- Email: [email protected]
Using electropolymerized non-conducting polymers to develop enzyme amperometric biosensors
Review
TRENDS in Biotechnology
Vol.22 No.5 May 2004
Using electropolymerized non-conducting polymers to develop enzyme amperometric biosensors Miao Yuqing, Chen Jianrong and Wu Xiaohua College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China
The response performance of amperometric biosensors can be likened to that of an uncovered electrode because the non-conducting polymer films used to develop these biosensors are very thin (10–100 nm), owing to their self-limited growth. The non-conducting polymer films also have favorable permselective properties, which could be used to eliminate possible electrochemical interference in samples. Composite structures or materials that include non-conducting polymers of, for example, phenol and its derivatives, phenylenediamines, and overoxidized or electroinactive polypyrrole could be used to optimize the biosensors. This article will discuss these issues, as well as the use of quartz crystal microbalance in the study of non-conducting-polymer-based biosensors. Enzyme amperometric biosensors are attracting widespread attention in clinical, environmental, agricultural and biotechnological applications. To achieve high sensitivity and fast response time, suitable immobilization methods for both enzyme and mediator must be explored. In recent years, electropolymerized polymers used for generating biosensors have received much attention. Unlike conventional immobilization strategies for biosensors, electropolymerization has no limit in terms of the geometry and area of the electrode, and offers advantages with respect to thickness control, reproducibility and the uniformity of the polymer film on electrode surfaces with more complex geometries [1]. In addition, electropolymerization permits simple electrode regeneration and can easily be extended to the production of microbiosensors. Electropolymerization of conducting polymers, such as polypyrrole (PPy), polyaniline, polyacetylene, polyindole, polythionine and polythiophene, has been studied extensively for the development of biosensors [2 – 4]. This is because these polymers have a high conductivity and stability in both air and aqueous solution. Also, the thickness of the electropolymerized film and the amount of immobilized enzyme can be controlled easily during electropolymerization. Non-conducting polymers are emerging as a novel support matrix for the immobilization of biomolecules because they offer impressive advantages, including Corresponding author: Miao Yuqing ([email protected]).
excellent permselectivity and high reproducibility, in addition to most of the reported merits of conducting polymers. An example of a non-conducting polymer is the thin electropolymerized film of poly[1,3-diaminobenzene (DAB)], which can be used to eliminate electrochemical interference from ascorbate, urea, acetaminophen and other oxidizable species. However, until now, the use of non-conducting polymers for developing enzyme amperometric biosensors has not been reviewed extensively [5,6]. Non-conducting polymers for biosensors By definition, the non-conducting polymers reviewed here are those with high resistivity that have been prepared by electropolymerization. The growth of such polymers is self-limited and the film that is formed is much thinner than typical conducting polymer films. Because the thickness of non-conducting polymers is only 10 – 100 nm, substrates and products diffuse rapidly to and from the enzyme [7,8]. Also, the non-conducting films are permselective, which might be useful in preventing interfering species from approaching or contaminating the electrode surface. Therefore, fast response time and high selectivity could also be expected for non-conducting-polymer-based enzyme biosensors. Non-conducting polymers of phenol and its derivatives The use of non-conducting polymers of phenol and its derivatives for the development of biosensors has been reported [9,10]. The electropolymerized films of phenol are produced by ortho- or para- coupling of phenolate radicals generated by oxidation of the phenylate anion. Subsequent reactions produce oligomers and, finally, poly(phenylene oxide) films are polymerized on the surface of the electrode [7,8] (Figure 1). The electropolymerization of other phenol derivatives (e.g. 3-nitrophenol, pyrogallol, 4-hydroxybenzenesulfonic acid and bromophenol blue) is similar to that of phenol. Bartlett et al. [11] reported that the electropolymerization of phenol at a platinum electrode surface gave a thin-layer film with a thickness of 38 nm (25-mM phenol, sweep range of 0.00– 0.95 V vs standard calomel electrode). A glucose biosensor was fabricated by repetitive potential cycling in an aqueous solution of phenol and glucose oxidase (GOx) [11]. This electropolymerized non-conducting film enables rapid diffusion of substrate and product,
www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.03.004
228
Review
TRENDS in Biotechnology
Vol.22 No.5 May 2004
Figure 1. The electrooxidation processes of phenol. Electropolymerized films of phenol are produced by ortho- or para-coupling of phenolate radicals generated from the oxidation of the phenylate anion. Subsequent reactions produce oligomers and, finally, poly(phenylene oxide) films are polymerized.
and exhibits selectivity towards interfering species. Similar research was also carried out by Calvoa et al. [10]. 3-Aminophenol can be electropolymerized on a carbonpaste electrode to immobilize ferrocene and horseradish peroxidase for the development of an H2O2 biosensor [8]. Both the hydroxyl and amino groups of 3-aminophenol seem to participate in the electropolymerization, and the formed polymer film might also be a non-conducting polymer. This biosensor is not influenced by easily oxidizable species such as L-ascorbic acid and uric acid. Glucose biosensors can also be created by immobilizing GOx on an electropolymerized poly(2-aminophenol) (PAP) film on a platinized glassy carbon electrode [12]. Poly(2-aminophenol) is a ladder polymer with a phenoxazine-like chain structure. The kinetics of charge transport in a PAP-coated electrode has been investigated by AC-impedance [13], and the probable redox reaction of the protonated polymer is shown in Figure 2. The conducting potential range of PAP has also been studied by Ortega [14], whose results show that PAP exhibits conducting properties at potentials more negative than the formal potential of the polymer. The conducting nature of PAP is due to polarons. Nakabayshi et al. examined five phenol derivatives (phenol, 3-aminophenol, 3-methyphenol, 3-nitrophenol and 1,3-dihydroxybenzene) [7]. 3-Aminophenol was found to be the most suitable monomer for creating amperometric glucose biosensors. Both hydroxyl and amino groups were involved in the electropolymerization and, consequently, larger amounts of GOx could be immobilized in this polymer film, which resulted in higher sensitivity to glucose. Also, this glucose biosensor minimized contributions from the easily oxidizable compounds uric acid and L-ascorbic acid. Eddy et al. also studied non-conducting polymers of phenol and several phenol derivatives (phenol, acetaminophen, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4-aminophenol, 1,3,5-trihydroxybenzene and 1,2,3-trihydroxybenzene) electropolymerized onto platinum anodes [15]. This study illustrates that polyphenolmodified and polyphenol-derivative-modified electrodes produce responses that are selective and exceptionally fast, with performances similar to that of uncovered electrodes. The modified electrodes also provide an alternative to standard polymeric membranes, as H2 N +
O
O
+ N H2
well as an advantageous one-step process for enzyme immobilization. Polymerization of functionalized monomers enables a more specific chemical interaction between the film and the enzyme to be immobilized. This method has been used with monomers bearing specific groups, such as tyramine, which can enhance the interaction between polymers and electroactive species through the formation of covalent bonds between either the amino and aldehyde or amino and carboxyl groups [16]. The tyramine electropolymerization mechanism is associated with phenol-group oxidation and subsequent radical formation (Figure 3). Glucose oxidase is covalently attached to free amine groups on the polytyramine film using the coupling reagents 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide [17] (Figure 3). This method of constructing enzyme biosensors produces a more reproducible and stable device. Non-conducting polymers of phenylenediamines Non-conducting membranes from polyphenylenediamines (PPDs) have been of particular interest because their thin, dense films lead to both fast response and high H2O2 or O2 selectivity. Moreover, these polymers are inert against reactive chemical species (e.g. H2O2). Jang et al. reported the electropolymerization mechanism for poly(1,2-DAB) [18], and the film of electropolymerized poly(1,2-DAB) has been analyzed by an impedimetric technique [19]. Also, GOx has been entrapped in a poly(1,2-DAB) film through the polymerization of 1,2-DAB on platinum-coated carbon fibers [20]. In this investigation, the potential was swept from 0.0 V to 0.8 V at 50 mV s21, and a diffusion –kinetic model was presented for an enzyme-modified microcylinder electrode. The amount of GOx entrapped in poly(1,2-DAB) was determined by X-ray photoelectron spectroscopy [21]. A poly(1,2-DAB)film-coated platinum electrode has been used to develop an in vivo electrochemical flow-injection system with online microdialysis sampling for the simultaneous monitoring of L-lactate and glucose in rat brain [22]. The system was free from any interference by oxidizable species or proteins present in the dialysate. One of the major problems with implantable glucose devices is the system response, namely fibrin formation and clotting on exposure to blood. To combat this, heparin
H
H+ + e –
.N+
O
O
+ N H2
H+ + e–
H N +
O
O
+ N H
TRENDS in Biotechnology
Figure 2. The probable redox reaction of the protonated poly(2-aminophenol) polymer. Electrochemical oxidation of the polymer is equivalent to its dehydrogenation. www.sciencedirect.com
Review
TRENDS in Biotechnology
Tyramine O–
OH
O– – H+
– e– –H+ (CH2)2–NH2
(CH2)2–NH2
O
(CH2)2–NH2
O O
n
O HOOC
GOx
Polytyramine–GOx
GOx
(CH2)2–NH2 Polytyramine TRENDS in Biotechnology
Figure 3. The polymerization of tyramine and the covalent attachment of glucose oxidase (GOx) to the polymer backbone. The attachment of GOx to free amine groups on the polytyramine film uses the coupling reagents 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride and N-hydroxysuccinimide (NHS).
was co-immobilized with GOx during the electrochemical formation of a non-conducting poly(1,2-DAB) film to develop the implantable glucose biosensor [23]. The incorporated heparin retained its bioactivity within the polymer network. Such electropolymeric co-entrapment imparts both biocatalytic and anticoagulation activities onto the transducer and, thus, greatly improves the performance of the sensor on exposure to whole blood. The anti-contaminating properties of poly(1,3-DAB)covered electrodes was also studied by Daly et al. [24]. The results show that these electrodes perform well in buffer solution, urine, plasma and serum samples, although not so well in blood. Poly(1,4-DAB) has also been employed as an entrapment medium for detecting glucose [25]. The specificity of the biosensors for possible interfering species such as lactose, sucrose and urea was tested, and no discernible signal was detected above the background current. Different techniques for film electropolymerization (of 1,2-, 1,3- and 1,4-DAB), such as cyclic voltammetry and chronoamperometry, were compared by Currulli et al. [26]. The techniques employed depend in the nature of the immobilized enzyme. Poly( p-chlorophenylamide) (PCPA) was also used to develop biosensors for the amperometric determination of glucose [27]. It was found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Non-conducting polymers of overoxidized PPy The use of conducting PPy to develop enzyme biosensors has been widely reported because of its high conductivity and stability, as well as the simplicity and flexibility of the immobilization procedure. Recently, a non-conducting PPy has emerged as a novel material for biosensor construction. A glucose biosensor, based on GOx immobilized on an overoxidized PPy (PPyox)-modified platinum electrode, has www.sciencedirect.com
229
been investigated [28]. The PPyox is a non-conducting, permselective polymer membrane with excellent interferent rejection. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. The use of PPyox has also been reported for the development of a cholesterol biosensor in a flow system [29,30]. Electroinactive PPy, prepared from an aqueous solution of pyrrole and NaOH, was used to develop a potentiometric biosensor for urea [31,32]. This enzyme-immobilized PPy-film electrode showed a stable potential response to urea, based on the pH response of the electroinactive PPy-film electrode.
n
EDC + NHS (CH2)2–NH–CO
Vol.22 No.5 May 2004
Composite structures of non-conducting polymers for the development of biosensors Composite materials that include non-conducting polymers have been used frequently to increase the amount of immobilized enzyme or to prevent interference. However, when the immobilization of enzymes is attempted during electropolymerization of non-conducting layers, a problem emerges. Only a limited number of enzymes can be immobilized because the self-limiting non-conducting polymer is very thin. The presence of poly-L-lysine and glutaraldehyde during electropolymerization of poly(1,3-DAB) improves sensitivity by increasing the amount of enzyme immobilized [1,33]. The amino groups of poly-L-lysine assist in attaching GOx to an electrode coated with a polyion-complex layer comprising poly-L-lysine and poly(4-styrenesulfonate) [34]. Covalent bonds are formed between glutaraldehyde and the residual (non-reacting) amino groups at the surface of the poly(1,3-DAB) layer and, thus, might help increase the amount of immobilized enzyme [34]. Klinchan et al. used two-step immobilization to construct a sucrose biosensor [35]. Glucose oxidase was immobilized on a poly(1,3-DAB) film by electropolymerization. Invertase and mutarotase were then immobilized by co-crosslinking with bovine serum albumin and glutaraldehyde on a poly(1,3-DAB)– GOx electrode. In addition, composite polymers have been reported to protect the immobilized enzyme or decrease possible interference [27,36]. A biosensor modified with a composite polymer was reported in a flow-injection analysis system for the detection of galactose in human plasma [36]. The composite polymer showed improved selectivity to H2O2 compared with both of its individual polymeric components – Nafion and a copolymer of DAB and resorcinol. The composite polymer minimized the effect of possible interference from urate, ascorbate, and acetaminophen. Poly( p-chlorophenylamide) and Nafion were combined to develop a composite polymer (PCPA – Nafion) for the immobilization of GOx [27]. On its own, as a nonconducting polymer, PCPA can reduce the influence of electroactive interferents. The introduction of the inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability. A new type of miniaturized glucose sensor has been developed. Its structure consists of an inner layer of two electropolymerized non-conducting films and an outer
230
Review
TRENDS in Biotechnology
bilayer of teflon and polyurethane films [37]. Glucose oxidase is entrapped during the electropolymerization of a highly interference-resistant poly(1,3-phenylenediamine) (PMPD) film. A second PMPD film causes no significant decrease in the accessibility of glucose to the immobilized enzyme, and the relatively adhesive outer layer of the sensor enables a stable current response. Because of high permeability, information regarding enzyme activity can be obtained without serious error, despite the presence of the outer layer between the enzymes and the solution. Recent reports show that substituted naphthalenes – especially 5-amino-1-naphthalene (5A1N) – are promising candidates for use as monomers of highly selective nonconducting polymers [38,39]. A bilayer composed of poly5A1N and PMPD has been reported as a thin membrane composite for miniaturized glucose biosensors [39]. Successive electropolymerization of poly-5A1N and PMPD created a bilayer (with a thickness of tens of nanometers) that showed excellent selectivity and permeability to H2O2, as well as low activity loss of immobilized enzymes, while effectively rejecting possible interfering species such as ascorbic acid and acetaminophen. Vidal et al. compared cholesterol amperometric biosensors with enzymes entrapped in electropolymerized layers of diaminonaphthalene (DAN) derivatives and PPy polymers [40]. Seven configurations of monolayer or multilayer films were prepared from pyrrole, 1,8-DAN and 1,5-DAN monomers. The results show that sensitivity and selectivity depend greatly on hydrophobicity, permeability, compactness, thickness and the type of polymer used. Overoxidized PPy – poly[o-phenylenediamine(o PPD)] bilayer biosensors have been developed with either GOx or cholesterol oxidase entrapped within an inner PPy layer, onto which a non-conducting o PPD film is then electrodeposited [30]. The analytical performance of this type of biosensor in detecting glucose and cholesterol was compared with that of a PPy single-layer sensor. The combined exclusion properties of PPy and o PPD enabled the PPy –enzyme –o PPD configuration to increase the selectivity of the biosensor towards interfering electroactive species that are frequently present in biological samples. Using QCM to study non-conducting-polymer-based biosensors The growth of non-conducting polymers is self-limited because of their high resistivity and because, consequently, the films tend to be very thin (10 –100 nm) [41]. As a result, the film structures are difficult to investigate using a standard optical microscope or even a scanning electron microscope. The demand for closer inspection of the electropolymerization and immobilization processes, as well as of enzyme distribution, is growing because such non-conducting films have been reported to show excellent functions suitable for developing enzyme biosensors. A quartz crystal microbalance (QCM) consists of an AT-cut quartz crystal disk (a plate cut from a crystal of quartz in such a way that the plate contains the X-axis and makes an angle of , 358 with the optic or Z-axis) sandwiched between two metal electrodes. An applied oscillating electric field induces an acoustic wave. The www.sciencedirect.com
Vol.22 No.5 May 2004
resonant frequency is shifted proportionally by mass change (e.g. because of adsorption of material to the electrode) at the crystal surface. Therefore, this is a well-established method for measuring small changes in mass. A more recent application of the QCM is its coupling with electrochemical analysis (using an electrochemical QCM; EQCM). This could be used to measure mass changes associated with electrochemical processes such as adsorption or electrodeposition. An EQCM was used to investigate the immobilization process of GOx in the poly(1,3-DAB) network [1]. The poly(1,3-DAB) film was prepared by potential scanning from 0.2 to 0.8 V. The frequency change for each cycle decayed rapidly, as did the repeated potential cycle, which corresponded to the growth of a high-resistance film on the electrode surface. This, in turn, blocked the access of 1,3-DAB to the electrode surface. Glucose oxidase was captured in a poly(1,3-DAB) layer , 50-nm thick, and was found to distribute mostly in the outer part of the polymer film. A 514-Hz frequency increase corresponded to 541-ng immobilized GOx. Similar research has been reported by Si et al. They used QCM to monitor the frequency change due to the immobilization of protein on a poly(1,3-DAB)– polyaniline film [42]. Poly(o-aminophenol) films deposited on platinum electrodes have also been studied by quartz crystal impedance [43]. Concluding remarks The main advantage of using non-conducting polymers to create enzyme biosensors is their favorable permselective properties. In addition, high sensitivity, fast response time and good reproducibility can be expected because of the ultra-thin non-conducting film used. However, the main drawback of this method is the limited amount of immobilized enzyme during electropolymerization of nonconducting layers, which is because the self-limiting non-conducting polymer film is very thin. Composite structures or materials that include non-conducting polymers are useful for enhancing the amount of immobilized enzyme and decreasing the interference by easily oxidized species in the sample. Also, PPyox can be employed as a non-conducting polymer. The relatively recent development of this immobilization strategy could have a profound effect on future research. Acknowledgements This material is based upon work funded by Zhejiang Provincial Natural Science Foundation of China, grant number M203106.
References 1 Chung, T.D. et al. (2001) Reproducible fabrication of miniaturized glucose sensors: preparation of sensing membranes for continuous monitoring. Biosens. Bioelectron. 16, 1079– 1087 2 Vidal, J-C. et al. (2003) Recent advances in electropolymerized conducting polymers in amperometric biosensors. Microchim. Acta 143, 93 – 111 3 Gerard, M. et al. (2002) Review: application of conducting polymers to biosensors. Biosens. Bioelectron. 17, 345– 359 4 Saxena, V. and Malhotra, B.D. (2003) Prospects of conducting polymers in molecular electronics. Curr. Appl. Phys. 3, 293 – 305 5 Cosnier, S. (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films: A review. Biosens. Bioelectron. 14, 443 – 456
Review
TRENDS in Biotechnology
6 Palmisano, F. et al. (2000) Amperometric biosensors based on electrosynthesised polymeric films. Fresenius J. Anal. Chem. 366, 586 – 601 7 Nakabayshi, Y. et al. (1998) Amperometric glucose sensors fabricated by electrochemical polymerization of phenols on carbon paste electrodes containing ferrocene as electron transfer mediator. Anal. Sci. 14, 1069 – 1077 8 Nakabayashi, Y. and Yoshikawa, H. (2000) Amperometric biosensors for sensing of hydrogen peroxide based on electron transfer between horseradish peroxidase and ferrocene as a mediator. Anal. Sci. 16, 609 – 613 9 Carelli, I. et al. (1996) Electropolymerization of hydroxybenzene and aminobenzene isomers on platinum electrodes to assemble interference-free electrochemical biosensors. Electrochim. Acta 41, 1793 – 1800 10 Calvoa, E.J. and Danilowiczb, C. (1997) Amperometric enzyme electrodes. J. Braz. Chem. Soc. 8, 563– 574 11 Bartlett, P.N. and Cooper, J.M. (1993) A review of the immobilization of enzymes in electropolymerized films. J. Electroanal. Chem. 362, 1 – 12 12 Zhang, Z. et al. (1996) A glucose biosensor based on immobilization of glucose oxidase in electropolymerized o-aminophenol film on platinized glassy carbon electrode. Anal. Chem. 66, 1632 – 1638 13 Terubisa, K. et al. (1998) Charge-transport processes at poly-oaminophenol film electrodes: electron hopping accompanied by proton exchange. Electrochim. Acta 43, 723– 731 14 Ortega, J.M. (2000) Conducting potential range for poly(o-aminophenol). Thin Solid Films 371, 28 – 35 15 Eddy, S. et al. (1995) The modification of enzyme electrode properties with non-conducting electropolymerized films. Biosens. Bioelectron. 10, 831 – 839 16 Cordas, C.M. et al. (2002) EQCM study on the polytyramine modified electrodes for the preparation of biosensors. In Nanostructured Materials and Coatings for Biomedical and Sensor Applications (Gogotsi, Y.G., ed.), pp. 371 – 376, Kluwer Academic Publishers 17 Situmorang, M. et al. (1998) Electrodeposited polytyramine as an immobilization matrix for enzyme biosensors. Biosens. Bioelectron. 13, 953 – 962 18 Jang, D-H. et al. (1995) Electropolymerization mechanism for poly (o-phenylenediamine) (PPD) and its electrocatalytic behavior for O2 reduction. Bull. Korean Chem. Soc. 16, 392 – 397 19 Martinusz, K. et al. (1997) Impedance analysis of poly(o-phenylenediamine) electrodes. J. Electroanal. Chem. 433, 1 – 8 20 Somasundrum, M. and Aoki, K. (2002) The steady-state current at microcylinder electrodes modified by enzymes immobilized in conducting or non-conducting material. J. Electroanal. Chem. 530, 40 – 46 21 Griffith, A. et al. (1996) Probing enzyme polymer biosensors using X-ray photoelectron spectroscopy: determination of glucose oxidase in electropolymerized films. Biosens. Bioelectron. 11, 625– 631 22 Yao, T. et al. (2003) Simultaneous determination of glucose and L-Lactate in rat brain by an electrochemical in vivo flow-injection system with on-line microdialysis sampling. Anal. Sci. 19, 61 – 65 23 Wang, J. et al. (2000) One-step electropolymeric co-immobilization of glucose oxidase and heparin for amperometric biosensing of glucose. Analyst 125, 1431– 1434 24 Daly, D.J. et al. (2000) The use of electrochemically grown polymers on metallized electrodes to reduce electrode fouling in biological matrices. Biochem. Soc. Trans. 28, 89– 94 25 Ekinci, E. et al. (1996) Electrochemical synthesis and sensor application of poly(1,4-diaminobenzene). Synth. Met. 79, 57 – 61 26 Curulli, A. and Palleschi, G. (2000) Construction and application of
www.sciencedirect.com
Vol.22 No.5 May 2004
27
28
29
30
31
32
33
34
35
36
37 38
39
40
41
42
43
231
highly selectively sensors and biosensors using non-conducting electropolymerized films. In Proceedings of The 2nd Workshop on Chemical Sensors and Biosensors (Mazzei, F. and Pilloton, R., eds), pp. 439– 444 Xu, J. et al. (2002) Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion. Anal. Chim. Acta 463, 239 – 247 Guerrieri, A. et al. (1998) Elelctrosynthesized non-conducting polymers as permselective membranes in amperometric enzyme electrodes: a glucose biosensor based on a co-crosslinked glucose oxidase/overoxidized polypyrrole bilayer. Biosens. Bioelectron. 13, 103– 112 Vidal, J.C. et al. (1999) In situ preparation of a cholesterol biosensor: entrapment of cholesterol oxidase in an overoxidized polypyrrole film electrodeposited in a flow system; determination of total cholesterol in serum. Anal. Chim. Acta 385, 213 – 222 Vidal, J.C. et al. (1999) In situ preparation of overoxidized PPy/o PPD bilayer biosensors for the determination of glucose and cholesterol in serum. Sens. Actuat. 57, 219 – 226 Schinichi, K. et al. (1997) Potentiometric biosensor for urea based on electropolymerized electroinactive polypyrrole. Electrochim. Acta 42, 383– 388 Osaka, T. et al. (1996) High-sensitivity urea sensor based on the composite film of electroinactive polypyrrole with polyion complex. Sens. Actuat. 35-36, 463 – 469 Karalemas, I.D. and Georgiou, C.A. (2000) Papastathopoulos D.S., construction of a L-lysine biosensor by immobilizing lysine oxidase on a gold-poly(o-phenylenediamine) electrode. Talanta 53, 391– 402 Mizutani, F. et al. (1998) High-throughput flow-injection analysis of glucose and glutamate in food and biological samples by using enzyme/ polyion complex-bilayer membrane-based electrodes as the detector. Biosens. Bioelectron. 13, 809 – 815 Klinchan, S. et al. (2002) Construction of sensor chips by electrochemical polymerization techniques for sucrose determination. The Journal of KMTMB 12, 12 – 16 Stoecker, P.M.W. and Yacynych, A.M. (1995) Galactose biosensors using composite polymers to prevent interferences. Biosens. Bioelectron. 10, 359– 370 Yang, H. et al. (2002) Glucose sensor using a microfabricated electrode and electropolymerized bilayer films. Biosens. Bioelectron. 17, 251–259 Murphy, L.J. (1998) Reduction of interference response at a hydrogen peroxide detecting electrode using electropolymerized films of substituted naphthalenes. Anal. Chem. 70, 2928 – 2935 Chung, T.D. (2003) Electropolymerized thin bilayers of poly-5-amino1-naphthol and poly-1,3-phenylendiamine for continuous monitoring glucose sensors. Bull. Korean Chem. Soc 24, 291 – 294 Vidal, J.C. et al. (2003) Comparison of biosensors based on entrapment of cholesterol oxidase and cholesterol esterase in electropolymerized films of polypyrrole and diaminonaphthalene derivatives for amperometric determination of cholesterol. Anal. Bioanal. Chem. 377, 273– 280 Malitesta, C. et al. (1990) Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film. Anal. Chem. 62, 2735– 2740 Si, S.H. et al. (1995) Electrochemical quartz crystal microbalance study on electropolymerization of m-phenylenediamine: effects of aniline and polyaniline. Electrochim. Acta 40, 2715– 2721 Ortega, J.M. (1998) Studies of poly(o-aminophenol) by quartz crystal impedance measurements. Synth. Met. 97, 81 – 84
TRENDS in Biotechnology
Vol.22 No.5 May 2004
Using electropolymerized non-conducting polymers to develop enzyme amperometric biosensors Miao Yuqing, Chen Jianrong and Wu Xiaohua College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, China
The response performance of amperometric biosensors can be likened to that of an uncovered electrode because the non-conducting polymer films used to develop these biosensors are very thin (10–100 nm), owing to their self-limited growth. The non-conducting polymer films also have favorable permselective properties, which could be used to eliminate possible electrochemical interference in samples. Composite structures or materials that include non-conducting polymers of, for example, phenol and its derivatives, phenylenediamines, and overoxidized or electroinactive polypyrrole could be used to optimize the biosensors. This article will discuss these issues, as well as the use of quartz crystal microbalance in the study of non-conducting-polymer-based biosensors. Enzyme amperometric biosensors are attracting widespread attention in clinical, environmental, agricultural and biotechnological applications. To achieve high sensitivity and fast response time, suitable immobilization methods for both enzyme and mediator must be explored. In recent years, electropolymerized polymers used for generating biosensors have received much attention. Unlike conventional immobilization strategies for biosensors, electropolymerization has no limit in terms of the geometry and area of the electrode, and offers advantages with respect to thickness control, reproducibility and the uniformity of the polymer film on electrode surfaces with more complex geometries [1]. In addition, electropolymerization permits simple electrode regeneration and can easily be extended to the production of microbiosensors. Electropolymerization of conducting polymers, such as polypyrrole (PPy), polyaniline, polyacetylene, polyindole, polythionine and polythiophene, has been studied extensively for the development of biosensors [2 – 4]. This is because these polymers have a high conductivity and stability in both air and aqueous solution. Also, the thickness of the electropolymerized film and the amount of immobilized enzyme can be controlled easily during electropolymerization. Non-conducting polymers are emerging as a novel support matrix for the immobilization of biomolecules because they offer impressive advantages, including Corresponding author: Miao Yuqing ([email protected]).
excellent permselectivity and high reproducibility, in addition to most of the reported merits of conducting polymers. An example of a non-conducting polymer is the thin electropolymerized film of poly[1,3-diaminobenzene (DAB)], which can be used to eliminate electrochemical interference from ascorbate, urea, acetaminophen and other oxidizable species. However, until now, the use of non-conducting polymers for developing enzyme amperometric biosensors has not been reviewed extensively [5,6]. Non-conducting polymers for biosensors By definition, the non-conducting polymers reviewed here are those with high resistivity that have been prepared by electropolymerization. The growth of such polymers is self-limited and the film that is formed is much thinner than typical conducting polymer films. Because the thickness of non-conducting polymers is only 10 – 100 nm, substrates and products diffuse rapidly to and from the enzyme [7,8]. Also, the non-conducting films are permselective, which might be useful in preventing interfering species from approaching or contaminating the electrode surface. Therefore, fast response time and high selectivity could also be expected for non-conducting-polymer-based enzyme biosensors. Non-conducting polymers of phenol and its derivatives The use of non-conducting polymers of phenol and its derivatives for the development of biosensors has been reported [9,10]. The electropolymerized films of phenol are produced by ortho- or para- coupling of phenolate radicals generated by oxidation of the phenylate anion. Subsequent reactions produce oligomers and, finally, poly(phenylene oxide) films are polymerized on the surface of the electrode [7,8] (Figure 1). The electropolymerization of other phenol derivatives (e.g. 3-nitrophenol, pyrogallol, 4-hydroxybenzenesulfonic acid and bromophenol blue) is similar to that of phenol. Bartlett et al. [11] reported that the electropolymerization of phenol at a platinum electrode surface gave a thin-layer film with a thickness of 38 nm (25-mM phenol, sweep range of 0.00– 0.95 V vs standard calomel electrode). A glucose biosensor was fabricated by repetitive potential cycling in an aqueous solution of phenol and glucose oxidase (GOx) [11]. This electropolymerized non-conducting film enables rapid diffusion of substrate and product,
www.sciencedirect.com 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.03.004
228
Review
TRENDS in Biotechnology
Vol.22 No.5 May 2004
Figure 1. The electrooxidation processes of phenol. Electropolymerized films of phenol are produced by ortho- or para-coupling of phenolate radicals generated from the oxidation of the phenylate anion. Subsequent reactions produce oligomers and, finally, poly(phenylene oxide) films are polymerized.
and exhibits selectivity towards interfering species. Similar research was also carried out by Calvoa et al. [10]. 3-Aminophenol can be electropolymerized on a carbonpaste electrode to immobilize ferrocene and horseradish peroxidase for the development of an H2O2 biosensor [8]. Both the hydroxyl and amino groups of 3-aminophenol seem to participate in the electropolymerization, and the formed polymer film might also be a non-conducting polymer. This biosensor is not influenced by easily oxidizable species such as L-ascorbic acid and uric acid. Glucose biosensors can also be created by immobilizing GOx on an electropolymerized poly(2-aminophenol) (PAP) film on a platinized glassy carbon electrode [12]. Poly(2-aminophenol) is a ladder polymer with a phenoxazine-like chain structure. The kinetics of charge transport in a PAP-coated electrode has been investigated by AC-impedance [13], and the probable redox reaction of the protonated polymer is shown in Figure 2. The conducting potential range of PAP has also been studied by Ortega [14], whose results show that PAP exhibits conducting properties at potentials more negative than the formal potential of the polymer. The conducting nature of PAP is due to polarons. Nakabayshi et al. examined five phenol derivatives (phenol, 3-aminophenol, 3-methyphenol, 3-nitrophenol and 1,3-dihydroxybenzene) [7]. 3-Aminophenol was found to be the most suitable monomer for creating amperometric glucose biosensors. Both hydroxyl and amino groups were involved in the electropolymerization and, consequently, larger amounts of GOx could be immobilized in this polymer film, which resulted in higher sensitivity to glucose. Also, this glucose biosensor minimized contributions from the easily oxidizable compounds uric acid and L-ascorbic acid. Eddy et al. also studied non-conducting polymers of phenol and several phenol derivatives (phenol, acetaminophen, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 4-aminophenol, 1,3,5-trihydroxybenzene and 1,2,3-trihydroxybenzene) electropolymerized onto platinum anodes [15]. This study illustrates that polyphenolmodified and polyphenol-derivative-modified electrodes produce responses that are selective and exceptionally fast, with performances similar to that of uncovered electrodes. The modified electrodes also provide an alternative to standard polymeric membranes, as H2 N +
O
O
+ N H2
well as an advantageous one-step process for enzyme immobilization. Polymerization of functionalized monomers enables a more specific chemical interaction between the film and the enzyme to be immobilized. This method has been used with monomers bearing specific groups, such as tyramine, which can enhance the interaction between polymers and electroactive species through the formation of covalent bonds between either the amino and aldehyde or amino and carboxyl groups [16]. The tyramine electropolymerization mechanism is associated with phenol-group oxidation and subsequent radical formation (Figure 3). Glucose oxidase is covalently attached to free amine groups on the polytyramine film using the coupling reagents 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide [17] (Figure 3). This method of constructing enzyme biosensors produces a more reproducible and stable device. Non-conducting polymers of phenylenediamines Non-conducting membranes from polyphenylenediamines (PPDs) have been of particular interest because their thin, dense films lead to both fast response and high H2O2 or O2 selectivity. Moreover, these polymers are inert against reactive chemical species (e.g. H2O2). Jang et al. reported the electropolymerization mechanism for poly(1,2-DAB) [18], and the film of electropolymerized poly(1,2-DAB) has been analyzed by an impedimetric technique [19]. Also, GOx has been entrapped in a poly(1,2-DAB) film through the polymerization of 1,2-DAB on platinum-coated carbon fibers [20]. In this investigation, the potential was swept from 0.0 V to 0.8 V at 50 mV s21, and a diffusion –kinetic model was presented for an enzyme-modified microcylinder electrode. The amount of GOx entrapped in poly(1,2-DAB) was determined by X-ray photoelectron spectroscopy [21]. A poly(1,2-DAB)film-coated platinum electrode has been used to develop an in vivo electrochemical flow-injection system with online microdialysis sampling for the simultaneous monitoring of L-lactate and glucose in rat brain [22]. The system was free from any interference by oxidizable species or proteins present in the dialysate. One of the major problems with implantable glucose devices is the system response, namely fibrin formation and clotting on exposure to blood. To combat this, heparin
H
H+ + e –
.N+
O
O
+ N H2
H+ + e–
H N +
O
O
+ N H
TRENDS in Biotechnology
Figure 2. The probable redox reaction of the protonated poly(2-aminophenol) polymer. Electrochemical oxidation of the polymer is equivalent to its dehydrogenation. www.sciencedirect.com
Review
TRENDS in Biotechnology
Tyramine O–
OH
O– – H+
– e– –H+ (CH2)2–NH2
(CH2)2–NH2
O
(CH2)2–NH2
O O
n
O HOOC
GOx
Polytyramine–GOx
GOx
(CH2)2–NH2 Polytyramine TRENDS in Biotechnology
Figure 3. The polymerization of tyramine and the covalent attachment of glucose oxidase (GOx) to the polymer backbone. The attachment of GOx to free amine groups on the polytyramine film uses the coupling reagents 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride and N-hydroxysuccinimide (NHS).
was co-immobilized with GOx during the electrochemical formation of a non-conducting poly(1,2-DAB) film to develop the implantable glucose biosensor [23]. The incorporated heparin retained its bioactivity within the polymer network. Such electropolymeric co-entrapment imparts both biocatalytic and anticoagulation activities onto the transducer and, thus, greatly improves the performance of the sensor on exposure to whole blood. The anti-contaminating properties of poly(1,3-DAB)covered electrodes was also studied by Daly et al. [24]. The results show that these electrodes perform well in buffer solution, urine, plasma and serum samples, although not so well in blood. Poly(1,4-DAB) has also been employed as an entrapment medium for detecting glucose [25]. The specificity of the biosensors for possible interfering species such as lactose, sucrose and urea was tested, and no discernible signal was detected above the background current. Different techniques for film electropolymerization (of 1,2-, 1,3- and 1,4-DAB), such as cyclic voltammetry and chronoamperometry, were compared by Currulli et al. [26]. The techniques employed depend in the nature of the immobilized enzyme. Poly( p-chlorophenylamide) (PCPA) was also used to develop biosensors for the amperometric determination of glucose [27]. It was found that PCPA, as a non-conducting polymer, can largely reduce the influence of electroactive interferents. Non-conducting polymers of overoxidized PPy The use of conducting PPy to develop enzyme biosensors has been widely reported because of its high conductivity and stability, as well as the simplicity and flexibility of the immobilization procedure. Recently, a non-conducting PPy has emerged as a novel material for biosensor construction. A glucose biosensor, based on GOx immobilized on an overoxidized PPy (PPyox)-modified platinum electrode, has www.sciencedirect.com
229
been investigated [28]. The PPyox is a non-conducting, permselective polymer membrane with excellent interferent rejection. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. The use of PPyox has also been reported for the development of a cholesterol biosensor in a flow system [29,30]. Electroinactive PPy, prepared from an aqueous solution of pyrrole and NaOH, was used to develop a potentiometric biosensor for urea [31,32]. This enzyme-immobilized PPy-film electrode showed a stable potential response to urea, based on the pH response of the electroinactive PPy-film electrode.
n
EDC + NHS (CH2)2–NH–CO
Vol.22 No.5 May 2004
Composite structures of non-conducting polymers for the development of biosensors Composite materials that include non-conducting polymers have been used frequently to increase the amount of immobilized enzyme or to prevent interference. However, when the immobilization of enzymes is attempted during electropolymerization of non-conducting layers, a problem emerges. Only a limited number of enzymes can be immobilized because the self-limiting non-conducting polymer is very thin. The presence of poly-L-lysine and glutaraldehyde during electropolymerization of poly(1,3-DAB) improves sensitivity by increasing the amount of enzyme immobilized [1,33]. The amino groups of poly-L-lysine assist in attaching GOx to an electrode coated with a polyion-complex layer comprising poly-L-lysine and poly(4-styrenesulfonate) [34]. Covalent bonds are formed between glutaraldehyde and the residual (non-reacting) amino groups at the surface of the poly(1,3-DAB) layer and, thus, might help increase the amount of immobilized enzyme [34]. Klinchan et al. used two-step immobilization to construct a sucrose biosensor [35]. Glucose oxidase was immobilized on a poly(1,3-DAB) film by electropolymerization. Invertase and mutarotase were then immobilized by co-crosslinking with bovine serum albumin and glutaraldehyde on a poly(1,3-DAB)– GOx electrode. In addition, composite polymers have been reported to protect the immobilized enzyme or decrease possible interference [27,36]. A biosensor modified with a composite polymer was reported in a flow-injection analysis system for the detection of galactose in human plasma [36]. The composite polymer showed improved selectivity to H2O2 compared with both of its individual polymeric components – Nafion and a copolymer of DAB and resorcinol. The composite polymer minimized the effect of possible interference from urate, ascorbate, and acetaminophen. Poly( p-chlorophenylamide) and Nafion were combined to develop a composite polymer (PCPA – Nafion) for the immobilization of GOx [27]. On its own, as a nonconducting polymer, PCPA can reduce the influence of electroactive interferents. The introduction of the inner Nafion membrane not only further eliminates the influence of ascorbic acid on the sensor response but also increases electrode stability. A new type of miniaturized glucose sensor has been developed. Its structure consists of an inner layer of two electropolymerized non-conducting films and an outer
230
Review
TRENDS in Biotechnology
bilayer of teflon and polyurethane films [37]. Glucose oxidase is entrapped during the electropolymerization of a highly interference-resistant poly(1,3-phenylenediamine) (PMPD) film. A second PMPD film causes no significant decrease in the accessibility of glucose to the immobilized enzyme, and the relatively adhesive outer layer of the sensor enables a stable current response. Because of high permeability, information regarding enzyme activity can be obtained without serious error, despite the presence of the outer layer between the enzymes and the solution. Recent reports show that substituted naphthalenes – especially 5-amino-1-naphthalene (5A1N) – are promising candidates for use as monomers of highly selective nonconducting polymers [38,39]. A bilayer composed of poly5A1N and PMPD has been reported as a thin membrane composite for miniaturized glucose biosensors [39]. Successive electropolymerization of poly-5A1N and PMPD created a bilayer (with a thickness of tens of nanometers) that showed excellent selectivity and permeability to H2O2, as well as low activity loss of immobilized enzymes, while effectively rejecting possible interfering species such as ascorbic acid and acetaminophen. Vidal et al. compared cholesterol amperometric biosensors with enzymes entrapped in electropolymerized layers of diaminonaphthalene (DAN) derivatives and PPy polymers [40]. Seven configurations of monolayer or multilayer films were prepared from pyrrole, 1,8-DAN and 1,5-DAN monomers. The results show that sensitivity and selectivity depend greatly on hydrophobicity, permeability, compactness, thickness and the type of polymer used. Overoxidized PPy – poly[o-phenylenediamine(o PPD)] bilayer biosensors have been developed with either GOx or cholesterol oxidase entrapped within an inner PPy layer, onto which a non-conducting o PPD film is then electrodeposited [30]. The analytical performance of this type of biosensor in detecting glucose and cholesterol was compared with that of a PPy single-layer sensor. The combined exclusion properties of PPy and o PPD enabled the PPy –enzyme –o PPD configuration to increase the selectivity of the biosensor towards interfering electroactive species that are frequently present in biological samples. Using QCM to study non-conducting-polymer-based biosensors The growth of non-conducting polymers is self-limited because of their high resistivity and because, consequently, the films tend to be very thin (10 –100 nm) [41]. As a result, the film structures are difficult to investigate using a standard optical microscope or even a scanning electron microscope. The demand for closer inspection of the electropolymerization and immobilization processes, as well as of enzyme distribution, is growing because such non-conducting films have been reported to show excellent functions suitable for developing enzyme biosensors. A quartz crystal microbalance (QCM) consists of an AT-cut quartz crystal disk (a plate cut from a crystal of quartz in such a way that the plate contains the X-axis and makes an angle of , 358 with the optic or Z-axis) sandwiched between two metal electrodes. An applied oscillating electric field induces an acoustic wave. The www.sciencedirect.com
Vol.22 No.5 May 2004
resonant frequency is shifted proportionally by mass change (e.g. because of adsorption of material to the electrode) at the crystal surface. Therefore, this is a well-established method for measuring small changes in mass. A more recent application of the QCM is its coupling with electrochemical analysis (using an electrochemical QCM; EQCM). This could be used to measure mass changes associated with electrochemical processes such as adsorption or electrodeposition. An EQCM was used to investigate the immobilization process of GOx in the poly(1,3-DAB) network [1]. The poly(1,3-DAB) film was prepared by potential scanning from 0.2 to 0.8 V. The frequency change for each cycle decayed rapidly, as did the repeated potential cycle, which corresponded to the growth of a high-resistance film on the electrode surface. This, in turn, blocked the access of 1,3-DAB to the electrode surface. Glucose oxidase was captured in a poly(1,3-DAB) layer , 50-nm thick, and was found to distribute mostly in the outer part of the polymer film. A 514-Hz frequency increase corresponded to 541-ng immobilized GOx. Similar research has been reported by Si et al. They used QCM to monitor the frequency change due to the immobilization of protein on a poly(1,3-DAB)– polyaniline film [42]. Poly(o-aminophenol) films deposited on platinum electrodes have also been studied by quartz crystal impedance [43]. Concluding remarks The main advantage of using non-conducting polymers to create enzyme biosensors is their favorable permselective properties. In addition, high sensitivity, fast response time and good reproducibility can be expected because of the ultra-thin non-conducting film used. However, the main drawback of this method is the limited amount of immobilized enzyme during electropolymerization of nonconducting layers, which is because the self-limiting non-conducting polymer film is very thin. Composite structures or materials that include non-conducting polymers are useful for enhancing the amount of immobilized enzyme and decreasing the interference by easily oxidized species in the sample. Also, PPyox can be employed as a non-conducting polymer. The relatively recent development of this immobilization strategy could have a profound effect on future research. Acknowledgements This material is based upon work funded by Zhejiang Provincial Natural Science Foundation of China, grant number M203106.
References 1 Chung, T.D. et al. (2001) Reproducible fabrication of miniaturized glucose sensors: preparation of sensing membranes for continuous monitoring. Biosens. Bioelectron. 16, 1079– 1087 2 Vidal, J-C. et al. (2003) Recent advances in electropolymerized conducting polymers in amperometric biosensors. Microchim. Acta 143, 93 – 111 3 Gerard, M. et al. (2002) Review: application of conducting polymers to biosensors. Biosens. Bioelectron. 17, 345– 359 4 Saxena, V. and Malhotra, B.D. (2003) Prospects of conducting polymers in molecular electronics. Curr. Appl. Phys. 3, 293 – 305 5 Cosnier, S. (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films: A review. Biosens. Bioelectron. 14, 443 – 456
Review
TRENDS in Biotechnology
6 Palmisano, F. et al. (2000) Amperometric biosensors based on electrosynthesised polymeric films. Fresenius J. Anal. Chem. 366, 586 – 601 7 Nakabayshi, Y. et al. (1998) Amperometric glucose sensors fabricated by electrochemical polymerization of phenols on carbon paste electrodes containing ferrocene as electron transfer mediator. Anal. Sci. 14, 1069 – 1077 8 Nakabayashi, Y. and Yoshikawa, H. (2000) Amperometric biosensors for sensing of hydrogen peroxide based on electron transfer between horseradish peroxidase and ferrocene as a mediator. Anal. Sci. 16, 609 – 613 9 Carelli, I. et al. (1996) Electropolymerization of hydroxybenzene and aminobenzene isomers on platinum electrodes to assemble interference-free electrochemical biosensors. Electrochim. Acta 41, 1793 – 1800 10 Calvoa, E.J. and Danilowiczb, C. (1997) Amperometric enzyme electrodes. J. Braz. Chem. Soc. 8, 563– 574 11 Bartlett, P.N. and Cooper, J.M. (1993) A review of the immobilization of enzymes in electropolymerized films. J. Electroanal. Chem. 362, 1 – 12 12 Zhang, Z. et al. (1996) A glucose biosensor based on immobilization of glucose oxidase in electropolymerized o-aminophenol film on platinized glassy carbon electrode. Anal. Chem. 66, 1632 – 1638 13 Terubisa, K. et al. (1998) Charge-transport processes at poly-oaminophenol film electrodes: electron hopping accompanied by proton exchange. Electrochim. Acta 43, 723– 731 14 Ortega, J.M. (2000) Conducting potential range for poly(o-aminophenol). Thin Solid Films 371, 28 – 35 15 Eddy, S. et al. (1995) The modification of enzyme electrode properties with non-conducting electropolymerized films. Biosens. Bioelectron. 10, 831 – 839 16 Cordas, C.M. et al. (2002) EQCM study on the polytyramine modified electrodes for the preparation of biosensors. In Nanostructured Materials and Coatings for Biomedical and Sensor Applications (Gogotsi, Y.G., ed.), pp. 371 – 376, Kluwer Academic Publishers 17 Situmorang, M. et al. (1998) Electrodeposited polytyramine as an immobilization matrix for enzyme biosensors. Biosens. Bioelectron. 13, 953 – 962 18 Jang, D-H. et al. (1995) Electropolymerization mechanism for poly (o-phenylenediamine) (PPD) and its electrocatalytic behavior for O2 reduction. Bull. Korean Chem. Soc. 16, 392 – 397 19 Martinusz, K. et al. (1997) Impedance analysis of poly(o-phenylenediamine) electrodes. J. Electroanal. Chem. 433, 1 – 8 20 Somasundrum, M. and Aoki, K. (2002) The steady-state current at microcylinder electrodes modified by enzymes immobilized in conducting or non-conducting material. J. Electroanal. Chem. 530, 40 – 46 21 Griffith, A. et al. (1996) Probing enzyme polymer biosensors using X-ray photoelectron spectroscopy: determination of glucose oxidase in electropolymerized films. Biosens. Bioelectron. 11, 625– 631 22 Yao, T. et al. (2003) Simultaneous determination of glucose and L-Lactate in rat brain by an electrochemical in vivo flow-injection system with on-line microdialysis sampling. Anal. Sci. 19, 61 – 65 23 Wang, J. et al. (2000) One-step electropolymeric co-immobilization of glucose oxidase and heparin for amperometric biosensing of glucose. Analyst 125, 1431– 1434 24 Daly, D.J. et al. (2000) The use of electrochemically grown polymers on metallized electrodes to reduce electrode fouling in biological matrices. Biochem. Soc. Trans. 28, 89– 94 25 Ekinci, E. et al. (1996) Electrochemical synthesis and sensor application of poly(1,4-diaminobenzene). Synth. Met. 79, 57 – 61 26 Curulli, A. and Palleschi, G. (2000) Construction and application of
www.sciencedirect.com
Vol.22 No.5 May 2004
27
28
29
30
31
32
33
34
35
36
37 38
39
40
41
42
43
231
highly selectively sensors and biosensors using non-conducting electropolymerized films. In Proceedings of The 2nd Workshop on Chemical Sensors and Biosensors (Mazzei, F. and Pilloton, R., eds), pp. 439– 444 Xu, J. et al. (2002) Glucose biosensors prepared by electropolymerization of p-chlorophenylamine with and without Nafion. Anal. Chim. Acta 463, 239 – 247 Guerrieri, A. et al. (1998) Elelctrosynthesized non-conducting polymers as permselective membranes in amperometric enzyme electrodes: a glucose biosensor based on a co-crosslinked glucose oxidase/overoxidized polypyrrole bilayer. Biosens. Bioelectron. 13, 103– 112 Vidal, J.C. et al. (1999) In situ preparation of a cholesterol biosensor: entrapment of cholesterol oxidase in an overoxidized polypyrrole film electrodeposited in a flow system; determination of total cholesterol in serum. Anal. Chim. Acta 385, 213 – 222 Vidal, J.C. et al. (1999) In situ preparation of overoxidized PPy/o PPD bilayer biosensors for the determination of glucose and cholesterol in serum. Sens. Actuat. 57, 219 – 226 Schinichi, K. et al. (1997) Potentiometric biosensor for urea based on electropolymerized electroinactive polypyrrole. Electrochim. Acta 42, 383– 388 Osaka, T. et al. (1996) High-sensitivity urea sensor based on the composite film of electroinactive polypyrrole with polyion complex. Sens. Actuat. 35-36, 463 – 469 Karalemas, I.D. and Georgiou, C.A. (2000) Papastathopoulos D.S., construction of a L-lysine biosensor by immobilizing lysine oxidase on a gold-poly(o-phenylenediamine) electrode. Talanta 53, 391– 402 Mizutani, F. et al. (1998) High-throughput flow-injection analysis of glucose and glutamate in food and biological samples by using enzyme/ polyion complex-bilayer membrane-based electrodes as the detector. Biosens. Bioelectron. 13, 809 – 815 Klinchan, S. et al. (2002) Construction of sensor chips by electrochemical polymerization techniques for sucrose determination. The Journal of KMTMB 12, 12 – 16 Stoecker, P.M.W. and Yacynych, A.M. (1995) Galactose biosensors using composite polymers to prevent interferences. Biosens. Bioelectron. 10, 359– 370 Yang, H. et al. (2002) Glucose sensor using a microfabricated electrode and electropolymerized bilayer films. Biosens. Bioelectron. 17, 251–259 Murphy, L.J. (1998) Reduction of interference response at a hydrogen peroxide detecting electrode using electropolymerized films of substituted naphthalenes. Anal. Chem. 70, 2928 – 2935 Chung, T.D. (2003) Electropolymerized thin bilayers of poly-5-amino1-naphthol and poly-1,3-phenylendiamine for continuous monitoring glucose sensors. Bull. Korean Chem. Soc 24, 291 – 294 Vidal, J.C. et al. (2003) Comparison of biosensors based on entrapment of cholesterol oxidase and cholesterol esterase in electropolymerized films of polypyrrole and diaminonaphthalene derivatives for amperometric determination of cholesterol. Anal. Bioanal. Chem. 377, 273– 280 Malitesta, C. et al. (1990) Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film. Anal. Chem. 62, 2735– 2740 Si, S.H. et al. (1995) Electrochemical quartz crystal microbalance study on electropolymerization of m-phenylenediamine: effects of aniline and polyaniline. Electrochim. Acta 40, 2715– 2721 Ortega, J.M. (1998) Studies of poly(o-aminophenol) by quartz crystal impedance measurements. Synth. Met. 97, 81 – 84