huang, H.J., 1998. An amperometric urea biosensor based on a polyaniline-peruorosulphonated ionomer composite electrode. Anal. Chem. 70, 3946 3951.

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359 Gregg, B.A., Heller, A., 1990. Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications. Anal. Chem. 62, 258 263. Groom, C.A., Luong, J.H.T., 1993. Improvement of the selectivity of amperometric biosensors by using a permselective electropolymerized lm. Anal. Lett. 26, 1383. Gros, P., Bergel, A., 1995. Improved model of a polypyrrole glucose oxidase modied electrode. J. Electroanal. Chem. 386, 65 73. Guiseppi-Elie, A., Wallace, G.G., Matsue, T., 1997. Chemical and biological sensors based on electrically conducting polymers. In: Skotheim, T., Elsenbaumer, R., Reynolds, J.R. (Eds.), Handbook of Conducting Polymers, 2nd ed. Marcel Dekker, New York, pp. 963 991 Chapter 34. Gun, J., Lev, O, 1996. Sol-gel derived ferrocenyl-modied silicategraphite composite electrode: wiring of glucose oxidase. Anal. Chim. Acta 336, 95 106. Hammerle, M., Schuhmann, W., Schmidt, H.L., 1992. Amperometric polypyrrole enzyme electrodes: effect of permeability and enzyme location. Sens. Actuat. B 6, 106 112. Harsanyi, G., Dobay, R., Visy, Cs., 1999. Conducting polymer based electrochemical sensors on thick lm substrate. Electroanalysis 11, 804 808. Hart, A.L., Turner, A.P.F., 1996. On the use of screen and ink-jet printing to produce amperometric enzyme electrodes for lactate. Biosens. Bioelectron 11, 263 270. Heeger, A.J., 1986. In: Skotheim, T.A. (Ed.), Handbook of Conducting Polymers, vol. II. Marcel Dekker, New York, p. 729 and references therein. Heller, A., 1990. Electrical wiring of redox and enzymes. Acc. Chem. Res. 23, 128 134. Hirose, S., Hayashi, M, Tamura, N., Kamidate 1983. Determination of urea in blood serum with use of immobilized urease and a microwave cavity ammonia monitor, Anal. Chim. Acta, 151, 377 382. Ikeda, T., Hamad, H., Miki, K.and, Senda, M., 1985. Glucose oxidase immobilized benzoquinone-carbon paste electrode as a glucose sensor. Agric. Biol. Chem. 49, 541 543. Ivory, D.M., Miller, G.G., Sowa, J.M., Shacklette, L.W., Chance, R.R., and. Baughman, R.H, 1979. Highly conducting chargetransfer complexes of poly(p-phenylene). J. Chem. Phys. 71, 1506 1507. Iwakura, C., Kajiya, Y., Yoneyama, H., 1988. Simultaneous immobilization of glucose oxidase and a mediator in conducting polymer lms. J. Chem. Soc. Chem. Commun., 1019 1020. Kajiya, Y., Sugai, H., Iwakura, C., Yoneyama, H., 1991. Glucose sensitivity of polypyrrole lms containing immobilized glucose oxidase and hydroquinone sulphonate ions. Anal. Chem. 63, 49 54. Kanazawa, K.K., Diaz, A.F., Geiss, R.H., Gill, W.D., Kwak, J.F., Logan, J.A., Rabolt, J.F., Street, G.B., 1979. Organic Metals: polypyrrole, a stable synthetic metallic polymer. J. Chem. Soc. Chem. Commun., 854 855. Karyakin, A.A., Vuki, M., Lukachova, L.V., Karyakina, E.E., Orlov, A.V., Karpachova, G.P., Wang, J., 1999. Processable polyaniline as an advanced potentiometric pH transducers. Anal. Chem. 71, 2534 2540. Kawai, T., Kuwabara, T., Wang, S., Yoshino, K., 1990. Secondary battery characteristics of poly(3-alkylthiophene). Jap. J. Appl. Phys. 29, 602 605. Kittlesen, G.P., White, H.S., Wrighton, M.S., 1984. Chemical derivatization of microelectrode arrays by oxidation of pyrrole and N-methyl pyrrole. Fabrication of molecule-based electronic devices. J. Am. Chem. Soc. 106, 7389 7396. Koncki, R., Radomska, A., Glab, S., 2000. Potentiometric determination of dialysate urea nitrogen. Talanta 52, 13 17. Kranz, C., Wohlschlager, H., Schmidt, H.L., Schuhmann, W., 1998. Controlled electrochemical preparation of amperometric biosen-

357

sor based on conducting polymer multilayers. Electroanalysis 10, 546 552. Kroschwitz, J.I., 1988. In: Kroschwitz, J.I. (Ed.), Electrical and Electronic Properties of Polymers. Wiley, New York. Krutovertsev, S.A., Sorokin, S.I., Zorin, A.V., Letuchy, Y.A., Antonova, O.Y., 1992. Polymer lm based sensors for ammonia detection. Sens. and Actuat. B 7, 492 494. Kumar, A., Rajesh, Chaubey, A., Grover, S.K., Malhotra, B.D., 2001. Immobilization of cholesterol oxidase and potassium ferricyanide on dodecylbenzene sulfonate ion doped polypyrrole lm. J. Appl. Polym. Sci. 82, 3486 3491 Lepsenyi, I, Reichardt, A., Inzelt, G., Harsanyi, G., 1999. Highly sensitive and selective polymer based gas sensors, Proc. of twelfth European microelectronics and packaging Conf., Harrogate, UK, pp. 301 305. Lewenstam, A., Babacka, J., Ivaska, A., 1994. Mechanism of ionic and redox sensitivity of p-type conducting polymers. Part I. Theory. J. Electroanl. Chem. 368, 23 31. Lewis, T.W., Wallace, G.G., Smyth, M.R., 1999. Electrofunctional polymers: their role in the development of new analytical systems. Analyst 124, 213 219. Livache, T., Roget, A., Dejean, E., Barthet, C., Bidan, G., Teoule, R., 1995. Biosensing effects in functionalized electroconducting conjugated polymer layers: addressable DNA matrix for the detection of gene mutations. Synth. Met. 71, 2143 2146. Lopez-Avila, V., Hill, H.H., 1997. Field analytical chemistry. Anal. Chem. 69, 289R 305R. Lu, W., Zhao, H., Wallace, G.G., 1995. Pulsed electrochemical detection of proteins using conducting polymer sensors. Anal. Chim. Acta 315, 27 32. Malitesta, C., Palmisano, F., Torsi, L., Zambonin, P.G., 1990. Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) lm. Anal. Chem. 62, 2735 2740. Marco, M.P., Barcelo, D., 1996. Environment applications of analytical biosensors. Measurement Sci Technol. 7, 1547 1572. Minehan, S., Marx, K.A., Tripathi, S.K., 1994. Kinetics of DNA binding to electrically conducting polypyrrole lms. Macromolecules 27, 777 783. Minot, M.J., Peristein, J.H., 1971. Mixed valence square planar complexes: a new class of solids with high electrical conductivity in one dimension. Phys. Rev. Lett. 26, 371 373. Mirmohseni, A., Price, W.E., Wallace, G.G., 1993. Electrochemically controlled transport across conducting polymer composites basis of smart membrane materials. Polymer Gels and Networks 1, 61 77. Mosbach, K, Danielsson, B, 1981. Thermal bioanalyzers in ow streams enzyme thermister devices. Anal. Chem. 53, 83A 94A. Mulchandani, A., Wang, C.L., 1996. Bienzyme sensors based on poly(anilinomethylferrocene)-modied electrode. Electroanalysis 8, 414 419. Nishizawa, M., Matsue, T., Uchida, I., 1992. Penicillin sensor based on a microarray electrode coated with pH-responsive polypyrrole. Anal. Chem. 64, 2642 2644. Nou, R., Frank, A.J., Nozik, A.J, 1981. Stabilization of n-type silicon photoelectrodes to surface oxidation in aqueous electrolyte solution and mediation of oxidation reaction by surface attached organic conducting polymers. J. Am. Chem. Soc. 103, 1849 1850. Nou, R., Tench, D., Warren, L.F., 1980. Protection of n-GaAs photoanodes with photoelectrochemically generated polypyrrole lms. J. Electrochem. Soc. 127, 2310 2311. Osaka, T., Komaba, S., Fujino, Y., Matsuda, T., Satoh, I., 1999. High sensitivity ow injection analysis of urea using composite electropolymerized polypyrrole-polyion complex lm. J. Electrochem Soc. 146, 615 619. Pal, P., Nandy, D., Misra, T.N., 1994. Immobilization of alcohol dehydrogenase enzyme in a Langmuir Blodgett lm of stearic

358

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359 Schuhmann, W., Lehn, C, Schmidt, H.L, Grundig, B., 1992. Comparison of native and chemically stabilized enzymes in amperometric enzyme electrodes. Sens. and Actuat. B 7, 393 398. Shirakawa, H., Louis, E.J., MacDiarmid, A.G., Chiang, C.K., Heeger, A.J., 1977. Synthesis of electrically conducting organic polymers: halogen derivatives of polyacetylene, (CH)x. J. Chem. Soc. Chem .Commun., 578. Singhal, R., Chaubey, A., Pande, K.K., Malhotra, B.D., 2000. Immobilization of Glucose oxidase on Poly(3-Hexyl Thiophene) LB lms for application to Glucose biosensor in 4th National Conference on Solid State Ionics, 3 5 March, 2000 at Indian Institute of Technology, Mumbai. Situmorang, M., Hibbert, D.B., Gooding, J.J., 2000. An experimental design study of interferences of clinical relevance of polytyramine immobilized enzyme biosensors. Electroanalysis 12, 111 119. Situmorang, M., Gooding, J.J., Hibbert, D.B., Barnett, D., 1998. Electrodeposited polytyramine as an immobilization matrix for enzyme biosensors. Biosens. Bioelectron. 13, 953 962. Stephens, S.K., Cullen, D.C., Warner, P.J., 1998. Biosensors for novel rapid assay methods in the food industry. New Food 1, 47 51. Stredansky, M, Pizzariello, A., Stredanska, S., Miertus, S., 1999. Determination of D-fructose in food stuffs by an improved amperometric biosensor based on a solid binding matrix. Anal. Commun. 36, 57 61. Strike, D.J., de Rooij, N.F., Koudelka-Hep, M., 1993. Electrodeposition of glucose oxidase for the fabrication of miniature sensors. Sens. Actuat. B 13, 61 64. Sun, Z., Tachikawa, H., 1992. Enzyme-based bilayer conducting polymer electrodes consisting of polymetallopthalocyanines and polypyrrole-glucose oxidase thin lms. Anal. Chem. 64, 1112 1117. Sung, W.J., Bae, Y.H., 2000. A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant. Anal. Chem. 72, 2177 2181. Svorc, J., Miertu, S., Katrlik, J., Stredansk, M., 1997. Composite transducer for amperometric biosensors. The glucose sensor. Anal. Chem 69, 2086 2089. Tatsuma, T., Watanabe, T., Watanabe, T., 1993a. Polypyrrole bioenzyme electrodes with glucose oxidase and peroxidase. Sens. Actuat. B 14, 752 753. Tatsuma, T., Watanabe, T., Watanabe, T., 1993b. Electrochemical characterization of polypyrrole bioenzyme electrodes with glucose oxidase and peroxidase. J. Electroanal. Chem. 356, 245 253. Teasdale, P.R., Wallace, G.G., 1993. Molecular recognition on conducting polymers: basis of electrochemical sensing technology. Analyst 118, 329 334. Tourillon, G., Garnier, F., 1983. Stability of conducting polythiophene and derivatives. J. Electrochem. Soc. 130, 2042 2044. Trojanowicz, M., vel Krawczyk, T.K., 1995. Electrochemical biosensors based on enzymes immobilized in electropolymerized lms. Mikrochim. Acta 121, 167 181. Trojanowicz, M., vel Krawczyk, T.K., Alexander, P.W., 1997. Organic conductings as active materials in electrochemical chemosensors and biosensors. Chem. Anal. 42, 199 213. Trojanowicz, M., Matuszewski, W., Szczepanczyk, B., Lewenstam, A., 1993. In: Guilbault, G.G., Mascini, M. (Eds.), Uses of Immobilized Biological Compounds. Kluwer, Dordrecht, pp. 141 150. Umana, M., Waller, J., 1986. Protein modied electrodes: the glucose oxidase/polypyrrole system. Anal. Chem. 58, 2979 2983. Unwin, P.R., Bard, A.J., 1992. Ultramicroelectrode voltammetry in a drop of solution: a new approach to the measurement of adsorption isotherms at solid liquid interface. Anal. Chem. 64, 113 119. Urban, G., Jachimowicz, A., Kohl, F., Kuttner, H., Olcaytug, F., Kamper, H., Pittner, F., Mann-Buxbaum, E., Schalkhammer, T., Prohaska, O., Schonauer, M., 1990. High resolution thin lm

acid. Its application as an ethanol sensor. Thin Solid Films 234, 138 143. Pandey, P.C., 1992. Membrane electrodes as biosensor. Bull. Electrochem. 8, 212 221. Pandey, P.C., 1988. A new conducting polymer-coated glucose sensor. J. Chem. Soc. Faraday Trans. I 84, 2259 2265. Pandey, P.C., Mishra, A.P., 1988. Conducting polymer-coated enzyme microsensor for urea. Analyst 113, 329 331. Paul, E.W., Ricco, A.J., Wrighton, M.S., 1985. Resistance of polyaniline lms as a function of electrochemical potential and the fabrication of polyaniline-based microelectronic devices. J. Phys. Chem. 89, 1441 1447. Porter, R.A., 2000. Investigation of electroplated conducting polymers as antibody receptors in immunosensors. J. Immunoassay 21, 51 64. Price, W.E., Wallace, G.G., Zhao, H., 1994. Effect of counter ion on transport across polypyrrole membranes. J. Membr. Sci. 87, 47 56. Rabolt, J.F., Clarke, T.C., Kanazawa, K.K., Reynolds, J.R., Street, G.B., 1980. Organic metals: poly(p-phenylene sulphide) hexauoroarsenate. J. Chem. Soc. Chem. Commun., 347 348. Ramanathan, K., 1995a. Application of some conducting polymers to biosensor, Ph.D Thesis, IIT Delhi, India. Ramanathan, K., Ram, M.K., Malhotra, B.D., Murthy, A.S.N, 1995b. Application of polyaniline-Langmuir-Blodgett lms as a glucose biosensor. Mater. Sci. Engg. C 3, 159 163. Ramanathan, K., Mehrotra, R., Jayaram, B., Murthy, A.S.N., Malhotra, B.D, 1996a. Simulation of electrochemical process for glucose oxidase immobilized conducting polymer. Anal. Lett. 29, 1477 1484. Ramanathan, K., Ram, M.K., Verghese, M.M., Malhotra, B.D., 1996b. Dielectric spectroscopic studies on polypyrrole glucose oxidase lms. J. Appl. Polym. Sci. 60, 2309 2316. Ramanathan, K., Sundaresan, N.S., Malhotra, B.D., 1995c. Ion exchanged polypyrrole-based glucose biosensor: enhanced loading and response. Electroanalysis 7, 579 582. Ramanathan, K., Pandey, S.S., Kumar, R., Gulati, A., Murthy, A.S.N., Malhotra, B.D., 2000. Covalent immobilization of glucose oxidase to poly(o-amino benzoic acid) for application to glucose biosensor. J. Appl. Polym. Sci. 78, 662 667. Ratcliffe, N.M., 1990. Polypyrrole-based sensor for hydrazine and ammonia. Anal. Chim. Acta 239, 257 262. Rogers, K.R., 1998. Biosensor technology for environment measurement. In: Meyers, R.A. (Ed.), Encyclopedia of Environmental Analysis and Remediation. John Wiley and Sons, New York, pp. 755 768. Sadik, O., Wallace, G.G, 1993. Pulsed amperometric detection of proteins using antibody containing conducting polymers. Anal. Chim. Acta 279, 209 212. Santhanam, K.S.V., 1998. Conducting polymers for biosensors: rationale based on models. Pure Appl. Chem 70, 1259 1262. Santhanam, K.S.V., Gupta, N., 1993. Conducting-polymer electrodes in batteries. TRIP 1, 284 289. Saoudi, B., Jammul, N., Abel, M.L., Chehimi, M.M., Dodin, G., 1997. DNA adsorption onto conducting polypyrrole. Synth. Met. 87, 97 103. Saraswathi, R., Gerard, M., Malhotra, B.D., 1999. Characteristics of aqueous polycarbazole batteries. J. Appl. Polym. Sci. 74, 145 150. Schmid, R.D., 1987. Preface, in Biosensors International Workshop, R.D. Schmid, (Ed.), 10, VCH, Weinheim, Federal Republic of Germany. Schuhmann, W., 1995. Conducting polymers and their application in amperometric biosensors. Mikrochim. Acta 121, 1 29. Schuhmann, W., Kranz, C., Huber, J., Wohlschlager, H., 1993. Conducting polymer-based amperometric enzyme electrodes. Towards the development of miniaturized reagentless biosensors. Synth. Metals 61, 31 35.

M. Gerard et al. / Biosensors & Bioelectronics 17 (2002) 345 359 temperature sensor arrays for medical applications. Sens. Actuat. A 21 23, 650 654. Verghese, M.M., Ramanathan, K., Ashraf, S.M., Malhotra, B.D., 1998. Enhanced loading of glucose oxidase on polyaniline lms based on anion exchange. J. Appl. Polm. Sci. 70, 1447 1453. Wang, J., Jiang, M., Aantonio, F., Mukherjee, B, 1999. New labelfree DNA recognition based on doping nucleic acid probes within conducting polymer lms. Anal. Chim. Acta 402, 7 12. Wrighton, M.S., 1986. Surface functionalization of electrodes with molecular reagents. Science 231, 32 37. Wring, S.A, Hart, J.P., 1992. Chemically modied carbon-based electrodes and their application as electrochemical sensors for the analysis of biologically important compounds. Analyst 117, 1215 1229. Xie, B., Khayyami, M., Nwosu, T., Larsson, P., Danielsson, B., 1993. Ferrocene mediated thermal biosensor. Analyst 118, 845 848.

359

Yamato, H., Ohawa, M.and, Wernet, W., 1995. A polypyrrole/three enzyme electrode for creatinine detection. Anal. Chem. 67, 2776 2780. Yang, Q., Atanasov, P., Wilkins, E., 1999. Needle type lactate biosensor. Biosens. Bioelectron. 14, 203 210. Yang, Y., Shaolin, M., 1997. Bioelectrochemical responses of polyaniline horseradish peroxidase electrodes. J. Electroanal. Chem. 432, 71 78. Yon Hin, B.F.Y., Lowe, C.R., 1992. Amperometric response of polypyrrole entrapped bienzyme lms. Sens. Actuat. B 7, 339 342. Yon-Hin, B.F.Y., Sethi, R.S., Lowe, C.R., 1990. Multianalyte microelectronic biosensors. Sens. Actuat. B 1, 550 554. Zinger, B., Miller, L.L., 1984. Timed release of chemicals from polypyrrole lms. J. Am. Chem. Soc. 106, 6861 6863.