Electrochemical polymerization is a simple and at- tractive approach for the immobilization of enzymes at electrode surfaces. The process, which is also referred.
Journal of Electroanalytical Chemistry, 362 (1993) 1-12
1
JEC 02882
A review of the immobilization of enzymes in electropolymerized
films
P.N. Bartlett Department of Chemistry, University of Southampton, Southampton, SO9 5NH (UK)
J.M. Cooper Department of Electrbnics and Electrical Engineering, University of Gksgow, Glasgow, G12 8QQ (UK) (Received 24 February 1993)
Abstract The use of electrochemically polymerized films to imkobilize enzymes at electrode surfaces is reviewed, and the interpretation and modelling of the results from these studies is discussed.
1.Introduction Electrochemical polymerization is a simple and attractive approach for the immobilization of enzymes at electrode surfaces. The process, which is also referred to as electrochemical immobilization, involves the electrochemical oxidation of a suitable monomer. from .a solution containing enzyme to form a conducting or non-conducting polymer on the surface of the electrode. The electropolymerization solution should preferably be an aqueous solution with a neutral pH in order for the biological component to be incorporated into the polymer film in a suitable form. ” The process can be governed by the electrode potential, and therefore allows accurate control of the polymer film thickness and hence the amount of enzyme entrapped in close proximity to the electrode surface. In addition, since ekctropolymerization occurs locally at the electrode surface, it can be used to confine an enzyme precisely at an electrode without cross-immobilizing it on a neighbouring electrode. This property makes it a suitable procedure for the fabrication of arrays of enzyme microelectrodes. It is also possible to use this technique as a method to build up multilayer structures, either with one or more enzymes layered within a single polymer, or with one enzyme within a multilayered copolymer. Finally, there is the prospect that it may be possible to use electropolymerization as a method of arranging 0022-0728/93/$6.00
for direct electron transfer between the electrode and the active centre of the enzyme via ‘molecular wires’. However, it should be emphasized at the outset that, to date, there are very few cases in which this has been clearly demonstrated. As a consequence of these useful characteristics, the entrapment of enzymes within electropolymerized films has become an increasingly important technique for electronically addressing biological molecules. Consequently, the technique has found a wide range of applications in the fields of both biosensors and molecular electronics [1,2], as well as in the study of heterogeneous enzymic catalysis within membranes. The aim of this review is to outline the mechanism and the kinetic consequences of the immobilization of enzymes in conducting and non-conducting polymers, and to highlight some of the problems which have been encountered in this field. A significant proportion of the research reviewed in this paper has been concerned with the study of polypyrrole (Ppy) films for the imniobilization of glucose oxidase (GOx, EC 1.1.3.4) at electrode surfaces. This is partly because the development of an amperometric enzyme electrode (or biosensor) based upon GGx has become a prime commercial goal, with a variety of biomedical applications in glucose measurement, and partly because GGx is a very robust enzyme and as a consequence provides a stable “model” system. In addition to the investigation of the GGx/Ppy 0 1993 - Elsevier Sequoia S.A. All rights reserved
2
P. Bartlett, .I. Cooper / Review of enzymes immobilized in electropolymerized j&s
TABLE 1. Immobilization of enzymes in electropolymerized
films
Gxidase enzymes + conducting polymers Glucose oxidase (EC 1.1.3.4) Poly+yrrole)
A variety of 3- and N-substituted pyrrole derivatives (e.g. N-carboxy homopolymers and heteropolymers were grown by cycling between -0.3 and + 1.6 V vs. Ag/AgCl
Enzyme immobilized by carbodiimide bonding through carboxy groups; glucose was detected at high current densities, with pennselectivity against interferents, e.g. ascorbate
[21,741
0.5 M monomer + 0.1 M KCI (unbuffered) + 500 U cme3 GOx; potentiostatic growth at 0.61 V vs. Ag/AgCl, ca. 5 pm thick on Pt or glassy carbon electrode
H,O, was found to degrade the polymer films; I-/MoWI) was used to detect H202 indirectly at -0.04 V vs. Ag/AgCl via I, reduction
t431
0.2 M monomer + 0.13 mM Gox, potentiostatic growth at 0.76 V, 2.5 h on printed Pt electrode
H2G2 was detected at 0.66 V vs. Ag/Agcl, benzoquinone at 0.36 V vs. Ag/AgCl or PMS at 0.31 V vs. Ag/AgCl as mediators
1411
0.1 M monomer + 0.1 M KCl (unbuffered) + 50030000 U cmm3 GC$ potentiostatic growth at 0.66 V vs. Ag/AgCl resulted in 850 mC cm-’ on Pt electrode
Differential pulse voltammetric peak at - 0.36 V vs. Ag/AgCl at pH 5.3 was assigned to the reduction of enzyme
1231
0.32 M monomer + 0.1 M KC1 f 500 U cmm3 GGx at 0.65 V vs. SCE with Pt electrode
H,O,
1341
destroys the conductivity of the ftis
Incorporation response
of Pt microparticles
improves the
1751
0.2 M monomer + 0.1 mM FCC&H (pH 6.3); potentiostatic growth at 0.8 V vs. SCE using Pt electrode gave 200 mC cm-*
Glucose detected at 0.4 V vs. SCE due to entrapped FcCO,H as a mediator species
1391
Polymer chemically grown in a photo-cross-linked _PVA layer containing GGx and Fc
Glucose detected at 0.2 V vs. SCE
WI
0.2 M monomer + 180 U cmv3 GGx + 0.1-10 mM hydroquinone sulphonate; potentiostatic growth at 0.8 V vs. SCE at a Pt electrode, deposition time ca. 7min
Glucose was detected at 0.35 V vs. SCB with hydroquinone sulphonate entrapped in film acting as mediator
1191
Enzyme adsorbed on to preformed polymer films by dipping in lo-50 mg cmq3 GGx solution
H,O, or Fc were used for. glucose detection; also used covalently attached Fc
W31
0.2 M pyrrole + 1, mg cmm3 GGx + 0.1 M phosphate buffer (pH 7.01 at 1.0 V vs. Hg/HgSO, (0.5 M H2S04)
Assay performed at 0.58 V vs. Hg/HgSO, (0.5 M HsSO;) in 0.1 M phosphate buffer containing 2 mM benzoquinone as a mediator
Ml
GGx covalently attached to Ppy after film deposition
Used in flow injection analysis
1221
0.05 mg cme3 GGx/035 M pyrrole, grown at 0.5 ~1A with no supporting electrolyte
Bilayer structure formed by the deposition of PC followed by GGx/Ppy; glucose was detected at 0.5 V vs. SCE at a PC layer (stability 1 month)
1721
0.1 M pyrrole for IO mC at 0.7 V vs. Ag/AgCI; GGx derivatized by coupling the surface lysines to N-(2-carboxyetl@pyrrole
High enzyme loading and better stability
1731
0.4 M pynole + 250 U cme3 GGx at 0.7 V vs. Ag/AgCl for 300 mC cm-*
Glucose detected at 0.7 V vs. Ag/AgCl; used in flow i&&on analysis; overoxidized Ppy is permselective and prevents fouling of the electrode surface
1711
Ppy grown in track-etched membranes with GGx
Direct electron transfer claimed; glucose detected independently of oxygen concentration at 0.35 V vs. Ag/Agc1
136,371
P. Bartktt, I. Cooper / Reviewof enzymes itnndiked in el~ctropolymerized @ns
3
TABLE 1 kontinued) Glucose oxidase (EC 1.1.3.4) PolyWmethyl pyrrole)
0.05 M monomer + pH 7.2 phosphate buffer with 0.1 M TEAm, films were grown at 0.8 V vs. SCE
Analysis of the effect of enzyme loading and film thickness on response showed that H,O, was detected at Pt surface and not at the polymer (at 0.8 V vs. SCE)
[331
Polyp-methyl pyrrole)
0.05 M monomer + pH 7.2 phosphate buffer with 0.1 M TEATFB and various concentrations of [Fe(CN),14-; films were grown at 0.79 V vs. SCE
Analysis of the effect of enzyme [Fe(CN),14-/3concentrations; glucose detected at 0.45 V vs. SCE via the mediator, which reacts at the Pt electrode surface and not at the polymer
1201
Fc-substituted PPY
Copolymer of pyrrole and Fc-substituted pyrrole grown by cycling between -0.4 V and 0:96 V vs. Ag/AgCl
Glucose detected at 0.26 V vs. Ag/AgCh mediation after 2 days
WI
Poly(aniline)
0.2 M monomer in 1.2 M HCl at 600 mV vs. SCE, polymer reduced at -500 mV to remove anions and then GOx entrapped as apolyanion
Glucose detected at 0.6 V vs. SCE
[761
0.02 M monomer + 0.15 M phosphate buffer (pH 7.2) + 0.1 M TEATFB + l-10 mg cmm3 enzyme; potentiostatic growth at 1.4 V vs. SCE on Pt electrode
Very thin films with poor stability; H202 detected at 0.9 V vs. SCE
WI
0.1 M monomer +O.l M phosphate buffer (pH 7) + 2.0 mg cmm3 enzyme; potentiostatic growth at 1.24 V vs. Ag/AgCl for 30 s at Pt electrode
Decrease in 0, reduction current at -0.54 V vs. Ag/AgCl was monitored
[491
0.1 M aniline (PH 1.1); growth by cycling -0.2 to +0.7 V vs. SCE at 100 mV s-t with l-4 mg cmW3 GGX
Glucose measured at pH 5.5; not clear which species responsible for the mediation
[SOI
Glucose not detected at 0.7 V vs. SCE, competition between electrode and catalase for H202; direct electron transfer is unlikely
[351
Dopamhte and related catecholamines detected amperometrically at 0.7 V vs. SCE, good response time and favourable selectivity
[31
Various phenols detected air-saturated buffer
V vs. SCE in
1701
FcC02H used as a mediator; cholesterol detected at 0.4 V vs. SCE, co-immobilised cholesterol esterase in some films was used to detect the cholesterol ester
[671
H202 detected at 0.15 V vs. Ag/AgCl; suggests that pyrrole oligomers may mediate HRP electron transfer
1681
Uric acid detected at 0.35 V vs. SCE
[771
GGx + catalase (EC 1.11.1.6) 30 mM pyrrole + 2.5 mg cmm3 GGx + 3.2 mg cmm3 pw catalase at 0.7 V vs. SCE Polyfphenol) oxidase/whole cell (EC 1.14.18.1) Whole cells (banana) or poly(pheno1) oxidase enPPY trapped by potentiostatic growth at 0.91 V vs. SCE. from 0.1 M monomer in phosphate buffer (pH 7.0) with 0.1 M KCl Tyrosinase (EC 1.14.18.1) at electropolymerized N-substituted amphiphilic pyrrole (at 0.75 V vs. SCE) Cholesterol oxidase (EC 1.1.3.6) Galvanostatic growth (10 PA cmM2 to 1 mA cn~-~) PPY from 0.1 M pyrrole + 0.5 mM FcC02H + 14 U cmm3 enzyme (pH 8.0) with no added electrolyte Horseradish peroxidase (EC 1.11.1.7) 50mMpyrrole+60mMKCl+0.6mgcm-3HRP PPY deposited on Sn02 at 0.1 mA cmm2 Uricase (EC 1.7.3.3) Poly(aniline) Ekyme adsorbed into polymer film after growth by oxidation at 0.6 V vs. SCE
enzyme electrode, a-wide range of other enzymes, and
other co&wting and non-conducting polymers, have also beepl investigated (see Table 11, demonstrating the high level of interest in this research topic at present.
at -0.2
loss of
Although not fully comprehensive, Table 1 sets out to illustrate the variety of these investigations and to higbligbt ,some of the impormnt research papem in this area.
4
P. Bartlett, J. Cooper / Review of enzymes immobilized in electropolymerized j&s
TABLE 1 (continued) Dehydrogenase enzymes -I-conducting polymers Dehydrogenases (NAD +/NADH) Polymer of RMIII) complexed pyrrole grown potenPPY tiostatically at 0.82 V vs. SCE in acetonitrlle and TBAP
Film reduces enxymically generated NAD’ in solution; no enzyme incorporated in polymer film, although there is obvious potential to do so
[551
NADH re-oxidized in Ppy + enzyme film at 0.1 V vs. SCE (but not at Ppy film), possibly direct electron transfer; faster rate of oxidation with mediators
1561
NQS mediated the amperometric detection of glucase at 0.2 V vs. SCE, response was independent of oxygen and linear to > 20 mM glucose
[541
Glutamate dehydrogenase (EC 1.1.4.47) 0.1 M monomer + 3 mM NADP + 10 mM PMS + 60 PPY U IX-~ GLDH in 0.1 M KCI; 0.8 V vs. Ag/AgCl, 880 mC cm-’ on Pt
Glutamate detected at 0.3 V vs. SCE; PMS was mediator for the oxidation of NAD(P)H
[521
Alcohol dehydrogenase (EC 1.1.1.71) 0.1 M monomer + 25 mM NADH + 100 mM Mel4Y dola’ blue + 15 mg cmm3 enzyme- in 0.1 M KCl; potentiostatic growth, 0.95 V, 1 C cm-3 deposited on Pt electrode
Ethanol detected at 0.25 V vs. Ag/AgC$ Meldola blue used as mediator for the oxidation of enzymitally produced NADH
[511
Direct electrochemistry vs. Ag/AgCl
of FDH observed at 0.6 V
[69]
Site-to-site electron transfer between electrode and bound ferredoxin at -0.8 V vs. SHE in aqueous solution
[781
Glucose detected at 1.56 V vs. Ag/AgCl; data analysis is incorrect because wrong model is used
[47]
Detection of H,O, at 0.8 V vs. SCE, rapid response, good stability on storage
1651
These tilms have also been used to reduce interference from other solution species
[79]
50 mM monomer + phosphate buffer (pH 7.0) + 510 mg cmm3 enzyme; potentiostatic gtowth at 1.4 V vs. SCE at Pt &&rode
Detection of H,O, at 0.8 V vs. SCE; good stability, response saturates at ea. 0.1 M glucose
I481
GQx in a range of different poly-substituted nol) films
Detection of H,O, at 0.8 V vs. SC@ data analysis using thin-film model
[27]
NADH dehydrogenase (EC 1.6.99.-j 0.1 M monomer + 2.0 mM mediators galvanostatiPPY tally; Fc and quinone derivatives incorporated
Glucose dehydrogenase (EC 1.1.4.47) 0.1 M monomer + 10 U cme3 GDH + 0.1 mM Pl?Y NAD’+ 0.1 mM, fis with NQS grown galvanostatically
Fructose dehydrogenase CPQQ enzyme EC 1.1.99.11) 0.1 M pyrrole t 0.1 M KCl at 0.7 V vs. Ag/AgCl PPY Miscelhaneous enzymes + conducting polymers
Redox proteins PPY
Coelectropolymerlzation of cysteinyl-derived pyrroles and alkylammonium pyrrole at 0.7 V vs. Fc+/Fc; reduction of S-S by D’IT and exposure to MeCN, ferrodoxin clusters ionically bound
Oxidase enzymes + non-conducting polymers Glucose oxidase (EC 1.1.3.4) Poly(indole) 20 mM monomer in MeCN + 0.1 M TEAP f 0.15 mM GOx; grown potentiostatically at 1.36 V vs. Ag/AgCl for 1 h at Pt electrode Polyto-phenylene-diamine)
Polfiphenol)
5 mM monomer in pH 5.2 buffer (I =c0.2) + 500 U cme3 GOx, grown potentiostatically at 0.65 V vs. SCE at Pt elkctrode
(phe-
2. Biolqgieal materials used in electropolymerization studies
A vaxiety of different biological materials lyve been immobilized into either conducting or non-conducting
electropolymerized films, ranging in size from whole cells [3] to protein fragments [41. However, most of the work has been carried out using the entrapment of enzymes for the development of amperometric biosenSOTS.
P. Bartlett,J. Cooper / Reviewof enzymesiinmobilizedin electropolymerized films
5
\
TABLE 1 (continued) Glucose oxidase (EC1.1.34) GQx and o-amino acid oxidase (EC 1.4.3.3) 25 pm diameter Pt electrode; films grown at 0.9 V/SCE for 5 min from 3.15 mg cmm3 GOx + 0.1 M TEATPR + 0.05 phenol (pH 7.0) Electroplated
Pt
5 mM K,PtCl, + 8% enzyme + 0.1 M phosphate buffer (pH 7.4) cycled between - 0.75 and + 0.15 V using a glassy carbon electrode
GGx and o-amino acid oxidase immobilized at’ Pt microelectrodes; enzyme also preadsorbed onto electrode prior to growth; polyphenol films block interference from ascorbate and urate
[66]
Detection of H,O, at 0.7 vs. Ag/AgCh flow injection analysis systems
El
used in
A similar method has been used with carbon fibre electrodes and with Pt electrodes
lW811
PMS, phenazine methosulphate, PVA, polyfvinyl alcohol); PC, polymetallophthalocyanines; TEATPR, tetraethylammonium tetraflavoborate; HRP, horseradish peroxidase; TRAP, tetrabutyhumnonium perchlorate; NQS, naphthoquinonesulphate; GLDH, glutamate dehydrogenase; GDH, glucose dehydrogenase; PDH, fructose dehydrogenase; DTT, dithiothreitol; T&W, tetraethylammonium perchlorate
Of the many classes of enzyme available to the investigator, research has necessarily centred on the redox enzymes. These proteins catalyse the conversion of the substrate between the reduced or oxidized state, which can subsequently be detected at the electrode. Although entrapped redox enzymes within polymers have generally been studied using amperometric techniques, other electrochemical techniques have also been investigated, particularly conductimetry or potentiometry Dl. The redox enzymes, when classified by function, are dominated by two of the largest groupings of enzymes, the oxidase enzymes and the dehydrogenase enzymes. Like all redox proteins, these enzymes are characterized by having a prosthetic group which contains a covalently bound nucleotide (e.g. FAD), a complex quinone (e.g. PQQ) and/or a coordinated transition metal, usually iron (e.g. haem), molybdenum, copper, or zinc. The prosthetic group is positioned within the polypeptide matrix of the enzyme, often close to the protein’s surface to enable a redox interaction with the substrate. The prosthetic group, in conjunction with its surrounding amino acid structure, acts as the enzyme’s “active site”. The generalized reaction scheme for redox enzymeelectrodes is. substrate( RED) + enzyme( OX), + product( OX) -t enzyme( RED)
( 1)
enzyme( RED) + cosubstrate( OX) + enzyme( OX) + coproduct( RFD)
(2)
The oxidase enzymes use oxygen, which acts as an electron acceptor, as their primary co-substrate. GOx, obtained from Aspergillus niger, is an example of an oxidase enzyme. It is relatively large, with a relative molar mass of ca. 1.86 kDa and a hydrodynamic radius of 4.3 run. It is a dimeric flavoprotein composed of two
polypeptide chains, and is covered with a glycoprotein coat. The enzyme has one FAD prosthetic centre associated with each monomer. The poor direct electrochemistry of GOx observed both at bare and chemically modified electrodes [61, including polymer-modified electrodes, suggests that the FAD is buried deep within the enzyme molecule at a distance beyond which electron tunnelling can readily occur ( 2 1 nm). GOx catalyses the oxidation of glucose, with a subsequent two-electron equivalent reduction of a single molecule of oxygen, resulting in the production of one molecule of H,O,: glucose + GOx( OX) + gluconolactone
+ GOx( RED) (3)
GOx(RED)
+ 0, + GOx(OX)
+ H,O,
(4)
In general, oxidase enzymes entrapped within polymer fihns can be assumed to be dependent upon the presence of oxygen, unless an alternative electron acceptor is present. As a consequence, in the generalized scheme for an oxidase/polymer enzyme electrode involving the two-electron reduction of oxygen it is assumed that H,O, will be enzymically produced: H,O, + 2H++ 0, + 2e-
(5)
Under suitable conditions the rate. of H,O, production is directly related to the substrate concentration. The H,O, can be measured by electrochemical oxidation at potentials above about 0.7 V vs. SCE. Equations (l)-(5) outline the basis on which many glucose biosensors operate and also represent the principle upon which many generic-oxidase-based polymer electrodes (fabricated using either conducting or non-conducting films) function. The dehydrogenase enzymes differ from the oxidase enzymes in that in vivo, in place of oxygen, they use a soluble physiological nicotinamide cofactor as either an
6
P. Bartlett, 3. Cooper / Review of enzymes immobilized in electropolywierizedf&s
electron acceptor (i.e. NAD+) or an electron donor (i.e. NADH). In vitro, the dehydrogenase enzymes may use non-physiological mediators, as is the case for many of the PQQ enzymes. The direction of the dehydrogenase-enzyme-catalysed reaction is highly dependent on pH, and by decreasing the pH it is possible to drive the “back” reaction in the “forward” direction. Both oxidase and dehydrogenase enzyme catalysis can be mediated by a wide variety of inorganic species which act as alternative electrons acceptors. The most common of these are ferrocene and its derivatives, which have been used for the development of a number of electrochemical assays [7-91. The use of such mediators, incorporated into electropolymerized films as ions, ligands or through covalent attachment of the molecule to the monomer prior to electropolymerization, is discussed later in the text. Several examples are given in Table 1. 3. Electroehemlcal pdymerlzation presence of GOx
of pyrrole in the
A major motivation for the entrapment of glucose oxidase in electrodeposited polymers has been the development of amperometric glucose electrodes. Most of this work has concentrated on the study of GGx in Ppy, and it is for this reason that it is used in this paper to illustrate the underlying mechanisms of enzyme entrapment and applications of the entrapped enzyme. Pyrrole can be electropolymerized to form electrically conducting Ppy films at positive potentials greater than +600 mV vs. SCE, depending on the reaction conditions and the electrode surface. Films are generally grown at a fiied potential or by cyclic voltammetry, the potentiostatic method having the advantage that the thickness of .the polymer film is more easily controlled. The gross morphology of the film, and hence many of its physical characteristics, depend upon a range of variables including the nature of the underlying electrode, the speed of deposition, the ionic and polyionic species present, and the solution pH [lo-141. The general scheme for the electrochemical polymerization of pyrrole is shown in Fig. 1. The first step involves the oxidation of the pyrrole monomer I to give the radical cation II. This radical cation can either react with a second radical cation to give the dimeric species III or can react with a neutral monomer molecule, followed by further oxidation of the dimer to give III. LOSSof two protons then produces the dimer IV. Further oxidation and coupling reactions of this type lead to oligomers and eventually to the deposition of a polycationic polymer at the electrode [15]. Although the precise details of these steps are stili unclear, it is important to note that the radical cation II
Fig. 1. Mechanism for the electrochemical polymerization of pyrrole.
and radical cations derived from the dimeric or oligomeric species can undergo reactions with nucleophiles present in solution to give addition products which will block further polymer growth. As a result, the conditions for the polymerization process will become less favourable as the pH of the solution is increased owing to an increased probability of attack by OH-. This would also suggest that strongly nucleophilic counter-ions such as Cl- should be avoided. Another feature of this mechanism which has, important consequences for the immobilization of enzymes is that protons are liberated within the growing film during electropolymerization. This will cause the reaction solution to become increasingly acidic, particularly within the boundary layer of the film at the electrode surface. If the polymer growth solution is not buffered the large local fall in the pH at the electrode-sohttion interface could result in denaturation of the enzyme and therefore should be avoided. Addition of GGx to the polymerization solution has little effect on the polymerization process itself but generally leads to an increase (by about a factor of 2)
P. Bortlctt,J. Cooper / Review of enzymes immobilized
in the oxidative current, all other conditions remaining the same. A similar effect has been observed in the presence of other polyanions [I61. Since GGx has an isoelectric point p1 of 4.2 at a pH above this value, the enxyme is negatively charged. At pH 7.0, for example, the enzyme carries an overall charge of between -9 and - 10 [17]; Since the Ppy is deposited as a polycation, electrostatic interactions between the polymer and the enzyme will play an important part in the incorporation of the enzyme into the growing film. Support for this view is provided by three pieces of evidence: firstly, that GGx can be adsorbed onto preformed Ppy films [18]; secondly, that when Ppy films containing glucose oxidase are grown in the presence of sulphonated hydroquinone [19] or ferrocyanide [20], the amount of incorporated enzyme and the enzymic activity of the films decreases as the concentration of anion increases; thirdly, that it is very difficult to incorporate positively charged proteins into Ppy films. Alternative methods of incorporating enzymes into the polymer matrix have been described. As well as adsorbing the enzyme on to the preformed Ppy films [18], it can be preadsorbed on to the electrode surface prior to the initiation of the electropolymerization process [20]. If the polymer/enzyme film which is subsequently grown is relatively thin, this will increase the overall concentration of the enzyme and may increase the current response. This latter technique is particularly well suited to the design of enzyme electrodes based on non-conducting polymers, where the polymer film is of limited thickness. As an alternative to physical adsorption, GOx has also been covalently immobilized using carbodiimide onto a wide range of preformed 3- and N-derivatized carboxylate pyrroles [21], or N-amino-substituted pyrroles [22]. This technique may increase current densities at a given glucose concentration by as much as twentyfold when compared with the best of the results obtained elsewhere. However, the authors make no claims as to direct electron transfer between the enzyme and the polymer and cite the metallic anode surface as the position of electrocatalytic oxidation of
inelectropolymebzed fdms
7
polymer and GOx [23], it appears that the process is generally inefficient unless the enzyme is partially denatured during immobilization 1241. One problem in testing for direct electrochemical oxidation of the enzyme by a polymer is that very small amounts of mediator species, either oxygen or some other species, can be very efficiently cycled within the film. Under such circumstances, it would appear that direct oxidation was occurring when in fact mediator recycling was ensuring that the enzyme was efficiently re-oxidized. Therefore it is essential that investigators should be very rigorous in excluding oxygen from the polymer film before claiming direct re-oxidation of the enzyme by the polymer. One way of understanding the enzymic processes and their reaction sites within the polymer film is’ to model the combined effects of substrate and product diffusion and of enzyme kinetics and to compare the predictions of these models with experimental observation. It is important to recognize that the enzyme kinetics for reactions within a polymer film are likely to be different from those for the homogeneous solutions [251. It is probable that the immobilization of the enzyme will decrease the turnover rate of the enzyme and increase K,. It is also important to consider the effects of partition of both reactants and product into the film, it is quite possible that the concentration of substrate within the electropolymerized film will be very different from the bulk solution concentration. Figure 2 is a diagram-
Membrane
Electrode
A
k,
E’
Sample
P
kn
H202.
N-carboxylate derivatized Ppy films have also been shown actively to exclude ascorbate, bioactive amine proteins and sulphydryl-containing peptides as interferents [21], and this is an additional advantage of this approach. 4. The kinetic behaviour of immobilised GOx within a PPY
fh
Although it has been claimed. that there is a possibility of obtaining direct electron transfer between the
.xX B&B Db
E
S
Fig. 2. The general kinetic scheme for an immobilized enxyme electrode. S and P are the substrate and product respectively, apd A/B and E/E’ represent the mediator and enxyme redox couples. The rate terms k, and k, describe the kinetics for the enzyme substrate and enzyme mediator reactions. K, and D, are the paitition and diffusion coefficients for species within the film.
8
P. Barzlett, J. Cooper / Review of enzymesimmubilizedin ekxtrop~merized fibs
matic iliustration of the physi&chemical processes which are iwAved in the enzymic turnover of substrate to product within a polymer film. Such processes include maw ttxuts~ of substrate and product either to or from the film; partition of these species across the polymer-solution interface, transport of reactants and products within the film (by diffision) and electrochemical reaction with enzymic products at the electrode surface. The effects of migration of charged species within the film are usually ignored. In studying such multistep processes it is advantageous to arrange the reaction conditions so that a single step, such as mass transfer or the heterogeneous electron transfer process at the electrode surface, controls the overall rate. For this reason many of the investigations of the kinetics of these systems have made use of the rotating-disc electrode so that the effects of mass transfer in the bulk solution can be controlled and calculated [26]. The study of these complex reactions has generally involved looking at limiting cases to provide approximate solutions to-non-linear relationships. In the case where the polymer film is thin compared with the reaction layer thickness, so that there is no polarization in the substrate concentration through the film, it is relatively easy to derive expressions for the current as a function of the enzyme kinetics ‘and substrate and mediator concentrations [27,28]. When the films are thicker, so that the variation of concentration of the substrate through the film must be taken into account, the problem is more difficult. Approximate solutions have been presented which are applicable to certain limiting cases [26] and digital simulation has been used to explore some aspects of the behaviour of the system [29-311. These studies suggest that the behaviour can be quite complex, and it is essential in any kinetic study to investigate the dependence of the current not only on the substrate concentration but also on changes in film thickness, enzyme loading and mediator concentration before coming to any conclusions about either the site or mechanism of reaction. This is overlooked in too many studies. Details of the kinetic analysis can be found in a recent review [32]. An example of this approach is provided by work on the oxygen mediation for the GOx catalyzed oxidation of glucose in,.a polyWmethylpyrrole) fihn [33]. In this case, investigation of the effect of enzyme loading and film thickness on the amperometric response shows that the H,O, generated by the enzyme reaction is detected by oxidation at the underlying platinum electrode and not by direct oxidation on the polymer, as has been suggested elsewhere for the GOx/Ppy enzyme electrode. A characteristic feature of a system where the mediator species are freely diffusing and
must react at the underlying platinum electrode, rather than within the polymer fii, is that the emzymef polymer electrode exhibits an optimum film thickness at which the response is a maximum [26]. This, occurs because there is an increased response with increasing film thickness as the total quantity of entrapped enzyme increases. In contrast, thick films will tend to show a decreased response because the substrate entering the film tends to react at the outside surface of the polymer. The result of this is that reduced mediator (product) is more easily lost to the bulk solution rather than diffusing through the film to be detected at the underlying electrode. A further implication of ‘this model, which has been demonstrated experimentally, is that the current will depend on the mass transport of the reduced mediator away from the electrode surface and into the bulk solution [26,33]. Further evidence against’ a direct electrochemical mechanism for enzyme re-oxidation is given in a ldetailed study of GOx immobilized in Ppy films by B6langer et al. [34], who have shown that the conductivity of the film is destroyed during the reaction of the polymer with the enzymically produced H,Op. Finally, evidence that the response of GOx/Ppy electrodes is due to H,O, can be obtained from the fact that when catalase is co-immobilised with GOx into a Ppy film, no glucose dependent response is obtained [35]. Catalase is an efficient scavenger of H,O,, and at equimolar concentrations with GOx removes the peroxide before it reaches the anode. However, there have been several claims for either direct oxidation of the enzyme at Ppy [23,36,37] or poly(N-methylpyrrole) [24]. Koopal et al. [36,371 have grown Ppy films inside track-etch membranes to produce conducting microtubules onto which GOx has been irreversibly adsorbed by subsequent adsorption and drying. These films are able to re-oxidize !the reduced form of GOx (FADH,) directly at 0.35 V vs. Ag/AgCl, and are independent of oxygen coneentration. In this case it is possible that the direct electron transfer between the polymer and the enzyme occurs as a result of the manner in which the Ppy filrn is grown and subsequently treated with enzyme, and is a function of the physical (rather than the chemical) interaction with the enzyme. It is known that the properties of Ppy grown in confined spaces can’ be different from those of bulk material [38] and it is possible that this is an important factor. Further detailed studies of these systems seem warranted. For instance, it is not clear why, in this case, the reaction of Ppy with H,O, does not seem to be a problem. It is also interesting to consider the work of de Taxis du Poet et al. [24] on GGx entrapped in poiyW-methylpyrrole) films. In this case, in common with other
P. Bartlett, J. Cooper / Review of enzymes immobilized in electropolymehzed fums
workers, they found no evidence of direct electrochemistry for films grown at 25°C. However, when they prepared films at 50°C they found evidence for direct electrochemistry and observed that the selectivity of the enzyme was significantly reduced. They rationalized these observations by suggesting that at 50°C the enzyme is partially denatured, so that it is entrapped within the film in a more open form allowing access of the polymeric chains to its active site. The fact that, in general, the mechanism for the detection of oxidase enzyme activity within polymer films at electrodes is mediated by H,O, has important analytical consequences for these systems. Since H,O, can only be detected at potentials greater than about 0.65 V vs. Ag/AgCl, the enzyme electrodes, if used in real analytical situations (e.g. in biological fluids), are liable to.problems of low signal-to-noise ratios because of electro-oxidation of interferents such as ascorbate. A number of groups have tried to resolve these problems associated with H,Oz detection by using artificial mediators such as ferrocene derivatives [18,39,4O], ferricyanide [20], phenazine methosulphate [411, benzoquinone [41,42], sulphonated benzoquinone [19] and iodide in the presence of a molybdenum(W) catalyst [43]. Ppy copolymerised with a ferrocene-substituted pyrrole has also been employed [441. The use of such artificial mediators has the advantage that the electrodes can be operated at lower overpotentials, thus reducing the effects of interferent species and eliminating, or greatly reducing, dependence on oxygen concentration. Most of these studies have not addressed the question of where the reaction between the artificial mediator and the electrode occurs. Although it is often claimed that the mediator is re-oxidized on the polymer, direct evidence to support this assertion is sparse. Bartlett and coworkers have performed detailed studies of the behaviour of GOx immobilized in polyWmethylpyrrole) films using both ferrocene monocarboxylic acid [45] and ferricyanide [20] as the mediator species. In both cases their results indicate that the polyW-methylpyrrole) films are insulating and that there is no evidence for the direct re-oxidation of either mediator on the polymer. This is a curious result since these mediators are electroactive at poly(Nmethylpyrrole) films which do not contain GOx. It is important to consider the effect that H,O, has on films of Ppy and its derivations, since it appears that GOx, as well as catalysing the oxidation of glucose in the presence of a mediator, can also catalyse the reduction of oxygen to H,O, in the presence of certain mediators M as electron acceptors: 0, + 2M + 2H+4
H,O, + 2M+
(8)
9
H,O, produced by this reaction can react with Ppy and destroy the conductivity of the film. Although there have been claims for the direct oxidation of mediators on the conducting Ppy chains, clear evidence to support these claims is sparse. In many cases the results can be equally well explained by diffusion of the mediator species to, and re-oxidation on, the underlying electrode. In addition to Ppy and polyW-methylpyrrole), other conducting polymers have been used for enzyme immobilization, including poly(thiophene) [46l, poly(indole) [47] and polyianiline) [48-501 films, Poly(thiophenes) and polfiindole) films have the possible disadvantage that the enzyme/polymer film must be grown from aprotic solvents, conditions which may denature some enzymes. GOx/poly(aniline) films have been grown from aqueous solutions, although acidic solutions (around pH 1) are necessary for the electropolymeriza~ tion and this can also adversely affect the enzyme. A recent study by Cooper and Hall [501 suggests that direct electron transport between GOx and poly-(aniline) is a possible mechanism for the glucose dependent response. Ppy has also been used to entrap a number of dehydrogenase enzymes, including alcohol dehydrogenase 1511,glucose dehydrogenase [52] and lactate dehydrogenase [5]. These enzymes are dependent upon the soluble cofactor, nicotinamide adenine dinucleotide, which can exist either in the oxidized form (NAD+) or the reduced form (NADH). Since NADH cannot be oxidized directly at Ppy films, these enzyme electrodes depend upon the catalytic re-oxidation of NADH using a mediator either entrapped within the film or incorporated as a ligand. Examples of such mediators include ferricyanide [53], napthoquinonesulphonate [54] and rhodium pentamethylcyclopentadienyl bipyridine chloride 1551. The direct oxidation of NADH at Ppy coated electrodes has been achieved at potentials as low as 0.1 V vs. SCE by the incorporation of the enzyme NADH dehydrogenase into the conducting polymer film [56]. These results indicate that there is direct but slow re-oxidation of the enzyme by the conducting polymer. The rate of re-oxidation of the enzyme is considerably enhanced by the incorporation of ferrocene or quinone derivatives as mediators within the films [56]. 5. Imaging of enzymes within conducting polymers using scanning tunnelling microscopy Yaniv et al. 1571 have used scanning tunnelling microscopy WlM) to image GOx immobilized within a Ppy film. They found that the electropolymerization potential has an effect on the appearance of the Ppy +
10
P. Bartlett, J. Cooper / Review of enzymes inmddized
GGx fihns and they observed “black holes” within the the continuous fibrous structure of Ppy which they attribute to clusters of enzymes. The number of clusters increases with increasing potential of electropolymerization of the enzyme-loaded film. Polymer fti grown on graphite surfaces in the absence of the enzyme were more uniform in appearance, and GOx/Ppy films grown at lower potentials (e.g. 0.7 V vs. Ag/AgCl) showed a more even distribution of enzyme within the film (judged by the distribution of the “black holes” in the STM images). In general, however, these experiments have only produced low resolution images with little structural information about either the enzyme or the polymer matrix available, and are therefore open to some degree of subjective interpretation. This is probably’ due to the fact that the scanning tip is easily embedded in the Ppy resulting in a weak dependence of current on tip displacement. 6. Noa-amducting enzymes
pdymers for the immobilization of
The fact that some conducting polymer films become non-conducting in the presence of enzymically produced H,O, has been discussed above. This has prompted investigators to use the electropolymeriza-
Fig. 3. Mechanism for the electrochemical polymerization of phenol.
in electropoiyme~
jBn.9
tion of non-conducting polymers to immobilize enzymes at electrode surfaces. The growth of non-conducting polymer is self-limiting, and ‘consequently the fihns tend to be very thin (10-100 nm), generally resulting in fast diffusion of substrates and products to and from the enzyme. As well as incorporating relatively high amounts of enzyme, these films have been shown to be able to reject interferent species and hence can improve selectivity. The electropolymerization of phenols proceeds in a manner similar to that for pyrrole, (Fig. 3). Oxidation of the phenylate anion V at the electrode produces a radical VI which can either react with impurities or other species present in solution or can react with a further molecule of phenol to give the predominantly #uru-linked dimeric radical VII. Further oxidation of WI and loss of a proton gives the neutral dimer Ma. Subsequent reactions then produce oligomers and finally polymeric material at the electrode surface, It should be noted that me&-linked species such as IX can also be formed, depending on the substitution pattern of the phenol monomer. Thus poly(pheno1) itself is probably a mixture of para- and meta-linked units. The electrochemical polymerization of phenol differs from the polymerization of pyrrole in that the films which are produced in aqueous solution are insulating. As a consequence the fihn thicknesses are selflimiting (10-100 14. However, the films are generally continuous and free from defects such as pinholes [58], and they have been used for corrosion protection 1591, as permselective films 160-621 and as pH sensors [63]. In common with Ppy films, electropolymerized phenol films have a number of features which make them attractive for the immobilization of enzymes. Fimtly, the films can be grown under electrochemical control from aqueous buffered solution at neutral PH. Secondly, a wide variety of derivatized phenols are available which allow some control over the physical characteristics of the films. These insulating ftis are also permselective, a feature which could be useful in pmventing interfering species from reaching the electrode surface or fouling the electrode if used as biosensors. In a recent study Sasso et al. [64] used electropolymerized films of l,Zdiaminobenzene, a monomer which gives films with properties very similar to those of the phenols, to reduce interferences in a glucose sensor. Malitesta et al. [65] have also used the same monomer to immobilize GChr at a platinum electrode. Bartlett et al. [27] have investigated the immobilization of GOx at platinum electrodes in electropolymerized films using five different phenols. Since these films are very thin, it is possible to assume that there is no concentration polarization &thin the film, and to
P. Bartlett,.I. Cooper / Review of enzymes immobilized inelectropo~merized j3n.r
analyse the response to glucose according to a simple model which takes amunt of the glucose/enzyme kinetics. In this way a quantitative comparison of the different films can be made. 7. conchlsion This review illustrates the large variety of work which has been carried out in the field to date. There are undoubtedly many unanswered problems, particularly with respect to the question of how fast reversible direct electron transfer between proteins and conducting polymers can be obtained. Studies to date have amply shown that many polymers and many enzymes can be used and that the resulting films will respond to the substrate for that enzyme. More detailed mechanistic studies are now required to establish clearly the kinetics for the immobilized enzyme reactions, the role of the electropolymerized matrix and the precise nature of the mediation and mechanism of electron transport from the electrode to the enzyme active site. References T. Matsue, M. Nishizawa, T. Sawaguchi and I. Uchida, J. Chem. Sot. Chem. Commun., (1991) 1029. P.N. Bartlett and P.R. Birkin, Anal. Chem., 65 (1993) 118. M. Deshpande and EA. Hall, Biosensors Bioelectron., 5 (1990) 431. S. Cosnier and C. Innocent, J. Electroanal Chem., 338 (1992) 339. A.G. Dubinin, F. Li, Y. Li and J. Yu, Bioelectrochem. Bioenerg., 25 (1991) 131. K. Narasimluun and LB. Wingard, Anal. Chem., 58 (1986) 2989. A.E.G. Cass, G. Davis, G.D. Francis, H.A.O. Hill, W.J. Aston, I.J. Higgins, E.V. Plotkin, L.D.L. Scott and A.P.F. Turner, Anal. Chem., 56 (1984) 667. 8 K. Di Gleria, M.J. Green and H.A.O. I-Ml, Anal. Chem., 58 (1986) 1203. 9 J.M. Dicks, W.J. Aston, G. Davis and A.P.F. Turner, Anal. Chhn. Acta, 182 (1986) 103. 10 A.F. Diaz and J. Bargon in T.A. Skotheim (Ed.), Handbook of Conducting Polymers, Vol 1, Marcel Dekker, New York, 1986, p 81. 11 S. Kuwabata, J. Nakamura and H. Yoneama, J. Electrochem. Sot., 137 (1990) 1788. 12 Q. Pei and R. Qian, J. Electroanal. Chem., 322 (1992) 153. 13 R. Qian, J. Giu and D. Shen, Synth. Met., 18 (1987) 13. 14 A. Witkowski, M.S. Freund and A. Brajter-Toth, Anal. Chem., 63 (1991) 622. 15 G.K. Chandler and D. Pletcher, Specialist Periodic Reports, Electrochemistry, Vol. 10, Royal Society of Chemistry London, 1985, p 117. 16 T. Shim&u, A. Ohtani, T. Iyoda and K. Honda, J. Electroanal. Chem., 224 (1987) 123. 17 R. Bentley in P.D. Boyer, H.A. Hardy and H. Myrback (Ed.%), The Enzymes, Vol. 7, Academic Press, New York, 1973p. 567. 18 J.M. Dicks, S. Hattori, I. Karube, A.P.F. Turner and T. Yokozawa, Ann. Biol. Chn., 47 (1989) 607.
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P. Bartlett, J. Cooper / Review of enzymes immobilized in electrcpolymerized j%ns
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