Reduction of Interference Response at a Hydrogen Peroxide

Anal. Chem. 1998, 70, 2928-2935

Reduction of Interference Response at a Hydrogen Peroxide Detecting Electrode Using Electropolymerized Films of Substituted Naphthalenes Lindy J. Murphy

Section of Endocrinology and Metabolic Medicine, Imperial College School of Medicine, St. Mary’s Hospital (Mint Wing), Praed Street, London, W2 1NY, UK

The abilities of electropolymerized films formed from various substituted naphthalenes to prevent the oxidation of the plasma interferences ascorbate, acetaminophen, and urate at a hydrogen peroxide detecting platinum electrode are reported. Films prepared from 2,3-, 1,5-, and 1,8-diaminonaphthalene and 5-amino-1-naphthol were compared with films prepared from o-, m-, and p-phenylenediamine for their permselectivity and stability. The films were quick to prepare (15 min) and insulating. All films exhibited reduced permeability to hydrogen peroxide (except poly(o-phenylenediamine)) and interferences. Lowest permeabilties to hydrogen peroxide, ascorbate and acetaminophen were obtained with poly(2,3diaminonaphthalene) (39 ( 16, 0.5 ( 0.1, and 0.2 ( 0.1%, respectively) and poly(5-amino-1-naphthol) (20 ( 10, 0.2 ( 0.1, and 0.2 ( 0.1%, respectively), although these films had longer response times to hydrogen peroxide (60 and 120 s, respectively). These films also had the highest permselectivities to hydrogen peroxide against the common interferences ascorbate and acetaminophen (>80 ( 40:1 and 250 ( 160:1 for poly(2,3-diaminonaphthalene) and >80 ( 35:1 and 100 ( 35:1 for poly(5-amino-1-naphthol)). The changes in permeabilities of these films were determined for 21 days, and while all films experienced some deterioration, the poly(substituted naphthalene) films maintained lower permeabilities to all species compared to the poly(phenylenediamine) films. There are many published examples of biosensors based on the electrochemical oxidation of hydrogen peroxide produced by the interaction between analyte (substrate) and an oxidase enzyme. This type of biosensor has been widely investigated because of the relative ease of construction and absence of any components such as mediator that could leach from the electrode. However, the oxidation of other species (known as interferences) present in the analyte matrix (which could be plasma, serum, or the tissue surrounding an implanted electrode) is a key problem. The major endogenous interferences found in biological matrixes are ascorbate and urate, while the exogenous species acetaminophen is also a potent interference if present.1 These give rise to a nonspecific oxidation current in addition to the hydrogen 2928 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

peroxide derived oxidation current. Failure to reduce the oxidation of interferences can result in overestimation of the analyte concentration, and consequently, the development of different techniques for reducing the effect of interferences is an active area of biosensor research. Prevention of oxidation of acetaminophen has been particularly difficult to achieve, and failure to do so can lead to gross overestimation of the biological species being measured.1 One approach to reducing the effect of interferences is the use of polymeric films over the electrode surface.2 These films are intended to be permeable to hydrogen peroxide while rejecting interferences and preventing them from reaching the electrode. Polymeric films are generally one of two types: solvent-cast or electropolymerized. Solvent-cast films giving good rejection of acetaminophen have recently been reported, based on PVC and polyether sulfones3 or Nafion and cellulose acetate.4 However, it can be difficult to obtain a coat of uniform thickness if thin films are required or if the electrode surface is not simple, and the thickness of these coats can result in long response times. The second type of polymer film is formed by electropolymerizing a film directly onto the electrode surface. The films are generally insulating and hence the thickness of the film is selflimiting, since access of the monomer to the electrode surface is blocked as film formation proceeds. Consequently, the films are generally thin and have fast response times, and immobilization of enzyme is possible by electropolymerizing the monomer in the presence of the enzyme. The use of nonconducting electropolymerized films of o-phenylenediamine has been widely reported. These films are simple and quick to prepare, but variable success in the rejection of acetaminophen has been reported, with both good5 and poor6 rejection being observed. Several other electropolymerized films have been reported to be impermeable to (1) Moatti-Sirat, D.; Velho, G.; Reach, G. Biosens. Bioelectron. 1992, 7, 345352. (2) Emr, S. A.; Yacynych, A. M. Electroanalysis 1995, 7, 913-923. (3) Benmakroha, Y.; Christie, I.; Desai, M.; Vadgama, P. Analyst 1996, 121, 521-526. (4) Zhang, Y.; Hu, Y.; Wilson, G. S.; Moatti-Sirat, D.; Poitout, V. Reach, G. Anal. Chem. 1994, 66, 1183-1188. (5) Centonze, D.; Malitesta, C.; Palmisano, F.; Zambonin, P. G. Electroanalysis 1994, 6, 423-429. (6) Moussy, F.; Harrison, D. J.; O’Brien, D. W.; Rajotte, R. V. Anal. Chem. 1993, 65, 2072-2077. S0003-2700(97)01182-7 CCC: $15.00

© 1998 American Chemical Society Published on Web 06/09/1998

acetaminophen, e.g., polyphenol,7 poly(m-phenylenediamine),8 copolymers of m-phenylenediamine and resorcinol9 or o-phenylenediamine and resorcinol,10 and overoxidized films of polypyrrole,11 although some require long coating times (several hours) to be efficient. This paper reports the ability of electropolymerized films of the substituted naphthalene compounds, 2,3-diaminonaphthalene (2,3-DAN), 1,5-diaminonaphthalene (1,5-DAN), 1,8-diaminonaphthalene (1,8-DAN), and 5-amino-1-naphthol (5A1N), to reject the major interferences, in particular acetaminophen. These films are compared to those prepared from o-, m-, and p- phenylenediamines (1,2-, 1,3-, and 1,4-diaminobenzene (o-, m-, and p-PD)). It was hoped that the extra benzene ring on each substituted naphthalene molecule (compared to phenylenediamine) would confer greater hydrophobicity on the films formed from these materials. This could result in a lower degree of solvation, closer packing of the polymer chains, and reduced diffusion of the solvated interference species through the film. The ability of poly(substituted naphthalene) films to reject interference species has not been previously reported. There are only a few reported studies of electropolymerized films of the various diaminonaphthalenes and 5A1N used in this work. These studies have used experimental conditions different from those used here and so can act only as a guide to the present work, since film morphology is often dependent on experimental conditions. Oxidation of 1,8-DAN is reported to result in only one amino group being oxidized,12 in comparison with 2,3-DAN13 and 1,5-DAN14 for which both amino groups are oxidized. For 5A1N, only the amino group is reported to be oxidized, and the hydroxy group does not take part in the electropolymerization reaction.15 In comparison, oxidation of o-PD results in oxidation of both amino groups,16 while for p-PD only one amino group is oxidized.17 There are no reported mechanistic studies of the oxidation of m-PD. The different positions of the amino groups on the benzene or naphthalene molecule, and the different number of amino groups oxidized per monomer, result in polymers of varying morphology. Poly(2,3-DAN) is reported to be a ladder polymer containing phenazine rings,13 similar to poly(o-PD).16 Poly(1,5DAN) is also suggested to be a ladder polymer, with some alternative structures occurring.18 Poly(1,8-DAN) is reported to have head-to-tail coupling at the para position relative to the (7) Eddy, S.; Christie, I.; Ashworth, D.; Purkiss, C.; Vadgama, P. Biosens. Bioelectron. 1995, 10, 831-839. (8) Reynolds, E. R.; Yacynych, A. M. Biosens. Bioelectron. 1994, 9, 283-293. (9) Geise, R. J.; Adams; J. M.; Barone, N. J.; Yacynych, A. M. Biosens. Bioelectron. 1991, 6, 151-160. (10) Manowitz, P.; Stoecker, P. W.; Yacynych, A. M. Biosens. Bioelectron. 1995,10, 359-370. (11) Palmisano, F.; Centonze, D.; Guerrieri, A.; Zambonin, P. G. Biosens. Bioelectron. 1993, 8, 393-399. (12) Skompska, M.; Hillman, A. R. J. Chem. Soc., Faraday Trans. 1996, 92, 41014108. (13) Oyama, N.; Sato, M.; Ohsaka, T. Synth. Met. 1989, 29, E501-E506. (14) Jackowska, K.; Skompska, M.; Przyluska, E. J. Electroanal. Chem. 1996, 418, 35-39. (15) Pham, M.-C.; Mostefai, M.; Simon, M.; Lacaze, P.-C. Synth. Met. 1994, 63, 7-15. (16) Chiba, K.; Ohsaka, T.; Ohnuki, Y.; Oyama, N. J. Electroanal. Chem. 1987, 219, 117-124. (17) Compton, R. G.; King, P. M.; Reynolds, C. A.; Richards, W. G.; Waller, A. M. J. Electroanal. Chem. 1989, 258, 79-88. (18) Jackowska, K.; Bukowska, J.; Jamkowski, M. J. Electroanal. Chem. 1995, 388, 101-108.

oxidized amine group,14 while in poly(5A1N) coupling is via the amino group to the ortho or para position with respect to the amino group.15 Poly(p-PD) gives 1,4-coupling, via the amino group.19 There is no reported structure for poly(m-PD), although a structure can be proposed based on the effect of the electrondonating amino groups. These activate the benzene ring to electrophilic attack at positions ortho and para to the amino group, and for m-PD, this would result in double activation at the 2-, 4-, and 6-positions. Consequently one would expect coupling to occur adjacent to the amino group at the 4- and/or 6-positions, while coupling to the 2-position would be unfavorable because of steric hindrance. It is not possible to predict with any accuracy the relative permeabilities of these polymeric films from their proposed structures. However, a more hydrophobic, less solvated film or a film with highly ordered polymer chains could be expected to have closer packing of the polymer chains and lower permeability. This suggests poly(2,3-DAN) and possibly poly(1,5DAN) would be the least permeable polymers. EXPERIMENTAL SECTION Chemicals and Solutions. 2,3-DAN (>98%), 1,5-DAN (>98%), and 1,8-DAN (∼99%) were obtained from Fluka Chemicals. 5A1N (97%), o-PD (99%), m-PD (99+%), and p-PD (99+%) were obtained from Aldrich Chemical Co. AristaR buffer reagents were obtained from Merck Ltd. Hydrogen peroxide (30% solution), ascorbic acid, uric acid, and acetaminophen were obtained from Sigma Chemical Co. All solutions were made up with deionized water obtained from an Elgastat Option 4 water purifier. All experiments were carried out in pH 7.4 phosphate buffer containing Na2HPO4 (0.1 M) and NaCl (0.15 M), balanced with concentrated HCl. All solutions were prepared with this buffer, with the exception of the solutions of substituted naphthalenes used for electropolymerization (see below). Stock solutions used were 10 mM ascorbate, 10 mM acetaminophen, and 3 mM urate. Hydrogen peroxide solution was 1000-fold dilute (∼8.82 mM). The precise concentration of the 30% hydrogen peroxide solution was determined by density, i.e., weight of known volume (1.11 g cm-3 at 20 °C for a 30% solution according to Sigma). Apparatus. Amperometry was performed using a Bioanalytical Systems, Inc. model LC-4C amperometric controller (BAS Technicol, Stockport, U.K.). Current-time transients were recorded using a Gould BS272 YY′T chart recorder. Cyclic voltammograms were performed using modules constructed in-house by Mr. John Hooper and were recorded with a Gould series 60000 XYT chart recorder. Preparation of the Microdialysis Electrode. The majority of the work reported here was carried out with a microdialysis electrode, which is shown schematically in Figure 1. The microdialysis electrode is a novel configuration of the frequently used hydrogen peroxide detecting enzyme electrode. The electrode has been successfully used to measure glycerol20 and glutamate,21 by infusing the appropriate enzyme solution into the dialysis fiber surrounding the platinum working electrode. However, in the work reported in this paper, no enzyme is used and (19) Adams, R. N. Electrochemistry at Solid Electrodes; Marcel Dekker: New York, 1969; Chapter 10. (20) Murphy, L. J.; Galley, P. T. Anal. Chem. 1994, 66, 4345-4353. (21) Albery, W. J.; Boutelle, M. G.; Galley, P. T. J. Chem. Soc., Chem. Commun. 1992, 12, 900-901.

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Figure 1. Schematic diagram of the microdialysis electrode (not drawn to scale). The electrodes are formed from lengths of Tefloncoated wire, with 4-mm Teflon stripped from either end to expose the metal. One end is soldered to a gold contact, while the other end is used as the electrode. The contacts are held at the top of the electrode in a block formed from epoxy resin set in a rectangular mould.

the microdialysis electrode simply functions as a convenient selfcontained unit, with the working, counter, and reference electrodes in solution contact within the same housing. This enables the working electrode to be dipped from one solution to another without disconnecting the electrodes, which reduces the stabilization time between test solutions. This particular electrode configuration has been used in this work since it is a continuation of previous work which used the microdialysis electrode for the measurement of glycerol.20 Microdialysis electrodes were purchased from Sycopel International Ltd. (Boldon, Tyne and Wear, U.K.). The electrodes were constructed to our design and specifications. Cuprophan dialysis fibers, MWCO 5-10 000, were obtained from a Gambro dialysis cartridge (Gambro Ltd.). The electrodes were supplied dry. Immediately prior to use, buffer was infused into the electrode at a flow rate of 0.5 µL min-1 using a Carnegie Medicin syringe drive. The flow was stopped once the electrode was filled, and the internal solution was stationary throughout all experiments. The dialysis fiber of the filled electrode was then completely immersed in buffer solution (4 mL) held in a small beaker, and the solution stirred with a magnetic flea. When in use, the platinum working electrode potential was held at +650 mV with respect to the internal Ag/AgCl reference electrode. When not in use, the dialysis electrodes were stored with the dialysis fiber immersed in buffer at 4 °C. 2930 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

It should be noted that the precise area of the platinum electrode is unknown. However, since the diameter of the platinum wire was 50 µm, and the length of the exposed platinum was ∼4 mm, the area of the electrode was estimated to be 6 × 10-3 cm2. Constant Potential Electropolymerization. Phenylenediamine solutions (5 mM) were prepared using pH 7.4 buffer, and substituted naphthalene solutions (5 mM) were prepared using 0.2 M NaCl, balanced to pH 1.0 with concentrated HCl. The dialysis fiber of the microdialysis electrode was immersed in the monomer solution, which was deoxygenated with nitrogen for 15 min. Electropolymerization then took place at +650 mV vs Ag/ AgCl for 15 min, directly onto the platinum electrode in situ within the dialysis fiber, while continuing to deoxygenate with nitrogen. The electrode was then placed in fresh pH 7.4 buffer and allowed to stabilize. The electrode was taken to be stabilized when the background current remained constant for 10 min. The amount of charge passed during electropolymerization was calculated from the area under the curve of the current-time profile. Efficiency of Electropolymerized Films. This was assessed using microdialysis electrodes. The response of a bare working electrode to hydrogen peroxide was determined from the gradient of the electrode response to five successive 8.8 µM aliquots of hydrogen peroxide stock solution. The responses to 100 µM ascorbate, 100 µM acetaminophen, and 300 µM urate were also determined by addition of a single aliquot of stock solution to fresh buffer solution. The dialysis fiber of the microdialysis electrode was then placed in fresh buffer for 10 min prior to constantpotential electropolymerization following the method above, after which it was placed in fresh buffer and the background current allowed to settle. Once the background current was stable, the response of the coated working electrode to hydrogen peroxide and interferences was determined as before for the bare electrode. Stability of Electropolymerized Films. Some of the coated electrodes prepared in the above section were tested on subsequent days for response to hydrogen peroxide and interferences. Day 1 was taken as the day on which electropolymerization took place. The electrodes were stored in pH 7.4 buffer at 4 °C when not being tested. Cyclic Voltammetry of the Monomers. This was performed using a platinum disk electrode (1-mm diameter) made in house. The electrode was cleaned by polishing manually with 0.05 µM alumina, followed by washing with deionized water. The electrode potential was then cycled in pH 7.4 buffer between -1.8 to +1.8 V vs SCE at 100 mV s-1 for at least 1 h, using a platinum gauze as counter and a saturated calomel reference electrode (SCE). The electrochemical cell was a glass pot with two sidearms, containing 20 mL of solution. Cyclic voltammetry of the monomers was performed with the solutions described above. Solution was deoxygenated for 15 min with nitrogen prior to commencing cyclic voltammetry at a scan rate of 50 mV s-1, between -150 and 1300 mV vs SCE. The initial scans were commenced at 0 mV, scanning in a positive direction. Scanning Electron Microscopy. Films were grown onto platinum wire of 50-µm diameter (Goodfellow Cambridge Ltd., Cambridge, U.K.) by constant-potential electropolymerization as above. After coating, the wires were washed with buffer and

Table 1. Physical Characteristics of Films Grown from Various Monomers monomer

105Qa/C

o-PD 49 ( 15 m-PD 6.9 ( 0.6 p-PD 110 ( 20 2,3-DAN 25 ( 5 1,5-DAN 5.8 ( 0.3 1,8-DAN 99 ( 5 5A1N 7.2 ( 3.4

film density of thicknessh/ Eox,1b/mV Eox,2d/mV monomer22/ (vs SCEc) (vs SCEc) g cm-3 nm ng +0.20 +0.30 +0.05 +0.55 +0.50 +0.50 +0.40

e +1000 e +1100 +1050 +850 +900

f 1.1421 f 1.0968 1.4 1.1265 f

4 4 2 4 4 2 2

150-230 28 650-980 160 27 1200 66-98

a The data are given as the mean values ( SD (n ) 3). These data are from the same experiments given in Table 2. b The potential at which the first oxidation of the monomer species commences, seen from cyclic voltammetry. c The potential of the SCE is -60 and -30 mV vs Ag/AgCl at pH 1.0 and 7.4, respectively. d The potential at which the second oxidation wave or peak commences, seen from cyclic voltammetry. e Second oxidation wave or peak not observed. f Where density is not known, it is assumed to be 1< density < 1.5. g The number of electrons per oxidation of monomer (see introduction for number of amino groups oxidized for each monomer species). h Error bars omitted since thicknesses are only estimated.

Figure 2. Typical current-time profiles obtained during electropolymerization of the (A) phenylenediamine and (B) substituted naphthalene monomers. The electrode was held at +650 mV vs Ag/ AgCl, and the platinum electrode was in situ within a microdialysis electrode. Legend: (A) Solid line, o-PD; dotted line, m-PD; and solid line with circle, p-PD. (B) Solid line with circle, 2,3-DAN; dotted line, 1,5-DAN; solid line with square, 1,8-DAN; and solid line, 5A1N.

allowed to air-dry. Sections of wire were inspected with a JEOL JSM T220A scanning electron microscope. RESULTS AND DISCUSSION Electropolymerization of the Monomers. The electropolymerized films of phenylenediamines and substituted naphthalenes were grown from monomer solutions in pH 7.4 phosphate-buffered saline and pH 1.0 KCl solution, respectively. A solution of greater acidity was required for solubilization of the substituted naphthalenes, presumably because of their greater hydrophobicity. Typical current-time (i-t) transients obtained during electropolymerization of the films are shown in Figure 2. All monomers showed a decrease in current with time, indicating that the films

formed at the electrode are insulating and block the electrode surface to any further electropolymerization. The i-t profiles for p-PD and 1,8-DAN give a slight shoulder within the first 60-90 s, possibly due to a nucleation process occurring. The i-t profile for poly(2,3-DAN) film formation differed from those for all the other monomers, exhibiting a much slower decrease in current with time. This is probably because the oxidation potential for 2,3-DAN is the most positive of all the monomers tested (see Table 1), so that, at the electropolymerization potential of +650 mV vs Ag/AgCl, the overpotential for the oxidation of the monomer at the electrode is small and film formation proceeds more slowly. Electropolymerization of 2,3-DAN at the higher potential of +850 mV gave the same i-t profile as that observed for the other monomers at +650 mV, and the permeabilities of various species through poly(2,3-DAN) prepared at these two potentials were not significantly different. This suggests the morphology of the poly(2,3-DAN) film is independent of electrode potential between +650 and +850 mV, and it is valid to compare poly(2,3-DAN) films prepared at +650 mV with the other polymeric films. After electropolymerization, the coated electrodes were placed in fresh buffer and left to stabilize. The films were taken to be stabilized when the background current remained constant for 10 min. Poly(1,8-DAN) took significantly longer to stabilize compared to the other films and after 5 h gave a background current of 3.0 nA, which continued to decrease with time, indicating the film was not fully stabilized. For the other films, the stabilization times were between 2 and 4 h, with poly(o-PD) stabilizing most rapidly (2 h) and poly(1,5-DAN) least rapidly (4 h). The background currents for the stabilized films (other than poly(1,8-DAN)) were between 1 and 2 nA. Estimation of the Film Thickness. An estimate of the film thickness can be made from the amount of charge passed during electropolymerization, by making the following assumptions: (a) The polymerization process proceeds with 100% efficiency; i.e., no oxidized monomers or oligomers are lost to solution. This last assumption might not hold well for some of the films, such as poly(1,8-DAN). The very slow stabilization of background current (>5 h) observed with this film could have been due to Analytical Chemistry, Vol. 70, No. 14, July 15, 1998

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the loss of soluble oligomers from the film. (b) The density of the polymer is the same as that of the monomer. For those monomers whose density is unknown, a range of density from 1 to 1.5 g cm-3 is used, which covers the range of known densities of the monomers. (c) Each amino group undergoes one oxidation reaction only. It has been reported that the amino group can undergo two oxidation steps. Initial oxidation results in formation of a radical cation on the amino group, which deprotonates and undergoes another oxidation followed by coupling to form -CNH-C- bonds. Overall this is a two-electron, two-proton process. A second oxidation is also possible at higher potentials to give -C-NdC- bonds.19 Cyclic voltammetry of the monomers was performed to determine the potential at which the first oxidation step occurs and also whether a second oxidation step occurs. Oxidation potentials are given in Table 1. It can be seen that it is a reasonable assumption that the potential used during electropolymerization (+650 mV) is not sufficiently extreme for the second oxidation of the amine to take place. Estimated values of film thickness are given in Table 1. The films can be divided into roughly three groups, with the thinnest films obtained with m-PD and 1,5-DAN (∼30 nm), the thickest films formed from p-PD and 1,8-DAN (∼103 nm), and the films formed from the other monomers falling somewhere between (∼102 nm). It is noticeable that the calculated film thickness for poly(o-PD) is significantly thicker than usually quoted (10 nm23), perhaps due to different experimental conditions or significant loss of oxidized species from the film. It should be noted that the estimated film thicknesses do not take into account the degree of solvent swelling in the films, which will depend on the type of monomer used. The films were examined by scanning electron microscopy of coated platinum wire (50-µm diameter), on the surface of the wire and in cross section. Poly(1,8-DAN) was clearly visible as a smooth, uniformly thick film of thickness 600 nm. This is 50% of the estimated thickness, possibly because of substantial loss of soluble oligomers to solution (see above). Poly(p-PD) was also visible as a lumpy film of thickness 700 nm (which is within the estimated range) and with particles ranging in size from 1 to 10 µm. The thickness of the other films could not be determined because they were too thin (