The modification of enzyme electrode properties with non-conducting electropolymerised films

Biosensors & Bioelectronics lO (1995) 831-839 The modification of enzyme electrode properties with non-conducting electropolymerised films S. Eddy, K. Warriner, I. Christie, D. Ashworth, C. Purkiss & P. Vadgama University of Manchester, Department of Medicine, (Clinical Biochemistry), Hope Hospital, Eccles Old Road, Salford M6 8HD, UK. Tel: 0161 787 5991 Fax: 0161 787 7432. Abstract: Phenol and several phenol derivatives have been electropolymerised on to platinum anodes. The selective properties of the modified electrodes were dependent on the final polymeric structure. Although essentially nonconducting, dual phenolic films could be deposited on electrode surfaces. This was achieved by the initial deposition of a porous polymer formed from, phenol red, followed by the more diffusion limiting film of oxidised phenol. The resulting composite film showed the combined selective characteristics of the two component polymers. Attempts to enhance polymeric film selectivity via the incorporation of charged or neutral surfactants are outlined. The characteristics of glucose oxidase incorporated into (poly)phenol films are discussed. Keywords: amperometry, enzyme entrapment, glucose determination, modified electrodes, phenols, selectivity, surfactant INTRODUCTION Amperometric enzyme electrodes have received considerable attention in recent years (Yacynych, 1992). Enzyme incorporation to form a highly selective biological recognition layer considerably extends the analytical range of the base electrochemical transducer offering a route to the reagentless analysis of many solutes of biological interest including glucose, lactate, and acetaminophen. Practical realisation has involved several approaches including the use of membrane technology to modify solute access to the enzyme layer (Vadgama, 1990; Koochaki et al., 1993), provide a biocompatible surface (Turner et al., 1991; Davies et al., 1992; Higson et al., 1993), 0956-5663/95/$09.50 and confer a reduction in interference (PaUeschi et al., 1986). In this way direct whole blood assay has been possible with minimum sample dilution and preparation. Membrane materials commonly used by our group for a range of diffusion control and selectivity function have included polyaryl sulphone, cellulose acetate, and microporous polycarbonate, and polyurethane (Christie & Vadgama, 1994). Many such preformed membranes present significant diffusion distances for solutes, up to 10/xm, and therefore can add significantly to response time. For most purposes resulting time to steady state is acceptable, but it is valuable to extend the technology to the use of thinner membrane phases. Furthermore, coatings would be of possible advantage in 831 S. Eddy et al. diminishing diffusion of passivating species to the underlying electrode in blood (Higson et al., 1993). Electropolymerised films based on polyphenol (Bartlett & Whitaker, 1987), polyaniline (Cooper & Hall, 1992; Pud, 1994), polyindole and polypyrrole (Fortier et al., 1990) have been seen as an alternative to conventional membranes (Wang et al., 1989). The oxidation of species at an anode can lead to modified electrode behaviour with beneficial properties, including optimised selectivity (Sasso et al., 1990; Groom & Lvong, 1993) and protection of the electrode surface from passivation, in the past this oxidation was seen as disadvantageous and referred to as electrode fouling. Interest in modified electrodes has tended to centre on electron conducting or redox polymers (Foulds & Lowe, 1986; Pud, 1994), but non-conducting polymers are an ideal membrane equivalent (Christie & Vadgama, 1993). Because the growth of non-conducting polymers is selflimiting, the resulting films formed are very thin (10-100 nm; Bartlett & Cooper, 1993). The response time of these modified electrodes can in principle, approach that of a bare electrode (Christie & Vadgama, 1993). Phenol has been especially studied in this regard with a mechanism for electrochemical polymerisation proposed based on polymerisation through simple radical initiation (Bartlett & Cooper, 1993). Other advantages are that such films are usually free from defects such as pinholes, and are potentially robust, indeed they were originally used for their anti-corrosion properties (Subramanian, 1980). Phenol films have been shown to have favourable perm-selective properties for hydrogen peroxide over interferents of clinical significance in amperometric assays, notably ascorbate, acetaminophen, and uric acid (Christie & Vadgama, 1993). This selectivity appears to be based predominantly on size exclusion, the film thus acting as a molecular sieve. Such films can also be used to immobilise enzymes (Bartlett & Whitaker, 1987; Bartlett & Whitaker, 1987; Bartlett et al., 1992), moreover with mild conditions for electropolymerisation entrapment, enzyme activity can be retained more readily (Bartlett & Cooper, 1993). Electrochemical polymerisation is a simple and attractive means of enzyme immobilisation at an electrode surface. The process requires simply electrochemical oxidation of a suitable monomer from solution containing enzyme. The process more832 Biosensors & Bioelectronics over can be governed by polarisation potential and duration which can allow accurate control of the film thickness and the amount of enzyme entrapped (Bartlett & Cooper, 1993). Precise confinement of an enzyme at an electrode without cross-immobilisation at a neighbouring electrode, would be a particularly favourable aspect in the production of microelectrode arrays (Bartlett & Caruana, 1992; Bartlett & Caruana, 1994). The present study was concerned with a comparative examination of phenolics notably phenol, phenol red and 4-aminophenol. Polymer formation on platinum, subsequent selectivity, and ability to retain enzyme, using glucose oxidase (GOx) as a model, is discussed. GOx was chosen for entrapment as it is a very robust enzyme and therefore provides a more stable system to study. Glucose is also an important analyte both in medicine and the food industry. EXPERIMENTAL Apparatus The electrochemical cell used was obtained from Rank Bros. (Bottisham, Cambs., UK). This consisted of a 2 mm diameter platinum anode, surrounded by a 12 mm diameter annular Ag/ AgC1 pseudoreference. Polarisation at +0.85V vs. Ag/AgCl was achieved with a variable voltage source, also equipped to measure current in the range 0.1 nA to 2.0 mA (Department of Chemistry Workshop, University of Newcastle-UponTyne). The output was recorded using a Goertz Metrawatt SE120 (Austria) strip chart recorder. Membranes used to cover the electrode surface were secured with an O-ring held in place by the sample chamber when the cell was assembled. Membrane fabrication Cellulose acetate, 37.8% acetyl content (Aldrich, Gillingham, UK), membranes were formed from 1 ml of 2% w/v solution in acetone, cast on a 5 × 5 cm glass plate, manually rotated for several minutes while the solvent evaporated, and then left for at least 2 h before use. PVC membranes were cast from solutions of 0.06 g PVC (200,000 Daltons, BDH) and 150/xl isopropyl myristate (Fluka, Buchs, Switzerland) in 5 ml tetrahydrofuran poured into a Petri dish, (covered and left Biosensors & Bioelectronics at room temperature for the solvent to evaporate; - 4 days). Coating and measurement procedures Experiments were performed in buffered solution containing, per litre, 2.44 g NaH2PO4.H20, 7.5 g Na2HPO4, 3 g NaCI, and 0.6 g disodium EDTA adjusted to pH 7.4 with 1 M HCI. Phenol (Anal. grade) and phenol red were obtained from Sigma Chem Co (Poole, Dorset), 4-aminophenol from Fluka. All solutions were prepared in buffer. Electrodes were cleaned by scrubbing with 0.3/zm alumina then a drop of concentrated nitric acid was placed on the anode for 15 min followed by rinsing in distilled water. The cell was conditioned for several hours in buffer at +0.85V vs. Ag/ AgCI until a constant baseline was achieved ( - 5 nA). To prevent damage to the films from the action of a rotating stirrer bar, an overlying high permeability cellulosic dialysis membrane from a haemodialysis cartridge (Gambro, Lund, Sweden) was employed. Coatings were formed through the dialysis membrane by continued polarisation of the anode (+0.85V vs Ag/AgC1) in the presence of a 5 mM buffered solution of the coating species, 0.5 mM for phenol red. A potential of +0.85V vs. Ag/AgCI was found to electropolymerise all the phenolics used, without causing raised background currents due to electrolysis of water. Coating was initiated by the placement of 1 ml of the coating species in the cell chamber, and terminated by its removal and replacement with buffer. Coating times were between 1-24 h depending on the phenolic species used. The response to 0.1 mM ascorbate was used to assess the integrity of the resulting films. The surfactants used in order to modify phenolic films were deoxycholic acid, cholic acid, Noctadecyl-N,N-dimethyl-3-ammonio-2propanesulphonate, and n-decyl-b-D-maltopyranoside, purchased from Sigma. Surfactant was present at a concentration of 10 mM in the phenolic solution, and the coating procedure as above performed. Cyclodextrin (Sigma) was present at 1% (w/v) in the phenol solution. Enzyme entrapment Glucose oxidase (E.C. 1.1.3.4, Type VII, 180, 000 U/g) from Aspergillus niger was obtained from Sigma. Varying enzyme quantities were dissolved in 1 ml of the phenolic solutions, and Modification of enzyme electrode properties the coating procedure followed as above. After completion a cellulosic dialysis membrane was carefully laid over the film to prevent damage by the action of the stirrer. Enzyme laminate GOx solutions were made up containing 30 mg/ ml enzyme (Sigma) and 200 mg/ml BSA (Sigma). 10/xl of this solution was combined with 5/zl 5% w/v glutaraldehyde when required for deposition in enzyme laminates. The procedure for laminate formation was common to all polymer membranes. 5 txl of enzyme/BSA/glutaraldehyde solution was sandwiched between two 1.5 × 1.5 cm membranes. The laminate was then compressed between two microscope slides for 10 min until the cross linking was complete, then rinsed in buffer before being placed in the electrochemical cell. Responses to glucose, hydrogen peroxide, and interferents were determined by adding stock solutions to buffer solution in the cell. All solutions were prepared daily except glucose which was prepared at least 24 h in advance to allow for mutorotation and then stored at 4°C. The pH of buffer was altered by the addition of 1 M HC1 or NaOH solution, to obtain solutions with a pH range between pH 2-8 for the investigation of the effect of pH on enzyme entrapped within a phenolic film. RESULTS Electropolymerised film studies The following study was initiated to demonstrate the properties of phenolic films, the intention was not to optimise procedures for specific applications. Monophenol deposition in stirred solution gave current decay curves which confirmed that electropolymerisation was a self-limiting process (Fig. 1). Similar curves were seen at microporous membrane protected platinum anodes. The anodic current observed with phenol shows a rapid initial increase followed by a decrease until a stable baseline current was reached ( - 5 nA). Current vs. time profiles were similar irrespective of the oxidisable phenolic derivative employed. In addition to the loss of response to the modifying species due to film formation there 833 S. Eddy et al. Biosensors & Bioelectronics trode (Table 1). This latter effect applied to modifier species with two or more hydroxy groups or both hydroxy and amine substituents of the aromatic ring. Application of 4-AP films did not affect linearity of response up to at least 1.6 mM hydrogen peroxide (Fig. 2). Response times were similar to that of the bare electrode (<20 s). Also phenol responses were retained enabling later work to include a subsequent electropolymerisation. A more complex charged phenolic monomer gave a different behaviour on electropolymeris- lOO 80 .~= 60 ~ 4o 2O 0 5 10 15 Time 20 25 30 i i 35 40 (rain) 60 4-AP Fig. 1. Current vs. time profile during coating of Pt anode from 5 mM phenol solution (+0.85V vs. Ag/ AgCI, pH 7.4 phosphate buffer, electrode overlaid by dialysis membrane). Uncoa'(ed coated 50 40 ~esponse [~A) 3.0 was a strong solute selectivity shown for some polyphenolic layers (Table 1). With the phenol film, both ascorbate and acetaminophen signals are greatly attenuated compared to that of hydrogen peroxide. With some films however, ascorbate and acetaminophen responses were not decreased and the hydrogen peroxide response was significantly increased compared with that of the bare elec- 20 1.0 0 04 08 12 I6 Modified electrode responses expressed as % of unmodified response to each analyte (0.1 mM) Phenol Acetaminophen Dopamine 1,4-Dihydroxybenzene 1,3-Dihydroxybenzene 1,2-Dihydroxybenzene Hydrocaffeic acid 4-Aminophenol 1,3,5 Trihydoxybenzene 1,2,3 Trihydoxybenzene 834 24 Fig. 2. Responses of hydrogen peroxide at uncoated and 4-AP coated Pt anode (+0.85V vs. Ag/AgCl, pH 7.4, electrode overlaid by dialysis membrane). TABLE 1 Effects on electrode responses of 1-h duration electropolymerisation from 5 mM solution (+0.85 V vs. Ag/AgC1, pH 7.4, electrode overlaid by dialysis membrane). Modifier 2C [H2021 (m~Al Ascorbate Acetaminophen Hydrogen peroxide 4Aminophenol 0.13 2 2 95 21 70 99 54 70 0.12 60 30 121 84 143 148 100 156 26 90 146 182 177 179 167 205 223 25 75 96 105 107 120 135 107 123 94 94 140 119 Biosensors & Bioelectronics ation. Phenol red appeared to discriminate on the basis of solute charge, such a resolution of solute diffusion is shown for 0.1 mM solutions of hydrogen peroxide and interferents at an electrode modified by a 24 h electropolymerisation period (Fig. 3). The results clearly show that acetaminophen, a neutral species, and 4-AP a cation at pH 7.4, permeate freely through phenol red whereas anionic ascorbate does not. Baseline currents obtained after extensive phenol red polymerisation were higher than that with phenol films. An electrode modified by a phenol red polymeric layer following extensive polymerisation could still be further modified by the subsequent electropolymerisation of phenol. However phenol red could not be deposited after a phenol film had formed. The former produced a dual film modified electrode (Fig. 4), where response to hydrogen peroxide is exceptionally high but response to ascorbate and acetaminophen are both reduced substantially, though not to the extent found with 4-AP/phenol dual coatings - H202 ascorbate being 139:1 for the phenol red/ phenol whereas a ratio of 346:1 was found for 4-AP/phenol, despite the H202 response for phenol red/phenol being larger by a factor of two. 500400- 300- Response(nA) 200- Modification of enzyme electrode properties ELECTRODE RESPONSES FOR SUCCESSIVE PHENOL RED AND PHENOL 800 ~ I 750 ~ I ,oot 650 ,-% J i "-°°°t ~ Hydrogen peroxide "~550 o~.250 T J,~ I, Ace,tammophen rr200 ~o I ~ '°°1 O I ll~ ~ ......... I \ 20 40 60 80 100 T i m e {rain) Phenol red coating I Phenol coating Fig. 4. Electrode responses during interrupted coating of phenol, after initial phenol red coating (+0.85V vs. Ag/AgCI, p H 7.4, electrode overlaid by dialysis membrane, 0.1 mM solutions). A series of surfactants were included in the prepolymer solution in order to achieve possible co-entrapment of high molecular weight surfactant for film modifying. Whether for charged or neutral surfactants there was little or no significant effect on permeability of coatings towards the model charged/neutral diffusing species (Table 2). These surfactants did increase polymerisation time and in the cases of N-dodecyl-n, N-dimethyl3-ammonio-l-propanesulfonate and n-decyl b-Dmaltopyranoside totally inhibited film formation (Table 2). This effect was likely to be the result of surface adsorption of surfactant on the anode because in the case of cholic acid exposure of the electrode to surfactant prior to phenol electropolymerisation led to identical I-t profiles. Interestingly addition of cyclodextrin enabled discrimination of ascorbate and urate (Table 2). Enzyme electrode studies 100- 0- 8 Fig. 3. Responses at a Pt anode (+0.85V vs. Ag/AgCI, pH 7.4, electrode overlaid by dialysis membrane), to 0.1 mM solutions after coating of anode by 24-h exposure to 5 mM phenol red (+0.85V vs. Ag/AgCI, pH 7.4, electrode overlaid by dialysis membrane). (a) Enzyme/membrane laminate Because of the enhanced hydrogen peroxide responsiveness of 4-AP modified electrodes, these were chosen for use with subsequent prolonged phenol electropolymerisation in order to effect selectivity. Table 3 shows favourable selectivity towards glucose when an enzyme laminate is placed above the films, compared with an electrode with only a phenol film, comparison is also shown for a more conventional enzyme electrode with the 835 S. Eddy et al. Biosensors & Bioelectronics TABLE 2 Effect on phenolic film formation of the present of modifier species in the prepolymer solution. Responses after 1-h electropolymerisation (+0.85 V vs. Ag/AgCI pH 7.4). Modified electrode responses expressed as % of unmodified response to each analyte (0.1 mM) Modifier Cyclodextrin Deoxycholic acid Cholic acid N-Dodecyl-N,Ndimethyl-3-ammonio-1propanesulfonate n-decyl/3-dmaltopyranoside Ascorbate Acetaminophen Hydrogen peroxide Urate 7 41 11 103 20 39 2.6 107 20 77 26 104 138 10 8 99 91.5 106 107 94 TABLE 3 Selectivity ratios of glucose biosensors for 1 mM solutions acetaminophen, ascorbate and 5 mM glucose. Selective barrier Glucose : Acetaminophen Cellulose acetate Electropolymerised phenol Electropolymerised 4Aminophenol then phenol (b) Enzyme in electropolymerised film Enzyme could be entrapped in both single 4-AP and double layer (4-AP and phenol) (Fig. 6) films on electrodes. A very rapid initial linear response phase was found, decaying to a reduced but finite slope after approximately 12 s. These kinetics were seen with all enzyme electrodes, irrespective of phenolic monomer, single or double layers, where the enzyme was entrapped in electropolymerised film. For the conventional electropolymerisation of phenol Glucose : Ascorbate 2:1 100 : 1 100 : 1 30 : 1 >600 : 1 >600 : 1 (a) enzyme immobilised between preformed, polymeric microporous polycarbonate and cellulose acetate membranes (laminates shown in Fig. 5). Table 3 shows the selectivity of the dual electropolymerised film structures for glucose vs. ascorbate and acetaminophen. For both latter species selectivities were superior to a conventional cellulose acetate membrane. 001urn PCM 001pro PCM Enzyme Enzyme Cellulose acetate Electrode 0 01!ira PGM Enzyme 0 05#m PCM 0 05urn PCM Phenol coating Phenol coating II 4-AP coating E~ectrode Electrode Fig. 5. Membrane and film structures of enzyme electrodes for glucose. 836 40 30 c (b) 2o lO 0 ~ 0 4 -, 8 i , , 12 16 20 Time (sec) Fig. 6. Current vs. time profiles for (a) 20 mM and (b) 10 mM glucose. Enzyme entrapped in 4-AP film with subsequent phenol polymerisation (no covering membrane). Maximum rates of response 2300 and 1200 nA/min, respectively. Biosensors & Bioelectronics Modification of enzyme electrode properties the presence of FAD in the cell chamber at neutral pH. This resulted in a calibration curve for glucose very similar to that seen before exposure to acid pH, with also the same time dependent behaviour. No change in ascorbate and acetaminophen exclusion was noted after acid treatment and similarly the permeability of the enzyme/phenol film towards H202 remained unchanged. 3(10 250200Response (after 5rain nA) 150100500 0 1'0 2'0 3'0 4'0 50 IGlucosel (mM) DISCUSSION Fig. 7. Responses to glucose obtained with glucose oxidase entrapped within a phenol film, either before (n = II) or after (m = 0), exposure to p H 2 phosphate buffer (+ 0.85V vs. Ag/AgCl, p H 7. 4 phosphate buffer). alone in the presence of GOx, an arbitrary 5 min current was used to construct the calibration curve shown in Fig. 7. The effect of solution pH on the behaviour of the phenol entrapped enzyme system was also investigated; Fig. 8 (n = II) shows the pH profile between pH 2-8. The optimum pH was 6. However if the enzyme electrode was pre-exposed for 5-15 min to pH 2 buffer, the pH curve was altered with no apparent optimum observed (Fig. 8 (m = O). The signal to 10 mM glucose remained constant between pH 3 to pH 8. Furthermore the stir dependence of the electrode was entirely eliminated. Lost activity could be regained by 300 250200Response (nA) 15010050- o! i i i i 3 4 5 6 pH Fig. 8. Response to 10 m M glucose at different p H values (n = II) before and (m = (3) after the electrode incorporating Gox in a phenol film was exposed to p H 2 phosphate buffer (+0.85V vs. Ag/AgCI). Electropolymerised film studies The non-conductive polymer film formed on electrode surfaces imparts advantageous characteristics which can be influenced by use of the appropriate phenol derivative. That is, basic phenol films give a degree of selectivity against electroactive species such as ascorbate and acetaminophen which is thought to be predominantly via a size exclusion effect (Christie & Vadgama, 1993) more so than charge exclusion. In contrast amino phenols and di-phenolics are less selective, probably due to a more porous structure related to polymer branch chain formation. Where additional enhancement of the response to hydrogen peroxide occurs (Fig. 2), this phenomenon may be explained by modification of the platinum oxide by the presence of the phenolic coating (Christie et al., in press). Hydrogen peroxide anodic reactions have been reported to be strongly influenced by the nature of the platinum oxide layer (Conway & Novak, 1979). Films formed from phenol red (tri-aromatic) show generally greater responses to hydrogen peroxide than phenol. In addition phenol red films were also found to discriminate between acetaminophen and anionic ascorbate. This selectivity probably resides in the cationic nature of the polymer film. The porous nature of phenol red polymer permits the formation of dual films with probably the phenol monomer being able to freely permeate to the underlying platinum electrode enabling a further polymerisation reaction. Phenol red/phenol film combinations demonstrated much of the selectivity of the basic polyphenol but also had the maintained hydrogen peroxide response attributed to the phenol red polymer. Such characteristics may be due to the formation of a less dense phenol film as a consequence of an open phenol red structure. 837 S. Eddy et al. Enzyme electrode studies The co-immobilisation of GOx during the polymerisation process is favoured due to the mild reaction conditions employed (Bartlett & Cooper, 1993). It is reasonable to assume that GOx enzyme is located within the film at multiple depths in the polyphenol matrix and the surface. Upon exposure of the enzyme electrode to acidic solutions the surface G O x becomes inactivated which permits dominance of the internal matrix entrapped GOx. The inner polymer matrix located enzyme is clearly protected from H ÷, indicated by the p H independence (Fig. 8), and by its survival in pH 2 solution. The response in this instance is comparable to that observed with GOx immobilised in membrane laminates, being steady state and stir-independent (Vadgama, 1990). The deactivation of surface enzyme may be marginally due to denaturation but loss of the prosthetic group is the most probable important factor; indeed exposure to pH 2 is the basis for the formation of apo-GOx (Bergmeyer, 1985). The lack of proton permeability in GOx loaded phenolic films contrasts access to glucose, this therefore warrants further study, and suggests phenol may have some charge discriminating properties when the film is formed in the presence of enzyme. Any contribution to extended operational lifetime as a result of enzyme protection from adverse bulk solution matrices remains to be ascertained. CONCLUSION This study has illustrated the ease with which polyphenol (and derivatives) can modify electrodes that demonstrate responses that are selective, yet exceptionally fast, and of magnitude similar to that of the bare electrode. The electrodes are also stir independent and immune from pH changes. Non-conducting films provide an alternative to standard polymeric membranes with the advantageous one step process of enzyme immobilisation. ACKNOWLEDGEMENT The authors wish to express their gratitude to EPSRC for their generous financial support during this study. 838 Biosensors & Bioelectronics REFERENCES Bartlett, P.N. & Cooper, J.M. (1993). A review of the immobilisation of enzymes in electropolymerised films. J. Electroanal. Chem., 362, 1-12. Bartlett, P.N. & Caruana, D.J. (1992). Microelectrodes for the detection of glucose based on GOx immobilised in a poly(phenol) film. Analyst, 117, 1287. Bartlett, P.N. & Caruana, D.J. (1994). Electrochemical immobilisation of enzymes part VI. 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