Ferrocenylethyl Phosphate: An Improved Substrate for the Detection of

Anal. Chem. 1996, 68, 4141-4148

Ferrocenylethyl Phosphate: An Improved Substrate for the Detection of Alkaline Phosphatase by Cathodic Stripping Ion-Exchange Voltammetry. Application to the Electrochemical Enzyme Affinity Assay of Avidin Benoıˆt Limoges* and Chantal Degrand

Equipe d’Electrochimie Organique, Thermodynamique et Electrochimie en Solution, URA 434, Universite´ Blaise Pascal de Clermont-Ferrand, 24 Avenue des Landais, 63177 Aubie` re, France

The ferrocenylethyl phosphate disodium salt was synthesized and used as a new substrate for alkaline phosphatase (AP). The enzyme-generated ferroceneethanol was selectively and sensitively detected at a Nafion filmcoated electrode by anodic preconcentration of the ferricinium salt, followed by cyclic voltammetry. The accumulated ferricinium units could be expelled from the polymer film in their neutral form by cathodic stripping, and so the Nafion-modified electrode could be reused for more than 10 measurements with a standard deviation less than 3%. Values of 0.75 mM for the Michaelis constant and 1.42 µmol s-1 (mg of protein)-1 for the maximal velocity were found. The regenerable Nafioncoated electrode was employed for the indirect detection of AP down to 2 × 10-12 M and for the noncompetitive heterogeneous enzyme assay of avidin, whose detection limit was 5 × 10-12 M. Alkaline phosphatase (AP) is one of the most commonly used labels in enzyme immunoassays and related affinity sensing methods,1-3 due to its high turnover number, low cost, and broad substrate specificity. This enzyme hydrolyzes orthophosphoric monoesters into alcohols, and its concentration is indirectly determined by the amount of alcohol generated. The detection methods the most frequently associated with AP are spectrophotometric techniques.1-5 However, the electrochemical detection devices have been successfully applied to AP enzyme immunoassays,6-15 and good detection limits are achieved with small (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (2) Ishikawa, E. Clin. Biochem. 1987, 20, 375-385. (3) Porstmann, B.; Porstmann, T.; Evers, U. J. Immunol. Methods 1985, 79, 27-37. (4) Fisher M.; Harbron, S.; Rabin, B. R. Anal. Biochem. 1995, 227, 73-79. (5) Christopoulos, T. K.; Diamandis, E. P. Anal. Chem. 1992, 64, 342-346. (6) Wehmeyer, K R.; Halsall, H. B.; Heineman, W. R.; Volle, C. P.; Chen., I. W. Anal. Chem. 1986, 58, 135-139. (7) Tang, H. T.; Lunte, C. E.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1988, 214, 187-195. (8) Yu, Z.; Xu, Y.; Ip, M. P. C. J. Pharm. Biomed. Anal., 1994, 12, 787-793. (9) Risphon, J.; Gezundhajt, Y.; Soussan, L.; Rosen-Margalit, I.; Hadas, E. In Biosensor Design and Application; Mathewson, P. R., Finley, J. W., Eds.; ACS Symposium Series 511; American Chemical Society: Washington, DC, 1992; pp 59-70. (10) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369-1377. (11) Mc Neil, C. J ; Higgins, I. J.; Bannister, J. V. Biosensors, 1987/88, 3, 199209. S0003-2700(96)00576-8 CCC: $12.00

© 1996 American Chemical Society

sample volumes when a sensitive electrochemical technique is coupled to liquid chromatography or flow injection analysis.6-8 Moreover, the affinity reagent can be directly attached onto the electrode surface,9,10 providing an assay where no separation step is required.10 The current trend in electrochemical enzyme immunoassays is to detect very low amounts of electroactive enzyme product using simple nonflow instrumentation, with the electrode sensor directly immersed in the assay solution.9-15 The advantage is to provide relatively simple, inexpensive, and rapid assays using portable low-cost devices with disposable electrode sensors, able to be performed on site, i.e., without requiring sample transfer to an analytical laboratory.14 However, the detection at a bare electrode is not sufficiently sensitive and is prone to fouling,6 and the choice of substrate/product couples suitable to an electrochemical detection is restricted with respect to AP labels. Up to now, the couple p-aminophenyl ester phosphate/p-aminophenol has been widely used,7-10,12,13,15 because it possesses the required electroanalytical features for the selective detection of the enzyme product at a bare electrode.7 Recently, we developed an alternative electrochemical detection method based on a selective Nafion film electrode, which amplifies the detection of the enzyme product and concomitantly discriminates the substrate signal, although it possesses the same oxidation potential as the product.16,17 The substrate N-ferrocenoyl-6-amino2,4-dimethylphenyl phosphate sodium salt was utilized, and the corresponding phenol derivative was generated enzymatically and then entrapped selectively and irreversibly in the film as a ferricinium salt by applying an anodic potential. Despite its high sensitivity and low detection limit of AP (3 × 10-14 M), this new method has the limitation that the Nafion film electrode can be used only once, because the product is strongly retained within the polymer, but also because of the simultaneous electropolymerization of phenolic radicals during the course of the anodic preconcentration.16 A first possible solution to this drawback is (12) Frew, J. E.; Foulds, N. C.; Wilshere, J. M.; Forrow, N. J.; Green, M. J. J. Electroanal. Chem. 1989, 266, 309-316. (13) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 1559-1563. (14) Athey, D.; Ball, M.; McNeil, C. J. Ann. Clin. Biochem. 1993, 30, 570-577. (15) Treolar, P. H.; Nkohkwo, A. T.; Kane, J. W.; Barder, D.; Vadgama, P. M. Electroanalysis 1994, 6, 561-566. (16) Le Gal La Salle, A.; Limoges, B.; Degrand, C. J. Electroanal. Chem. 1994, 379, 281-291. (17) Le Gal La Salle, A.; Limoges, B.; Degrand, C. Anal. Chem. 1995, 34, 12451253.

Analytical Chemistry, Vol. 68, No. 23, December 1, 1996 4141

the use of a disposable sensor of low cost, and so a renewable Nafion carbon paste electrode was recently prepared in this group18 and successfully applied to the AP assay.19 Another possibility is to select a new anionic substrate which leads to an enzyme product less hydrophobic than N-ferrocenoyl-6-amino-2,4dimethylphenol. In this context, we have prepared the sodium salt of ferrocene ethyl phosphate ester (FcEtOPO32-) as a new

AP substrate, which leads to ferroceneethanol (FcEtOH) by enzyme hydrolysis. We show that this latter molecule has the ability to be accumulated into the polymer as a ferricinium salt and then to be released from the film in its neutral form by cathodic stripping, and so the Nafion-modified electrode can be reused. A regenerable Nafion-coated electrode is thus available for AP assays, and its potential application to electrochemical enzyme affinity assay is illustrated in the noncompetitive heterogeneous enzyme assay of avidin. EXPERIMENTAL SECTION Materials and Reagents. A 5 wt % Nafion solution (EW 1100) and ferrocene methanol were purchased from Aldrich. Ethylene chloro phosphate was obtained from Lancaster. Maxisorp Nunc tubes were provided from Polylabo. Affinity-purified avidin from egg white, alkaline phosphatase from bovine intestinal mucosa (AP, 2600 units mL-1, VII-S, No. P-5521), biotinylated alkaline phosphatase (B-AP, 500 units mg-1, and biotin content 5.2 mol (mol of protein)-1, No. P-8024), lyophilized biotin--amidocaproylbovine serum albumin (B-BSA, biotin content 8.9 mol (mol of albumin)-1, No. A-6043), bovine serum albumin (BSA, fraction V, No. A-3059), sodium azide, and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma. Tween 20 was purchased from Fluka and the other buffer constituents from Prolabo. Deionized and doubly distilled water was used to prepare the buffered aqueous solutions. Synthesis of Ferrocenylmethylpyridinium Iodide. We repeated the procedure given by McNeil and Bannister for the synthesis of ferrocenylmethyl phosphate ester,20 and we obtained, as expected, an oil which we dissolved in a 1:1 mixture of ethanol/ water containing NaI. The solvent was evaporated, and the residue was dissolved in hot ethyl acetate. After a few seconds under sonication, a yellow precipitate appeared. The solid was filtered, washed with ether, and dried to afford the ferrocenylmethylpyridinium iodide salt: mp 175-8 °C (lit.21 mp 173.5-4.5 °C); NMR 1H (400 MHz, CD3SOCD3) δ 9.25 (s, 2H), 8.62 (s, 1H), 8.20 (s, 2H) (pyridinium), 5.70 (s, 2H) (CH2), 4.65 (s, 2H), 4.34 (s, 2H), 4.31 (s, 5H) (ferrocenyl). Anal. Calcd for C16H16FeIN: C, 47.44; H, 3.99; N, 3.46; I, 31.33. Found: C, 45.79; H, 3.90; N, 3.39; I, 30.23. (18) Rapicault, S.; Limoges, B.; Degrand, C. Anal. Chem. 1996, 68, 930-935. (19) Rapicault, S.; Limoges, B.; Degrand, C. Electroanalysis, in press. (20) McNeil, C. J.; Bannister, J. V. European Patent Application 86308781.3, 1986. (21) Nesmeyanov, A. N.; Pervalova, E. G.; Reshetova, M. D. Izv. Akad. Nauk SSSR, Ser. Khim. 1966, 2, 335-337.

4142

Analytical Chemistry, Vol. 68, No. 23, December 1, 1996

Synthesis of Ferroceneethanol and Ferrocene Ethyl Phosphate Ester. The starting material, (ferrocenylmethyl)trimethylammonium iodide salt, was prepared according to the literature method,22 and FcEtOH was produced according to the procedure given by Lednicer et al.:23 mp 41-2 °C (lit.23 mp 41-2 °C); NMR 1H (400 MHz, CD SOCD ) δ 4.60 (t, 1H) (OH), 4.14 (s, 7H), 4.07 3 3 (s, 2H) (ferrocenyl), 3.55 (q, 2H) (CH2O), 2.48 (t, 2H) (CH2). Ester phosphate FcEtOPO3Na2 was synthesized according to the general method described by Thuong and Chabrier.24 Ferroceneethanol (1.15 g, 5 mmol) and triethylamine (0.703 mL, 5.05 mmol) were dissolved in 10 mL of dried benzene. Ethylene chlorophosphate (0.735 g, 5 mmol) in 1.5 mL of benzene was then slowly added to the mixture with continuous stirring, and then stirring was maintained for 4 h. After the reaction was completed, the solution was filtrated and evaporated to dryness in a rotary evaporator to give an oily residue. To this crude ethylene phosphate ethylferrocene was added NaCN (0.49 g, 10 mmol) in dry DMF (3 mL), and the mixture was heated to 70-90 °C for 12 h under reduced pressure (30-60 mmHg). Afterward, the reaction mixture was lyophylized (2 days under vacuum) to afford a brown viscous oil which was dissolved in 20 mL of hot EtOH, and then acetone was added to precipitate a yellow powder 0.76 g (47%): NMR 1H (400 MHz, CD3SOCD3) δ 4.25 (s, 5H), 4.28 (s, 2H), 4.02 (s, 2H) (ferrocenyl), 3.45 (q, 2H) (CH2O), 2.50 (t, 2H) (CH2). Anal. Calcd for C13H13FePO4Na2‚2H2O: C, 36.95; H, 4.39; P, 7.94. Found: C, 36.61; H, 4.31; P, 8.62. Buffers and Solutions. Phosphate-buffered saline (PBS; 4.3 mM NaH2PO4, 15.1 mM Na2HPO4, and 120 mM NaCl, pH 7.4) and Tris buffer (TB; 50 mM Tris, 1 mM MgCl2, 50 mM NaCl, and 0.02% (w/v) NaN3, pH 9.0) were prepared. A coating solution (15 mM Na2CO3, pH 9.6) and a blocking buffer [0.3% (w/v) BSA in PBS] were used for the avidin assay. Avidin standard solutions in PBS (0.1-10 µg mL-1) were prepared from a concentrated solution (1 mg mL-1 in PBS), and a B-AP solution (1 unit mL-1) was prepared from a B-AP stock solution (100 units mL-1 in PBS, 1% BSA, and 0.02% NaN3). A stock solution 10-3 M of FcEtOPO3Na2 was prepared by dissolving 0.80 mg of the salt in 2 mL of TB. This solution was stored at 4 °C for up to 2 weeks. Fresh substrate working solutions were prepared just before use by dilution (50-fold) of the stock solution in TB. Equipment and Electrochemical Measurements. An EG&G PAR 273 potentiostat interfaced to an IBM XT 286 computer system with a PAR M270 software was used for cyclic voltammetry (CV). The electrochemical experiments were carried out at room temperature in a small one-compartment glass cell with a working volume of 1 mL. The Nafion film-coated GC electrode surface was prepared as already described.25 The film thickness was controlled by varying the concentration of the Nafion solution (0.1-1%) and the syringed droplet volume, and it was estimated by assuming a Nafion density of 1.58 g cm-3.26 A platinum wire counter electrode and an Ag/ AgCl (0.05 mol L-1 NaCl) reference electrode were introduced into the cell in addition to the modified working electrode. (22) Lombardo, A.; Bieber, T. I. J Chem. Educ. 1983, 60, 1080-1081. (23) Lednicer, D.; Lindsay, J. K.; Hauser, C. R. J. Org. Chem., 1958, 23, 653655. (24) Thuong, N. T.; Chabrier, P. Bull. Soc. Chim. 1975, 9/10, 2083-2088. (25) Limoges, B.; Degrand, C.; Brossier, P.; Blankespoor, R. L. Anal. Chem. 1993, 65, 1054-1060. (26) Mauritz, K. A.; Hora, C. J.; Hopfinger, A. J. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1978, 19, 324-329.

For the accumulation procedure, the rotating disk-modified electrode was exposed to the solution and immediately rotated at 600 rpm for 5 min (the electrode is rotated to enhance mass transfert). During this exposure time, the cationic exchange was accomplished by applying a potential positive to the ferrocene oxidation, i.e., typically 0.3 V, before the CV curve was recorded at the stationary Nafion electrode. The Nafion film regeneration procedure was carried out at a rotating electrode by applying a cathodic potential of -0.1 V for 2 min unless otherwise stated. Determination of Alkaline Phosphatase Using Ferrocene Ethyl Phosphate Ester as Substrate. To (990 - x) µL of TB were added x µL of AP solution (520 units L-1 diluted in TB) and 10 µL of FcEtOPO32- (10-3 M in TB) into the electrochemical cell. The stopwatch was then immediately started, and the modified electrode was introduced into the cell where the enzyme reaction proceeded at room temperature. At defined time intervals during the course of the enzyme reaction, the electrode was rotated at 600 rpm under polarization at -0.1 V for 2 min and 0.3 V for 5 min, before the CV curve (potential scan rate of 100 mV s-1) was recorded at the stationary Nafion electrode. The resulting cathodic peak current was taken as the analytical response. Determination of the Enzyme Kinetic Constants for the Ferrocene Ethyl Phosphate Ester. The enzyme hydrolysis of FcEtOPO32- was followed by assaying inorganic phosphate release from the adsorbance of a blue phosphomolybdate complex. The procedure is similar to the method applied by Hall and Williams27 and is as follows: to (985 - x) µL of TB were added x µL of FcEtOPO32- (5 × 10-2 M in TB) and 16 µL of AP solution (3 × 10-8 M in TB). The substrate concentrations ranged from 5 to 0.05 mM, and the final enzyme concentration per assay was ∼5 × 10-10 M. The enzyme reaction was allowed to proceed for 10 min, and then 100 µL of acetic acid (2 M) was added to stop the enzyme reaction and bring the pH to ∼4. Then, 50 µL of ascorbic acid (10% solution), 50 µL of ammonium molybdate (10% solution), and 3 mL of acetate buffer at pH 4 were added to the mixture. The color was allowed to develop for 10 min, and the adsorbance was measured at 660 nm with a Kontron spectrophotometer (Uvikon, Model 941). The assay was calibrated against the standard inorganic phosphate solutions. The Michaelis constant Km and the maximal velocity Vm were deduced from the linear least-squares regression of the Lineweaver-Burk plot.28 Heterogeneous Enzyme Assay of Avidin. Immobilization of B-BSA on the walls of reagent tubes was carried out by distributing 1 mL of B-BSA (5 µg mL-1 in coating solution) to each Maxisorp NUNC tube kept at 4 °C for 15-20 h. The tubes were washed once with PBS (1.05 mL), incubated for 1 h with 1 mL of blocking buffer, and washed three times again with PBS. Samples (1 mL) containing known concentrations of avidin (0.110 µg mL-1) were added to the tube and incubated for 30 min at room temperature. The tubes were washed three times with PBS (1.05 mL), a B-AP solution (1 mL, 1 unit mL-1) was added, and the resultant mixture was incubated for 30 min at room temperature. The tubes were washed three times with Tris buffer (1.05 mL) and then a substrate working solution (1 mL of 20 µM FcEtOPO32- in TB) was added. After incubation for 2 h at room (27) Hall, A. D.; Williams, A. Biochemistry 1986, 25, 4784-4790. (28) Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56, 658-666. (29) Nesmeyanov, A. N.; Reshetova, M. D.; Perevalova, E. G. Izv. Akad. Nauk SSSR, Ser. Khim. 1967, 12, 2746-2748. (30) Dixneuf, P. Tetrahedron Lett. 1971, 19, 1561-1563.

temperature, 800 µL of solution was transferred into a glass tube and mixed with 200 µL of PBS. Each solution was then successively assayed using the accumulation procedure. RESULTS AND DISCUSSION An attempt to synthesize the ester phosphate of ferrocene methanol by applying the patented procedure given by McNeil and Bannister, i.e., phosphorylation of ferrocene methanol in pyridine with POCl3,20 failed. The elementary analysis of the compound obtained, its melting point, and its NMR spectrum indicate that it corresponds to a ferrocenylmethylpyridinium salt (see the Experimental Section) whose synthesis under similar conditions is described elsewhere.21 Clearly, the procedure described in ref 20 does not lead to ferrocenylmethyl phosphate, but to a ferrocenylmethylpyridinium salt. This is why we tried another phosphorylation method24 to synthesize the ester phosphate of ferrocene methanol, but it failed again. In fact, it was not surprising, because it was previously shown that the hydroxyl group of ferrocene methanol derivatives can be easily removed to produce a highly stabilized carbocation29,30 inclined to a nucleophilic attack. To circumvent this problem, ferroceneethanol was selected, because the formation of a stabilized cation is prevented during the phosphorylation step, and so the synthesis of the corresponding phosphate ester sodium salt was successfully achieved by using the general phosphorylation method described by Thuong and Chabrier.24 Behavior of Ferroceneethanol at a Nafion-Coated Electrode. The CV behavior of FcEtOH was compared at a Nafion film-coated electrode and a naked glassy carbon electrode, under conditions advantageous for the enzyme reaction of AP, i.e., in pH 9.0 TB solution containing 1 mM MgCl2.6-15 Figure 1A shows the continuous CVs recorded after immersion for 2 min of a Nafion-coated electrode in a 2 × 10-5 M FcEtOH solution and at an applied potential of 0.3 V. The anodic potentiostatic conditions allow for the generation of ferricinium cation, which is immediately trapped by ion exchange within the polymer film and thus accumulated. The first recorded cyclic curve shows that the anodic peak current (ip,a) on the reverse sweep is much lower than the cathodic peak current (ip,c), which shows that the neutral form of FcEtOH tends to be released from the film. Indeed, the signal continuously decreases upon repetitive cycling due to a continuous release of the neutral form of ferrocene from the polymer until it reaches a steady state limit (not shown). This limit corresponds to an equilibrium between the preferred incorporation of the ferricinium and the expulsion of the ferrocene (neutral form). A similar decrease was previously observed for ferrocene and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy,31 but it was slower in the former case and faster in the latter case. This can be explained by an intermediate balance of the hydrophobic/ hydrophilic nature of the redox molecules. If the accumulation is performed at a potential of -0.2 V where the ferrocene remains in its neutral form, the resulting signal is similar to the steady state limit and does not differ significantly from a bare electrode. This shows that neutral FcEtOH has a low affinity for the polymer whereas its oxidized cationic form possesses a higher partition coefficient. This property makes it possible to regenerate the Nafion film by cathodic polarization as shown in Figure 1B, where curve a is the CV signal obtained after anodic preconcentration (31) Limoges, B.; Degrand, C.; Brossier, P. J. Electroanal. Chem. 1996, 402, 178-187.


4143

Figure 2. Dependence of the peak current (scan rate, 100 mV s-1) on the accumulation time for several film thickness of 0.23 (1), 0.63 (2), 0.78 (3), 1.10 (4), 1.56 (5), 1.88 (6), 2.50 (7), and 3.20 µm (8). The accumulation proceeded at 0.3 V and 600 rpm in TB solution of 1 µM FcEtOH. Between each measurement the modified electrode was cleaned for 2 min at -0.1 V and 600 rpm. Figure 1. CV curves (scan rate, 100 mV s-1) at the same Nafion film electrode (film thickness, 1.6 µm) immersed in TB solution (pH 9.0) of 20 µM FcEtOH (A) and of 2 µM FcEtOH (B). The accumulation step (5 min at 0.3 V) at a rotated electrode (600 rpm) was followed by (A) continuous cycling (30 cycles) and (B) cathodic cleaning (2 min at -0.3 V and 600 rpm): (a, c, d) after accumulation step; (b) after cleaning.

of FcEtOH (10-6 M in TB) for 5 min and curve b the resulting CV signal after cathodic polarization for 2 min. Two more accumulation/detection/regeneration cycles (curves c and d) were successively carried out, and the corresponding CVs overlaid almost perfectly curve a. It is clear from these results that the accumulated ferricinium ethanol molecule can be completely released under its neutral form from the Nafion polymer. Only a small cathodic signal remains visible on curve b due to the presence of the electroactive species in solution. Hence, the modified electrode can be reused for several measurements for an analytical purpose. Repetitive accumulations (cleaning pretreatment at -0.3 V for 2 min followed by preconcentration at 0.3 V for 2 min and 600 rpm) were carried out at a same Nafion film electrode (film thickness of 0.8 µm) immersed in TB solution of 5 µM FcEtOH. The relative standard deviation of the peak current was only ∼3% for the first 10 measurements, and it was mainly associated with a systematic error deviation (average decrease of 0.7% per measurement). Moreover, this slow decrease of the peak current was enhanced when the electrode was left to dry in air between each measurement, and consequently, the modified electrode was held in a pure TB solution between experiments. This decrease cannot be attributed to a redissolution of the Nafion film,31 but it seems likely that it results from a slow microstructural change of the Nafion polymer, which would be consistent with a previously published study.32 Indeed, it was shown that the microrheology of Nafion films changes with the electrochemical mass transport of molecules such as p-hydroquinone.32 Moreover, it was observed33 that ionic transport within the [Os(bipy)2(PVP)10(32) Muramatsu, H.; Ye, X.; Ataka, T. J. Electroanal. Chem. 1993, 347, 247255. (33) Clarke, A. P.; Vos, J. G. J. Electroanal. Chem. 1993, 356, 287-293.

4144 Analytical Chemistry, Vol. 68, No. 23, December 1, 1996

Cl]2+/+ redox polymer [bipy ) 4,4′-bipyridyl, PVP ) poly(vinylpyridine)] induces a very slow polymer relaxation which, consequently, provides a “memory” effect that depends on the previous history of the polymer. By analogy with these results, the Nafion polymer would have a memory of its previous history after repeated accumulation/regeneration periods, probably due to slow polymer relaxation. A study using an electrochemical quartz crystal balance should clarify this. A plot of log [ip,c ] vs log [v], where v is the scan rate, was performed at an electrode with a 1.6 µm Nafion film thickness immersed in TB solution of 1 µM FcEtOH. For each measurement the accumulation step (5 min at 0.3 V and 600 rpm) was preceded by a cleaning period (2 min at -0.1 V and 600 rpm). At low scan rates (v < 200 mV s-1), ip,c varies linearly with v(log[ip,c] ) 0.995 log[v] + 0.939 when ip,c is expressed in µA and v in V s-1), which indicates a thin-film behavior (finite diffusion). At higher scan rate, ip,c vs v1/2 is linear (log[ip,c] ) 0.453 log[v] + 0.668), and so semi-infinite diffusion conditions prevail. Nafion Film Thickness. We have previously shown31 that the sensitivity at the Nafion-coated electrode can be modulated by the film thickness, the preconcentration time, and the film processing, and we have found that there exists an optimum film thickness which depends on the nature of the incorporated molecule, i.e., its Nafion/water partition coefficient and its diffusion coefficient in the polymer phase. We took advantage of the reusable Nafion-modified electrode to study thoroughly these parameters in the case of FcEtOH and, consequently, to determine the film thicknesses providing the highest sensitivity for a defined accumulation time. Figure 2 shows the influence of the accumulation time on ip,c for different film thickness, l, and Figure 3 gives a plot of ip,c vs l for a constant accumulation time of 5 min (corresponding to the dashed line in Figure 2). A constant-current is reached rapidly for the thinner Nafion films in Figure 2. On the other hand, a long time is necessary for the thickest films. The constant current results from the equilibrium distribution (34) Espenscheid, M. W.; Chatak-Roy, A. R.; Moore, R. B.; Penner, R. M.; Szentirmay, M. N.; Martin, C. R. J. Chem. Soc., Faraday Trans 1 1986, 82, 1051-1070. (35) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1980.

would be measured without film and it is given by

is ) 0.62nFACsDs2/3ν-1/6ω1/2

(3)

where ν is the kinematic vicosity, ω is the rotation speed of the electrode, and Ds is the diffusion coefficient of FcEtOH in solution. Current if represents the limiting current through the film and it is expressed by

if ) nFACsKDf /l

(4)

Df is the diffusion coefficient of FcEtOH in the polymer film. Current iχ must be considered when the extraction of FcEtOH is not at equilibrium and it is given by

iχ ) nFACsχf Figure 3. Peak current vs Nafion film thickness for a constant accumulation time of 5 min at 0.3 V and 600 rpm in TB solution of 1 µM FcEtOH. Inset: plot of peak current vs the inverse of film thickness (thicker film, >1.5 µm). Error bars represent the standard deviation for peak currents at three different electrodes. The straight lines were obtained by linear regression fitting.

(5)

(partition coefficient, K)34 of the electroactive species between the solution and the polymer. Assuming that the cathodic peak current is associated with a finite diffusion for the considered scan rate (100 mV s-1), it is expressed by35

where χf is the heterogeneous rate of mass transfer across the film/solution interface. Relation 2 was previously applied in the study of the accumulation of FcTMA+ under open circuit at the Nafion film electrode.38 In the case of thick films and short accumulation times, the equilibrium partition is not reached, and so it can be considered that all the FcEtOH molecules that reach the electrode surface are oxidized and thus entrapped by ion exchange within the film. In the limit case of high ω values the term 1/is becomes negligible, and so the whole limiting current can be approximated by

ip ) (n2F2/4RT)vVCf

1/il ) 1/if + 1/iχ

(1)

where n, F, R, and T have their usual meaning, V is the film volume (V ) Al, with A the electrode disk area), Cf is the concentration of ferricinium ethanol in the Nafion film polymer (Cf ) KCs, with Cs the concentration of FcEtOH in the solution). The limiting peak current is thus directly proportional to the concentration Cf of ferricinium ethanol accumulated within the film. When the extraction equilibrium is reached, the concentration in the film remains constant whatever the film thickness, and so the peak current is directly proportional to the volume of the film and, consequently, to the film thickness, as shown by the ascending linear part of the curve for thin films in Figure 3. It is possible to deduce the partition coefficient K for ferricinium ethanol from the slope of the regression line, and so the calculation provides a value of ∼1500. The same K value (1500) was obtained for the (ferrocenylmethyl)trimethylammonium iodide salt (FcTMA+) in Nafion by a coulometric technique.36 For the descending part of the curve in Figure 3, a linear relationship is observed between ip,c and 1/l (see the inset), which can be reasonably explained as follows. During the accumulation step at the rotated disk electrode polarized at 0.3 V, an overall limiting current il is flowing and it is controlled by the maximum rate at which the electroactive species can penetrate through the film to reach the electrode surface. This current is related to three characteristic currents is, if, and iχ by37

1/il ) 1/is + 1/if + 1/iχ

(2)

Current is corresponds to the steady state Levich current that

(6)

Assuming that χf is constant during the preconcentration step, relation 6 can be simplified as

il ) if + cst

(7)

In other words, il is inversely proportional to 1/l, and thereby the corresponding amount of ferricinium units entrapped within the film must be inversely proportional to the film thickness. The ip,c values measured after 5 min of accumulation for thick films reflect this amount, which would explain the linear relationship between ip,c and 1/l (inset of Figure 3). Moreover, we have verified that ip,c does not change with the rotation speed within the range 300-3200 rpm, which justified a posteriori the approximated relation 6. Figure 3 shows that the best sensitivity for the detection of FcEtOH is obtained for a film thickness of ∼1.6 µm. This value is higher than the values found previously with other ferrocene derivatives (∼0.4 µm) corresponding to ferrocene-labeled drugs,31 probably because FcEtOH is a smaller molecule which would diffuse more rapidly through the polymer film and/or would possess a lower partition coefficient. The value of the potential applied during the preconcentration time has a strong influence upon the sensitivity. The peak current (accumulation of 5 min) of FcEtOH (1 µM in TB) slightly (36) White, H. S.; Leddy, J.; Bard, A. J. J. Am. Chem. Soc. 1982, 104, 48114817. (37) Leddy, J.; Bard, A. J.; Maloy, M. T.; Save´ant, J. M. J. Electroanal. Chem. 1985, 187, 205-227. (38) Fan, Z.; Harrison, D. J. Anal. Chem. 1992, 64, 1304-1311.


4145

Table 1. CV Characteristics of FcEtOPO32- and FcEtOH at a Bare GC Electrode (C ) 10-3 M, v ) 100 mV s-1, pH 7.4)

FcEtOPO32FcEtOH

∆Ep (mV)

E1/2 (mV)

ip,a (µA)

ip,a/ip,c

90.8 97.0

36.0 75.8

10.30 11.45

0.990 0.987

Figure 4. CV curves (scan rate, 100 mV s-1) at a same Nafion film electrode immersed in TB solution of 10 µM FcEtOPO32- (A); 1 µM FcEtOH (B); and 10 µM FcEtOPO32- + 1 µM FcEtOH (C). For each measurement the accumulation step (5 min at 0.3 V and 600 rpm) was preceded by a cleaning period (2 min at -0.1 V and 600 rpm). Inset: effect of the increasing concentration of FcEtOPO32- on the peak current of FcEtOH (1 µM in TB). The measured value corresponds to the peak current obtained after substraction of the FcEtOPO32- blank signal.

increased from 0.6 to 0.8 µA as the applied potential was raised from 0.1 to 0.3 V, then it became constant between 0.3 and 0.5 V, and the current value decreased abruptly as the applied potential was higher than 0.55 V to reach a low intensity of 0.2 µA at 0.7 V. These results are consistent with a previous study performed with other procationic compounds.31 An accumulation potential of 0.3 V was chosen for this study. Nafion Film Selectivity. The CV characteristics [difference of the peak potentials ∆Ep ) (Ep,a - Ep,c), half-wave potential E1/2 ) (Ep,a + Ep,c)/2, peak currents ip,a and ip,c] of FcEtOPO32- and FcEtOH at a bare GC electrode are indicated in Table 1, which shows that the substrate/product couple FcEtOPO32-/FcEtOH is not adapted for enzyme assays at a bare GC electrode. Indeed the two molecules have close E1/2 values and comparable current responses. Hence, it is clear that an enzyme assay is not possible since the electrochemistry of the parent compound and of the hydrolysis product is not sufficiently different. However, the substrate/product couple is a good candidate at a Nafion-modified electrode, as is depicted in Figure 4. The polyanionic film acts as a good electrostatic barrier against the ester phosphate (Donnan exclusion) since FcEtOH (curve A) is preferentially preconcentrated compared with FcEtOPO32- (curve B). In Figure 4, the peak intensity with FcEtOPO32- is lower than with FcEtOH although the ester phosphate is 10 times more concentrated. In other words, the ratio ip,c /Cs is ∼90 times higher with the alcohol than with the ester phosphate. Curve C of Figure 4 indicates that the reduction peak of FcEtOH decreases when some substrate is added; i.e., the higher the ester phosphate concentration in solution, the lower the FcEtOH detection peak (inset in Figure 4146 Analytical Chemistry, Vol. 68, No. 23, December 1, 1996

Figure 5. Calibration curves of FcEtOH at a bare electrode (A) and at the same Nafion film electrode (B, C). For curves B and C, the accumulation/cleaning procedure was the same as in Figure 5. Curve C was obtained in the presence of FcEtOPO32- and for a total concentration (FcEtOPO32- + FcEtOH) of 10 µM. The dashed line corresponds to the blank signal of 10 µM FcEtOPO32- at the Nafion film electrode, and error bars represent the standard deviation for two peak current measurements.

4). A plausible explanation would be a slow electrochemical crossreaction taking place at the interface film/solution or inside the film, between the accumulated ferricinium salt and the substrate molecule during the preconcentration step. Indeed, Table 1 indicates that the E1/2 potentials of FcEtOH and its ester phophate are favorable to such electron exchange. Therefore, the quantity of ferricinium ethanol entrapped within the film during the accumulation time should be lowered. Another possibility is that FcEtOPO32- may likely exert a barrier against FcEtOH diffusion toward the modified electrode. A rather low concentration of substrate (10-5 or 2 × 10-5 M) was further selected in AP assays to minimize this effect. Sensitivity. Figure 5 compares the calibration curves of FcEtOH (logarithm scale) at a bare electrode (curve A) and at a reused Nafion film electrode (curve B, more than 20 measurements with the same modified electrode). A detection limit (signal/noise ) 3) of 2 × 10-8 M FcEtOH was achieved at a Nafion film electrode, whereas the linear response range (slope 1) was relatively narrow (5 × 10-8-10-6 M) since the slope decreased at higher concentration. The sensitivity (linear part of the curve) at the modified electrode is amplified 90 times compared with the bare electrode. It was useful to examine the calibration curve of FcEtOH in the presence of FcEtOPO32- since the ester phosphate interferes on the FcEtOH accumulation (Figure 4). The total concentration was kept constant (10-5 M) in order to mimic the enzyme conditions when FcEtOH is catalytically generated from FcEtOPO32(curves C in Figure 5). The peak current response of FcEtOH at low concentrations is lower than in the absence of FcEtOPO32(compare curves B and C), as expected from the ester phosphate interference, and surprisingly, it leads to a linear relationship up to 10-5 M. A detection limit (signal/blank ) 3) of ∼2 × 10-7 M FcEtOH could be estimated by extrapolation of curve C in Figure

Figure 6. CV curves (scan rate, 100 mV s-1) at a same Nafion film electrode immersed in TB solution containing 10 µM FcEtOPO32- (04) and 7.8 × 10-11 M AP (1-4), and for an incubation period of 7 (1), 22 (2), 37 (3), and 52 min (4). The accumulation/cleaning procedure was the same as in Figure 5.

5 and by considering the blank signal of 10-5 M FcEtOPO32(horizontal dashed line). AP Assay. Figure 6 shows the successive CV curves obtained at a Nafion-coated electrode immersed in a solution containing AP (7.8 × 10-11 M) and FcEtOPO32- (10-5 M) in TB (pH 9.0) at room temperature. As expected, the peak current increases during the course of the enzyme reaction due to the hydrolysis of FcEtOPO32- to FcEtOH. A series of experiments was carried out at various AP concentrations (CAP), and the results are plotted in Figure 7. The peak current of curves 1-4 increases linearly with the incubation time, which is consistent with a pseudo-firstorder kinetic reaction. The deviation observed at high CAP (curve 5) indicates a decrease of the enzyme reaction rate when a significant amount of ester phosphate is transformed. The slope δip,c/δt was determined for each curve in Figure 7, and a graph δip,c/δt vs CAP was plotted and a linear relationship was thus obtained (inset in Figure 7). A detection limit of 2 × 10-12 M AP is estimated, which is ∼100 times less sensitive than previously obtained with N-ferrocenoyl-6-amino-2,4-dimethyl phosphate as substrate.16 This difference is due to the well-known lower enzyme hydrolysis rates of the aliphatic ester phosphates compared with the aromatic ester phosphates.27 To confirm this, the enzyme kinetic constants were determined for FcEtOPO32- (see the Experimental Section), and so values of 0.75 mM for the Michaelis constant Km and 1.42 µmol s-1 (mg of protein)-1 for the maximal velocity Vm were found. The pseudo-first-order catalytic constant kcat found to be 227 s-1 was derived from Vm, assuming an enzyme molecular weight of 160 000. The Km value is typical of an aliphatic ester phosphate27 and is ∼2 orders of magnitude higher than the Km values obtained for aromatic ester phosphates, which is a disadvantage from an analytical view point since higher amounts of enzyme product are generated when Km is low. However, the possibility to perform the assay directly in the basic working

Figure 7. Reaction progress curves for AP at a Nafion film electrode immersed in TB solution containing 10 µM FcEtOPO32- and different AP concentrations of 0 (0), 3.7 × 10-12 (1), 7.8 × 10-12 (2), 2.3 × 10-11 (3), 7.8 × 10-11 (4), and 2.3 × 10-10 M (5). The accumulation/ cleaning procedure was the same as in Figure 5. Inset: kinetic calibration curve of AP.

solution is an advantage over the previous system for which a supplementary pH change step was necessary.16,17 Moreover, this advantage offers the possibility of performing the accumulation step during the whole enzyme incubation period, which should increase the sensitivity and compensate at least partially for the slower enzyme hydrolysis. Heterogeneous Enzyme Assay of Avidin. The development of avidin-biotin technology has unified and simplified a variety of affinity sensing methods, and the avidin-biotin couple has been introduced in various indirect labeling procedures for immunoassays.1,39,40 The first reason is the remarkable specific affinity between the two molecules (dissociation constant of ∼10-15).39 A second reason is the presence of four identical subunits on avidin, each of them being able to bind one biotin, and so an amplified signal can be generated. A third reason is the exceptional stability of avidin protein, amenable to a wide variety of modifications and conjugations with many compounds, without disturbing the biotinbinding properties. Among the variety of assays available in the field of the avidinbiotin technology, we have focused the last part of this work on the quantitative determination of avidin. The assay is based on a “sandwich” heterogeneous assay,39 and it is a good model for the analytical evaluation of the technique coupled to the FcEtOPO32substrate. A representative avidin assay is shown in Figure 8. As expected, the peak current increases with the concentration of (39) Bayer, E. A.; Ben-Hur, H.; Wilchek, M. In Methods in Enzymology; Wilchek, M., Bayer, E. A., Eds.; Academic Press: New York, 1990; Vol. 184, Chapter 23, pp 217-222. (40) Re´my, I.; Brossier, P. Analyst 1993, 118, 1021-1025.


4147

Figure 8. Calibration curve of avidin (see the Experimental Section for details).

avidin. Nonspecific interactions were negligible considering the low blank current response (mainly due to the substrate detection). The current tends to be constant at high avidin concentration, due to the saturation of B-BSA on the tube wall. The effective range of quantification was varied within 2 orders of magnitude, and the sensitivity was approximately the same as the colorimetric AP assay of avidin with p-nitrophenyl phosphate as substrate under similar incubation conditions.39 The detection limit was estimated to be 5 × 10-12 M avidin after an enzyme incubation time of 2 h. The sensitivity and the detection limit are good compared with the colorimetric method if we consider that the enzyme hydrolysis is slower for FcEtOPO32- than for p-nitrophenyl phosphate, which is known to be one of the best substrates for AP in term of activity.27 Conclusion. The stripping analysis procedure of FcEtOH at a regenerable Nafion-modified electrode is promising for the indirect determination of very low concentrations of AP or AP label. The results reported in this study and in the previous works16,17 establish that a large variety of phosphate esters is suitable for the sensitive electrochemical detection of AP, insofar as they form cationic or procationic electroactive products by AP (41) Vreeke, M.; Rocca, P.; Heller, A. Anal. Chem. 1995, 67, 303-306.

4148


hydrolysis. Moreover, these results confirm the possibility of discriminating the electrochemical detection of an anionic substrate from its neutral enzyme product at a selective Nafion electrode, although their electrochemistry is similar. The practical cathodic cleaning of the Nafion electrode allows one to reuse the same electrode. The sensitivity can be increased by prolonged enzyme incubation as the parent compound selected for this study and its hydrolysis product are stable. Indeed curve 0 of Figure 7 indicates that spontaneous hydrolysis of FcEtOPO32- is very slow. Moreover, the peak current of 1 µM FcEtOH in TB did not change significantly after more than 24 h at room temperature. This is an advantage compared with the unstable p-aminophenol usually employed.7-10,12,13,15 The sensitivity and the detection limit are adequate for many common assays, though not at the cutting edge of immunoassays. Further improvements in sensitivity can be expected by utilizing microelectrodes, and this possibility is presently being explored. Moreover, lower current responses can be fulfilled if FcEtOH is quantified at a Nafion film electrode inserted in an amperometric cell for high-performance liquid chromatography or flow injection, although the analysis complexity and cost are increased. The avidin-biotin-based procedure allows flexibility in the detection method employed. This makes it particularly suitable for further developments of electrochemical detection systems in immunoassay. Moreover, the avidin-biotin couple can be directly used for immobilization of reagents on the electrode surface to provide an affinity sensor, where no separation step is required.41 ACKNOWLEDGMENT We gratefully acknowledge Professor P. Brossier at the Faculte´ de Pharmacie, Universite´ de Bourgogne, for his useful comments and suggestions relative to this work. Received for review June 12, 1996. Accepted September 10, 1996.X AC9605760 X

Abstract published in Advance ACS Abstracts, October 15, 1996.

Ferrocenylethyl Phosphate: An Improved Substrate for the Detection of

Recommend Documents